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NATURE AT WORK: ONGOING SAGA OF EVOLUTION

Publication of The National Academy of Sciences, India

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CONTENTS Title

Page No.

Foreword Preface

v vii

Prof. Asis Datta, President, The National Academy of Sciences, India

Legacy Continuum 1.

The descent of humans and the Darwinian unification of all life P. Dayanandan

2.

Ida: A link to human evolution V.P. Sharma

21

3.

Darwin’s theory of evolution: Survival of nature’s fit! Veena Tandon and Gaurangi Maitra

33

4.

The life and research of JBS Haldane in India with special reference to Charles Darwin Krishna R. Dronamraju

5.

Charles Darwin: A driving force for humanity toward agnosticism Amit Sharma

1

51 59

Viruses, Microbes and Fungi 6.

Phylogeographic evolution of plant viruses Anupam Varma and Shelly Praveen

75

7.

Evolution of HIV-1 in India Pradeep Seth

93

8.

Darwin and microbial evolution T.K. Adhya and M. Patra

9.

Evolutionary relationships among cyanobacteria, algae and plants: Revisited in the light of Darwinism Radha Prasanna and B.D. Kaushik

10.

Biodiversity, phylogeny and evolution of fungi C. Manoharachari, I.K. Kunwar and S. Vishnuvardhan Reddy

103

119 141

Insects: Evolution in Action 11.

The origin of reproductive isolating mechanisms is an important event in the process of speciation: Evidences from Drosophila B.N. Singh

159

12.

Adaptive radiation and insects V.V. Ramamurthy and Asha Gaur

175

13.

Insights from mosquito evolution: Patterns, tempo and speciation Karamjit Singh Rai

197

14.

The saga of pollination biology Rajesh Tandon and H.Y. Mohan Ram

219

Genomics 15.

Darwinian evolution and post developments in genomics Arun Kumar Sharma

16.

Pathogen pressure and molecular evolutionary genetics of innate immunity genes in humans Partha P. Majumder

241

249

Unique Case Studies 17.

Macroevolution in relation to the drift models of the Indian plate Ashok Sahni and R.S. Loyal

18.

Testing the melanism-desiccation hypothesis: A case study in Darwinian evolution Ravi Parkash

279

Origin and evolution of human malaria parasite, P. falciparum and P. vivax Nidhi Datta and V.S. Chauhan

307

19.

267

20.

Evolutionary trends in soil-inhabiting Mahlaqa Choudhary and M. Shamim Jairajpuri

21.

Evolution of the cerebral cortex in amniotes: Anatomical consideration of neuronal types U.C. Srivastava and R.C. Maurya

329

Medicinal and aromatic plants: A case example of evolving secondary metabolome and biochemical pathway diversity Suman P.S. Khanuja, Tripta Jhang and Ajit Kumar Shasany

355

Conservation of Himalayan bioresources: An ecological, economical and evolutionary perspective Lok Man S. Palni and Ranbeer S. Rawal

369

22.

23.

Color Images Section

319

395

Foreword Charles Robert Darwin was born on 12th February, 1809 in Shrewsbury, England. Darwin shares his birthday with U.S. President Abraham Lincoln. Both were crusaders against slavery: Darwin disliked slavery and Lincoln abolished it. Darwin was a born naturalist and showed keen interest in nature from the very beginning. A breakthrough came when he was selected as a naturalist on the H.M.S. Beagle ship. His five year voyage on the Beagle started in 1931 and was completed in 1936. This was followed by publication of his research findings that challenged creationist views of the church. Darwin conducted a study of fossils and geological records and concluded rightly, that all life forms emerged over millions of years of evolution through the force of natural selection. In 1959 Darwin published his work on evolution in a book titled “On the Origin of Species by Means of Natural Selection or the Preservation of Favored Races”. The book was received as a scientific bomb shell and has since changed the human understanding of life forever. Today Darwin’s ideas on evolution provide foundation to modern biology. Darwin died of a heart attack on the 19th April 1882 and was buried in Westminster Abbey near the grave of Sir Isaac Newton. The scientific community is celebrating Darwin’s bicentenary worldwide in honor of his ingenuity, scientific thought, conviction and courage. Coinciding with the bicentenary celebrations are three major discoveries in paleontology, namely Darwinius masillae from Germany, skeleton of winged Dinosaur from China and fossilized Dinosaur eggs from India. The National Academy of Sciences, India (in short NASI) at Allahabad is paying tribute to British naturalist Charles Darwin by compilation of articles titled “Nature at Work: Ongoing Saga of Evolution”. Twenty three contributory articles bring out many facets of evolution and human understanding in this book. Articles begin with the Darwin’s legacy of the descent of man, ‘Ida’ the link to human evolution, theory of evolution, Haldane in India, and agnosticism. The second section describes the evolution of microbes, viruses and fungi affecting health and environment worldwide. Next section deals with the evolution of insects for understanding basic biology, adaptations, speciation, and interplaying with plants in pollination. The section on Genomics brings out the post-Darwinian developments in biology providing proof of unification at the molecular level and the discoveries of the cause of many genetic disorders. Finally there are seven unique case studies representing various facets of macro and micro Darwinian evolution taking examples from malaria, nematodes, amniotes, v

and implications for health and well being, hunger and food, bio-resources and conservation of biodiversity. In summary the book brings out the universality of Darwin’s concept of evolution ending in unification of life without exception from gross to molecular basis of the “Tree of Life”, a continuum. Many friends have been of great help in the preparation of this book. I wish to acknowledge the help and suggestions inter alia, 2009 Council of the NASI, Prof. M.G.K. Menon, Prof. H.Y. Mohan Ram, Prof. K.S. Rai, Prof. Anupam Varma, Dr. Amit Sharma, Prof. Veena Tandon, Prof. P. Dayanandan, Prof. R.C. Sobti, Dr. Mrs. Manju Sharma, and of course all the authors and co-authors for their excellent work on evolution. I am particularly grateful to the team of Springer India Pvt. Ltd. for their commitment and hard work that made it possible to bring out this excellent book on Darwin’s bicentenary. Finally, on my behalf and the NASI, I wish to thank Prof. Ananda Chakrabarty, distinguished Professor at the University of Illinois College of Medicine at Chicago, for the release of this book on 14th December 2009 during the 79th Annual Session of the Academy hosted by Calcutta University, Kolkata. Vinod P. Sharma CRDT, IIT, Delhi - 110016 Dated: 17th October, 2009

vi

Preface It is a pleasure that the National Academy of Sciences, India (NASI) Allahabad is bringing out a book titled “The Nature at Work: Ongoing Saga of Evolution” to celebrate bicentenary of Charles Robert Darwin. The book is edited by Dr. V.P. Sharma, former President, NASI. Charles Darwin was an English naturalist who realized and presented compelling evidences that all species of life have developed over time from common ancestors. Darwin’s scientific discoveries are the unifying theories of the life sciences. The theory of evolution really does explain everything in biology. The phenomena that Darwin understood in broad brush strokes can now be accounted for in the precise language of DNA. He established himself as an eminent geologist/biologist through his observations, theories and ideas and became famous as a popular author. This book is primarily for those who want to understand various theories of Charles Darwin. We have selected the most important parts of his writings and have added learned annotations. These annotations are highly precious and useful, especially for academics. This book is divided into five themes each clearly written by the eminent dignitaries/scientists of India. The text is well written and factual without being overwhelming. I found the information very interesting and well presented. We desire to express our gratitude to all the contributors who were kind enough to provide us relevant information on highly specialized areas of modern understanding of evolution and unification of life. I also believe that the book would provide an impression of his theories along with some new ideas which will serve as an inspiration and challenge for the professionals and enthusiast of science, young and old alike. A pressus book for personal possession, the libraries and an important item of gift of lasting value. Asis Datta President, NASI, New Delhi Dated: 14th October, 2009

vii

1 The descent of humans and the Darwinian unification of all life P. Dayanandan Department of Botany, Madras Christian College, 1/2 First Cross, MES Road, Tambaram, Chennai - 600 059 [email protected]

Abstract: The biological revolution that Darwin initiated has given us a view of an earth that was once devoid of life and where life originated and evolved from simpler pre-existing forms over immense periods of time. Darwin saw human beings as an integral part of this evolutionary history. How, when and where life originated remains an inscrutable challenge to science. However, discoveries in every branch of biology since the time of Darwin have affirmed the essential unity of life, including the 30 million or more extant species and more than 600 million extinct species. Darwin’s graphic representation of relationships between organisms is now employed to construct phylogenetic trees at all levels of biological organization. A Neanderthal fossil was the only hominin remain known at the time of Darwin. Today paleoanthropologists work with more than 20 species of hominins. The study of these fossil ancestors combined with the molecular dating techniques based on the analysis of mutations in mitochondrial and Y-chromosomal DNA have vindicated Darwin’s worldview of the origin of humans in Africa, as well as his suggestion that the great apes are the closest living ancestors of Homo sapiens. The discovery that all human beings on earth are descendants of one woman, the African ‘Eve’ who lived not more than 200,000 years ago has extended the Darwinian biological worldview into a unifying philosophical perspective. It is now possible to determine the mitochondrial and Y-chromosomal branch (haplogroup) of any human being with comparative ease. This helps in tracing the possible time and route of migration of our ancestors. The emerging consensus that our ancestors left Africa as recently as 70,000 years ago has profound implications for history, culture, linguistics, psychology and human predicament. The unification of all life, including human life is perhaps the most enduring contribution of Darwin’s evolutionary theory. Human beings are yet to fully comprehend the significance of this unification. Perhaps there is a lesson in Darwinism for how we humans should deal with each other and treat other life and nature with which we have an organic and primeval connection. Keywords: African ‘Eve’, Darwin, Evolution, Grand unification, Haplogroups, Human phylogeny, Mitochondrial DNA, Out of Africa, Tree of life, Y-chromosome

V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

2

Nature at work: Ongoing saga of evolution

Introduction One hundred and fifty years ago Darwin concluded the Origin of Species with his magnificent view of life on earth as one of organic relationship developed over immense time scales from one or a few forms of life: “There is grandeur in this view of life .... from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved.”1 Charles Wallace certainly shared an identical and independently developed evolutionary view of life. However, Wallace could not reconcile himself with Darwin’s views on the origin of human beings and how natural selection could account for the mental capacities and moral sensibilities of humans. Darwin formulated his theory based on extensive field work, meticulous observations and documentation and relentlessly pursued his views for nearly 50 years from the beginning of his exploration on the Beagle. Meanwhile the Origin of Species, first published in 1859, went through six editions. In the Origin of Species all that Darwin revealed about human evolution was this cryptic statement: “light will be thrown on the origin of man and his history.” Four years later in 1863, Thomas Huxley, who was a friend and ardent supporter of Darwin, applied the concept of evolution by natural selection to humans in his book Evidence as to Man’s Place in Nature.2 In his 1871 Descent of Man, the second most influential of his numerous publications, Darwin revealed that human beings too, were an unequivocal product of evolution.3 This in essence was a grand unification of all life. Implicit in the evolutionary theory is the still grander unification of life with nonliving matter. The origin of life was an abominable mystery then, and it continues to be no less a daunting challenge today in spite of the 1953 Miller-Urey experiment.3,4 During the past 150 years the best of biology has provided answers to the two fundamental questions that Darwin struggled with, namely the mechanism of inheritance and the process by which heritable variations occur. In turn biologists have come to assert with Dobzhansky that: “Nothing in biology makes sense, except in the light of evolution.”5 In this chapter we focus on how science has updated this extraordinary story of life on earth and the place and history of the human species; a story that was first narrated so eloquently by Darwin. In essence this updated story tells us that: life has a history measured in millions and billions of years; all species have descended in a branching pattern from common ancestors; populations can change over time and evolve into species; biodiversity is a product of evolution; all life, including the estimated 30 million living species and 600 million extinct species are related to each other; human beings who share some 500 genes with all living things are genetically related to all other life; modern humans evolved as recently as 250,000 to 200,000 years ago and all people living today share common descent from one woman, the ‘most recent maternal common ancestor’, who lived perhaps as recently as 200,000

The descent of humans and the Darwinian unification of all life

3

years ago. There is indeed grandeur in this view of life. Perhaps there can be no better tribute to Darwin in this bicentenary year than what paleoanthropologists and human evolutionary biologists and molecular geneticists are accomplishing to unravel human origins and their migratory routes by peering into fossil bones and DNA of chromosomes and mitochondria.

Darwin’s trees and unification of life Darwin made the first attempts at drawing ‘tree of life’ diagrams that initiated a powerful method of visualizing the very concept of evolution. As early as 1837/1838 Darwin drew trees (Figs.1–3) to clarify to himself the branching pattern of evolutionary divergence and extinction of species. Twenty years later another sketch of a ‘tree of life’ was the only illustration that appeared in the Origin of Species (Fig. 4). As the pre-Darwinian artificial systems of classification were replaced by classifications based on explicitly evolutionary principles, a variety of phylogenetic trees were constructed. As the available characters expanded from morphological and anatomical to biochemical and molecular data, sophisticated analytical tools such as computer assisted statistical methods of parsimony, maximum likelihood, least squares, neighbor-joining and Bayesian inference are now used in cladistics

Figures 1–3. Darwin drew these diagrams 20 years before the publication of the Origin of Species. In Figures 1 and 2 the branches represent living species and the dotted lines the extinct ancestors. Darwin noted that this tree of life should perhaps be “called the coral of life” with dead branches representing transitional forms that cannot be seen. Darwin’s comments for Figure 3 emphasized the importance of the extinction of species: “I think case must be that one generation then should be as many living as now. To do this and to have many species in same genus (as is) requires extinction. Thus between A and B immense gap of relation. C and B the finest gradation, B and D rather greater distinction” (From Darwin’s Notebook B: Transmutation of species, 1837–1838. http://darwin-online.org.uk/)

4

Nature at work: Ongoing saga of evolution

Figure 4. This diagram of the divergence of taxa is the only diagram that appeared in all six editions of the Origin of Species in the chapter on ‘Natural Selection’. The branching trees were used to explain how eleven species (A–L) of a genus can evolve and produce living lineages (A and I). Species F has remained unaltered as a living fossil. All other lineages have gone extinct. Darwin’s concept of evolutionary timescales was immense. He wrote: “The intervals between the horizontal lines in the diagram may represent each a thousand generations; but it would have been better if each had represented ten thousand generations”

and molecular systematics to construct evermore instructive trees. The legacy of Darwin’s “ever branching and beautiful ramifications” is all too evident in the new generation of trees of life such as the hyperbolic and cladogenetic trees and trees based on genome size.6 All modern phylogenetic trees of life, as the one shown in Figure 5, stand testimony to Darwin’s grand unification of all life. Trees such as this are now constructed as specialists working on various groups of organisms cooperate and pool their findings based on classical and molecular data. Darwin’s basic idea of branches diverging from common ancestors characterizes all such trees. In Figure 5 all life is shown to emerge from a common ancestor that first gave rise to Bacteria and the common ancestor of Archaea and Eucarya. The common ancestor of plants, protists, fungi and animals lived in the distant past. Plants share a more recent common ancestor with the characean green algae. Surprisingly, recent studies reveal that fungi and animals shared a common ancestor and hence fungi are more closely related to animals than plants.

The descent of humans and the Darwinian unification of all life

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Figure 5. Phylogeny of living organisms including major endosymbiotic events. (Courtesy: Dr. J. Peter Gogarten, Department of Molecular and Cell Biology, University of Connecticut)

Most biologists now consider that endosymbiosis7 was a powerful mechanism responsible for the evolution of some major groups during early periods. Darwin, of course, was not aware of this evolutionary force. The diversification of all eukaryotes became possible only after a common ancestor of Eucarya acquired a Proteobacterium-like cell as the mitochondrial symbiont. The acquisition of cyanobacterial photosynthetic endosymbionts was responsible for further evolution and diversification of protists and plants. Interestingly, green and red algal cells became secondary plastids in at least six different endosymbiotic episodes. We now have a view of life where Darwin’s unification is seen to be further fortified by endosymbiotic exchanges that unite many millions of species, including humans. In effect, all eukaryotes are chimeras. In a surprising historical twist, a billion years after our ancestral eukaryotes acquired mitochondria, scientists are now probing the DNA of these bacteria-turned-mitochondria to trace human origins and the routes of migration from the African homeland.

Africa and the great apes Darwin looked towards Africa for the progenitors of humans. In his 1871 Descent of Man Darwin reasoned: in any great region living species are closely related to extinct forms; chimpanzee and gorilla are the nearest allies of Homo sapiens;

6

Nature at work: Ongoing saga of evolution

Africa is where these great apes are living today; Africa must have been formerly inhabited by extinct apes related to the chimpanzee and gorilla; therefore “it is somewhat more probable that our early progenitors lived on the African continent than elsewhere.”8 The only human ancestral fossil known when Darwin published the Descent of Man was a Neanderthal skull discovered in 1856 in the Neander Valley, Germany. Some earlier finds from Belgium were later identified as Neanderthal fossils. Darwin died in 1882. Later discoveries confirmed that Neanderthals were indeed members of human lineage and they were once widespread in Europe. This prompted a search for fossil hominins leading to the discovery of Homo erectus in Java (1890) and China (1920s) and Australopithecus africanus in Zambia (1924). Since the 1960s when Louis and Mary Leakey made initial discoveries at Olduvai in Northern Tanzania, increasingly abundant fossils are being unearthed. Darwin would not be surprised if he were to see this array of fossils linking modern humans to ancestral forms in Africa. With more than 20 species of hominins already described and more that are likely to be unearthed, Darwin can have a hearty laugh at his caricature as a missing link! It is of interest to learn that recent molecular studies of hominoids (humans, gibbons, and the great apes - orangutan, chimpanzee and gorilla) have led to reassessment of affinities and proposals for new classification schemes.9 Indeed, some have argued for considering the chimp (Pan) as a member of the genus Homo. Studies on single nucleotide polymorphism reveal that human DNA is about 98.4% identical to that of chimpanzees. In traditional classification schemes only humans and their fossil ancestors are included in the family Hominidae and are referred to as hominids. The discovery of closer genetic affinity between humans and chimps has broadened the concept of hominids to include lineages of humans and the great apes (chimps, gorillas and orangutans) as members of the family Hominidae. The lineages of humans and the African great apes (but not orangutans) are all members of a subfamily Homininae. In this new classification scheme humans (Homo) and related bipedal fossil genera in human lineage such as Ardipithecus, Australopithecus, Paranthropus and Kenyanthropus, and possibly the early genera Sahelanthropus and Orrorin are members of the tribe Hominini, hence the common name hominins. Human beings are members of the primates group. In this bicentenary year of Darwin a 47 million-year-old fossil, Darwinius masillae, nicknamed Ida, is on display and reveals the deep ancestry of humans tracing all the way back to prosimians such as lemurs (See chapter 2 by Prof. V.P. Sharma in this volume). As shown in Figure 6 primate evolution began about 55 million years ago (mya), in early Eocene epoch. Humans and apes are tailless and share similar brain and body organization as well as complex social life. Several fossils collectively referred to as Proconsul that lived 27–17 mya in Africa and presumed to be early apes or their ancestors suggest that the Old World monkeys and the apes diverged from

The descent of humans and the Darwinian unification of all life

7

a common ancestor some 20-18 mya, in the early Miocene epoch. It was during the terminal Miocene epoch that the ape lineages separated from each other and the evolution of hominins accelerated. Molecular studies of hominoids suggest that the gibbons (Family Hylobatidae) and great apes (Family Hominidae) lineages diverged about 18–12 mya. The African great ape hominid lineage diverged from the orangutan hominid lineage about 12 mya. The Indian fossil Ramapithecus is considered to be an early genus in orangutan ancestry. Some believe that Pierolapithecus catalaunicus that lived about 13 mya might have been a common ancestor of the great apes and humans. The fossils Nakalipithecus from Kenya and Ouranopithecus from Greece appear to resemble the common ancestor of gorilla, chimpanzees and

Figure 6. A simplified family tree of primate evolution

humans. The human lineage emerged between 8 and 5 mya when first the gorilla and then the chimpanzee lineages separated from the hominin tribe. The earliest hominins might have been Sahelanthropus (7 mya) and Orrorin (6 mya). While chimpanzees and humans are closest living relatives, humans did not evolve from extant chimpanzees; both lineages had evolved independently over the past 6–7 million years. Further discussion in this chapter is devoted to a brief summary of evolution of the hominins leading to the emergence of modern humans Homo sapiens.

Descent of humans Although primates were present in Africa, Eurasia and the New World, the evolution of hominins was confined to Africa. The idea of a separate creation of human ‘races’ – known as polygeny – with its racist overtones is now discredited.

8

Nature at work: Ongoing saga of evolution

Some anthropologists still subscribe to a related concept of multiregionalism according to which speciation of different ‘races’ occurred after human-like species migrated out of Africa and settled in different regions of the world.10 We follow the emerging consensus views of ‘monogenesis’ and ‘Out of Africa’ according to which human evolution, including the emergence of anatomically modern humans (H. sapiens) occurred in Africa. During this period a few species of Homo (H. ergaster, H. erectus, H. antecessor and H. heidelbergensis) did move out of Africa within the last 2 million years but did not contribute to any further evolution of humans. The Out of Africa model is synonymous with the Recent Single-Origin Hypothesis, Replacement Hypothesis and Recent African Origin. Our own species evolved in Africa and a small group migrated out of Africa and colonized the rest of the world. The tree shown in Figure 7 is a graphic representation of the evolutionary relationship among the hominin fossils described so far. Paleoanthropology is one of the most active fields of research in biology, and specialists offer different interpretations of evolutionary relationships.11 Taxonomy of fossil hominins varies as some specialists tend to split and others to lump the different taxa. The 7 million-year-old Sahelanthropus tchadensis (Toumai), first described in 2001 from Chad, might have been a bipedal hominin. Some claim this to be a possible common ancestor of human and chimpanzee lineages. Our own genus, Homo, appeared about 5 million years later. Three other genera are known from Africa during the in-between period, namely Orrorin (6 mya) followed by Ardipithecus (5.5–4.4 mya) and Australopithecus (4–2 mya). A. afarensis, first described from Hadar, Ethiopia in 1973 might have been the ancestor of our genus Homo. Homo habilis, first discovered in 1962 in the Olduvai Gorge in Tanzania, lived from 2.4–1.4 mya in East and South Africa, and closely resembled Australopithecus. Nicknamed ‘handy man’, H. habilis was the first hominin to have made and used stone tools known as ‘choppers’ in the early Paleolithic period, more than 2 mya. Evolution of hominins continued in Africa eventually resulting in the emergence of anatomically modern H. sapiens, about 200,000 years ago. Yet H. sapiens was not the first hominin to leave Africa. H. ergaster (1.8 mya) closely followed by H. erectus and later H. antecessor and H. heidelbergensis (=H. rhodesiensis) left Africa bringing the Paleolithic technology to Eurasia. H. heidelbergensis and H. neanderthalensis with a thick skull, prominent brow ridges and non-prominent chin but with brain size (1200–1400 cc) similar to modern humans are sometimes referred to as “archaic H. sapiens”. Darwin’s insightful prediction that our earliest progenitors would be found in Africa turned out to be true. It might surprise us that in a matter of 4 million years the continent witnessed the rapid evolution of four genera (Australopithecus, Kenyanthropus, Paranthropus and Homo) and nearly 20 species of hominins; and

The descent of humans and the Darwinian unification of all life

9

Figure 7. An interpretative tree of relationship among hominins with time-scale. The connecting lines indicate well-supported relationship between the species

more will surely be discovered. Darwin also reasoned that the absence of any human remains in older fossil strata must indicate that the evolution of humans must have occurred recently and exceptionally rapidly. Several factors seem to have influenced human evolution. Climate change that resulted in the conversion of tropical forests into woodlands and savannah grasslands might have influenced the Miocene ancestral apes to descend from trees and become bipedal, thereby changing their foraging habits and freeing their forelimbs to make tools. Ice ages during the past 2 million years might have acted as selective agents in human physiological and physical adaptations and influenced the patterns of migration as hominins moved in search of food plants and animals. Brain development and the extraordinary ability to shape tools with hands and the mutual influence between brain development and the need to respond to the changing environment have all contributed to unique human qualities such as planning, innovation, abstract thinking and symbolic behavior. It appears that during the past 10,000 years humans have been evolving 100 times faster than in the past. The Neolithic revolution, which resulted in excessive consumption of starch and milk, urbanization, poor sanitation, epidemic diseases and changing food habits all seem to have operated and continue to operate as selective agents.12 Homo neanderthalensis lived until about 30,000 years ago in Europe while the small-sized H. floresiensis, discovered in 2004 in Indonesia, might have become extinct as recently as 15,000 years ago. Since the Neanderthals and modern humans

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Nature at work: Ongoing saga of evolution

were contemporaries in parts of Europe, researchers have wondered if there were gene exchanges between them. Recently the mitochondrial DNA (mtDNA) from fossil bones of H. neanderthalensis were extracted and compared with the DNA sequence of H. sapiens. It appears that the sequences are very different and the two remained as separate species ever since they evolved from a common ancestor, some 660,000 years ago until the Neanderthals went extinct about 30,000 years ago.13 The only genus and species of hominin that is living today and the only species that ever occupied all the continents is H. sapiens. During the past 20 years molecular geneticists have revealed a remarkable story of the origin and spread of humans, a story of unification of all human beings through a bacterial endosymbiont, the mitochondrion, and recluse Y-chromosome!

Humanity unified - the mitochondrial African mother Human beings, all 6.5 billion now living and the billions more that will be added, are remarkably like each other with 99.9% genetic similarity. A mere 0.1% difference is responsible for the diversity we observe. We are not a highly variable species. This similarity is due to our recent origin in Africa and the presumed small size of the population that left Africa and colonized the rest of the globe. Within the last 20 years a remarkable and unexpected story of our ancestry has emerged and is now profoundly affecting the way we think of ourselves. In an important paper published in 1987, Cann, Stoneking and Wilson reported their analysis of mtDNA samples of 147 people from five geographic populations. Using restriction mapping techniques to detect polymorphism and parsimony methods to infer a common ancestor they came to the conclusion: “All these mtDNAs stem from one woman who is postulated to have lived about 200,000 years ago, probably in Africa. All the populations examined except the African population have multiple origins, implying that each area was colonized repeatedly.”14 This ancestral woman is now popularly known as ‘African Eve’ and ‘Mitochondrial Eve’. Subsequent studies15 have confirmed the findings of Cann et al. although the estimated age of this ‘mother’ is reported to be in the range of 150,000–250,000 years before present. Since the mtDNA now found in all human mitochondria do not undergo recombination, theoretically they all can be traced back to a single root, the so-called ‘coalescent point’. This point represents the Mitochondrial Eve. The implications of these studies are: modern humans evolved in Africa; about 200,000 years ago an ancestral woman in East Africa passed on her mitochondria to her offspring; transmitted to every generation only through the female line to both sons and daughters, her mtDNA is now present in all people on earth; this woman is the matrilineal most recent common ancestor (MRCA) of all people who ever lived since then, and the more than 6 billion people who are living today in all continents, including Africa. The matrilineal MRCA (= African Eve, Mitochondrial Eve) was not the only woman living at that time in Africa. For various reasons the

The descent of humans and the Darwinian unification of all life

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mtDNAs of other women were not passed on to subsequent generations; perhaps these other women did not beget daughters or their female lineages died out. It is likely that through their sons their genes still occur in the nuclear DNA of people living today. The mtDNA of our matrilineal MRCA is found in people outside of Africa also because it was carried by her female descendants when some people left Africa about 100,000 years later to colonize all other continents.

And a Y-chromosomal African father While a mitochondrion is inherited only through the mother, the Y-chromosome is passed on to the next generation only through the father. The difference between the two is that while both men and women receive mitochondria from their mothers, the Y-chromosome is passed on only to males. As with mtDNA it is possible to trace back the root – the coalescent point – of all Y-chromosomes found in males since most of Y-chromosomal DNA (YDNA) does not recombine during meiosis. A study16 of YDNA polymorphism in a worldwide sample of men using methods similar to the analysis of mtDNA revealed that the root of the human male family tree was placed in Africa, just like the female root. However, the date of the putative patrilineal MRCA, the ‘Y-chromosomal Adam’, was just 60,000 years. Again this does not imply that there were no other men around; only that the Y-chromosome of this male MRCA is now found in all men, in all continents. The more recent date (60,000–90,000 years) for the patrilineal MRCA is explained on the basis of human sexual behavior where the variance of reproductive success is higher for men. Unlike women, who all have more equal opportunities to have children, a few men can mate with more women and pass on their genes. The Y-chromosomal lineages of men who lived before the MRCA were lost. The mitochondria of one woman and the Y-chromosomes derived later from one man in Africa were passed on to people who spread throughout Africa, and through a group that migrated out of Africa to all other people in non-African continents. One would expect greatest genetic diversity in the DNA of mitochondria, Y-chromosomes, as well as other chromosomes in Africa, as indeed is shown by several studies.17

Wandering humans and their DNA trees and haplogroups The circular mtDNA inherited through mothers and the male-specific YDNA both carry mutations that are used for understanding human origins and for constructing trees from which the geographical origin and migration of human populations could be inferred.18 Since the mutations occur at a fairly regular rate it is possible to estimate the time when populations branched off using molecular dating techniques. mtDNA and most of YDNA do not recombine and hence genes are not shuffled as they are in autosomal chromosomes. mtDNA offers the advantage of small size of only 16,569 base pairs and 37 genes. These advantages are now widely

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exploited to discover an ever increasing number of polymorphic sites. Single nucleotide polymorphism (SNP) due to point mutations can be easily identified with restriction mapping. Contemporary human populations have accumulated a large number of mutations in mtDNA since the time of Mitochondrial Eve, the MRCA.19,20 Mitochondria also possess short Hyper Variable Regions (HVR1, 2 and 3) which have rates of mutation 100 times greater than that of the nuclear genome. The sequence of nucleotides in these regions (e.g. HVR1: 16,001–16,569) can be compared with a Cambridge Reference Sequence to determine specific markers that help assign individuals to unique mitochondrial haplogroups. Haplogroups are branches on the family tree of groups of people who share a set of genetic markers. All members of a haplogroup share a marker that first appeared in the MRCA of that group. Y-chromosome polymorphism is now increasingly used to understand the origin and migration of human populations. YDNA consists of many millions of nucleotides and provides scope for examining thousands of mutations. Besides SNPs the Y-chromosomes also carry non-coding microsatellites or Short Tandem Repeat (STR) regions consisting of repetitive DNA sequences. These repetitions lead to copying errors resulting in addition or subtraction of repeat units. Each STR is designated by a DYS number (DNA Y-chromosome Segment number). Analysis of markers on YDNA provides necessary information to assign individuals to Y-chromosomal haplogroups. Mitochondrial and Y-chromosomal haplogroups

Figure 8. A simplified tree of mitochondrial haplogroups. Haplogroups L4, L5, L6 and L7 are not shown. Haplogroups M, N and R can be considered as ‘Super haplogroups’ since they have many subclades. Some of the defining markers are shown. Marker mutations may occur as SNPs in coding regions or in HVR1 and HVR2 regions. The most ancient haplogroups L0–L3 are African; only during the colonial period people with these markers have been moved to non-African continents

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are now routinely analyzed in several laboratories around the world. A number of private testing companies also analyze DNA samples and provide information on genealogies and geographic origins. The Genographic Project21 is an ambitious worldwide project undertaken by the National Geographic Society with geneticist Spencer Wells to map the human family tree and trace the migratory routes of our ancestors.22 The Y-chromosome Consortium23 at the University of Arizona carries out collaborative studies on YDNA variations and nomenclature of Y haplogroups. Family trees constructed using mitochondrial markers provide a graphic picture of deep human matrilineal ancestry (Fig. 8). Three main groups, designated by letters L, M and N, and many subgroups are known. The earliest branching of the tree occurred in Africa about 200,000 years ago when the L0/L1 split occurred somewhere in East Africa. Today L0 is widespread among the San and Mbuti people in Africa. L1 and its branch L2 are also confined to Africa. Only after M and N macrohaplogroups branched off from L3 was there any major migration of the female ancestor out of Africa. Thus the woman in whose mtDNA a mutation first occurred, to found the L3 haplogroup branch, is known as the ‘Eurasian Eve’ since her descendants were the first to move out of Africa. Today the M group derived from L3 is found in East Africa, Asia, Melanesia, Australia and the Americas. The

Figure 9. Y-chromosomal haplogroups showing the defining markers. Haplogroups A and B are African branches with most diverse lineages dating back to 60,000–50,000 years. Major haplogroups C, F and K occur in India especially as exclusive subclusters of H, L and R

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N group, also born in Africa, later migrated out of Africa and moved northwards spreading to many parts of Asia, Europe, India and the Americas. Haplogroup R that branched off from N is now found in East and West Eurasian populations, accounting for nearly 75% of the lineages. The YDNA family tree also reveals ancient splits that occurred in our male lineage in Africa (Fig. 9). Haplogroup A defined by the marker M91 is perhaps the deepest of our branches, now confined to Africa and prevalent among the Khoisan, Ethiopians and Nilotes. Haplogroup B with marker M60 is also confined to Africa mostly among the Pygmies and Hadzabe. Descendants of B with a marker M168 (‘Eurasian Adam’) were perhaps the first males to leave Africa more than 60,000 years ago. A man born to this migrant group had the marker M130 defining the haplogroup C. His descendants are considered to have moved along the coast into India, and moved beyond to Southeast Asia and finally into Australia. M130 is now found in <5% of the Indian population mostly along the coastal belt in Maharashtra, Tamil Nadu, Andhra Pradesh, Orissa and West Bengal. Thus both mtDNA and YDNA polymorphism provide information useful for tracing human migratory patterns.24

Out of Africa and into India The Indian Subcontinent was a decisive juncture in the early migration of hominins. Long before the entry of modern humans, other species of Homo had left Africa and spread to many parts of Eurasia, as attested by fossil remains and early Paleolithic stone tools. The Indian subcontinent too, had its share of early hominin migrants dating back to 2 million years.25 Oldowan tools have been found at Riwat in Soan valley in Western Punjab and in the Siwalik rocks of Himachal Pradesh. Early Acheulean stone tools in Hungsi valley in Karnataka are dated to be 350,000-years-old. Similar stone tools are also known from Attirampakkam near Chennai. Fossil remains of early hominins are rare in India; a few remains found in the Central Narmada valley are believed to be at least 500,000-year- old.26 These remains may belong to either H. erectus or H. heidelbergensis. Earliest fossil remains of modern humans are found in Africa, Middle East and Australia. The ‘Omo skull’ from Ethiopia is dated to be about 195,000-yearsold. Human remains found in Eastern Mediterranean regions and in the Qafzeh and Skuhl caves of modern Israel suggest that modern humans moved into this extended area of the African continent as early as 110,000 years ago. However, the descendants of these early settlers disappeared about 50,000 years ago. The area was then colonized by the Neanderthals and only much later by another wave of modern humans. Paleoanthropologists believe that the first wave of modern humans left Africa only about 66,000–70,000 years ago.27 The genetic diversity now found in all non-African countries is a legacy of this early group of H. sapiens

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who left Africa. After 2,000 generations the descendants have multiplied to more than 5 billion people now living in non-African countries. The Y-haplogroup C, defined by the marker M130 was the earliest clan to migrate through India into Sri Lanka, Southeast Asia and eventually Australia.28 mtDNA studies also show that the people of New Guinea and Australia belong to the M and N haplogroups that left Africa through the ‘southern coastal route’ more than 50,000 years ago.29 Although early migrants traveled through India, the oldest fossils of modern humans have been discovered so far only in Australia (50,000 years), Thailand (37,000 years) and Sri Lanka (30,000 years). The mtDNA haplogroup M that branched off from L3 about 60,000 years ago and its variants occur in nearly 30% of the Indian population. (The author of this chapter belongs to an East Eurasian subgroup M* carrying markers that first appeared some 50,000 years ago in his maternal ancestor; M* constitutes 30–50% of the gene pool in Pakistan and Northwest India.) It is well established that Africa is genetically the most diverse continent in the world, which is a reflection of nearly 250,000 to 200,000 years of accumulation of polymorphism in mtDNA as well as in nuclear genome. While no other continent can match this diversity, India appears to be the second richest region in genetic diversity. The reasons for this diversity appear to be: a) early settlement of people who first left Africa, b) subsequent entry of people at various periods from the Northeast and Northwest, c) a process of stratification of the society, and d) a countervailing process of exogamy and intermixture of various groups. Sexual selection might also have played a role in the diversity we observe. Singh, who identified 4,693 major communities and more than 50,000 small endogamous groups, described a typical Indian as a “migrant par excellence”.30 The Anthropological Survey of India recognized 418 tribal communities in India who belong to Andamanese, Austro-Asiatic, Dravidian, or Tibeto-Burman language families.31 Seventy-five of these indigenous communities have been identified by the government and described using the unfortunate label, “Primitive Tribal Groups”. Others have described them as ‘relic’ tribes implying that they were the earliest settlers. Indeed the Onges and Jarawas of the Andaman Islands share many characteristics with the Khoisan and Pygmies of Africa suggesting that they were among the earliest groups, if not the earliest, to leave Africa, traverse Indian mainland and settle in the Islands.32 Besides the tribal communities, hundreds of different caste, outcaste and religious communities live in India. During the past 10 years this rich diversity has been under intense investigation by human geneticists. Sophisticated molecular and statistical techniques are used to detect new markers and to assign people to various haplogroups, determine gene flows, and trace relationships between various communities and their past migratory history and linguistic affinities.33

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The Y-haplogroups common in India include: C with marker 130; H with the ‘Indian marker’ M69; the ‘Indian Clan’ L (M20) found in 50% of South Indian men, and different subgroups of haplogroup R. The R group defined by the marker M207 descended from the central Asian clan marker M45. Descendants of M45 populated most of Europe and North America. R1A1 (M17) that occurs in 35% of Hindi-speaking men and 10% of Dravidian-speaking men is considered as the Indo-European marker associated with the migration into India of IndoEuropean language speakers, about 10,000 years ago. Haplogroups H, L and R2 are exclusive to the Indian Subcontinent, with F* nearly so. Subgroup O3e is associated with Tibeto-Burman speakers and O2a lineage is found among AustroAsiatic speakers. The focus of this chapter is on Darwin and his unification of life. The rapidly growing field of phylogeography of mtDNA and YDNA haplogroups cannot be covered in any detail here. The brief reference to phylogeography is made in order to highlight the exciting potential it has to unify people by reminding them of their shared common ancestry. (The author belongs to haplogroup H with the marker M69 which first appeared as a mutation in his male ancestors more than 30,000 years ago, somewhere in the Middle East!) Investigation of more than 200 Indian populations34 is now shedding light on the genetic basis for ethnic diversity in India. As additional markers are added and techniques for the study and statistical analysis of polymorphism of autosomal chromosomes are developed, many questions that have puzzled classical anthropologists, historians, sociologists and linguists about the peopling of India, the biological and cultural diversity, stratification of the society, and susceptibility and resistance to diseases will be answered. The dialog between human molecular geneticists and classical scholars is likely to result in new insights and revision of cherished concepts in anthropology and history.35 A recent study has suggested that most Indians are descendants of two genetically divergent ancestral populations, the ‘Ancestral South Indians’ (ASI) and ‘Ancestral North Indians’ (ANI). The latter is genetically close to Middle Easterners, Central Asians and Europeans while the ASI lineage has no close affinity to any outside group except the ancient isolate of indigenous Andaman Islanders.36

“Light will be thrown on the origin...” Darwin answered his own visionary statement of throwing light on the origin and history of humans by writing his second famous book, Descent of Man. This completed his grand unification of the biological world, genetically and temporally. Darwin also suggested another category of unification in the human family. He was keenly interested in the origin of language and its relation to human intellect. He also explained the process of biological evolution using the evolution of language

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as a metaphor. His observation on human evolution and language was farsighted: “If we possessed a perfect pedigree of mankind, a genealogical arrangement of the races of man would afford the best classification of the various languages now spoken throughout the world”. Statistical methods that help construct human family trees are also used to construct trees of major language families, tracing historical linguistic structural relations in some cases to 20,000 years ago.37 Since the “Out of Africa” date is <100,000 years perhaps all languages, too, will be traced to their roots in Africa someday. Darwin scrupulously adhered to methods of scientific enquiry and would not entertain any place for supernatural powers in the process that led to the evolution of humans endowed with amazing “powers of observation, memory, curiosity, imagination, and reason”.38 The human species may have developed through a cold and purposeless process of evolution but Darwin was not amused by the racism and discrimination that tore apart the unity and dignity of the human species; he attributed ignorance and self-interest to such divisive attitudes.39 Darwin loathed slavery as much as his abolitionist grandparents Erasmus Darwin and Josiah Wedgwood did, almost a 100 years before him. For Darwin, abolition of slavery was the ‘highest good’ even if it entailed a war that might result in ‘a million horrid deaths’. In this bicentenary year of Darwin, two well-known Darwin scholars and editors of his book and authors of his biography – Adrian Desmond and James Moore – have suggested40 that Darwin might have found encouragement in the common descent of humans from apes as proof in support of the essential unity of all human groups and as a challenge to racist ideologies. Theodosius Dobzhansky in his book on evolution41 quoted Einstein: “The most incomprehensible thing about the world is that it is comprehensible.” The majesty of Darwin’s theory is that we now comprehend a certain unity in a world that still is mysterious in many other ways. For Darwin the unified biodiversity had a certain splendor and dignity. In concluding the Origin of Species he wrote: “When I view all beings not as special creations, but as the lineal descendants of some few beings which lived long before the first bed of the Silurian system was deposited, they seem to me to become ennobled”.42 Darwin was acutely aware of past extinctions as a natural process. He also visualized life on earth as “an entangled bank, clothed with many plants of many kinds, with birds singing on the bushes with various insects flirting about....and dependent on each other in so complex a manner”.42 Darwin was the great unifier of all life. Today he would be appalled to see how the human species is now the cause of the sixth major extinction of the diversity life. Perhaps he might have some insight into the puzzling predicament of a living earth now endangered by one of its own manifestations, human beings with extraordinary intellect and reasoning power. Darwin also shaped the course of science by secularizing it. Mayr pointed out that Darwin “presented 50 or 60 biological phenomena easily explained by

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Nature at work: Ongoing saga of evolution

natural selection but quite impervious to any explanation under special creation, and equally inexplicable to so-called intelligent design.43 Free from dogmas, modern science has made impressive progress in many fields since Darwin. One such achievement is the illumination of immense time scales that we are helped to comprehend. This time scale for life on earth extends to nearly 4 billion years, close to the time of the formation of the earth. Someday when scientists discover how life originated from non-living matter, Darwin’s grand biological unification will merge with the unification that physicists seek of the fundamental forces of the universe. Acknowledgements: I am grateful to Dr. V.P. Sharma for inviting to me to contribute this chapter for a book that commemorates Charles Darwin. I thank Mr. Leslie Lawrence for his patient help with creating the Figures. Thanks to Dr. J. Peter Gogarten for kind permission to use his instructive phylogenetic tree of living organisms. As always, my wife Anne, encouraged, supported and helped me in many ways during the short time I had to write this chapter.

References 1.

Darwin C (1999) In: The Origin of Species by Means of Natural Selection or The Preservation of Favoured Races in the Struggle for Life. London: Murray; 1859. Reprint New York, Bantam Books

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Glendorff N, Xu Y, Labeden B (2008) The last universal common ancestor: emergence, constitution and genetic legacy of an elusive forerunner, Origin and early evolution of life Edited by Dr Eugene V Koonin. Biology Direct 3:29

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Glansdorff N (2000) About the last common ancestor, the universal life-tree and lateral gene transfer: a reappraisal. Mol Microbiol 38(2):177–85

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Dobzhansky TH (1973) Nothing in biology makes sense except in the light of evolution. Am Biol Teacher 35:125–129

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Wood BA, Richmond BG (2000) Human evolution: taxonomy and paleobiology. J Anat 197:19–60

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Ward P (2008) What will become of Homo sapiens? Sci Am India 4:44–49

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Serre D, Langaney A, Chech M, et al. (2004) No evidence of Neandertal mtDNA contribution to early modern humans. PLoS Biol 2(3):E57

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Cann RL, Stoneking M, Wilson AC (1987) Mitochondrial DNA and human evolution. Nature 325:31–36

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Ingman M, Kaessmann H, Paabo S, Gyllensten U (2000) Mitochondrial genome variation and the origin of modern humans. Nature 408:708–713

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Underhill PA, Shen P, et al. (2000) Y chromosome sequence variation and the history of human populations. Nature Genetics 26:358–361

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Tishkoff SA, et al. (2009) The genetic structure and history of Africans and African Americans. Science 324:1035–1044

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Stoneking M (2008) Human origins. The molecular perspective. EMBO Rep 9:546–550

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Behar DM, Villems R, et al. (2008) The Dawn of Human Matrilineal Diversity. Am J Hum Gen 82:1130–1140 (http://www.pubmedcentral.nih.gov/articlerender. fcgi?artid=2427203#app2)

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Gonder MK, Mortensen HM, Reed FA, de Sousa, Tishkoff SA (2006) Whole-mtDNA Genome Sequence Analysis of Ancient African Lineages (http://mbe.oxfordjournals.org/ cgi/content/full/24/3/757)

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The National Genographic Project. (www.nationalgeographic.com/genographic)

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Wells S (2007) Deep Ancestry: Inside the Genographic Project. National Geographic Society, Washington DC; Wells S (2002) The Journey of Man: A Genetic Odyssey, Penguin Books India (P) Ltd., Delhi

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The Y-chromosome Consortium. (http://ycc.biosci.arizona.edu/)

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Oppenheimer S (2004) In: The Real Eve: Modern Man’s Journey Out of Africa. Carrol and Graff, New York

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Habib I (2001) In: Prehistor. Tulika Books, New Delhi

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Sankhayan AR (2005) New fossils of Early Stone Age man from Central Narmada Valley. Curr Sci 88:274–707

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Stanyon R, Sazzini M, Luiselli D (2009) Timing the first human migration into eastern Asia. J Biol 8:18

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Wells S: The Journey of Man, A Genetic Odyssey by Spencer. p 123–125, http://www. google.com/search?hl=en&q=Wells+S%3A+The+Journey+of+Man%2C+A+Genetic+O dyssey.+p+123%E2%80%93125&btnG=Search&aq=f&oq=&aqi=

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Hudjashov G, Kivisild T, Underhill PA, Endicott P, Sanchez JJ, Lin AA, Shen P, Oefner P, Renfrew C, Villems R, Forster P (2007) Revealing the prehistoric settlement of Australia by Y chromosome and mtDNA analysis. Proc Natl Acad Sci USA 104:8726–8730

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Singh KS (2002) In: People of India: an introduction. OUP, New Delhi

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Mandal H, Mukherjee S, Datta A (2002) India: An Illustrated Atlas of Tribal World. Anthropological Survey of India, Kolkata

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Thangaraj K, Singh L, Reddy A, Rao V, Sehgal S, Underhill P, Pierson M, Frame I, Hagelberg E (2003) Genetic Affinities of the Andaman Islanders, a vanishing human population. Curr Biol 13:86–93

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Majumder PP (2008) Genomic inferences on peopling of South Asia. Curr Opin Genet Develop 18:280–284. Krithika S, Maji S, Vasulu TS (2007) For a good review of previous publications see Molecular Genetic Perspective of Indian Populations: A Y-Chromosome Scenario. In: Anthropology Today: Trends, Scope and Applications. BhasinV, Bhasin MK (Eds.), Anthropologist Special, Kolkatta 3:385–392

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34.

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Pitchappan RM, Kavitha VJ, Wells SR (2008) Genomic Implications of Dravidian Cultural Evolution. (www.firstgreatmigrations.org/.../DR-Pitchappan-19%20JUNE%202008.doc)

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Reich D, Thangaraj K, Patterson N, Price AL, Singh L (2009) Reconstructing Indian population history. Nature 461:489–494

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Mindell DP (2009) Evolution in the Everyday World. Sci Am India 4:58–65

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Howard J (2006) Darwin - A Very Short Introduction. OUP, New Delhi

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Desmond AJ, Moore JR (2009) Darwin’s Sacred Cause: How a Hatred of Slavery Shaped Darwin’s Views on Human Evolution. Allen Lane, London

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2 Ida: A link to human evolution V.P. Sharma Centre for Rural Development and Technology, Indian Institute of Technology, Hauz Khas, New Delhi - 110016 [email protected]

Abstract: Year 2009 is being celebrated as the bicentenary of British naturalist Charles Robert Darwin’s birth as well as 150 years of his book “On the Origin of Species by Means of Natural Selection, or The Preservation of Favored Races in the Struggle for Life” in short “On the Origin of Species”. Darwin is hailed as one of the greatest scientist who put forth the theory of gradual evolution and branching of all life forms. Unfortunately, then fossil records were incomplete to produce evidence of human evolution. For 200 years paleobiologists were looking for any evidence of a transitional fossil to showcase the evolution of monkeys, apes and human. The story of Ida began in 1983 when she was collected from Messel pit in Germany by an unknown collector, and displayed in his home for 20 years before deciding to sell. The fossil Ida was preserved in nature for ages in the Germany’s Messel pit, a crater rich in Eocene Epoch fossils. Ida lived when major changes were taking place on earth, the dinosaurs had become extinct, the Himalayas were forming and a range of mammals thrived in vast jungles all over the world. Prof. Jørn Hurum of the Natural History Museum, University of Oslo, Norway bought the fossil for 1 million USD, assembled top international team of experts, studied the fossil secretly for 2 years, and satisfied themselves as to the genuineness of the fossil. Finally Prof. Jørn Hurum announced the discovery of 47 million years old fossil - a link in the evolution of monkeys, apes and man. He named the fossil “Ida” and her scientific name is Darwinius masillae (named in honor of Charles Darwin’s bicentenary). Ida will remain for long a subject of intense research and discussion almost throughout the world. The fact that all life forms have common ancestors at some point of time is a well established universal reality as unfolded by research on DNA. Keywords: Darwin, Evolution, Darwinius masillae (Ida), Paleobiology, Primate, Religion

Introduction Prof. Jørn Hurum of the Natural History Museum, University of Oslo, Norway announced the discovery of Ida - the missing link that led to the evolution of monkeys, apes and man (Ida, pronounced “ee-dah”). This breakthrough V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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announcement was well timed coinciding with the bicentenary celebrations of English naturalist Charles Darwin. It is noteworthy to mention that Darwin for the first time gathered evidence of evolution by natural selection, and said “that species change over time through the selective retention of beneficial traits, gradually evolving into new species. In this way, all species alive today are descended from common ancestors”. In November 1859 Darwin published his ground breaking theory “On the Origin of Species by Means of Natural Selection, or The Preservation of Favored Races in the Struggle for Life” in short “On the Origin of Species”.1 At that time there were major gaps in the fossil records. Darwin acknowledged that the lack of fossil evidence of the intermediate transitional fossils is a serious problem between species posed a problem, and “this was the most obvious and gravest objection which can be urged against my theory”.1 Two years later in 1861 the first transitional fossil Archaeopteryx2, 3 was discovered. Thereafter, evidence has been collected that unambiguously brings out the intermediate form in major animal groups. For example, the finding of Archaeopteryx has supported the intermediate forms between dinosaurs and birds. As Prof. Jørn Hurum explains, Darwin said a lot about transitional species and how they were missing from the geological record. Quoting Darwin he said “that if a transitional species is never found, his whole theory will be wrong”. Explaining the importance of Ida, Prof. Hurum said that “for the last 50 years we’ve gained a deeper and deeper understanding of species that show transitions between large groups of mammals. Ida is very comparable to some of the most significant fossils that have been described like Lucy,4 the Neanderthals,5 Tyrannosaurus rex6 and Archaeopteryx.3 It’s a really important specimen that will become an icon of evolution. So I think Darwin would be really happy about this specimen”.

The discovery of “Ida” Fossil Ida was unearthed in 1983 by a private fossil hunter from Grube Messel pit near Frankfurt, Germany (Fig. 1). The village Messel is located about 35 km south-east of Frankfurt and the word masilla is the name of a 9th century monastery where the fossil was found. The Messel site has yielded hundreds of superbly preserved fossils including eight species of crocodile, 20 or so snakes, more than 60 specimens of pygmy horse, more than 1,000 bats, insects with colored pigments on their wings still visible, the largest ant ever to crawl the planet, and eight fragmentary primate specimens. Messel is now UNESCOs world heritage site. At the time of Ida’s finding, the German collector (anonymous) had no idea of how valuable the fossil is. The fossil was preserved in a polyster resin and hung for 20 years in his home, and admired like the painting of Vincent Willem van Gogh. The fossil was split and sold in two parts separately; a lesser part was purchased for the private museum in Wyoming, USA. Prof. Jørn Hurum, was the first to recognize the significance of the fossil. The fossil was reassembled in 2007

Ida: A link to human evolution

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Figure 1. Left: An artist’s reconstruction of the Eocene period ecosystem shown on left side. Credit: Christopher Klein and Mark Hallett. At the upper left corner is an adapoid primate, Europolemur, a close relative of Darwinius. Right: Messel pit site in Germany-UNESCOs world heritage site. Credit: Atlantic Productions http://blogs.ngm.com/blog_central/2009/05/darwiniusand-the-real-missing-link.html

and now belongs to the Natural History Museum, University of Oslo, Norway.7 In 2006 Thomas Perner, a private collector facilitated the deal of Ida. He showed the fossil to Prof. Hurum at the annual Hamburg Fossil and Mineral Fair in Germany. Prof. Hurum was amazed at the “most beautiful fossil worldwide”. This to him looked like a blue print of human evolution. The asking price was more than $1 million (£660,000), almost 10 times the normal value of such a fossil. After 6 months of negotiations Thomas Perner brokered a $1 million (£629,000) deal of the 47 million years old fossil in a vodka bar in Hamburg. An international team of scientists headed by Prof. Hurum at the Natural History Museum have studied this fossil for 2 years before making an announcement. The fossil is named “Ida” after Prof. Jørn Hurum’s daughter who in maturity is equivalent to his 6-year-old daughter Ida. Ida’s scientific name is Darwinius masillae given in honor of English naturalist Charles Darwin. The discovery of the 95% complete ‘lemur monkey’ Ida is described by experts in detail, and their final conclusion was that Ida is completely genuine and authentic (Fig. 2). The fossil was amazing and awesome. Such a well preserved specimen has never been encountered anywhere in the world. The fossil belongs to Eocene era and Ida (she) lived in semi-tropical forest 47 million years ago. Ida was preserved in the oil shale. This shale consists of soft finely stratified rock of mud or clay and easily splits. The fossil is 95% complete and perfectly preserved. In contrast “Lucy” which was thus far the most reliable fossil evidence for direct descendent of man. Lucy was found in Ethiopia, is 3.18 million years old and only 40% complete. Ida’s fossil shows soft body outline as well as the preserved gut contents of seeds, fruits and leaf but no insects. Ida when died was a juvenile female, 6–9 months in age, 58 cm (23 inch) overall length of the body and about 24 cm (9.4 inch) tail. Ida as an adult would have weighed about 1 kg, but she was probably 60% of her adult body weight. Ida was perhaps a nocturnal with large eye holes in the skull.

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Figure 2. (Left) Dr Jens Franzen and Dr Jørn Hurum studying Ida the fossil. Photograph: Sam Peach/Atlantic Productions, and (Right) Fossil Ida: Photo credit: Atlantic Productions Ltd.

She is female as she does not have a penis bone. She has talus foot bone present in the humans. She has nails rather than the claws. Her teeth are similar to those of monkeys. Her forward facing eyes are similar to humans. This allows her 3D vision and ability to judge distances. Ida has five fingers on hands, short arms and legs. It lacks a grooming claw and fused row of teeth known as a toothcomb a characteristic feature of Lemur. It is notable to mention that Ida fossil is 20 times older than other fossils used as evidence of human evolution. Prof. Hurum said that the impact of Ida on the world of paleontology will be “somewhat like an asteroid falling down to earth”. Because Ida fossil was so complete, it was also described as a “Rosetta stone”.8 “This fossil “promised to change everything that we thought we understood about the origins of human life”.9–11 The discovery of fossil Ida ends 200 years search of the evidence of evolutionary history of Homo sapiens. The team headed by Prof. Hurum announced the discovery of Ida on May 19, 2009 at the American Natural History Museum, New York. Announcing the discovery Hurum said “It is something about owning a part of history, that you know when you show it to people it will change things ... It is a powerful feeling to have something ... nobody knows about.” Prof. Hurum declared that Ida was “the most complete fossil before human burial”. Hurum showed pictures of the Mona Lisa and the Rosetta stone, making a point about the importance of “Ida”, and he called it the Holy Grail and the lost ark of paleontology.12 Sir David Attenborough said, while narrating the documentary on “Ida” at BBC. “This little creature is going to show us our connection with the rest of all the mammals - with cows and sheep, and elephants and anteaters.” “The more you look at Ida, the more you can see, as it were, the primate in embryo.13” Because she is so complete, the scientists who have studied Ida describe her as a “Rosetta stone” for making sense of early primate evolution. “[She is] the eighth wonder of the world14” said Jens Franzen,9 at the Senckenberg, who is another co-author of the paper, describing the fossil in the journal PLoS ONE. Further Hurum said: “This is the first link in human evolution. A find like this is something for all humankind. It tells a part of our evolution that’s been hidden so far”. “This

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is our Mona Lisa and it will be ... for the next 100 years.13,15” “This little creature is going to show us our connection with all the rest of the mammals. The link they would have said until now is missing ... it is no longer missing.” Renowned broadcaster and naturalist Sir David Attenborough. The day after the Ida launch UK-based Guardian newspaper featured a front-page article under Sir David Attenborough’s personal by-line which is reproduced below.16 “Humanity is very egocentric. We are fascinated with ourselves. I’m not sure that it is a particularly nice characteristic but we are. When we look round at the natural world, there is often an ulterior motive. We desperately want to know where we came from...” ...and later continued, as part of the layout of a special two-page spread about the discovery. “...The more you look at Ida the more you can picture, as it were, the primate in embryo. She represents the seed from which the diversity of monkeys, apes and ultimately every person on the planet came...”

Importance of Ida The fossil Ida or Darwinius, was officially revealed on May 19, 2009 at New York’s American Museum of Natural History. On May 20, 2009 a day after the announcement in New York, Google’s search page featured “Ida”, and on clicking the logo, one automatically reaches at the “missing link” (Fig. 3). Simultaneously a book, a movie, a press release, news reports, TV special and an interactive web site were all launched to tell the world that in Ida the “missing link” of the man’s evolution has been found.17

Figure 3. Darwinius masillae, the fossil known as “Ida” was displayed on Google’s search page

The Ida fossil is more similar to anthropoids, the group that includes apes, monkeys and humans. Ida lived around the time when primates split in to prosimians or the non-human branch who have survived as the modern lemurs; and the monkeys, apes and humans evolved from the other branch known as anthropoids. Ida has anthropoid features placed within the primate family tree. Prof. Jørn Hurum said she is perhaps a transitional fossil between primitive lemur-like primates and the monkeys, including the human lineage. “This is the first link to all humans – truly a fossil that links world heritage,17 ” he said: “This fossil rewrites our understanding of the evolution of primates... It will probably be pictured in all textbooks for the next 100 years”.15 As a primate, Ida is part of our own order, and able to throw light on our own ancestry. She comes from

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Figure 4. Sketch of Ida are published by the research team on the Public Library of Science website. Creative Commons Attribution License Franzen et al.9

a highly significant age—the time when the anthropoids, our own subgroup of primates, were branching off from the prosimians” (Fig. 4). Darwin was ridiculed for his thesis on evolution. On the November 17, 1877, Charles Darwin was awarded an honorary doctorate by Oxford University. As he headed towards the podium, unruly undergraduates unfurled a giant banner of half-man half-monkey titled “Missing Link”. Such was the feeling against his monumental contribution to science. The notion that humans were connected to other animals was causing hurt in the Christian society (Fig. 5). When Darwin told the Bishop of Worcester’s wife about his theory of evolution, she remarked: “Descended from the apes! My dear, let us hope that it is not true, but if it is, let us pray that it will not become generally known”. On the bicentenary of Darwin “Pope has now argued that man descended from apes, and natural selection was the means by which some species prospered while others failed”. In 2009 Rev Dr Malcolm Brown, Director Church of England issued this statement “Charles Darwin: 200 years from your birth, the Church of England owes you an apology for misunderstanding you and, by getting our first reaction wrong, encouraging others to misunderstand you still. We try to practice the old virtues of ‘faith seeking understanding’ and hope that makes some amends.”18

Discussion The theory of evolution by natural selection as outlined by Charles Darwin is now widely accepted. During Darwin’s period, fossil records were few and that was the biggest obstacle in convincing skeptics. In the last 150 years fossil records have unambiguously proved Darwinian evolution. But during his period Darwin had to face the wrath of the Church of England for his theory of evolution as it was considered against the divine creation. Today as we celebrate the bicentenary of

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Figure 5. Caricature of Charles Darwin as an ape published in The Hornet, a satirical magazine 22 March 1871. University College London Digital Collections, Author unknown. This caricature shows Darwin with the body of an ape. As “Darwinism” became widely accepted in the 1870s, caricatures of Charles Darwin with an ape or monkey body symbolized evolution19

this great man his views on religion stand vindicated by the Church of England.18 There are still missing specimens in the chain of evolution of various animals and plants. The search for more fossil evidence would profoundly affect the young minds. Even for atheists it may be recalled that Genesis 1 states, “And God said, let the earth bring forth the living creature” and “Let the waters bring forth abundantly.” This allows the teaching that God let the earth and waters do its thing and create life, which would not contradict Darwinism or religion. As Editor in Chief, National Geographic magazine Bill Allen states in his letter from the Editor “National Geographic aims to explore the world, often by highlighting scientific concepts such as evolution. Is this approach necessarily at odds with faith, which lies beyond the possibility of scientific proof? No. Just as religion did not disappear after Galileo demonstrated that the earth is not at the center of the solar system, evolution does not exclude God from our origins, the ‘mystery of mysteries’—a 19th century astronomer’s description borrowed by Darwin himself”.19 Jørn Hurum explains evolution for all life forms, “When we say that Ida is related to all the monkeys and apes that live today, at the same time we’re saying that they all have separate lines of evolution. Chimpanzees, gorillas, orangutans and lemurs all have the same amount of evolution as humans do. We just evolved in different directions. The beauty about the theory of evolution is that we all have a starting point somewhere, and then the ancestors go in different directions (Fig. 6). What we see with specimens like Ida is something about the

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Figure 6. Showing the place of Ida Drawinius masillae in the primate family. Humans first walked on earth 190,000 years ago in the 4.5-billion-year history of earth. http://image.guardian.co.uk/ sys-files/Guardian/documents/2009/05/20/Primate_tree.pdf

deep evolution of all primates - we have a common ancestor somewhere down the timeline. All of us.” Primates have evolved 55 million years ago and between 40 and 50 million years ago a branching took place splitting apes, monkeys and humans from the second group that includes lories and lemurs. Ida lived during the Eocene period. This period began about 55 million years ago and by this time 10 million years had lapsed since the dinosaurs had become extinct. Darwinius masillae “Ida” lived some 47 million years ago in the middle Eocene (in Greek “New Dawn”) period, and dinosaurs disappeared some 18 million years ago before Eocene. It was in the Eocene period that the first large land mammals appeared on the earth. This was the period when warm-blooded animals continued their evolutionary transition to the biodiversity in animal life we see today. Decent of man continued selecting for favorable mutations resulting in modern man (Fig. 7).20 Ida may belong to our own primate ancestry that led to monkeys, apes and man, but the announcement of “Ida - the missing link” has become controversial. It may be noted that evolution proceeds via incremental changes. The fossil record of each change is incomplete. Skeptics are still cautious in accepting this

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Figure 7. Evolution of monkeys, ape and Man20

as the missing link.21 Primatologists interested in evolution were conspicuously absent at the news conference and most of them believe that Ida was the ancestor of lemurs, not humans. John Fleagle Professor at Stony Brook University, New York who reviewed the manuscript before publication state “that the fossil is not a lemur but a link in the evolution of man”. Ida’s full significance would emerge only after it has been debated extensively among the paleontologists in the coming years. While final verdict will take time, it is safe to avoid the word “missing link”. Ida will remain an important discovery of great importance as there are very few fossil records from this Eocene (in Greek “New Dawn”) period. Finally as we go to press, debate on the “Ida’s” place in evolution is becoming murkier. A group of paleontologists writing in Nature (Lond.) claim that 47 million years old fossil Darwinius masillae belongs to lemurs and lorises. Nonetheless we believe that not in too distant a future such an undisputed fossil in the lineage of monkey, ape and man would be found;22,23 and until such time DNA story24 has already clinched the issue. Darwin’s theory of evolution has stood test of time, and has proved to be of invaluable help in explaining the various new pathogens causing pandemics. Work of Watson and Crick24 that every organism carries a 4-letter code of DNA25 common to all life, vindicated Darwin’s theory of evolution. For example, chimpanzee and human genomes are highly related. Such similarities based on genome analysis of various animals and plants have proved to be a boon in understanding common descent. Further advanced genetic research is marching ahead to solve the problem of hunger and health in man and animals. The evolution of various microbes is causing serious human health problems world over. In the last decade or two about 30 new microbes or their malignant forms have appeared and caused high morbidity and mortality, and heavy economic losses. The examples are plague, AIDS, Ebola virus, SARS, H1N1 viral influenza, tuberculosis, malaria, West Nile virus, Japanese encephalitis et al. By the process of mutations and natural selection they acquire resistance to chemicals that were initially very useful.

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These organisms change to new and more dangerous forms, and some also jump from animals to man. To watch the entire panorama of plant and animal life, consequences of biodiversity loss, and emergence of lethal organism is mind boggling. Darwin would have been thrilled to see “the nature at work”.

References 1.

Darwin C (1859) In: On the Origin of Species by Means of Natural Selection, or the Preservation of Favored Races in the Struggle for Life. 1st edition, John Murray, London. http://darwin-online.org.uk/content/frameset?itemID= F373&viewtype=text&pageseq=1, retrieved 2009–01–09

2.

Ostrom JH (1976) Archaeopteryx and the origin of birds. Biologic J Linnean Soc 8: 91–182

3.

Nedin C (1999) All About Archaeoptery. http://www.talkorigins.org/faqs/archaeopteryx/ info.htmlx

4.

They Didn’t Love Lucy. New York Times, March 13, 2009. http://www.nytimes. com/2009/03/19/arts/artsspecial/ 19bust.html?_r=2&pagewanted=all

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Tattersal I, Jeffrey H, Schwartz (1999) Hominids and hybrids: The place of Neanderthals in human evolution. PNAS 96(13):7117–711

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Erickson GM, Makovicky PJ, Currie PJ, Norell MA, Yerby SA, Brochu CA (2004) Gigantism and comparative life-history parameters of tyrannosaurid dinosaurs. Nature 430(7001):772–775

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Andrew L (2009) Celebrity fossil primate: missing link or weak link? Science 324: 1124–1125

8.

“It’s really a kind of Rosetta Stone,” commented study co-author Professor Philip Gingerich, of the Museum of Paleontology at the University of Michigan, Science Daily, May 19, 2009. http://www.sciencedaily.com/releases/ 2009/05/090519104643.htm

9.

Franzen JL, Gingerich PD, Habersetzer J, Hurum JH, von Koenigswald W, et al. (2009) Complete Primate Skeleton from the Middle Eocene of Messel in Germany: Morphology and Paleobiology. PLoS ONE 4(5):e5723.

10.

World Heritage Fossil: Science Daily, May 19, 2009. http://www.sciencedaily.com/ releases/2009/05/ 090519104643.htm

11.

The Guardian [UK] “To get a glimpse of the Ida fossil, the media make monkeys of themselves”. http://www.guardian.co.uk/science/2009/may/19/ida-fossil-primatemedia-us

12.

Ida: The Holy Grail of Missing Links? http://www.arn.org/blogs/index.php/2/2009/05/20/ ida_the_holy_grail_of_evolutionary_specu_1

13.

National Geographic Magazine. September, 2009

14.

Randerson J. Ed Pilkington in New York Deal in Hamburg bar led scientist to Ida fossil, the ‘eighth wonder of the world’ guardian.co.uk, Tuesday 19 May 2009. http://www.guardian. co.uk/science/2009/may/19/fossil-ida-missing-link-discovery

15.

“Backgrounder to Ida Fossil.” The Hindu, May 20, 2009. http://www.hindu.com/ holnus/008200905201041.htm

16.

http://www.age-of-the-age.org/evolution/darwinius_masillae.html#ida_darwinius_masillae_ study_background_story

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17.

World heritage fossil. Science Daily, May 19, 2009. http://www.sciencedaily.com/ releases/2009/05/ 090519104643.htm

18.

Church of England. http://www.telegraph.co.uk/news/newstopics/religion/2910447/CharlesDarwin-to-receive-apology-from-the-Church-of-England-for-rejecting-evolution.html

19.

Janet BE (2002) The Power of Place. Vol. 2, Jonathan Cape, Charles Darwin, London

20.

www.edb.utexas.edu/.../socialcognitive.htm

21.

O’Leary. Scientific American quietly disowns Ida “missing link” fossil. 21 July 2009. http://www.uncommondescent.com/intelligent-design/scientific-american-quietly-disownsida-missing-link-fossil/

22.

Rex D (2009) Fossil primate challenges Ida’s place. Nature 46:1040

23.

Seiffert ER, Perry MGJ, Simons EL, Boyer DM (2009) Conversant evolution of arthropoidlike adaptations in Eocene adapiform primates. Nature 461:1118–1121

24.

Watson JD, Crick FHC (1953) Molecular structure of nucleic acid-a structure for deoxyribose nucleic acid. Nature 171(4356):737–738

25.

Watson JD, Crick FHC (2003) Molecular structure of nucleic acids A Structure for deoxyribose nucleic acid. Am J Psychiatry 160:623–624

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3 Darwin’s theory of evolution: Survival of nature’s fit! Veena Tandon* and Gaurangi Maitra** *Department of Zoology, North-Eastern Hill University, Shillong - 793022, Meghalaya, India [email protected], [email protected] **Assam Rifles Public School, Laitkor, Shillong - 793022, Meghalaya, India [email protected]

Abstract: Even after 200 years after his birth, Charles Robert Darwin and his work are still central to any discussion on evolution. What is the relevance of evolution as Darwin perceived it? What were the pre-Darwinian, Darwinian and post-Darwinian pictures? Among the ancient pre-Darwinian theories Aristotle’s ‘Scala Naturale’ and the ‘Special Creation’ theory are perhaps the most important, whereas Lamark’s theory of ‘Evolution’ and Lyell’s theory of ‘Uniformitarianism’ are perhaps the most important modern ones, before Darwin’s work. His voyage of circumnavigation on the Beagle lead to pertinent observations: the similarities of structures between fossils and living forms in the same area; the presence of related but different species in adjacent areas of the same continent; the resemblance of species on isolated islands to those of the nearest continent and differences between species on closely adjacent islands of the Galapagos Archipelago in relation to their modes of life and feeding ; natural selection and survival of the fittest. The conclusions arising out of these and other observations were published as “The Origin of Species by means of natural selection or the preservation of Favored Races in the Struggle for Life”. But Darwin’s theory could not explain how the variations favored by natural selection initially arose or were inherited; the lack of intermediate forms and the long time frame needed for the changes to occur were two further objections to it. However, in the post-Darwinian era beginning with the rediscovery of Mendelism to modern day molecular biology, and the inputs from radioactive dating, these and other queries have not only been answered but they also strongly vindicate Darwin’s theory that has indeed stood the test of time and has emerged as the most convincing and fittest of all! Man has always wondered where he came from, wondered what his origins were and wondered spellbound at the wonderful variety of life around him! Was he a demigod, a spectator or participant in the natural scheme of things? May be on a star studded night, when man looked up at the sky and wondered, he became mankind. It is probably this single quality of wonder, more than anything else that made Charles Darwin synonymous with evolution in particular and biology in general. Even two hundred years after his birth, Charles Robert Darwin and his work are still central to any discussion on evolution. Darwin was an ‘evolutionary’, with a M.A. degree in theology and with an unquenchable V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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thirst for natural history! Darwin carefully marshaled his facts for more than two decades after his return from the famous Beagle voyage before publishing his magnum opus in 1859. Through this book, he set out to provide evidences that all life is interrelated and life forms are mutable; that they evolve from the simple to complex forms directed by nondeterministic natural selection; and the changes that make the organism suited to its environment and are passed onto the progeny result in descent with modification. The theory made change in organisms an irrevocable part of life. It created a controversy, because to accept this change would be going against certain religious dogma. But change is an inescapable fact of life. Is it therefore true that “nothing in biology makes sense except in the light of evolution”?1 In the century and half since publication of this book, the rapid advances in Biology have lead to investigating the very molecules that make up life! From the first detailed description of the nucleus in 1830 to the elucidation of the human genome is a mere 170 years! In this quickly progressing scenario, what is the relevance of evolution as Darwin perceived it? What were the pre-Darwinian, Darwinian and post-Darwinian pictures? And, how are they relevant today? Keywords: Darwin, Evolutionary theory, Natural selection, Variation, Neo-Darwinism, Origin of Species, Evidences from molecular biology

Evolution in the pre-Darwinian times Every human society had and continues to have its personal story of origin. Some of these are codified into written religious texts. Unlike a scientific theory whose strength lies in the fact that it should be falsifiable, religious theories suffer from the burden that to falsify is sacrilege! Added to this is the immutability of creation preached by most Semitic religions. Thus the traditional Judeo-Christian maintained that there was a special and independent creation of every species and life forms do not change through time from generation to generation. These creationists’ views were widely held, even by scientists, right into the early nineteenth century. On the other hand, the non-Semitic, especially Vedic religions, are closer to natural reality in their cyclical view of life. Thus immutability did not become an issue with them. Born in 384 BCE, Plato’s student Aristotle in his ‘History of Animals’ classified organisms in relation to a hierarchical ‘Ladder of Life’, placing them according to complexity of structure and function.2 Aristotle’s philosophy would thrive for centuries before being challenged. In AD 164, Galen, personal physician to the Roman Emperor, Marcus Aurelius, developed the belief that everything in the universe was created by God. The divine design was revealed in the form and organization of the human body.3 The struggle for existence was apparently first described by Al-Jahiz in the ninth century.4

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In his book ‘The Wisdom of God’ John Ray, the seventeenth century naturalist, who defined the species and genus, cited innumerable examples of the perfection of organic mechanism. Many examples of useful adaptations and design in nature were suggested by him, such as the structure of the eye, the hollowness of the bones, the camel’s stomach and the hedgehog’s armor.5 John Ray’s work influenced the leading biological scientist of the mid 18th century, Carolus Linnaeus. The latter’s most important contribution to science was his logical classification system for all living things which he proposed in his book ‘Systema Naturae’, first published in 1735. He apparently believed that he was just revealing the unchanging order of life created by God. Late in his life, however, he was troubled by the fact that plant hybrids could be created by cross pollination. These were varieties that had not existed before. Linnaeus stopped short of concluding that these plants had evolved.6 Buffon, in his ‘Historie Naturelle’, a 44 volume encyclopedia describing everything known about the natural world, wrestled with the similarities of humans and apes and even talked about common ancestry of Man and apes. Although Buffon believed in organic change, he did not provide a coherent mechanism for such changes. He thought that the environment acted directly on organisms through what he called ‘organic particles’. In his publication ‘Les Epoques de la Nature’ he openly suggested that the planet was much older than the 6,000 years against what was proclaimed by the church,7 and discussed concepts very similar to those in Charles Lyell’s ‘uniformitarianism’, which were formulated 40 years later.8 Erasmus Darwin, the paternal grandfather of Charles Darwin and an eminent speculative biologist, believed that evolution has occurred in living things including humans, in his poem Zoonomia. But his mechanisms of change were unclear; he also suggested that the earth and life on it must have been evolving for millions of ages before the commencement of the history of humankind.9 The first evolutionist who confidently and very publicly stated his ideas about the processes leading to biological change was a French protégé of the Comte de Buffon. He was Jean-Baptiste Chevalier de Lamarck who believed that microscopic organisms appeared spontaneously from inanimate materials and then transmuted, or evolved, gradually and progressively into more complex forms through a constant striving for perfection. The ultimate product of this goal-oriented evolution was thought by Lamarck to be humans. He believed that evolution was mostly due to the inheritance of acquired characteristics as creatures adapted to their environments and that creatures could develop new organs or change the structure and function of old ones as a result of their use or disuse. This idea was clearly put forward publicly in ‘Philosophie Zoologique’, published in 1809.10 Georges Curvier, the famous French comparative anatomist and paleontologist had identified skeletons of the extinct mammoths, pterosaurs and the giant lizard

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Mosasaurus. The proof of change lay before his eyes! Yet this undoubtedly great paleontologist rejected evolution on the grounds that he knew of no fossil forms between existing species and the 5,000 year old mummies! Cuvier had travelled to Egypt with Napoleon and seen mummified animals. He attributed sudden catastrophic changes (or Catastrophism) to account for changes in the Earth’s surface and extinction of animals.11 Cuvier’s theory would be superseded by the theory of Uniformitarianism with the publication of the first of the three volumes of Charles Lyell’s ‘The Principles of Geology: Being an Attempt to Explain the Former Changes of the Earth’s Surface, by Reference to Causes now in Operation’ in 1831. Uniformitarianism used the uniformity of nature as a basis for investigation with the present being a key to the past. In Lyell’s own words, “the animate and inanimate world, of which we find memorials in the Earth’s crust, may be similar both in kind and degree to those which are now in progress”.12

Inferences from the voyage Darwin joined the second survey expedition of the ship HMS Beagle that sailed from England on December 27, 1831. He carried the first volume of Lyell’s publication with him on this voyage. Originally planned to last two years, the journey lasted almost five– the Beagle did not return until October 2, 1836. Darwin spent most of this time exploring on land as the ship proceeded with its survey work. Darwin’s notes made during the voyage illustrate observations on Biology, Geology and Paleontology. These lead to the main inferences drawn from the voyage. Among the fossil haul in the region Punta Alta, Argentina was a large piece of the tessellated covering like that of the armadillo, but of gigantic size,13 which Darwin immediately realized must belong to an enormous Armadillo, living species of which genus were so abundant there. This first real pointer to evolution came from the Pampean formations of South America indicating the similarities of structures between fossil and living forms in the same area! Darwin noted how the rhea (a flightless ostrich-like bird), despite their similarity, hardly overlapped in their geographic distribution within the same continent. Instead, they seemed to replace each other as one travelled north to south; whereas the larger Rhea americana (also known as the greater or American rhea) was found beyond the Rio Negro in the Northern Patagonian Pampas, on the southern plains this was replaced by a related but smaller and distinct species (now called Rhea pennata or the lesser rhea or Darwin’s rhea).14 Thus, there was the presence of related but different species in adjacent areas of the same continent (Figs. 1 and 2). The islands that Darwin visited added a wealth of information. He found that 23 of the 26 species of land birds in the Galapagos had a close affinity with the South American mainland.15 European land shells were common to

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Figure 1. Rhea americana range. Source: Wiki- Figure 2. Rhea pennata range. Source: Wikimedia Commons media Commons

the Madeira Islands and its adjoining islet of Porto Santro. These observations made Darwin write, “such colonists would be liable to modification– the principle of inheritance still betraying their original birthplace”. 14 Indicating that the species of the isolated islands showed resemblance to those of the nearest continent. Darwin found the finches of Galapagos Archipelago, agreeing on many points – a peculiar structure of their bill, short tails and general form and in their plumage (Fig. 3). All species, excepting two, fed in flocks on the ground, and had very similar habits.13 Darwin found it very remarkable that this group showed a near perfect gradation in beak structure. The original beak was any hardly different from that of a warbler pointing clearly to the fact that all the finches had a common ancestor. It illustrates how natural selection has led to the evolution of a common ancestral species into a variety of forms adapted to different ecological niches.

1. Geospiza magnirostris 2. Geospiza fortis 3. Geospiza panrula 4. Geospiza olivacea Finches from Galapagos Archipelago

Figure 3. Darwin’s Finches. Source: Wikimedia Commons

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Formulation and publication of the theory Darwin returned from his voyage in 1836. The findings of the voyage were summed up in his Journal of Researches, published in 1839. The facts supporting the notion that species change were inescapable. He began the first of his notebooks on transmutation within a year of his return, in 1837. Darwin admitted ‘descent with modification’ and concluded that nature was in an open continuous process of ‘becoming’. Having accepted Lyell’s theory of uniformitarianism, it seemed logical to Darwin that change in land forms should be echoed in the flora and fauna, otherwise they would be doomed to perish. The multitude of living forms around him was a testimony to their ability to change with the environment. But how was this change directed? In October 1838, Darwin read Malthus’s famous treatise,’ An Essay on the Principle of Population’.15 It at once struck him that under natural circumstances favorable variations would tend to be preserved, and unfavorable ones to be destroyed. The result of this would be the formation of new species! With this insight Darwin had the frame work of his theory in place. He combined the forces of blind competition and random variation to explain development of design in living organisms. This neatly cut out the need for a designer or God. Knowing how controversial this matter was, he perhaps feared to rush into publication, where men like Buffon and Cuvier had feared to tread. In 1858, sick with malaria in the jungles of Malaysia, Alfred Russell Wallace posted Darwin a theory of evolution that he called “On the Tendency of Varieties to depart indefinitely from the Original Type”! This essay contained exactly the same theory as Darwin’s.15 Shocked as he was at having his life’s work preempted, Darwin did not dare to tear up a letter from a sick man and deny its existence. This joint paper entitled, ‘On the Tendency of Species to form Varieties and on the Perpetuation of Varieties and Species by Natural Means of Selection’ was read out on 1st July 1958, in the Linnaean Society! There was no significant reaction from the audience. The first edition of ‘The Origin of Species by Means of Natural Selection or The Preservation of Favored Races in the Struggle for Life’ was published on November 24, 1859. ‘The Origin’ in its opening chapters introduces variation under domestication and in nature and provides evidence to show how the constant intra and inter specific competition lead to the struggle for existence leading to natural selection or the survival of the fittest. Darwin also showed how species of the same genus vary in an analogous manner. Hybridism, the geological record, geographical distribution and their effect on variation is supported by a wealth of data. The finale is magnificent “There is a grandeur in this view of life, with its several powers, having being originally by the Creator into a few forms or into one; and that whilst this planet has gone on cycling according to the fixed laws of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been and are being evolved”.14 ‘The Origin’ did not seem to negate the role of a creator at this point, but did negate a single act of uniform creation.

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Reaction to ‘The Origin of Species’ The public reception to the book was very mixed. The Church of England scientific establishment went against the book, while the Anglicans supported Darwin’s natural selection as an instrument of God’s design. The Revd. John Stevens Henslow, the botany professor whose natural history course Charles had joined thirty years earlier, gave faint praise to ‘The Origin’ as ‘a stumble in the right direction’ but distanced himself from its conclusions.16 The most famous confrontation took place at the public 1860 Oxford Evolution debate during a meeting of the British Association for the Advancement of Science, when the Bishop of Oxford, Samuel Wilberforce argued against Darwin’s explanation. In the debate that followed Joseph Hooker argued strongly for Darwin and Thomas Huxley established himself as ‘Darwin’s bulldog’– the fiercest defender of evolutionary theory on the Victorian stage.17 Two famous nineteenth century comparative anatomists, Louis Agassiz at Harvard University and Richard Owen in England, surprisingly did not support the theory. Revd. Adam Sedgwick, geologist at the University of Cambridge who had taken Darwin on his first geology field trip, could not see the point in a world without God. The famous traveller in Africa and missionary David Livingstone could see no struggle for existence on the African plains.16 Many scientists of the day welcomed Darwin’s theory enthusiastically. Hewett Cottrell Watson, a famous nineteenth century English botanist, plant ecologist and evolutionist hailed Darwin as the “greatest revolutionist in natural history of this century”. Professor Robert Edmund Grant, who had shown him the study of invertebrates when Darwin was a student at the University of Edinburgh and who was still teaching Lamarckian evolution at University College London dedicated a book to Darwin. Asa Gray, the famous American botanist arranged the first US Edition of ‘On the Origin of Species’. Gray was a staunch supporter of Darwin in America, and collected together a number of his own writings to produce an influential book, ‘Darwiniania’.16 Sir Charles Lyell, the leader of the Uniformitarianism Theory in geology took almost a decade to completely accept Darwin’s theory.

Objections to the theory But whatever the reception, certain valid questions remained. Natural selection could account for the success of well developed adaptations but had no explanation for the initial stages of these developments, the development of the eye and the peacock’s feather were the most glaring examples of this lack of explanation. The absence of intermediate forms and the gaps in the fossil records being the other valid objections. The theory also demanded an extremely long time for small variations to accumulate. This was incompatible with the then estimated age of the earth. The most serious flaw was perhaps that Darwin had no satisfactory mechanism to show how variations were passed on to further generations.18

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Objections overruled! Initially the publication ‘The Mutation Theory’ (1900–1903) by Hugo De Vries gave rise to the proposition that mutation and not natural selection was responsible for evolution. De Vries’ theory that evolution, especially the origin of species, might occur more frequently with such large-scale changes than via Darwinian gradualism, basically suggesting a form of saltation became one of the chief contenders for the explanation of how evolution worked. At the same time Gregor Mendel’s trailblazing work in 1900, demonstrated in pea plants that inheritance follows definite rules. Each organism has two genes per trait, which segregate when the organism makes male and female gametes. Each gene in a pair is distributed independently during the formation of gametes and this clearly indicated as to how variations could be passed on and therefore supported Darwin’s theory and provided an answer to one of the main objections against it, i.e., how variations could be passed on! During 1910–1915, T.H. Morgan’s team from the fly room in Columbia University deduced the existence of genes, linked them to inheritance and mapped their location on chromosomes. This further strengthened the notion that natural selection was less important than mutations. Fisher, Haldane and Wright developed models to show that mutations only provide raw materials on which natural selection must work to produce speciation. Work on radiometric analysis that began in the early 1900s finally put the accepted age of Earth of 4.55 billion.19 This data helped to further the cause of Darwin’s theory by providing enough time for variations or mutations to accumulate. Darwinism was thus beginning to reemerge as neo-Darwinism. Darwinism only gained from the tremendous surge in Biology, especially in the work leading up to the proof that DNA was the genetic material. This opened the doors to the ‘Secret of Life’! 1953: The Double Helix structure of DNA was revealed by Francis Crick, James Watson and Maurice Wilkins, based on X-rays taken by Rosalind Franklin. Francis Crick’s suggested mode of DNA replication and Central Dogma of Biology solved the puzzle how variation are inherited. The new field of Molecular Biology was born and it proved Darwin was right! The decades following DNA structure elucidation gave us the vital genetic code, restriction enzymes, reverse transcriptase and DNA fingerprinting. It culminated in complete sequencing of the human genome.

Support to Darwinian postulates from modern biology Variation under domestication The effect of a Mendelian factor/or present day gene could be slight or powerful. Maize, the new world gift to humankind, has an almost unrecognizable ancestral,

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Figure 4. Teosinte vs. Maize. Source: teaandscones.wordpress.com/../page/3

wild form called toesinte. This bushy plant evolved into the Maize with its single stalk, due to mutations in the regulatory area of the one gene controlling cell division pattern during stem development. The change from the hard unappetizing small kernels to the soft, exposed kernels we are used to biting into is again due to change in a second developmental gene (Fig. 4). Mesoamericans bred the maize from toesinte by artificial hybridization with no access to all this special knowledge and in relatively few steps.20 Differences between ancestral and modern day forms have always been good support for the fact that species do and can change. Another South American plant that shows great variation under domestication is the tomato. In two thousand years of domestication, the original variety (Fig. 5) has given rise to 7500 varieties, with a whole spectrum of taste, color, texture and size. It is also interesting that in moving from its home in Peru, its pollinator, a species of halictid bee, did not move with them. Modern day cultivars have therefore been selected for self pollination. The frequent problem of blossom drop in domestic cultivars of tomato shows that attempted self pollination is not a great success. This could be due to a natural mechanism for preventing self pollination. In the flower the stigma extends well beyond the anthers, and it grows into that position a couple days before the anthers begin pollen shedding. Wild halictid bees, the best wild pollinators in field situations, are very sensitive to pesticides and cannot be introduced in crop fields. With the native pollinator absent, the other bees that might possibly serve are in short supply. Bumblebees are the second best, with honeybees a distant third, when they work the tomato blossoms.21 Darwin wrote in ‘The Origins’ “When we compare the individuals of the same variety or subvariety of our older plants and animals, one of the first points which strikes us is, they generally do not differ more from each other than do the individuals of any one species or variety in the state of nature”.14

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Convergent evolution The Moray eels (Muraena retifera) can eat prey, very much larger than their body. At the University of California, Davis (USA) Rita Mehta, an expert on snakes’feeding behavior, teamed up with fish biomechanist Peter Wainwright. Having worked on snakes, they looked for a similar mechanism in this eel. And surely enough, they found the moray eel using a second set of jaws to both restrain and transport prey! The eel’s pharyngeal jaws reach up from behind the skull to grasp prey in the oral jaws – mechanically similar to the ratcheting mechanisms used in snakes (Fig. 6). Similar adaptations found in animals widely separated phylogenetially, is a classical case of convergent evolution. It allows a fish and snake to be part of striking similar morphological, behavioral and ecological convergence!22 Darwin wrote in ‘The Origin’ “That mere physiological importance of an organ does not determine its classificatory value, is almost proved by the fact that in allied groups, in which the same organ, as we have every reason to suppose, has nearly the same physiological value, its classificatory value is widely different”.14

Figure 5. Wild tomato from Chile. www.usda. gov/is/pr/2005/051230,tomatoes. photo credit to Carl M Jones, University of California, Davis

Figure 6. Eel ratcheting jaws. blog.ngm.com/ blogcentral/2009/03/jaws-two.html

Adaptive radiation The works of twentieth century evolutionary biologists have shown beak and body size to change with climatic changes and they record possible examples emerging new species.29 In this era of molecular genetics, the various genes that are switched on in the developing beaks of finch chicks have been checked out. The gene for calmodulin (a molecule involved in calcium signaling that is vital in many aspects of craniofacial development and metabolism) is expressed more strongly in the long and pointed beaks of cactus finches than in the beaks of other species. An elongation of the upper beak, similar to that seen in cactus

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finches is seen when the levels of calmodulin were boosted artificially.24 The bone morphogenetic protein gene 4 [BMP4] induces cartilage and bone formation and also acts in mesoderm induction, tooth development, limb formation and fracture repair in humans. BMP4 is expressed at the putative site of fusion in the mid facial region. Its malfunction leads to the most common craniofacial birth defect (i.e., clefts of the lip and/or palate) in humans. BMP4 also plays a critical role during early embryonic development in the craniofacial region of chicks. In cichlid fish of Lake Victoria, the algae scrapping jaws have been modified into thick powerful jaws capable of crushing open snail shells.25 The BMP4 gene has been found responsible for this development. This same gene is found most strongly expressed in the large ground finch (Geospiza magnirostris) in the Galapagos Islands. The finch uses this robust beak to crack open large seeds and nuts (Fig. 7).24 The BMP4 gene provides an excellent example of the same gene responsible for the same function in different classes of vertebrates, thus strongly vindicating Darwin’s theory that all species have a common origin. Struggle for existence Human fossils indicate that tuberculosis (TB) has been around for more than half a million years. Today it claims two million lives annually and affects two billion people. This is due its resistance to antibiotics and mutlidrug regimes (MDR). This MDR-TB form has changed into the extensively drug resistant type XDRTB. This new form, first found in the 2006 KwaZulu, Natal outbreak has spread to 49 countries by 2008. New antibiotics to treat both types are being produced and undergoing clinical trials; SQ109 inhibits cell wall synthesis and PA-842 slows its cell division. But in vitro and in vivo result correlation have never been promising for TB. Like a bacterium worth its salt, Mycobacterium always finds a way out through evolution! With its genome sequenced in 1998, the new field of chemical genomics is being used to combat its evolutionary wizardry. Using bioinformatics as a tool the final goal is to replicate the bacterium in silico and thereby study its complete dynamics in a computerized cell free system and nullify its disease causing potential.26 Is it not also showcasing evolution of human ability? Necessity is surely the mother of invention in both the bacterium and human being. The real problem lies in not knowing the complete human - Mycobacterium interaction story. Darwin wrote in ‘The Origin’ “It is therefore good to give any one species advantage over another. Probably in no single instance should we know what to do. This ought to convince us of our ignorance on the mutual relations of all organic beings; a conviction as necessary, as it is difficult to acquire”.14

The descent of man In 1974, anthropologists working in a maze of ravines at Hadar in northern Ethiopia

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Figure 7. BMP4 at work: scienceblogs.com/.../06/nytimes_on_evodevo.php

followed a small clue on a slope. They found nearly 40% skeletal remains of a hominid, or humanlike creature that lived around 3.2 million years ago. Based on its small size, and pelvic shape, they concluded that it was female.27 It was later identified as Australopithecus afarensis and it is an ape closely related to human beings (Fig. 8). Here was an intermediate form in the story of human evolution! Something both Cuvier and Darwin missed seeing! Katherine S. Pollard provides significant insights into chimp-human dissimilarity despite 99% similarity. Using the huge computer cluster at the

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University of California, Santa Cruz and a self designed computer programme, Pollard highlights some sequences that are uniquely human. Human accelerated region 1 (HAR1) is a group of 118 bases in that part of our genome that is dissimilar from that of the apes. This region has hardly changed in vertebrates whose genomes are available to us. Between the chimp and chicken, which diverged some 300 million years ago, there is a two base difference in this sequence; whereas between the human and chimp, which diverged only 6 million years ago, there is a difference of 18 bases! HAR1 is required for the formation of a healthy cerebral cortex (Fig. 9). That HAR1 is not a protein encoding region but encodes RNA was proved by experimental work from a UC group at Santa Cruz. Thus, it is also the first documented example of a RNA-encoding sequence that seems to have been positively selected, showing that natural selection, Darwin’s bedrock for mechanism of evolution, can occur at the nucleotide level. To a Hindu mind, the letters HAR1 look uncannily like the word hari (meaning the preserver) an allusion not very far off the mark, considering the fact it controls the development of the vital cerebral cortex! Its malfunction is linked to congenital lissencephaly (smooth brain) and adult schizophrenia.28 Similarly, HAR2 has an ancestral version in the ape that does not promote the same level of wrist and thumb development as its modern version does in humans. It seems to be responsible for the uniquely

Figure 8. Female homonid skeleton. Source: Wikimedia commons

Nature at work: Ongoing saga of evolution

Figure 9. HAR1 makes us human?

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human dexterity that leads to tool making and technology.28 This only underscores Darwin’s postulates of common descent and shows diversity and adaptation arise from natural selection. Therefore, in 1860, TH Huxley was right to end the famous Oxford debate against Darwinism by apparently saying that he was not ashamed to have a monkey for his ancestor, but he would be ashamed to be connected with a man who obscured the truth. This was in reply to Bishop Samuel Wilberforce’s question as to whether it was through his grandmother or his grandfather that Huxley considered himself descended from a monkey.17

Beyond Darwin Darwinian medicine applies evolutionary data to our understanding of health problems and health promotion. Matching disease to genotype can result in customized medicine. Darwinian medicine tells us inconveniences like fever, diarrhea, vomiting, actually help in cleansing out microbial infections! Vaccines against human papilloma virus and improved hepatitis C vaccines have been developed by a process called directed evolution. Mixing segments of 20 different human interferons has given us a jigsaw that is 250,000 times more effective against viral replication than the normal interferon. Uniquely human complex problems like air traffic control, stock portfolio options, electronic circuits, optimum combinations of medicines, orbits of communication satellites for minimizing signal loss are finding answers from evolutionary or genetic algorithms that are often less symmetrical than conventional designs but far more effective! Critical areas like ecosystem services on which the life and livelihood of this planet depends could benefit tremendously from evolutionary methods and knowledge.29

Conclusion Religious dogma held center stage till Charles Darwin did a Copernicus act for biology and rewrote the script for the play of life. In retrospect it seems that only a blind person would be incapable of learning from the billions of years of nature’s experiments revealed on the voyage. The real reward from the Beagle voyage was the huge data base on which generations continue to work. It gave Darwin the raw material, with which he synthesized his theory of Organic Evolution, set out in the ‘Origin of Species’. The theory of evolution propounded in this volume has not only survived but has vigorously prevailed in the century and half since its publication. No small tribute to its ‘fitness’! Darwin’s theory is a foundational pillar of modern science at par with relativity and quantum theory. Darwin, like all original thinkers, marched to his own drumbeat. He rejected ‘Scala Naturale’ for branching evolution, accounted for design without designer via natural selection and embraced uniformitarianism. Today natural selection has basic relevance

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from basic science to practical pursuits, from biotechnology to forensic science. We still have questions to answerHow common is natural selection? How far does it occur at the molecular level? What is the origin of genetic variation on which natural selection works? Nature’s open book and evolution only underline the philosophy - the only permanent thing in life is change. Therefore, when evolution and change are synonymous, we must acknowledge that “Nothing in biology makes sense except in the light of evolution”.1

References 1.

Dobshansky T. Theory of evolution. http:// bio.classes.ucsc.edu/bio20c/evolution/Lecture1. Html

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Bynum WF (1975) The great chain of being after forty years: an appraisal. In: History of Science.

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Spanenburg R, Moser DK (1999) From the ancient greeks scientific revolution. In: The History of Science. Universities Press (India) Limited

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Bayrakdar M (1983) Al-Jahiz and the Rise of Biological Evolutionism. (Third Quarter) The Islamic Quarterly, London

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Ray J. The Wisdom of God Manifested in the Works of the Creation. http://www.jri.org. uk/ray/wisdom_links.html.

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Linnaeus Carolus. http://www.biodiversitylibrary.org/bibliography/542. 10th Edition (2 volumes) online at the Biodiversity Heritage Library

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James Barr (1987) Biblical Chronology: Legend or Science. The Ethel M. Wood Lecture 1987. Delivered at the Senate House, University of London on 4 March 1987. London: University of London, 1987. Pbk. ISBN: 7187088644. pp 1

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Leclerc, Georges-Louis, Comte de Buffon. http://www.eoearth.org/article/Leclerc,_GeorgesLouis,_Comte_de_Buffon

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Darwin E. Zoonomia; or the Laws of Organic Life. http://www.gutenberg.org/ etext/15707

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Jay GS (2002) The Structure of Evolutionary Theory. Belknap Harvard, Harvard

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Martin JS, Rudwick (1997) In: Georges Cuvier, Fossil Bones, and Geological Catastrophes. The University of Chicago Press

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Uniformitarianism (1982) In: Encyclopedia Britannica Macropedia Volume, 15th edition, University of Chicago

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Darwin CR (1989) In: Voyage of the Beagle. Browne J, Neve M (Eds.), Penguin Books

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Darwin CR (1989) In: The Origin of Species. Modern Library Paperback Edition

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Darwin CR: In: The Autobiography of Charles Darwin With the Original Omissions Restored. Edited and with appendix and notes by his grand-daughter Nora Barlow. London: Collins. pp 118, 1809–1882

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Reaction to Darwin’s theory. http://en.wikipedia.org/wiki/Reaction to Darwin’s theory

17.

Lucas JR (1979) Wilberforce and Huxley: A Legendary Encounter. Historical J 22(2): 313–330

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18.

Miller J (1986) In: Darwin for Beginners. Writers and Readers Cooperative in association with Unwin Paperbacks

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Claire P (1956) Age of meteorites and the earth. Geochimica et osmochimica Acta 10(4):230–237

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Kingsley DM (2009) From atoms to traits. Scient Am 4(1):33

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Tomato: [ http://en.wikipedia.org/wiki/Tomato]

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Mehta RS, Wainright PC: Raptorial Jaws in the throat help moray eels swallow large prey. [http://www.nature.com/nature/journal/v449/n7158/abs/nature06062.html]

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Stix G (2009) Darwin’s Living Legacy. Scient Am 4(1):14

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Abzhanov A, Kuo WP, Hartmann C, Grant BR, Grant PR, Tabin CJ (2006) The calmodulin pathway and evolution of elongated beak morphology in Darwin’s finches. Nature 44: 563–567

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Terai Y, Morikawa N, Okada N (2002) The Evolution of the Pro-Domain of Bone Morphogenetic Protein 4 (BMP4) in an Explosively Speciated Lineage of East African Cichlid Fishes. Mole Biol Evol 19:1628–1632

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Barry CE, Cheung MS (2009) New tactics against tuberculosis. Scient Am 4(3):52–57

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Johanson D, Maitland E (1981) In: Lucy: The Beginnings of Huhumankind. Simon and Schuster, New York

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Pollard KS (2009) What Makes Us Human? Scient Am 4(5):34–37

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Mindell DP (2009) Evolution in the Living World. Scient Am 4(1):58–65

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4 The life and research of JBS Haldane in India with special reference to Charles Darwin Krishna R. Dronamraju Foundation for Genetic Research, P.O. Box 27701-0, Houston, Texas, USA 77227 [email protected]

Abstract: JBS Haldane (1892–1964) was a polymath, a great scientist who made important contributions to multiple sciences such as Physiology, Genetics, Biochemistry, Statistics, Biometry, Cosmology and philosophy, even though he did not possess an academic degree in any science. His most important contribution to science was the mathematical theory of evolution which combined the evolutionary process defined by Darwin and the science of genetics founded by Mendel. Haldane moved from London to live and work in India in 1957. As a Research Professor at the Indian Statistical Institute in Calcutta, Haldane directed research on local fauna and flora, which followed the Darwinian tradition. In 1962, he moved to head the Genetics and Biometry Laboratory in Bhubaneswar but died in 1964 because of cancer. Haldane’s research and teaching in India benefited many young scientists, partly because of his great knowledge, but also because of the encouragement he provided by emphasizing the dignity and value of scientists to society. Haldane was a fierce critic of science administration and science policy. Haldane advised the Defense research organization in India in problems related to deep sea diving, and adaptation to extremes of cold temperatures and atmospheric pressures, while breathing toxic mixtures of various gases, based on knowledge gained by painfully experimenting upon himself. We were very proud that Haldane who chose to spend his last years in India. Both Haldane and his wife (scientist Dr Helen Spurway) applied for Indian citizenship which was granted in 1961. Thus, he fulfilled the promise he made to his friends in London that he would die as an Indian. Keywords: Darwin, Mendel, Biometry, Animal behavior, Human genetics, Statistics, Paleontology, Epidemiology, Speciation, Population genetics, Natural selection

Introduction JBS Haldane (1892–1964) was born on 5th November 1892, in Oxford, England. He was a distinguished British scientist who migrated to live and work in India in 1957. Haldane was invited by Prof. Prasanta Chandra Mahalanobis, Director V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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of the Indian Statistical Institute in Calcutta, to be a Research Professor at the institute. He was known as a polymath, an individual who possessed great intelligence and knowledge of multiple disciplines. In 1962, Haldane moved to Bhubaneswar to direct the Genetics and Biometry Laboratory which was set up especially for him by the Chief Minister of Orissa Mr. Biju Patnaik. Unfortunately, he was diagnosed with cancer in 1963 and was operated in London, but the cancer recurred and Haldane died on 1st December, 1964.1 Haldane directed my PhD in genetics from the Indian Statistical Institute. Mrs. Haldane (Dr Helen Spurway) was also a scientist who specialized in genetics and animal behavior. I worked closely with the Haldanes at Calcutta and Bhubaneswar. We traveled together to attend several scientific meetings, for instance, to Sri Lanka, Israel, UK, France and Italy, etc.

Background Haldane was a great popularizer of science and contributed numerous articles to newspapers and popular magazines in several countries including India. Some of these essays were recently published in a book which I have edited.2 Many of his essays were written while traveling on trains and planes. Haldane always carried a pad and pen and started writing as soon as he had a few minutes to spare. He never possessed a degree in any branch of science, yet he became one of the great and influential scientists of the 20th century. Haldane, who was popularly known as ‘JBS’ or ‘Prof’ or simply ‘Haldane’ was a polymath, that is to say, a genius who possessed almost universal knowledge. He made important contributions to several sciences as well as to philosophy, religion, logic, popular writing, and ethics. In science, Haldane’s contributions covered physiology, genetics, biochemistry, statistics, biometry, cosmology and history and philosophy of science. As a popular writer of science, Haldane covered even more subjects, including zoology, physics, chemistry, mathematics, geology, geography, astronomy, meteorology, politics, psychology, non-violence, military affairs, theology and literature. He was a marxist and supporter of Soviet science from about 1937 to 1950 but abandoned that position when the science of genetics was suppressed in the Soviet Union under the pseudo-scientist Lysenko who was supported by Stalin. Haldane’s distinguished relatives include his father John Scott Haldane, physiologist at Oxford University, his uncle Viscount Haldane who was Minister for war and Chancellor of the Exchequer in Prime Minister Asquith’s cabinet, his great uncle Burdon Sanderson, first Waynflete Professor of Physiology at Oxford University, his sister (Lady) Naomi Mitchison, prolific writer of numerous books in fiction, non-fiction, travel, history, politics and other subjects. His three nephews are distinguished scientists in the life sciences. Haldane’s scientific reputation is based on his extensive series of papers on the mathematical theory of Darwinian natural selection (evolution). The independent contributions of Haldane and

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R.A. Fisher in England and Sewall Wright in the United States led to the establishment of the branch of genetics called ‘population genetics’. Haldane’s classic work, The Causes of Evolution3 was a founding document of this process. However, Haldane’s scientific work was much broader than that of his colleagues. In addition to his monumental work in genetics, Haldane made important contributions to enzyme kinetics in biochemistry. In physiology, Haldane is best remembered for his daring experiments in testing the effects of poisonous gas mixtures, cold temperatures and higher atmospheric pressures in which he employed himself as the ‘guinea pig’. They were often painful experiments, causing convulsions and injuries. He also conducted many diving experiments to investigate a submarine accident for the British Navy, once again employing himself as his own ‘guinea pig’. He suffered permanent injury because of rapid decompression during the experiments and the back pain haunted him rest of his life.4

Science in India Haldane’s research in India was primarily influenced by the Darwinian tradition. His major contribution to science was his mathematical theory of evolution (natural selection) which was an interpretation of Darwin’s theory of evolution in the context of Mendelian genetics. During the years: 1924–1934, Haldane’s series of mathematical papers on this subject became a foundation for the new scientific discipline called ‘population genetics’. However, after his arrival in India, Haldane saw Darwin from a new perspective.5 Much of our early research was concerned with a detailed analysis of ‘meristic variation’ in ‘Bateson’s6 terminology. As Haldane explained to our visitors, all our research projects followed the Darwinian tradition. They were summarized in my book: Haldane: The Life and Work of JBS Haldane with special reference to India.1

Animals have rights and duties Haldane5 stated that Christian theologians drew a sharp distinction between humans and other species. Hence, Darwin’s declaration that human beings have descended from animals created a furor among Europeans. Whereas many eastern religions such as Hinduism, Buddhism and Jainism have long recognized that animals have rights and duties. Indian art and religion have depicted divinity in various animal forms and kindness towards other species has long been an accepted tradition, at least in theory if not in practice always, in India. There are no parallels in Europe and North America. There is still some opposition to teaching evolution in some Christian communities, especially in the southern United States. Not only did

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Haldane interpret Darwinism from the Indian philosophical and religious point of view, but he also developed several research projects on Indian species of plants and animals which are in the Darwinian tradition. As his students, we were all encouraged to conduct research on different projects in that tradition. There were certain characteristics which are common to all these projects: (a) Much of our research involved measuring biological variation, especially ‘meristic’ variation, that is to say, variation in like parts such as petal numbers in flowers, venation in leaves, number of teeth, or bilateral spotting in caterpillars, etc. (b) research involved local resources as much as possible, (c) the projects did not require much financial support, (d) the methods employed were simple but the results had scientifically profound consequences, (e) the observations must be quantified wherever possible, (f) the project should be flexible enough to facilitate the incorporation of any new and unexpected results and the resulting change in direction or methods, (g) it is necessary to have several ongoing projects simultaneously to achieve quick results as there will always be some lines of pursuit which may not bear fruit, and (h) the results should be published immediately. Some of the projects were an imitation of Darwin’s experiments. One such example is the estimation of the volume of soil brought to the surface by earthworms per acre per year. It is a sound research project in ecology and agriculture. S.K. Roy conducted experiments with mixtures of rice varieties planted in plots which gave increased yields of seeds in some cases.6 Another project, which was my research interest, is the investigation of color preferences of Lepidoptera among different colored flowers and their impact on speciation.7 By their preferential visits to different varieties of a plant species differing in flower color, insect pollinators can create reproductive isolation within a species. By means of increasing such isolation (for instance, it could happen if there is a new flower color mutant appearing in the plant population) further divergence can follow, ultimately leading to complete isolation and speciation. Such speciation has been called ‘sympatric speciation’, as opposed to ‘geographic speciation’ where such geographic factors as floods, earthquakes and climate change can result in reproductive barriers within a species. Haldane believed that such research projects are following the Darwinian tradition and would have interested Darwin greatly. Another colleague T.A. Davis8 pursued research on coconut palms, and discovered that palms with left-handed coiling in their crowns produce more nuts than those with right-handed spiraled crowns. Haldane stated that they were what Darwin called ‘fools’ experiments. S.D. Jayakar and Mrs. Haldane (Dr. Helen Spurway) collaborated with me to conduct biometrical investigations of the nest-building activity of the solitary wasp, Sceliphron.9 Anthropologist A.K. Ray10 conducted population studies of toe anomalies and their possible genetic basis. Haldane commented that such studies are easier to follow in India because many people have bare feet or wear sandals, unlike western countries where shoes and boots are worn by many.

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Consequences of Darwinism Haldane regarded Darwin’s work in botany as his most original and important contribution to science. That work includes books on insectivorous plants, climbing plants, sexuality in plants, and inbreeding in plants. Charles Darwin is best known for his work on evolution; however, that subject was also discussed by many others including his grandfather, long before Darwin. His experiments on plants, although not so well known, are original. Haldane encouraged us to follow Darwin’s research in botany. But, Haldane wondered what Darwin’s attitude which led to his discoveries was. Darwin was most open minded in his attitude towards all living beings. Haldane wrote: “Darwin did not draw a sharp line between earthworms and the old gentlemen who had failed to interest him in mathematics at Cambridge. From the biographical account of his son Francis, we know that Darwin was totally immersed in his experiments and observations. Indeed, he was so dedicated that he used human attributes when describing his experimental animals and plants. Haldane5 explained that such dedication is characteristic of saints in ancient India. He stated that a saintly attitude with total dedication results in excellent science and it is needed today in India. Haldane5 drew attention to Aristotle’s logic and his difficulties in classifying animals which he based on similarities. Darwin, on the other hand, foreshadowed a logic based on differences. In the Origin of Species, Darwin11 wrote: “Systematists will have only to decide (not that this will be easy) whether any form be sufficiently constant and distinct from other forms, to be capable of definition, and if definable whether the difference be sufficiently important to deserve a specific name... Hence, without rejecting the consideration of the present existence of intermediate gradations between any two forms, we shall be led to weigh more carefully and to value higher the actual amount of difference between them.” Haldane regarded Darwin’s statement as the starting point for a whole new program in statistics.

Figure 1. JBS Haldane before his death

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There are ethical consequences of Darwinism which have not always been desirable. Darwin was led to the theory of natural selection by reading Malthus on population growth. Although Darwin pointed out the importance of physiological adaptation, he left the erroneous impression that the struggle for life was analogous to war and economic competition in the human species. Consequently, Darwinism was used to justify wars and persecution. Social Darwinism is an undesirable and unwanted consequence of Darwinism. On the other hand, Darwinism has reminded us of our responsibility towards other species. It opened up a whole new aspect of our social behavior, the attitude we should adopt towards other species which are related to us, although distantly. Haldane believed that an Indian perspective of Darwin offers a corrective to the ‘western’ point of view. Additional information can be found in my book: Selected Genetic Papers of JBS Haldane.12 Science and ethics are discussed in Haldane’s Daedalus Revisited.13

Impact on Indian science The foremost impact of Haldane’s activities in India was to raise the social awareness of scientists and their value to society. As an outspoken scientist of great importance, Haldane utilized his position to express publicly what he thought about the state of science in India. He was a fierce critic of the science administration in India and pointed out many of its deficiencies. He fought for greater independence for younger scientists, lesser beaurocratic complications in science administration, increased support for scientific research by the Central Government, and greater dignity and respect for the scientific workers in society. He pointed out the need to raise the quality of research and the teaching standards. Haldane’s efforts were applauded by the younger generation. Many of them believed that he was voicing their grievances fearlessly when they could not do so themselves. Haldane’s emphasis on the utilization of local resources for research opened a new tradition in the 1960s when many Indian scientists returning from Europe and North America were complaining of lack of facilities in India. Haldane, on the other hand, suggested several research projects which could be followed in India without utilizing expensive equipment or great expense. All our research projects met these criteria which were defined by Haldane1. Yet, the results were of profound importance and interest. They followed the Darwinian tradition. India had produced great scientists of its own; some examples are J.C. Bose, C.V. Raman, P.C. Mahalanobis and Homi Bhabha among others. Haldane’s migration to India added another dimension. We were fortunate that he chose to live and work in India during his last years. He saw the resources of India — the fauna and flora, from a fresh perspective. Consequently, many younger scientists who listened to his pronouncements with an open mind continued to follow the traditions started by Haldane long after his death in 1964. Today, his impact can

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be seen in several sciences, such as ecology, population genetics, animal behavior, human genetics, biometry, statistics, paleontology, epidemiology and several other biological and medical sciences. One branch of research which benefited India’s Defense Department was Haldane’s contributions to human physiology which was concerned with adaptation to cold temperatures and higher atmospheric pressures, as well as his great work in diving physiology and inhalation of various toxic gases. Haldane’s advice is of great interest to both underwater research of the Navy and also to the physiological problems encountered in space flight.14 The fundamental physiological principles enunciated by Haldane and his father are still important today in many fields and are followed by scientists all over the world. Two such examples are the use of heart-lung machines in surgery and the development of a cure of tetany. To conclude, the impact of Haldane’s great genius will be felt in India and the rest of the world for many years to come.

References 1.

Dronamraju KR (1986) Haldane: The Life and Work of JBS Haldane with Special Reference to India. Aberdeen (UK): Aberdeen University Press (1985). (A summary was published in my review: “On some aspects of the life and work of John Burdon Sanderson Haldane, FRS in India. Notes and Records of the Royal Society of London) 41:211–237

2.

Dronamraju KR (Ed.) (2009) What I Require from Life: Writings on Science and Life from JBS Haldane (with a Foreword by Sir Arthur C Clarke), Oxford University Press, Oxford

3.

Haldane JBS (Ed.) (1932) In: The Causes of Evolution. Longmans. Green, London

4.

Dronamraju KR (Ed.) (1968) In: Haldane and Modern Biology. Johns Hopkins University Press, Baltimore

5.

Haldane JBS (1959) In: An Indian Perspective of Darwin. Centennial Review, Michigan State University,

6.

Roy SK (1960) Interaction between rice varieties. 57:137–152

7.

Dronamraju KR (1960) Selective visits of butterflies to flowers, a possible factor in sympatric speciation. Nature 186:178–179

8.

Davis TA (1963) The dependence of yield on asymmetry in coconut palms. J Genet 58:186–215

9.

Spurway H, Dronamraju KR, Jayakar SD (1964) One nest of Sceliphron madraspatanum. J Bombay Nat Hist Soc 61:1–26

10.

Ray AK, Haldane JBS (1965) The genetics of a common Indian digital abnormality. Proc Nat Acad Sci 53:1050–1053

11.

Darwin C (Ed.) (1859) In: On the Origin of Species. John Murray, London

12.

Dronamraju KR (Ed.) (1990) In: Selected Genetic Papers of JBS Haldane. Garland Publishing Inc., New York

13.

Dronamraju KR (Ed.) (1995) In: Haldane’s Daedalus Revisited. With a Foreword by Nobel laureate Joshua Lederberg. Oxford University Press, Oxford

14.

Haldane JBS (1960) Physiological problems at high pressure. Aero Medical Society J 1–10

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5 Charles Darwin: A driving force for humanity toward agnosticism Amit Sharma Structural and Computational Biology Group, ICGEB, New Delhi - 110067, India [email protected]

Abstract: Charles Darwin’s insights into evolution of biological life are perhaps the most significant contributions in the realm of human existence. There have been many other startling scientific discoveries, and indeed many are yet to come, but arguably none have caused such intense, visceral reactions in mankind like Darwinian ideas. Nearly a century and a half after the unbiblical Origin of Species appeared, the fact of biological evolution as a step-wise, slow, blind, non-religious phenomenon continues to awe many, while riling the religious bigots. More significantly, Darwinian forays into evolution of life have laid the foundation for modern scientific investigations, and have profoundly guided and influenced all spheres of biological research. The role of ‘evidence’ to back up human ideas has become a mainstay in evolved thought. A direct result of this quest for ‘evidence’ is the devaluation of religious claims. Rejection of all religious neurosis is however insufficient now, and its possibly time to adopt ‘evidence-based existence’. The short essay here deals with some such issues. Keywords: Atheism, Darwin, Dogmas, Evolution, Religion All great truths begin as blasphemies. —Bernard Shaw

When interviewed,1 an African prostitute revealed that although she had had on average ~10 clients a day over the past 25 years (many of whom had been HIV positive and had subsequently died), she had miraculously remained HIV free. The interview ended with her final statement where she felt “thankful to god for saving her from HIV”. The irony here may be lost on the devout but genetic evidence has shown that it was not the divine this lady should have thanked, but rather a rare mutation in her genome that probably arose ~700 years ago1. This mutation makes human cells resistant to infection by HIV, and may have evolved to resist bubonic plague or smallpox at the time. Poignantly, this genetic roulette makes it likely that her genes, which can withstand the HIV rampage in Africa, V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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may spread over the next generations in the population and save many from HIV. I hope this story illustrates two points – (a) the power of modern biology in light of evolutionary processes, (b) the ludicrous nature of religious belief to explain such biological oddities (wonders). This essay deals with the implications of Darwin’s evolutionary discoveries for understanding our existence.1–3 Accounts of Darwin’s biographical life may be found elsewhere in this book, or indeed on various websites dedicated to Charles Darwin. In particular, this essay deals with the dichotomy between our new understanding of biological life (derived from Darwin’s works) and the alternative explanations for life’s origins (derived from the axis of monotheistic and older religious systems). Revolutions in science over the past many centuries, including Copernican and Darwinian discoveries, have provided unambiguous, testable, rational and unbiased answers to understandable human inquiries about emergence of both our universe and life in it. However, the sway of dogmatic beliefs on humanity continues to rise – and despite more than 150 years of Darwinian revolution his insights remain topics of debate. An excellent example of the continuous nature of debate on Darwinian insights is the launch of this very book. Clearly, a lot remains to be learnt and accepted about Darwin, evolutionary biology and the inherent contradictions between Darwinian ideas and varied versions of religious stories. Note in contrast that Copernican and Galilean scientific revolutions have been more widely accepted and there are, hopefully, no books commissioned that still debate whether earth is at the centre of our universe (although again, there must be a band of zealots who think differently). In part, lack of general acceptance of Darwinian insights into the origins of biological life stem from deep seated hubris humans have had – fueled further by religious delusions. Most dogmatic belief systems remain essentially in complete denial of scientific discoveries, perhaps because the latter directly threaten or overthrow the schizophrenias that religions like to perpetuate. I have at least, as I hope, done good service in aiding to overthrow the dogma of separate creations. —Charles Darwin

Part I: On Darwin – the master iconoclast Charles Lyle, father of geology, profoundly influenced Charles Darwin. Charles Lyle had proposed (correctly) that the earthly landscapes were not formed over a few thousand years (as has been wrongly advocated by dominant religious systems) but instead over millions of years by gradual change and as a result of natural (physical) forces.1 Darwin read up on Charles Lyle’s geological insights while on Beagle, and there is little doubt that this must have influenced Darwin tremendously. To dwell on the vastness of time since earth formed and life

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~4.5 billion years

Cyanobacteria

Earth birth

Eukaryotes

Single celled

Scale

Multicellular

Mammals Vertebrate

Time

500 million years 50 million years 5 million years

= represents time required for continuous re-incarnation of a 100 year old human more than 50,000 times Figure 1. A linear perspective on the length of time (horizontal bar) since earth originated and since various forms of life evolved. Note that for the first ~2000 million years there was only bacterial life. The vertical bars represent times when respective life forms appeared. The scale bars represent time periods and the smallest speck equates ~5 million years. If a human lived for 100 years then this smallest speck will contain more than 50,000 reincarnations, back to back. The scale of time since life originated on earth and started to evolve is almost unfathomable by everyday human markers of time

evolved is challenging to say the least (Fig. 1). In a way, Darwin’s insights may be considered very similar to Lyle’s – the difference essentially lies in the resolution of objects studied. Just like in Lyle’s (geological) universe, in the Darwinian world also there is the (biological) landscape, the eons of slow change and the natural force (natural selection) that orders objects (life). Many a long dispute among divines may be thus abridged: It is so; It is not so. It is so; it is not so. —Benjamin Franklin

Darwin’s discoveries on the evolution of life and mechanisms therein have influenced most if not all scientists. Fundamental research in biology during the last 150 years has developed within the framework of evolutionary biology – and has led to revelation of the genetic (DNA) basis of life. The key tenets of Darwinian1–3 and modern evolutionary biology4–6 are: (1) Random mutations are the central players that drive evolution, (2) Harmful mutants are deleted and beneficial variations tolerated by force of natural selection, (3) Evolution moves

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forward by collecting small, useful variations in a gradual manner over a vast length of time, (4) Process of evolution has remained the same over time, and (5) All biological life (extinct and extant) may be drawn as a ‘tree of life’ – as depicted by Darwin in the Origin of Species. The first two points are the mainstay of Darwinian ideas. Satan hasn’t a single salaried helper; the Opposition employs a million. —Mark Twain

Mutations are considered to be random occurrences but only in the sense that there is no foresight (i.e. the system is unaware of beneficial mutations a priori). The key insight into the random mutations that have driven evolution is that these alterations are not ‘directed’ or ‘guided’. Mutations are in general random and occur rarely.4–6 But most importantly, mutations are blind to whether they will end up benefiting the organism or not. The next step - natural selection - is far from ‘random’. These twin processes of ‘change’ and then ‘fixation’ iterate and thereby underpin evolution of life. Many organisms have existed for billions of years without any significant (genomic) change. Others may evolve rapidly under various biological or environmental stresses. In either case, natural selection watches over and approves those best adapted. In humans, juggling of parental genes during sexual reproduction provides enough fuel for natural selection to keep working on the resulting progeny. However, the saga of variation (mutations) versus fixation (natural selection) does not play out evenly. Once organisms have ‘adjusted/adapted’ to their environmental stresses, they may still incur random mutations but these variations will not be policed by natural selection unless a new selective pressure is applied on the system. An example of this is the genetic discovery of resistance amongst some humans to infection by HIV.7 This genetic background in ‘select’ humans has probably existed for ~700 years but it is only the recent appearance of HIV infection in humans that these ‘mutant’ genetic profiles may start to play a role in natural selection. Therefore, stochastic variation, without direction or purpose, fuels evolution. Herein, chance begins to surface as a key ingredient in evolutionary processes, but not without natural selection watching over it.8–11 The introduction of ‘chance’ in matters of life is unsettling for creationists, who like to believe that humans or even animals are “engineered” biological specimens. But as science has shown over and over again, chance and chance alone is responsible for the diversity in the living and in the dead. Evolution is driven forth by genetic randomness which is followed by determinism via natural selection. Mechanistically, all biological diversity has therefore been generated by force of natural selection which works upon random genetic tinkering. These evolutionary processes are terrifically creative and powerful, as evidenced by the wonders of flora and fauna. The Darwinian idea of evolution by natural selection constitutes a fundamental law of nature which governs life. This Darwinian revelation has thankfully obviated any requirement

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for solace in whimsical and mythical stories of life’s origins as divine gifts. But natural selection is also blind, emotionless, directionless and in itself aimless and perpetual. By the development of vaccines, drugs, surgeries and health care systems, modern medicine aims to fight back the ruthless and stoic force of natural selection. With our new understanding of natural selection as a power that both creates and destroys, aimlessly, we have and must continue to protect those who are genetically or otherwise weak and will be therefore the first victims of natural selection. No amount or effort spent on religious intercourses will rescue children and the frail from opportunistic viruses, bacteria and parasites (which altogether are only doing what their genomes drive them to do). Only man’s ingenuity and rigorous commitment to reason, rationally and science can protect all of us from the blind force of natural selection. We can allow satellites, planets, suns, universe, nay whole systems of universes to be governed by laws, but the smallest insect, we wish to be created at once by special act. —Charles Darwin

Natural selection cannot anticipate future environments on earth. Therefore, the set of existing organisms can never be fully prepared for environmental catastrophes that await life. An outcome of this is the extinction of those species which cannot overcome environmental adversity. This failure to survive, in modern terms, can be attributed to the genomes which are unable to withstand geological vagaries or biological mishaps (infections, diseases and so on). In biological evolution on earth, extinction of species has been a major feature. Earth may presently have up to 10 million species, yet more than 90% of species that have ever lived on earth are now extinct.8–11 Once again, the creationist doctrines fail to satisfactorily address why a divine creator will firstly bother to create millions of species and then allow them to perish. The Darwinian explanation for extinct life is once again simple, elegant and at once convincing – biological life goes extinct as a function of environmental or biological assaults for which their inheritance deems them ill-equipped. Therefore, the so-called Darwinian theory of evolution is not a theory at all. Evolution happens – this is a fact. The mechanism of evolution (Darwin proposed natural selection) is also amply supported by scientific data. Indeed, to date no single zoological, botanical, geological, paleontological, genetic or physical evidence has refuted either of the central two main Darwinian ideas. To those free of religious bigotry, Darwinian laws are acceptable just like the laws proposed by Copernicus, Galileo, Newton and Einstein – sets of natural laws that explain natural phenomenon in the universe. The spectacle of what is called religion, or at any rate organized religion, in India and elsewhere, has filled us with horror, and I have frequently condemned it and wished to make a clean sweep of it. —Jawaharlal Nehru

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Darwin’s original tree of life, which can be viewed in other chapters of this book, remains a stunning depiction of extinct and extant life. Creatures who ‘made it’ through the lines are present today and those where lines ceased did not (extinction). The other striking implications of this tree of life include (1) questioning the idea of progress in evolution, and (2) wondering whether there is a single route to the biological present. All modern day biological life is equally evolved, and most organisms have been evolving for a lot longer than humans. All that natural selection has ensured is the passing over of genetic contents from one organism to its subsequent generations. Darwin’s original tree of life has been verified by genomic studies in the past two decades.12 None of the modern scientific genomic discoveries have refuted the core of Darwinian ideas. From the tree of life discussion, it follows naturally that all organisms can be traced back, most probably, to one ancestor in whom life originated billions of years ago. A man has no reason to be ashamed of having an ape for his grandfather. If there were an ancestor whom I should feel shame in recalling, it would be a man of restless and versatile intellect, who, not content with success in his own sphere of activity, plunges into scientific questions with which he has no real acquaintance, only to obscure them by an aimless rhetoric, and distract the attention of his hearers from the point at issue by eloquent digressions and skilled appeals to religious prejudice. —Charles Darwin

A key question in evolutionary biology has been regarding complexity – either organizational and/or genomic.8–11 Will it be fair to understand that life has been getting more complex since its inception ~3.5 billion years ago? The likely answer here is no. It has been shown that many ancestral genomes were comparable in genomic content to the modern ones. It even seems that some organisms may have undergone gene loss and genome shrinking as possible modes of evolution. Therefore, excessive genomic cargo does not correlate with biological complexity (compare the number of genes between humans, mice and rice). Again, it was Darwin’s genius to tie together the most complex and the simplest of organisms into one interconnected thread of life. Darwin provided the idea that the apparent grandeur of life in nature, regardless of its biological size and complexity, had a natural basis and did not require any metaphysical justifications. Devout believers are safeguarded in a high degree against the risk of certain neurotic illnesses; their acceptance of the universal neurosis spares them the task of constructing a personal one. —Sigmund Freud

Chimpanzees are the closest living relatives of humans and our common ancestor probably lived ~6–8 million years ago (Fig. 2). Humans and chimps belong to the hominid group which also has other charming members (some

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Figure 2. Top panel - shows the striking structural parallels between hands of humans and his cousins. Structural features like these are in abundance when one compares anatomies in related (and at times unrelated) biological creatures. Genomic studies of the underlying genetic basis (DNA) for these similarities confirm the anatomical observations. Lower panel – a family album of humans with closest cousins. Gorilla and chimpanzee genomes are ~99% identical to the human genome – a fact that seals the debate on human evolution from ape-like ancestors

extinct) like gorillas and orangutans. There is no single evolutionary line that leads to humans, rather there are branches and more branches and furthermore more branches.8–11 This kind of speciation requires thousands of years making it difficult to observe. In another sobering thought, it seems that there were up to five different hominin species that existed previously – now only homo sapiens sapiens does. All people today are classified as Homo sapiens sapiens and these likely arose ~200,000 years ago from earlier species (Fig. 3). Data from fossils indicate that modern humans likely evolved from archaic humans in East Africa. Modern human fossils from Asia suggest that the present species may have been in

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Asia around 60,000 years ago. It seems that human population started to grow fast ~40,000–60,000 years ago. This growth was concomitant with migration into new areas on earth and other developments. Incredibly, the present world population has reached close to 6.6 billion people, although it is abundantly clear that earth with its finite natural resources cannot support so many. Migration levels, gene flow and genetic intermixing today are probably the highest they have ever been in history – best examples are populations of cosmopolitan cities worldwide. Clearly, the drama of Darwinian evolution is very much on-going, both in terms of genetic variations and forces of natural selection. There are several examples of this already, and include development of genetic resistance to infectious agents like malaria parasites and HIV. The growing and potent field of comparative genomics is revealing features of the physical principle (i.e. DNA) that drives evolutionary processes at an ever increasing pace.12 In time, we shall know how different the present human race already is from those humans who existed 10,000–30,000 years ago. Millions of humans of varied genetic stocks and ancestries co-exist in modern cities the world over. On one hand, this pool represents an exciting DNA cauldron which may ensure appearance of enough mutations that may come in handy for humans. However, these DNA broths also provide fertile ground for contagious diseases – potentially adding new selective pressures. DNA neither knows nor cares. DNA just is. And we dance to its music. —Richard Dawkins

~Millions of years

Human ancestors

5–4

Ardipithicus ramidus

4

Australopithecus anamensis

4–2.7

Australopithecus afarensis

3–2

Australopithecus africanus

2.2–1.6

Australopithecus robustus

2.2–1.6

Homo habilis

2.0–0.4

Homo erectus

0.4–0.2

Homo sapiens archaic

0.2–0.03

Homo sapiens neandertalensis

0.2–present

Homo sapiens sapiens

Figure 3. Shown here is immediate human ancestry after breaking away from the apes many millions of years ago. Note the time required for a new species to arise. The past ~half a million years represents a gradual evolution of modern humans from homo sapiens arachaic

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Figure 4. Web of life based on genome sequencing of numerous biological specimens. These diagrams place all organisms whose DNA has been sequenced on a relatedness map. The three kingdoms of life – namely, bacteria, archaea and eukaryotes are shown along with representative organisms from each. Revelation of encoded DNA information from 1000s of organisms now supports the tree of life view that Darwin had proposed. These data suggest that all biological life is related and likely originated from a common ancestor via transfer of DNA through eons. The dot indicates position of humans on this genetic web along with the number of genes present in humans

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The physical basis for transmission of evolutionary processes was still not discovered at the time of Darwin. We now know it to be DNA, and it binds all life together (Fig. 4). Our ancestors have passed on, in an immaculately unbroken fashion, their DNA to subsequent generations. Past humans who could not procreate before their death failed this Darwinian odyssey, and their DNA is no more amongst us. One of the last experiments that Charles Darwin conducted was on cockles. In 1882, Walter Crick (a shoe maker keen on molluscks) contacted Darwin about a cockle he had found hitchhiking on a water beetle. This find invoked Darwin’s curiosity and eventually resulted in a study published in Nature in 1882 titled along the lines of “On the dispersal of freshwater bivalves”. A few days later Darwin died, and a few years later (in 1903) Walter Crick also died.13–15 However, exactly 50 years hence, not too far from where Darwin had written the Origin of Species, Walter Crick’s grandson and avowed atheist Francis H.C. Crick discovered the structure of DNA – the very physical principle that has enacted evolution of life over billions of years. If revealed religions have revealed anything it is that they are usually wrong. —Francis Crick

Part II: On sadistic prescriptions Not only is there no God, but try getting a plumber on weekends. —Woody Allen

The two Charles’s - Lyle and Darwin - had made it perfectly feasible for humans to exist without recourse to any divine dogmas, to wonder about life and the universe without seeking flimsy, prejudiced and contradictory explanations for existence of our universe and its life. However, religious beliefs have continued to enslave humankind the world over. In a sense, religious beliefs aim to overrule the essence of ‘nature’ in us, to control the encoded DNA that propels us, in favor of ‘nurturing’. The latter provides not only flexibility and but also a window of opportunity to propagate just about any dogmatic system. It may be considered that almost all religious dogmas are essentially un-Darwinian – their effort is to abolish natural diversity in humans and to replace it with a sterile uniformity. But uniformity in biological life is contradictory to Darwinian facts. Life is in constant dynamism both as a result of environmental vagaries and underlying genetic differences. The worst vice of the fanatic is his sincerity. —Oscar Wilde

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Figure 5. A linear view of the past 80,000 years of existence of homo sapiens sapiens. The start of the arrival of various religious dogmas is indicated (~4000 years ago). Recent 500 years have witnessed evolution of the most significant scientific ideas to date. This period has provided the deepest and greatest discoveries regarding our place in the universe. These scientific discoveries have debunked prevalent dogmatic beliefs and nullified any role of the divine in physical or biological evolution of our universe. Lower panel - note the average life expectancy of humans in the past 10,000 years. It is evident that growth of religious ideas and practices had no impact on increased life expectancy (or quality) in humans. The significant jump in human life span (and in quality) has happened directly as a result of developments in science (more specifically, of evidence-based medicine)

Creationist ideas take away the beauty of discoveries that bright human minds make in the realm of science. The effort of creationists is to provide a divine explanation for existence and evolution of the physical and the biological universe. But this effort is constantly, almost daily, being thwarted by scientific discoveries (Fig. 5). When sufficiently challenged on scientific grounds, the devout seek

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refuge in illusionary dichotomies like ‘science and religion are separate spheres of life’. In fact, there is nothing intellectual, cerebral, scientific, or intelligent in any of the religious movements about either the evolution of biological life or about the physical universe. The religious existence that is idealized by many devout is suffocating, revengeful, painful, and full of vacuous religious practices not to mention prohibitions, orders, sermons, restrictions and regulations. Worst of all, many a religious lifestyles are justified on the schizophrenic concept of heaven and hell. Religious prescriptions have included (and still do in many societies) incarceration, starvation, laceration, mutilation, strangulation, suffocation, crushing, choking, burning, garroting, stoning, flogging, amputation, and crucifixion. A quick survey of modern times shows that the most troubled, backward, violence-ridden, illiterate, undemocratic, misogynistic, non-egalitarian and scientifically backward nations also happen to be very religious. This is not a simple coincidence, and the underlying message is clear – people still adhere to religious explanations for origins of physical and biological life no matter how contradictory these are to facts. A quick preview of sociological facts on the extent of human suffering on earth is sufficient to put even the most religious of human minds into a contemplative mode about the creator. The divine creation and its subjects seem to be replete with misery, starvation, violence, rape, child torture, genocides, massacres, droughts, famines and disease epidemics. The ‘cure’ for apostasy may be death. Yet, religions are followed blindly, and their fictitious tales about development of the universe are actively propagated. Contrast this scenario with the elegant, beautiful, consistent, cogent, testable and evidence-based understanding of biological and geological life on earth provided by Darwin and Lyle. We can make a lot more sense of human travails in light of evolution of both the physical and the biological universe. Darwin’s discoveries allow us to reject the religious and spiritual basis for human misery and to confront earthly realities by accepting our place in this evolutionary tussle. Incurably religious, that is the best way to describe the mental condition of so many people. —Thomas Edison

In a recent survey of the Indian scientific workforce (from most if not all top Indian institutes), it was revealed that ~88% were convinced about the theory of evolution.16 But the good news ends here. It appears that only ~25% of the polled declared themselves as either agnostic or atheistic. Many of the polled seem to value religious myths and/or are open to various beliefs. In the same study, it was discovered that ~50% of the surveyed scientists feel there is efficacy in homeopathy and also in praying (perhaps both will be even better). More than 10% of the polled believed in palmistry, horoscopes, vaastu, astrology and faith healing. Close to 40% of the interviewees felt it was acceptable to seek divine blessings before

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launch of rockets. Finally, ~24% and 38% of the scientific workforce believed that holy people and god can perform miracles, respectively of course. Such blinding adherence is likely a direct function of religious conditioning from childhood. In India in general, besides participation in religious systems, there is wide public acceptance of additional ‘belief systems’ including crystal healing, stone healing, reiki, distance healing, faith healing, magnetic therapy, numerology etcetera. Such brazen acceptance of systems which lack evidence reflects a sad albeit sound conditioning. The mechanism of propagation of such conditionings is rather clever however. Religious doctrines and other belief systems victimize children (and the naïve) first and foremost as part of a grand conditioning program. This routine is clearly meant to offset the genetic programming in children, which probably tends to question rather than obey initially. Religious brain-washing and conditioning in the irrational also aims at neutralizing the inherent genetic differences in humans. In order for this religious indoctrination to work, it is essential that the nascent minds of children are addressed (or overpowered) – time lost at this stage may result in transition of children’s minds into young thinking adults and possible rejection of the suffocating religious myths thus fed. It is due to this that one finds that although many parents are happy for their children to decide on their ‘lives’ (careers, spouses, etc.) once children have attained adulthood and maturity, the same parents will not wait that long for religious subjugation of their children. The precious, impressionable, malleable, flexible minds of young children are the easiest targets for intellectual asphyxiation by rigorous religious conditioning with the ulterior motive of propagating myths about divinity. It is feasible that if indoctrination of children into belief systems ceased from hereon, religions will likely dwindle to insignificance within a few human generations. One is often told that it is a very wrong thing to attack religion, because religion makes men virtuous. So I am told; I have not noticed it. —Bertrand Russell

Contrary to religious beliefs in man’s perfection (or perfection in the work of the imaginary creator), there are numerous imperfections evident in humans as a result of Darwinian evolution. Cleary, most organisms are not perfectly adapted to their environments, and struggle for survival continues on a daily basis on earth. Rudimentary organs showcase the dynamic nature of natural selection, as these organs are essentially of little or no value to the biological individual at present. From a creationist perspective, these vestigial flesh and bones smack either of incompetence of the creator, or at best of wastefulness and poor design. Humans exemplify some of these ‘design faults’. A list of potential defunct organs in humans include appendix, paranasal sinuses, vomeronasal organ, coccyx, extrinsic ear muscles, toes, neck rib, Darwin’s point, third eyelid, palmaris muscle, subclavius muscle, male nipples, wisdom teeth, erector pili, thirteenth rib, male

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uterus and female Vas Deferens. Evolution by natural selection on the other hand provides very satisfactory answers for these ‘extra’ organs, as these organs were probably of value to our ape-like ancestors or even earlier. Far from being the ultimate specimen of divine’s creativity, the reality of homo sapiens sapiens reflects ‘manufacturing’ on an ‘off’ day. Furthermore, humans seem to have no special status on earth (1) arguably in terms of being the most complex, (2) or of continued existence for the longest period, or (3) as most efficient biological machines, or (4) as the best ‘designed’ creature. Except human brain, every other human organ most likely has a superior homolog in another animal species on earth. Therefore, humans are not the pinnacle of evolution. We are far from perfect. We are also increasingly susceptible to new diseases and infections. Modern molecular biology has provided ample evidence for clear genetic linkages for numerous human ailments including heart disease, diabetes, infections, cancer, obesity, stroke and neurodegenerative disorders. These may be justified as divine retribution for agnostics or atheists but only flippantly. Darwinian insights into human evolution warn us that in the absence of medical science, modern humans will likely have a very precarious existence – left at the mercy of natural selection. Darwinian principles have shown us that divine miracles do not create life, do not nurture life and do not destroy life. Indeed, the object of life is uncomfortably balanced between environmental pressures and genetic wherewithal. Most of evolved creatures remain imperfect as all that natural selection cares about are the differential selective advantages that arise as a result of random mutations. Never mind perfect, even a fraction of normal vision or hearing or extra running prowess may be sufficient to tide a creature over its rivals. This daily, endless and directionless struggle is being closely watched and monitored by the force of natural selection and not by an imaginary, benevolent albeit inept ‘engineer’. At a molecular level, our DNA reflects that we are not much more than vehicles for its transfer to subsequent generations. Our DNA today is a collection of ‘noise’ made in passage over eons past – aberrations that despite their blindness allowed DNA propagation in those environmental conditions. A most striking and at once humbling depiction of biological life is presented by the so-called tree of life which places all biological beings on an evolutionary network – here man rests in peace amongst and between his biological relations and without a damn stamp of ‘miracle’ about him (Fig. 4). All extinct and extant life can trace its origins to one or few original organisms. It is the genetic cargo in those ancient organisms that has resulted in us of today. These linkage ideas have been verified by the description of genomic web of life based on DNA sequencing of numerous biological agents.12 Which is it: is man one of God’s blunders, or is God one of man’s blunders? —Friedrich Wilhelm Nietzsche

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Expectedly, after the death of Charles Darwin stories began to float about his so-called death-bed conversion to theism and even retraction of his theory.13–15 Darwin’s daughter Henrietta and son Francis both refuted such ludicrous claims – although I shall argue that even in the event that Darwin had a fit of undeserving sympathy for religion and possibly some trepidation toward the end of his life - even then we need not fall prey to the same. We don’t need know conversions. Darwin’s achievements and his genius helped man realize his material nature. Since Darwin, his ideas have also provided a backbone for all theoretical life sciences and also for modern medicine. Darwinian laws of mutation and natural selection reign over our deepest insights into modern biology. Ironically, those in denial of Darwinian principles seldom fail to utilize the benefits of modern medicine or for that matter any scientific advancement. The urge to cling to empty faiths is strong in many, despite the fact that only science rescues mankind. Darwin provided a materialistic basis for biological life, taking away its pretensions of links with divinity. Since Darwin, it is clear that biological life has no guiding forces, no accountability, no forgiver, no heavenly monitoring system, no loving overseer, no voyeuristic interferer, no spatial benefactor, no (moral) hero and no sympathetic angel. Darwin’s insights provide a driving force for humanity to withdraw from time-warped, non-scientific, irrational and poor attempts at explaining human life via divinity. With and after Darwin we can embrace science, reason, rationality and evidence-based existence without recourse to religious distortions. Darwinian discoveries have obviated the need for bogus explanations for origin of life once and for all. Darwin has made agnosticism or even atheism a human necessity. So, let’s revel and let our DNA rebel.

A short list of some facts relevant to this essay ● ● ● ● ● ● ● ● ● ● ●

Earth and proximal stars are ~4.6 billions years old. Earth was shaped by geological and physical forces during this time. Liquid water on earth is ~3.6 billion years old. Life originated in simple forms close to ~3 billion years ago. Organized, multicellular life is ~800 million years old. There were no birds or mammals 250 million years ago. Dinosaurus and humans could not have co-existed as 60 million years separate them. Major life forms of the past are no longer living. All present forms of life arose from ancestral forms that were different. Humans and modern apes have common ancestors. All biological life is related and shares a common genetic basis.

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● Religious dogmas appeared in the most recent 2–5% of the time since homo sapiens sapiens have been around. ● Clearly, the ‘creator’ needed evolution of language before ‘appearing’. ● Human life expectancy has increased as a function of modern medicine. Clearly, the ‘creator’ engineered low life span and unhealthy humans to start with. ● There is no evidence for better ‘upkeep’ of the devout over agnostics and atheists. ● To date, there is no evidence for any miracle.

References 1.

Richard Dawkins Series – ‘Genius of Charles Darwin’ and ‘Dawkins on Darwin’

2.

Darwin C (Ed.) (1871) In: The Descent of Man, and Selection in Relation to Sex. John Murray

3.

Darwin F (Ed.) (1887) Charles Darwin’s Autobiography. Basic Books, New York

4.

Koonin EV (2009). Nucleic Acids Res 37(4):1011–1034

5.

Haldane JBS (1932) In: The Causes of Evolution. Longmans, Green & Co., London

6.

Huxley JS (1942) In: Evolution: The Modern Synthesis. Allen and Unwin, London

7.

Zolopa AR, Cllavel F, Soriano V. “HIV infection and antiretroviral resistance”.

8.

Avise JC (2006) In: Evolutionary Pathways in Nature: A Phylogenetic Approach. Cambridge University Press, Cambridge, UK

9.

Dawkins R (1976) In: The Selfish Gene. Oxford University Press, Oxford

10.

Dawkins R (2004) The Ancestor ’s Tale: A Pilgrimage to the Dawn of Life Houghton Mifflin

11.

Dawkins R (1986) The Blind Watchmaker. Longman

12.

Interactive Tree of Life can be viewed at http://itol.embl.de/

13.

Barlow N (1958) In: The Autobiography of Charles Darwin Collins. London

14.

L. Eiseley (1958) In: Darwin’s Century: Evolution and the Men Who Discovered It, Doubleday. New York

15.

Hodge MJS, Radick G (Eds.) (2003) In: The Cambridge Companion to Darwin. Cambridge University Press, Cambridge

16.

International Survey Worldviews and Opinions of Scientists India (2007–08) by Ariela Keysar, Barry A Kosmin and Dr N Innaiah. For information on Institute for the Study of Secularism in Society and Culture see www.trincoll.edu/secularisminstitute.

6 Phylogeographic evolution of plant viruses Anupam Varma* and Shelly Praveen** Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi - 110012, India *[email protected], **[email protected]

Abstract: Viruses, discovered about four decades after the publication Darwin’s treatise ‘The Origin of Species’, are important tools to address the questions related to the evolution of species. Viruses are unlikely to have existed in the pre-cellular environment, as they require the complex host cell system to replicate and are inert outside such cells. Viruses might have coevolved with their natural hosts. Some of the present day viruses could be escaped host genes, as the genome of some of such viruses is found to be integrated with host genome. However, there is no reason to believe similar origin of all viruses. The viruses that might have originated in one group of organism may occur in another group. Some viruses replicate both in plants and insects as their hosts, leaving the question open about their origin. Whatever the origin, biologically, morphologically and molecularly similar viruses seem to have evolved in diverse pathosystems suggesting convergent evolution. High rates of mutations, genomic recombination, intensification of agricultural practices, exchange of germplasm, and human intervention in inadvertently introducing exotic viruses to new environments and host genes for susceptibility have played key roles in the evolution of present day viruses. In this article, we briefly discuss phylogeographic evolution of some of the important plant viruses like badna-, begomo-, poty- and tospoviruses, which have emerged as a severe constraint in improving crop productivity in the tropics and sub-tropics. Keywords: Phylogeographic, Evolution, Plant viruses

Introduction Viruses, discovered about four decades after the publication of the treatise “The Origin of Species by Means of Natural Selection or Preservation of Favoured Races in the Struggle for Life” by Charles Darwin in in 1859, are emerging as important tools to address the questions related to the evolution of species raised by Darwin. Viruses and the viroids, the simplest known replicating systems, have been an attractive subject of speculation about their origin for long. Although V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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the viruses are unlikely to have existed in primitive forms in the pre-cellular environment, as they require the complex host cell system to replicate and are inert outside such cells, the possibility of their pre-cellular origin cannot be ruled out. Origin of viruses by degeneration of cells of obligate cellular parasites seems improbable as the viruses use the host protein synthesizing machinery, generally lack membranes and do not multiply by binary fission. It is logical to assume that the present day viruses might have originated in their natural hosts and may have found niche in a diverse host system. Some of them could be host genes that escaped, as the genome of some viruses is integrated with host genome. However, there is no reason to believe similar origin of all viruses, and the viruses that might have originated in one group of organism may occur in other group. Some viruses replicate both in plants and insects as their hosts, leaving the question open about their origin. Whatever the origin, biologically, morphologically and molecularly similar viruses seem to have evolved in diverse pathosystems suggesting convergent evolution. The size of the genome of plant viruses varies from 250 nt for viroids, the smallest known pathogens to about 30 kbp for the largest plant infecting reoviruses. The viruses appear to have co-evolved with their hosts. Therefore, chances are that the viruses infecting insects might have evolved along with insects about 400 millions year ago, plant viruses around 200 to 40 million years ago, and mammalian viruses about 80 million years ago. However, so far, there is no evidence of the occurrence of viruses in the fossils. The advent of efficient detection techniques may soon unravel the possible fossilized viruses. Bunya-, Reo- and Rhabdoviruses, which infect vertebrates, invertebrates and insects, may have had a common origin in insects. Interestingly, some insect viruses like Nilaparvata lugens reovirus and Rhopalosiphum padi virus, which do not infect plants, can be acquired by healthy insects from plants on which infected insects have fed. Insect viruses could have moved to plants and animals about 135 million years ago during the cretaceous period. Some of the viruses like the Tymoviruses might have moved across the contingent as a result of continental drift 138–80 million years ago. Co-evolution of plant viruses with their hosts is testified by the evolution of potato viruses like Potato virus X and Andean potato mottle virus in the Andean region of South Peru and Northern Bolivia, which are the centres of origin of potato.

Underlying factors for the evolution of viruses Many of the plant infecting viruses, like Tobacco mosaic virus, Cucumber mosaic virus and potato viruses, have been known for a long time and continue to cause serious disease problems in various parts of the world. Gradually many ‘new’ viruses have been discovered with the help of improved and efficient diagnostic techniques (Fig. 1). A large number of new viruses have been identified during the last two decades mainly due to the development of advanced molecular diagnostic

Phylogeographic evolution of plant viruses

Figure 1. Development of diagnostic techniques for plant viruses

Box 1: Underlying factors leading to emergence of new variants of viruses  Genotypic changes  Mutations  Recombinants  Pseudo recombinants  de novo gene acquisition  Host factors  Introduction of new crops or their genotypes susceptible to endemic viruses  Changes in the cropping system  Movement of viruses to new ecosystem  Vector factors  Environmental factors

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Figure 2. Emergence of viruses in tomato

techniques. This is amply illustrated by the sharp increase in the number of viruses infecting tomato in various parts of the world in the last decade (Fig. 2). With the expected development of diagnostic DNA Chips, the number may swell many folds. All the ‘new’ viruses may not be new but detected as a result of increased efforts and application of molecular diagnostics. Yet, many new strains and truly new viruses seem to have evolved due to various underlying factors (Box 1). Among these, high rates of mutation and frequent genomic recombination have played important roles in the evolution of viruses. Intensification of agricultural practices, exchange of germplasm, and human intervention in inadvertent introduction of viruses to new environments and introduction of genes for susceptibility in efforts to improve crop productivity have also been critical. These factors have resulted in wide geographical distribution of viruses and emergence of serious virus disease problems across the world. Here we examine phylogeographic evolution of some plant viruses which have emerged as major constraint in crop production particularly in the Indian sub-continent.

Badnaviruses Badnaviruses, bacilliform DNA viruses, belong to family Caulimoviridae. The importance of these viruses has been realized relatively recently, although the diseases caused by them have been in prominence for a long time. Emergence of Cacao swollen shoot virus (CSSV), the first Badnavirus described in 1947 is an excellent example of an inconsequential virus assuming a serious proportion due to the introduction of exotic plants in a new environment. In the first half of

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the last century cacao was introduced in West Africa from South America. Soon, CSSV, which occurs in indigenous wild plants, was transmitted by several species of mealy bugs in a semi-persistent manner to cacao causing devastating swollen shoot disease. To check the spread of CSSV, it is estimated that since 1945 over 100 million cacao trees have been cut down in Ghana alone. Several badnaviruses affecting banana, citrus and sugarcane in India have been characterized recently. Phylogenetic analysis of nucleotide sequences of their genomes and the genomes other badnaviruses shows their clustering according to their hosts rather than their geographic origin (Fig. 3). Citrus yellow mosaic virus (CMBV) was first detected in 1992 in Karnataka in Pomello plants, since then it has spread to Andhra Pradesh and Maharashtra apparently due to the distribution of infected plants. The viruses affecting citrus are subject of extensive research in all citrus growing regions of the world, but, CMBV has not been found in

Figure 3. Phylogeographic analysis of badnaviruses based on full length genome sequences: Banana streak Acuminata virus (BSAuV) DQ092436; Banana streak virus (BSV) NC008018; Banana streak Trichy virus (BSTrV) DQ859899; Banana streak GD virus (BSGDV) DQ451009; Banana streak golden finger virus (BSGFV) NC 007002; Banana streak Mysore virus (BSMysV) AF 214005; Banana streak OL virus (BSOLV) NC003381; Cacao swollen shoot virus (CSSV) NC001547; Citrus yellow mosaic virus – Acid Lime (CMBV-AL) EU489744; Citrus yellow mosaic virus - Pummelo (CMBV-PM) EU489745; Citrus yellow mosaic virus Rangpur Lime (CMBV-RL) DQ875213; Citrus mosaic virus Sweet Orange (CMBV-SO) AF347695; Commelina yellow mottle virus (ComYMV) X52938; Dioscorea bacilliform virus (DaBV) DQ822074; Dracaena mottle virus (DrMV) DQ473478; Kalanchoe top-spotting virus (KTSV) NC004540; Sugarcane bacilliform virus (SCBV) NC003031; Taro bacilliform virus (TaBV) AF357836; The isolates from India are shown by dark pink circles; these group with an isolate from China (BSGDV)

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any other country indicating its possible origin in India. Interestingly CMBV is phylogenetically closely related to CSSV. The two viruses could have a common progenitor. Experimental transmission of CMBV to cacao has not been tried, but so far there is no report of the occurrence of cacao swollen shoot disease in India, although cacao is grown in the region where CMBV is spreading, indicating biological differences between the two viruses. Banana streak virus (BSV), unlike CMBV , the viruses causing streak disease of banana are widespread, but all the isolates occurring in India and other countries, sequenced so far, cluster together. Kalanchoe top spotting virus (KTSV) also clustered with BSV isolates suggesting a possible common origin of these viruses, although they do not cross infect their natural hosts. Banana could have been the host of the ancestor of BSV and KTSV, and that independent host shifts could have occurred subsequently during the evolution of these viral species. Extensive spread of BSVs is facilitated by the global trade in planting material, and micropropagated from infected but apparently symptomless plants. Banana streak disease was first observed in 1958 in West Africa and subsequently in many banana growing countries. Significance of banana streak disease in the evolution of viruses was realized a decade ago when the molecular and cytogenic evidence showed integration of viral genome with the host genome.1 The integrated complete Banana streak OL virus (BSOLV) sequences found in banana cv. Obina l’Ewai (Musa AAB group) may have arisen by recombination between the host and viral DNA during their co-evolution. Such integrated sequences could cause episomal infection under certain conditions like the micro-propagation or development of inter-specific hybrids by crossing Musa acuminata (A genome) and M. balbisiana (B genome). Musa B genome seems to be the main source of BSV.2 In India, the genome sequences of commonly occurring Banana streak Mysore virus (BSMysV), have been found integrated with the genome of banana cv. Poovan (Musa AAB group), which is grown throughout the country. Integration of BSMysV genome with the genome of other cultivars is also expected as the entire germplasm of M. balbisiana seems to contain sequences of BSMysV. Similar integration of Badnavirus sequences could also occur in other hosts. Further work is required to understand the significance of integration of viral sequences with the host sequences and their role in the evolution of these viruses.

Begomoviruses Begomoviruses, belong to family Geminiviridae. The genome of begomoviruses consists of circular ssDNA encapsulated in twinned icosahedral particles, which are transmitted efficiently by whitefly Bemisia tabaci. The genome of some begomoviruses is monopartite consisting of single circular molecules of about 2.8 kb, known as DNA A, some have bipartite genome consisting of DNA A and another molecule of similar size known as DNA B. All the monopartite

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begomoviruses and some bipartite begomoviruses also have a betasatellite DNA of about 1.4 kb, which code for a single 13 kDa protein and depend on viral DNA A for replication. Some monopartite begomoviruses also have a self-replicating alphasatellite molecules coding for 36 kDa protein. Both types of satellite molecules depend on the helper virus for encapsulation and transmission. The significance of alpha-satellites is not known, but beta-satellites are shown to increase the virulence of the helper viruses. Interplay of these molecules with plant hosts and the vector whitefly has resulted in evolution of a large number of Begomovirus species. Begomoviruses have been broadly grouped as New World (NW) begomoviruses occurring in the Americas and Old World (OW) begomoviruses occurring in other parts of the world. The genome organization NW and OW begomoviruses is similar except that the latter also have an additional ORF AV2. NW begomoviruses need both DNA A and DNA B for infection whereas DNA A alone of OW begomoviruses can cause infection. Greatest diversity in begomoviruses and their satellites occurs in South-East Asia, indicating a possible centre of origin for these viruses.3 Phylogenetically, the begomoviruses originating in different continents like Africa, Asia and Americas form distinct clades. Amongst the Asian begomoviruses, those originating in India cluster separately indicating that begomoviruses have originated in different regions and are mainly evolving intra-regionally. A major reason for their geographical restriction is the absence of transmission of these viruses through true seeds, although some have spread through the intercontinental movement of vegetative propagules of plants and infected transplants.4 Begomoviruses have been a major constraint in Indian agriculture for a long time, but during the last two decades they have emerged as devastating pathogens of crops. Major contributory factors in the emergence of begomoviruses are the evolution of variants through mutation, recombination and pseudo-recombination. In addition, intensification of agricultural practices, introduction of susceptible genotypes for crop improvement, climate change and increase in vector population have also contributed to the emergence of begomoviruses in India and other countries.4 Begomoviruses associated with cotton leaf curl disease Cotton leaf curl disease (CLCuD) has been endemic in Africa for a long time, but in the Indian sub-continent, it was first observed in a small pocket in the Multan area in Pakistan about fifty years ago. It remained confined to that area, and first emerged in a serious proportion in 1988 in a newly released susceptible cotton var. S12 and by 1993, the disease affected 0.2 million ha in Pakistan. In India, it was first noticed in 1989 in a few plants of Gossypium barbadense in an experimental field at New Delhi (Fig. 4), where it was contained by rouging of symptomatic plants and avoidance of cultivation of G. barbadense. However, in 1994 and 1995, CLCuD appeared in epidemic proportion in commercial

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G. hirsutum fields in Punjab and Rajasthan along the border with Pakistan. The incidence of the disease formed a gradient, gradually declining in fields further away from the border. Emergence of CLCuD in epidemic proportion in those years was apparently caused by the viruliferous whiteflies blown in by dust storms from the adjoining CLCuD affected fields in Pakistan. The spread of CLCuD was restricted in the subsequent years by (a) cultivating G. arboeum, which is not affected by CLCuD and resistant varieties of G. hirsutum, (b) avoidance of cultivation of G. barbadense and its hybrids, which have been found to be most susceptible, (c) avoidance of cultivation of Okra in the CLCuD prone areas, as it can help in the buildup of inoculums, and (d) integrated management of weeds and vector Bemisia tabaci. Five Begomovirus species, Cotton leaf curl Alabad virus (CLCuD), Cotton leaf curl Gezira virus (CLCuGV), Cotton leaf curl Kokhran virus (CLCuKV), Cotton leaf curl Multan virus (CLCuMV), and Cotton leaf curl Rajasthan virus (CLCuRV), and their satellite DNA molecules are associated with CLCuD5. In India, CLCuD is caused by Cotton leaf curl Bangalore virus (CLCuBV), CLCuKV and CLCuRV. In Sudan, it is caused by CLCuGV and in Pakistan by CLCuAV, CLCuKV and CLCuMV. CLCuD also occurs in the backyard gardens in Karnataka. A new Begomoviruses species, tentatively named as CLCuBV, is associated with the disease in Karnataka. Fortunately, it has not spread to the commercial crops of cotton in Southern India. The CLCuD associated begomoviruses in India and Pakistan are highly divergent and form two distinct phylogenetic clusters. These viruses appear to have originated independently of CLCuGV, with which they share <75% sequence identify. Moreover, the isolates from the north-western parts of Indian sub-continent could have emerged as recombinants of endemic begomoviruses. Yellow mosaic disease of grain legumes Yellow mosaic disease (YMD) was first observed in the late 1940s in Lima bean

Figure 4. Cotton leaf curl caused by CLCuV

Figure 5. Golden mosaic disease of cowpea caused by MYMIV

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(Phaseolus lunatus) in Western India and in the late 1950s in mungbean in Northern India. Since then YMD has spread to all parts of the Indian Sub-continent affecting a variety of grain legumes.4 Cowpea is one of the important grain legumes in India. The cowpea varieties grown in India were immune to the begomoviruses causing YMD in other grain legumes, except a variant causing minor yellow fleck disease, but in 1978 a golden mosaic disease (Fig. 5) appeared in some germplasm lines introduced from West Africa and by 1984, it emerged as serious disease problem throughout Northern India. Like CLCuD, YMD in cowpea also emerged with the introduction of susceptible exotic germplasm.6 Two distinct bipartite begomoviruses have been identified to cause YMD of grain legumes in the Indian Sub-continent. In Northern India, YMD epidemic is caused by Mungbean yellow mosaic India virus (MYMIV) and in Southern India and Thailand by Mungbean yellow mosaic virus (MYMV). Several variants of MYMIV occur in different regions and crops in Northern India. The variant(s) of MYMIV causing golden mosaic disease in cowpea (CpGMD) must have evolved in the late 1970s when susceptible genotypes were introduced from West Africa. In western India CpGMD seems to be caused by pseudorecombination of genomic components, as the disease was shown to be caused by MYMIV DNA A in association with DNA B of MYMV, which was closely related to DNA B of an isolate MYMV from southern India.7 In 2004, a severe leaf distortion disease appeared in cowpea in Northern India. The infected plants developed yellow mosaic, downward leaf curling and enations. This disease was also caused by another variant of MYMIV in association with a betasatellite molecule.8 Analysis of sequences of DNA A and DNA B of a larger number of cowpea infecting isolates of MYMIV is required to identify the mutations, rcombinations and pseudorecombinations leading to increase in the host range of MYMIV. The begomoviruses causing yellow mosaic disease of grain legumes in India form a distinct group of begomoviruses, with MYMIV spreading in northern India and MYMV in southern India. YMDs have been mainly managed by the use of resistant varieties, but the life span of resistant varieties is limited due to fast emergence of resistant breaking viral variants.6 Begomoviruses affecting tomato Tomato is the natural host of largest number of begomoviruses, and the number is increasing at a fast rate; 52 begomoviruses were reported to cause diseases of tomato until 2003, and by 2006, the number swelled to >70 (Fig. 2). The diseases known by their generic names “tomato leaf curl” and “tomato yellow leaf curl” are most common. In India, tomato leaf curl disease (ToLCD) – known to occur for over sixty years – is widespread. Four distinct begomoviruses have been identified to cause ToLCD in India. Two, Tomato leaf curl Bangalore virus (TolCBV) and Tomato leaf curl Karnataka virus (ToLCKV) are monopartite and the other two, Tomato leaf curl Gujarat virus (ToLCGV) and Tomato leaf curl New Delhi

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virus (ToLCNDV) are bipartite begomoviruses. The distribution of these viruses suggests diverse origin of the begomoviruses causing ToLCD in India. ToLCGV and ToLCNDV have also been detected in weed hosts like, Datura metel, Solanum nigrum and wild sunflower, but not the other two viruses (ToLCBV and ToLCKV) in north-western India. Nearly 1000-fold increase in whitefly population and appearance of biotype-B of B. tabaci resulted in a severe epidemic of ToLCD in southern India in 1999. Limited surveys indicate that ToLCBV is distributed in Southern India, ToLCNDV in Northern India, whereas ToLCGV and ToLCKV have a wider distribution. Mixed infection with these viruses also occurs, including with ToLCKV, which is prone to recombination. Variability in tomato infecting begomoviruses seems to be arising due to genomic recombination. Several isolates of begomoviruses affecting tomato in India have been sequenced. Tomato infecting begomoviruses form two distinct branches.9 One branch is represented by the monopartite viruses isolated from southern India and another by the bipartite viruses isolated from northern India. Phylogenetically

Figure 6. Phylogeographic analysis of Tomato leaf curl viruses based on DNA-A genome: Tomato leaf curl Bangalore virus (ToLCBV) - keral I DQ852623; ToLCBV-keral II NC003891; Tomato leaf curl Gujarat virus ( ToLCGV) -AF449999; Tomato leaf curl New Delhi virus (ToLCNDV) - Himachal AM850115; ToLCNDV-India-Mild (ToLCNDV-Ind-m) U15016; ToLCNDV-IndiaSevere ToLCNDV-Ind-s U15015; Tomato leaf curl Iran virus (ToLCIV-I) NC005842; ToLCIV-II AY297924; Tomato leaf curl Karnataka virus (ToLCKV) BanII AF295401; ToLCNDV - Pakistan:Punjab AM258977; Tomato leaf curl Philippines virus (ToLCPhV ) NC005032; ToLCV-Puerto Rico AY134494; Tomato leaf curl Seychelles virus (ToLCSV-Sey) AM 491778; Tomato leaf curl Taiwan virus (ToLCTwV) NC003898; ToLCNDV Thai AB330079; Tomato leaf curl Vietnam virus (ToLCVV) NC004153

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these viruses differ from those occurring in South-East Asia, Australia, and South Africa. However, one branch of South and West Indian isolates represented by ToLKV is phylogenetically close to the Iranian isolates. The phylogenetic analysis, thus, showed emergence of distinct tomato infecting begomoviruses in different geographical regions (Fig. 6). The southern isolate TOLKV appears to be recombinant of viruses occurring in western and southern India. Interestingly in Northern India only ToLCNDV has been found. This virus seems to be most wide spread among the tomato infecting viruses from India, as it not only occurs in Pakistan but also in Thailand. Betasatellites are shown to be an important factor determining the virulence and evolution of begomoviruses. Considerable diversity has been found in betasatellites associated with tomato begomoviruses. India is a major producer of chilli (Capsicum annuum). The crop is susceptible to several viruses in various parts of the world. In 2004, a severe epidemic of chilli leaf curl disease (ChiLCD) destroyed the chilli crop in Jodhpur district of Rajasthan in India. The affected plants developed upward leaf curling, puckering, and leaf size reduction, stunting and produced no fruits. The epidemic was caused by the emergence of a variant of Chilli leaf curl virus (ChiLCuV), which shared 96.5% sequence identity with the ChiLCuV – [Pak:Mul] and is distantly related (59–68% sequence identity) to Cotton leaf curl Multan virus, Pepper yellow leaf curl Indonesia virus and Tomato leaf curl New Delhi virus.10 The virus must be endemic in the chilli growing areas of Rajasthan, but emerged in epidemic proportions due to weather conditions favouring build up of whitefly population.

Okra leaf curl disease Bhindi (Okra) Yellow vein mosaic disease (BYVMD) is endemic in India, but the crop was free of leaf curl disease (BLCuD), which commonly occurs in West Africa, until the late 1980s when it emerged after the introduction of okra genotypes from West Africa for crop improvement, particularly for breeding for resistance to BYVMD. Mixed infection by the viruses causing BYVMD and BLCuD is common in northern India. The plants infected by both the viruses are not productive. Such infections increase the chances of evolution of recombinants. Potyviruses Potyviruses, belong to family Potyviridae. They form the largest group of filamentous viruses. Many of the potyviruses are economically important and cause serious diseases throughout the world. Several potyviruses occur in India, causing serious diseases in a variety of crops like cucurbits, grain legumes, papaya, potato and sugarcane. Here we examine the phylogeographic evolution of Papaya ring spot virus (PRSV). PRSV is a serious constraint in the production of papaya in various parts of the world. In India, it was first found to occur in Pune region and

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Figure 7. Phylogeographic distribution of PRSV in India. (Source: Jyoti Sharma and A. Varma)

subsequently in various parts of Northern India. Gradually, it also spread to other parts of the country. Until 1972, PRSV was not found in regions south of Pune, but in mid 1970’s, the virus appeared in Karnataka. Papaya plantations in Tamil Nadu were free from this virus until 2001. During this period, popular papaya varieties like CO-2 and CO-7 were grown in the region. However, a couple of years later, the virus appeared in Tamil Nadu as well, in a very serious form, so much so that the experimental plantation of variety CO-7 of papaya at Coimbatore Agriculture University had to be completely uprooted. The virus also appeared in commercial plantations of papaya var. CO-2. The isolates which have been characterized from India11 show clustering according to geographical origin, except that an isolate phylogenetically related to a southern isolate was also found to occur in northern

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Figure 8. Phylogeographic analysis of Papaya ring spot viruses (PRSV) based on coat protein gene: PRSV – Australia (PRSV-Aus) U14736; PRSV – Bangladesh AY 423557; PRSV – Bangalore AF120270; ; PRSV – Brazil AF344640; PRSV – China AF243496; PRSV – Coimbatore AY687386; PRSV – Delhi AF063220; PRSV – Hyderabad AY238880; PRSV – Japan AB044339;PRSV Lucknow AY458620; PRSV – Philippines AF506894; PRSV Pune (mild) AF063221; PRSV Pune (severe) AY238881; PRSV – Raipur AY491011; PRSV – Ranchi AY458619; PRSV – Solan AF238883; PRSV – Thailand U14743; PRSV – USA D00595; PRSV – Varanasi AY238882; PRSV – Vietnam U14742; PRSV – West Bangal (PRSV-WB) AY238885

India. This could be due to the possible movement of infected transplants from southern to northern region. Similar spread also seems to have occurred for an isolate occurring in the western region (Fig. 7). The phylogenetic analysis also shows clustering of the isolates of Indian origin according to their geographical distribution (Fig. 8), except the clustering of PRSV-severe isolate originating from Pune with that the isolates originating in northern India. Interestingly PRSVmild isolate formed an independent branch; this isolate has been found useful in providing cross protection against the severe isolates. The isolates occurring in northern India group with the isolates from Australia, Brazil and USA, whereas, the isolate from Bangladesh seems to have separated from this cluster (Fig. 8). Similar relationship was also reflected in the comparison of full length sequences. Full length sequences of PRSV isolates from Delhi indicate genetic recombination of the Indian isolates with the American isolates.12 Based on the phylogeographic evidence it has been suggested that PRSV

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might have originated in India and spread through South-East Asia to eastern pacific region and also separately to the Americas, and from there to Australia, and Taiwan.13 PRSV is not transmitted through seeds, although there is a preliminary report from South-Asia of its seed transmission in papaya. Also, so far, there is no evidence of seed transmission of cucurbit infecting PRSV in cucurbit hosts. However, the movement of virus through transplants taken by the migrants from India to southeast Asia and eastern-Pacific Islands is a distinct possibility. Transplants could also been taken to the Americas. Sequences of more isolates from the path of spread of PRSV may provide clearer picture on PRSV evolution, and may explain the recombination between the Indian and American isolates. Recombination in PRSV is common. RDP (recombinantion detection programme) analysis of PRSV genome indicates that 5′ end of the genome is prone to recombination. Several sequences between 1–600 nt from the 5′ end of Indian PRSV seem to have recombined at 4 hot spots of isolates from Taiwan, 3–4 different hotspots of isolates from Thailand and China, 2 each from Hawaii, Mexico and Brazil. Interestingly the 5’ UTR seems to have moved around.14 Tospoviruses Tospoviruses belong to family Bunyaviridae, which has four other genera. Three of these genera, Orthobunyavirus, Nairovirus and Phlebovirus can replicate in vertebrates and vector insects. The fourth genus Hantavirus seems to have no vector. Tospoviruses, replicate both in vector thrips and host plants, and are emerging as the most destructive plant viruses in India and also other parts of the world. The tospoviruses occurring in Asia are most diverse.15 Generally, plant viruses do not kill their hosts, but the tospoviruses are the exception as they seem to be alien to plants. Tospoviruses have isometric particles of 80–120 nm containing tripartite genomic RNAs, designated as large, medium and small components. The genomic RNAs are tightly linked with ‘N’ protein, coded by the small RNA, forming ribonucleoproteins, which are encapsidated by a lipid membrane, consisting of two virus-coded glycoproteins and a membrane of host origin. Their structure suggests animal origin, finding a niche in plants. For a

Figure 9. Mung bean necrosis caused by GBNV

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Figure 10. Phylogeographic analysis of Tospoviruses based on nucleocapsid protein gene: Calla lily chlorotic spot virus (CCSV-Taiwan) AY867502; Capsicum chlorosis virus (CaCv-Thailand) NC008301; Gloxinia tospovirus (GTV-USA) AF059577; Groundnut bud necrosis virus (GBNV-India) AY871098; Impatiens necrotic spot virus (INSV-Japan) A109100; Iris yellow spot virus (IYSV-Netherland) AF001387; Melon severe mosaic tospovirus (MSMTV-Mexico) EU275149; Melon yellow spot virus (MYSV-Japan) NC008300; Peanut chlorotic fan spot virus (PCFSV-Taiwan) AF080526; Peanut yellow spot virus (PYSV-USA) AF013994; Physalis severe mottle virus (PhySMoV-Netherland) AF007151; Poligonum ring spot tospovirus (PoRSTV-Italy) EF445397; Tomato spotted wilt virus ( TSWVUSA) AY870391; Tomato yellow ring virus (TYRV-Iran) DQ810196; Tomato zonate spot virus (TZSV-China) NC010489; Watermelon bud necrosis virus ( WBNV-India) EU249351; Watermelon silver mottle virus (WsMoV-Japan) AB042650; Watermelon spotted wilt virus ( WBNVIndia) NC003843

long time tospoviruses were considered to be monotypic represented by Tomato spotted wilt virus (TSWV), distributed in the temperate and sub-tropical parts of the world, but now tospoviruses are represented by eight recognized and six tentative species, which are widely distributed and can infect about 1200 plant species; TSWV alone infects about 900 plant species. In India, occurrence of TSWV has not been detected so far, but two other tospoviruses - Groundnut bud necrosis virus (GBNV) and Watermelon bud necrosis virus (WBNV) - have emerged as the major pathogens of a wide range of crop plants, the natural host range of GBNV is wider, whereas WBNV seems to be restricted to cucurbits. In 2000, a severe epidemic GBNV occurred affecting groundnut in 0.25 million ha in Andhra Pradesh. This epidemic was caused by GBNV and an Ilarvirus (Tobacco

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streak virus (TSV). In subsequent years also, infection by these two viruses were common. Groundnut bud necrosis disease (GBND) caused by GBNV is a serious constraint in the production of groundnut throughout India. Occurrence of GBNV in groundnut has been known for a long time, but during the last decade the virus has also spread to other crops, like mungbean (Fig. 9), tomato and potato. One could argue that the virus was not detected earlier as the diagnostic tools were not available, but the diseases caused by tospoviruses are so destructive that these would not have been missed. For example, the destructive stem necrosis disease of potato, characterized by extensive stem and petiole necrosis, leaf distortions and stunting of plants. The disease is caused by GBNV. It emerged in central India in the early eighties. Since then, it has spread to other potato growing regions of the country, due to the introduction of susceptible genotypes in the GBNV endemic areas. GBNV is also fast spreading in tomato, particularly in southern and western India, and could assume serious proportion in these areas. WBNV emerged in the nineties as a devastating problem in watermelon cultivation in Karnataka. It has also spread to a limited extent in watermelon and cucumber in northern India and ridge-gourd in southern India.16 Other cucurbits may also be affected. Recently two other tospoviruses – Iris yellow spot virus (IYSV) and Capsicum chlorosis virus (CaCV) – are also reported to occur in the country. IYSV is fast spreading in onion growing areas in western India,17 and CaCV is emerging as a serious problem in chili pepper cultivation.18 Various genes of GBNV and WBNV genome are highly conserved sharing >95% identity. Phylogenetically tospoviruses occurring in Asia and the Americas form two major clusters.15 Three of the four tospoviruses occurring in India, i.e. CaCV, GBNV and WBNV, represent one branch of the Asian cluster, whereas IYSV represents another distinct branch, indicating a distant relationship from the other tospoviruses spreading in India. GBNV and WBNV have been found to occur only in India, whereas CaCV also occurs in Thailand and IYSV has a worldwide distribution. CaCV, GBNV and WBNV mainly infect plants grown through true seeds and tospoviruses are not seed-borne, which explains their restricted geographical distribution. In contrast to this IYSV infects bulbs crops, which are propagated by true seeds as well as vegetative propagules, which can carry viral infections. International trade in bulbs must have resulted in the wide distribution of IYSV, which seems to have originated in Europe. CaCV might have originated in Australasia, and introduced in India through infected transplants. CaCV has been detected in India only recently, but it might have been introduced many years ago and remained unnoticed. The two tospoviruses, GBNV and WBNV, which are endemic in India could have originated from CaCV. This analysis indicates that the tospoviruses occurring in India have originated along with some other tospoviruses reported from other parts of Asia (Fig.10). Some of these viruses also share sequences with tospovirus infecting Gloxinia in USA, suggesting genomic recombination amongst the tospoviruses. The oldest known tospovirus – Tomato

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spotted wilt virus (TSWV) does not occur in India, although the symptoms produced by GBNV on tomato look similar to those induced by TSWV. Absence of TSWV, which forms a separate branch of tospoviruses, suggests independent origin of TSWV and the two tospoviruses occurring in India. Ilarviruses During the last decade, TSV, an Ilarvirus has emerged as a destructive virus in a variety of crops particularly in southern India. The most serious incidence of the virus has been on the newly introduced hybrids of sunflowers. It has also spread to other crops like cucurbits, groundnut, legumes, cotton and okra. Emergence of TSV in India has not been understood. Phylogenetically isolates sequenced from India are close to other isolates found in other geographical regions, but the destruction caused by TSV to crops in India is unique. Ilarviruses are also emerging as constraint in the production temperate fruit crops like apple in the country.

Conclusions Phylogeographically economically important viruses occurring in the Indian Subcontinent fall in three broad groups. One group is represented by badnaviruses, which infect vegetatively propogated plants like banana and citrus and seem to have evolved slowly and form phylogenetic clusters according to their host species, rather than their geographical distribution. The second group is represented by begomoviruses, which mainly infect plants grown through true seeds, are more diverse and seem to have undergone rapid mutations and recombinations, resulting in the fast emergence of new viruses and strains. These viruses appear to have evolved within specific geographic regions and form phylogeographic clusters. The third group is represented by ilar-, poty- and tospoviruses, which also evolve geographically but unlike the second group these viruses, have evolved slowly as they are not prone to rapid mutations and recombinations, but some of them are wide spread due to the movement of infected planting material. The tospoviruses appear to be conserved with occurrence of limited variability; this could be due to the possible origin of these viruses in insects. Whether the limited variation occurred to them during their replication in insects or plants remains to be seen. Vector transmission and human interventions have played important roles in the evolution and spread of plant viruses. Further work and sequencing of full genomes of different groups of viruses are expected to throw more light in the understanding of evolution of viruses. This is important as viral disease problems are threatening food production particularly in the tropics and semi-tropics. Acknowledgments: The authors thank Ms. Priyanka Singh, Ms. Suneha Upadhyaya and Ms. T. Vinutha for their help in bioinformatics and Mr. Zile Singh for logistic support.

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References 1. 2. 3. 4. 5.

6.

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Harper G, Julian OO, Harrison JSH, Hull RG (1999) Integration of banana streak badnavirus into the Musa genome: molecular and cytogenetic evidence. Virology 255:207–213 Harper G, Hull R (1998) Cloning and sequence analysis of Banana streak virus DNA. Virus Genes 17:271–278 Muhammad Shah NR, Claude F (2009) Evolution of geminiviruses and their satellites. FEBS letters 583:1825–1832 Varma A, Malathi VG (2003) Emerging geminivirus problems: a serious threat to crop production. Ann Appl Biol 142:145–164 Malathi VG, Radhkrishnan G, Varma A (2003) Cotton. In: Virus and Virus-like Diseases of Major Crops in Developing Countries. Loeberstein G, Thattappilly G (Eds.), Kluwer Academic Publishers, London, UK Varma A, Dhar AK Mandal B (1992) MYMV transmission and control in India. In: Mungbean Yellow Mosaic Disease. Green Sk, Kim D (Eds.), Asian Vegetable Research and Development Centre, Taipei John P, Sivalingam PN, Haq QMI, Kumar N, Mishra A, Briddon RW, Malathi VG (2008) Cowpea golden mosaic disease in Gujarat is caused by a Mungbean yellow mosaic India virus isolate with a DNA B variant. Arch Virol 153:1359–1365 Rouhibakhsh A, Malathi VG (2005) Severe leaf crinkle disease of cowpea – a new disease of cowpea in northern India caused by Mungbean yellow mosaic India virus and satellite DNA-ß. Plant Pathol 54:259 Praveen S, Dasgupta A, Varma A (2004) Phylogenetic analysis and homologies of the replicase of tomato leaf curl geminiviruses: implications for obtaining pathogen derived resistance. Virus Genes 28:195–199

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Senanayake DMJB, Mandal B, Lodha S, Varma A (2006) First report of Chilli leaf curl virus affecting chilli in India. Plant Pathol 55:13 (http://www.bspp.org.uk/ndr/july2004/ 2004-25.asp)

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Jain RK, Sharma J, Sivakumar AS, Sharma PK, Byadgi AS, Verma AK, Varma A (2004) Variability in the coat protein gene of Papaya ringspot virus isolates from multiple locations in India. Arch Virol 149:2435–2442

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Mangrauthia SK, Parameswari B, Praveen S, Jain RK (2009) Comparative genomics of Papaya ringspot virus pathotypes P and W from India. Arch Virol 154:727–730

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Gibbs AJ, Mackenzie AM, Wei1 KJ, Gibbs MJ (2008) The potyviruses of Australia. Arch Virol 153:1411–1420

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Mangrauthia SK, Parameswari B, Jain RK, Praveen S (2008) Role of genetic recombination in the molecular architecture of Papaya ringspot virus. Biochemical Genetics 46(11):835–846

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Pappu HR, Jones, RAC, Jain RK (2009) Global status of tospovirus epidemics in diverse cropping systems: Successes achieved and challenges ahead. Virus Research (in press)

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Jain RK, Bag S, Umamaheshwaran K, Mandal B (2007) Natural infection by tospovirus of cucurbitaceous and fabaceous vegetable crops in India. J Phytopsthol 155:22–25

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Ravi KS, Kirkaru AS, Winter AS (2006) Iris yellow spot virus in onions: a new tospovirus record from India. Plant Pathol 55:288

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Krishnareddy M, Usha Rani R, Kumar A, Madhavi Reddy K, Pappu HR (2008) Capsicum chlorosis virus (Genus Tospovirus) infecting Chili pepper (Capsicum annuum) in India. Plant Dis 92:1469

7 Evolution of HIV-1 in India Pradeep Seth Seth Research Foundation, Gurgaon, Haryana, India [email protected]

Abstracts: Nearly 25 years after the discovery of the human immunodeficiency virus type 1 (HIV-1) effective control of the AIDS pandemic remains elusive. At the root of this challenge is the evolution of this virus to elude immune control. Error-prone nature of replication and retro-transcription is the hallmark of this virus. This fidelity of replication in HIV-1is due to the absence of proof-reading/repair and post-replicative error correction mechanisms that normally operate during replication of DNA Viruses. Advances in sequencing technology and expanded disease surveillance have allowed researchers to characterize the variation in HIV-1 around the world and within individual patient overtime. Although HIV-1 has been classified into distinct subtypes, the classification does not reflect dynamic genetic evolution of HIV-1 through which new strains are constantly emerging. The resultant viral diversity has implications for differential rates of disease progression in different geographical areas, differential responses to antiretroviral therapy (including the development of resistance), and vaccine development. In this review evolution of HIV-1 in India is discussed. Keywords: HIV, Evolution, Phylogenetic analysis, HIV-1 subtypes, HIV-1 diversity “Everything changes and moves and that movement is God.” —Leo Tolstoy in War and Peace- Book-14 Chapter XV

Introduction Since the unfolding of the AIDS epidemic in the early 1980s, there has been an increasing interest in the emergence and evolution of infectious diseases. It has become extremely important to investigate the factors that allowed new infections like HIV to appear, or older ones to reappear and then to track their spread through populations. These tracks form part of science of molecular epidemiology. Traditionally serology has been used to trace the spread of infectious diseases. These days comparative analysis of gene sequence data is being undertaken to V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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study spread of infectious diseases. Meaning thereby, phylogenetic-trees have become an important analytical tool to track the spread of infections through populations. Since DNA sequences provide the most detailed information possible for any organism in evolutionary studies, this information is recognized as an invaluable document of history of life on earth. There are two types of HIV: a highly virulent global type (HIV-1) and a somewhat less virulent strain HIV-2 found mostly in West Africa. Many African monkeys are commonly infected at high frequencies with HIV like viruses known as simian immunodeficiency viruses (SIV). Therefore, African monkeys are almost certainly the animal reservoir that ultimately gave us the HIV. HIV-1 and HIV-2 are most closely related to different primate species: HIV-1 to the viruses found in chimpanzees (SIV-CPZ) and HIV-2 to those found in sooty mangabey monkeys (SIV-SM). Sootey mangabey monkeys are likely direct source of HIV-2 since these are West African monkeys and HIV-2 also is found predominantly in West Africa. On the other hand, since chimpanzees are rarely infected with SIV-CPZ in the wild they are less likely to be the direct source of HIV-1. However, it is possible that an unknown SIV from other monkey species may be the ancestor of both HIV-1 and SIV-CPZ. Nonetheless, separation of HIV-1 and HIV-2 on phylogenetic tree suggests that they must have entered human populations on different occasions. HIV-1 genetic diversity over time is driven by two factors namely the high error rate of the viral reverse transcriptase and the rapid turn over of HIV-1 in infected individuals. Recombination events, pressures generated by the host immune responses, and antiviral drugs further contribute to differential viral genetic evolution. Globally circulating strains exhibit an extraordinary degree of genetic diversity, which may influence several aspects of their biology such as infectivity, transmissibility and immunogenicity. Molecular analyses of various HIV isolates reveal sequence variations over many parts of the viral genome. Sequences derived from these HIV-1 strains have historically been classified on the basis of their Phylogenetic relationship. The groups were originally named M (major), O (outlier), and N (non-M, non-O).1 The last two groups (N and O) remain essentially restricted to West Africa, whereas the M group comprises a number of viruses that dominate the global AIDS epidemic. Since HIV-1 M group began its expansion in humans roughly 70 years ago, it has diversified rapidly, now comprising a number of different subtypes and circulating recombinant forms. Based on the sequence of the envelope glycoproteins, genetic subtypes including CRFs (circulating recombinant forms) have been identified in group M, whereas subtypes within group O remain unidentified. Subtypes are genetically defined lineages that can be resolved through phylogenetic analysis of the HIV-1 M group viruses as well-defined clades or branches in a tree. CRF describes a recombinant

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lineage that plays an important role in the HIV-1 epidemic. The CRF members must share an identical mosaic structure, that is, they have descended from the same recombination event(s).

HIV-1 Genetic subtypes On the basis of their Phylogenetic relationships, group M viruses have been classified into nine subtypes or clades (A to K; except E and I). Virus strains representing the genetic subtypes E and I have not yet been found. The viruses originally identified as subtype E (the predominant group of viruses involved in heterosexual transmission in Thailand) and I (a small group of viruses from Mediterranean region) are now considered inter-subtype recombinants and have been termed CRF01_AE and CRF04_cpx, respectively. Disproportionate spread of different lineages of group M viruses has been taken to indicate that specific biological differences may exist among various subtypes. Therefore, the phylogenetic analysis of subtype sequences remains an important molecular epidemiological tool with which to track the course of group M pandemic. Existence of viral subtypes or clades may be the result of a ‘founder effect’ in which certain variants of the virus become founders of a subepidemic because they happen to be involved in an extensive transmission chain. In this scenario, the subtypes may be similar biologically even though they are genetically very different. Alternatively, it is likely that certain characteristics of subtypes allowed them to out-compete less-fit viral variants. Despite the lack of clear correlations subtypes and overt biological characteristics, other more subtle phenotypic distinctions have been reported such as the pattern of co-receptors usage. There is growing evidence that the subtype C has a preponderance of ‘non-syncytia inducing’ viruses which bind to CCR5 receptors in addition to CD4 receptors present on the target cells and lack ‘syncytia inducing’ viruses that use CXC4 and CD4 receptors to infect the target cells. Distinctive RNA secondary structure in the important regulatory domain, TAR is a property associated uniquely with subtype A and viruses having AE mosaic. Thirdly different subtypes differ in susceptibilities to antiretroviral drugs. In addition, difference between subtypes is reflected in subtype specific pattern of genetic variation. There is an elevated rate of non-synonymous substitution in the third variable loop of subtype D viruses, compared with other subtypes.2 Most interesting feature of these subtypes is geographic predilection of their distribution worldwide. It is possible that these subtypes may have spread through different populations at different times and by different routes. For example, subtype A is composed of two sub-subtypes (A1 and A2), both of which appear to have a widespread geographic distribution3 and is commonly found in sub-Saharan Africa and Russia where it is predominantly transmitted through heterosexual

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intercourse.4 It may be one of the oldest of all subtypes. In contrast, subtype B is associated with the HIV epidemic among homosexual men and injecting drug users in North and South Americas, Europe, Japan and Australia. The most prevalent HIV-1 subtype in the global epidemic is subtype C which is dominant in India, Ethiopia, South Africa, Zimbabwe, Botswana and China and is transmitted through heterosexual intercourse. Interestingly HIV-1 viral lineages of O and N are mainly confined to West Africa and phylogenetically these are separated from the other HIV-1 sequences suggesting multiple entry of the viruses into humans.

Subtype C: The expanding pandemic One of the most dramatic changes in the HIV/AIDS has been the rapid emergence and devastating spread of subtype C viruses. HIV-1 C accounts for 56% of all circulating viruses and is the most commonly transmitted subtype worldwide. The subtype C epidemic has now become the most predominant subtype in Southern African countries and Indian subcontinent where HIV prevalence is the highest in the world. The proportionate increase in C viruses relative to other HIV strains suggest that subtype C may be more easily transmitted or that it has a higher level of “fitness” at the population level. One possible explanation is that founder effects relating to the ongoing introduction of subtype C into new population groups with different host factors, or different social and sexual practices, may be responsible for the rapid spread. However, founder and host effects cannot account for the fact that C viruses are overtaking preexisting virus subtypes in several different geographical regions. It is increasing evident that additional (non-host) viral factors are also contributing to the rapid spread of HIV-1 C. Viral studies indicate that subtype C has distinct genetic and phenotypic properties that differentiate it from other HIV-1 subtypes. Subtype C viruses have an extra NF-κB binding site in the long terminal repeat,5 a prematurely truncated Rev protein or a 5-amino-acid insertion in Vpu6 may influence viral gene expression, altering transmissibility and pathogenesis of C viruses. Factors related to C viral entry and pathogenesis such as CCR5 and non-syncytium-inducing properties of C isolates, may also contribute to the increased spread of C viruses. Interestingly, though both subtype B and C are spreading exponentially in Brazil, the subtype C growth rate is about twice that of subtype B there; thus providing evidence of a different epidemic potential between two HIV-1 subtypes7.

Geographical distribution of HIV-1 subtypes Epidemiological and Phylogenetic studies have also shown that HIV-1 clades are unequally spread out throughout the world.8 The HIV epidemic in Africa began in the late 1970s and, during the late 1980s, gradually spread to the South of the continent. Though Africa is considered home of all HIV-1 subtypes, their

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spread to other continents is attributed to some groups of individuals, particularly travelers, who contribute to the initiation of local epidemics worldwide. These groups include, in particular, immigrants, IV drug users, tourists, truck drivers, military troops and seamen. The global view on the contribution of travel to HIV-1 spread usually derives from the prevalence of non-B subtypes in various countries.9 The prevalence of non-B infections has indeed markedly increased in recent years in several European countries.9,10 Recent immigrants from areas of high HIV-1 endemicity and European travelers have been shown to contribute in large part to the increase in the prevalence of non-B infections in western and northern Europe. There is no doubt that it is high risk human behavior and not occupation that determines HIV-1 infection risk. Predominance of subtype B in the western countries including Japan and Australia is attributed to transmission among homosexual men and is generally thought to be spreading separately from that among IV drug users and heterosexual individuals. However, in India subtype C predominance even among homosexual men and IV drug users suggests interplay of host genetic factors and the virus in determining geographical distribution of HIV-1 subtypes. Among the HIV-1 group M viruses, HIV-1 subtype C is by far the most prevalent HIV in the world and is linked to heterosexual transmission. It was first discovered in North-east Africa in the early 1980s11,12 and has since moved to the southern parts of Africa. In addition, the subtype C epidemic has spread to East and Central Africa where it is becoming predominant subtype.13,14 From Africa, it has spread to India, Brazil and South and Central China where is appears to have been introduced from India.15 In England and Wales, preponderance of subtype C infections has been observed among HIV-infected heterosexual STI clinic attendees, particularly in younger age groups, suggesting recent acquisition of this viral strain.16

The Indian scenario In India HIV infection was first reported in 1986 in six commercial sex workers in the State of Tamil Nadu and since then it has been reported from all the States and Union Territories. India now holds dubious distinction of accounting second largest number of HIV infections in the world following South Africa. With an estimated 2.5 million people living with HIV infection in adult population (15–49 years) by 2008, India accounts for 13% of global HIV prevalence.17 Tracking the epidemic and implementing effective programmes is made difficult by the fact that there is no one epidemic in India. Rather, there are several localized sub-epidemics reflecting the diversity in social-cultural patterns and multiple vulnerabilities present in the country. Though the overall national prevalence is low, six states have reached high prevalence (>1%): Manipur,

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Nagaland, Andhra Pradesh, Tamil Nadu, Karnataka and Maharashtra. Certain districts in Goa and Gujarat have also reported high prevalence. Sexual transmission is driving the AIDS epidemic in India. This route accounts for about 86% of HIV infections in the country. Remaining 14% are accounted for by other routes namely blood transfusion, mother-child transmission and IV drug use, particularly in North-east India. Over one-third of all HIV infections occur in young people in the age group 15–24 years. Early studies have indicated the presence of both HIV-1 and HIV-2 in India.18,19 Subsequent studies further emphasized a predominance of subtype C strains in India, which were found to cluster with South African isolates.20,21 Other HIV-1 subtypes, A and B, have been reported in India between 1980s and early 1990s among the recipients of blood and blood products and IV drug users, respectively, suggesting multiple introductions of HIV-1 in this country. Subtype A strains were found to be related to Central and East African subtype and subtype B strains obtained from Manipur were related to subtype B sequences circulating in Thailand.21 However, recent studies have clearly shown that subtype C strains have displaced subtype B in the IV drug users in that part of India.22,23 The trends across the country show that there is no explosive HIV epidemic in India as a whole. However, there are serious sub-national epidemics in various parts of the country with rapid spread and evidence of high prevalence of HIV among both sexual transmitted infections (STI) and antenatal clinic attendees in different sites located in States of Andhra Pradesh, Maharashtra, Tamil Nadu, Gujarat, Pondicherry, Assam, Bihar, Chhatisgarh, Delhi, Haryana, Himachal Pradesh, Kerala, Orissa, Goa and Manipur. In high prevalence states the epidemic appears to be spreading gradually from urban to rural areas and from high-risk behavior groups to the general population. The epidemic continues to shift towards women with an estimated 39% of the infected being women17. An explosive epidemic driven by intravenous drug use has unfolded in the state of Manipur (North-east India) bordering Myanmar and is close to the Golden Triangle composed of Thailand, East Myanmar and West Laos and is the hub of international drug trafficking. A recent study documents that two-thirds HIV infections in this region of India are caused by subtype C and subtype B (Thai B) accounts for 20% of infections.22 The presence of multiple subtypes circulating in Manipur suggests the likelihood of recombinant viruses evolving in this region. Indeed, this has been corroborated by a recent study which reported presence of B/C recombinants from this region.24 Apart from north-eastern states there are also sporadic reports of the presence of A/C and B/C recombinants from West and South India.25,26 The occurrence of HIV-1 recombination in nature is borne out by the identification of genomes that are recombinants between different HIV-1 subtypes.27 Some of these recombinant viruses have become fixed in the human

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population and are referred to as circulating recombinant forms (CRFs), and in at least a few cases CRFs have become the predominant strain in specific geographic areas of infection such as A/E recombinants in Thailand and B/C recombinants in parts of Southeast Asia and China. HIV-1 recombinants are estimated to contribute to 10–40% and 10–30% of the infections in Africa and Asia, respectively. The identification of subtypes and CRFs provides a means of tracking dissemination of the pandemic worldwide. To delineate the molecular features of HIV-1 strains circulating in India, Phylogenetic analyses of sequences of Indian subtype C isolates along with a small number of subtype sequences from other countries revealed that almost all sequences from India form a distinct lineage within subtype C (CIN). Overall CIN lineage sequences were more closely related to each other (level of diversity, 10.2%) than to subtype C sequences from Botswana, Burundi, South Africa, Tanzania and Zimbabwe (range 15.3 to 20.7%). Suggesting thereby, much of the current Indian epidemic is descended from a single introduction into the country.28 In an assessment of the Phylogenetic relationships among subtype C sequences from eleven different countries including India, an overall star-like phylogeny was observed.29 Unlike sequences from South Africa and Botswana, which are scattered in numerous lineages, almost all sequences from India formed a monophyletic lineage, which is lying close to the sequences. Sequences from India generally clustered together more than sequences from other countries. Genetic characterization of the virus during the early seroconversion stage is crucial as the virus isolated is closely related to the transmitted strain and hence immunologically naïve. Phylogenetic analyses of Indian subtype C envelope sequences obtained from early seroconverts indicated that the Indian sequences not only clustered within the C clade but also clustered away from the African subtype C sequences.30 Moreover, a recent study demonstrating lower diversity within immunodominant epitopes and a tight clustering of Indian isolates29 suggested that production of a vaccine particularly against Indian subtype C may not be an unattainable and daunting task.

References 1.

Simon F, Mauclere P, Roques P, Loussert-Ajaka I, Muller-Trutwin MC, Saragosti S Georges-Courbot MC, barre-Sinoussi F, Brun-Vezinet F (1998) Identification of a new human-immunodeficiency virus type 1 distinct from group M and group O. Nat Med 4:1032–1057

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Korber B, Theiler J, Wolinsky S (1998) Limitation of a molecular clock applied to considerations of the origin of HIV-1. Science 280:1868–1871

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Gao F, Vidal N, Li Y, Trask SA, Chen Y, Kostrikis LG, Ho DD, Kim J, Oh MD, Choe K, Salminen M, Robertson DL, Shaw GM, Hahn BH, Peeters M (2001) Evidence of two distinct sub-subtypes within the HIV-1 subtype A radiation. AIDS Res Hum Retroviruses 17:675–688

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Bobkov AF, Kazennova EV, Selimova LM, Khanina TA, Ryabov GS, Bobkova MR, Sukhanova AL, Kravchenko AV, Ladnaya NN, Weber JN, Pokrovsky VV (2004) Temporal trends in the HIV-1 epidemic in Russia: Predominance of subtype A. J Med Virol 74: 191–196

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Jeeninga RE, Hoogenkamp M, Armand-Ugon M, de Baar M, Verhoef K, Berkhout B (2000) Functional differences between the long terminal repeat transcriptional promoters of human immunodeficiency virus type 1 subtypes A through G. J Virol 74:3740–3751

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Gao F, Robertson DL, Carruthers CD, Morrison SG, Jian B, Chen Y, Barré-Sinoussi F, Girard M, Srinivasan A, Abimiku AG, Shaw GM, Sharp PM, Hahn BH (1998) A comprehensive panel of near-full-length clones and reference sequences for non-subtype B isolates of human immunodeficiency virus type 1. J Virol 72:5680–5698

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Salemi M, de Oliveira T, Soares MA, Pybus O, Dumans AT, Vandamme AM, Tanuri A, Cassol S, Fitch WM (2005) Different epidemic potentials of the HIV-1B and C subtypes. J Mol Evol 60:598–605

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UNAIDS (2004) AIDS Epidemic Update. www.unaids.org

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Thomson MM, Najera R (2001) Travel and the introduction of human immunodeficiency virus type 1 non-B subtype genetic forms into Western countries. Clin Infect Dis 32: 1732–1737

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Couturier E, Damond F, Roques P, Fleury H, Barin F, Brunet JB, Brun-Vézinet F, Simon F (2000) HIV-1 diversity in France, 1996-1998. The AC 11 laboratory network. AIDS 14:289–296

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Salminen MO, Johansson B, Sönnerborg A, Ayehunie S, Gotte D, Leinikki P, Burke DS, McCutchan FE (1996) Full-length sequence of an Ethiopian human immunodeficiency virus type 1 (HIV-1) isolate of genetic subtype C. AIDS Res Hum Retroviruses 20:1329–1339

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McCormack GP, Glynn JR, Crampin AC, Sibande F, Mulawa D, Bliss L, Broadbent P, Abarca K, Pönnighaus JM, Fine PE, Clewley JP (2002) Early evolution of the human immunodeficiency virus type 1 subtype C epidemic in rural Malawi. J Virol 76: 12890–12899

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Neilson JR, John GC, Carr JK, Lewis P, Kreiss JK, Jackson S, Nduati RW, Mbori-Ngacha D, Panteleeff DD, Bodrug S, Giachetti C, Bott MA, Richardson BA, Bwayo J, Ndinya-Achola J, Overbaugh J (1999) Subtypes of human immunodeficiency virus type 1 and disease stage among women in Nairobi, Kenya. J Virol 73:4393–4403

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Vidal N, Peeters M, Mulanga-Kabeya C, Nzilambi N, Robertson D, Ilunga W, Sema H, Tshimanga K, Bongo B, Delaporte E (2000) Unprecedented degree of human immunodeficiency virus type 1 (HIV-1) group M genetic diversity in the Democratic Republic of Congo suggests that the HIV-1 pandemic originated in Central Africa. J Virol 74:10498–10507

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Yu XF, Chen J, Shao Y, Beyrer C, Lai S (1998) Two subtypes of HIV-1 among injectiondrug users in southern China. Lancet 351:1250

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Tatt ID, Barlow KL, Clewley JP, Gill ON, Parry JV (2004) Surveillance of HIV-1 subtypes among heterosexuals in England and Wales, 1997–2000. J Acquir Immune Defic Syndr 36:1092–1099

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National AIDS Control Organization. website. www.naco.nic.in

18.

Dietrich U, Grez M, von Briesen H, Panhans B, Geissendörfer M, Kühnel H, Maniar J, Mahambre G, Becker WB, Becker ML, et al. (1994) HIV-1 strains from India are highly divergent from prototypic African and US/European strains, but are linked to a South African isolate. AIDS 7:23–27

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Grez M, Dietrich U, Balfe P, von Briesen H, Maniar JK, Mahambre G, Delwart EL, Mullins JI, Rübsamen-Waigmann H (1994) Genetic analysis of human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2) mixed infections in India reveals a recent spread of HIV-1 and HIV-2 from a single ancestor for each of these viruses. J Virol 68:2161–2168

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Voevodin A, Crandall KA, Seth P, al Mufti S (1996) HIV type 1 subtypes B and C from new regions of India and Indian and Ethiopian expatriates in Kuwait. AIDS Res Hum Retroviruses 12(7):641–643

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Maitra A, Singh B, Banu S, Deshpande A, Robbins K, Kalish ML, Broor S, Seth P (1999) Subtypes of HIV type 1 circulating in India: Partial envelope sequences. AIDS Res Hum Retroviruses 15:941–944

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Mandal D, Jana S, Bhattacharya SK, Chakrabarti S (2002) HIV type 1 subtypes circulating in eastern and northeastern regions of India. AIDS Res Hum Retroviruses 18:1219–1227

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Gupta RM, Prasad VVSP, Rai A, Seth P (2005) Analysis of HIV type 1 subtype C full-length gag gene sequences from India: Novel observations and plausible implications. AIDS Res Hum Retroviruses 21:1066–1072

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Tripathy SP, Kulkarni SS, Jadhav SD, Agnihotri KD, Jere AJ, Kurle SN, Bhattacharya SK, Singh K, Tripathy SP, Paranjape RS (2005) Subtype B and subtype C HIV type-1 recombinants in the northeastern state of Manipur, India. AIDS Res Hum Retroviruses 21:152–157

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Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, Ingersoll R, Sheppard HW, Ray SC (1998) Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 73:152–160

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Siddappa NB, Dash PK, Mahadevan A, Desai A, Jayasuryan N, Ravi V, Satishchandra P, Shankar SK, Ranga U (2005) Identification of unique B/C recombinant strains of HIV-1 in the southern state of Karnataka, India. AIDS 19:1426–1429

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Malim MH, Emerman M (2001) HIV-1 sequence variation: Drift, shift and attenuation. Cell 104:469–472

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Shankarappa R, Chatterjee R, Learn GH, Neogi D, Ding M, Roy P, Ghosh A, Kingsley L, Harrison L, Mullins JI, Gupta P (2001) Human immunodeficiency virus type 1 env sequences from Calcutta in eastern India: Identification of features that distinguish subtype C sequences in India from other subtype C sequences. J Virol 75:10479–10487

29.

Khan IF, Vajpayee M, Prasad VV, Seth P (2007) Genetic diversity of HIV type 1 subtype C env gene sequences from India. AIDS Res Hum Retroviruses 23:934–940

30.

Kalpana A, Srikanth T, Abhay J, Sushama J, Swarali K, Ramesh P (2004) gp120 sequences from HIV type 1 subtype C early seroconverters in India. AIDS Res Hum Retroviruses 20:889–894

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8 Darwin and microbial evolution T.K. Adhya and M. Patra Central Rice Research Institute, Cuttack - 753006, Orissa, India [email protected]

Abstract: In the 200th year of Charles Darwin’s birth anniversary and 150th year of the publication of the ‘Origin of Species’, biologists are trying to re-evaluate the concept of the evolution as propounded by Darwin in the light of the knowledge gathered through recent developments in genomics and proteomics. Since no technique existed for the isolation of bacteria and the tremendous diversity of the microbial world was still largely unknown, at the time Darwin wrote his masterpiece, concept of microbial evolution was not widely discussed. This was further compounded by the lack of a theoretical basis of the concept of species in prokaryotes as microbial lineages show substantial variation in population structure, ranging from essentially clonal to nearly sexual. Comparative genomic studies have revealed that prokaryotic genetic material are under continuous selective pressure that have profoundly influenced its organization indicating that the gene pool available in the microbial world is far larger than previously thought. As per established knowledge of microbial genetics, local DNA sequence changes, segment-wise rearrangement of genomic DNA sequences by recombinational reshuffling and horizontal gene transfer are the evolutionary processes shaping the genome of the prokaryotes. Modern knowledge of the molecular biology of the genes and their expression has provided a strong input in understanding the genome-wide map of selection, linking gene variation to phenotype and ecology. Keywords: Prokaryotes, Microbial speciation, Natural selection, Microbial evolution, Molecular evolution, DNA rearrangement, Horizontal gene transfer

Introduction The year 2009 marks the 200th anniversary of Charles Darwin’s birth and the 150th anniversary of his most influential publication ‘On the Origin of Species by Means of Natural Selection’1 (Fig. 1). Darwin’s ideas have influenced biology ever since and transformed the biological sciences in a way much similar that of Copernicus, Galileo and Newton, who centuries earlier transformed the physical science. Darwin’s thinking on how species evolved, his ideas on ‘survival of the fittest’ and selection during evolution are the guiding principles of nearly all V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Figure 1. Title page of The Origin of Species

aspects of life science. The concept of natural selection – as the guiding principle of adaptive evolution – is Darwin’s seminal contribution. It provides a worldly explanation of nature’s operations that contrasted sharply with the traditional beliefs of supernatural interventions that predominated before ‘The Origin’. Darwin is deservedly credited as the originator of modern theory of evolution. In ‘Origin of Species’, he gathered the best available evidences for evolution. He explained how natural selection works, discussed the role of hereditary variation, the mechanisms of which were not well understood in Darwin’s time, and also considered the possible objections to his theory. Darwin’s clear elucidation of natural selection launched a revolutionary dimension in biology wherein organismal traits could be studied and interpreted as products of natural forces amenable of rational scientific scrutiny. Scientific studies of natural selection are now more popular and powerful than ever and they have revealed the evolutionary origins of numerous biological features and taxa. A major limitation in Darwin’s characterization of evolution concerned the hereditary mechanism, an aberration that the modern biology began to rectify early in the 20th century by incorporating Mendelian genetics and population genetics in the dictum of evolutionary synthesis.2 Today, in the era of genomics, biologists routinely extend studies of natural selection and evolution of features to the level of DNA. Genomic investigations are also providing fresh insights into the ancient mystery of the origin of species. Characteristically, ‘The Origin’ mentions little detail about the evolution of reproductive barriers, which

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under the modern biological species concept are keys to the understanding of speciation process.

Darwin and microbiology It is commonly assumed that Darwin had nothing to say about microbes and was ignorant of or at best indifferent to microbes and microbial evolution. 3 Microbiology was a nascent science when Darwin published his ‘Origin of Species’. No techniques existed for the isolation of bacteria and the tremendous diversity of the microbial world was still largely unknown. The ‘germ theory’, microorganisms as the causal agent for diseases, would not be developed until many years later. Although Darwin did not draw heavily on the microbial world for observational data in his own work, he used his and others’ knowledge of microorganisms to sustain his arguments about natural selection. Three main themes emerged from these observations: (a) biogeographical patterns and processes, (b) distinction between evolution through natural selection and evolution as progressive complexification, and (c) continuous nature of evolution operating on all life-forms. Microbial biogeography Biogeography had formed the insight of Darwin’s evolutionary theories and he considered the biogeography of microbial life to be very similar to that of macroorganisms. Natural selection eventually became the ultimate explanation of the biogeographic distribution of different life-forms. The idea of microbial diversity was used by Darwin as evidence for his argument about dispersability which he saw as central to the geographical distribution of species.4 He preferred ‘dispersal’ explanations to those involving geological disruption, such as break-up of land bridge due to ‘continental shift’. Darwin made several observations and inferences about the dispersal mechanisms of plants and animals and successfully worked them into his all-encompassing explanation of natural selection. Microbes and their distribution were just as explicitly adjoined by this thesis. Evolution of microbes by natural selection Very little was known about microbial physiology when Darwin developed his theory of natural selection. Nevertheless, taxonomy of microbes existed and these began to grow systematically after the establishment of Ferdinand Cohn’s scheme of classification.5 However, the inherent handicap of largely invisible and always confusing microbial diversity made it less pressing to explain the microbial component than the abundant new discoveries of obvious diversity of plants, animals and the fungi. These visible entities formed the primary factual

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basis for Darwin’s theory of natural selection albeit his intention of covering all life forms in his theory of natural selection. In fact, in responding to criticisms that the simplicity of microorganisms disproved his theory of natural selection, Darwin added an argument about the adaptiveness of microbial organization to the third edition of ‘The Origin’.6 Continuity of evolved microbial complexity Darwin’s argument was that an evolutionary record could be detected in the more simply organized life-forms, such as microbes, and also their simplicity must not confuse the natural historians to their organization complexity. The complexity, at least in the functional level that has been confirmed with the current knowledge of biology, led Darwin to speculate on the continuous theory of evolution and the common ancestry of all living organisms. In other words, Darwin saw in today’s microbes a record of the very early life-forms. Suggesting this link between early and current life from bacteria onwards, was an inevitable consequence of accepting descent with modification.

Darwin’s views on the evolution of microbes In his discussions on the microbial world, Darwin made at least three major evolutionary claims: (1) that all living entities, no matter how different they seem from animals and plants, do undergo natural selection; (2) that microbes demonstrate that evolution is not a progression from simple to complex; and (3) that microbes and their adaptive capacities are very important biological phenomena to understand if the history of evolution on Earth is to be understood. These dicta are crucial to the understanding of the evolutionary theory put forth by Darwin. Today, in the background of huge explosion in knowledge in molecular biology and genomics, Darwin’s basic principles still holds sway. Darwin’s discussion of microbes thus is the, testing of a fundamental framework to see whether it can accommodate all forms of life and all the life’s history. In this context, Darwin’s investigations of microbes gave his theory cardinal support and the widest possible application. To establish his theory as firm as possible with the largest of audience, Darwin focused instead upon the visible and the obvious.

Concept of speciation in prokaryotes Unlike in the reproduction of complex eukaryotes, cell fusion and recombination are not the necessary steps in the reproduction of prokaryotes. As a result, early models for understanding evolution and speciation in prokaryotes often focused on clonality and periodic selection. According to such models, all individuals within a species resemble each other because they descend from a single

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ancestor that bested its siblings by virtue of some beneficial mutation(s) fixing not only the favored mutation but the entire genome in which it first occurred. In practice, bacterial lineages show substantial variation in population structure, ranging from essentially clonal (e.g. Salmonella) to nearly sexual (e.g. Neisseria gonorrhoeae). The basis of the taxonomic hierarchy is the species. However, the concept of a prokaryote species still lacks a theoretical basis, and all existing definitions are pragmatic ones, such as, for example: ‘A species consists of an assemblage of individuals (or, in micro-organisms, of clonal populations) that share a high degree of phenotypic similarity, coupled with an appreciable dissimilarity from other assemblages of the same general kind’, or: ‘a collection of strains showing a high degree of overall similarity, compared to other, related groups of strains’.7 Ward (1998)8 called for the establishment of a ‘natural’ species concept for the prokaryotes, based on ‘evolutionary species’, being defined as lineages evolving separately from others and with their own unitary evolutionary roles and tendencies. The species definition is so extremely subjective because one cannot accurately determine and define such concepts as ‘a close resemblance’, ‘essential features’, or how many ‘distinguishing features’ are sufficient to create a species. In-depth essay on the current thoughts on the species concept for prokaryotes was recently published.9 The species should ideally be described as ‘a category that circumscribes a (preferable) genomically coherent group of individual isolates/strains sharing a high degree of similarity in (many) independent features, comparatively tested under highly standardized conditions’ (the phylo-phenetic species concept). Our present species concept is, to a large extent, based on the recommendations published in 1987 by a committee of experts,10 recently confirmed and extended by a new ad hoc committee.11 Species classification is based on a combination of diagnostic phenotypic features and genomic properties. The questions of whether clusters of strains observed in the bacterial world share the dynamic properties attributed to the eukaryote species, and whether the mechanisms driving the origin of new species in bacteria might be shared with the eukaryotes, were addressed.12 He concluded that, despite basic differences in the nature of the genetic exchange between bacteria and eukaryotes, bacterial species share many of the fundamental properties of eukaryotic species. In case of the prokaryotes, recombination depends on vectors such as bacteriophagemediated transduction or plasmid mediated conjugation, and is therefore limited by the host ranges of the respective vectors. Moreover, restriction endonuclease activity can greatly reduce the rate of recombination. Cohan12 further assessed the potential of the formation of new species within the prokaryote world as a result of the formation of new ‘ecotypes’. On the basis of multilocus sequence typing he defined an ecotype as a set of strains using the same or very similar ecological niches, such that an adaptive mutant from within the ecotype outcompetes all other

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strains of the same ecotype. The potential for speciation is very large indeed for the following reasons: ● Speciation in prokaryotes requires only ecological divergence (versus both reproductive and ecological divergence in sexual eukaryotes). ● The extremely large population size of prokaryotes makes rare mutation and recombination events much more accessible to a population than is the case for macro-organisms. ● A prokaryote species is open to gene transfers from many taxa, even those distantly related, so it can take up existing adaptations from anywhere in the prokaryote world. ● Recombination events have a localized nature, as only a small fraction of the donor’s genome is integrated. This allows for transfer of a useful adaptation without co-transfer of other donor segments that would be deleterious for the recipient. This is in contrast with the processes of meiosis and fertilization in eukaryotes, which yield hybrids that are a 1:1 mix of both genomes. The physiology of bacteria may thus be more modular than is the case for macro-organisms in that a new adaptation might be accommodated with very little fitness cost.12 Direct molecular analyses of natural microbial populations reveal patterns that should compel microbiologists to adopt a more natural species concept that has been known to biologists for decades. The species concept can be utilized to address the larger issue of delineating the concept of microbial evolution as had already been done for eukaryotes.

Microbial evolution in the present context As compared to the diploid nature of mostly higher organisms, prokaryotic microorganisms, archaea, eubacteria and their viruses, are usually haploid and they rapidly reveal phenotypic consequences of genetic modification. This and the fast propagation of these micro-organism facilitate genetic variation which is a prerequisite for biological revolution. Indeed, mixed populations of parental forms and different genetic variants are the substrate for natural selection that is exerted by the living conditions encountered by the organisms. Aided by recent developments in the understanding of genomics, prokaryotes certainly provide an interesting opportunity to study the mechanisms of evolution as (i) they harbor a previously unsuspected diversity even within species and populations,13 (ii) they are found in small to very large population sizes,14 (iii) they can survive or prosper in most inhospitable environments, and (iv) they frequently acquire and use genetic material from distantly related organisms.

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Universality and diversity of prokaryotic genomes Comparative genomic studies have revealed that prokaryotic genetic material are under continuous selective pressure that have profoundly influenced its organization. Processes of replication, transcription and the regulation of gene expression all impact how genes are arranged along the genome. It has long been known that the asymmetric manner in which the prokaryotic genetic material is replicated, with a leading and a lagging strand, is correlated with many evolutionary features, such as differential bias between the two strands and location of essential genes. Interestingly, the intensity of these biases shows a strong phylogenetic inertia. The replication of prokaryotic genetic material is oriented from the origin (Ori) to the terminus (Ter). This is also correlated with several evolutionary features like (a) in many genomes, genes near the terminus of replication tend to have lower G+C content and also exhibit higher rate of evolution;15 (b) also, as a result of the replication process, genes located close to the Ori can be significantly amplified in dividing cells, in comparison to those closer to the terminus. Not only these genes are over-represented in the Ori region but the genomes of fast-dividing bacteria show evidence that rearrangements that would disrupt this association are counter-selected.16 In spite of these common principles of genome organization, comparative genomics has revealed a previously unexpected degree of diversity among prokaryotic genomes. One of the most remarkable examples of this diversity is the comparison of gene contents within and between species. All forms of life seem to share only a handful of genes, ~60,17 and these are mostly dedicated to translation. The genes for other fundamental factions, such as DNA replication, transcription or basic metabolism seem to be more sporadically spread in the tree of life. More interestingly, this diversity of genome content can be seen at every phylogenetic scale. It has been estimated that the core genome of13 -proteobacteria contains <300 genes18 and although all Escherichia coli and Shigella genome sequenced to date have >4,000 protein coding genes, these genomes share <3,000 genes. This variability has raised the question of how to define the genome of a species, and the concept of ‘pan genome’ was proposed as the sum of all genes found in a species. It has been shown that in Bacillus anthracis, the pan genome was found to be fully described with only 4 genomes, a possible testimony of the recent emergence of this species.19 However, in group B Streptococcus, the ‘pan genome’ was ‘open’ meaning the number of new genes was expected to be ~30 irrespective of whatever the number of genomes already present in the comparison. Also, the study of seven strains of E. coli also suggested an open ‘pan genome’ for this species. These results suggest that the gene pool available in the microbial world is far larger than previously thought.

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Source of genetic variability in prokaryotes Genetic variation being a prerequisite for biological evolution, mixed populations of parental forms and different genetic variants are the substrate for natural selection which is exerted by the living conditions encountered by the organisms. The mechanisms explaining the diversity of gene repertoires, that is the processes by which genes are gained and lost have been the subject of numerous studies. As per established knowledge on microbial genetics, three qualitatively different strategies contribute to the overall production of genetic variability20 (Fig. 2). The first of these strategies brings about local DNA sequence changes such as nucleotide substitutions, small deletions and small insertions of one or a few nucleotides or a limited scrambling of nucleotides. The second strategy is a segment-wise rearrangement of genomic DNA sequences by recombinational reshuffling. The third strategy is based on horizontal gene transfer (HGT) and results in the acquisition of foreign DNA sequences. These three major strategies to generate genetic variability differ from each other in the quality of their effects. With regard to their evolutionary impact, individual mechanisms belonging to the same strategy may substitute for one another, but not for a member of another type of strategy. Hence, evolutionarily well adaptable bacteria may be expected to be genetically equipped with at least one or several systems for each of the three evolutionary strategies. Like gain of genetic information, losses of unique DNA sequences are also important source of genetic variability. Before complete genomes from different strains of the same species or from closely related species were available,

Figure 2. A schematic view of the elements of molecular evolution of prokaryotic microorganisms (adapted from Arber 2000)

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nonfunctional genes or ‘pseudogenes’ were thought to be rare in bacteria. The first reports of a significant number of pseudogenes were in pathogens undergoing strong genome reduction such as Rickettsia prowazekii or Mycobacterium leprae, but free-living bacteria were believed to contain relatively few pseudogenes. However, a recent study based on comparisons based on closely related genomes has shown that E. coli MG1665 genome contains ~100 genes that are >80% shorter than their orthologs in other E. coli strains and are therefore likely to be pseudogenes.21 The most frequent causes of gene disruption are frame-shifts and truncations but some recent pathogens such as Yersinia pestis or Shigella flexneri exhibit a high proportion of pseudogenes due to introduction of ‘insertion sequence’ (IS) elements, probably as a result of relaxed selection pressure. Nevertheless, both gene loss and acquisition imparts variability in the prokaryotic population subjecting them to natural selection and further evolution. Local sequence change Local sequence changes might theoretically represent individual steps in the development of new biological functions. However, the mutational generation of a completely novel biological function may be extremely rare as long as natural selection cannot exert its pressure on an at least primitive function displayed by the product of the DNA sequence. The situation may change in the stepwise improvement of already available biological functions on which natural selection exerts its pressure. This is applicable to functional domains, entire genes as well as functional systems composed of different genes Evolutionary clocks used by the scientists to estimate evolutionary distances are based on local sequence changes often within a specific gene, for example a ribosomal RNA gene. Within functional genes lethal and heavily contra-selective mutations will of course not be maintained. Also, local sequence changes are known to affect different DNA regions with different efficiency. These reasons limit the precision of evolutionary clocks, but the method has its merits for the relative comparison of the evolutionary relations between different species. DNA rearrangements Gene conversion can also result from the intragenomic reshuffling of DNA sequences, if related sequences carried in the genome undergo recombination. Another often innovative and thus important effect of the DNA rearrangement strategy is the novel combination of available capacities by the fusion of different functional domains. Genomics reveal that different functional genes often share related sequence motifs and functional domains. Occasional DNA reshuffling is a good source for such fusion genes. The same holds true for the regrouping of expression control signals with different reading frames serving in protein

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production. DNA rearrangements can also bring about the duplication of DNA segments which may serve as substrates for further evolution. Similarly, deletion of DNA segments can also result from DNA rearrangement processes and can help in conjunction with natural selection to discharge the genome from inessential sequences. Bacterial population geneticists have known for some time that prokaryotic genomes do sometimes recombine,22 although early estimates suggested that rates of recombination were sufficiently low to be ignored when considering periodic selection events. Recent work has helped clarify the relationship between recombination and positive selection.23 Recombination and positive selection were both quantified in the Streptococcus core genome, and it was concluded that genes under positive selection are frequently recombined, a result also supported in a study of Listeria genome. Specifically, 78% of genes under positive selection in the Streptococcus pyogenes core genome were also inferred to be recombinant. Yet, of the genes identified as recombinant within this species, only a small fraction experienced positive selection. Therefore, although positively selected genes are frequently recombined, a substantial amount of within-species recombination shows no evidence of direct adaptive value. In other words, recombination within a species could be largely neutral while not so for recombination between species. Critical analysis of comparison between-species and within-species evolutionary events indicate 81% of all genes recombined between species also experienced positive selection, whereas only 4% of genes recombined within species also experienced positive selection. DNA acquisition The strategy of DNA acquisition allows microorganisms to share the evolutionary success of others. This strategy is very efficient and can result in essential new capabilities in a single step of acquisition. This is of particular relevance for accessory genes. Such genes may also open the possibility for a microorganism to populate new habitats. The evolutionary contribution of this strategy is thus considerable. This strategy can also affect functions of essential housekeeping genes by conversion upon horizontal transfer of DNA segments. In bacteria, horizontal gene transfer (HGT) is widely recognized as the mechanism responsible for the widespread distribution of antibiotic resistance genes, gene clusters encoding biodegradative pathways and pathogenecity determinants. HGT is also considered responsible for speciation and sub-speciation in bacteria Analysis of complete genome sequences is helping determine what types of genes are most prone to transfer. Recent studies using phylogenetic analysis of many genes conclude that genes which interact with many other genes (informational genes) are less prone to gene transfer than those with fewer interactions (operational

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genes). This was referred to as the ‘complexity hypothesis’.24 It is still not clear if complexity of interactions is the key factor here. For example, it is possible that sequence conservation of genes between species also influences the likelihood of transfer, which is probably what allows even rRNA genes to be transferred. In addition, because informational genes are more likely to be required for survival, they are probably less likely to be lost from a species. Therefore, there will be little opportunity to replace them with a homologous gene. In addition, some genes may even facilitate their own transfer. For example, genes could avoid the need for amelioration by carrying the features needed for their own expression. Possible examples include self-regulatory transcription factors (which could bind to their own promoter), amino-acyl tRNA synthetases (which have been shown to be prone to transfer and could help avoid codon usage problems), splicing factors, recombinases, and DNA polymerases. HGT is frequently invoked for those genes that have best matches to supposedly distant species. Lateral gene transfer can result in the addition of novel genes in a particular species — thus, in theory, gene transfers could be identified by detecting genes with uneven distribution patterns (e.g. if a particular gene is present in gram-positive bacteria and all plants but not other bacteria or eukaryotes). Some selected examples are: Thermophilic bacteria and archaea Analysis of the complete genomes of the bacteria Aquifex aeolicus and Thermotoga maritima and comparison with other complete genomes has led to the suggestion that large numbers of genes have been transferred between thermophilic bacteria and Archaea. In both cases, best-match methods have revealed that a high percentage of each proteome was most similar to Archaeal genes rather than bacterial genes, ~20% for A. aeolicus and 25% for T. maritime25. Transfers involving pathogen genomes A variety of studies have suggested the occurrence of extensive HGT in the history of many of the pathogen species for which complete genome sequences are now available. Analysis of nucleotide composition and codon usage has identified many regions of possible HGT in E. coli (up to 18% of the genome). Genome analysis suggests that a large percentage of the Xylella fastidiosa genome (~7%) appears to be derived from l-like phage. The finding that only a modest proportion of the genes in the Campylobacter jejuni genome had matches to genes from other -Proteobacteria, led to the suggestion that this species may have acquired many genes by HGT.

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Transfers involving whole genetic elements (small chromosomes, megaplasmids, plasmids) Analysis of complete genomes is revealing that many of the smaller genetic elements in microbes may have separate origins from the main chromosomes. For example, the linear plasmids of Borrelia appear to be prone to HGT among strains or species. Other proposed cases of discordant evolution of smaller genetic elements versus chromosomes include the Xylella fastidiosa plasmid, the megaplasmids and plasmid of D. radiodurans and the smaller chromosome of V. cholerae. The source of these smaller elements in each species remains unclear. Interestingly, phylogenetic analysis suggests that the megaplasmids of Rhizobia are only transferred among closely related strains or species and not across long distances. Antibiotic resistance: The immediate response The dissemination of antibiotic resistance genes among human and animal bacterial pathogens and associated commensal populations is the paradigm for HGT on a global scale. This is the best known example of very rapid adaptation of bacterial populations to a strong selective pressure. What has been learned is that this adaptation occurred (and still occurs if bacteria are challenged with a novel antibiotic) not by mutation of the menaced populations, but by acquisition and dissemination of, in most cases, simple antibiotic resistance genes by mobile genetic elements (Table 1). Nonetheless, the basis of antibiotic resistance development is formally simple: mobile genetic elements such as plasmids and transposons efficiently distributed the resistance determinants, singly or in clusters, among many genera and species of bacteria. Intergeneric boundaries were not respected, particularly the long-standing but only apparent distinction between Gram-positive and Gram-negative bacteria. Biodegradation pathways: delayed opportunistic response Based on studies with antibiotic resistance and its associated genes (e.g. genes for the metabolism of a number of sugars), it is not surprising that HGT has also led to the dissemination of gene clusters (operons) involved in the catabolism of xenobiotics in polluted environments. The selective pressures are similar to those of antibiotic resistance, but the process of degrading xenobiotic compounds requires more complex genetic systems, usually operons of ten or more genes, or even regulons of several operons with accompanying control circuits. An example of this can be found in the Sphingomonas aromaticovorans plasmid pNL1. This large conjugative plasmid contains 15 gene clusters directly associated with the catabolism or transport of aromatic compounds, allowing the host bacteria to

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Table 1. Mobile genetic elements involved in HGT in bacteria Mobile element

General features

Examples

Gene cassette

Mobility of ‘simple genes’ IntI1 Immediate constitutive IntI4 expression Extremely broad host range

Transposon

Mobility of genes and operons Very broad host range Loci screening for best performance

Tn21 Tn4651

Plasmid

Mobility of operons and regulons Immediate regulated expression Very broad host range

pNRG234a Nodulation plasmid of pNL1 rhizobium pCD1 Degradation of aromatic compounds Virulence determinants of Yersinia

CTX Bacteriophage Mobility of regulons ‘Maturation in chromosomes (via lysogeny) Narrow host range

Phenotype of specific element Antibiotic resistance (simple) Gene ‘maturation’ in Vibrio

Antibiotic resistance (complex) Toluene degradation

Virulence phage of Vibrio cholera

degrade compounds such as biphenyl, naphthalene, xylene and cresol. Although the dissemination of biodegradation pathways has not been analyzed to the same extent as that of antibiotic resistance, the available data clearly indicate that the genetic mechanisms responsible are likely to have been the same: plasmids and transposons distributed catabolic operons among a wide range of bacteria.26 It can be assumed that the degradation of xenobiotics by bacteria has intensified in the past century, as a result of the increased use of chemicals in industry and the waste disposal habits of humans, but the basic processes could have evolved much earlier. Significantly, degradative operons are also found integrated in the bacterial chromosome. In these cases, adjacent remnants of phage or plasmid genes remind us of the once-mobile nature of these operons. Armed with the awareness that HGT is so important in bacterial speciation, it is now possible to examine completely sequenced chromosomes in a new light and assess the extent of chromosomal DNAs of obscure lineage. Using E. coli and Salmonella as a model system, it has been shown that 17% of their genomes (i.e. ~800 kb), appears to have been acquired by HGT during the past 100 million

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years (the housekeeping genes of both organisms are ~90% identical). As the majority of this DNA was recruited recently, it is apparent that considerable genetic flux is occurring: the 234 detectable HGT events that have persisted are probably the tip of the iceberg of the thousands of mobile sequences that have entered and left any particular E. coli strain. Similarly, when the members of a well known collection of E. coli strains (the ECOR collection) were compared, they were found to be quite variable in the size and macro-organization of their chromosomes and plasmids. In summary, a significant proportion of the genome of any strain of a single bacterial species comprises fragments of DNA from various origins which, properly ‘nurtured’, can give rise to new bacterial species. The role of HGT seems to be crucial but one should not consider that gene exchanges have been so profound as to preclude the reconstruction of the history of life, in the sense of understanding how genomes have evolved to what they are. More integrative approaches combining information from species phylogenies, gene histories, ecology and cellular networks will be necessary to tell the chronicles of contemporary genomes. Involvement of non-genetic factors in the generation of genetic variants Nature also takes advantage of non-genetic factors for the production of genetic variants. Both intrinsic structural features of biological macromolecules and external influences, such as those by mutagens interacting with DNA are important. This is not limited to the strategy of local sequence changes but can also affect the other strategies of generation of variation. Another non-genetic factor influencing the production of genetic variations is the chance of random encounter. This plays its role in the interaction of enzymes with their substrate such as a site of recombination on DNA molecule. Random encounter is also clearly involved in the DNA acquisition strategy. During transformation, DNA molecule must encounter recipient bacteria, in transduction, the bacteriophage must encounter a host bacterium, and in conjugation, donor and recipient bacteria must encounter each other.

Conclusion Biological constituents of the process of biological evolution are likely to undergo biological evolution themselves. Identifying the signature of natural selection in microbial genomes can help to explore the unknown world of microbes. Which techniques can be used to identify positive selection depends on the rates and bounds of recombination in microbial populations. The first step in any study of natural selection in bacteria is to quantify the extent of recombination within a population before moving on to sequence-based tests. Once recombinant portions

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of the genome are identified, they can be tested for evidence of positive selection using diversity-based methods. Meanwhile, the non-recombinant clonal frame can be identified and tested for convergent evolution or excessive rates of functional substitutions. If possible, all tests should be performed genome-wide to estimate and control for demographic effects that might otherwise provide spurious evidence of positive selection. The modern knowledge of genomics and proteomics have provided a strong handle to the microbiologists and molecular biologists to understand the genomewide map of selection, linking gene variation to phenotype and ecology. Darwin was right in saying that many variations are minor but subtly alters protein structure or expression, but cumulatively, they leave a trail of footprints, which will finally lead us to a better understanding of the microbial world. Acknowledgment: This work was part of an in-house discussion-series held under the AMAAS project (theme: Microbial diversity and identification), funded by Indian Council of Agricultural Research, New Delhi. The senior author thanks Prof. Anupam Varma, for enthusing him to compile the stray thoughts in the form of this chapter.

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9 Evolutionary relationships among cyanobacteria, algae and plants: Revisited in the light of Darwinism Radha Prasanna* and B.D. Kaushik** *Division of Microbiology, Indian Agricultural Research Institute, New Delhi - 110012, India [email protected] **Netaji Subhas Institute of Technology, Dwarka, Sector 3, New Delhi - 110078, India [email protected]

Abstract: Algae represent an ancient group of plant like organisms, comprising a heterogeneous, polyphyletic assemblage of prokaryotes and eukaryotes, which have been primarily classified on the basis of their pigments, storage food material and cell wall characteristics. They have been playing a critical role in shaping the Earth’s atmosphere, recycling major elements such as C and N and mediating key biogeochemical processes and were the first to colonize land. Algae have been often conceptually and systematically linked to the land plants because they include some forms – such as seaweeds/kelps, which look much like plants, and as with plants – they are commonly sessile (attached/ rooted/occurring in mixed communities) and oxygenic photosynthesizers. DNA evidence suggests that the first eukaryotes (green plants) evolved from prokaryotes (through endosymbiotic events) between 2500 and 1000 million years ago. Among algae, blue green algae or cyanobacteria represent an ancient group of photosynthetic prokaryotes, whose ubiquity, metabolic flexibility and adaptive abilities have made them a subject of research worldwide. Their biological significance, especially as the prokaryotic interface between the primeval photosynthetic cell and present day plants is also well recognized. Darwin’s Theory of Evolution and Natural Selection have shaped much of our understanding of the evolution of this cosmopolitan group of algae and land plants. However, in the current scenario, with the advent of molecular phylogenetics and the expanding knowledge regarding the evolution of life through the use of bioinformatics, a thorough re-evaluation of algae and interrelationships with plants is needed. This compilation attempts to link Darwinism, Neo-Darwinism and Systemic Darwinism, with the rapidly generated information through modern molecular tools for a better understanding of evolution of land plants, as mediated through algae and endosymbiotic events combined with horizontal and lateral gene transfer processes. Keywords: Algae, Cyanobacteria, Endosymbionts, Evolution, Mitochondria, Plastids, Photosynthesis, rDNA V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Introduction Algae represent a morphologically and biochemically diverse assemblage of organisms, including cyanobacteria - which are amenable to a range of growth environments, culturing techniques and have applications in agriculture, industry and environmental management. This broad terminology includes both prokaryotes and eukaryotes, which exhibit tremendous diversity in metabolism, structure and habitat. Their ecological amplitude combined with their dynamic survival and adaptation strategies as primary colonizers and producers in the food chain make them one of the most significant contributors to the stability, fertility and maintenance of various ecosystems.1 It was more than three billion years ago, that the advent of primordial cyanobacteria marked a decisive turning point in the evolution of our planet by introduction of oxygen into a previously anoxygenic biosphere and bringing about global changes in the ionic composition of ancient water bodies.2 Their role in reclamation of wastewaters/wastelands, as pioneers in denuded/ degraded soils and as bioinoculants in rice based cropping systems is well established.3 In the present scenario, they have become prime targets for screening programs in search for novel compounds of potential pharmaceutical or diagnostic value and invaluable as tools for better understanding of diverse metabolic and evolutionary processes. Microalgal biotechnology is increasingly attracting attention in national and international research programs, as these organisms represent an unique and underexploited bioresource. Darwin’s Theory of Evolution is the widely held notion that all life is related and has descended from a common ancestor: the birds and the bananas, the fishes and the flowers – all related. Darwin’s general theory presumes the development of life from non-life and stresses a purely naturalistic (undirected) ‘descent with modification’, i.e. complex creatures evolve from more simplistic ancestors naturally over time. In a nutshell, as random genetic mutations occur within an organism’s genetic code, the beneficial mutations are preserved because they aid survival - a process known as ‘natural selection’. Such beneficial mutations are passed on to the next generation; over time, beneficial mutations accumulate and the result is an entirely different organism (not just a variation of the original, but an entirely different creature). For Darwin,4 the primary engine of evolutionary change was natural selection: “Furthermore, I am convinced that Natural Selection has been the main but not exclusive means of modification”. However, in the current scenario, Darwin’s Theory of Evolution seems to be a ‘theory in crisis’ in light of the tremendous advances made in molecular biology, biochemistry and genetics over the past fifty years. We now know that there are in fact tens of thousands of irreducibly complex systems on the cellular level. As molecular biologist Michael Denton wrote, “Although the tiniest bacterial

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cells are incredibly small, weighing >10-12 grams, each is in effect a veritable micro-miniaturized factory containing thousands of exquisitely designed pieces of intricate molecular machinery, made up altogether of one hundred thousand million atoms, far more complicated than any machinery built by man and absolutely without parallel in the non-living world”. Specified complexity therefore, pervades the microscopic biological world and Darwinism needs to be explored in the context of the rapidly generated information on genomics.5 This compilation therefore, focuses on the modern day significance of Darwinism in understanding algal evolutionary trends and interlinking with the evolution of land plants.

Concept of Darwinism Ancient Greek philosophers such as Anaximander postulated the development of life from non-life and the evolutionary descent of man from animal. While Darwin’s Theory of Evolution is a relatively young archetype; the evolutionary worldview itself is as old as antiquity. Charles Darwin simply brought something new to the old philosophy – a plausible mechanism called ‘natural selection.’ Natural selection acts to preserve and accumulate minor advantageous genetic mutations. Suppose a member of a species developed a functional advantage (it grew wings and learned to fly), its offspring would inherit that advantage and pass it on to their offspring. The inferior (disadvantaged) members of the same species would gradually die out, leaving only the superior (advantaged) members of the species. Natural selection is the preservation of a functional advantage that enables a species to compete better in the wild and can be considered the naturalistic equivalent to domestic breeding. Over the centuries, human breeders have produced dramatic changes in domestic animal populations by selecting individuals to breed. Breeders eliminate undesirable traits gradually over time. Similarly, natural selection eliminates inferior species gradually over time. Darwin’s Theory of Evolution is a slow gradual process. Darwin wrote “Natural selection acts only by taking advantage of slight successive variations; she can never take a great and sudden leap, but must advance by short and sure, though slow steps.” Thus, Darwin conceded that, “If it could be demonstrated that any complex organ existed, which could not possibly have been formed by numerous, successive, slight modifications, my theory would absolutely break down.” Such a complex organ would be known as an ‘irreducibly complex system’. The common mousetrap is an everyday non-biological example of irreducible complexity. It is composed of five basic parts: a catch (to hold the bait), a powerful spring, a thin rod called ‘the hammer,’ a holding bar to secure the hammer in place, and a platform to mount the trap. If any one of these parts is missing, the mechanism will not work. Each individual part is integral. The

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mousetrap is irreducibly complex and so are the present day biological systems which could not have evolved slowly, piece by piece. Darwin repeatedly wrote about the laws of variation, reproduction, correlation, and heredity and even about the laws of natural selection, the struggle for life, divergence of character, and extinction. The last sentence of On the Origin of Species established an analogy between Darwin’s evolutionary view of life and Newton’s ‘fixed laws of gravity’. Furthermore, in another well known passage he noted: “It is interesting to contemplate on entangled bank, clothed with many plants of many kinds, with various birds singing on the bushes, with various insects flitting about, and with worms crawling through the damp earth, and to reflect that these elaborately constructed forms, so different from each other, and dependent on each other in so complex a manner, have all been produced by laws acting around us”. (Darwin4 On the Origin of Species by means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life, Harvard Univ Press, Cambridge, MA.)

Modern day significance of Darwinism and the advent of Systemic Darwinism A major area of research on evolutionary biology focuses on individuality and adaptation across levels of selection and what ideally should we call as the unit of Darwinism and what should be its characteristics. The proper definition of a Darwinian individual constitutes one of the very fascinating and challenging areas of biology. In this context, two major clarifications have greatly abetted the understanding and fruitful expansion of the theory of natural selection and defining characteristics that define a Darwinian unit, in recent years. Firstly, the acknowledgement that interactors, not replicators, constitute the casual unit of selection; and the recognition that interactors are Darwinian individuals, and that such individuals exist with potency at several levels of organization (genes, organisms, demes and species in particular), thus engendering a rich hierarchical theory of selection in contrast with Darwin’s own emphasis on the organismic level.6 Some of the criteria that an entity must manifest to represent a Darwinian individual are as follows. The biological individual must exhibit a distinct birthpoint, distinct deathpoint and sufficient stability, followed by reproductive ability and interactive ability with the environment – these constitute the three basic properties of a Darwinian individual. Organisms, species and genes have been thoroughly investigated for their appropriateness in this context.

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The gene represents the most appropriate unit for marking evolutionary change for two reasons; firstly, the gene operates as a maximally faithful replicator across generations and as bookkeepers can therefore record the evolutionary history of populations as changing relative frequencies of these stable items. Secondly, genes become preferred units of bookkeeping because they represent the lowest-level individuals subject to clear and accurate recording in a genealogical hierarchy of inclusion. However, genes do not manifest sufficient stability or interactive ability as compared to organism or species; although they fulfil the other criteria. Organisms, on the other hand, although score well in terms of boundedness and functionality, but they have evolved powerful mechanisms to suppress selection at lower levels than itself. On defining Darwinian individuality across scales of size and time, the species prove most effective as Darwinian individuals in evolution, as compared to organisms or genes. The species individual, by not suppressing selection at lower levels, emerges as the suitable choice as a Darwinian unit because it maintains an excellent reservoir of traits and gains enormous flexibility that provides emergent fitness. This is very aptly illustrated by the studies of Tomitani et al.7 who integrated molecular phylogenetic, physiological, paleontological and geochemical data of cyanobacterial species and provided not only a calibration point, but also, very informative data which can further illuminate molecular evolutionary studies of deep nodes in the Tree of Life and thereby evolution of early organisms.

Systemic Darwinism Darwin’s 19th century, evolutionary theory of descent with modification through natural selection opened up a multidimensional and integrative conceptual space for biology. A significant amount of 21st century research focuses on systems (e.g. genomic, cellular, organismic, and ecological/global); systemic Darwinism8 is emerging in this context. It follows a ‘compositional paradigm’ according to which complex systems and their hierarchical networks of parts are the focus of biological investigation. Through the investigation of systems, Systemic Darwinism promises to reintegrate each dimension of Darwin’s original logical space. Moreover, this ideally and potentially unified theory of biological ontology coordinates and integrates a plurality of mathematical biological theories (e.g. self-organization /structure, cladistics/history and evolutionary genetics/function). Integrative Systemic Darwinism requires communal articulation from a plurality of perspectives. Although, it is more general than these, it draws on previous advances in Systems Theory, Systems Biology, and Hierarchical Theory. Systemic Darwinism would greatly further bioengineering research and would provide a significantly deeper and more critical understanding of biological reality. The concluding sentences of Darwin’s On the Origin of Species by Means of Natural

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Selection, or the Preservation of Favoured Races in the Struggle for Life (hereafter referred to as On the Origin of Species) sums this very effectively: “There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that whilst this planet has gone cycling on according to the fixed laws of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved”. Darwin’s ‘view of life’ as a conceptual space can be interpreted as consisting of three dimensions: explanatory pattern, levels of selection, and degree of difference among units of the same type. Each respective dimension is defined by a pair of poles: law and narrative explanations, organismic and hierarchial selection, and variational and essential thinking. Darwin’s integrative theory linking biological structure, history, and function was nursed and reared within this logical space. During the 20th century, the poles of each pair came to be seen as mutually exclusive. Systemic Darwinism, emerging at the beginning of the 20th century, promises to reintegrate Darwin’s view of life. Based on the theories of Darwin and the rapidly increasing documentation of fossil records vis a vis gene sequences, the evolution of algal lineages can provide useful indicators.

Evolution and paleontology of algae From tiny single-celled species one micrometer in diameter to giant seaweeds over 50m long, algae are abundant and ancient organisms which can be found in virtually every ecosystem in the biosphere. Algal taxonomists or phycologists, who specialize in their identification, naming and classification, believe that there exist 36,000–50,000 and possibly more than 10 million species of algae.9 Algae can best be defined as aquatic organisms (with exceptions) that are photosynthetic, oxygenic autotrophs, typically smaller (except kelps and few other genera) and less structurally complex than land plants. This rather inelegant definition allows the inclusion of cyanobacteria and other protists, which are otherwise distinguished by their prokaryotic structure. Fossil records show that algae were the first photosynthetic cellular organisms and all cytogenetic groups of plants and ultimately flowering plants may have arisen from algae. Chloroplasts are the right size to be descended from bacteria, reproduce in the same manner, by binary fission, and have their own genome in the form of a single circular DNA molecule. The enzymes and transport systems found on the folded inner membranes of chloroplasts are similar to those found on the cell membranes of modern cyanobacteria, as are their ribosomes. These similarities between cyanobacteria and chloroplasts suggest an evolutionary link between the two, and can be explained by the theory of endosymbiosis.

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The diversity of modern algal groups, and particularly of their chloroplasts, suggests that these endosymbiotic events were not unusual. Algal lineages: Linking the past with the future The oversimplified version of the story of algal evolution is tied to how they acquired their photosynthetic organelles or plastids. The first plants evolved from the engulfing of a photosynthetic prokaryote by an aerobic eukaryote. From this initial event two major plant groups evolved - the Green Algae and the Red Algae. After this primary endosymbiotic union, secondary and even tertiary endosymbioses occurred - algal cells themselves getting engulfed to give rise to other algal groups (Fig. 1). It is interesting to note that Chlamydia contain several plant-like genes which are attributed to horizontal transfer. On the other, Plasmodium and Trypanosoma represent apicomplexans, with residual or no plastids. The type of chlorophyll pigments together with the common algal carbon reserve materials, form a major basis for classifying algae. However, in recent times, cellular architecture, along with molecular sequences or genome architecture information has been utilized for classifying into several phyla (divisions). Modern algae comprise a range of organisms with very different structures but identical photosynthetic pigments. This suggests that very different host organisms have formed a symbiosis with the same photosynthetic cells. That is, the algal groups must have evolved through separate endosymbiotic events, i.e. polyphyletic, and the group as a whole is identified on the basis of a similar level of structure, rather than on its evolutionary origins.

Figure 1. Schematic outline of endosymbiotic events and evolution of diversity of life

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One of the several recent classification schemes divides algal species among eleven phyla, which includes Cyanophyta and Prochlorophyta, both containing oxygen–producing autotrophic prokaryotes and nine eukaryotic phyla.10 However, phylogenies based upon ribosomal RNA gene sequences suggest that the chlorophyll a and b are distributed among cyanobacterial taxa, rather than representing a coherent group, which suggests that, they should not be separated into different phyla.11 Based on monophyly (descent from a single ancestor) and molecular evidence, nine phyla have been distinguished – Cyanobacteria Glaucophyta, Euglenophyta, Cryptophyta, Haptophyta, Dinophyta, Ochrophyta, Rhodophyta and Chlorophyta. Modern ultrastructural and molecular studies have provided important information that has led to a reassessment of the evolution of algae.12 In addition, the dearth of fossil records for some groups of algae has hindered evolutionary studies, and the realization that some algae are more closely related to protozoa or fungi than they are to other algae came late, producing confusion in evolutionary thought and delays in understanding the evolution of the algae. Fossils of eukaryotes that resemble living brown algae have been found in sedimentary rocks from China that are 1700 million years old, while possibly the oldest photosynthetic eukaryote Grypania, and come from rocks 2100 million years old. Stromatolites, which are formed by cyanobacteria, provide living and fossil evidence of cyanobacteria going back 2700 million years. The order of algae with the best fossil record are the Dasycladales, which are calcified unicellular forms of elegant construction dating back at least to the Triassic Period. Some scientists consider the red algae, which bear little resemblance to any other group of organisms, to be very primitive eukaryotes that evolved from the prokaryotic blue-green algae.13 Evidence in support of this view includes the nearly identical photosynthetic pigments and the very similar starches among the red algae and the blue-green algae. Many scientists, however, attribute the similarity to an endosymbiotic origin of the red algal chloroplast from a blue-green algal symbiont. Other scientists suggest that the red algae evolved from the Cryptophyceae, with the loss of flagella, or from fungi by obtaining a chloroplast. In support of this view are similarities in mitosis and in cell wall plugs, special structures inserted into holes in the cell walls that interconnect cells. Some evidence suggests that such plugs regulate the intercellular movement of solutes. Ribosomal gene sequence data from studies in molecular biology suggest that the red algae arose along with animal, fungal, and green plant lineages.14 The fossil record for the algae is not nearly as complete as it is for land plants and animals. Red algal fossils are the oldest known algal fossils. Microscopic spherical algae (Eosphaera and Huroniospora) that resemble the living genus Porphyridium are known from the Gunflint Iron Formation of North America (formed about 1.9 billion years ago). Fossils that resemble modern tetraspores

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are known from the Amelia Dolomites of Australia (formed some 1.5 billion years ago). The best characterized fossils are the coralline red algae represented in fossil beds since the Precambrian time. Fossil Dinophyceae date from the Silurian Period (430 million years ago). Some workers consider at least a portion of the acritarchs, a group of cyst like fossils of unknown affinity, to be Dinophyceae, but few scientists agree with that view. The acritarchs occurred as early as 700 million years ago. The Chromophyta have the shortest fossil history among the major algal groups. Some scientists believe that the group is ancient, whereas others point out that there is a lack of data to support this view and suggest that the group evolved recently, as indicated by fossil and molecular data. The oldest chromophyte fossils, putative brown algae, are approximately 400 million years old. Coccolithophores, coccolith-bearing members of the Prymnesiophyceae, date from the Late Triassic Epoch (230 to 200 million years ago), with one reported from approximately 280 million years ago. Coccolithophores were extremely abundant during the Mesozoic Era, contributing to deep deposits such as those that constitute the white cliffs of southeast England. Most species became extinct at the end of the Cretaceous Period (65.5 million years ago), along with the dinosaurs, and indeed there are more extinct species of coccolithophores than there are living species. The Chrysophyceae, Bacillariophyceae, and Dictyochophyceae date from about 100 million years ago, and despite the mass extinctions 65.5 million years ago, many species still flourish. In Lompoc, California, US, their siliceous remains have formed deposits of diatomite almost 0.5 km (0.3 mile) in depth, while at Mývatn in Iceland, the lake bottom bears significant amounts of diatomite in the form of diatomaceous ooze, many meters in depth. Some of the green algal classes are also very old. Organic cysts resembling modern Micromonadophyceae cysts date from about 1.2 billion years ago. Tasmanites formed the Permian ‘white coal,’ or tasmanite, deposits of Tasmania and accumulated to a depth of several feet in deposits that extend for miles. Similar deposits in Alaska yield up to 150 gallons of oil per ton of sediment. Certain Ulvophyceae fossils that date from about one billion years ago are abundant in Paleozoic rocks. Some green algae deposit calcium carbonate on their cell walls, and these algae produced extensive limestone formations. The Charophyceae, as represented by the large stoneworts (order Charales), date from about 400 million years ago. The oospore, the fertilized female egg, has spirals on its surface that were imprinted by the spiraling protective cells that surrounded the oospore. Oospores from before about 225 million years ago had right-handed spirals, whereas those formed since that time has had left-handed spirals. The reason for the switch remains a mystery. The green algal classes are evolutionarily related, but their origins are unclear.2 Most consider the class Micromonadophyceae to be the most ancient group, and some fossil data support this view. The class

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Ulvophyceae is also ancient, whereas the classes Charophyceae and Chlorophyceae are more recent. The Euglenophyceae are believed to be an ancient lineage of algae that includes some zooflagellate protozoa, which is supported by ultrastructural and molecular data. Some scientists consider the colourless euglenophytes to be an older group and believe that the chloroplasts were incorporated by symbiogenesis more recently. The class Dinophyceae is of uncertain origin. During the 1960s and ’70s the unusual structure and chemical composition of the nuclear DNA of the Dinophyceae were interpreted as somewhat primitive features. Some scientists even considered the Dinophyceae to be mesokaryotes (intermediate between the prokaryotes and the eukaryotes); however, this view is no longer accepted. Their peculiar structure is considered as a result of evolutionary divergence, perhaps about 300 or 400 million years ago. The Dinophyceae may be distantly related to the chromophyte algae, but ribosomal gene sequence data suggest that their closest living relatives are the ciliated protozoa. It is likely that the Dinophyceae arose from nonphotosynthetic ancestors and that later some species of Dinophyceae adopted chloroplasts by symbiogenesis and thereby became capable of photosynthesis, although many of these organisms still retain the ability to ingest solid food, similar to protozoa. The origin of the chromophyte algae also remains unknown. Ultrastructural and molecular data suggest that they are in a protistan lineage that diverged from the protozoa and aquatic fungi about 300 to 400 million years ago. At that time, chloroplasts were incorporated, originally as endosymbionts, and since then the many chromophyte groups have been evolving. Fossil, ultrastructural, and ribosomal gene sequence data support this hypothesis. The Cryptophyceae are an evolutionary enigma. They have no fossil record, and phylogenetic data are conflicting. Although some workers align them near the red algae, because both groups possess phycobiliproteins in their chloroplasts, most scientists suggest that independent symbiotic origins for the red or blue colour of their chloroplasts could explain the similarity. Cryptophytes have flagellar hairs and other flagellar features that resemble those of the chromophyte algae; however, the mitochondrial structure and other ultrastructural features are distinct and argue against such a relationship. The Xanthophyceae may be even more recent, with fossils dating from about 20 million years ago, while fossil records of the remaining groups of algae, notably the Euglenophyceae and the Cryptophyceae, which lack mineralized walls, are negligible. Owing to their long history, cyanobacteria constitute an unusually diverse and heterogeneous group of prokaryotes, yet they form a coherent systematic group, which is distinct from other bacteria in many features. Cyanobacteria share a close evolutionary relationship with eukaryotes as they have the same photosynthetic

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pigments as the chloroplasts of algae and land plants. They comprise a remarkable group of organisms, whose ubiquity, antiquity and adaptability is unparalleled in biology. Their diverse morphologies coupled with independence for carbon, and nitrogen (in many forms), and resilience to extreme environmental conditions, underlies their widespread significance in the environment and in the evolution of life. These structurally simple organisms combine in themselves interesting facets of plant and bacterial metabolism, which is amenable to genetic exploitation, especially in enhancing our knowledge regarding phylogenetic and evolutionary interrelationships among prokaryotes and eukaryotes, and thereby evolution of life on this planet. Cyanobacteria are found everywhere - in marine, freshwater and terrestrial environments and as symbionts e.g. lichen - and contribute up to 50% of the atmosphere’s oxygen. To date, 17 cyanobacterial total genome-sequencing projects have been completed or are still in progress. From the 11 total cyanobacterial genome projects for which information is available, five were on marine cyanobacteria - and four of these were for extremely similar species - the Prochlorococcus, Synechococcus group of marine pico-phytoplankton. Such analyses have provided valuable information about the dynamics of microbial and plant genome evolution, biochemistry, physiology and ecology of photoautotrophic organisms. Cyanobacterial genomes reveal a complex evolutionary history, which cannot be represented by single strictly bifurcating tree for all genes, with all genes, belonging to any functional groups subject to transfer, without any specific bias. Interestingly, the proportion of metabolic gene transfers in interphylum transfers are more as compared to informational genes. Estimates have shown that about 4500 (18%) of all nuclear genes of extant land plants could derive from the cyanobacterial endosymbionts.7 The first complete genome sequences of marine cyanobacteria indicate unprecedented flexibility and dynamics and created a new level of insight into the ecology and molecular biology of these organisms.15 In this context, it may be interesting to re-visit the endosymbiotic theory in order to understand the evolution of plants from algae.

Endosymbiosis Chloroplasts, the sites of photosynthesis within plant cells, comprise a prominent and well-known class of plastids, sub cellular organelles with diverse, specialist functions in plant and algal cells.16 Mereschkowsky’s 1905 hypothesis which was initially greeted with skepticism or even derision, gained support from electron microscopical and biochemical studies which showed that plastids contain DNA, RNA and ribosomes, supplying a structural and biochemical basis for nonMendelian, cytoplasmic inheritance of plastid-related characters. Subsequent

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molecular genetic studies have demonstrated the ubiquity of plastid genomes and confirmed that their replication, transcription and translation closely resemble those of (eu) bacteria.17 The hypothesis that certain organelles of eukaryotic cells, in particular, the plant chloroplasts, evolved from bacteria is not that much younger than the Origin (Darwin 1895). It was proposed by several researchers in the late 19th century on the basis of microscopic study of plant cells that revealed conspicuous structural similarity between chloroplasts and cyanobacteria (then known as blue green algae) and was presented in a coherent form by Mereschkowsky in the beginning of 20th century. For the first two-thirds of the 20th century, this hypothesis of endosymbiosis remained a fringe speculation. However, this perception changed shortly after the appearance of the seminal 1967 publication of Sagan (Margulis) who summarized the available data on the similarity between certain organelles and bacteria, in particular, the striking discovery of organellar genomes, and came to the conclusion that not only chloroplasts but also the mitochondria evolved from endosymbiotic bacteria. Subsequent work, in particular, phylogenetic analysis of both genes contained in the mitochondrial genome and genes encoding proteins that function in the mitochondria and apparently were transferred form the mitochondrial to the nuclear genome, turned the endosymbiosis hypothesis into a well established fact. Moreover, these phylogenetic studies convincingly demonstrated the origin of mitochondria from a particular group of bacteria, the ά-proteobacteria. The major evolutionary role assigned to effectively unique events like endosymbiosis is, of course, incompatible with both gradualism and uniformitarianism.18,19 Chloroplasts are descendents of a cyanobacterial endosymbiont, but many chloroplast protein genes of endosymbiont origin are encoded by the nucleus.20 The chloroplast–cyanobacteria relationship is a typical target of orthogenomics, an analytical method that focuses on the relationship of orthologous genes. A pilot study of functional orthogenomics, combining bioinformatic and experimental analyses, was undertaken by Masayuki et al.21 to identify nuclear-encoded chloroplast proteins of endosymbiont origin (CPRENDOs). Phylogenetic profiling based on complete clustering of all proteins in 17 organisms, including eight cyanobacteria and two photosynthetic eukaryotes, was used to deduce 65 protein groups that are conserved in all oxygenic autotrophs analyzed but not in non-oxygenic organisms. With the exception of 28 well-characterized protein groups, 56 Arabidopsis proteins and 43 Synechocystis proteins in the 37 conserved homolog groups were analyzed. Green fluorescent protein (GFP) targeting experiments indicated that 54 Arabidopsis proteins were targeted to plastids. Expression of 39 Arabidopsis genes was promoted by light. Among the 40 disruptants of Synechocystis, 22 showed phenotypes related to photosynthesis. Arabidopsis mutants in 21 groups, including those reported previously, showed

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phenotypes. Characteristics of pulse amplitude modulation fluorescence were markedly different in corresponding mutants of Arabidopsis and Synechocystis in most cases, which led to the conclusion that physiological functions of orthologous genes may be different in chloroplasts and cyanobacteria. The gene sequences of the enzymes involved in the major steps of the carotenoid biosynthetic pathway in algae (Cyanobacteria, Rhodophyta and Chlorophyta) have been analyzed. Phylogenetic relationships among proteincoding DNA sequences were reconstructed by neighbor-joining (NJ) analysis for the respective carotenoid biosynthetic pathway genes (crt) in algae.22 The phylogenetic trees showed that the taxa of some genera have a closer evolutionary relationship with other genera in some gene sequences, which suggests a common ancient origin and that lateral gene transfer has occurred among unrelated genera. The phylogenetic analysis also revealed that lateral gene transfer may have taken place across algal genomes and the dN values suggest that most of the early crt genes are well conserved compared to the later crt genes.23 It is well established that the modern day chloroplasts were once free living cyanobacteria that became endosymbionts but molecular studies have yet to link plastids robustly with any particular group of contemporary cyanobacteria, leaving the precise lineage of cyanobacteria that gave rise to plastids unknown. Previous work has shown that many gene transfers to the nucleus have occurred during plastid evolution but estimates for the total number of genes that were transferred have been elusive. But the genomes of contemporary plastids encode only 5–10% as many genes as those of their free living cousins, indicating that many genes were either lost from plastids or transferred to the nucleus during the course of plant evolution.24,25 A comparison of 24,990 proteins encoded in the Arabidopsis genome to the proteins from three cyanobacterial genomes, 16 other prokaryotic reference genomes, and yeast was undertaken. Of 9,368 Arabidopsis proteins sufficiently conserved for primary sequence comparison, 866 detected homologues only among cyanobacteria and 834 other branched with cyanobacterial homologues in phylogenetic trees. Extrapolating from these conserved proteins to the whole genome, the data suggest that ≈ 4,500 of Arabidopsis protein-coding genes (= 18% of the total) were acquired from the cyanobacterial ancestor of plastids. These proteins encompassed all functional classes, and the majority of them are targeted to cell compartments other than the chloroplast. Phylogenetic analysis of proteins from Arabidopsis, three cyanobacterial genomes [Synechocystis sp. PCC6803, Prochlorococcus marinus, Nostoc punctiforme] other prokaryotic reference genomes and yeast, in addition to the phylogeny of 15 sequenced chloroplast genomes provided interesting information.24 Previous estimates have suggested that between 800 and perhaps as many as 2,000 genes in the Arabidopsis genome might come from cyanobacteria, but genome wide phylogenetic surveys that could provide direct estimates of this

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number are lacking.26,27 Analysis of 15 sequenced chloroplast genomes revealed 117 nuclear-encoded proteins are also still present in at least one chloroplast genome.24 A phylogeny of chloroplast genomes inferred from 41 proteins and 8,303 amino acid sites indicated that at least two independent secondary endosymbiotic events have occurred involving red algae and that amino acid composition bias in chloroplast proteins strongly affects plastid genome phylogeny. Therefore, it was concluded that the evolutionary process that transformed the cyanobacterial symbiont into a contemporary organelle involved both inheritance and invention. Such inheritances included photosynthesis, 70S ribosomes, cell division proteins, and, in some primitive plastids, a peptidoglycan wall. Important inventions included the protein import machinery, which permits the plastid to import nuclear-encoded proteins and hence to donate genes to the nucleus over evolutionary time. Contemporary chloroplast genomes encode between 60–200 proteins in various photosynthetic lineages and have thus undergone a process of severe genome reduction during the course of endosymbiosis, because contemporary cyanobacteria encode several thousand proteins. But plastids contain roughly just as many proteins as their free-living cyanobacterial cousins, current estimates suggesting that between 1,000 and 5,000 proteins in higher plants are targeted to plastids.

Evolutionary biology in the age of genomics The collection of completely sequenced genomes that is available on Darwin’s 200th anniversary consists of thousands of viral genomes, close to 1000 genomes of bacteria and Archaea, and close to 100 eukaryotic genomes.8,28,29 The importance of massive amounts of sequences for comparison is obvious because this material allows researchers to investigate mechanisms and specific events of evolution with the necessary statistical rigor and to reveal even subtle evolutionary trends. In addition, it is worth emphasizing that collections of diverse complete genomes are enormously useful beyond the sheer amount of sequence data. Although, certainly not all major taxa are adequately represented, this rapidly growing collection increasingly satisfies the demands of both micro evolutionary and macro evolutionary research. Complementary to the advances of traditional genomics is the more recent accumulation of extensive metagenomic data. Although metagenomics typically does not yield complete genomes, it provides invaluable information on the diversity of life in various environments and a means to understand life as it evolved. Horizontal gene transfer, the network of evolution and the forest replacing of the TOL (Tree of Life) Even long before the genomic era, microbiologists realized that bacteria had the capacity to exchange genetic information via horizontal gene transfer (HGT), in

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some cases, producing outcomes of major importance, such as antibiotic resistance (Raymond and Blankenship, 2003).30 Multiple molecular mechanisms of HGT have been elucidated including plasmid exchange, transduction (HGT mediated by bacteriophages) and transformation. These discoveries not withstanding, HGT was generally viewed as a minor phenomenon that is important only under special circumstances and, in any case, was not considered to jeopardize the concept of the TOL that could be reconstructed by phylogenetic analysis of rRNA and other conserved genes. This fundamental belief was challenged by early results of genome comparisons of bacteria and Archaea which indicated that, at least, in some prokaryotic genomes, a major fraction of genes were acquired via demonstrable HGT. The pathogenicity islands and similar symbiosis islands that comprise over 30% of the genome in many pathogenic and symbiotic bacteria are the prime case in point.31 Moreover, comparative analysis of the genomes of hyperthermophilic bacteria and Archaea suggested that even interdomain HGT can be extensive given shared habitats. The rate of HGT substantially differs from different genes depending on the gene functions, in part, according to the so called complexity hypothesis which posits that barriers might exist for HGT of genes encoding subunits of protein complexes because dosage imbalance and mixing of heterologous subunits resulting from such events could be deleterious. However, phylogenetic analyses indicate that even such genes, for instance, those for ribosomal proteins and RNA polymerase subunits, are not immune to HGT. The high prevalence of HGT in prokaryotes might, in part, explain the persistence of the organization of many operon hypotheses. This presents a notable case of a combination of selective (co regulation) and neutral (HGT) forces contributing to the evolution of a major aspect of genome organization. Eukaryotes are different from prokaryotes with respect to the role played by HGT in genome evolution. In multicellular eukaryotes, where germ line cells are distinct from the soma, HGT appears to be rare although not impossible. Under certain special circumstances, such as persistence of endosymbiotic bacteria in animals, transfer of large segments of bacterial genomes to the genome of the host is indeed common. Unicellular eukaryotes do seem to acquire bacterial genes between themselves on relatively frequent occasions. Far more crucial, however, is the major contribution of the genomes of the endosymbionts to the gene complements of all the eukaryotes.32 The discovery of mitochondria –like organelles and genes of apparent mitochondrial origin in all thoroughly characterized unicellular eukaryotes, essentially, ascertain that the last common ancestor of the extant eukaryotes already possessed the mitochondrial endosymbiont. In terms of their apparent phylogenetic affinities, eukaryotic genes that possess readily identifiable prokaryotic orthologs are sharply split into genes of likely archaeal origin (primarily, but not exclusively, components of information processing systems) and those of likely bacterial origin (mostly, metabolic enzymes and

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components of various cellular structures). It is often assumed on general grounds that the majority of ancestral ‘bacterial’ genes in eukaryotes are of mitochondrial origin but this is hard to demonstrate directly because in phylogenetic analysis, these genes cluster with diverse groups of bacteria. The symbiogenetic scenario according to which the α proteobacteria, ancestor of mitochondria invaded an archaeal host, and this event triggered eukaryogenesis including the formation of the signature structural features of the eukaryotic cells such as the endomembrane system, the cytoskeleton and the nucleus may be of relevance. Regardless of the exact role played by eukaryogenesis, there is no reasonable doubt that the gene complement of the eukaryotes is a chimera comprised of functionally distinct genes of archaeal and bacterial descents.28 Moreover, endosymbiosis apparently made substantial contributions to the gene complements of some of the individual major groups of eukaryotes. Thus, strong evidence was presented of massive HGT of thousands of genes from a cyanobacterial endosymbiont (the chloroplast) to the host (plant) genomes. Similarly, genes of apparent algal origin were detected in chromalveolates that engulfed a red alga in an act of secondary endosymbiosis.31 The observations of HGT as extensive, ubiquitous and occurring via multiple routes outline above lead to a fundamental generalization: the genomes of all life forms are collections of genes with diverse evolutionary histories. The corollary of this generalization is that the TOL concept must be substantially revised or abandoned because a single tree topology or even congruent topologies of trees for several highly conserved genes cannot possibly represent the history of all or even the majority of genes. Certainly, this conclusion is not to be taken as an indication that the concept of evolutionary tree introduced by Darwin4 should be abandoned altogether. First, trees have the potential to accurately represent the evolution of individual gene families. Secondly, there exist, beyond doubt, expansive part of life’s history for which congruent trees can be obtained for large sets of orthologous genes, and accordingly, the consensus topology of these tree qualifies as a species tree. Evolution of major groups of eukaryotes, such as animals or plants, is the most obvious case in point but tree like evolution seems to apply also to many groups of prokaryotes at relatively shallow phylogenetic depths. Evolution of life in its entirety is best depicted as: (i) A consensus tree of highly conserved genes that represents a ‘central trend’ in evolution, with HGT events, including massive ones associated with endosymbiosis, comprising horizontal connections between the tree branches (ii) A complex network where phases of tree-like evolution (with horizontal connections) are interspersed with ‘Big Bang’ phases of rampant horizontal exchange of genetic information that cannot be represented as trees in principle. In summary, comparative genomics and metagenomics reveal a vast,

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dynamic, interconnected world of selfish replicons that interact with genomes of cellular life forms and, over long spans of evolution, makes major contributions to the composition of chromosomes. In prokaryotes, the interaction between the bacterial and archaeal chromosomes and selfish replicons is so intensive, and the distinction between chromosomes and mega plasmids is blurred to such an extent that chromosomes are, probably, best viewed as ‘islands’ of relative stability in the turbulent ‘sea’ of mobile elements. In eukaryotes, especially, in multicellular forms that evolved the separation between the germ line and soma, the distinction between chromosomes and selfish replicons is sharper. Nevertheless, intragenomic mobility of selfish transposable elements is extensive, and intergenomic mobility, at least, within a species, is actually facilitated by sex, with bursts of transposable element propagation likely marking evolutionary transitions. The central role of mobile elements in genome evolution further undermines the TOL concept, although phylogenetic trees of individual hallmark genes can be highly informative for the reconstruction of the evolution of the selfish elements themselves. Modern genetic studies of plastid genomes have revealed that they encode only 60–200 proteins, while more than 5000 nuclear encoded gene products are targeted to plastids, and many chloroplast lacking genomes also reveal a great degree of similarity in their genome architecture.24,33 Therefore, the cyanobacteria have much greater implications in evolution, beyond plants. Conservation vis-a-vis fluidity of genomes A large body of information has been generated on the evolutionary conservation of gene sequences and structure versus the fluidity of gene composition and genome architecture. A fundamental observation supported by the entire body of evidence amassed by evolutionary genomics is that the sequences and structures of genes encoding proteins and structural RNAs are, generally, highly conserved through vast evolutionary spans. With the present collection of sequenced genomes, orthologs in distant taxa are found for the substantial majority of proteins encoded in each genome. For instance, recent genome sequencing of primitive animals, sea anemone and Trichoplax, revealed extensive conservation of the gene repertoire compared to mammals or birds, with the implication that the characteristic life span of an animal gene includes (at least) hundred millions of years. The results of extensive comparative analysis of plant, fungal and prokaryotic genomes are fully compatible with this conclusion.30 The striking conservation of gene sequences and structures contrasts the fluidity of the gene composition of genomes of all forms of life that is revealed by comparative genomics and evolutionary reconstruction. The (nearly) universal genes make up but a tiny fraction of the entire gene universe: altogether, this central core of cellular life consists of, at most, –70 genes that is, no more than 10% of the genes in even the smallest of the genomes of cellular life forms, but typically, closer to 1% of the genes or

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less. Although in each individual genome, the majority of the genes belong to a moderately conserved genetic ‘shell’ that is shared with distantly related organisms, within the entire gene universe, the core and shell genes (or more precisely, set of orthologous genes) are a small minority.34 Given this distinctive structure of the gene universe, evolutionary reconstruction inevitably yield a dynamic picture of genome evolution, with numerous genes lost and many others gained via HGT (mostly in prokaryotes), and gene duplication.35 Gene and genome duplication: The principal route of genomic innovation Gene duplications occurs throughout the evolution of any lineage but the rate of duplication is not uniform on large evolutionary scales, so that organizational transitions in evolution seem to be accompanied by bursts of gene duplication, conceivably, enabled by weak purifying selection during population bottlenecks. Perhaps, the most illustrative case in point is the emergence of eukaryotes that was accompanied by a wave of massive duplication, yielding the characteristic many-to-one co-orthologous relationship between eukaryotic genes and their prokaryotic ancestors. At the level of general concepts of evolutionary biology primarily concerned here, genomic studies on gene duplication lead to, at least, two substantial generalizations. First, the demonstration of the primary evolutionary significance of duplications including duplications of large genomes regions and whole genomes is a virtual death knell for Darwinian gradualism: even single gene duplication hardly qualifies as an infinitesimally small variation where WGD qualifies as a bonafide salutatory event. Secondly, the primacy of gene duplication with the subsequent (sometimes, rapid) diversification of the paralogs as the route of novel gene origin reinforces the metaphor of evolution as tinkerer: evolution clearly tends to generate new functional devices by tinkering with the old ones after making a backup copy rather than create novelty from scratch. Genome-wide quantification of selection and junk DNA: Distinct evolutionary regimes for different genomes There are major differences in the genome layouts between different lines of life evolution. Prokaryotes and, especially, viruses have ‘wall-to-wall’ genomes that consist, mainly, of genes encoding proteins and structural RNAs, with non coding regions comprising, with a few exceptions, no more than 10–15% of the genomic DNA. The genomes of unicellular eukaryotes have lower characteristic gene densities but, on the whole, do not depart too far from the prokaryotic principles, with most of the DNA dedicated to protein-coding, despite the distinct, exon-intron gene architecture. The genomes of multicellular eukaryotes are drastically different in that only a minority (a small minority in vertebrates) of the genomic DNA is comprised of sequences encoding proteins or structural

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RNAs. Generally, across the entire range of life forms, there is a notable negative exponential dependence between the density of protein-coding genes and genome size although significant deviations from this overall dependence are seen as well, particularly, in prokaryotes. Evolutionary genomics effectively demolished the straightforward concept of the TOL by revealing the dynamic, reticulated character of evolution where HGT, genome fusion, and interaction between genomes of cellular life forms and diverse selfish genetic elements take the central stage. In this dynamic worldview, each genome is a palimpsest, a diverse collection of genes with different evolutionary fates and widely varying likelihoods of being lost, transferred, or duplicated. So the TOL becomes a network, or perhaps, most appropriately, the Forest of Life that consists of trees, bushes, thickets of lianas, and of course, numerous dead trunks and branches. Whether the TOL can be salvaged as central trend in the evolution of multiple conserved genes or this concept should be squarely abandoned for the Forest of Life image remains as an open question. All the classical concepts have undergone transformation, turning into much more complex, pluralistic characterizations of the evolutionary process. Depicting the change in the widest strokes possible, Darwin’s paramount insight on the interplay between chance and order (introduced by natural selection) survived, even if in a new, much more complex and nuanced form, with specific contributions of different types of random processes and distinct types of selection. By contrast, the insistence on adaptation being the primary mode of evolution that is apparent in the Origin, but especially in the Modern Synthesis, became deeply suspicious if not outright obsolete; making room for a new worldview that gives much more prominence to non-adaptive process. The variability of the genome architectures presents an interesting dilemma to evolutionary biologists: do organisms possess unique genome architectures that are specifically adapted to satisfy unique functional demands of the respective organisms, or is evolution of genome architecture a mostly neutral process? Although local clustering of functionally related genes and other patterns suggestive of functionally relevant gene co-expression were repeatedly observed, these trends are relatively weak and by no means ubiquitous. Thus, the dominant factor in the evolution of genome architecture appears to be random, non-adaptive rearrangement rather than purifying or positive selection.

Conclusions The fundamental principles of molecular evolution have been established, and many specific observations of major importance and impact on the fundamentals of neo-Darwinism were made in the pre-genomic era, the rRNA based phylogeny being the premier case in point. However, the advent of full fledged genome

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sequencing quantitatively changed the entire enterprise of evolutionary biology and the availability of the treasure trove of genomic, metagenomic and post –genomic data has much altered our evolutionary concepts, especially in relation to Darwinism; and in some cases served to strengthen the significance of his views. The species-individual has emerged, rather than the organism, as a Darwinian interactor in selection at its own level, operating largely with cross-level exaptations arising from unsuppressed evolution of subparts (primarily organisms) at lower levels within itself. Such nonsuppression acts as a source of power by permitting species to draw upon a wider pool of features than organisms can access (for the organismic style of individuality enjoins active suppression of most selection at lower levels within itself). This can have wide ranging implications in understanding the evolution of land plants from algal ancestors. Beyond genomics and metagenomics, one of the hallmarks of the first decade of the new millennium is the progress of research in functional genomics and systems biology. These fields now yield high quality, genome-wide data on gene expression, genetic and protein-protein interactions, protein localization within cells, and more, opening new dimensions of evolutionary analysis, what is sometimes called Evolutionary Systems Biology. This new field of research has the potential to insights into the genome-wide connections between sequence evolution and other variables, such as the rate of expression, and often illuminate the selective and neutral components of the evolution of these aspects of genome functioning and the next decade may prove the most exciting in terms of evolutionary biology. The majority of the sequences in all genomes evolves under the pressure of purifying selection or, in organisms with the largest genomes, neutrally, with only a small fraction of mutations actually being beneficially and fixed by natural selection as envisioned by Darwin. Furthermore, the relative contributions of different evolutionary forces greatly vary between organismal lineages, primarily, owing to differences in population structure. Two centuries after Darwin’s birth, 150 years after the publication of his ‘Origin of Species’ and 50 years after the consolidation of the Modern Synthesis, comparative analysis of hundreds of genomes from many diverse taxa offers unprecedented opportunities for testing the conjecture of (neo) Darwinism and deciphering the mechanisms of evolution. Comparative genomics revealed a striking diversity of evolutionary processes that was unimaginable in the pre-genomic era. In addition to point mutations that can be equated with Darwin’s ‘infinitesimal changes’, genome evolution involves major contributions from gene and whole genome duplications, large deletions including loss of genes and entire genomic regions, various types of genome rearrangements, and interaction between genomes of cellular life forms and diverse selfish genetic elements. The emerging landscape of genome evolution includes the classic, Darwinian natural

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selection as an important component but is by far more pluralistic and complex than entailed by Darwin’s straightforward vision that was solidified in the Modern Synthesis. The key role played by algae, particularly cyanobacteria has been beyond doubt established in the mosaic of plant genomes. As Raven and Allen16 pointed out ‘What have the cyanobacteria not done for plants’ which implies subtly the significance of algae in the evolution and sustenance of life on Earth. Acknowledgements: The authors thank the ICAR funded projects and authorities of the Division of Microbiology, IARI, New Delhi, for providing necessary facilities for undertaking this study.

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Prasanna R, Kaushik BD (2005) Algal diversity from morphology to molecules. In: Microbial Diversity: Current Perspectives and Potential Applications. Satyanarayana T, Johri BN (Eds.), IK International, New Delhi

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Raven JA, Douglas AE (2003) Genomes at the interface between bacteria and organelles. Proc R Soc Lond B Biol Sci 358:5–18 Mereschkowsky C (1905) Uber Natur und Ursprung der Chromatophoren im Pflanzenreiche. Biologia Centralbl 25:593–604 Woese CR, Fox GE (1977) Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc Nat Acad Sci USA 74:5088–5090 Woese CR (1987) Bacterial evolution. Microbiol Rev 51:221–271 Gray MW (1992) The endosymbiont hypothesis revisited. Int Rev Cytol 141:233–357 Masayuki I, Makoto F, Kintake S, Naoki S (2009) Orthogenomics of photosynthetic organisms: Bioinformatic and experimental analysis of chloroplast proteins of endosymbiont origin in arabidopsis and their counterparts in synechocystis. Plant Cell Physiol 50(4): 773–788 Liang C, Zhao F, Wei W, Wen Z, Qin S (2006) Carotenoid biosynthesis in cyanobacteria: Structural and evolutionary scenarios based on comparative genomics. Int J Biol Sci 2(4):197–207 Ochman H, Lawrence JG, Groisman EA (2000) Lateral gene transfer and the nature of bacterial innovation. Nature 405:299–304 Martin W, Rujan T, Richly E, Hansen A, Cornelsen S, Lins T, Leister D, Stoebe B, Hasegawa M , Penny D (2002) Evolutionary analysis of Arabidopsis, cyanobacterial and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc Nat Acad Sci USA 99:12246–12251 Mulkidjanian AY, Koonin EV, Makarova KS, Mekhedov SL, Sorokin A, Wolf YI, Dufresne A, Partensky F, Burd , Kaznadzey D, Haselkorn R, Galperin Y (2006) The cyanobacterial genome core and the origin of photosynthesis. Proc Nat Acad Sci USA 103:13126–13131 Lopez-Juez E (2007) Plastid biogenesis, between light and shadows. J Exper Bot 58: 11–26 Zhang Y, Chen M, Zhou BB, Jerminn LS, Larkum WD (2007) Evolution of the inner light-harvesting antenna protein family of cyanobacteria, algae, and plants. J Mol Evol 64:321–331 Dagan T, Martin W (2006) Getting a picture of microbial evolution en route to a network of genomes. Philosophical Transactions of the Royal Society (London). B Biological Sciences 364:2187–2196 Koonin EV (2009) Darwinian evolution in light of genomics. Nucleic Acids Res 37: 1011–1034 Raymond J, Blankenship RE (2003) Horizontal gene transfer in eukaryotic algal evolution. Proc Nat Acad Sci USA 100:7419–7420 Gogarten JP, Townsend JP (2005) Horizontal gene transfer, genome innovation and evolution. Nat Rev Microbiol 3:679–687 Palenik B (2002) The genomics of symbiosis: Hosts keep the baby and the bath water. Proc Nat Acad Sci USA 99:11996–11997 Raven JA, Allen JF (2003) Genomics and chloroplast evolution: What did cyanobacteria do for plants? Genome Biol 4:209 Zhaxybayeva O, Gogarten JP, Charlebois RL, Doolittle WF, Papke T (2006) Phylogenetic analyses of cyanobacterial genomes: Quantification of horizontal gene transfer events. Genome Res 16:1099–1108 Lynch M (2007) In: The Origin of Genome Architecture. Sinauer Associates, Sunderland, MA

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10 Biodiversity, phylogeny and evolution of fungi C. Manoharachary*, I.K. Kunwar** and S. Vishnuvardhan Reddy*** Botany Department, Osmania University Hyderabad - 500 007, India *[email protected], **[email protected], ***[email protected]

Abstract: India is rich in biodiversity. One-third of fungal diversity of the globe exists in India. The early fossil record of the fungi is poor, as unlike other organisms, fungal structures do not fossilize well. Thus, theories on phylogeny of fungi are based on the morphological features of the extant fungi. From the beginning of the 20th century fungi were proposed to be monophyletic, assuming that all the fungi were derived from an algal ancestor that lost its ability to photosynthesize. This gave rise to the flagellate fungi, from which rest of the fungi evolved. The loss of flagella and the evolution of zygospore gave rise to the Zygomycotina. The uninucleate zygospores of the Edogonales gave rise to the Ascomycota. The link between them being a fungus resembling Dipodascopsis (Ascomycota). The unicellular yeasts and complex filamentous Ascomycota members having extended dikaryotic stage evolved from it, believed to be similar to modern day Taphrina. This Taphrina-like ancestor was believed to have given rise to the ancestral Basidiomycota. Monophyletic origin of fungi was followed by most of the mycologists till 1960s. But, some mycologists proposed polyphyletic origin, with red algae as origin of Ascomycota. In late 1960s Oomycota was separated from the fungi. Slime molds were also separated into a different kingdom as well. Fungi were separated into two kingdoms: All the flagellate fungi were placed in kingdom Protista (including Chytridiomycota and slime molds) and the remainder in kingdom Myceteae. Hypothesis of phylogeny of fungi has changed radically with the advent of molecular techniques, ultrastructural and biochemical studies. On the basis of these studies in the late 1980s chytridiomycetes, zygomycetes, ascomycetes and basidiomycetes were included in kingdom Fungi. Oomycota, hyphochytrids, labyrinthulids, thraustochytrids and slime molds were accommodated in pseudo fungi. In 2007 a new classification of kingdom fungi-based on recent molecular phylogenetic analysis and morphotaxonomy was proposed, having one subkingdom – Dikarya and seven phyla. Recently in the 10th edition of Dictionary of the Fungi (2008) three kingdoms are accepted viz. Chromista, Fungi and Protozoa. True fungi belong to kingdom fungi having six phyla – Ascomycota, Basidiomycota, Chytridiomycota, Glomeromycota, Microsporidia and Zygomycota. The review contributes to the understanding of phylogenetic hypotheses, evolutionary relationships and circumscription of the fungi. Keywords: Origin, Eukaryotes, Monophyletic, Polyphyletic, Molecular characters V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Introduction Fungi are highly specialized, non-chlorophyllous, heterotrophic, eukaryotic organisms with cell wall made up of chitin and β-glucans, nutrition absorptive, the absence of chlorophyll has enforced them to become saprophytes, parasites and symbionts. Thallus holocarpic or eucarpic, reproduce both asexually and sexually by producing spores characteristic of each group. The sexually produced fruit bodies are formed both in Ascomycotina and Basidiomycotina. Macroscopic fruit bodies are formed in mushrooms, puff balls, stink horns, bracket fungi, etc. Some fungi like mycorrhiza enter into symbiotic relationship with plants. Fungi are ubiquitous and widely distributed in nature. They are found growing on a variety of substrates having some organic matter and moisture. They are widely distributed in water, soil, on decaying plant and animal parts, etc. Fungi colonize, grow, multiply and survive on/in diversified habitats including extreme environments. Fungi are not only beautiful, but play a significant role in the daily life of human beings besides their utilization in industry, agriculture, medicine, food industry, textiles, bioremediation, natural cycling as biofertilizers and in many other ways. Fungal biotechnology has become an integral part of the human welfare.1 Biodiversity, the extent of biological variation on earth, has come to the fore as a key issue in science and politics since 1990s.2 This subject gained momentum in the 21st century in the light of global gene resources and environmental problems such as global climate change caused by the increase of CO2, loss of biodiversity, loss of tropical rain forests, acid rain, desertification, ozone depletion caused by freon gas and chemical pollution.3

Diversity spectrum The variety and galaxy of fungi and their natural beauty occupy prime place in the biological world and India has been the cradle for such fungi. Only a fraction of total fungal wealth has been subjected to scientific scrutiny and mycologists have to unravel the unexplored and hidden wealth. A conservative estimate provides 1.5 million fungal taxa in the world,2 and among these less than a tenth of the taxa in the kingdom fungi have been discovered.4 Recently Kirk et al.5 in the 10th edition of The Dictionary of Fungi gave a figure of 97,861 species which have been inventoried and described. One-third of fungal diversity of the globe exists in India. The number of fungi recorded in India is around 28,000 species, the largest biotic community after insects.1,6–8 About 350 new genera have been described from India, of which ≈32% were discovered by C.V. Subramanian of the University of Madras.5

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Fossil history and evolution of fungi The fossil record of fungi dates back to the early Proterozoic Eon some 1,200 million years ago. The existence of fossil fungi indicates their evolutionary significance besides solving certain phylogenetic complexities. The early fossil record of the fungi is meager. For much of the Paleozoic Era, the fungi appear to have been aquatic, and consisted of organisms similar to the extant chytrids in having flagellum-bearing spores. The fungi probably colonized the land during the Ordovician. Fungal fossils were common in the early Devonian, when they were abundant in the Rhynie chert. All modern classes of fungi were present in the late Carboniferous (Pennsylvanian Epoch). For some time after the PermianTriassic extinction event, a fungal spike, originally thought to be an extraordinary abundance of fungal spores in sediments formed shortly after this event, suggesting that they were the dominant life form at this time – in nearly 100% of the fossil records available from this period. However, the relative proportion of fungal spores relative to spores formed by algal species is difficult to assess, the spike did not appear worldwide, and in many places it did not fall on the PermianTriassic boundary. Charles Darwin (1809–1882) was an English naturalist whose theory of evolution through natural selection is one of the greatest contributions ever made to science in his book The Origin of Species.9 It forms the basis of modern evolutionary theory. In another book The Descent of Man10 he applied his theory to the evolution of man from a primitive monkey-like animal. Both the books aroused worldwide controversy but, scientists still accept his basic idea. The fungi are of great importance agronomically, bioindustrially, medically and biologically. In spite of such significance attached, our knowledge is poor about phylogeny and evolution within the fungi and between fungi and other organisms, as well as the taxonomic inventory of fungal species diversity. Bruns et al.11 opined their simple and frequently convergent morphology, their lack of a useful fossil record, and their diversity have been major impediments to progress in this field. In the past, phylogenetic speculation of the fungi was based mainly on comparative analyzes of morphological, ontogenetical and biochemical data.12–21 Kimura’s neutral theory of molecular evolution impacted studies of the phylogeny and evolution.22 The development of molecular biological techniques (particularly gene cloning, nucleic acid sequencing and polymerase chain reaction), proliferation of high performance computers, and improvement of molecular evolutionary analysis programs have extended studies on relatedness, phylogeny and evolution of organisms, including fungi, at the molecular level.23–25 Analyzes using molecular phylogenetics support a monophyletic origin of the fungi. The taxonomy of the fungi is in a state of constant flux, especially due to recent research based on DNA comparisons. These current phylogenetic analyzes

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often overturn classifications based on older and sometimes less discriminative methods based on morphological features and biological species concepts obtained from experimental matings.

Phylogenetic hypotheses, evolutionary relationships and circumscription of the fungi The monophyletic origin of “fungi” is the dogma that made mycologists to explore the fungi and their relationships.20,26–28 In this theory, it is thought that all the fungi were derived from an algal ancestor that lost its ability to photosynthesize and gave rise to the flagellate fungi. This ancestor was thought to be a member of the Chytridiomycota, which was morphologically similar to their modern counterparts and produced posteriorly, uniflagellated zoospores. Like many of its modern counterparts, this ancestral chytrid was aquatic, but over time, became more adapted to the terrestrial environment. The loss of flagella and the evolution of the zygospore gave rise to the Zygomycota whose members were believed to be morphologically similar to Mucor or Rhizopus, which produce large, single, multispored, columellate sporangia. As this group evolved, the number of sporangiospores in a sporangium became reduced and the columella was lost, thereby the sporangiole was evolved, with the most advance members producing single-spored sporangioles, e.g. Cunninghamella. This led to the evolution of the conidium, an asexual spore typically produced by the Ascomycota and Basidiomycota where an anamorph stage is produced. Another line that arose within the Zygomycota was that which included the Endogonales. The zygospores produced in this line were uninucleate. This line was believed to have given rise to the Ascomycota. The link between the Zygomycota and Ascomycota was made with a fungus that resembled Dipodascopsis (Ascomycota). The Taphrina-like ancestor gave rise to other members of Ascomycota and ancestral Basidiomycota, which diverged into two lines. One line produced microcyclic rusts, which produced only teliospores and basidiospores and led to the present-day rusts. The other line produced the ancestor of the present day Auriculariales, which had a poorly differentiated basidiocarp. This line gave rise to the remaining basidial and basidiocarp types in the Basidiomycota. Bessey29 believed that the Zygomycota were derived from the Oomycota rather than Chytridiomycota, based on similarities in morphology of their sporangia and that both phyla produced coenocytic mycelium. Bessey,29 Sachs,30 Denison and Carroll31 believed that the Ascomycota were derived from the Floridean red algae (Rhodophyta). Although Kohlmeyer,32 Kohlmeyer and Kohlmeyer33 did not advocate the direct derivation of the Ascomycota from the Rhodophyta, they postulated a hypothetical, common ancestor that gave rise to both phyla.

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Demoulin15 had a similar hypothesis, but believed that both the Ascomycota and Basdiomycota were derived from a red algal ancestor. During this old era, characteristics not previously incorporated into determining fungal phylogeny and new tools that became available brought forth new theories on the interrelationship of the different fungal taxa. Some new criteria that were considered in fungal phylogeny included: ● Cell biological data on mitochondrial cristae, organelle distribution, flagellation, mitosis. ● Biochemical data on cell wall constituents, amino acid synthesis and enzymology. This indicated that the Chytridiomycota should be classified in the kingdom Myceteae, with the fungi; the Oomycota should be in the kingdom Stramenopila with the Chrysophyta and Phaeophyta as proposed by Pringsheim34 and Kreisel.35 Although their theory did turned out to agree with this modern interpretation in phylogeny, they based their classification of the Oomycota only on superficial morphological similarities. The Hyphochytriomycota were also first classified in this group at this time. The different phyla of slime molds were also shown to be more distantly related than once believed, and also, it is noteworthy that the Rhodophyta is unrelated to the Ascomycota. Although phylogeny in the higher taxa of fungi seem to be more resolved, the lower taxa now seem to be more confused. DNA sequence analysis does not support the Dipodascopsis-like hypothetical ancestor that was once thought to link the Zycomycota to the Ascomycota. Thus, we no longer have a link between the two phyla. Another Ascomycota dogma that has been demonstrated to be incorrect is the monophyletic origin of the ascosporogenous yeasts. Members of Saccharomycetales no longer appear to be monophyletic and are more distantly related to other ascosporogenous yeasts. Phylogeny within the Basidiomycota has also become more confused. The relatedness of taxa within this phylum, based on the morphology of the basidium, which was the main character used to classify fungi in this phylum, is not in agreement with DNA sequence analysis. Instead, it appears that the septal pore apparatus is a better indicator of relationship between members of the Basidiomycota. Thus, with the exception of the Tremellales, all of the various basidial types, that are formed in basidiocarps now appear to be more closely related than once believed, i.e. Agaricales, Gasteromycetes, Auriculariales, Dacrymycetales, etc. are now believed to form a monophyletic group with the Tremellales as a sister group. The Ustilaginomycetes and Uredinomycetes are believed to be more distantly related.

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Recent aspects Among earlier phylogenetic speculations concerning fungi and related organisms that have been made during the past 20 years, Cavalier-Smith’s 36 theory is noteworthy. He provided a framework for a taxonomic system and phylogeny for the fungal kingdom that was based mainly on cell wall chemistry, biosynthetic pathways for lysine, type of motile cells, cellular ultrastructure and 5S rRNA sequence divergence. He included only the chytridiomycetes, zygomycetes, ascomycetes and basidiomycetes in the kingdom fungi. These four fungal groups are characterized by chitinous cell walls12 and the aminoadipic acid (AAA) lysine biosynthetic pathway.37 The oomycetes, hyphochytrids, labyrinthulids, thraustochytrids, and slime molds, which are cellulosic 12 and have the (diaminopimelic acid; DAP) lysine biosynthetic pathway,37 were excluded from the fungi. These first four major groups were accommodated by Cavalier-Smith36 in the pseudo fungi and the slime molds in the kingdom Protozoa.38 He also assumed that fungi and animals had a common ancestor, a choanociliate (choanoflagellte) protozoan. He concluded that the kingdom fungi is monophyletic and also that all major eufungal taxa in the Endomycota, Ascomycota and Basidiomycota, evolved from the Entomophthorales from a chytridiomycete ancestor by loss of cilia (flagella). Evidence from 18S rDNA sequence divergence put an end to a debate on the circumscription of the kingdom Fungi, i.e. whether oomycetes, hyphochytrids and chytrids were the “true” fungi, and confirmed the extent of “true” fungi.11,39 The “true” fungi are chytridiomycetes, zygomycetes, ascomycetes and basidiomycetes. The AAA lysine pathway and the presence of chitin in the cell walls strongly support the 18S rDNA sequence-based phylogeny. In general, the “true” fungi are hyphal, have cell walls through most or all of their life cycle and are exclusively absorptive in their nutrition. These “true” fungi form a monophyletic group, though below the ~95 level-based on bootstrap analysis, and are distinguished phenotypically from cellular slime molds (Acrasiomycetes), plasmodial slime molds (Myxomycetes) and the oomycetes (Oomycota).39 In this phylogenetic tree, the two groups of slime molds diverged separately, prior to the terminal radiation of eukaryotes. This is consistent with many differences between slime molds and fungi in form, function and life cycle. The oomycetes (Achlya, Lagenidium and Phytophthora), hyphochytrids (Hyphochytridium) and labyrinthulids (Thraustochytrium and Ulkenia) form a cluster with brown algae and diatoms. These organisms have heterokont flagella, contain chlorophylls a and c, and are classified within the Chromista sensu CavalierSmith,38 or the Stramenopila40 segregated from the Chromista. The labyrinthulids appear to be basal to other heterokont algae and oomycetes and hyphochytrids within the Chromista.41,42 Maximum likelihood phylogentic analysis of the 18S

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rDNA suggests that Plasmodiophora brassicae, a severe pathogen of crucifers, may be more closely related to the alveolates than to any of the fungi.43 Evolutionary relationships between the three kingdoms of complex eukaryotes, fungi, plants and animals, are still controversial, because of lack of solid fossil evidence. However, recent 18S rDNA phylogenies and amino acid sequence of elongation factor show that the closest relatives to fungi are animals, not plants.44–46 Nikoh et al.47 concluded that the kingdom Animalia is closely related to the kingdom Fungi and is distantly related to the kingdom Plantae. In contrast, on the basis of the ribosomal protein peptide and nucleotide sequences, Plantae and Animalia are sister clades and Fungi form a more distinct clade to them.48 More data on informative molecular characters are needed to solve these evolutionary problems. It is believed that the chytrids are the most primitive fungi within the “true” fungi, because they are zoosporic and gave rise to the Zygomycota and other higher fungi. Molecular phylogenetic studies of the chytridiomycetes and zygomycetes are limited because their economic interest is relatively low, pure cultures have not been achieved for many species, and even detection of some species is very difficult. However, such studies are indispensable to fill the gaps in knowledge of fungal evolution; rRNA sequence comparisons answered the debate as to whether flagellated fungi were “true” fungi. Early phylogenetic analysis of 5S rRNA sequences by Walker49 indicated that Basidiobolus was closely related to the chytrids. The Harpellales, an order of the Trichomycetes, was proposed to have a close relationship with the Kickxellales (Zygomycota) based on similarities in septal pore ultrastructure, cell wall structure, asexual reproductive apparatus, and serological affinity,50 but this interpretation conflicted with Walker’s49 5S rRNA sequence comparison. Gunderson et al.51 revealed that the 18S rRNA sequence-based phylogeny supported exclusion of the oomycetes. The sequence analysis of 18S rRNA genes suggested that the chytrid Blastocladiella emersonii and the higher fungi shared a common ancestor,52 and that the rumen anaerobic fungus Neocallimastix spp. should be assigned to the chytridiomycetes.53 Numerous hypotheses on phylogeny and evolution of the higher fungi, i.e. the ascomycetes, basidiomycetes and their anamorphs, have been proposed.20,23,54,55 Among these, Savile’s20 phylogenetic considerations of higher fungi have attracted the attention of a lot of mycologists. He suggested that Taphrina was the closest survivor of the common ancestor of the higher fungi. He suspected that two major lineages evolved from “Prototaphrina” a common ancestor. One major lineage led to the present day Taphrina and the ascomycetes, whereas other major lines led to the basidiomycetes (the Uredinales line and the parasitic Auriculariaceae line) through a “Protobasidiomycete.” In this phylogenetic scheme Taphrina was considered to be the most important fungus.

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Phylogenetic trees inferred from 18S rDNA sequence divergence indicate the existence of the two divisions, Ascomycota and Basidiomycota, among the higher fungi.38,56-59 Blackwell et al.60 presented phylogenetic tree diagram of members of kingdom fungi, based on the phylogenetic studies of a number of mycologists with the goal of producing a stable higher-level phylogenetic classification of fungi. Mushroom fossils were reported by Hibbett et al.61,62 the impact from these fungal fossil records is great to fungal phylogenetics and evolution but the complete connections are rare. On the other hand, DNA is the macromolecule that harbors evolutionary information as well as genetic information.63 Evolution, at the molecular level, is observable as nucleotide (or base) changes in the DNA and amino acid changes in proteins.64 The 18S rRNA gene divergence has been an especially useful molecular character to elucidate the identity of fungal taxa and their evolutionary relationships. If there are conflicts between the molecular and morphological characters, both molecular and morphological data should be re-examined, but often the morphological data have been misinterpreted. The anamorphic yeast Saitoella complicata and the phytopathogen Mixia osmundae provide examples. Recent 18S rDNA sequence analyzes indicate the existence of two divisions within the higher fungi, the sister groups Ascomycota and Basidiomycota. This relationship suggests that the ascus and the basidium, as meiosporangium are phylogenetically meaningful. The molecular phylogenies do not support the existence of the deuteromycetes as a distinct higher taxon. Instead they integrate both anamorphs and teleomorphs into a molecular phylogenetic tree. The genome of Saccharomyces cerevisiae has been sequenced (see Saccharomyces Genome Database, http:www.stanford.edu/Saccharomyces/). Fungal sequence of mitochondrial (chytrids, zygomycetes, dikaryomycetes, Oomycota)65 and nuclear genomes (Saccharomyces, Candida, Aspergillus, Magnaporthe, Neurospora) (see http://fungus.genetics.uga.edu:5080/main.html) provide new nucleic acid targets for phylogenetics and population genetics.66

Evolution of fungi within the groups Bessey,13,29 Cain14 and Savile20 summarized their own ideas, as well as those of others, about fungal evolution. Since then, enhanced evolutionary studies brought a number of systematists to study the diverse array of organisms grouped as protists. The resulting consideration of all of the protists led to more inclusive comparisons necessary to define monophyletic lineages. One notable example of this wider view resulted in the exclusion of Oomycota from fungi and the inclusion of this group in Stramenopila. The phylogenetic tree is based largely on DNA sequence analysis and ultra structural features.11,39,46,67 Several points of the hypothesis are important to emphasize that: (1) The acrasid slime molds (Acrasiomycota) diverged early in

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the eukaryotic lineage along with certain ameba. These groups are characterized in part by their discoidal mitochondrial cristae. (2) Plasmodial slime molds (Myxomycota) and dictyostelid slime molds (Dictyosteliomycota) are additional taxa with amoeboid stages that diverged independently of each other at a later time. Both groups have tubular mitochondrial cristae. (3) Another lineage of interest, Stramenopila, is viewed as a kingdom of morphologically diverse organisms, usually having a heterokont flagellar condition. Oomycota, Hyphochytriomycota and Labyrinthulomycota, as well as several groups of algae with chlorophylls a and c are in a monophyletic lineage. (4) The red algae diverged in a lineage distinct from any group ever considered to be fungi. This is important as evolution of Ascomycota from a red algal ancestor has been proposed on several occasions. (5) Fungi (Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota) are restricted to a monophyletic lineage with a close relationship to animals through a choanoflagellate-like ancestor. (6) Little information is available for Plasmodiophoromycota; however the group may share some features of the infection apparatus and basal body structure with certain ciliate protozoans. (7) The recognition of the polyphyly of organisms that previously have been grouped in the kingdom protista. The use of phylogenetic analysis methods in conjugation with DNA sequences is undoubtedly important for mycology. For example, Oomycota and Chytridiomycota had already been distinguished on the basis of the flagellation type, biosynthetic pathways, wall chemistry and the genetic code. However, the inclusion of a wide range of taxa in phylogenetic analyzes has certainly improved the hypothesis by showing the degree of divergence between these groups. Another lineage of interest the monophyletic fungi (Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota) is supported as monophyletic by sequence analysis.11,39,52 Many of the same morphological and biochemical characters that were used to separate fungi from Oomycota are shared among all the fungal phyla. The hypotheses of relationships within fungi are less well supported. There is general agreement of the sister group relationship of Ascomycota and Basidiomycota, however, the position of some taxa remains problematical.11,39 Few species of Chytridiomycota and Zygomycota have been included in sequencing studies to allow generalizations on the group as a whole, and few morphological or biochemical characters are known to support each as a monophyletic group. Chytridiomycota has been distinguished from the other fungi by the presence of a single posterior flagellum. However, when this character is considered in the context of studies employing phylogenetic analysis, the flagellate condition is recognized as a primitive character and cannot be used to support a monophyletic lineage of this group. In addition, another character, the zygospore used to distinguish Zygomycota, may not be unique to the group but may be shared with some Chytridiomycota.

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Oomycota diverged within the monophyletic group of Stramenopila, an idea suggested much earlier34 because of similarities in sexual reproduction but with little other evidence. The presence of flagellar hairs, tubular mitochondrial cristae, and β-1,3- or β-1,6-linked glucans as storage product all serve to strengthen the hypotheses.67 Oomycota, Hyphochytriomycota and Labyrinthulomycota have been included among Stramenopila on the basis of flagellation and DNA sequence analysis.41,42 Many types of characters may provide evidence of relationships. Thus far at the higher taxonomic levels, nucleotide characters have been very important because they extend across all of the taxa of interest.

Chytridiomycota The Chytridiomycota are commonly known as chytrids. These fungi are ubiquitous with a worldwide distribution. Chytrids produce zoospores that are capable of active movement through aqueous phases with a single flagellum, leading early taxonomists to classify them as Protists. Molecular phylogenies, inferred from rRNA sequences in ribosomes, suggest that the chytrids are a basal group divergent from the other fungal divisions, consisting of four major clades with suggestive evidence for paraphyly or possibly polyphyly.

Blastocladiomycota The Blastocladiomycota were previously considered a taxonomic clade within the Chytridiomycota. Recent molecular data and ultrastructural characteristics, however, place the Blastocladiomycota as a sister clade to the Zygomycota, Glomeromycota and Dikarya (Ascomycota and Basidiomycota). The Blastocladiomycetes are fungi that are saprotrophs and parasites of all eukaryotic groups and undergo sporic meiosis unlike their close relatives, the chytrids, which mostly exhibit zygotic meiosis.

Neocallimastigomycota The Neocallimastigomycota were earlier placed in the phylum Chytridiomycota. Members of this small phylum are anaerobic organisms, living in the digestive system of larger herbivorous mammals and possibly in other terrestrial and aquatic environments. They lack mitochondria but contain hydrogenosomes of mitochondrial origin. As the related chrytrids, neocallimastigomycetes form zoospores that are posteriorly uniflagellate or polyflagellate.

Zygomycota The Zygomycota, commonly known as ‘sugar’ and ‘pin’ molds, reproduce sexually with meiospores called zygospores and asexually with sporangiospores. Black

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bread mold (Rhizopus stolonifer) is a common species that belongs to this group; another is Pilobolus capable of ejecting spores several meters through the air. Medically relevant genera include Mucor, Rhizomucor and Rhizopus. Molecular phylogenetic investigations have shown Zygomycota to be a polyphyletic phylum with evidence of paraphyly within this taxonomic group.

Ascomycota The Ascomycota, commonly known as sac fungi or ascomycetes, constitute the largest taxonomic group within the Eumycota. These fungi form meiotic spores called ascospores, which are enclosed in a special sac-like structure called an ascus. This division includes morels, a few mushrooms and truffles, single-celled yeasts (Saccharomyces, Kluyveromyces, Pichia and Candida), and many filamentous fungi living as saprotrophs, parasites and mutualistic symbionts. Prominent and important genera of filamentous ascomycetes include Aspergillus, Penicillium, Fusarium and Claviceps. Many ascomycete species have only been observed undergoing asexual reproduction (called anamorphic species), but analysis of molecular data has often been able to identify their closest teleomorphs in the Ascomycota. Because the products of meiosis are retained within the sac-like ascus, ascomycetes have been used for elucidating principles of genetics and heredity (e.g. Neurospora crassa).

Basidiomycota Members of the Basidiomycota, commonly known as the club fungi or basidiomycetes, produce meiospores called basidiospores on club-like stalks called basidia. Most common mushrooms belong to this group, as well as rust and smut fungi, which are major pathogens of cereals. Other important basidiomycetes include the maize pathogen Ustilago maydis, human commensal species of the genus Malassezia, and the opportunistic human pathogen, Cryptococcus neoformans.

Glomeromycota Members of the form arbuscular mycorrhizae with higher plants and reproduce asexually. The symbiotic association between the Glomeromycota and plants is ancient, with evidence dating to 400 million years ago. The fungal symbionts of arbuscular mycorrhiza form a monophyletic group phylum Glomeromycota in the true fungi.68 The only member of this clade known to form a different type of symbiosis is Geosiphon pyriformis, which associates with cyanobacteria. Because none of these fungi has been cultivated without their plant hosts or cyanobacterial partners, progress in obtaining multigene

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phylogenies has been slow and the nuclear-encoded rDNA genes have remained the only widely accessible molecular markers, rDNA phylogenies have revealed considerable polyphyly of some glomeromycotan genera that have been used to reassess taxonomic concepts. Environmental studies using phylogenetic methods for molecular identification have recorded an amazing diversity of unknown phylotypes, suggesting considerable cryptic species diversity. Protein gene sequences that have become available recently have challenged the rDNA supported sister group relationship of the Glomeromycota with Ascomycota/ Basidiomycota.68 However the number of taxa analyzed with these new markers is still too small to provide a comprehensive picture of intraphylum relationships.

Are the fungi animals? Fungi are more closely related to animals than plants. The evolutionary origin of fungi is important in determining the phylogenetic relationships between fungi, animals and plants, and in questioning a previous view of the origin of life, which stated that photosynthetic organisms were the first to evolve since they were utilized by heterotrophs as a food source. The key evidence in support of a fungi-animalia clade includes analysis of protein sequences biosynthetic pathways, cytochrome systems, mitochondrial genetic material, biochemical and structural cellular features, glycoproteins, mode of nutrition, and storage of nutritive materials. The hypothesis that fungi evolved from algae, the ancestor of photosynthetic plants is not well supported, the hypothesis that fungi evolved independently of both plants and animals is also not supported. Fungi are most closely-related to animals than plants. The biochemical evidence supporting the hypothesis of a fungi-animalia clade is particularly striking. The protein sequences of fungi show homology to those of animals. Fungi also display similarities to animals in the biosynthesis of polyunsaturated fatty acids. Fungi also show similarities to animals in their cytochrome systems. Fungi share common morphological and structural cellular features with animals. Fungi lack chloroplasts, as do animals. The evidence that was shown in support of hypothesis regarding RNA and the mitochondrial genome particularly the translation of UGA as tryptophan codon, as well as the similarities in biosynthetic pathways of animals and fungi are especially convincing. There certainly is a need for further research, particularly regarding the composition of the cell wall, which is an evolutionary novelty for fungi since it contains both chitin and cellulose.

Recent classifications In the early 20th century, the importance of nucleus was recognized and based on its presence or absence the microorganisms were grouped as prokaryotes and eukaryotes under protista. Whittaker69 proposed five kingdoms in the classification

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Figure 1. Five kingdoms of living organisms proposed by Whittaker69 (from Whittaker69)

of living organisms viz. monera, protista, fungi, plantae and animalia (Fig. 1). Woese et al.70,71 classified life on this planet in three “domains”- Bacteria, Archaea, and the Eucarya, each containing two or more kingdoms. Fungi were placed in Eucarya along with animalia, plantae and others (not defined). Hibbett et al.72 proposed a new classification of kingdom fungi-based on recent molecular phylogenetic analysis and morphotaxonomy (Fig. 2). It was subdivided with one subkingdom – Dikarya and seven phyla – Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Glomeromycota, Microsporidia, Basidiomycota and Ascomycota, having ten subphyla, 35 classes, 12 subclasses and 129 orders. The clade containing Ascomycota and Basidiomycota is classified as the subkingdom Dikarya reflecting the putative synapomorphy of dikaryotic hyphae. The most dramatic changes in the classification with relation to earlier works are in the groups that have been traditionally included in the Chytridiomycota and Zygomycota. The Chytridiomycota is retained in a restricted sense, with Blastocladiomycota and Neocallimastigomycota also as segregate phyla of flagellated fungi. Taxa earlier included in Zygomycota

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Figure 2. Phylogeny and classification of fungi (from Hibbett et al.72)

are now placed among Glomeromycota, and several subphyla incertae sedis including Mucoromycotina, Entomophthoromycotina, Kickxellomycotina and Zoopagomycotina. Microsporidia are included in the fungi, but no further subdivision of the group is proposed.72 In the 10th edition of Dictionary of the Fungi by Kirk et al.5 three kingdoms are accepted viz. Chromista, Fungi and Protozoa. True fungi belong to kingdom fungi having six phyla – Ascomycota, Basidiomycota, Chytridiomycota, Glomeromycota, Microsporidia and Zygomycota; 36 classes, 140 orders, 560

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families, 8,283 genera (+5,101 synonyms) and 97,861 species. Unlike the earlier editions of the dictionary Deuteromycotina is not accepted as a formal taxonomic category. They are not a monophyletic unit, but are fungi which have either lost a sexual phase or which are anamorphs of the other phyla (mainly Ascomycota, some Basidiomycota). With modern molecular or ultra structural techniques such fungi can be assigned to existing taxa.5 Acknowledgements: The authors are grateful to CSIR and Ministry of Environment and Forests for financial support.

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11 The origin of reproductive isolating mechanisms is an important event in the process of speciation: Evidences from Drosophila B.N. Singh Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi - 221005, India [email protected]

Abstract: In the origin and maintenance of race and species, isolation is an indispensable factor and its role and importance have been recognized for a long time. Even Lamarck and Darwin pointed out that interbreeding of different populations may result in swampimg of the differences acquired during the process of evolutionary divergence. During the process of speciation the diverging populations must acquire some means of isolation so that the genes from one gene pool are prevented from dispersing freely into foreign gene pool. Reproductive isolating mechanisms prevent exchange of genes between Mendelian populations by genetically conditioned mechanisms which are intrinsic to the organisms themselves. Thus, the origin of reproductive isolating mechanisms is an important event in the process of cladogenesis (speciation). Drosophila characterized by rich species diversity (over 1500 described species) has been utilized in genetical, behavioral and evolutionary studies since the beginning of the last century as it is very good material for such studies. Different types of reproductive isolating mechanisms have been studied in Drosophila such as gametic isolation, ethological (sexual or behavioral) isolation, hybrid inviability and hybrid sterility. Among these four types of reproductive isolating mechanisms, ethological isolation is widespread in the genus Drosophila. Various techniques have been used by different investigators to test the degree of ethological isolation. In such studies, both types of ethological isolation, interspecific as well as intraspecific have been tested. In intraspecific sexual isolation tests, wild type and mutant strains of the same species have been utilized. The pattern of isolation as well as the degree of isolation has often been used to discuss the phylogenetic relationship among the species and also to elucidate the direction of evolution among closely related species of different species groups. There are different types of stimuli involved in mating behavior of Drosophila which provide basis for ethological isolation. There are interesting cases reported by Indian workers regarding the pattern of ethological isolation and the direction of evolution. Mechanisms of origin of reproductive isolating V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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mechanisms and also the models which have been proposed to predict the direction of evolution based on the mode of mating preference have been discussed while mentioning numerous examples of both kinds of incomplete sexual isolation, intra- and inter-specific. While considering different forms of post-zygotic reproductive isolation, hybrid sterility is the most common form and it is of special interest as its genetic basis may provide information about the mechanism of speciation. Key words: Reproductive isolating mechanisms, Drosophila, Phylogenetic relationship, Evolutionary sequence, Origin of isolating mechanisms

Introduction Even after 150 years of publication of Charles Darwin’s ‘On the Origin of Species by Means of Natural Selection’, his theory of evolution is important.1 Darwin’s theory of evolution has two components: (i) Descent with modification-all species living and extinct have descended from one or a few original forms of pre-existing species, and (ii) natural selection as causal agent of evolutionary change. Darwin also recognized that species not only evolve but also divide. Infact Darwin was one of the very few nineteenth century evolutionists whose major interest lay in the causal rather than the historical problems. The importance of isolation has been recognized for a long time. Lamarck and Darwin pointed out that interbreeding of different populations results in swamping of differences which they have acquired during the process of evolutionary change.2 Darwin who devoted so much of his life to the systematics of species, fully appreciated the significance of this level, as he made clear in choice of title for his great evolutionary classic ‘On the Origin of Species’.3 The idea of sympatric speciation was of course Darwin’s - an important process of biological diversity. Darwin saw sympatric speciation as the main if not the only engine of diversity. Darwin also thought that the species could arise either sympatrically, allopatrically or parapatrically.4 Dobzhansky realized that Darwin’s notion of speciation was implausible or at least incomplete, since ecologically distinct forms can not coexist without barriers to gene exchange. Dobzhansky then stressed the importance of reproductive isolating mechanisms. In his view, the problem of speciation was not the occupation of new niches but the origin of reproductive isolation. This problem was discussed in his seminal work Modern Synthesis, Dobzhansky’s Genetics and the Origin of Species.4 While discussing speciation and adaptation from Darwin to Dobzhansky, Schluter5 has pointed out that appreciation of the connection between adaptation and speciation began with Darwin when morphological concept of species largely prevailed. Darwin understood the importance of reproductive barriers between species but the study of speciation after the publication of his book focused mainly on the evolution of species differences, particularly of morphological traits but also of behavioral and

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other phenotypic traits.5 Dobzhansky remarked that without isolation evolution is impossible. Role of isolation in evolution was stressed by Wagner6 and in a modern form by Jordan.7 Races and species differ from each other in many genes and gene combinations. If these genetically different populations interbreed, these will breakdown and the differences between them will be swamped off. The gene pool of every species is discretely isolated from that of any other.8,9 Therefore, during the process of species formation the diverging populations must acquire some mechanisms of isolation so that the genes from one gene pool are prevented from dispersing freely into a foreign gene pool. Dobzhansky (1937, cited in Dobzhansky2) coined the term “isolating mechanisms” for different factors which alone or in combination prevent the gene flow between different Mendelian populations. Among different kinds of isolation, two types are easily distinguished: geographic (spatial) and reproductive isolation. In spatial isolation, species and races are allopatric and females and males of these populations which are separated by certain forms of geographical barriers do not mate hence mating is prevented. Such allopatric populations may or may not show reproductive isolation. However, Mendelian populations separated by geographical barriers may develop contact zone and gene exchange and gene deffusion may occur if the two populations have not developed certain mechanisms to keep them as separate gene pools. Reproductive isolating mechanisms prevent the exchange of genes by means of genetically conditioned factors which are intrinsic to the organism’s themselves.2 The different Mendelian populations may coexist sympatrically and they must be isolated reproductively other wise their identity could not be maintained due to gene flow between them and would merge into a single gene pool. Dobzhansky2,8 and Mayr3 have classified reproductive isolating mechanisms into different kinds supported by numerous suitable examples from variety of animal groups. There are two major classifications of reproductive isolating mechanisms: pre-mating and post-mating isolating mechanisms. Some authors have also used the terms pre-zygotic and post-zygotic isolating mechanisms (see Strickberger10; Hartle and Hallgrimsson11). According to Mayr,3 isolating mechanisms are the most important attributes a species has. Drosophila has proved to be very suitable material for studies on evolution and population genetics.8 With the development of modern genetics in Drosophila, interest has been focused on the question of role of isolating mechanisms in evolution. Different types of reproductive isolating mechanisms have been studied in the genus Drosophila. These are: ethological isolation, gametic isolation, hybrid inviability and hybrid sterility. Numerous examples are available in Drosophila which shows the operation of these kinds of isolating mechanisms. 2–4,12–16 Ethological (behavioral or sexual) isolation constitutes the most important class of reproductive isolation in animal species. It is a premating barrier to gene exchange

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in which the opposite sexes of different populations fail to mate due to behavioral incompatibility. Thus, it becomes a primary mechanism of reproductive isolation in the process of speciation. Genetic basis of sexual isolation in Drosophila has been demonstrated.17,18 The mate recognition system has been considered important species attribute which acts as cohesive factor19 and genetic events may be responsible for the appearance of different mate recognition systems. Ethological isolation has been studied extensively in the genus Drosophila and has been found to be wide spread.12–15 Many of the species tested show complete isolation. However, numerous cases of incomplete sexual isolation among closely related species have been found. When isolation is incomplete preference for homogamic matings occurs in most cases. In certain cases, males and females of different species may mate randomly. Different populations or stocks of the same species may show incipient isolation. The degree and pattern of ethological isolation have often been used to discuss the phylogenetic relationship between the species and also their evolutionary sequence. The present review summarizes briefly the different types of reproductive isolating mechanisms in Drosophila with more emphasis on the results of sexual isolation studies (inter- and intra-specific) with particular reference to the pattern of isolation and the models proposed on the basis of one-sided ethological isolation to predict the direction of evolution. In Drosophila, to test ethological isolation between species/strains, various techniques used are also described. Further, the mechanisms of origin of isolating mechanisms and the types of stimuli involved in mating behavior of Drosophila are also discussed. Further, hybrid sterility which is a common form of post-zygotic reproductive isolation in Drosophila is also discussed.

Types of reproductive isolating mechanisms In the genus Drosophila, various kinds of reproductive isolating mechanisms have been studied by numerous investigators beginning from Sturtevant, Dobzhansky, Mayr, Crow, Patterson, Stone, Mainland and others. These isolating mechanisms are: ethological isolation, gametic isolation, hybrid inviability and hybrid sterility. Ethological (behavioral or sexual) isolation In ethological barrier to gene exchange, females and males of different species/ populations do not mate as there is lack of mutual attraction due to behavioral incompatibility. It has been extensively studied in Drosophila and found to be widespread. With the development of modern genetics in Drosophila, interest has been focused on the evolutionary significance of isolating mechanisms. Although

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many species show complete isolation, there are numerous cases of incomplete sexual isolation, both between the species and within the species (for references see Dobzhansky2; Patterson and Stone12; Mayr3; Spieth and Ringo13; Chatterjee and Singh14; Singh15; Coyne and Orr4). There are a considerable number of studies that have demonstrated the existence of genetic variation for sexual isolation among conspecific populations either by direct observation or by selection for enhanced or reduced homogamic matings.17 Genetic basis of courtship songs which is the basis of sexual isolation has been demonstrated in D. pseudoobscura and D. persimilis.20 Mayr3 said “if we were to rank the various reproductive isolating mechanisms of animals according to their importance, we would have to place behavioral isolation far a head of all others”. Thus sexual isolation constitutes the most important class among different types of reproductive isolation in animal species and plays a key role in speciation4. In studies of sexual isolation, different techniques have been employed by researchers: (i) Multiple-choice: In this method, both sexes are given choice to mate with either their own strain or species or with the other. Females and males of both types are placed together hence both the types of matings, homogamic and heterogamic, are possible. Under this method, there are four combinations of matings - two homogamic and two heterogamic. This method is the most appropriate method because it gives the best approach to natural conditions. (ii) Male-choice: Here only males are given choice. Both the types of females are kept with one type of males. (iii) In this technique, the females are given choice. One type of females is confined with both types of males. (iv) No-choice: In this method, no choice is given to the flies. One type of males is confined with one type of females. Mating success can be scored by different methods. Nowadays, frequently direct observation method is used in which Elens and Wattiaux21 mating chamber is used. To measure the degree of sexual isolation, either isolation index or isolation estimate is calculated. Sexual isolation is caused due to assortative (non-random) mating between males and females of the same species or strains One-sided or asymmetrical isolation in may Drosophila species groups where preferential mating occurs in one direction only where as in the opposite cross males and females mate randomly. Thus, isolation may be symmetrical (both sided) or asymmetrical (one-sided). Ethological isolation is based on the production and reception of stimuli by male and female. On the basis of main sense organs involved, these stimuli have been classified into different types: visual, auditory, and chemical. A male Drosophila provides the female a set of stimuli during courtship. Based on these stimuli, the female is able to discriminate between its own and alien males and is stimulated for copulation. Role of visual stimuli in mating has been detected in different species of Drosophila. All species court during day light and some also court in darkness. It has been shown that presence

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or absence of light has pronounced effect on mating of Drosophila. Various species exhibit varying degrees of light dependency in their mating behavior. Auditory stimuli play more important role in isolating mechanisms than visual stimuli. Songs, calls and other acoustic signals have precision and specificity. Females may not become receptive for copulation until stimulated by appropriate wing vibration of conspecific males. Males of most Drosophila species produce wing vibrations which cause the production of sound termed as male courtship songs. With respect to these songs, there is variation in different species. The antennae of females are the main organs to receive auditory signals. The male sex songs are species specific. The inter pulse interval of male pulse songs is most important and is discriminated by females. Thus, it forms the basis of behavioral isolation. When incipient isolation occurs between different strains of the same species, variation in the male pulse songs has been observed in certain cases. Chemical stimuli which act at a distance (olfactory) or on contact often serve as isolating mechanisms. It has been shown that the olfactory stimuli in the form of pheromones are involved in mating behavior of Drosophila. In both sexes, receptors for olfactory stimuli are present on the third antennal segment. During courtship, the physical contacts between the male and female also results in the transmission of chemostimuli. Chemostimuli are exchanged during tapping of female by the male (see Chatterjee and Singh,14 1989). Recently, evidences for temporal, mechanical and habitat isolation between certain species pairs have been reported: temporal isolation between D. melanogaster and D. simulans, mechanical isolation between D. mauritiana female and D. simulans male and habitat isolation between D. sechellia and D. simulans (see Coyne and Orr4). Gametic isolation In gametic isolation, spermatozoa of one species are not attracted to the eggs or are poorly viable in sexual ducts of another species. Gametic isolation which is post mating as well as prezygotic barrier has been reported to occur in different species groups of Drosophila.22–25 In cross insemination, the mobility of sperm in ventral receptacle of females of foreign species is rapidly lost so fertilization does not take place. In certain cases, females become sterile due to insemination reaction in interspecific matings. Hybrid inviability Hybrid inviability is a post-mating and post-zygotic intrinsic reproductive barrier in which hybrids suffer developmental defects causing full or partial inviability4. A very good example of this type of isolation involves D. melanogaster and D. simulans, two closely related and sibling species.26 When D. melanogaster females are crossed with D. simulans males, only hybrid females appear because

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hybrid males die during larval to pupal transition. In the opposite cross, only hybrid males are produced and the F1 females die as embryo26.This shows that hybrid inviability may appear at different stages of development and may affect one sex only or both and it may be complete or partial4. Hybrid sterility The problem of hybrid sterility goes back to Aristotle who discussed at length the sterility of mule. Hybrid sterility is biologically not very efficient mechanism of reproductive isolation as it causes wastage of gametes of both species and of hybrids.2 While considering different forms of reproductive isolation, hybrid sterility is the most common form of post-zygotic reproductive isolation in Drosophila. Further, it is of special interest because its genetic basis may provide information about the mechanism of speciation. Since males in Drosophila are heterogametic as is the case in mammals, hybrid sterility is of special interest. Hybrid male sterility appears very quickly after species divergence as demonstrated by a large number of reports.27 The rapid appearance of hybrid sterility in males accounts for the majority of cases which follow Haldane’s rule. Sterility in hybrid males represents a well defined developmental system (namely, spermatogenesis) for genetic analysis. The study of evolutionary relationship among the species is not complete without testing hybridization among them. The first review of interspecific hybridization studies in animals was published by Haldane28 who catalogued all the case known at that time in which artificially performed crosses produced hybrids with a distortion of sex-ratio or fertility. Haldane also suggested that in hybrids when one sex is absent, rare or sterile that sex is heterogametic sex. This suggestion has come to be known as Haldane’s rule. However, exceptions to this generalization have been found.29 The total number of hybridization (interspecific) reported in the genus Drosophila is 266 involving a total of 191 different species.18,30 There is also variation in the results when successful hybridization occurs. It has been observed that when two species hybridize, larvae may be produced in F1 which die before pupation, development may proceed till pupal stage but adults are not produced or F1 adults are produced showing varying degree of fertility or sex-ratio distortion. Further, hybridization may occur in one direction or both directions. Only those species which could be cultured in the laboratory were used in hybridization studies. There are some reports of natural hybridization in Drosophila.31 In total, there are eight cases of natural hybridization in Drosophila.30 Thus natural interspecific hybridization in Drosophila is rare. The species which could be hybridized in laboratory remain completely isolated in nature. Hybridization could be achieved among closely related species belonging to the same species groups but no case of interspecific hybridization has been reported between the species belonging to different species group.18 Although there is variation in the results in different species groups, the most common finding is

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the occurrence of hybrids of both sexes in F1 and males becoming sterile. This observation in Drosophila supports Haldane’s rule because male is heterogametic. The different species groups of Drosophila in which hybrid sterility is known are repleta, virilis, melanica, funebris, guarani, mesophragmatica, cardini, willistoni, melanogaster, saltans, and quinaria (for references, see Singh16,18). There are six semi species of D. paulistorum which is regarded as a cluster of species status nascendi32 and these semi-species show varying degree of reproductive isolation from one another. Male sterility has been found in hybrids of these semi species. It is interesting to note that these semi-species of D. paulistorum are isolated by different degrees of pre-mating (behavioral) as well as post-mating reproductive barriers. Thus there are numerous cases of hybrid sterility particularly in males of Drosophila which is an important type of post mating or post-zygotic reproductive isolating mechanism. Generally, the species formation is marked by the appearance of reproductive isolating mechanism which is based on those genetic changes that differentiate incipient species up to the level when the exchange of genes is completely prevented. Since there are a large number of intercrossable species pairs generating sterile hybrids, hybrid sterility is potential force for cladogenesis. Drosophila has been very favorable material for the study of speciation genetics. A number of studies carried out on the genetics of hybrid sterility in Drosophila have shown that there are genetic loci involved in determining sterility in hybrids of closely related species. However, the results of different studies differ with respect to the number of loci involved and the pattern of interaction of X-chromosome, Y-chromosome and autosomes determine hybrid male sterility in different species pairs (see Singh18; Mishra and Singh33). One-sided sexual isolation and the direction of evolution There are numerous cases of clear cut one-sided sexual isolation between closely related species of Drosophila as well as between geographic strains/races of the same species. These findings are of much evolutionary significance and have often been used to discuss their phylogenetic relationships. Two models which are opposite to each other have been proposed based on behavioral asymmetry to predict the direction of evolution elucidating ancestral and derived nature of species/strains. Kaneshiro’s Model: Kaneshiro34 studied sexual isolation among different members of the planitibia subgroup of Hawaiian species of Drosophila. He found asymmetrical isolation among these species. Based on behavioral asymmetry, he suggested that ancestral females strongly discriminate against the derived males but the converse situation does not hold true. Kaneshiro predicted the direction of evolution for the planitibia subgroup as D. differens→ D. planitibia→ D. silvestris. Kaneshiros’ model was also applied to interpret the evolutionary sequence of different geographic strains of D. adiastola and D. silvestris based on asymmetrical

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mating preference.35,36 The phylogenetic relationship between D. takahashii and D. pseudotakahashii, two closely related allopatric species has been discussed by Dwivedi et al.37 on the basis of behavioral asymmetry and it was suggested by these authors that D. takahashii is ancestral to D. pseudotakahashii which agrees with Kaneshiro’ model. Ranganath and his students38,39 studied sexual isolation among D. nasuta nasuta and D. nasuta albomicans and also among the parental races and newly evolved cytoraces. They have also found behavioral asymmetry and have discussed their results in the light of different models to predict the evolutionary significance. Powell40 observed one-sided behavioral isolation in laboratory populations of D. pseudoobscura subjected to flush-crash cycles and the results were consistent with Kaneshiro’s model. Founder principle of Mayr41 has been hypothesized as the main factor to explain inter-island speciation of Hawaiian Drosophila.42 Kaneshiro’s model is also based on founder principle (via genetic drift). Several facets of male courtship behavior are lost due to genetic drift which occurs during a new invasion of previously uninhabited locality by a small number of migrants.34 Due to the loss of certain elements of courtship in males of new species, females of ancestral species show strong discrimination against the males of derived species since they contain only a part of total courtship pattern of conspecific males. On the other hand, the females of new species readily accept the males of older species because they identify the courtship pattern of these males since they contain all the elements of courtship pattern in the conspecific males. However, the original concept of loss of certain courtship elements in males of derived species has been modified43,44 by suggesting that courtship requirements of derived species females are simplified during early stage of founder events when the size of population is drastically reduced. Giddings and Templeton45 have redefined the model of Kaneshiro by suggesting that it would apply under specific conditions. Watanabe and Kawanishi’s model: Watanabe and Kawanishi46 studied sexual isolation among D. melanogaster, D. simulans and D. mauritiana. They also used the published data of the D. virilis group.23,47 Based on the asymmetrical isolation, they suggested that it is the females of derived species which do not mate with the males of ancestral species and the species whose females readily accept the courtship overtures of other is the ancestral species. Thus the direction of evolution is just opposite to that of Kaneshiro’s model. It is interesting to note that the direction of evolution among the species of virilis group based on the model of Watanabe and Kawanishi agrees well with the phylogenetic relationship suggested on the basis of cytological, morphological and electrophoresis studies. Watanabe and Kawanishi argued that every subpopulation is always exposed to the danger of invasion by migrants of the ancestral population. If the gene flow from the ancestral population to the new subpopulation is restricted, the new population can survive against the danger of contamination with genes from the

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original population and can retain its identity. Thus, the first step for the initiation of speciation is the failure of mating between derived females and ancestral males which is the most important change for the creation of new species.46 Thus there is fundamental difference between the two models: Kaneshiro’s model is based on allopatric speciation but the Watanabe and Kawanishi’s model is based on sympatric speciation. Ancestral and derived status of the species has been discussed on the basis of behavioral asymmetry in virilis group and D. arozonensis and D. mojavensis species pairs and the direction of evolution agrees with Watanabe and Kawanishi’s model. Evidence in favor of Watanabe and Kawanishi’s model also comes from the phylogeny of willistoni group established on the basis of electrophoresis studies.48 Behavioral asymmetry observed between D. bipectinata and D. parabipectinata by Singh et al. 49 also lends support to Watanabe and Kawanishi’s model. D. bipectinata is considered ancestral to D. parabipectinata. Singh and Chatterjee50 observed asymmetrical isolation among geographic strains of D. ananassae and the results obtained by them also support Waranabe and Kawanishi’s model. Thus, both models having predictable value regarding the direction of evolution are supported by the results of different studies. However, Markow51 suggested that the evolutionary relationship proposed in these models are not the general concomitant of the evolutionary process although a consistent relationship having predictable value might exist in certain species groups. The relationship may depend on ecological and evolutionary history of a group and evolutionary events may occur differently in different groups. These models have also been criticized by Wasserman and Koepfer.52 Ehrman and Wasserman53 suggested that mode of mating preference alone can not be used to predict the direction of evolution as there may be more than one mechanism for producing asymmetrical mode of mating preference. Origin of reproductive isolating mechanisms According to the biological species concept, advocated by the founders of Modern Synthesis, ‘the question how new species evolve’ can be substituted by a more answerable question’ how reproductive isolation is established between populations’.54 Different theories have been proposed to explain how reproductive isolating mechanisms originate between populations. Muller55 suggested that reproductive barriers to gene exchange appear as a side effect of genetic divergence because populations adapted to different environments acquire genetic differences which lead to reproductive isolation. Thus, isolating mechanisms might originate as a consequence of random genetic drift or an accidental byproduct of genetic divergence in allopatric populations as they adapt to different environments.

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Since many genes are pleiotropic, such divergent populations might also exhibit behavioral differences. According to Carson,42 there is role of random genetic drift through founder effect in the origin of reproductive isolation which serves as barrier to gene flow. Thus reproductive isolation may originate allopatrically (or peripatrically) as a byproduct of reorganization of gene pool. Based on this principle, Cason42 has explained the evolution of Hawaiian species of Drosophila. It is interesting to note that there are more than 500 species of Drosophila found on Hawaiian Islands and their evolution has occurred through founder principle of Mayr41. Powell40 conducted laboratory experiments in D. pseudoobscura and based on his results, he extended evidence in support of flush-crash speciation theory of Carson.56 Strong assortative matings developed in cage populations of D. pseudoobscura which were passed through successive bottlenecks in population size. These studies also support Muller’s model regarding the origin of isolating mechanisms. Dobzhansky8,57 gave more importance to natural selection acting on appropriate genetic variability when the allopatric populations that has an incipient isolation become sympatric. When two allopatric populations become sympatric, natural selection acts to develop and strengthen the barriers to gene exchange between these populations whose hybridization results in reproductive wastage. Thus there is reinforcement of isolation by natural selection in sympatry. There are a number of examples available in Drosophila which support these models. As suggested by Dobzhansky, selection for premating isolation occurs in sympatry, it is expected that the degree of isolation would be stronger in sympatry than between allopatric populations of related species. A number of studies have been reported in Drosophila which corroborate Dobzhansky’s model. For example, results reported by Ehrman58 in semi-species group of D. paulistorum and by Wasserman and Koepfer59 in D. arizonensis and D. mojavensis. Sexual isolation appeared as a byproduct of natural selection between a population of D. mojavensis and its sibling species D. arizonensis.60 Sexual isolation has been induced by artificial selection between ebony and vestigial mutants of D. melanogaster.61 The degree of ethological isolation between different species/strains may be altered by artificial selection in the laboratory (see Chatterjee and Singh14). On the other hand, there are reports which extend evidence in favor of Muller’s model. There are cases which show that degree of behavioral isolation is stronger in allopatric populations than sympatric ones. This does not support the hypothesis of reinforcement of sexual isolation by natural selection. Similarly, results in this regard have been reported by different investigators.34,35,62 Singh and Chatterjee50 found high degree of isolation in isofemale lines than mass culture stocks of D. ananassae which also demonstrated that isolation in isofemale lines was caused due to random genetic drift resulting from founder effect.

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Conclusions Although Drosophila melanogaster was described in scientific literature in 1830 by German Entomologist JW Meigen63 and these flies were known to Ancient Greeks, they were used for the first time in experimental studies by FW Carpenter64 in 1905 to test reaction of these flies to light, gravity and mechanical stimulation and by W Castle65 in 1906 for studies on effects of inbreeding on fecundity. Results of these studies showed that it was suitable organism for experimental work and brought it to the attention of TH Morgan who used these flies for the first time for genetical studies and proposed the theory of linkage.66 In the experiments conducted by Sturtevant,67 non-random mating was detected due to differences in the vigor of mutant and wild type flies extending evidence for sexual isolation.68 These observations suggest that Drosophila was used for behavioral, genetical and evolutionary studies about 100 years ago. The researches carried out during the past 100 years concerning behavior, genetics and evolution in Drosophila support the Darwin’s theory of evolution which was main thesis of his book “On the Origin of Species” published 150 years ago. The importance and role of reproductive isolation in the process of cladogenesis was emphasized by Darwin which include temporal, habitat and behavioral barriers. This is evident from the following lines from his book: Finally, then I suppose that a large number of closely allied or representative species, now inhabiting open and continuous areas, were originally formed in parts formerly isolated; or that varieties became in fact isolated from haunting different stations, disliking each other, breeding at different times, and so as not to cross (see Stauffer69). Thus the origin of reproductive isolating mechanisms is an important event in the process of speciation which is evident from the work done in Drosophila. The foremost architect of synthetic theory of evolution and leading evolutionary geneticist of his time, Theodosius Dobzhansky70 remarked that “Nothing in biology makes sense except in the light of evolution”.

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12 Adaptive radiation and insects V.V. Ramamurthy* and Asha Gaur** Division of Entomology, Indian Agricultural Research Institute, New Delhi - 110012, India *[email protected], **[email protected]

Abstract: The fundamental aspect of principle of divergence has emerged in different spheres of evolutionary biology as propounded by Charles Darwin. Adaptive radiation is an aspect of evolutionary biology encompassing microevolution and macroevolution, explaining the principles of divergence, taking into account the theories of natural selection and struggle for life. In addition to the classical example of Darwin’s finches in the Galapagos Islands, there are many others, of which the phytophagous insects are a prominent one that could be examined in detail to explain the different paradigms of adaptive radiation. With this example the evolutionary process of natural selection can be explained in its totality when one ancestral species gives rise to two or more species with different kinds of feeding characteristics and other core biological phenomena. There are many criteria that can be adopted to establish that adaptive radiation has taken place and all these focus toward aspects of microevolution and macroevolution. Many times the ecological, reproductive, stratigraphic, barriers due to abiotic factors are the ones that are focused toward establishing the facts on adaptive radiation. There are evidences that pluralistic diversity in insects and their species, genetic and ecological diversity is mainly due to successful adaptive radiation. The macroevolutionary patterns on insect host plant evolution are enormously varied due to the special features of wings and metamorphosis in addition to characteristics of migration and dispersal. The inordinate fondness for beetles and other phytophagous insects exhibited by nature and discussed in detail is mainly due to adaptive radiation as explicit through sister group analysis done in the large insect groups. It is also established that the evolutionary rates in these insect groups are much enormous as exhibited by the Chrysomeloidea and Curculionoidea. There are examples like the cotton stem weevil, other gall-inducing Coleopterans and other insects wherein the coevolution on the insect plant surface had been brought out and integrated with molecular phylogenetic studies to establish the bridge between microevolution and macroevolution as envisioned by Charles Darwin. These are shedding light on the underlying causes of the patterns of diversification in phytophagous insects. It is very clear that such efforts are providing the ultimate appraisal of Darwin’s bridge between microevolution and macroevolution as components of adequate radiation. All these finally testify that the treatise ‘On the Origin of Species’ by Darwin is a living document that contains wealth of ideas, which if subjected to modern synthesis and innovative appraisal can provide multiple answers to many intriguing questions in V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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evolutionary biology, especially those relating to ‘inordinate fondness for beetles’ and the adaptive radiation in the phytophagous insects. Keywords: Adaptive radiation, Coleopterans, Coevolution, Criteria, Insects, Phytophagy, Sister group analysis

Introduction The fundamental truth of Charles Darwin’s principle of divergence has emerged in different facets of evolutionary biology. There are many evolutionary processes in the form of character displacement or some models of sympatric speciation, which had been discovered independently in different contexts over a century after the publication of ‘On the Origin of Species’ by Darwin.1 There is compelling evidence for biotic interactions in at least some extinction events, and close relationship between divergence and extinction. These find enough support for Darwin’s proposals especially as a viable link between microevolution and macroevolution. Understanding of microevolution and the macroevolution, and the bridge between these is possible only through integration of ecology, evolution and the role of history in shaping the diversification or decline of lineages. There is a need for increased integration between these fields to build a bridge between microevolution and macroevolution, to reinvent the Darwin’s mechanism of the turnover of species resulting from their evolution and interactions. The historical patterns of diversification of lineages which can now be mined from molecular phylogenies, are shedding light on the underlying causes of these patterns. There are compelling evidences that Darwin has to be revisited again to understand the evolution and the adaptive radiation. Needless to state the insects, in particular, the phytophagous ones will provide the optimum examples and case studies for such discoveries.

Adaptive radiation Adaptive radiation is an evolutionary process by which a single ancestral species evolves into several new species within a relatively short time in a specific geographic domain. The new species that evolve have evolutionary advantages to occupy an ecological niche and even gets specialized for a definite role in a habitat. Thus adaptive radiations describe the process by which a group of organisms adapts to diverse variety of situations. Naturally this involves coevolution with other organisms in the geographical domain focused toward their feeding and other essential life processes. This also encompasses the changes that are envisaged in the changed ecological environments, other life processes triggering dispersal, processes of natural selection, competition and struggle for survival. According

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to Darwin,1 the varieties of forms that are observed arose because “As many more individuals of each species are born than can possibly survive; and as consequently, there is a frequently recurring struggle for existence, it follows that any being’s if it vary however slightly in any manner profitable to itself, under the complex and sometimes varying conditions of life, will have a better chance of surviving and thus naturally selected. From the strong principle of inheritance, any selected variety will tend to propagate its new and modified form”. This explains the adaptive radiation very well even in the current contexts.

Evolution and adaptive radiation An evolutionary process by which new species emanate through natural selection, encompasses changes in the traits of organisms or their populations over time so that the modified forms are successful and establish themselves in an environment. These changes occur as a result of genetic mutations leading to the changes getting passed on to surviving generations. The populations or groups of populations result in new species with better advantage in the specific environment leading to a successful adaptive radiation. The mechanisms which lead to this kind of adaptive radiation include the divergence due to geographic isolation or due to other causes including inherent, genetic and or extraneous and other environmental factors. The divergence, when exhaustive and the barriers becoming exclusive, leads to the process of speciation being successful and adaptive radiation leading to modifications takes place. These modifications are endured as a result of the need for survival and struggle for existence in an ecological niche. Further these are focused toward profitability to a species under the complex and varying conditions of life through a process of natural selection as explained by Darwin.1 Also such an adaptively radiated species tends to propagate itself in its modified form for its sustainability, and the latter is mainly through its efforts for coevolution with other organisms in its selected habitats.

Criteria for adaptive radiation The modifications in the genetic, biological and ecological characteristics leading to the adaptive radiation have their own criteria to establish that it has taken place. There are many criteria that can be followed and these include the factors and barriers leading to reproductive isolation and ecological isolation. In any study resorting to establish that adaptive radiation has taken place, an insight into the critical areas of genetic or other kinds of reproductive isolation, in particular those of the barriers that result in the isolation, is essential. The changes in the genetic, reproductive and ecological aspects of populations and species in an ecological niche and the degree to which these changes elicit a suitable response in the biological phenomena and characteristics will be the yardstick for evaluating the essential criteria.

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The exploitation of ecological niches and their extent is an important factor toward adaptive radiation. Especially islands or isolated pockets that have specific sets of environmental conditions will trigger this kind of exploitation and the populations or groups of species that are successful in this endeavour will undergo adaptive radiation. The physical features such as water and other stratigraphic factors will lead to barriers, isolation of populations, and their adaptive radiation. In addition, there will be dispersal barriers due to factors like mountains, which are likely to radiate the populations at or near the limits of the range of dispersal. The rapid genesis of new species through sympatric or allopatric speciation is yet another criterion that can be adopted to evaluate species radiations and their divergence. In case of phytophagous species, the coevolution, in particular, in terms of pollination relationships becomes important. In species with broad geographical ranges, and wide dispersal abilities in view of large and wide habitat range and growth forms, the adaptive radiation can be gauged through the habitat ranges and their extent. Many biotic factors within the range of populations of a species can also be used as criteria. Especially when these factors relating to biology, development, growth and reproduction, and variations in feeding and other behavioral characteristics, which are crucial for the life of an organism, get altered, there is an upheaval in the biological aspects leading to radiation of the important biological processes. Hence, an insight into these characteristics and their dynamics will be important criteria for evaluating adaptive radiation. These biological changes will imply changes in the ecological aspects, and the biotic and abiotic factors when examined will provide the criteria for evaluating the origin and maintenance of adaptive radiation.

Adaptive radiation and Charles Darwin Adaptive radiation as implied by Charles Darwin through his classical studies on the South American finch like birds at the Galapagos Islands is a concept that can be applied more usefully. It is a concept defining the way in evolution of variety of species taking place from a single ancestral species. It is a process by which variety of species develop from their ancestral species wherein each gets adapted to requirements of new way of life. Most of the examples like the one explained by Darwin are over relatively short periods of time, when a closely related group of species has radiated in areas recently colonized by the ancestors of the group. The examples include Darwin’s finches on the Galapagos Islands, the honey creepers of the Hawaiian Islands, the cichlids of the eastern African lakes and the fruit flies of the Hawaiian Islands. Explaining one such example will elaborate the adaptive radiation in its entirety and the Darwin’s finches is a classical one (Fig. 1). About 1000 km west

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Figure 1. Few Darwin’s finches in the Galapagos Islands

of Ecuador is the Galapagos archipelago, which is known for surfacing a few million years ago. This archipelago was colonized by South American finch like birds probably 0.5 to 1.5 million years ago. This ancestral species gave rise to 13 duly recognized modern species of birds on the Galapagos. These 13 modern species have been tested by showing that all these are derived and closely related to one another than to any related species outside the islands. These members of the modern bird community have thus evolved from a single ancestral species rather than from multiple invasions of the island, with one species getting derived from each invasion. It has been found that these 13 species are adapted to eat different food, with related variations in their beak forms. Some of these have larger beaks while others have smaller, those which have them larger are as a result of modification required for cracking nuts, while those with smaller beaks are as a result of modifications required for gathering seeds with their beaks. One of these is a “woodpecker” finch, which lacks a true woodpecker’s powerful beak. Its beak performs the functions similar to that of a true woodpecker through using small sticks to probe for insects in a tree hole. Thus one of its species has individually adapted for a specific type of feeding and food through behavioral, functional and structural adaptations, and thereby behaves like a true woodpecker. Five of the 13 Galapagos finches are insectivorous, one is a vegetarian tree finch, three are ground dwelling seed eaters, two feed on cacti and two are warbler like. All these can be revealed through their modifications in their beaks and attributing a food or feeding behavior is easily possible in view of adaptation to different foods hence it is called adaptive radiation in terms of concept.

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Principle of divergence A single ancestral species evolves into a variety of forms through adaptations to different types of food, that too in the shortest possible time. Naturally, there are extreme similarities among these modern species which are fundamentally unique, owing to their evolution from a common ancestor resulting in a homologous group of organisms in a habitat as that existing in Galapagos archipelago. Thus in the process of adaptive radiation, the new species are evolving, and these evolve apart from each other, and the evolution is through divergence of characters. While diverging due to the new requirements, the ancestral species splits into two or more modern, derived species. This lineage splitting is a unique phenomena as regards speciation and it is opposite of convergence. The splitting leads to divergence of species and evolutionary lines, necessitated mainly due to the change in the requirements of food and feeding, or behavioral, ecological and other biological characters. The major cause is thus achieving less competition for food, and derives an advantage through a process of natural selection. Charles Darwin refers to this as “the principle of divergence” and this concept explains the tree of life that is the fundamental of evolutionary classification.

Adaptive radiation and insect biodiversity The biodiversity comprising of species, genetic and ecological diversity when analyzed indicates that insects are the most diverse and are the characteristic examples explaining adaptive radiation. This adaptive radiation stems from the fundamental fact that the insects and their host plants represent the major part of terrestrial diversity. Insect plant coevolution is the most discussed of all examples of diversification of living organisms, as phytophagous insects account for approximately 40% of all described insects. By far the most influential model of insect plant diversification is based on the hypothesis of insect plant coevolution, leading to interpretations of adaptive radiation as a life phenomenon in insects.2 The macroevolutionary patterns on the insect host plant evolution especially toward plant defense and host affiliations provide a model for explanations on the adaptive radiation. This is absolutely empirical as insects and plants have diversified over roughly the same intervals in the geological eras in which the flowering plants have evolved in consonance with the insects, especially toward pollination, seed dispersal and other activities. The diversification of insects, as in the case of Darwin’s finches in the Galapagos Islands, had happened due to the requirements of adaptation and coevolution consequent upon changes in herbivory and phytophagous feeding behavior. It has been documented that rather than accumulating herbivores at a rate proportional to their geographic area of distribution or biomass, some plant

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groups pose apparent chemical barriers to potential herbivore colonists, and seem accessible to relatively few insect lineages, possibly preadapted by use of chemically similar or related host plants.3 It has also been observed that longterm success of such coevolutionary associations and adaptive radiations leading to these patterns are influenced by ecological conditions in a long term. The inordinate fondness for beetles as explained by the well-known biologist JBS Haldane is a case to be discussed as it has been seen that diversity of insects and plants especially in tropics is an extraordinary example of successful adaptive radiation. This can be seen from the data on the world totals of described living species in the different orders of insects4 (Table 1). Some details of diversification in insects, with a focus on the phytophagous groups, are presented herein.

Metamorphosis and wings in adaptive radiation of insects The fundamental aspects of transition in insects and their adaptation have hovered around the differences in metamorphosis and wings in addition to score of other specific characters. In case of insects with complete metamorphosis, this transition has been focused toward resource partition wherein an insect will base its different components of life, namely the ones required for growth and development separated from those concerned with reproduction. The ability of the holometabolous insects with larval stages entirely differing from the adults in both structure and habits is one such adaptive radiations leading to successful and efficient utilization of resources especially food and shelter. In fact the development of tunneling behavior as an adaptive radiation in these insects is an ecological advantage and specialization toward an exclusive habitat. The development of wings and associated modifications in the legs is an additional feature which adds to the triggering of adaptive radiation toward their dispersal and migration in occupying specific ecological niches and to survive the process of natural selection. The evolution of plants has presented a new adaptive zone for insects to exploit. As insects have evolved means to exploit plants as food, through their varied mouth parts and their elaborate modifications, plants have evolved counter measures which led to greater diversification of plants. When this diversification has happened to the plants in the same geological era in which the insects have also undergone their diversification, the processes had got related and it had become mutualistic in terms of coevolution through adaptive radiation. In fact the successful pollination and seed dispersal of many flowering plants has coevolved along with insects as successful pollinators and movers. Despite the plants evolving defenses the insects have learnt to coexist through physiological adaptations for feeding on plants protected by toxic secondary compounds. The insects like tobacco aphid and the tobacco hornworm are some such examples, where there had been long-term effects of coevolution

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Table 1 World totals of described living species of Class Insecta Order20

Described Species4

Archaeognatha

504

Zygentoma

527

Ephemeroptera

3046

Odonata

5680

Dermaptera

1967

Notoptera

39

Plecoptera

3497

Embiodea

458

Zoraptera

34

Phasmatodea Orthoptera

2853 23,616

Mantodea

2384

Blattaria

4565

Isoptera

2864

Psocoptera

5574

Phthiraptera

5024

Thysanoptera

5749

Hemiptera

100,428

Coleoptera

359,891

Raphidioptera

225

Megaloptera

337

Neuroptera

5704

Hymenoptera Mecoptera

144,695 681

Siphonaptera

2048

Strepsiptera

603

Diptera

152,244

Trichoptera

12,868

Lepidoptera

156,793

TOTAL

1,004,893

as a result of adaptive radiation taking place rather more swiftly, and in a subtle manner. It has been demonstrated that phytophagy is an evolutionary innovation especially in beetles, when famous biologist JBS Haldane had stated that God

Adaptive radiation and insects

Figure 2. Adaptive radiation in major phytophagous insect orders

183

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has an inordinate fondness for beetles. There had been multiple processes of coadaptation and cospeciation in these coevolutionary phenomena, taking place at the microevolutionary and macroevolutionary aspects. These aspects lead to a variety of forms from a single ancestral stock often after colonizing an island group or restricted specialized habitats or through entering a new adaptive zone.

Adaptive radiation and phytophagy The adaptive radiation in phytophagous insects if explored will indicate that plants initially represented a new unexploited adaptive zone for insects. With the flowering plants getting diversified in the paleozoic era, this new unexploited adaptive zone became the foreground for the transformation of current day highly evolved insects especially the endopterygotes which have acquired a tunneling behavior for feeding or other essential biological purposes in their larval stages. Also, the resources got partitioned for the purpose of growth and development and delineated from those required for their reproduction in the adult stage. In fact, in these insects which have a pupal and transition stage, this delineation gets demarcated and the adaptive zone put forth by the flowering plants get well exploited toward the diversity. There are evidences that accelerated diversification across many independent insect groups have occurred and adaptive shifts had been repeatedly associated with phytophagous insect groups, in particular the Chrysomeloidea and Curculionoidea, which are the most successful of the herbivorous groups.

Sister group analysis and adaptive radiation Examples of adaptive radiation are enormous in insects and sister group analysis of adaptive radiation had proved this. When a lineage moves from an ancestral adaptive zone to a new one, adaptive shifts occur and such shifts are enormous in the most dominant phytophagous insect groups especially the coleopterans. It has been proved through analysis of sister groups that the differences in diversity in these could be used as a yardstick for evaluating adaptive radiation. If the sister group that has undergone adaptive shift is consistently more diverse than the original sister group that remains in the original adaptive zone, it can be confirmed that the adaptive radiation has been successful in causing divergence. It had been shown that higher plant feeding is found in nine orders of insects and it has probably arisen at least 50 times in just the extant forms with known habits (Fig. 2). The present phylogenetic identification allows identification of 13 pairs of sister groups, one of which feeds on higher plants and the other which does not5 (Table 2). It has also been conclusively established that the phytophagous lineage is more diverse than its presumed nonphytophagous sister group. Thus the phytophagous feeding habit is associated with increased diversification due to the

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Figure 3. Darwin’s view of microevolution and macroevolution1

processes of cospeciation and a coadaptation representing the macroevolutionary and microevolutionary processes. Most plant and insect ecologists believe that plant secondary compounds have evolved as defense against herbivory by phytophagous insects and that insects in turn have evolved behavioral and physiological adaptations to counter the effects of toxic secondary plant compounds. Such coadaptive responses between insects and plants also may be reflected in the extent of cospeciation of these groups.

Microevolution, macroevolution and adaptive radiation The relationship between microevolution and macroevolution must be understood to explain adaptive radiation. The microevolution or adaptation is always linked with macroevolution or speciation, phylogeny of the organisms above species level and the development of complex organs to effectively reach the diversification and achieve divergence. Many times this linkage or bridge is never understood by the evolutionary biologists as it cannot be explored in one’s lifespan and hence hypotheses are only possible. Hence, it is a major source of conflict between science and religious beliefs.6 Biologists often forget that Charles Darwin offered a way of resolving this issue, and his learned views hold good now and are required to be revisited. For Darwin, the interactions that define the struggle for existence and shape how organisms evolve were diverse, including competition, predation, parasitism, disease and pollination.

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Figure 4. Cotton stem weevil Pempherulus affinis (a) damage; (b) gall; (c) grub feeding on the internal tissues; (d) grub; (e) pupa; (f) adult

All these hold good for the phytophagous insects and as cause for their adaptive radiation Darwin illustrated the combined action of his principle of descent with modification, the principle of divergence and extinction in his ‘On the Origin of Species’ and this explains the link between microevolution and macroevolution1 (Fig. 3). Many of the resultant organisms go extinct, but some persist, becoming modified and improved descendants as in the case of many phytophagous insect groups of the present day. These always strive for their perpetuation so that they drive some of their close relatives to extinction and to fill the unoccupied insect plant interface, leading to cladogenesis where the splitting of one ancestral species leads to more than one descendant. Darwin had rightly propounded that the processes of such diversification and extinction can explain the gaps that are seen among the living species.

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Table 2 Sister group diversity comparisons on independent phytophagous insect lineages5 Primitively phytophagous lineage Taxon

Sister group

Approximate Taxon no.of species (phytophages only)

Hemiptera

Joppeicidae

Tingidae (? + Thaumastocoridae)

1,800 15 ≤1,815

Miridae

10,000 Isometopidae

Trichophora

>5,000 Aradidae

Approximate no. of species (excluding phytophages)

Sign of difference in diversity 1

+

60

+

1,000

+

7 1,200 190 ≤1,397

+

400

+

Coleoptera Elateridae + Cebrionidae

Scarabaeidae sensu lato, minus Geotrupinae Aphodiinae, and Scarabaeinae

9,000 Cerophytidae and/ 170 or 9,170 Eucnemidae and/or Throscidae 14,000 Geotrupinae or Aphodiinae/ Scarabaeinae

3,200

Languriinae

410 Lobarinae+ Erotylidae–

200 1,500 1,750

Epilachninae

700 Coccinellini

250

+

≤10,000

+

10,000 Panorpida

80,000

_

140,000 Trichoptera

7,000

+

80

+

1,733

+

200

+

Phytophaga Hymenoptera (Symphyta only) Lepidoptera

130,000 non-phytophagous Cucujoidea

Diptera Chloropidae Oscinellinae +Chloropinae

1,350 Siphonellopsinae 860 2,210

Tephritidae sensu stricto

4,000 All or part of nonphytophagous Tephritidae sensu lato

Agromyzidae

2,000 Clusiidae

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Figure 5. Adaptive radiation through host plants – cotton stem weevil case study

Adaptive radiation and evolutionary rates Phytophagous insects, which could be used as a classical model for explaining the adaptive radiation, are also unique models that can be utilized to explain evolutionary rates. In case of well-known phytophagous insect groups namely Coleoptera, in particular Chrysomeloidea, it has been shown that there are consistently different rates of evolutionary changes involving different kinds of characters of biological and ecological significance. These affect the cospeciation and coadaptation in relation to a particular host plant and also these changes become consistent with passage of time. Depending upon the rates of host use, the rates of evolutionary change varies and in fact this variation on the rates of change leads to diversification and divergence.7 Specificity on host tissues are well known in plant feeding insects, particularly in the diverse orders Lepidoptera,8,9 Coleoptera,10 and Hymenoptera.11,12 It has also been shown that this specificity extends to the whole host species leading to specialization and this has been widely accepted. There are many such examples of host specializations due to the specific behavioral and physiological adaptations.13,14

Gall-inducing Coleoptera and adaptive radiation Cotton stem weevil – a case study An ideal example that could be discussed as a case study in the context of biogeography and adaptive radiation in gall-inducing Coleoptera is that of cotton

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Figure 6. The diversity of larval stages in Coleoptera16

stem weevil Pempherulus affinis (Fig. 4). This species is widely distributed in India, from Kerala in the far south to Rajasthan in the north, and from Tamil Nadu and Andhra Pradesh in the east to the Gujarat in the west, as far as the Indian subregion goes. P. affinis is also known from adjacent countries, viz. Thailand and Myanmar, extending up to the Philippines, indicating its resourcefulness in terms of populations specializing on various species and varieties of gossypium cultivated in these areas and countries. It has also been documented that this species is adapting to other Malvales specific to this region namely Malvastrum spp., Sida spp., and other such weed plants found in the cotton agroecosystems. P. affinis is a typical example for a gall-inducing Coleoptera, which has diversified itself adapting to adverse conditions in the absence of its most preferred host plant viz., cotton, the gossypium spp., in spite of its specialized gall-inducing

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Table 3 Contrasts of coleopterans associated with gymnosperms vs angiosperms10 Contrasts Primitively gymnosperm associated taxon 1 Nemonychidae

2 3

OxycoryninaeAllocoryninae AseminaeSpondylinae

4

Palophaginae

5

OrsodacninaeAulacoscelidinae

Diversity Primitively angiosperm associated taxon 85 AttelabinaeRhynchitinae, Apioninae, CurculionidaeRhynchophoridae 30 Belinae 78 Lepturinae, LamiinaeCerambycinae 3 MegalopodinaeZeugophorinae 26 Remaining Chrysomelidae

Diversity

44002

150 25000

400 33400

behavior. Detailed explorations in the region extending up to Thailand in the east and beyond Gujarat in the west may reveal its distribution extending toward other regions of the Orient and the adjoining Palearctic regions. This is due to the fact that their documented host plants have an extensive distribution in these regions (Fig. 5). Coleoptera and angiosperms feeding While indexing 22 species of Buprestidae and 15 species of Cerambycidae it was concluded that gall induction occurs almost exclusively in the most advanced superfamilies Chrysomeloidea and Curculionoidea.15 As far as Curculionoidea are concerned, gall-inducing behavior is restricted to the phanerognathous groups only, which have their feeding and oviposition behavior characterized by scooping, digging, burrowing or mining activities. The evolutionary trends discussed indicate that gall-inducing habit increases in the course of evolution of the Coleoptera, and has become established in three phyletic lineages namely Buprestoidea, Chrysomeloidea, and Curculionoidea.15 The phylogeny of the Phytophaga, the largest and oldest radiation of herbivorous beetles was reconstructed, using molecular data.10 The results of these analyses have been used to interpret the role of angiosperms in beetle diversification. It has been shown that repeated origins of angiosperm feeding beetle lineages are associated with enhanced rates of beetle diversification, indicating a series of adaptive radiations. Collectively

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191

these radiations represent nearly half of the species in the order Coleoptera and a similar proportion of herbivorous insect species. These inferences will apply equally to gall inducing Coleoptera of the Orient and eastern Palearctic. It was concluded that there has been disproportionate rise in the diversity of post Cretaceous phytophagous beetles reflecting the likelihood of exponential rise in angiosperm diversity.10 Five independent contrasts of Coleoptera groups associated with gymnosperms versus angiosperms when analyzed have yielded a positive relationship in favour of the hypothesis that angiosperm feeding is associated with enhanced diversity (Table 3). It has also been shown that the addition of remaining (mostly weevils) subfamilies, not yet sequenced, will bring the total number of species to 135,000. The current affiliations of the oldest beetle lineages with preangiospermous seed plants support the hypothesis that these lineages retain affiliations that were formed early in the Mesozoic, before the diversification of flowering plants. The diversification of phytophagous beetles, thus, is consistent with the coevolutionary models that ascribe differences in the present diversity of insect and plant groups to evolutionary changes or characters (which affect their ecological interactions).2,3 Combined evidences from phylogenetic estimates and from the fossil record show a pronounced conservation in the evolution of beetle plant associations, which is important for the implication that plants might escape herbivory via key innovations. Thus gall inductions or other associations of coleopterans with plant galls might be concluded as such innovations toward coevolution. Curculionidae and Chrysomelidae proliferation of life history traits Correlated with angiosperm feeding is the proliferation of life history traits, in the Curculionidae and Chrysomelidae. In contrast with the strobilus feeding of conifer and cycad associated ancestors, diversification of the subfamilies that attack flowering plants has been accomplished by larval folivory, leaf mining and seed and root feeding in addition to gall induction, which exemplify the concept of adaptive radiation. The diversity of the larval stages of the coleopterans is a testimony to this fact16 (Fig. 6). This fact can be discussed in the light of the propositions that most of the pollen and nectar feeders are generalists, whereas gall-inducing insects are specialists and remain tied closely to their food plants.17 The specialized gall-inducing habit among insects is considered to have arisen from leaf mining Diptera and Microlepidoptera, which, over time, are purported to have sought ‘new’ food sources in concealed, and thus, ‘protected’ environments. This proposition derives support from the fact that gall-inducing habit originated from the behaviour of the tunneling Lepidoptera and Coleoptera. The success of gall-inducing Coleoptera like the other non gall inducing Coleoptera seems to have been enabled by the rise of flowering plants,

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and through many different evolutionary routes, in addition to maintaining the conservative host plant associations. These propositions hold good for the gall inducing and gall associated Coleoptera found in the Oriental and eastern Palearctic regions as well. Further exploration of the ecology and behavior of gall inducing Coleoptera will no doubt provide new insights into the evolution of this taxon, in particular throwing light on the coevolution between the gall inducers and their host plants, in these biogeographic regions. Gall-inducing coleopterans and their coevolution Taking into account the correlation between the richness of gall inducing insects and their host plants, the current estimated number of gall inducing insect species ranges between 21,000 and 210,000, with an average of 132,930 species. Gall inducers represent a gap in the taxonomic knowledge especially among insects in the tropical regions of the Orient and eastern Palaearctic. Although there had been several attempts to bridge this gap, it has not been achieved in its entirety, especially with regard to Coleoptera, which is one of the largest insect groups. Its members have interactions other than gall induction too, unlike other insects, due to their specialized feeding and reproductive behaviours. Under many situations it is difficult to delineate their specific roles and understand them, which contribute to widening of the gaps in the knowledge. Also, there are many species which are economically important in view of the proliferation by way of gall induction. Many species interact with other gall inducing organisms, forming an ecological community. Due to this complexity, many attempts to interpret the evolution of gall inducing Coleoptera, especially at the higher taxonomic levels have remained inconclusive. Chrysomeloidea and Curculionoidea: evolutionary trends Gall induction occurs almost exclusively in the most advanced Chrysomeloidea and Curculionoidea. The evolutionary trends indicate that gall inducing habit seems to increase in the course of evolution of the Coleoptera, and became established in three phyletic lineages namely Buprestoidea, Chrysomeloidea and Curculionoidea. Elucidation of the role of angiosperms in beetle diversification have shown that repeated origins of angiosperm feeding beetle lineages are associated with enhanced rates of beetle diversification. Groups associated with gymnosperms versus angiosperms, when analyzed yielded a positive inference in favour of the hypothesis that angiosperm feeding is associated with enhanced diversity. These inferences equally apply to the gall inducing coleopterans. The diversification of phytophagous beetles is consistent with the coevolutionary models which ascribe differences in the present diversity of insect and plant groups to their ecological interactions. Also there is a pronounced conservation

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in the evolution of beetle plant associations. This is important for the implication that plants might escape herbivory via key innovations. Gall inductions as coevolutionary innovations Gall inductions or other associations of coleopterans with plant galls might be concluded as innovations toward coevolution. The specialized gall inducing habit among insects is considered to have arisen from leaf mining Diptera and Microlepidoptera which over time have adapted themselves toward achieving efficiency in resource utilization or partition. This proposition derives support from the fact that gall inducing habit could have originated from tunneling behaviour as a novelty in the metamorphosis of a holometabolous insect. The success of gall inducing coleopterans thus seems to have been enabled by the rise of flowering plants, and through many different evolutionary routes, in addition to maintaining the conservative host plant associations. The information accumulated on the Coleoptera as gall inducers and interactors in galls has shown that such evolutionary behaviour is true with regard to the fauna of the Oriental and eastern Palearctic regions too. The little information that has been brought out by the present contribution indicates that tremendous scope exists for such explorations on the gall inducing coleopterans. No doubt such attempts will provide new insights into the evolutionary information, in particular toward the coevolution between the gall inducers and their host plants.

Conclusions The concept of reproductive isolation being the central part for the biological species derives its merit from the principle of divergence propounded by Darwin. Especially in case of phytophagous insects and their adaptive radiation, the fundamental truth of his principle of divergence has emerged in different aspects of their diversification at the insect plant interface as has been demonstrated in the phytophagous Coleoptera and in the case of gall inducing coleopterans and other insects by different workers.10,17,18 The compelling evidences now emerging in terms of molecular ecology in case of insect groups like Chrysomeloidea and Curculionoidea by the far the largest of the herbivorous groups of animals as explained in terms of their ecological interactions and fossil evidences on coevolution have proved that Darwin’s principle of divergence can be discovered independently in different contexts, especially in these large insect groups. These interactions leading to evolutionary changes in the form of microevolution and macroevolution and coupling of information gathered through fossil evidences on insects and plants finds enough support for Darwin’s ideology and provides the missing link between the cospeciation and coadaptation, and also convincingly explain the diversification as a result of adaptive radiation.

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The information about the historical aspects of adaptive radiation in insects in terms of insects and flowering plants diversification in the different geological eras through fossil evidences and coupling of these with historical patterns of diversification of lineages mined now through molecular phylogenies are shedding light on the underlying causes of the patterns of diversification in phytophagous insects. It is very clear that such efforts are providing the ultimate appraisal of Darwin’s bridge between microevolution and macroevolution as components of adequate radiation. All these finally testify that the ‘On the Origin of Species’ by Darwin is a living document that contains wealth of ideas, which if subjected to modern synthesis and innovative appraisal can provide multiple answers to many intriguing questions in evolutionary biology especially those relating to ‘inordinate fondness for beetles’ and the adaptive radiation in the phytophagous insects.

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Boving AG, Craighead FC (Eds.) (1931) In: An Illustrated Synopsis of the Principal Larval Forms of the Order Coleoptera. The Entomological Society, Brooklyn, New York

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Raman A (2007) Insect induced plant galls of India: unresolved questions. Curr Sci 92:748–756

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Raman A, Burckhardt D, Harris KM (2009) Biology and adaptive radiation in the gall inducing Cecidomyiidae on Mangifera indica in the Indian subcontinent. Trop Zool 22:27–56

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Ramamurthy VV (2007) Faunistic, ecological, biogeographical and phylogenetic aspects of Coleoptera as gall inducers and associates in plant galls in the Orient and eastern Palearctic. Orient Ins 41:93–119

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Grimaldi DA, Engel M (Eds.) (2005) In: The Evolution of Insects. Cambridge University Press, Cambridgess

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13 Insights from mosquito evolution: Patterns, tempo and speciation Karamjit Singh Rai Biological Sciences, University of Notre Dame, IN, USA, and Zoology, School of Life Sciences, Guru Nanak Dev University, Amritsar, India [email protected]

Abstract: A great deal of information has accumulated on chromosome morphology and evolution, heterochromatin distribution and differentiation, and molecular structure, organization and evolution of genomes in the mosquito family Culicidae. Whereas numerically the haploid chromosome number (n = 3) has remained virtually unchanged, extensive variation exists at different levels of genomic structure and organization. A number of trends in genome evolution emerge when these data are considered in light of cladistic phylogenies of Culicidae and its sister families. Anophelinae have heteromorphic sex chromosomes, a small genome size and repetitive elements are distributed in a longperiod interspersion pattern. In contrast, Culicinae have homomorphic sex chromosomes and repetitive DNA is organized in a short-period interspersion pattern. There has been a general increase in genome size during the evolution of culicine taxa. The most likely hypothesis for the evolution of sex chromosomes and genome organization in Culicidae would be that homomorphic sex chromosomes and a long-period interspersion pattern are ancestral in lineages leading to Toxorhynchitinae and Culicinae. Larger genomes developed in subsequent culicine lineages through accumulation of short-period interspersed repetitive elements. Heteromorphic sex chromosomes evolved early in the evolution of Anophelinae and a long-period interspersion pattern was retained. An alternative route may be that Culicidae arose from a Chaoborid Mochlonyx-like ancestor with heteromorphic sex chromosomes and possibly short-period interspersion. This would require the loss of heteromorphic sex chromosomes in the lineage leading to Toxorhynchitinae and Culicinae and ‘shedding’ of repetitive elements in the lineage leading to Anophelinae. Several interesting patterns have emerged from studies of C-banding and the distribution of heterochromatin in Culicidae and phylogenies derived from these studies are supported by the modern cladistic analyses. Intensive multi-point linkage map studies suggest that recombination frequencies/ genome have remained relatively constant over the course of culicid evolution such that Anophelinae with relatively small genome size has a linkage map of similar size to Aedini. As a consequence, taxa in Anophelinae have a higher amount of recombination V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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per haploid genome size than Culicinae. Undoubtedly, family Culicidae represents one of the best studied systems of genome evolution in animals, and to ascertain the mechanics of sympatric v/s allopatric speciation in closely related group of species. Extensive variation in vector competence to arboviral and other pathogens exists within and between species. Although the genetic basis of susceptibility or refractoriness of mosquito populations to certain parasites has been known for more than half a century, the underlying molecular mechanisms controlling such differential expression have been resolved relatively recently. Apparently, arboviral susceptibility in mosquitoes is under polygenic control. However, major genes for susceptibility of mosquito vectors to malaria have been identified and mapped. Keywords: Mosquito evolution, Zoogeography, Chromosomal differentiation, Genome structure, Organization and evolution, Reproductive isolation, Speciation, Vector competence

Introduction Charles Darwin published his seminal work ‘On the Origin of Species’ in 1859.1 A century later, in the Spring quarter of 1959, as part of the Darwin Centennial Celebration, the University of Chicago, USA offered an interdisciplinary Advance Graduate Level course/Symposium (Anthropology 425-Zoology 425) involving a galaxy of leading biologists, geneticists, evolutionists, anthropologists, theologians and philosophers of the time to discuss, dissect and reconcile Darwin’s monumental theory of Natural Selection with what till then were largely disparate worlds of Science and the Christian church. Among other luminaries, the invited participants included renowned British evolutionist, Sir Julian Huxley, F.R.S., the American Noble Laureate geneticist, Herman J. Muller, Ernst Mayer (Harvard), George Gaylord Simpson (Amer. Museum of Natural Hist.), Theodosius Dobzhansky (Columbia U.), G. Ledyard Stebbins (UC, Davis), E.B. Ford, F.R.S. (Oxford), Noble Laureate ethologist, Nikolaas Tinbergen (Leiden and Oxford) and the Naturalist Marston Bates (Rockefeller Foundation). I had joined the Department of Botany, University of Chicago as Charles Hutchinson Fellow working for my Ph.D. in the previous fall quarter of 1958. I recall with considerable pride enrolling and participating in this admittedly thought provoking and high decibel Course/Symposium in the Spring Quarter of 1959. Personally, this resulted in a clearer understanding of the issues involved from the formal presentations, and the very vocal give and take discussions. At the end of the week, I thought all the stakeholders including the theologians, by and large, began to accept the reality of the evolutionary theory. Half a century later to have the opportunity to revisit this theme is, therefore, highly gratifying and nostalgic. This presentation briefly highlights the patterns and tempo of geological, taxonomic, genomic and ethological differentiation and speciation within and

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among populations of selected taxa of mosquitoes, in the context of their often distinctly contrasting geographical distribution, observed by members of my research laboratory during this period at the University of Notre Dame, USA, located some 100 miles east of my Alma mater, the University of Chicago. The family Culicidae is comprised of more than 3,400 mosquito species, many of which are major vectors of human disease. In view of their importance as vectors, many mosquito genera and species have been the subject of intensive cytological, genetic, evolutionary and molecular investigations over the last half a century.2-5 The purpose of this review is to highlight the salient features of the following studies as they relate to the patterns, pathways and tempo of intraand inter-specific differentiation at various levels of genetic organization and evolutionary relationships of various mosquito taxa: (1) Mosquito systematics employing cladistic analyses of morphological and molecular characters to estimate phylogenetic relationships among sister families to Culicidae and among Culicidae subfamilies, tribes, genera, subgenera and species. (2) Karyotypes and their evolution emphasizing that the number of chromosomes has remained at a constant 2n = 6 despite a relatively ancient origin of Culicidae, the evolution of both homomorphic and heteromorphic sex chromosomes and evidence of extensive chromosomal repatterning in the speciation process. (3) Molecular genome size and organization in Culicidae and considered in light of current phylogenetic relationships. Genome evolution is also reviewed in the context of extensive data on heterochromatin distribution and in terms of the linkage maps established through various recent intensive genome mapping protocols in Culicidae. (4) Ethological differentiation, reproductive isolation and speciation in sympatry versus allopatry, and (5) Brief observations concerning variation in and genetics of vector competence.

Mosquito systematics, zoogeography and speciation The family Culicidae, which includes all mosquitoes, is divided into three subfamilies, Anophelinae, Toxorhynchitinae, and Culicinae.6-10 Anophelinae includes three genera, the Neotropical Chagasia (4 species), the Australasian Bironella (9 species in 3 subgenera), and the nearly cosmopolitan Anopheles with some 422 species grouped in 6 subgenera. Toxorhynchitinae includes a single genus, Toxorhynchites with 76 species. Culicinae is by far the largest subfamily subdivided into 10 tribes, 33 genera, 117 subgenera and includes about 2,925

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described species. The total number of genera, subgenera and species in Culicidae currently stand at 37, 129, and 3,436, respectively.10 The genus Aedes, which includes some 962 species grouped in 43 subgenera, is one of the best studied and has been the primary focus of the author’s laboratory.3,4,5 Based on the available fossil record, and zoogeographic evidence involving past intercontinental connections and faunistic composition, it has been suggested that mosquitoes had evolved by the Jurassic, approximately 210 million years ago (MYA).11 This is about the time continental drift began.12 The continental break up led to fragmentation and geographical isolation of populations. This may have been accompanied by great ecological flux that promoted rapid speciation.13 Ross14 proposed that a burst of Culicinae lineages arose approximately 120 MYA. By the end of the Cretaceous, some 65 MYA, the generic composition of family Culicidae was well established. New Zealand has been in its present position of isolation for approximately the last 50 million years.17 With the exception of three species, Aedes notoscriptus, Aedes australicus, and Culex quinquefasciatus the present day mosquito fauna of New Zealand is relict and endemic. This provides circumstantial evidence that the genus Aedes existed prior to the island’s separation from Australia and that it was probably widely dispersed during the Cretaceous which began 145 MYA.15 Fossils of family Culicidae (Culex, Aedes) and its sister family Chaoboridae are well known from the Eocene (Tertiary) and Oligocene which began 60 and 55 MYA respectively.16 The phylogenetic relationship of Culicidae relative to other nematocerous dipteran families has been evaluated using modern cladistic analysis.5 Munstermann and Conn18 have reviewed the impact of molecular biology and cladistic analysis on systematics of selected taxa of Culicidae with particular emphasis on Aedes and Anopheles species. Phylogenies have been estimated with morphological characters19 and nucleotide sequences from the 18S and 5.8S nuclear ribosomal DNA (rDNA)20 and 28S rDNA.21 In summary, data from these analyses consistently support Chaoboridae-Corethrellidae as sister taxa to Culicidae. All analyses support Anophelinae as the basal clade in Culicidae and are consistent in placing Toxorhynchitinae as basal to the Culicinae. Within Culicinae, the tribe Sabethini is basal to Culicini and Aedini. All datasets support a monophyletic relationship between Culicini and Aedini.

Comparative karyotypes, chromosome morphology and evolution Chromosomal karyotypes have been established for a relatively large sample involving some 20 genera, 35 subgenera, and more than 300 species in family Culicidae.4,22-25 One of the most remarkable findings of this survey is that despite the ancient origin of the group and despite extensive repatterning of the genome

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involving translocations and inversions, 2,26 the basic chromosome number (2n = 6) has remained unchanged. The only exception, Chagasia bathana (2n = 8) of the subfamily Anophelinae, possesses three autosome pairs and a heteromorphic pair of sex chromosomes.28 All other anophelines possess two pairs of generally metacentric chromosomes of unequal size and one pair of heteromorphic sex chromosomes that often show extensive polymorphism in overall length and of the quantity and quality of heterochromatin differentiation among various species.23 The position of the centromeres in the heteromorphic X and Y chromosomes in Anophelinae varies from subtelocentric or acrocentric to submetacentric and metacentric.2931 In contrast, species of the subfamilies Toxorhynchitinae and Culicinae all possess three pairs of homomorphic metacentric and/or slightly submetacentric chromosomes: a pair of small chromosomes, a pair of large chromosomes and a pair of intermediate-sized chromosomes.3,22,24,32 In culicine mosquitoes, sex is determined by a gene at a single locus. Females are homozygous recessive at this locus while males are heterozygous for a dominant allele.33,3 In species in which linkage group-chromosome correlations have been made, the shortest chromosome contains the sex-locus and is therefore sex-determining.32,35,36 Differences clearly exist in overall lengths and arm ratios of individual chromosomes, both within and between species.3,37 Total chromosomal length among various genera varies almost five-fold, from 8.2 microns in Anopheles quadrimaculatus to 39.3 u in Aedes alcasidi. Within the genus Aedes, there is a three-fold variation in chromosome length.5 Genetic mapping data show that groups of allozyme loci have remained linked and collinear, particularly around the centromere regions, in a variety of culicine taxa but that these linkage groups have translocated and are inverted extensively involving the arms of the three culicine chromosomes.26,38 The extensive variation in chromosome number in most Diptera taxa belies the extreme conservation found in Culicidae. For example, chromosome number ranges from n = 3 to 7 in the genus Drosophila,39 and from n = 3 to 8 in the genus Glossina.40 In family Muscidae, most species possess six pairs of chromosomes; however six species have only five pairs each.41 Nevertheless, certain other Dipteran families such as Simulidae42 and Sarcophagidae also show extensive conservation of chromosome number, although some exceptions do occur.39 No logical explanation exists for the extraordinary conservation of the haploid chromosome number in Culicidae. The evolution of sex chromosomes Current dogma suggests that heteromorphic sex chromosomes evolved from virtually identical homologues. Both the theory43 and considerable experimental evidence suggest that it is the gradual accumulation of repetitive sequences on the

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Y chromosome followed by loss of recombination between the heteromorphic pair that leads to the differentiation of X and Y chromosomes. Theory predicts eventual loss of function and extinction of the Y chromosome.44-47 This directionality is generally referred to as the ‘Rise and Fall of the Y Chromosome’.45 Evolution of a heteromorphic Y-chromosome may have occurred only once or possibly may have been reversed in the evolution of sex chromosomes in Culicidae. The primitive Nematocera families Tipulidae and Dixidae possess homomorphic sex chromosomes. However, the sister families Chaoboridae-Corethrellidae contain genera with homomorphic (Eucorethra, Corethrella, Chaoborus) and heteromorphic (Mochlonyx) sex chromosomes.24 If homomorphy was ancestral in Culicidae then it was retained in the lineages leading to Toxorhynchitinae and Culicinae while heteromorphy probably evolved early in the evolution of Anophelinae and was retained in all taxa. This scenario is in conformity with the current dogma concerning the evolution of sex chromosomes.47 Alternatively, if as proposed by Rao and Rai (1987a), Culicidae arose from a Mochlonyx-like ancestor, then Anophelinae retained heteromorphic sex chromosomes, while homomorphic sex chromosomes evolved through euchromatinization or loss of the Y in Toxorhynchitinae and Culicinae.24

Hetrochromatin: Localization, variation, and expression The application of Giemsa C-and other banding procedures to somatic and meiotic chromosomes has provided important insights concerning linear differentiation and evolution of chromosomes in Culicidae. Studies have been conducted in 36 species belonging to seven genera of Culicinae (Aedes, Mansonia, Culiseta, Armigeres, Sabethes, Wyeomyia, Toxorhynchites) including 28 Aedes species,24,48,49 three species of Culex50 and several Anopheles species.29-31, 51-53 C-banding patterns were also studied in representative species of Tipulidae, Dixidae, and Chaoboridae in order to examine how chromosomes have evolved in these families.24 These studies established that the distribution of heterochromatin is markedly different in anopheline and culicine mosquitoes particularly in the heteromorphic sex chromosomes. All species showed the presence of heterochromatin around the centromeres of the autosomes although there are often large inter- and intra- specific differences in amounts of the same. Using different banding techniques, three types of heterochromatin were identified on the basis of staining characteristics in the pericentromeric regions in the Culicini species Culiseta longiareolata.53,54 In addition to centromeric bands, the autosomes in certain species such as Aedes bahamensis,24 and the long arms of the sex chromosomes in An. atroparvus55 possess telomeric C bands also. The organization of heterochromatin is markedly different among the two homologues of the sex chromosome pair in most Aedes species as well as between

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anopheline and culicine mosquitoes. Motara and Rai48,49 reported two distinct types, constitutive and facultative heterochromatin, in Aedes mosquitoes. The former is present around the centromere region of all the three chromosome pairs and the latter in an interstitial position on one of the arms of the female-determining (m) chromosome in most Aedes species. The intercalary band is located proximal to the centromere in Ae. annandalei and in telomeric position on both the male and female determining chromosomes in Ae. vittatus. Aedes mascarensis, Ae. katherinesis, Ae. excrucians, Ae. stimulans, Ae. cinereus and Ae. triseriatus lack the intercalary band. The fact that these species belong to three different subgenera suggests that heterochromatinization of particular segments is species specific. The male-determining chromosome (M) in Ae. aegypti lacks even the centromeric heterochromatin. The constitutive and facilitative heterochromatin replicate at different times in the cell cycle.56 Unlike Aedes, the intercalary heterochromatin is not present on the femaledetermining chromosome in Armigeres subalbatus or Toxorhynchites splendens but on an arm of one of the autosomes (chromosome II in the former and chromosome III in the latter).24 Rai et al.3 suggested a possible evolutionary derivation of the various heterochromatin patterns observed in Aedes species. The overall patterns observed among various genera are also suggestive of the role chromosome repatterning played in genome evolution. The expression of the intercalary C-band on the sex chromosome in a particular species varies as a function of the genetic background in which it is placed. This was revealed by Giemsa C-banding of the F1 hybrids and progeny of certain backcrosses between two closely related species, Aedes aegypti and Ae. mascarensis.48 Crosses involving Ae. aegypti females and Ae. mascarensis males produced F1 progeny in which the expression of the distal intercalary C-band on the female-determining (m) chromosome of Ae. aegypti was suppressed in both the males and the females. This indicated that the distal region of the femaledetermining (m) chromosome represented by the heterochromatic C-band was derepressed and that it became euchromatic. When F1 males from this cross were backcrossed to Ae. aegypti females, a proportion of the sons developed into intersexes and differed from normal males in their C-banding pattern. Thus, it was possible to relate abnormal sexual development of adult males in the backcross progeny to a selective activation of a discrete chromosomal locus on the male determining chromosome of their fathers.48 Reciprocal crosses (Ae. mascarensis females X Ae. aegypti males) gave expected results. The reversible genetic regulation of the facultative C-band apparently represents selective control of a chromosomal segment of one species (e.g. Ae. aegypti) through genetic interaction with another, Ae. mascarensis.48 Such genetic regulation, which was also observed in progeny of crosses involving Ae. katherinensis and Ae. hebrideus,24 may be widespread among Aedine mosquitoes and may help protect species integrity.

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In anopheline species, the heteromorphic chromosomes often show extensive differences in the amount, distribution and types of heterochromatin. The Y chromosome may be entirely heterochromatic in most Anopheles species while the X-chromosomes may be heterochromatic from < 1/2 to > 3/4 of their length even among closely related species. Furthermore, several of these species, for example, the Hyrcanus group (subgenus Anopheles), the maculatus group (subgenus Cellia) and others are polymorphic for the size of the X chromosome and for the amount of heterochromatin.29,30,31 Such differences are diagnostic and allow unambiguous identification of species whose polytene chromosome banding pattern is virtually homosequential.57 Presumably, different densities of the Giemsa bands on the X and the Y chromosomes in these species reflect different types of constitutive heterochromatin.58 Four satellite DNAs defined on Hoechst 3325S CsCl density gradients are similarly reflective of the presence of different types of heterochromatin in the An. stephensi genome.59 In conclusion, there seems little doubt that changes in amounts, types, and location of heterochromatin are associated with mosquito speciation, particularly in the subfamily Anophelinae and Culicinae. Dynamics of highly repetitive DNA sequences Intraspecific and interspecific variation in the sequence and abundance of eight highly repeated DNA sequences obtained from a population of Ae. albopictus was determined by dot-blot hybridization with genomic DNA of three species each of the Ae. albopictus and Ae. scutellaris subgroups in the Ae. albopictus group.60 The copy number differed widely from 0 (absent) to 810,000 both within and among species, and without any correlation with genome size. Extensive sequence divergence was also observed. The results suggest rapid and asynchronous turnover and evolution of these highly repeated DNA elements. In situ chromosomal localization of four cloned, repetitive DNA fragments (H-76, 61, H-19 and H-85) indicated that they are dispersed throughout the lengths of the three haploid chromosomes in all Aedes species examined.61,62 Although the sequences homologous to these cloned repetitive DNA fragments are present in other culicid genera, Haemagogus equinus, Tripteroideres bambusa and Anopheles quadrimaculatus, significant differences in their abundance and distribution were observed.61,62 Unlike such dispersed pattern in Aedes, Satellite 1 was localized to the heterochromatic arms of the X and the Y chromosomes and the centromere regions of chromosome 3 in Anopheles stephensi.59 Similarly, a highly repetitive DNA clone isolated from Aedes albopictus (H115) was shown to be located at an intercalary position on chromosome 1 in all Aedes species examined. 63 Southern hybridization of this DNA fragment with genomic DNA of An. quadrimaculatus on the other hand showed a dispersed pattern.

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An important difference in chromosome organization with regard to heterochromatin distribution between anophelines and most culicines may be critical in determining whether polytene chromosomes can be easily mapped. There is generally a good resolution of individual bands on each of the euchromatic chromosome arms in the anophelines, while culicines are largely refractory to this type of analysis. In anophelines, apparently much of the heterochromatin is clustered around the centromeres of each of the three pairs of chromosomes resulting in the formation of a chromocenter in polytene chromosome preparations. Of the eight mosquito genera in which polytene chromosome morphology has been studied, Anopheles alone possesses a chromocenter. All other genera (Aedes, Culex, Mansonia, Toxorhynchites, Orthopodomyia, Wyeomyia, Sabethes) lack a distinct chromocenter. Nevertheless, Orthopodomyia pulcripalpis64 and Sabathes cyaneus65 have yielded well resolved polytene chromosomes. This suggests that these taxa have long-period interspersion and may be more basal in culicid evolution. Furthermore, as indicated above, repetitive DNA constitutes a large proportion of the genome in culicine mosquitoes. Since this DNA undergoes latereplication during the S period,56 such dispersed sequences may conceivably act like micro-chromocenters thereby preventing effective separation of individual chromosomes. In examining karyotypes and C-banding patterns in species of Tipulidae, Dixidae, Chaoboridae, and Culicidae, Rao and Rai (1987a) 24 concluded that Culicidae arose from a chaoborid Mochlonyx-like ancestor and that the Anophelinae and Culicinae evolved along separate lineages from a common ancestral stock. The Chagasia karyotype was considered to be primitive for Anophelinae while the Toxorhynchites karyotype was considered primitive for Culicinae. The cladistic analyses discussed above support this proposal.

Genome size and general genome organization Interspecific variation and genome organization Considerable effort has been expended in recent years to determine haploid nuclear DNA amounts in the superfamily Culicoidea.66-69 This has been done through quantitative cytophotometry of Feulgen-stained primary spermatocytes and in a few cases through analyses of renaturation kinetics of nuclear DNA.69-71 As a result, haploid genome sizes have been established for 44 species belonging to 13 genera of mosquitoes and related Culicoidea families. Genome size is generally small in Anophelinae (0.23–0.29 pg/haploid genome).66,71,79 The single species, Toxorhynchites splendens examined in subfamily Toxorynchitinae possesses intermediate size genome of 0.62 pg as do Sabethes cyaneus and Wyeomyia smithii (Sabethini). The haploid genomes of Culex species examined ranged from 0.54 to 1.02 pg and those of Culiseta species (Culicini)

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from 0.92 to 1.25 pg. Armigeres subalbatus and Haemagogus equinus (Aedini) contained 1.24 pg and 1.12 pg, respectively. At the generic level, the cosmopolitan genus, Aedes showed more than three-fold variation in nuclear DNA amounts with the Polynesian species Ae. pseudoscutellaris and Aedes cooki (belonging to the Ae. scutellaris subgroup in the subgenus Stegomyia) possessing the lowest genome size of 0.59 pg while Ae. zoosophus (subgenus Protomacleaya) possessed the highest genome size of 1.9 pg among the 23 species examined.25,68 Placed in the context of phylogenetic relationships discussed earlier, these values suggest a general increase in genome size during the evolution of Culicidae. Black and Rai69 demonstrated that all classes of repetitive DNA sequences increased linearly in amount with total genome size. Furthermore, linear regression analysis of a fairly large data set involving 28 species belonging to 11 genera of the superfamily Culicoidea showed a highly significant positive correlation between total chromosomal length and haploid genome size.25 Nevertheless, eight-fold variation in haploid genome size was accompanied by only an approximate fivefold variation in the total chromosomal length indicating that DNA amounts have increased almost twice as much as the increase in chromosomal size. Studies using reassociation kinetics have provided information on genome organization in Anophelinae and Culicinae.69-71 Genome organization refers to the amounts, complexity and dispersion of repetitive elements in a genome. Two basic forms of genome organization have been described in eukaryotes.72 The first type is termed short-period interspersion and describes a pattern wherein single copy sequences, 1,000–2,000 bp in length, alternate regularly with short (200–600 bp) and moderately long (1,000–4,000 bp) repetitive sequences. This characterizes genome organization in the majority of animal species and was found in the culicine species Culex pipiens, Aedes aegypti, Ae. albopictus and Ae. triseriatus.74 The second type of genome organization is termed long-period interspersion and describes a pattern of long (> 5,600 bp) repeats alternating with very long (> 13,000 bp) uninterrupted stretches of unique sequences. Repeats in Anopheles quadrimaculatus69 and An. gambiae71 follow a long-period interspersion pattern. Genome organization is of the long-interspersion type in Chironomus tentans73 but has not been determined in sister families Chaoboridae-Corethrellidae. However, haploid DNA amounts of 0.47, 0.55 and 0.40 picograms (pg) were observed in the three principal genera Corethrella, Mochlonyx and Chaoborus respectively.68 In insects, long period interspersion is characteristic of most species with small genome sizes (0.1–0.5 pg/haploid genome) while short period interspersion tends to be associated with larger genomes with larger amounts of repetitive DNA.74 It is difficult to predict genome organization in ChaoboridaeCorethrellidae based on genome size because they fall into the upper limit for long interspersed species. Thus there remain two competing hypotheses for ancestral

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genome evolution in Culicidae. It is possible that long period interspersion was ancestral in Culicidae and was retained in the lineage leading to Anophelinae while larger genomes developed through accumulation of short period interspersed repetitive elements in Culicinae. The alternative hypothesis is that Culicidae arose from a short period interspersed species and, while that organization was retained in the Culicinae, repetitive elements were shed and became organized into a long period interspersion pattern in the Anophelinae. This is the scenario considered by Rao and Rai68 who proposed a phylogeny of the superfamily Culicoidea based on haploid chromosome numbers and genome sizes. They suggested that the line that possibly gave rise to Anophelinae from Mochlonyx-like ancestor underwent many deletions of highly repetitive DNA. However, there is little corroborating, empirical evidence for this. These arguments suggest that long-period interspersion may be ancestral in Culicidae. However, the hitherto unknown genome organization in Chaoboridae or Corethrellidae must be determined to test this hypothesis. Furthermore, analysis of genome organization in Sabethini and Toxorhynchitinae is imperative to determine when short-period interspersion arose in culicine evolution. Intraspecific genome size variation Studies of intraspecific variation in genome size in the author’s laboratory revealed unequivocally that DNA amounts are not fixed within species.67,75,80 An analysis of 47 geographic populations of Ae. albopictus from 18 countries showed a 2.5-fold variation in DNA amounts ranging from 0.62 pg in the Koh Samui population from Thailand to 1.66 pg in a population introduced to the continental United States from Houston, TX. Furthermore, extensive variation existed among and within populations from contiguous geographic locations. Genome size was independent of geographic origin in the various populations examined75. Using DNA-reassociation kinetics, Black and Rai 69 showed that the intraspecific variation in DNA content in two strains of Ae. albopictus was due mainly to highly repetitive DNA sequences. Further, MacLain et al.76 showed that populations of Ae. albopictus that were significantly different in DNA content also varied in the frequency of different classes of highly repetitive DNA. Thus, the variation in DNA content among populations of Ae. albopictus appears to be due mainly to repetitive DNA sequences that are under rapid change. This suggests that the amount of repetitive DNA is dynamic in Ae. albopictus and probably other mosquito species as well. Significant variation in haploid DNA content has also been observed among several species of invertebrates77 and vertebrates.78 Genome size also varies with the duration of generation time80 and body size.77 Cavalier-Smith (1985) proposed that variation in DNA amount is subject to natural selection and plays an adaptive

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role.79 Although exact function(s) of highly repetitive DNA have long been debated, biologically significant roles have been ascribed in various species. For example, it has been suggested that the proportion of repetitive DNA in Plasmodium berghei may be directly correlated with mosquito infectivity.81 Saturated linkage maps In recent years, simultaneous use of hundreds or thousands of molecular genetic markers in one or a few crosses has allowed a virtual revolution in construction of the so-called saturated linkage maps simultaneously. This technology has allowed a number of different laboratories to construct multi-point linkage maps of entire mosquito genomes. This was first accomplished by constructing a linkage map of Aedes aegypti using 50 RFLP markers from 42 random cDNA clones, 3 random genomic clones and 5 cDNAs of known origin.82 The lengths of chromosomes I, II and III were 49 cM, 60 cM, and 56 cM respectively (165 cM total). Antolin et al. (1996) constructed a linkage map of Ae. aegypti using Single Strand Conformation Polymorphism (SSCP) analysis of 94 RAPD markers.83 The lengths of linkage graphs I, II and III were 52 cM, 58 cM and 57 cM, respectively (168 cM total), remarkably similar to the cDNA map. Mutebi et al. (1997) constructed a linkage map of Ae. albopictus using SSCP analysis of 68 RAPD markers.84 The lengths of chromosomes I, II and III were 54 cM, 67 cM, and 104 cM respectively (225 cM total). Severson et al. showed that cDNA markers are colinear in Ae. aegypti and Ae. albopictus.85 These studies using molecular markers suggest a large (57 cM) increase in the recombinational size of the Ae. albopictus genome. Furthermore, most of this appears to be due to increased recombination on chromosome III. It is uncertain whether these differences are due to variation in DNA amount or differences in the distribution and frequency of chiasmata on chromosome III of the two species. Aedes species are known to vary widely in chiasmata distribution and frequency.86,87 The lengths of the three linkage maps involving morphological and enzyme loci calculated from observed chiasmata frequencies were 62, 86 and 80 cM, respectively (total 228 cM in Ae. aegypti).88 However, large stretches of all three linkage maps were devoid of any markers particularly on linkage group III on which the 17 observed markers were clustered in a 44-unit map while the chiasmata based model predicts an 80-unit map. A linkage map of Armigeres subalbatus constructed using 26 RFLP markers involving cDNA clones from Ae. aegyti resulted in overall lengths of linkage group I, II and III as 51, 72, 58, respectively (181 cM total) and except for one marker, the linear order was the same as in Ae. aegypti.89 A similar RFLP linkage map has been constructed for Culex pipiens using a relatively small sample of 21 cDNA clones from Ae. aegypti. The comparative linkage maps for chromosomes II and III in Cx. pipiens and Ae. aegypti reflect whole arm translocations.27

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Zheng et al. (1996) mapped 131 microsatellite markers in Anopheles gambiae. Chromosomes I, II and II were respectively 49 cM, 72 cM and 94 cM in length (215 cm total).90 Integration of RAPD markers into this map increased the overall density of markers without affecting the overall length.91 It is instructive to consider linkage map size, an indication of the amount of recombination on individual chromosomes, relative to the genome sizes discussed earlier in this review. The observed total linkage map sizes are: 165 cM in Ae. aegypti, 225 cM in Ae. albopictus, 166 in Cx. pipiens, 181 in Ar. subalbatus, and 215 cM in An. gambiae. These do not correspond in any way to the respective genome sizes of 0.83, 0.86–1.32, 0.54–1.02, 1.12 and 0.27 pg/haploid genome respectively in these species. The relationship of physical to recombination distance is approximately 3–6 Mbp DNA/cM in Ae. aegypti, Ae. albopictus and the two other culicine species studied, and 1.2 Mbp DNA/cM in Anopheles gambiae.5 Thus there appears to be little relationship between genome size and recombination frequency. The frequency of recombination remains high in An. gambiae despite having a genome size 1/3 to 1/5 the size of the Aedes and other culicine species genomes. DNA reassociation kinetic analysis has shown that the amount of repetitive DNA sequences in culicine species is generally much higher than that in Anopheles species.69-71 Since recombination is considerably restricted in chromosomal regions rich in repeated DNA sequences,92 overall, Anopheles would be expected to show higher recombination rates. Also, this predicts a closer relationship between physical and linkage maps in Anopheles. Furthermore, the fact that the size of the linkage maps do not vary by more than 60 cM in these three species suggests that the number of chiasmata have remained relatively constant despite increases in genome size and chromosome length in the evolution of Culicidae.

Ethological differentiation, reproductive isolation and speciation Analyses of these inter-related areas involved several Aedes species with particular emphasis on those that comprise the Aedes (Stegomyia) scutellaris group. This group is divided into two subgroups: the scutellaris subgroup consisting of some 34 species which are largely allopatric in distribution on the islands of the South Pacific, generally endemic to single island or groups of island; and the albopictus subgroup with 11 described species having a predominantly sympatric distribution in Southeast Asia.3 This distribution suggests that speciation in the former subgroup occurred after gene flow was interrupted by the development of oceanic barrier. A comparison of allozyme and morphological relationships based on numerical taxonomic methods showed that adult morphological traits and allozymes are congruent with geographical distributions and classical taxonomic assignment in the Ae. scutellaris subgroup and certain other (Stegomyia) species. Furthermore

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allozyme relationships at the group and subgroup levels were consistent with previous taxonomic treatments and all species fell into independent and cohesive clusters.93 Genetic comparisons were also based on interspecific hybridization compatibilities and the levels of fertility and viability of the hybrids experimently produced in the laboratory among the Ae. scutellaris group and certain other Aedes species.3,94 The development of reproductive isolation in the island-dwelling species has been largely a random event such that no association was found between Nei’s unbiased genetic distances and compatability.95 The relative tempo of speciation was estimated by comparing patterns of allozyme relationships with morphology, hybridization, and geological history in allopatric island-dwelling mosquitoes.95 Assuming that molecular evolution occurs at a relatively constant rate, and that Nei’s genetic distance roughly estimates time since divergence, calibration of a known geologic event, e.g. the separation of two islands in the South Pacific, was used to correlate zoogeography with allozyme and ethological differentiation and to obtain reasonable estimates of geological time frames of the onset and tempo of speciation events in the group. A molecular clock, based on the ratio between experimentally determined genetic distances between two given species and divergence time (one unit of Nei’s distance = 11 million years) inferred from a known geologic event allowed estimates of geologic time frames ranging from 3 mya to approx 7 mya for the observed speciation events in the group following their geographic isolation.95 In addition, ethological divergence and reproductive isolation among eight species in the South Pacific Ae. sutellaris subgroup was investigated by giving females a simultaneous choice between males of their own species and males of another species thereby determining isolation indices. The degree of ethological isolation between species pairs thus measured was associated with the timing of their geographic isolation following the fragmentation of the Outer Melanesian Arc 2–10 mya and on other published studies of allozyme variation and percent egg hatch from interspecific hybridization.96 To answer the critical question whether sympatry between closely related but non-interfertile species can promote the evolution of prezygotic (ethological) reproductive isolation, three sibling species of the Ae. albopictus subgroup and six strains of Ae. albopictus with known dates of laboratory colonization were employed. The data showed that selection for reinforcement of ethological isolation can occur in sympatric populations where strong postmating reproductive isolation already exists.97 In the case of Ae. scutellaris subgroup species the production of fairly fertile hybrids in the laboratory among several species pairs and no hybrid breakdown in two filial generations suggest that allopatry preempts imposition of any selection pressure for the development of reproductive isolating mechanisms.

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In conclusion, the data from Ae. scutellaris group species allowed important insights concerning the onset, tempo, and intensity of reproductive isolation and genetic differentiation, in relation to their contrasting distribution patterns, with geological time frames.

Variation in and genetics of vector competence Extensive variation exists within and among species in their ability or inability to transmit important arboviral and other diseases such as malaria and filariasis. Hardy et al.98 presented an exhaustive review of intrinsic factors that affect vector competence of mosquitoes for arboviruses. Although the underlying molecular pathway of this fundamental vector characteristic is not yet clearly delineated, extensive data from laboratories around the world, including those from Notre Dame, have unequivocally established that differential ability to transmit a particular pathogen can be a characteristic of individuals or populations as well as of species. Furthermore, a particular geographic strain is often associated with different profiles of oral susceptibility and horizontal transmission rates. For example, Boromisa et al.99 examined vector competence of eight geographic strains of Aedes albopictus from N. America and Asia and one North American strain of Ae. aegypti to Fiji strain of Dengue-1 serotype virus. The Asian strains were derived from known recent dengue non-endemic, endemic, and epidemic history. All strains tested except the Houston strain, showed high susceptibility to midgut infection. However, there were marked differences in percent disseminated infection and transmission rates among the various strains tested. It is ironic that whereas single genes for aphid and leafhopper transmission of plant viruses have been known for more than half a century, the goal of establishing genetic basis of susceptibility of any of the mosquito transmitted arboviruses has remained illusive. This likely is due to complexity of viral transmission by mosquito species, involving several seemingly independent steps and barriers such as ingestion, replication, and dissemination of the virus from the hemocoel, the salivary glands and finally the ability to transmit. In all probability, these interrelated steps are controlled by different genetic factors and their interaction with the intrinsic environment of the specific vector population. Furthermore, numerous extrinsic environmental factors doubtlessly also come into play. In a presumptive genetic study, strain differences in basal lamina thickness, assessed by measurement of transmission electron micrographs, and disseminated infection rates of DEN-1 virus were compared among three laboratory strains of Ae. albopictus.100 Mean basal lamina thickness for the NEW ORLEANS and the HOUSTON strains were significantly greater than those for the OAHU strain, which exhibited a higher disseminated infection rate than the former two. Although the basal lamina thickness among the F1 progeny of reciprocal crosses of the

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OAHU and HOUSTON strains were intermediate between the parental strains, they were too variable to be useful in genetic studies. Measurements of basal laminae among individuals of the NEW ORLEANS strain, with disseminated or nondisseminated infections, failed to demonstrate a role of basal lamina thickness as a modulator of DEN-1 virus dissemination across the midgut epithelium of the Ae. albopictus, in contrast to the demonstration of this phenomenon, apparently of considerable importance in the Ae. triseriatus – La Crosse virus system.101 In contrast to the above paradox in delineating genetic basis of arbovirus vector competence, the genetics of mosquito – Plasmdium interaction has been well characterized. The susceptibility of Anopheles mosquitoes to Plasmodium infection may range from 100 percent where all mosquitoes transmit malaria to complete refractoriness. Furthermore, the infection success of Pl. falciparum has been shown to be variable depending upon the geographic origin of both the parasite and the mosquito.102 The molecular interactions of malarial parasite with their host tissues within the mosquito have been resolved and the molecular mechanisms responsible for pathogen destruction, such as melanotic encapsulation and immune peptide production have been characterized.103 Three genes responsible for the encapsulation of the malarial parasite in An. gambiae have been physically localized to discreet regions of polytene chromosome complement.104 Acknowledgements: I thank Prof. V.P. Sharma to invite me, on behalf of the National Academy of Sciences of India, to contribute an overview of the patterns of evolutionay differentiation and the mechanics of speciation in mosquitoes in connection with the Academy’s commemoration of the bicentennial of Sir Charles Darwin’s birth on February 12, 1809.

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14 The saga of pollination biology Rajesh Tandon* and H.Y. Mohan Ram** *Department of Botany, University of Delhi, Delhi - 110 007, India [email protected] **INSA Honorary Senior Scientist, 194, SFS DDA Flats, Mukherji Nagar, Delhi - 110 009, India [email protected]

Abstract: Developmental events leading to seed set in plants are broadly categorized into three phases of pollination biology: (1) formation of male and female gametes (2) dispersal and deposition of pollen grains on the pistils of individual of the same species and (3) pollen – pistil interaction leading to double-fertilization. Whereas the first and the third are under genotypic control, the second is largely influenced by the ecological conditions. Charles Darwin was among the first to recognize the functional components of pollen transfer or pollination. In accordance with the theme, we highlight the various aspects of pollination biology pertaining to the second event, which are fascinating and require careful observations. Pollination is a fundamental and vital process in the perpetuation of angiosperms. The process of transfer of pollen to a receptive stigma of the same plant species is accomplished with utmost accuracy amidst various challenges such as immobility of plants and the vagaries of environment that carry the pollen. Flowering plants have accommodated both biotic and abiotic means of pollen transfer. A vast array of floral adaptations such as showiness, colouration, nectar guide-marks on petals, nectaries and floral rewards ensure the targeted delivery of pollen to a conspecific stigma. The combination of floral adaptations – structure, aggregation, scent and display–result in an interplay of selection pressures that maximizes pollination efficiency. Consequently, every forager that visits a flower to seek rewards may not be a pollinator but a robber. Besides the structural organization of the flower, its functional morphology, blooming time, longevity and the innate genetic mechanisms further contribute to screening for fidelity in pollination. As pollination is a mutualistic interaction, the ecological domain has a crucial role in the timing of its occurrence. There are genetic consequences of the pattern of pollen-mediated gene flow in a community. On the other hand, a pollinator is only a forager which opportunistically seeks for a reward and relies on flowers of the other species, when a particular species ceases to bloom. Thus, studies on plant–pollinator interaction are now pursued at different levels of organization with growing emphasis on conserving and maintaining the networks. This paradigm shift arises because there is perceptive apprehension that habitat loss, fragmentation of ecosystems, notably V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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forests, dislocation of overlapping of flowering time and the life cycles of pollinators due to changing climatic conditions and anthropogenic activities are likely to threaten mutualistic interactions. Keywords: Pollination, Pollination syndrome, Generalization and specialization, Floral cues, Biotic Pollination, Abiotic pollination, Mutualism

Introduction Sexual reproduction in flowering plants essentially follows a sequence of events involving transfer of pollen grains (carrying two male gametes) onto a conspecific stigma, growth of the pollen tube through the style and ovary and subsequent release of the two male gametes into the embryo sac and eventually the fusion of one male gamete with the egg (female gamete) to form a zygote and the other male gamete with the polar nuclei to trigger endosperm (double fertilization). The development of male (pollen) and female (egg) gametes and their interaction (pollen–pistil interaction) are under the control of mating genotypes with the exception of self-pollination. The transfer of pollen (pollination) from dehisced anthers to pistil largely involves biotic and abiotic components of the ecosystem. Pollination is a crucial and complex ecological process. Being immobile, plants themselves are not capable of carrying the pollen. Nevertheless, the inability of plants to move has not restricted them to exchange genes among different individuals, as they have successfully accommodated a variety of organisms and abiotic means as carriers (modes of pollination) to transfer pollen from anthers to stigma of the same species. Among these modes of pollination, biotic pollination (zoophily) occurs in nearly three-fourths of the flowering plants which rely on around 2,00,000 animal pollinator species.1 Although abiotic pollination does not play a predominant mode, it occurs in grasses, bamboos, sedges and palms growing in a wide range of habitats. That the dynamics of pollination is complex can be experienced by conducting a single study on pollination biology in any flowering plant. It is not merely an event of transfer of pollen to the stigma of the same species; rather an infallible display of an intricate mechanism that brings connectivity, accuracy and coherence of biological events to ensure multiplication, variation, perpetuation and adaptation of plants. Modularity of plants is another feature that accentuates the complexity in pollination mechanism, as pollen may arrive from the flowers borne on different braches (ramets) of the same plant (geitonogamy) or from other plants (xenogamy). Consequently, the breeding system and eventually the genetic structure of a species population is influenced by pollination pattern; types, availability and behavior of the foragers; arrangement and opening of flowers in an inflorescence; functional

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morphology of the flowers including the cues and rewards (pollen, nectar and wax) for attracting the floral foragers and the time of maturation of anthers and pistil (dichogamy). Schmid has brought out an excellent review of the work on pollination biology done for over two centuries and has traced the evolution of approaches providing invaluable information.2 Even the summary of the enormous information and insights contained in Schmids’s account is difficult to accommodate in this paper. The contribution of the Indian biologists to the fundamental and applied aspects of pollination biology has been discussed in detail by Shivanna and Mohan Ram.3 This essay outlines the aspects of plant-pollinator interaction, highlights the current trends of pollination ecology and exemplifies some of the recent research carried out in India. This is a small but sincere effort to honour the legendary biologist Charles Darwin, whose bicentenary is being observed throughout the world. His work has stimulated biologists around the world and has dramatically changed our understanding and outlook on organic evolution and has brought respectability to biology as an autonomous science.3a

Resurgence of interest in pollination ecology The present day interdisciplinary field of pollination ecology is largely a legacy of the fascinating records on pollination gifted to posterity by Sprengel and Charles Darwin.4–7 Kölreuter was the first to establish the notion that insects are crucial for seed set in certain plants.8 He even carried out artificial hybridization between two plants. Later, Darwin (1859, 1862) used pollination as an example for establishing his theory on adaptations and co-evolution.5 His books on floral biology and pollination—The various contrivances by which orchids are fertilized by insects5; The effect of cross and self-fertilization in the vegetable kingdom6 and The different forms of flowers on plants of the same species,7 attest to his sharp powers of observation connecting the meaning of form and function and his deep understanding of the significance of pollination in flowering plants. One of the most astounding observations made by Darwin was the prediction of a possible pollinator that would pollinate a foot and half-long spurred orchid, Angraecum sesquipedale, in Madagascar.5 Although he could not locate the pollinator, what foretold was later successfully confirmed in 1903 with the identification of a moth, Xanthopan morgani predicta which had a 22-cm-long tongue to sip out nectar seated deep in the spur of the orchid and effect pollination in the process. These studies elaborated the functional significance of the morphology of the flower and have triggered unending studies on exploring the pollinators and their ingenious contrivances. The saga still continues and the secrets appear never ending. By the middle of the twentieth century the unification of approaches used in studying the functional floral morphology and the evolutionary aspects

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gave fresh impetus to pollination biology.9 Several books such as Flower pollination in the Phlox family,10 The Principles of Pollination Ecology11 and the edited volumes – Handbook of experimental pollination biology;12 and Pollination biology13 – have been instrumental in the resurgence to investigate and validate various aspects of pollination biology with novel approaches. Recently, the techniques to pursue pollination biology have been updated in compilations—Pollen biology: a laboratory manual14; Pollination ecology: a practical approach15; Techniques for pollination biologists16 and Field methods in pollination ecology.17 In the past one decade, there is renewed drive to protect mutualism in the tropics so that loss of diversity in the fragmented areas could be arrested.18 This is particularly so because tropical areas abound with outbreeders (including self-incompatible and dioecious or subdioecious species, particularly trees), with obligatory reliance on pollinators.19 Pollination biology in India has been largely pursued as a component of reproductive biological studies. Considering the extent of diversity and the potential of pollination in the maintenance of agroecosystems,20 the information on pollination from India is abysmally little.21 In recent years there has been increased realization to conserve bioresources in the hotspot regions of India. Several economically important species including those which provide non wood forest products (NWFPs) and timber have been investigated.22–27 Certain oleo-gum-resin yielding trees such as Acacia senegal, Boswellia serrata, Butea monosperma, Commiphora wightii and Sterculia urens from the dry deciduous ecosystems have shown interesting pollination syndromes, sexual systems and breeding strategies. In a community-wide survey of pollination modes in the trees of Kakachi forest in Western Ghats, Devy and Davidar28 have shown that ~75% of the trees are specialized to a single pollinator group and the remaining are polylectic; the diversity in pollination modes is comparable to other lowland tropical forests of the world. At present, there are no studies from India, which have covered the entire diversity of life-forms to establish the community level pollination-systems and the pollinator networking.

Biotic pollination A great majority of plant species are pollinated by animals (zoophily) rather than water (hydrophily) or wind (anemophily). This is obvious due to the co-occurrence of enormous plant and animal diversity in the tropical and subtropical regions where resources are abundant. Pollinators in general seek for rewards among which pollen and the nectar are the primary resources. Besides these, some foragers collect wax, oils, resins as floral rewards;29–31 others find a brooding place for their own reproduction and survival.

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Entomophily is most widespread and among these, bees (tribe Aipdae) being the most important and efficient pollinators.18,32 More than 66% of the 1500 crop plant species of the world are pollinated by bees, which essentially account for 15–30% of food production.33 It is believed that the social behavior of maintaining a huge colony and the need to provide pollen and honey to developing larvae have contributed towards the evolutionary specialization of the body parts in the bees. Among the honeybees, the giant Asian honeybee, Apis dorsata (Fig. 1C), is the predominant pollinator and is also capable of foraging in full moon light conditions in the night.34,35 Other Apidae members – Apis mellifera, Apis cerana, Apis florea and Trigona spp. are the other important pollinators of plantation crops in the tropics.36,37 Strobilanthes kunthianus is a semelparous species that comes to flower once every 12 years in the southern part of Western Ghats (Kerala/Tamil Nadu border). Mass flowering in the species attracts a variety of insects comprising bees and butterflies. However, only Apis cerana indica and certain ants are the frequent foragers and are the efficient pollinators ensuring 100% pollination and high seed set.38,39 Flowering behavior in bamboos has always been a botanical curiosity as it affects several dynamic features of the ecosystem. Bees are believed to be the major pollinators in the species so far studied,40 although the role of wind can not be ruled out in many of them. For example, Jijeesh et al. have reported wind pollination in Pseudoxytenanthera monadelpha.41 Butterflies frequently visit a number of herbaceous and woody flowering plants. However, only a few plant species are effectively pollinated. In Western Ghats, it has been shown that four out of the 86 tree species are primarily pollinated by the butterflies.28 Anitha and Prasad observed ~16 species of butterflies in gregariously flowering S. kunthianus, but none appeared to be involved in pollination.38 In India, ornithophily was reported in a few species belonging to families Bignoniaceae, Fabaceae, Loranthaceae, Malvaceae, Myrtaceae and Verbenaceae.42 Subramanya and Radhamani have reviewed the reports on plants that are visited by birds and bats to forage their flowers.43 Nearly 60 species of birds visit around 100 species and in the majority (~80%), more than one species of birds visit the flowers. However, only a few are believed to aid in pollination44 and others may act as nectar robbers.45 Thus, merely foraging or visiting of birds is not an indication of successful ornithophily in a plant. For example in B. monosperma, out of seven species of visiting birds, only the purple sunbird (Nectarinia asiatica) is effective in bringing about pollination.25 Most of the birds are reported to be nectar robbers.46 Thus, to establish the obligate necessity of birds as pollinators, it is essential to analyze their foraging behavior, pollen load and importantly, whether or not their visit to unpollinated flowers results in fruit-set. Among the variety of animal pollinators, there are unbelievable pollinators such as cockroaches,47 mosquitoes48 and lizards.49 Even giraffes were suggested to

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be pollinators of the knob thorn Acacia (A. nigrescencs).50 However, a subsequent study has shown that foraging by giraffes leads to low fecundity and only bees are effective.51 A frequently cited mode of pollination in the text-books is by snails and slugs. Malacophily is among the rarest instances of pollination.52 Snails are largely implicated as pests of crops and other plants as they eat away the fleshy floral organs. However, in Volvulopsis nummularium (Convolvulaceae), a rainy season prostrate herb that occurs in Delhi region, the pollination mechanism is present as a strange guild involving a terrestrial garden snail (Lamellaxis gracile) (Fig. 1D) and honeybees (Apis cerana indica), both of which contribute to successful cross-pollination and fruit set.52 On cloudy days the bee visits were lacking and on the bright sunny days the snails come first followed by the bees. Although the plants are self-compatible, obligate autogamy is prevented by ensuring crossing, mediated by snails and bees. This is the first report on malacophily in India. There is need to explore the possibility of more cases of malacophily in the wet and humid areas where snails are abundantly found. Thrips were mainly considered phytophagous insect pests. While demonstrating the significance of change in the colour of flower in an inflorescence of a particular variety of Lantana camara, Mathur and Mohan Ram53 established the role of thrips in pollination (thripophily). Several instances of thripophily have been reported from the members of Asteraceae, Solanaceae and Fabaceae54, reaching to the conclusion that thripophily is a common phenomenon in nectarproducing flowers and tropical forest trees of the genus Shorea in Malaysia.55 Although mediation of pollinators is mandatory in obligate outbreeders, their role is essential in certain self-compatible plants as well. For example, certain self-compatible leguminous crops exhibit autosterility because of the presence of a thick cuticle over the stigma. In these plants, visit by insects (tripping) causes rupturing of the cuticle over the stigma to release the stigmatic exudates. The latter facilitates germination of self-pollen.56 Conversely, insect visitation may induce dehiscence of anthers. In Incarvillea emodi (Bignoniaceae), nectar foraging by Apis mellifera, pressurizes the appendages of anthers to release the pollen grains and carries them while moving out of the flower.57 Pollen shedding is negligible in the absence of the pollinator.

The question of legitimacy Faegri and Pijl proposed a beautiful correlation between the flower types that animals prefer to visit.11 This relationship is termed “pollination syndrome” and essentially refers to suites of flower characteristics (morphology, colour, nectar and/or pollen as food) that attract a particular pollinator to visit them and discourage others from accessing the rewards or visit them. This means that bright

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Figure 1. (A) Flower colour signals and pollination. Top view of a variety of Lantana camara showing unopened pink-tipped flower buds in the centre, freshly opened yellow flowers ready to be pollinated, surrounded by post-pollinated flowers that have changed colour to prevent the foragers Figure 1. (B–E) Diversity in pollination modes. (B) Bat pollination in Oroxylum indicum, photographed late 9 in the evening using a tele-lens and a flash). A short-nosed bat, Cynopterus sphinx, approaching a flower bud in the inflorescence. Assured of the source of nectar, the bat develops constancy with the flowers and forcibly opens the corolla to sip the nectar: (C) The giant Asian honey bee, Apis dorsata, is the most frequent pollinator of many wild and domesticated plants. This picture shows a honeybee foraging the male flowers of kusum, Schleichera oleosa, a source of lac resin: (D) Enlarged view of a terrestrial garden snail (Lamellaxis gracile) with a conspicuous shell foraging the flower of a garden weed (Volvulopsis nummularis). The snail consumes the anthers and pollen but does not damage the pistil. Pollination occurs when the snail transfers the pollen deposited on the shell to the stigma of the same or of another flower resulting in seed-set. (E) Pre-anthesis cliestogamy in Polypleurum stylosum (Podostemaceae). Disposition of stamens and the pistil in the flower of the aquatic plant (spathe has been removed). The anthers are entangled with the bilobed stigma to facilitate autogamy. See text for details

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coloured flowers with copious nectar that lack fragrance and bloom during the day time are likely to be visited by birds to result in pollination, and such plants are called ornithophilous. Similarly plants pollinated by ants (myrmecophily), beetles (cantherophily), flies (myophily), dung and carrion flies (sapromyophily) honeybees (melittophily), butterflies (psychophily), moths (phalaenophily), snails and slugs (malacophily), reptiles, birds (ornithophily) rodents (therophily), marsupials and bats (cheiropterophily) are characterized by their respective pollination syndromes. Among these, the bats, moths, geckos and owls are associated with nocturnal pollination.58 Some experimental studies have shown that the syndrome concept holds true in certain cases and it usually helps in designing the field-based experiments59,60. Most of the flowers with restricted floral phenologies and complex structure may follow such a specialized relationship. For example, in Oroxylum indicum occurring in North India, there is a specialized relationship and the interaction follows the concept of pollination syndrome. The flowers are bat-pollinated (cheiropterophilous), dull-coloured, emit a fetid smell and bloom only for a short period in the night. As the floral phenology is confined to night time, bats gain access to the long erect racemes above the canopy (Fig. 1B). The structural organization of the flower, the placement and angle of the flower on the inflorescence axis allow only a suitable sized bat – Cynopterus sphinx to legitimately forage the flower and prevent over-sized bats to do so.61 In Malaysia and Thailand, a different species of bat, Eonycteris spelaea, with similar size and foraging behavior has been reported earlier.62, 63 This study establishes that O. indicum has strong affiliation of the plant for bat pollination and the principal pollinator of specialized flowers may vary in time and space. Similarly, Ravenala madagascariensis (Strelitziaceae), popularly known as the traveller’s tree, is shown to be pollinated by lemurs in Madagascar,64 while elsewhere its flowers are either explosively bird pollinated65 or foraged by large squirrels in Brazil (personal communication). In India, this plant produces seeds in Wayanad, (Kerala) (personal observation). The pollinator is yet to be established. The extent of extreme one-to-one relation, known as nursery pollination syndrome, is exhibited by a few species such as Ficus and their agaonid wasp pollinators66 and Yucca and Tegiticula,67 the moth pollinator. In nursery pollination syndromes, the flowers become the brooding site for pollinators. These extreme specializations are fragile as both the counterparts may perish if one were to disappear. However, recent studies have shown that the syndrome concept is imperfect in many cases, as several pollinators of distant taxonomic groups may visit the same flower with equal efficiency. Additionally, the syndromes are defined as we perceive the flowers and their colours and not as the pollinators see them.68 In Butea monosperma, popularly known as the “flame of the forest” for usually possessing bright India-orange coloured flowers, is “conceptually” meant

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for bird pollination. Detailed pollination ecology of the species has established that successful pollination requires legitimate foraging (pollination in return of rewards without damaging the flower) because the nectar is deep seated in the keel petals and the margin of the keel is the only opening to access it.25 Among a variety of birds, only the purple sunbirds (Nectarinia asiatica) are successful to legitimately do so. Besides the sunbirds, the common Three-striped squirrel, Funambulatus tristiatus, is equally effective in pollination. Instances of co-pollination in the otherwise specialized flower-bearing plants are increasingly mounting, indicating that a generalized relation involving one plant and many pollinators is believed to be of more common occurrence.69–71 Finally, the mismatching in the components of the syndrome concept is exemplified by the instances of ambophily. In Plantago lanceolata, 72 Hormathophylla spinosa,73 Buxus balearica74 exhibit ambophily, in which flowers are pollinated by insects as well as wind.75 A recent assessment of syndrome concept based on six communities from three continents has shown that pollination syndrome does not describe the diversity of floral phenotype or predict the pollinators of plant species.60

Floral display and cues Flowers advertise in a number of ways to establish a relation with the pollinators and optimize pollination efficiency. Besides the size and arrangement of flowers in an inflorescence, scent and pigmentation of petals are the crucial attractants.11,76–78 A small solitary flower may not have the same effect on a forager than a group of small flowers with compacted arrangement in an inflorescence. In such plants, the inflorescence serves as pollination unit.79 In acacias, individual flowers are very small; their compact arrangement into a long spike or globose inflorescence not only imparts a mass effect but also results in intense fragrance around the flowers and rapid pollination.80 Repeated visits by the foragers make them learn the cues81 to develop floral constancy.6, 82 Constancy with flowers by the foragers is acquired by learning experience and leads to targeted pollination of flowers of the conspecific plants while minimizing the loss of pollen. Floral constancy is more pronounced in mature foragers suggesting that they learn flower handling initially by visiting a number of flower forms and memorizing before narrowing on to a particular one or visit similar forms in different species.83 By using mutant lines in snapdragon (Antirrhinum spp.) that lacked only the conical cells on the petals, Whitney et al. showed that the conical cells assist in the landing of the bumblebees.84 The foraging bumblebees could use colour cues to discriminate between the petals with flat surface from those having conical cells.

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Besides the major floral contrivances that result in various forms, pigmentation on the petals has a significant effect on pollination efficiency. This has been aptly demonstrated by developing near isogenic lines (NIL) in two sister species of monkeyflowers (Mimulus spp.).85 The wild-type M. lewsii has pink flowered flowers and is pollinated by bumblebees, whereas M. cardinalis is red-flowered and is pollinated by hummingbirds. The Yellow Upper (YUP) allele at a single locus controls the yellow carotenoid pigments in the petals of pink flowered M. lewsii. NIL Substitution to alternative alleles in these species showed that M. cardinalis NILs with M. lewsii YUP allele had dark pink flowers which attracted 78-fold more bee visits than by hummingbirds. The M. lewsii NILs with yup allele had yellow-orange flowers which attracted 68-times more visits by the hummingbirds than on the wild type. Flowers often convey information about the presence or absence of rewards by means of changing their petal colour. In Lantana (Fig. 1A), the freshly opened flowers are yellow and attract thrips, butterflies or even birds (such as red-vented bulbul) to nectar rich yellow flowers whereas the pink flowers lack nectar and the change in the colour is caused by pollination and production of delphinidine glucoside.86 Change in colour is not always unidirectional. In Desmodium setigerum, tripping initiates change in colour from lilac to white. If the flower remains unpollinated even after tripping, it reverses its colour back to deep turquoise or lilac for a second time to receive pollen.87 Preference to yellow colour by other insects such as bumblebees was demonstrated in an ingenious experiment conducted by Chitka and Walker.88 They employed untrained bumblebees bred in dark for generations and exposed them to large prints of famous paintings – “Sunflowers” by Vincent van Gogh, “A vase of flowers” by Paul Gauguin, “Pottery” by Patrick Caulfield and “Still life with a beer mug” by Fernand Léger. van Gogh’s sunflowers with predominant yellow colour proved most attractive to the bees. Olfactory cues (fragrance) are an important attractant to pollinators, which intensify at the peak time of blooming. The floral scent emanates from the secretory tissue termed osmophores,89 which may contain 1–200 volatile compounds.90 A selective role of scent in attracting the pollinators and repelling the non-pollinators has been suggested by many workers.91,92 Attraction may be particular type of species or rewards91 and even to the unpollinated flowers for a second chance of pollination.93 Floral scents in many orchids (~30,000) that offer no reward94,95, mimic the pheromones of female insects to sexually deceive the male insect pollinators.96 In an Australian orchid, Chiloglottis trapeziformi,97 a novel compound, chiloglottone mimics the pheromone of the female wasp (Neozelboria cryptoides) to sexually deceive the males. In Ophyris sphegodes, by using gas chromatography-electromagnetic detection methods, Schiestl et al. have shown that there are 15 compounds which elicit electroantennographic

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responses in male antennae and copulation attempts in the male bees (Andrena nigroaenea).98 The experiment demonstrated that these compounds were similar to those present in the cuticular wax for protection. Generally the bird-pollinated flowers lack fragrance.11 However, flowers of several hummingbird pollinated species contain volatiles in their floral headspace, which are common to vegetative parts.99 These studies indicate that during the course of evolution the compounds present in the vegetative parts assumed the function of attracting pollinators in one way or the other.

Nocturnal pollination studies in India Research on nocturnal pollination is beginning to be taken up in India. In earlier studies,100 only cursory observations were made on visits made by bats to flowers. Whether or not their visits lead to fruit-set, was not reported. This is understandable because of challenges posed by nocturnal observations, poor technology for photographing/filming in darkness and above all by assuming that bats are probably more efficient diaspore dispersers than pollinators.28 Out of presumably 28 bat visited plants43 (Subramanya & Radhamani 1993), only two – Ceiba pentandra (Bombacaceae),101,102 and recently Oroxylum indicum (Bignoniaceae) have been investigated from India.61 Most bees are diurnal pollinators. However, recent work103 has demonstrated that carpenter bee, Xylocopa tenuiscapa, is capable of effecting pollination in dark in the self-incompatible Heterophragma quadriloculare. Somanathan and co-workers have shown that a nocturnal bee Xylocopa tranquebarica from the seasonal cloud forest in India can fly under starlight conditions and is even endowed with colour vision under extremely dim light.104

Pollination constraints in the introduced crops Many plants suffer from serious losses in fruit and seed production due to inadequate pollination. In hermaphroditic species, the combined effect of the distribution pattern of individuals in the population and the extent of index of self-incompatibility limits fecundity. In dioecious plants, seed set may vary due to skewed distribution of sexes in the population and if the species is wind-pollinated, inclement weather conditions may also affect the yield. Absence of legitimate pollinators of plants in the wild may also adversely affect fruit-set.57 Yield can be sustained in crop plants through artificial pollination or by increasing the pollinator visitation rates. In the East African oil palm (Elaeis guineensis) presently grown on a commercial scale, fruit-set is accomplished by wind as well as by insect-pollination. In Malaysia, fruit yield was initially low because the specific weevil pollinator, Eledobious kamaroonicus, had not been

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introduced along with the crop. Introduction of imported weevils drastically improved the fruit-set in the plantations. Oil palm exhibits temporal dioecism at the population level, as it bears male and female inflorescences in alternate years. The yield depends upon the extent of balanced sex expression in a season. In bad years, when there are few male plants in the plantation or under inclement weather conditions, the yield is low. Work done on oil palm in India has shown that assisted pollination sustains the regular yield.105 Oil palm pollen grains respond to long term storage for 8–10 years under cryo (–198°C) conditions.106 The other approach to improve insect visitation rates include enhancement of native pollinator populations. Significant improvement in the yield was noticed in clover (Trifolium pratense) after the nesting sites of its pollinator, Bombus spp. were increased around the crop field.107,108 Pollination may become a major constraint due to competition between native wild bees with the introduced colonies of domesticated honeybees, rendering poor yield at places where agricultural fields are managed in the vicinity of natural populations. Intensification of agricultural fields and increased reliance on domestic bees may adversely affect the native pollinator diversity in a particular area. For example in the Western Ghats, lesser cardamom (Elettaria cardamomum) is extensively planted with areca nut as a cash crop. On the basis of observation made at three sites, Sinu and Shivanna showed that the wild stingless bee, T. iridipennis serves as the main pollinator of cardamom at sites where the competition with domesticated honey bees is low.37 Urgency to conserve the native pollinators is being realized as insurance because of serious decline in the populations of domestic bees such as Apis mellifera, owing to diseases and heavy use of insecticides.109,110

Abiotic pollination In this category, wind pollination (anemophily) is predominant and water pollination (hydrophily) occurs in a few genera.111,112 Wind pollination Wind pollination is found in nearly 10% of the families in flowering plants.113 Based on phylogenetic analyses, it is believed that anemophilous plants are derived from their entomophilous ancestors114,115 and probably at 65 independent times.116 In general, wind-pollinated plants produce unisexual flowers, copious amount of dry pollen and occupy more open and wind dominated habitats.75 However, in tropical environments airborne pollen have also been recorded from entomophilous, especially the buzz pollinated species117 or plants with ambophilous floral traits.118 In these plants foraging by insects facilitates dispersal of pollen grains in the air but the may not actually pollinate the flowers.119 However, in certain Indian grasses

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pollen transfer to stigma has been observed.120 In closed tropical conditions wind pollination may not always correlate completely with anemophilous syndrome. In wind-pollinated Cicca acida and Emblica officinalis floral traits are conducive enough for successful release and trap airborne pollen but in the deciduous Madhuca indica and evergreen Mimusops elengi, they do not.121 As natural conditions vary in the distribution range of these species, it is important to assess their breeding systems, particularly the extent of autogamy, from different areas to establish the prevailing adaptive value of their floral traits. Unlike animal pollinated trees, wind-pollinated species suffer from acute pollination limitation in natural populations especially if the plants are sparsely distributed and are self-incompatible.122 In ambophilous oil palm, the airborne pollen density varies according to the number of male plants in the plantation. During the male dominated phase pollen density is limited up to 30 m from the source.105 The dispersal distance may vary according to the source height of the plants123 and habitat conditions. There is paucity of information on the pollination dynamics of wind-pollinated species form India. Individual contribution to total fecundity from wind and biotic pollination is important but difficult to gather from tall trees, as they can not be contained for establishing the exclusivity of modes or limitation of pollination as in the herbaceous species. Friedman and Barrett recorded that at least in nine out of ten herbaceous wind-pollinated plants, pollen was not a limitation.122 Water pollination Hydrophily occurs in nearly 30 genera18 and is believed to be a rare phenomenon. Many aquatic plants such as Cabomba, Nymphoides, Hottonia, and Potamageton exhibit entomophily, rather than hydrophily. Depending upon whether the flowers are exposed to air before pollination or remain submerged, hydrophily is either ephydrophily or hyphydrophily, respectively. Hyphydrophily is usually shown by marine angiosperms such as Amphibolis antartica, Zostera marina and Najas. Ephydrophily is more common and reported in plants such as Lemna trisulca;124 Vallisneria;125 Ruppia, Callitriche, Elodea and Hydrilla.126 In Ceratophyllum, plants are completely submerged and pollen grains are not released until the abscised anthers float up to the surface of water. The germinating pollen grains are released after the dehiscence of anthers and the elongated pollen tubes sink to effect pollination (Mohan Ram and Sehgal).126a In Callitriche palustris and C. heterophylla,127 the unisexual flowers are borne on the same plant, but in separate leaf axils. When the plants are completely submerged, the pollen grains have been noticed in a few cases to germinate inside the anthers of the partially developed male flowers and their tubes traverse through the filament and reach the ovule of the pistillate flower to effect fertilization; this mechanism is known as internal geitonogamy.

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Podostemaceae, inhabiting fast-flowing steams, rivers and cataracts attached firmly to the rocks, is the largest family of aquatic angiosperms and its members occur in the tropical and the sub-tropical regions of the world. Pollination system in Podostemaceae has been studied in a few species and variously interpreted. Since the majority of species have small, naked flowers with closely placed stamen and pistil, spontaneous autogamy seems to be the most probable mechanism.128–130 However, there have been reports of insect pollination in Mourera and Tulasneantha131 and speculations of wind pollination in Marathrum rubrum132 and some new world Podostemaceae.133 Cleistogamy and pre-anthesis cleistogamy (Fig. 1E) have also been reported from Podostemaceae134-136 which further corroborate the general occurrence of self-pollination in the family. Unfortunately, the family has not received the much warranted attention and still remains a neglected group of plants for further studies. Mutualism at peril The success of pollination event in a species is believed to be an evolutionarily stabilized mutualistic episode and in a community, pollination systems of different species contribute to a pollination network. As the collection of rewards from the flowers is intricate in the ecological network of animal-food web,137 even minor changes in the environment may disproportionately affect the dynamics of pollination systems. Effect of climate change, particularly global warming, is being best witnessed in the shifts in the flowering period of many plant species.138,139 Although detailed long-term phenological observations in the tropics are deficient,138 recent changes in the tropical cloudiness140 and monsoon dynamics141 may seriously affect the reproductive biology of many plant species in the tropical belt. Several tree species in northern India such as, Rhododendron and Reinwardtia are reported to have advanced their blooming period by nearly a month. In many tropical deciduous trees of India, in which the reproductive strategies have developed in response to monsoonic bioclimate,142 early flowering (Madhuca latifolia, Mangifera indica) to prolonged flowering (as in Cassia fistula) has been observed (personal observation). Such changes and shifts are likely to create gaps in the flowering episodes at the community level that may allow invasive plants to occupy the flowering niche. As the invasive species are known to proliferate intensely, the divergence in mutualism is likely to affect the native plant species. Many invasive plants such as Ageratum conyzoides, Chromolaena odorata, Eichhornia crassipes, Eupatorium adenophorum, Ipomoea carnea, Lantana camara, Mikania micrantha, Mimosa invisa, Parthenium hysterophorus and Prosopis juliflora have naturalized in several parts of India and are threatening the natural communities. Chromolaena odorata has invaded in the entire buffer area of silent valley (personal observation). The impacts of such plants on the

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pollinator diversity are yet to be analyzed and strategies are to be developed to eradicate their dominance in the species rich areas. Conclusion Evolution and the subsequent radiation of flowering plants is a reflection of their ability to adapt to a variety of habitats and their diversity and often flexibility, in the modes of reproduction. The enormous diversity in flowering plants amazed Charles Darwin and he discussed many of his careful observations based on evolution of co-adaptive traits in plants and their pollinators. The fascinating observation made by him on pollination mechanism in orchids is still an inspiration to pollination biologists. The dependence of flowering plants on pollinators, including a variety of animals and abiotic modes for pollen mediated gene flow has motivated biologists to use interdisciplinary approaches to explore the fascinating area of pollination biology. Amidst fresh challenges such as - habitat modification, climate change declining biodiversity, assessment of genetic resources in related gene pools, management of agroecosystems while sustaining future food security,143 gene flow from the genetically modified organisms and threats from the invasive plants, there is need to recombine conventional approaches with powerful molecular tools to design future course of investigation in pollination biology. It is ironical that Indian efforts on pollination biology mismatch the magnitude of the country’s biodiversity and its deep concern on the conservation of the flora and enhanced crop production. Acknowledgment: We thank Ms. Priyanka Khanduri for her able assistance in the preparation of this chapter.

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15 Darwinian evolution and post developments in genomics Arun Kumar Sharma Center of Advanced Study, Department of Botany, University of Calcutta, Kolkata - 700019, India

Abstract: The status of Darwin’s theory of Natural Selection in ‘Origin of Species’ and the impact of recent developments in genetics on evolution form the theme of the paper. Despite the conflicting views of the role of mega and micro evolution as visualized by Darwin, his theory, followed by theory of inheritance and chromosome theory of heredity form the very foundation of our knowledge of evolution and continuity of life from generation to generation. In the post Darwinian phase, the importance of the regulatory sequence in the complex expression of genes controlling evolution has been emphasized. The role of high amount of non coding repeat sequences as well as mobile sequences in evolution has been discussed. Finally, the emergence of RNA as the primitive molecule of life and the present role of small RNAs in defence, growth, differentiation and indirectly evolution of species have been indicated. Keywords: Evolution, Natural selection, Post-Darwinian genetics

Basic concept The discourse on evolution has all along been a topic of special interest to any biologist dealing with the origin of life itself. Since the origin of living organisms, and gradually the diversification to eukaryotic systems, Darwin’s theory of Natural Selection and Origin of Species1 and later the Descent of Man stood the test of time and almost universally accepted. The origin of species arising through random minute variations, and selection of the fittest through struggle for existence were visualized. Darwin fully believed that evolution follows a tree like growth with different branches of tree representing separate forms of life.2 However, even after the publication of the ‘Origin of Species’, 150 years ago, there have been nine different species concept, and according to some even more. Each concept has its merits and demerits but the most universally accepted is that ‘complete reproductive isolation’ is the crucial parameter for future of V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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speciation.3,4 Mayr’s concept of species5 involves ‘potentially inbreeding natural populations which are reproductively isolated from other such groups’6 concurs with the genic view of speciation on principle, that is, the evolution of reproductive isolation barrier with divergence as the key factor. However the importance of allopatry in reproductive isolation is an important factor as well. Fortunately, within a few years of Darwin’s publication of the paper in Origin of Species, Gregor Mendel’s theory of inheritance was published which formed the base of our knowledge of heredity. Just as Darwin’s theory envisaged the continuity of life through preexisting ones, Mendel’s ideas provided a base for their inheritance from generation to generation.

Darwin and Mendel There has, however, been efforts at post-Darwinian enthusiasm to bring together Mendel’s laws of heredity7 and Darwin’s on evolution. Several authors were in favour of Mendel but some biometricians accepted Darwinian selection but rejected Mendelism. Views were also expressed that natural selection as ineffective and evolution as occurring through macro mutation or rather gene change with major effect. Hybrids and polyploidy too in speciation were known to be widespread in plants. Even Ernst Mayr and Theodosius Dobzhansky suggested major changes rather than a continuum. In any case, (i) the laws of inheritance of Mendel,7 (ii) chromosome theory of heredity,8 and (iii) the establishment of gene concept,9 along with natural selection, without underestimating the importance of genetic drift and isolation, form the very foundation of our understanding of evolution – the origin and development of life forms in this planet. The later discovery of the double helical nature of the gene by Watson and Crick10 in 1953 and genetic code showing that the code is same in lilies, mosquito and man, brought distinct evidence of life – all united in a common descent. Evidence of extra planetary life beyond earth, however, cannot be ruled out. In later years, the chemical nature of the genes, and gene action, and their role in the expression of characters were lucidly manifested in the Central Dogma of DNA → RNA → Protein. The genetic code, and the simplicity embodied in it, provided the very chemical basis of evolution enshrined in Darwin-Mendelian concept. Undoubtedly the Laws of inheritance, Chromosome Theory of Heredity and Gene concept provided a solid foundation of genetic and chemical basis of both mega and microevolution of Darwin. However, the modality of bringing alterations both major and minor, has been greatly influenced by the gene structure of eukaryota on the one hand and novel gene sequences, forming major components of chromosome structure on the other. Of these, Repeated DNA sequences11–13 and Transposons14–16 need special mention because of their role in evolution.

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Recent developments on genetics with impact on evolution Regulatory sequences The coding regions of genes are no doubt the materials for evolution, but simultaneously evidences came up indicating profound role of regulatory DNA — the ‘cis element’ as a crucial factor in evolution. As genes control all characters, the expression of a character is the result of a series of chemical reactions set up and triggered at the gene level. Moreover, as demonstrated, each character is controlled by many genes, which are not necessarily located at the adjacent site and even at the same chromosome. The expression is thus coordinated by regulatory sequences – the cis elements – lying often far away from the target genes. The mutations in regulatory elements, the ‘cis elements’17 underlie many alterations and may provide the organism to have a long life and such DNA may not necessarily be located very near the genes. It is evident that mutations in coding regions and cis-regulatory changes both play a role in evolution. This is true also for several mammals which are similar in genomic content and proteins, but the difference is more due to the product of changes emanating out of – when, where and to what degree these genes function and express. Arguments have been put forward that (Sean Carrol, University of Wisconsin, vide Pennisi17 that genes involved in body patterns and plans are so wide ranging, affecting variety of tissues, at different stages of development, that a mutation in their coding region may create catastrophe. But rather changes in ‘cis’ elements which often bring about coordination and control have specific limited effect. Microevolutionary changes can be traced to cis-regulatory sequences too. Each element may serve as the base for a peculiar ‘transcription factor’, stimulating or multiplying gene expression. Because of this effect of modulation, there can be innumerable cis element combinations which allow genes to express in specific time, space and degree. There are several cases in plants where cis elements have proved to be very important. Teosinte the ancestor of modern maize had several stalks as compared to single stalk of corn. This change has been shown to be linked to a difference in DNA sequences17 – several thousand bases away for the gene ‘teosintebranched’. This indicates the role of non-coding ‘cis’ elements in corn evolution. As each character is the result of multiple effects – a mutation in cis-regulatory sequence much removed from the original site is crucial for evolution. This fact has been taken to explain why chimpanzee and man are so similar in genomic content but difference in general, is due to the function and expression of the genes at different time, stage and extent, that is, when, where and to what degree, these genes should express.

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In the light of the evidences obtained so far, the role of regulatory sequences such as transcription factors in species adaptation and evolution can hardly be overestimated. These ideas are still in a nascent state but the importance of coding and regulatory sequences have been clearly demonstrated in the evolution of species.

Gene structure The single gene – single protein concept of Beadle and Tatum18 needs to be looked at with the establishment of split nature of the gene made up of intron (non essential) and exon (essential) sequences.19 Despite the fact, that normal messenger processing following transcription is the rule, alternate splicing resulting in different combinations of exons as recorded to occur under certain conditions, result in more than one protein from a single gene locus. Latest studies, indicate that exons from two different genes may also undergo fusion to yield a fused transcript.20 Further complexity is added by RNA editing whereby the Guide RNA incorporates different nucleotides in the messenger transcript, before processing after transcription. As a result, the resulting protein is quite different from the protein, coded in the primary transcript from the master template of DNA. All such variations result in alterations in polypeptide structure, ultimately generating from a single gene locus. In addition, there may be ‘Post Translational Modifications’ as well which make Proteome more complex, deviating from the total message coded in the primary transcript. The variations in the structural make up of gene product significantly influences the expression of new characters involved in origin of species in Darwinian concept.

Repeat sequences of DNA Along with the further development in the understanding of structure and expression of genes, the chromosomes of eukaryotes in general, harbor huge mass of repeated or amplified DNA sequences11 occupying the major segments of chromosomes and are often termed as ‘Selfish’ or Junk21 with not much appreciable effect,13 as well as Dynamic need mention.22,23 It is presumed that these sequences multiply and exist as the cell environment is congenial for their survival. Moreover, the intergenic repeat sequences, structural obesity at the chromosome level as well as the non coding sequences and transcripts, call for a deeper analysis of their role in chromosome evolution. Conserved noncoding sequences in human system are associated with a large number of genes and their very survival for a long period of evolution may indicate a selective value. The location of some of these sequences has also been such as to indicate their possible role in brain functioning, may be even wiring of brain cells, cell signalling and growth.24 In the plant system, the repeated sequences occupy the major portion of the genome.25

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Their role in the control of non specific functions have been established.23,26 They also confer adaptation to extremely stress environment recorded in different plant genera.27 The importance of adaptation is one of the key factors in the evolution of species is evident.

Mobile Sequences Further to non-coding, repeats performing some non specific functions the mobile genes – transposons, initially studied by Barbara McClintock, capable of jumping from one segment to another has led to complexity in the concept of genome structure – its functioning and even evolution.14–16 In the plant system, in maize, transposon activity has been responsible for increasing genome size from 1 to 2.4 billion bases. In the chromosome structure, the transposons have been shown to be present in the flanking region of centromeres and telomeres and may serve as epigenetic regulators of the genome. Almost half of the human genome consists of mobile elements (LINE) which can jump around the genome. Some of these mobile elements add to diversity and are vital for regulation and human evolution.28 Mobile elements present in 50% of Primate genome indicate strong diversity and influence on evolution of genome vis-à-vis speciation.29

Gene Order: Synteny Lately new ideas have come up which have a profound influence on concepts and mechanics of species evolution. The comparative analysis of cereal genomes reveals that they are composed of a few similar genomic building blocks with conserved sequences. By simple rearrangement of these blocks, and amplification of some of the repetitive sequences contained within them, it is possible to reconstitute the chromosome complements of rice, wheat, maize, sorghum, millet and sugarcane, from the cleavage of a single chromosome.30,31 This similarity in the genetic maps of several plant genomes and the order of DNA sequences is termed ‘synteny’.30 There is striking collinear conservation in the gene order, between wheat, maize, sorghum, sugarcane, millet and rice, even though they differ in size, chromosome number and taxonomic distance. The existence of an ancestral order of all the DNA sequences possibly in one chromosome in the grasses, if not in the entire plant kingdom has been tentatively presumed. Similar syntenic arrangements have been recorded in animal system as well. The role of synteny in evolution of species can hardly be overestimated.

RNA world The most significant development in Post Darwinian Mendelian phase is the understanding of the RNA world.32–34 It is visualized that the first molecule to

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appear on earth in the primitive, dark anaerobic inhospitable world about 4 billion years back was a RNA molecule. The DNA came much later about, 3.6 billion years back where the role of RNA molecule became principally as the messenger. Lately, new classes of RNA have been discovered namely, micro RNA (miRNA) and Short interfering RNA (siRNA).35–37 Micro RNAs These RNAs, 20 to 25 bp long, are coded by the genome and couple with mRNA for silencing. Its role in transcription, silencing, viral resistance and metazoan evolution is now well recognized.38–40 The role of short RNAs in checking transposon activity is also demonstrated. In fact, later findings may show that41 there is a direct correlation of increasing morphological complexity with micro RNA number viz., sponges (8), sea anemone (50), Drosophila (147) and human (677). The antiquity of micro RNA can hardly be questioned as their origin is rather evident in metazoan evolution before multicellularity emerged.

Conclusion All these developments at the post-Darwinian era in the recent past, ranging from single nucleotide polymorphism to split genes, repeat DNA and mobile sequences, have direct bearing on the minute variations which Darwin witnessed as well as major changes. But the discovery of the RNA world – the initiator RNA molecule of evolution and later the tiny world of RNA as mentioned above surpass all others in their impact on evolution at the post-Darwinian phase. It is visualized now that non coding RNAs may represent a rich substrate for innovations in evolution in eukaryota.42 More startling is the fact, that despite their established antiquity almost prior to metazoan evolution these molecules are exerting at present a profound influence on growth, differentiation and indirectly the evolution which Darwin visualized, through their involvement in chromatin dynamics and gene regulation.

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16 Pathogen pressure and molecular evolutionary genetics of innate immunity genes in humans Partha P. Majumder Indian Statistical Institute and TCG-ISI Centre for Population Genomics, 203, B. T. Road, Kolkata - 700108, India [email protected]

Abstract: We report results of an extensive and comprehensive study of genetic diversity in 12 genes of the innate immune system in a population of eastern India. Almost half of the 548 DNA variants discovered was novel. DNA sequence comparisons with human and chimpanzee reference sequences revealed evolutionary features indicative of natural selection operating among individuals, who are residents of an area with a high load of microbial and other pathogens. The haplotype structures in India are significantly different from those of European-American and African-American populations, indicating local adaptation to pathogens. Most of the human haplotypes are many mutational steps away from the ancestral (chimpanzee) haplotypes, indicating that humans may have had to adapt to new pathogens. We have tested the opposing views concerning evolution of genes of the innate immune system that (a) being evolutionary ancient, the system may have been highly optimized by natural selection and therefore should be under purifying selection and (b) the system may be plastic and continuing to evolve under balancing selection. We have found that in these genes, there is (a) generally an excess of rare variants (b) high, but variable, degrees of extended haplotype homozygosity, (c) low tolerance to nonsynonymous changes and (d) essentially one or a few high-frequency haplotypes, with star-like phylogenies of other infrequent haplotypes radiating from the modal haplotypes. Purifying selection is the most parsimonious explanation operating on these innate immunity genes. This genetic surveillance system recognizes motifs in pathogens that are perhaps conserved across a broad range of pathogens. Hence, functional constraints are imposed on mutations that diminish the ability of these proteins to detect pathogens. Keywords: DNA resequencing, Single nucleotide polymorphism, Haplotype, TLR, Defensin, CAMP, MBL2, Neutrality tests, Haplotype networks, Extended haplotype homozygosity, Purifying selection

Introduction The September-October 1997 issue of American Scientist carried a review of the book titled “Darwin’s Black Box: The Biochemical Challenge to Evolution.” V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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The book is authored by Michael Behe, and the review was written by Robert L. Dorit. I reproduce, verbatim, some sentences (italicized for convenience) from this review: “Will you honestly tell me (and I should be really much obliged) whether you believe that the shape of my nose was ordained and ‘guided by an intelligent cause’?” In exasperation, but not without humor, Charles Darwin posed this question to Charles Lyell in 1860, the year Darwin dealt with the maelstrom unleashed by the publication of The Origin of Species. In both the popular and the scientific press, Darwin had to contend with the wrath of those for whom the notion of a living world based on accident, time and natural selection was simply too disquieting. Look around, Darwin’s critics argued, and see the evidence of design. And where there is design, there must be a designer. Today, when we are able to look around at the molecular level, we find patterns; patterns that are consistent with the theory of evolution propounded by Darwin; not the handiwork of a designer. Some of our recent findings on patterns of variation at the molecular level in genes of the innate immune system are reported in this article, and we examine whether and how these patterns can be explained in the context of the Darwinian theory. Vertebrates defend themselves against pathogens using the immune system. The vertebrate immune system has two distinct arms – the innate and the adaptive. Invertebrates only possess the innate immune system. They lack the mechanisms for generating diverse antigen receptors by recombination. Therefore, invertebrates do not possess B and T cells. The innate immune system responds very quickly (usually within minutes) to an infection. The proteins involved in this system detect broad cues from invading pathogens and responds. Diverse and evolutionarily conserved families of Pattern Recognition Receptors (PRRs) of the innate immune system detect broad and conserved patterns that differ between pathogenic organisms.1 The innate immune system provides signals to the adaptive immune system to initiate response. The adaptive immune response usually becomes effective after several days or weeks after infection, but provides antigenic specificity required for complete elimination of the pathogen and generation of immunologic memory. Quick recognition of broad signatures of pathogens by the innate immune system leads to the rapid mobilization of immune effector and regulatory mechanisms that provide the host with three critical advantages2: (i) initiating the immune response (both innate and adaptive) and providing the inflammatory and co-stimulatory context for antigen recognition; (ii) mounting a first-line of defense, thereby holding the pathogen in check during the maturation of the adaptive response; and (iii) steering the adaptive immune system towards the cellular or humoral responses most effective against the particular infectious agent. Important genes of the innate immune system include cathelicidin antimicrobial peptide (CAMP), defensins, mannose binding lectin (MBL2) and toll-like receptors (TLRs). CAMP is one of the major antimicrobial peptides of

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the human innate immune system in the intestinal tract.3 Cathelicidins are small (12–100 amino acids) cationic peptides that possess broad-spectrum antimicrobial activity, and share features with defensins.3 While humans and mice each possess a single cathelicidin, other mammalian species, such as cattle and pigs, express many different cathelicidins.4 The defensins code for small cationic, cysteinerich peptides. These cationic peptides exhibit a broad-spectrum of activity against gram-positive and gram-negative bacteria, fungal species and viruses,5 by interacting with negatively charged molecules on the surface of pathogens and permeating their membranes. On the basis of the position and bonding of six conserved cysteine residues, defensins in vertebrates are divided into two categories, designated as - and -defensins.6 While -defensins are produced primarily by intestinal Paneth cells and neutrophils, the -defensins are primarily produced by epithelial cells. MBL is a calcium dependent serum protein that plays a role in the innate immune response by binding to carbohydrates on the surface of a wide range of pathogens.7 It is also an important component of the complementactivation pathway for activation of macrophages by forming membrane-attack complexes (MACs). The TLRs are a class of single membrane-spanning, noncatalytic receptors that recognize structurally conserved molecules derived from microbes once they have breached physical barriers such as skin or intestinal tract mucosa.8 They are characterized by an extracellular leucine-rich repeat (LRR) domain for ligand recognition and an intracellular tail that bears a homology to the conserved interleukin-1 receptor (TIR) for signal transduction. Ten TLRs (TLR1–TLR10), each with its own ligand specificity, have been identified. Diversification of the genes of the innate immune system have taken place during evolution possibly in response to the diversification of microbes, especially pathogenic microbes. Gene families, such as TLRs, evolved by gene duplication and individual members of these families have evolved different but related functions, possibly to protect the host against a larger set of diverse pathogens. Individual members of these gene families also exhibit high levels of polymorphism, which is consistent with Haldane’s (1949)9 prediction that maintenance of polymorphisms in genes governing host-pathogen interaction is driven by rapid rates of microbial evolution. Indeed, evidence for overdominant selection (heterozygote advantage) has been documented at the major histocompatibility complex class I loci, in which the rate of non-synonymous (amino acid altering) nucleotide substitutions have been found to be significantly greater than synonymous substitutions in the antigen recognition site.10 With respect to the genes of the innate immune system, however, there is debate whether these genes are continuing to evolve or whether being evolutionarily ancient they have been highly optimized by natural selection.11 Specific polymorphic variants in innate immunity genes have been found to be associated with human diseases (12–15) and some investigations have been carried out on the mode and tempo of evolution of some genes of the innate immune system.12–15

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Nature and extent of variation in innate immunity genes in India We have assayed inter-individual variation at the DNA level in 12 genes of the innate immune system. These genes were: CAMP, defensins (DEFA4, DEFA5, DEFA6 and DEFB1), MBL2 and TLRs (TLR1, TLR2, TLR4, TLR5, TLR6 and TLR9). DNA resequencing of these genes was carried out on samples collected, with written informed consent, from 171 individuals belonging to Indo-European speaking groups resident in eastern India. On each sample, ~75 kb of DNA was resequenced. A total of 548 variations were detected, of which 229 were polymorphic. No polymorphic variant was observed in CAMP. Most variants (528; 96%) were single nucleotide changes. However, 20 (4%) changes were single- or multi-nucleotide insertions/deletions (INDELs). One tri-allelic variation (alleles: T, A and G) was also detected in TLR1 (rs4540055). A large number (259 of 548; 47%) of the detected variants were novel, i.e. unreported in dbSNP (build 126) prior to our submission to dbSNP. Of these novel variations, ~10% were polymorphic. From these data it is clear that the observed frequencies of DNA variants and polymorphisms are, respectively, ~7.4 and ~3.1 per kb (further details are provided in Bairagya et al.16). Among the 548 variations observed, 92 were in coding regions, of which 50 were non-synonymous. Of these 92 variants in coding regions, 21 (23%) were polymorphic; 11 (52%) were non-synonymous and 10 (48%) were synonymous. Since population genetic theory suggests that the most frequent allele is the oldest17 and therefore likely to be the ancestral allele, we compared our data at the variable positions with the corresponding data from the chimpanzee reference sequence (UCSC Genome Browser; March 2006 assembly). While the major allele at the majority (412 of 544; 76%) of variable sites in our data coincided with the ancestral (chimpanzee reference sequence) allele, at the remaining 132 (24%) sites the major allele is not the ancestral allele. Such observations have been made earlier.18 It is not unlikely to observe non-ancestral alleles to be major alleles in humans, when these pertain to loci that are influenced by natural selection. Indirectly, our observation lends support to natural selection acting on the innate immunity genes.

Extensive global variation in haplotype structures Haplotypes were reconstructed using genotype data at the polymorphic loci and their frequencies were estimated. For most genes, three or four haplotypes were in high frequencies. For most genes, the Human Reference Sequence Haplotype (build 36) was not the most common haplotype in the Indian study population and for some genes (e.g. DEFA4) the reference sequence haplotype was not even

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observed. The haplotype diversity values were very high (from 62% for TLR9 to 88% for DEFA6) for most genes. The ratio of SNPs to haplotypes varied from 0.41 (DEFA6) to 2.08 (TLR6). Haplotypes based on some specific loci in MBL2 are known to be associated with MBL serum concentration variability.7 MBL serum concentration influences susceptibility to and clinical course of many infectious and chronic diseases.19 The relevant details about these haplotypes and their frequencies observed in our Indian study population are presented in Table 1. Recently,14 it has been shown that there is a strong continental structuring of haplotype frequencies at two nonsynonymous sites (rs4986790, Asp > Gly and rs4986791, Thr > Ile) in TLR4. This structuring was inferred to be due to the interaction between our innate immune system and the infectious pressures in particular environments during the outof-Africa migration of modern humans. The observed percent frequencies of the Asp/Thr, Asp/Ile, Gly/Thr and Gly/Ile haplotypes in our data were, respectively, 86.52, 2.33, 1.16 and 9.94. For the variants in these innate immunity genes that were also assayed in the HapMap project (10 genes), we compared our data with the HapMap data (HapMap Data Release 21a/phaseII 07 January, on NCBI B35 assembly, dbSNP build 125). We found that there are three nucleotide positions (1 in DEFB1, rs2978873; 1 in TLR1, rs1873196; and 1 in TLR2, rs4696480) that are monomorphic in all four HapMap populations (Chinese [CHB], Japanese [JPT], African [YRI], Caucasian [CEU]), but are polymorphic in our study population. None of these variants is in a coding region, however. On the other hand, there are 26 positions that are polymorphic in at least one of the four HapMap populations, but are monomorphic in our study population. There is also a tremendous variation in allele frequencies at highly polymorphic loci (i.e. allele frequencies ranging between 0.2 and 0.8 in at least one population) among the HapMap populations and our study population from India. There is also considerable variation in haplotype frequencies (Fig. 1), calculated on the basis of loci that were common to HapMap and our study populations. (Because of lack of data in HapMap database for TLR5, haplotype frequencies could not be compared for this gene.)

Search for natural selection We have used these data to to assess the nature and extent of selective forces in shaping the contemporary genetic diversity of these genes. In particular, we sought to test whether overdominant selection operates on the TLR genes, because TLR proteins contain LRR domains (characterized by a segment LxxLxLxxNxL, in which “L” is Leu, Ile, Val, or Phe and “N” is Asn, Thr, Ser, or Cys and “x” is any amino acid) that are responsible for molecular recognition.20 Similar to the antigen recognition site of the MHC locus, individuals who are heterozygous at the variant

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Figure 1. Frequencies of haplotypes >10% in the study population (IND) and the four HapMap populations. (Haplotype identification and estimation of haplotype frequencies were based on SNPs that are common among these populations. Because of lack of data in HapMap database for TLR5, haplotype frequencies could not be compared for this gene. Haplotypes with frequencies <10% have been pooled into a single category)

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sites in these LRR coding regions may be capable of expressing various types of microbial recognition peptides and will therefore be expected to enjoy a selective advantage in a population exposed to a diverse array of microbial pathogens. We have tested for selective neutrality, using four different statistics, for the 12 innate immunity genes. CAMP showed statistically significant deviation from neutrality by all four statistics. Deviation from neutrality was not detected by most of the statistics for the defensin genes or for MBL2. For TLR genes, significant deviations were detected by three of the four statistics for TLR2, TLR4 and TLR9, but not for TLR1, TLR5 or TLR6. For all the four genes (CAMP, TLR2, TLR4 and TLR9) that showed significant deviations from neutrality, the values of the test statistics were negative, indicative of purifying selection. Interestingly, none of these genes show statistically significant evidence of selection in the European-American (EUR) or the African-American (AFR) populations (Table 1 of Mukherjee et al.).21 This indicates that local selection may be operating in regions with a high microbial load, although haplotype diversity values are similar for these genes among EUR and AFR and the present study population (Table 1 of Mukherjee et al.).21 This scenario is also consistent with local selective sweep, although this is unlikely in view of the results presented later. Contrary to our expectations, the TLR genes do not show signatures of overdominant selection. When dominant haplotypes in a genomic region increase to high frequencies under the impact of natural selection, it is expected that there will be increased homozygosity at an extended genomic region surrounding this region. For CAMP, DEFA4, DEFA5, DEFA6, TLR2, TLR4 and TLR9, we have found evidence of extended homozygosity. The remaining genes did not show any such evidence. It is noteworthy, that except for DEFA4 and DEFA5, the remaining four genes showed significant evidence of purifying selection. For both DEFA4 and DEFA5, even though three of the four tests of neutrality were not statistically significant, the values of the statistics were negative for all the four tests, consistent with purifying selection.

Haplotype networks reveal purifying selection Under a population expansion model, a star-like phylogeny of haplotypes is expected.22, 23 Balancing selection, on the other hand, is expected to retain multiple lineages for a long time, resulting in a network in which there are some high-frequency clusters and some small-frequency clusters with long branches.24 Strong purifying selection is expected to retain only a single haplotype, but under weak purifying selection one may expect a small number of haplotypes with moderate frequencies, separated by short branches. We constructed medianjoining networks25 of haplotypes depicting their phylogenetic relationships. For CAMP, essentially there was one haplotype (frequency = 94.41%), which was same as the chimpanzee haplotype; the remaining seven haplotypes were sporadic

19.59

None

+

Not polymorphic in the study population.

*Non-synonymous change.

HYPD LYQC LYPB

1.46 23.98

53.22

HYPA LYQA Intermediate LYPA Low LXPA

High

G (H) C (L) C (L)

G (H) C (L) C (L) C (L) G (Y) G (Y) G (Y)

G (Y) G (Y) G (Y) C (X) C (P) T (Q) C (P)

C (P) T (Q) C (P) C (P) T (D) C/T C/T

C (A) C (A) C (A) C (A) G/A G/A A (B)

G/A G/A G/A G/A

G/A A (C) G/A

G/A G/A G/A G/A

Table 1 Frequencies of individuals in the Indian study population who express MBL2 at different levels, and the underlying haplotypes Level Haplotype % Detailed definition of haplotype of MBL (using frequency rs# (location) expression traditional in study Nucleotide (traditional allelic nomenclature) allelic population rs1800450* rs1800451* rs11003125 rs7096206 rs7095891 rs5030737*,+ nomenclature) (Exon 1) (Exon 1) (Exon 1) (Promoter) (Promoter) (5’UTR)

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– observed in only a few individuals – and each was separated by one mutation from the dominant haplotype. This is indicative of a strong purifying selection operating on CAMP. Haplotype networks for the defensin genes (DEFA4, DEFA5, DEFA6 and DEFB1) essentially showed star-like phylogenies consistent with expectations under a population expansion model. However, among these the networks for DEFA4 and DEFB1 revealed some features that are more expected under balancing selection, than under purifying selection. DEFA4 had two high frequency (26.47–26.76%) haplotypes that were almost equidistant from the ancestral (chimpanzee) haplotype. DEFB1, on the other hand, had three high frequency haplotypes (14.04–17.25%) separated by long branches with one another; all these haplotypes were also very distant from the chimpanzee haplotype. Haplotype networks of the TLR genes, except TLR5 are consistent with purifying selection, each with one or a few modal haplotypes separated by short branches. The chimpanzee haplotype is separated by many mutational steps from the modal human haplotype for most of the TLR genes. Networks of 3 genes with contrasting patterns are presented in Figure 2. Haplotypes based on some specific SNPs in MBL2 are known to be associated with MBL serum concentration variability.7 MBL serum concentration influences susceptibility to and clinical course of many infectious and chronic diseases.19 As mentioned earlier, while the frequencies of haplotypes in our study population associated with high (53%) and deficient (20%) levels of MBL serum concentration were comparable to those observed worldwide (49% and 22%), the frequencies of intermediate (1.5%) and low (24%) haplotypes were significantly different from the global frequencies (15% and 13%).13 The significantly higher frequency of the low-secretor haplotype (and, consequently, a lower frequency of intermediate-secretor haplotypes) is noteworthy. The haplotype network for MBL2 (Fig. 3), the nodes of which have been identified with secretor status, shows an interesting pattern. The ancestral (chimpanzee) haplotype is closer (8 mutational steps away) to the modal haplotype associated with low MBL secretion. There are two high-frequency nodes, separated by a long branch, each of which is associated with high MBL secretion. Local star-like phylogenies appear around all the high-frequency nodes irrespective of MBL secretor status. These features are indicative of balancing selection, although the test for neutrality was not rejected by any of the statistics. The Pattern of non-synonymous versus synonymous changes reveal purifying selection and provide no evidence of overdominant selection on TLR genes For all innate immunity genes, the rate of synonymous change is higher than non-synonymous change (Table 2), as expected under purifying selection. Thus, the expectation that overdominant selection may be operating on the TLR genes was not supported by our data.

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Figure 2. Contrasting haplotype networks of three genes of the innate immune system [Each node represents a haplotype. The sizes of nodes are proportional to haplotype frequencies; numbers within a node represent observed number of haplotypes. Lengths of the edges joining nodes are proportional to the number of mutational differences between the corresponding haplotypes.]

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Figure 3. Haplotype network of MBL2 [For explanation of this figure see Figure 2. H, I, L and N represent the expression levels (high, intermediate, low and none, respectively) of MBL2; see Table 1.]

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It is noteworthy that the rate of non-synonymous changes (that is, number of non-synonymous changes per kb) in the TLR genes was the lowest in the EUR population (Fig. 4). In the Indian population, this rate was the highest in the TIR domain (the cytoplasmic toll/interleukin-1 receptor (TIR) signaling domain that is instrumental in inducing a signaling cascade upon recognition of specific ligands by TLRs) compared to the remaining regions. The TIR domain in the TLR genes may, therefore, be under stronger impact of natural selection than other regions.

Figure 4. Rates per kb of non-synonymous changes in the LRR and TIR domains and in other regions of the TLR genes among Indians (IND), European-Americans (EUR) and African-Americans (AFR)

Table 2 Rates of non-synonymous (dN) and synonymous (dS) substitutions per site and their ratios for innate immunity genes Gene* dN dS dN/dS CAMP 0.068 0.092 0.737 DEFA4 0.005 0.049 0.098 DEFA5 0.024 0.032 0.800 DEFA6 0.005 0.043 0.110 DEFB1 0.000 0.021 0.003 MBL2 0.004 0.012 0.341 TLR1 0.006 0.011 0.609 TLR2 0.002 0.011 0.200 TLR4 0.002 0.006 0.409 TLR6 0.008 0.028 0.272 TLR9 0.004 0.018 0.227 *Chimpanzee sequence for TLR5 is unavailable.

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Conclusions The extensive variation in the innate immunity genes observed in India is possibly an outcome of their exposure to a variety of infectious diseases. Compared to the average of one SNP occurring in 1.91 kb26 or one variant per 1.3 kb27 in the autosomal genome, the densities of SNPs (>3 SNPs per kb) and variants (>7 variants per kb) in the innate immunity genes in the India population were significantly higher (p < 0.02). Even compared to the more recent estimate28 of one SNP per 0.88 kb, the SNP density observed in this study is higher. There is, of course, variation in SNP density across autosomal chromosomes. The range of variation (see Table 1, Ref. 26) is one SNP per 1.19 kb (chromosome 22) to one SNP per 2.18 kb (chromosome 19); thus, the SNP density in the innate immunity genes observed in the Indian population exceeds the highest density observed for any autosomal chromosome in the human genome. In fact, at the genome-wide level, there are very few genomic regions where SNP density exceeds 2.5 SNPs per kb (see Fig. 1 of The International HapMap Consortium, 2007). Thus, compared with all existing data, the extent of genetic variation in the human innate immunity genes observed in India is very high. We have found that most of the innate immunity genes have been influenced by purifying selection, even though the strengths of the signatures of purifying selection are variable. For most genes, only one or two haplotypes were present in high frequencies; evolutionary variants that have arisen to create new haplotypes were either eliminated or were present at very low frequencies, being perhaps transient to being eliminated. Non-synonymous variations in these genes are not tolerated, as indicated by the ratios of rates of non-synonymous to synonymous changes being <1, that have been found to be 1.31 and 1.66, respectively, for exons on human chromosomes 21 and 22.29 Interestingly, most of the modal haplotypes were very dissimilar to the ancestral (chimpanzee) haplotype, perhaps indicating that humans have had to adapt to a different set of pathogens since divergence from their nearest common ancestor about 5 million years ago. Contrary to our expectation stemming from past studies on MHC genes10 that overdominant selection would operate, particularly in the LRR domains of the TLR genes that are important for pathogen recognition, we did not find any compelling evidence to support our expectation. However, it is interesting that compared to the EuropeanAmerican and African-American populations, the Indian population harbors a large number of unique non-synonymous variants in the LRR and TIR domains of the TLR genes. Perhaps this is a reflection of the greater diversity of pathogens to which the Indian population is exposed, particularly in the region from where individuals were recruited into this study. Our major result that purifying selection operates on genes of the innate immune system is in striking contrast with previous findings on genes of the

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adaptive immune system, such as genes of the major histocompatibility complex or immunoglobulin genes. The adaptive immunity genes are under either balancing or positive selection.10,24 A simple explanation of our finding of purifying selection operating on the genes of the innate immune system is that this genetic surveillance system recognizes motifs in pathogens that are conserved across a broad range of pathogens. Therefore, functional constraints are imposed on mutations that diminish the ability of innate immunity proteins to detect pathogens. Thus, this system in the host does not co-evolve with the mutations in genomes of pathogens. We note that purifying selection has also been detected in innate immunity genes in Drosophila melanogaster30 and Daphnia31 and in some genes of the TLR family in humans.32, 33 In summary, our study has revealed that (a) although there is considerable genetic and haplotypic diversity in the innate immune system, one or a few haplotypes are modal, indicating a high degree of optimization of the innate immune system;11 (b) the signatures of selection on these genes are variable, but purifying selection is the most dominant mode of selection, indicating that there is low tolerance to non-synonymous changes; and (c) there may be local variations in the nature of selection that is perhaps modulated by local differences in pathogen diversities and loads, implying that the extent of optimization of the innate immune system can be local and not necessarily global. Acknowledgements: This study was conducted in collaboration with the National Institute of Cholera and Enteric Diseases, Kolkata; The Centre for Genomic Application, New Delhi; and Research Triangle Institute International and Duke University, North Carolina, USA. It was financially supported by NIAID, National Institutes of Health, USA; Contract No. HHSN200400067C.

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Verdu P, Barreiro LB, Patin E, Gessain A, Cassar O, Kidd JR, Kidd KK, Behar DM, Froment A, Heyer E, Sica L, Casanova J-L, Abel L, Quintana-Murci L (2006) Evolutionary insights into the high worldwide prevalence of MBL2 deficiency alleles. Hum Mol Genet 15:2650–2658

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Ferrer-Admetlla A, Bosch E, Sikora M, Marques-Bonet T, Ramirez-Soriano A, Muntasell A, Navarro A, Lazarus R, Calafell F, Bertranpetit J, Casals F (2008) Balancing selection is the main force shaping the evolution of innate immunity genes. J Immunol 181:1315–1322

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Bairagya B, Bhattacharya P, Bhattacharya SK, Dey B, Dey U, Ghosh T, Maiti S, Majumder PP, Mishra K, Mukherjee S, Mukherjee S, Narayanasamy K, Poddar S, Sarkar Roy N, Sengupta P, Sharma S, Sur D, Sutradhar D, Wagener DK (2008) Genetic Variation and Haplotype Stuctures of Innate Immunity Genes in Eastern India. Infec Genet Evolu 8:360–366

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Hacia JG, Fan JB, Ryder O, Jin L, Edgemon K, Ghandour G, Mayer RA, Sun B, Hsie L, Robbins CM, Brody LC, Wang D, Lander ES, Lipshutz R, Fodor SP, Collins FS (1999) Determination of ancestral alleles for human single-nucleotide polymorphisms using highdensity oligonucleotide arrays. Nat Genet 22:164–167

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Nuytinck L, Shapiro F (2004) Mannose-binding lectin: laying the stepping stones from clinical research to personalized medicine. Personalized Med 1:35–52

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Matsushima N, Tanaka T, Enkhbayar P, Mikami T, Taga M, Yamada K, Kuroki Y (2007) Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors. BMC Genomics 8:124

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Mukherjee S, Sarkar-Roy N, Wagener DK, Majumder PP (2009) Signatures of natural selection are not uniform across genes of innate immune system, but purifying selection is the dominant signature. Proc Natl Acad Sci USA 106:7073–7078

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Takahata N, Nei M (1990) Allelic genealogy under overdominant and frequencydependent selection and polymorphism of major histocompatibility complex loci. Genetics 124:967–978

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Tanaka T, Nei M (1989) Positive Darwinian selection observed at the variable-region genes of immunoglobulins. Mol Biol Evol 6:447–459

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Jiggins FM, Hurst GDD (2003) The evolution of parasite recognition genes in the innate immune system: Purifying selection on Drosophila melanogaster peptidoglycan recognition proteins. J Mol Evol 57:598–605

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Little T, Colbourne J, Crease T (2003) Molecular evolution of Daphnia immunity genes: Polymorphism in a Gram-negative binding protein gene and an α-2-macroglobulin gene. J Mol Evol 2004 59:498–506

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Tapping RI, Omueti KO, Johnson CM (2007) Genetic polymorphisms within the human Toll-like receptor 2 subfamily. Biochem Soc Trans 35:1445–1448

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17 Macroevolution in relation to the drift models of the Indian plate Ashok Sahni* and R.S. Loyal** Centre of Advanced Study in Geology, Panjab University, Chandigarh, India *[email protected], **[email protected]

Abstract: Macroevolutionary processes, that operate at larger than gene-level evolution, seeks to explain evolutionary processes and patterns of higher taxonomic categories and their phylogenesis. Species multiplication and their radiations, involving several examples of evolutionary novelties are being focused through refinement of DNA sequencing, which are suggestive of dating divergence events. Trends towards linking genetic shifts through space and time are increasingly being associated with paleontology, tectonics and climatic patterns. The fossil records are now better supplemented through information on rifting and colliding landmasses, which help explaining the biogeography and biodiversity changes. The Indian raft exemplifies admixtures of the Cretaceous endemic and Laurasiatic affinities; while the collision of India-Asia landmasses is initiated by thermal maxima phases of Late Palaeocene-Early Eocene; and mammalian diversification happened in the northern Indian landmass in response to progressive land emergence and Tethyan sea withdrawals. Strong trends in fluctuations in biodiversity of mammals in response to orbital forcing and climatic shifts are observed during periods of glaciation and preglaciation during Miocene and later intervals. Keywords: Macroevolution, Charles Darwin, Molecular Clocks, Indian raft, India-Asia collision, Climatic change, Biodiversity changes

Introduction About 150 years ago, Charles Darwin synthesized enormous data on biodiversity, (while gathering impressive informations on fossil animals and plants and their extant populations) aboard HMS Beagle, and is accredited with persuasively explaining the way in which tremendous diversity of various forms of life evolved and advantaged in response to ecological ‘pressures’ and adaptations. The basic essays on evolution synthesized into The Origin of Species (1859) is a very extensive commentary on geographic and reproductive isolation, and natural selection as central lemma in the new species’ origins and diversifications of populations. V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Much as theologists’ dogmas gradually became redundant during the earlier part 19th century, Darwin, had the fortune of having been under enormous academic influence of William Smith, accredited for his law of faunal succession, whereby faunas have succeeded each other closely through time and their successions implied intervals of enormity of geological and ecological changes. Sir Charles Lyell, Darwin’s ultimate mentor and a progressive scientist, synthesized continuity of geological phenomena (‘Gradualism’) into uniformitarianism, which James Hutton, earlier (ca. early 18th century) had envisioned as kind of complex earth events (geological cycles) persisting even today. These geological studies, particularly those of Lyell1 in his work “Principles of Geology” that formed a strong basis that led Darwin to ultimately publish his theory in 1859 with much greater scientific bases and convictions. These complex historical chronicles provoked numerous writers projecting an intimate biographic accounts on Darwin - one of these is Janet Browne,2 who mentions T.H. Huxley’s remark, a tribute, rather in her book (pp. 4) “Charles Darwin - The Power of Place” as the one “who was one of the kindest and truest men that it was ever my good fortune to know.” One cannot refrain from mentioning hereafter another profound opening remark, though reeking of pithy, yet being subtle, by Browne (pp. 3)2: “If Charles Darwin had spent the first half of his life in the world of Jane Austen, he now stepped forward into the pages of Anthony Trollope.” Meanwhile, Darwin had laid great emphasis in evolutionary theory on mere competitions between species and other biological phenomena.3 Nevertheless, little was known during the times of Victorian era about the aforementioned planetary scale of plate movements, sealevel fluctuations and how the biotas and their habitats respond in form and function to these grand kinematic phenomena, and much less on molecular changes through time.

Mega scale events and molecular clocks Much stimulation towards global abiogenic processes, primarily related to earth’s growth to its modern plate dimensions and configurations has been associated with evolutionary processes and patterns, both at the macro- and micro-scale, extending upto even population and their turnovers. As organisms react differentially to climatic and ecologic changes, fossil records reflect changes in phylogenetic and form alterations, including those of lineages.4 The macroevolutionary scale, though very close to the micro-scale in evolution, broadly, is associated with larger than small scale gene-based evolutionary changes, and encompasses higher taxonomic categories, populations and their phylogenetic trends than the species. At this scale, there are evolutionary trends in a clade, with various determining factors such as variation in origination,

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extinction and diversification patterns, reflecting upon adaptations of species and their multiplication.5 Species turnovers are high, such as during Quaternary, because of differential survival of species,5 e.g. the zebra Equus oldowayensis had a critical and restricted niche, while the extant elephant Loxodonta africana and Equus grevi and had a variable and broader diets, migratory and social patterns.5 Above the species level, rock records display many significant evolutionary patterns such as Cambrian radiations, and origination of several marine forms; such evolutionary themes have gained macroevolutionary importance.6 A possible close link between micro- and macroevolution has been a subject of extensive researches and fierce debates through refinement of DNA sequencing and molecular evolutionary studies, which reflect on the patterns of dating divergence events, through phylogenic and fossil record data. A term ‘phylogeography’ was coined to integrate genetic time scale informations, climates, paleontology and tectonics to interpret biogeographic shifts in space and time, diversification times and phylogenetic trees.7–14 The molecular timescales, particularly divergence timings would enhance and supplement incomplete fossil records, providing rigorous examinations for macroevolutionary processes. 15 Patterns of time divergence based on molecular estimates suggests that timing of avian-mammalian diversification coinciding more with the rifting of the landmasses in the Cretaceous and not the ecological factors.16 Further, the role of climatic variations in relation to genetic responses like changes in gene patterns and provide significant clues to providing models of present and future worldwide climate that might affect extant organisms and their diversity changes.4,17 Interestingly, based on DNA phylogenetic studies and protein sequence data, the reconstruction of Afrotheria, comprising of one-third of ancient mammals, evolved during geographic isolation of African landmass was established and suggestive of occurrence of placental mammals in the Cretaceous, ca. 100 ma.18 Several endemic and surviving faunal components include proboscideans, elephant shrews, sirenians, aardvarks and others.19 Earlier, macroevolutionary patterns of divergence times amongst mammalians and other vertebrates were established from calibration of molecular clocks reflecting on more than 100 million years of diversification of atleast five lineages of placental mammals.15 Further, changes in Milankovitch-governed oscillations (of earth’s orbit) every 10–100 thousand years, with attendant climatic shifts, have also played a significant role in mass extinctions and clad diversification processes, e.g. the gene flow rates are closely tied to clade diversifications; and geographic ranges of various clades have a functional relations to diversity of endemic species and Milankovitch climatic shifts.20 The first overwhelming evidences of macroevolutionary aspects of life, diversification of life into myriads of form modifications and the modern day

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biogeographic distributions of the biotas are some of the few understood concepts with better clarity, if viewed in the light of plate-governed (like tectonic breakups) geospatially oriented vicariance models with patterns of continual biogeographic dispersals (or physical convergence), ecologic and climate shifts throughout the earth history into descendant biotas. Such themes are becoming more relevant at the interface of life and earth sciences, integrative themes poring huge and assuring data that plate tectonics (a paradigm that explains enormous landmass and ocean movements) and evolutionary patterns are, infact tied together. The ca. 200 millions of duration history of Gondwanaland through most of the Mesozoic, is a case in point, as it reflects continual rifting, rafting and cruising continent-islands through sundered oceans. The Gondwanaland breakups with resultant barriers, and dispersal mechanisms provide constraints on phylogenic patterns and reflect on the past biogeographic relationships.14 The abundant fossil biotas of this time are suggestive of growing body of evidences favoring the landmass-ocean configurations, the attendant climatic and sea level changes through interrelaionships of rock and water-air responses. These macroevolutionary issues on a planetary level, therefore, need to be raised and debated to be modeled into rationales at this opportune time, closely coinciding with nearly 150 years of Darwin’s centenary celebrations. And further, significant aspects of past and modern vertebrate biogeographic patterns have had continual responses to tectonic moulding of spatio-temporal land and seaway pathways of corridors, filters and sweepstakes.21,22 To cite the case in point, the related breakup and collisions of land and oceanic assemblies are implied to account for lesser biodiversities of reptilian assemblages of Gondwanaland throughout existence of more than 200 million years, in contrast to larger numbers of mammalian animals throughout Cenozoic (ca. 65 million years) era of land breakups and periods of fast drift rates.23 Various patterns of equid dispersal routes and vicariance homogenization and diversity radiance through respective plate models of convergence and breakup are also known.24 This work, in the latter interval would highlight modeling of evolutionary diversity with drift models of the Indian plate. Breakup of Pangaea is a one of the major tectonic factors in understanding of biogeographic patterns and the organisms.16

The Indian raft and the cretaceous Indian breakup A major reorganization in the biogeographic distribution, the turnovers and ecological shifts available for the faunal components of the Indian plate occurred in response to Indian landmass’s separation and ultimate drift from the Gondwanaland.25 Intense tectonic activity led to the initiation of new sedimentary peri-cratonic basins, shelves, episodes of flood basalt extrusion and its sedimentary intercalations of intertrappeans, and creation of Indian Ocean and a neo-Tethyan

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ocean. The rifting of India-Sychelles around 100 million years back began from Australia-Antarctica; at around 80 million years, India’s separation began from Madagascar (Fig. 1). The continental Jurassic of India, associated with India’s initial rifting phase shows both Laurasiatic and Gondwanic affinities, though stronger paleobiogeographic and vicariance patterns with the latter are also known.26 The piscean faunas recovered from the Creataceous of central Indian intertrappean shows the presence of a nearshore selachian Igdabatis, also known from Niger, W. African. A frog fossil Nasikabatrachus sahyadrensis,27 known from western Ghats, indicates endemism, after India split from Seychelles around Late Maastrichtian.28,29 Molecular divergence data indicates that this form split from that of Sooglossidae of Seychelles’ at terminal Cretaceous. Earlier, pelobatid and discoglossid frogs with Lauriastic affinities were known.30 Pelumadusid turtles Kurmademys and Sankuchemys suggest floating Indian raft phase in Maastrichtian. Sauropod dinosaurs show a Gondwanic affinity. Mammalian faunas, such as Deccanolestes during the drift phase and show both Laurasian and Gondwanic affinities. Besides siluroid fishes also belong to the same time interval. An extant form of siluroid Scleropages formosus inhabiting south-east Asia, another freshwater adapted Sumatran form, was subjected to molecular phylogenic studies; the divergence times from the molecular clock suggest that it was transported towards Sumatra, from northwards across the Tethys on the Indian landmass, probably through terrestrial freshwater habitats.31 Since Cretaceous, this form vicariantly diverged from Australasia towards towards Eurasia on the Indian plate or smaller subducted blocks.

Early Eocene (Drift phase of the Indian Plate) Early Eocene time represents a pre-collisional phase. The early part of the Paleogenes witnessed significant changes in global climate and ocean circulation as reflected in sedimentary responses, evolutionary turnovers and extinctions recorded in marine and terrestrial stratigraphic records. Regarding the reconstruction of paleobiogeography, modern orders of mammals appeared in the north, during Late Palaeocene Thermal Maxima, though no clear evidence is available to test this idea.32 However, these biotic and climatic responses have never been studied up to now in high resolution. Changes in the climatic and biotic patterns have been associated with heat effects that have been linked broadly to India-Asia collision and the Tethyan closure processes.33 During the latest Palaeocene epoch, a relatively brief global warming occurred, referred to as Late Palaeocene Thermal Maxima resulting in formation of warm saline bottom waters, linked with Tethyan closing. High resolution boundary analysis of various Paleogene units is essential for precise biozonation

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Figure 1. Sketch showing evolutionary events India-asia collision. A. 84-65 ma; biota mainly Gondwana derived. B. ca 55ma; Isolation leads to endemism as India becomes a drifting island subcontinent. C. ca 50ma; collision leads to Asian elements and results in admixture of biotas

and reconstruction of the paleobiogeography. These data are applicable along with biotic turnovers in quantification of paleocommunity structures, which can then be applied to draw vicariance/dynamic scenarios. Models of syncollisional and collisional histories are ultimately built on the basis of attendant climatic and geologic events, of which LPTM (Late PalaeoceneThermal Maxima) with short-

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lived warming event (Late Palaeocene-Early Eocene) evidenced by a negative carbon excursion, is characterized by a significant turnover and dispersals of the mammalian taxa in the fossil record. Several Early Eocene localities have been studied in parts of Jaisalmer and Barmer basins, and two papers have been published on biostratigraphic and biocorrelative aspects; and another one on mineralogic and geologic constraints on depositional cyclicity of the bentonitic facies. Well preserved selachian assemblages34 referable to Notorhynchus, Galeorhinus, Galeocerdo, Physogaleus, ?Sphyrna, Rhinobatus, Dasyatis and Myliobatis. Associated fish remains include? Osteoglossidae, Enchodus and Diodon. A lagoonal, nearshore (infra-littoral) depositional sequence, with a shallow watersediment interface and extremely slow rate of settling of clay facies is suggested. Paleoclimatic studies have been facilitated in the Palaeocene and Early Eocene of Surat and Kutch, and a warm climate of 32oC was suggested. The biostratigraphic zonation and community structures established in this basin, forms an extremely useful data, for extending these studies to sub-Himalayan and other basins. Work carried out on the Subathu Formation35–40 generated a wealth of diverse vertebrate faunas got known (Hexanchus, Procarcharodon, Isurus cf. I. spallanzani, Alopias cf. A. vulinus, Galeocerdo latidens, Hemipristis, Galeorhinus, Rhinobatus, Dasyatis vicaryi, Pycnodus toliapicus, Stephanodus lybicus, Diodon). An in-depth phylogenetic analysis of these vertebrates, suggests their affinities to lie with those from Thanetian beds of Niger, and from Palaeocene-Eocene beds of north Africa. It39 was further suggested that the bilateral migrations of the marine vertebrates along the marine corridors of the Tethys, which by Early Eocene, had narrowed, with the northward movement of the Indian plate These, however throw light on marine nearshore environments. More paleobiogeographic and paleoenvironmental data center on nearshore and terrestrial vertebrates and invertebrates. An Early Eocene initial contact may have been possible that lead to southerly migrations onto the Indian plate. The Late Palaeocene (syncollisional India-Asia phase) is considered to be an extremely anomalous episode in the earth history, following climatic and biotic perturbations at the terminal Cretaceous caused by impact and/or volcanicrelated events. A gradual restoration of equable climates, evolutionary radiation and increased diversity of marine and terrestrial biotas are some of the global characteristic events of Late Palaeocene-Early Eocene times, during which Neotethys shallowing advanced with a negative carbon isotope excursion.41 As earth experienced globally uniform temperatures, marine productivity reached its Cenozoic maximum during the Late Palaeocene (early Chron C25r), reflected by flourishing calcareous plankton and epicontinental benthic foraminiferal, ostracode, marine invertebrate and vertebrate faunas. Such records are signatured in the Subathu Formation.34,35,42 However, these biotic and climatic responses have never been studied up to now in high resolution. Further, more

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complexity in these signatures has been introduced by the tectonic and climatic fallouts of the collisional phases, which exhibit temporal overlap and have operated in opposite directions relative to the isotopic record and climate. The associated chemical and heat influxes implicated from the India-Asia collision, modified the earth’s climatic and biogeochemical cycles. During the latest Palaeocene epoch, a relatively brief global warming occurred, referred to as Late Palaeocene Thermal Maxima (resulting in formation of warm saline bottom waters, linked with Tethyan closing). These data are applicable along with biotic turnovers in quantification of paleocommunity structures, which can then be applied to draw vicariance/dynamic scenarios. The drift and collisional histories and their models (such as related to the India-Asia landmasses during LPTM) have been derived from attendant climatic, sedimentologic and other geologic events; alongwith significant turnovers and phenomena of mammalian originations and dispersals.

Collisional phase of the Indian plate The southwardly prograding foreland basin with its accumulated sub-Himalayan sediments provide a storehouse of information on precollisional, collisional and postcollisional histories of the Tethyan closure. Distinct episodes of Tethyan convergence reflect on the paleontological, climatic, sedimentologic and sea level changes. The time-transgressive Paleogene sections of Subathu, Dagshai/Murree/Dharamsala/Kasauli formations, alongwith coeval sediments of Jaisalmer-Barmer and Cambay basins would be studied. The current theme of the project is based on the fact that fossil turnovers and biogeographic belts through time have been largely controlled by climate, which in turn are influenced by plate movements, which would ultimately govern the course of evolutionary histories. Newer discoveries made in the early Subathu Formation comprises of diverse assemblages of larger foraminifera, ostracodes, fishes (sharks, rays, pycnodontids, tetraodontids), crocodilian and mammalian assemblages; with terrestrial assemblages concentrated upsection in the upper Subathus and Dagshais. The Palaeocene-Early Eocene sections, concomitant with the initial India-Asia collision (Late Palaeocene Thermal Maxima) seems to be related to the appearance and geographic spread of mammalians faunas on the northern continents. Initial continental collision between India and Asia caused significant changes in global climate and biota, despite the fact that its timing and biogeographic consequences are uncertain. Moreover, paleomagnetic, geophysical and regional structural studies reflecting on the initial collision in the sub-Himalayas have not been initiated even upto now. Further, the time-constraining complete depositional sequences are absent; and paleontologic finds, and zonations have largely been done in structurally complex and overturned sections.

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For the past 3 years, detailed mapping, sedimentologic studies and sequence stratigraphic analysis has been carried out in parts of Himalayan peripheral foreland basin of north-west India by the investigator. Further, it43 is suggested that the deposition of basinal sediments took place due to progressive upliftment of the basin, causing regression at the Subathu-Dagshai contact. The aforementioned biogeographic data currently suggest chronology of the IndiaAsia collision; therefore, a closer study of the global climatic constraints through time would help in generating sufficiently precise data as to constrain this event. The Middle Eocene (Upper Subathu/Murrees) represents a red continental facies stratigraphically succeeding the lower Subathu marine facies, suggestive of a progradational regressive package of fluvially-induced sediments, formed in response to progressive upliftment of the basin. Notable in these sediments, is the preservation of these mammals related to the collisional phase are referable to brontotheres, tapiroids, perissodactyls, dichobunids, tillodonts, rodents and hyaenodonts. In this regard, our work40 shows presence of Uppermost Subathu rodent faunas, viz. Birbalomys woodi, B. sondari, B. ibrahimshahi, Basalomys vandermeuleni, B. ijlsti, Chapattimys wilsoni, cf. Advenimus bohlini and cf. Petrokoslovia. Land emergence in response to collision is indicated. The Foreland basinal studies centering on the India-Asia collision are extremely important to assess the hypothesis of tectonic forcing on the Paleogene climate and biotic responses. Tectonism, particularly implying the India-Asia collision induces far-reaching changes in the geochemical cycling of carbon and oxygen in marine and terrestrial environments and attendant climatic and biotic changes, such as enhancement of the greenhouse effect (a hothouse phenomenon) due to exhuming of the organic carbon. The climate responses in relation to the geological upheavels reflect changes in community patterns and habitat shifts, which strongly are associated to faunal turnovers.44 The Eocene-Oligocene climatic framework is marked by “icehouse effect”, sequential framework with an “icehouse effect”, global extinctions have been superimposed on a gradually cooling earth due to development of Antarctic glaciation. The collisional and post-collisional scenarios with attendant development of the foreland basin resulted in invasion of diverse perissodactyl and other mammal groups.32 An Oligocene-early Miocene climatic response to orbital forcing is concentrated at all Milankovitch frequencies.45 Information on climatic records is strongly documented at the 406 thousand years period eccentricity band (of glaciation) over a 3.4 million years period of 20–23.4 million years ago as well as in the 125- and 95-ky bands for nearly 1.3 million years period (21.7–23.0 Ma).45 ‘Obliquity and eccentricity’ are associated with these periods of glaciation45 and low seasonality. A decline in unglulate and carnivore biodiversity patterns during late Miocene interval is possibly related to the tectonic rise of the Tibetan landmass and the attendant global cooling ca. 8–13 million years; before this interval major diversification is seen around 17 million years.46

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Briggs JC: The biogeographic and tectonic history of India. J Biogeog 30:381–388

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Sahni A, Prasad GVR (2008) Geodynamic evolution of the Indian plate: Conseqssuences for dispersal and distribution of the biota. Golden Jubilee Mem Geol Soc India 66:203–225

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Prasad GVR, Rage JC (1991) A discoglossid frog in the Late Cretaceous (Maastrichtian) of India. Further evidence for as terrestrial route between Indian and Laurasia in the latest Cretaceous. C R Acad Sci Paris 313:273–278

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Kumazawa Y, Mutsumi N (2000) Molecular phylogeny of osteoglossids: a new model for Gondwanan origin and plate tectonic transportation of the Asian Arowana. Molec Biol Evol 17:1869–1878

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Clyde WC, Khan IH, Gingerich PD (2003) Stratigraphic response and mammalian dispersal during initial India-Asia collision:evidence from Ghazij Formation, Baluchistan, Pakistan. Geology 31:1097–1100

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Loyal RS (1990) Lithostratigraphy of the basal Subathu Formation (Upper PalaeoceneMiddle Eocene) exposed in the stratotype, Kuthar River, Subathu, Himachal Pradesh, India, with introductory notes on physiography and tectonics. Tertiary Res Leiden 12(1):1–16

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Kumar K, Loyal RS (1987) Eocene ichthyofauna from the Subathu Formation, northwestern Himalaya, India. J Palaeont Soc India 32:60–84

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Kumar K, Loyal RS, Srivastava R (1997) Eocene rodents from new localities in the type Subathu Formation, Himachal Pradesh, northwestern Himalaya, India. J Geol Soc India 50(4):461–474

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Magiongalda R, Dupius C, Smith T, Steurbaut E, Gingerich PD (2004) Palaeocene-Eocene carbon excursion in organic carbon and pedogenic carbonate:direct comparison in a continental stratigraphic section. Geology 32:553–556

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Blas XPI, Loyal RS, Schleich HH, Agraser EL (2004) Pristichampsine cranial remains from the basal redbed facies of the Subathu Formation (Himachal Pradesh, India) and some palaeobiographical remarks. Palarch nl Vert Paleont, Amsterdam 3(1):1–8

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18 Testing the melanism-desiccation hypothesis: A case study in Darwinian evolution Ravi Parkash Department of Genetics, Maharshi Dayanand University, Rohtak - 124001, India [email protected] Abstract: In ectotherms, the occurrence of melanism within populations, across populations or closely related species is pervasive in nature. Many hypotheses have been proposed to explain diverse patterns of body color variations or polymorphisms in insects (e.g. thermoregulation, cryptic coloration, sexual selection, pathogen resistance, etc.). Since the body temperature of ectotherms is dependent on their surrounding environment, melanisation should play an important role in these organisms. The thermal melanism hypothesis states that melanic or dark individuals should be at a fitness advantage under cold conditions while lighter individuals are better adapted under warmer conditions. There is overwhelming support for this hypothesis in diverse taxa (beetles, butterflies and flies) from the temperate parts of the world. However, it is not clear why melanics occur in the tropics? We tested the role of desiccation stress in the maintenance of color polymorphism in tropical populations of different Drosophila species on the Indian subcontinent. We tested several predictions which are most significant in the field of evolutionary ecology. First, if desiccation resistance evolves through changes in cuticular permeability, the target of natural selection might be cuticular components either cuticular lipids or cuticular melanisation. Thus, species specific contrasting levels of desiccation resistance might correspond with varying levels of body melanisation across species. Secondly, we tested whether a thicker cuticle (either due to melanisation or cuticular lipids) leads to lesser cuticular water loss and higher desiccation resistance across seasons (autumn vs. winter). Thirdly, we examined, mechanistic basis of evolving desiccation resistance on the basis of differential rates of cuticular water loss in assorted darker and lighter phenotypes from a given population. Finally, if body melanisation confers desiccation resistance, dark morph is expected to prevail under dry season while the reverse may occur for light morph under humid conditions/season. Further, humidity changes (dry vs. wet) impact mating propensity (assortative matings) among different body color morphs. Thus, there are several evidences in favor of melanism – desiccation hypothesis. In this short review, an attempt has been made to sum up the available information on the role of varying levels of humidity as natural selection agent in maintaining body color polymorphism in diverse Drosophila species populations on the Indian subcontinent. V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Keywords: Body color polymorphism, Seasonal adaptations (autumn and winter), Genetic and plastic effects, Dark and light morphs, Desiccation resistance, High humidity stress, Cuticular water loss, Mating propensity, Assortative matings, Latitudinal, Altitudinal and longitudinal populations, Drosophila species.

Darwinian theory of natural selection A new era of Biology began on November 24, 1859, the day Charles Darwin published ‘On the Origin of Species by Means of Natural Selection’. Charles Darwin acknowledged natural selection as the principal driving force for evolution of living organisms. His evolutionary theory was largely based on the understanding of natural selection and its consequences for origin of species. According to Darwinian Theory of Natural selection, three conditions are essential for evolution of a trait. Firstly, the trait must be phenotypically variable. Secondly, such variations must be heritable. Thirdly, phenotypic variations must be seen by natural selection and should confer fitness benefits. For example, evolution of insecticide resistance in nature are due to a single gene mutation that is rare but once it arises, it spreads among populations due to higher fitness of the resistant variant.1 In successive generations, environmental factors filter (or edit) heritable variations. Organisms with traits favored by the environment tend to produce more offsprings than organisms without such traits. This differential reproductive success results in favored traits being disproportionately represented in the next generation. Thus, increases in the frequencies of favored traits in populations are an important source of evolutionary modifications or adaptations. The most direct validation of Darwin’s theory is provided by numerous studies which have documented evolution by natural selection in natural as well as laboratory populations.2–4 Thus, Darwin theory of evolution has withstood the test of time and has always stimulated several new research findings. We present a case study in Darwinian evolution on the role of body melanisation in desiccation stress resistance in some Drosophila species populations from the Indian subcontinent.

Adaptations to climatic stresses An organism in the natural world experiences daily and seasonal cycles of changes in humidity, temperature and photoperiod. These climatic variations impact the demography of populations and exert variable selection pressures over space and time. In order to understand evolutionary and ecological changes in ectothermic insects, it is pertinent to know how fast is adaptive evolution of ecologically relevant traits and to what role does phenotypic plasticity play in adaptations to geographical and seasonal changes? We analyzed evolution of resistance to environmental stress and its implications at different levels of biological

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organization. Stress influences individual’s physiology as well as genetic variation at the population level through the process of natural selection. To investigate these different levels of stress effects, we integrated approaches from ecology, evolution, ecophysiology and genetics. For investigating the mechanisms of stress resistance, how resistance evolves and what conflicting factors constrain its evolution, experiments were conducted with Drosophila species which are well studied both in genetics and evolutionary biology. Population studies were undertaken to study genetic differentiation as a response to environmental stress and synergistic effects of different stress factors. The interactions between life history and stress resistance were investigated along with responses to stress and their alteration by artificial selection. We have shown some novel findings on the role of melanisation - desiccation hypothesis to explain why melanics occur in the tropics. In the past, we mainly focused on (a) opposite clines for desiccation and starvation tolerance which was a novel report in face of generally accepted parallel evolutionary changes for these traits based on laboratory selection studies.5 Thus, we showed that field data could be quite different than laboratory investigations; (b) we have shown impact of body melanisation on heat vs. cold resistance as well as for desiccation resistance in populations and species from quite different habitats6,7; (c) Finally, we have shown that a warm adapted species (D. jambulina) lacks genetic structure for different quantitative traits.8 This species has adopted a different strategy to cope with significant seasonal variations in humidity by changing body color morph frequency. These results are quite different than what we have observed in several other Drosophila species on the Indian subcontinent. We studied the role of genetic variations in stress response mechanisms and on the interactions between stress resistance and life history traits. So far, Drosophila species and populations from extreme regions of temperate vs. tropics have been investigated to show genetic differentiation for stress related traits. There is growing concern that future studies should focus on the following: (a) studies on quantitative genetic variations should be carried out with freshly collected individuals from natural populations; (b) use of isofemale line technique in place of mass cultures or strains borrowed from fly stock centers; (c) analysis of several geographical populations at well defined temperature or humidity gradients along latitude or altitude or longitude; (d) analysis of fitness consequences of desiccation resistance patterns; (e) to identify the possible mechanisms that increase the stress resistance; (f) to examine correlated changes in various stress related traits on the basis of laboratory selection experiments as well as through field data (within population analysis); (g) to study the effects of individual climatic variables or in combination on the stress related traits under laboratory set up. Finally, the results need to be verified across populations and species from different continents. We have investigated the following aspects in light of Darwinian Theory of natural selection:

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● Comparison of desiccation tolerant and sensitive species on the basis of water balance mechanisms and body melanisation patterns. ● To examine the role played by genetic and plastic effects on stress related traits as well as body melanisation due to seasonal variations. ● To assess the impact of climatic stress on mating behavior between dark and light morphs.

Impact of climatic variables on phenotypic evolution According to Darwinian Theory of Evolution, environmental factors filter or edit heritable variations for a given trait. Environmental conditions are never static but change temporally as well as spatially. All living systems harbor genetic variability for a given trait so as to cope with varying environmental conditions. Thus, various climatic variables such as temperature, humidity etc. act as selection agents for editing heritable variations for different traits in diverse ectothermic taxa. Across the Globe, average annual temperature is negatively correlated with increasing latitude on a broader scale. By contrast, environmental changes in relative humidity show no such consensus with increasing latitude on different continents i.e. we encounter regions which are hot and humid vs. hot and dry; cold and humid vs. cold and dry. However, Indian subcontinent (8–33ºN) provides a large geographical range covering localities with diverse climatic conditions which favor adaptations of specific phenotypic variations. Southern and northern localities fall under tropical and subtropical climatic conditions because tropic of Cancer passes almost midway through the subcontinent. For investigating climatic adaptations, occurrence of seasonal changes on the Indian subcontinent (as we move from south to north) is responsible for providing a regular but changing climatic pattern according to latitude. Such a climatic pattern does not occur on many continents including Africa and Australia. The analysis of climatic data on the Indian subcontinent demonstrates that average yearly temperature (Tave) along the north-south transact is not variable (25.24–27.1ºC) and poorly correlated with latitude (r = 0.25) (Fig. 1 A). Thus, average temperature does not vary but seasonal variations increase with latitude for the Indian subcontinent. Latitudinally, southern peninsular localities face more humid conditions while northern localities face drier conditions due to seasonal variations (Fig. 1 B). The amplitude of thermal variations can be considered on the basis of coefficient of variation of mean monthly temperatures on a yearly basis (Tcv) which vary from 3.19% in Trivandrum (south) to 38.10 Shimla (north) and seasonal variations increase significantly from south to north (Fig. 1 C & D). Evidences in favor of thermal melanism in butterflies, moths and beetles have been reviewed by Majerus.9 Melanisation changes along altitude and latitude are in agreement with the thermal budget hypothesis (TMH) i.e. black body surface

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Figure 1. Changes in climatic variables (A: Tave.; B: RH; C: Tcv) of ten localities differing in latitude (from south to north) on the Indian subcontinent. Monthly variations in Tave for one southern, one central and one northern locality are shown in D

better absorbs solar radiation in order to maintain optimum body temperature under colder conditions. Direct evidences of differential effects of solar radiations on melanics vs. typicals have been obtained for wild and laboratory populations of beetles and butterflies.10,11 Unlike temperature, the role of humidity as a selection agent has not been considered for different quantitative traits in various Drosophila species so far. A single study has used laboratory selection to determine the evolutionary effects of relative humidity, temperature and their interactions on adult wing area (body size) in D. melanogaster.12 In that study, after 20 weeks of laboratory selection, low RH lines had significantly greater wing area than high RH lines. Thus, body size in D. melanogaster can evolve rapidly in response to humidity selection. Another study has shown rainfall as the significant determinant for wing polyphenism (wet and dry forms) in Bicyclus butterflies from Africa.13 Thus, it may be argued that evolutionary responses for different quantitative traits might result due to humidity selection in the tropics.

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Thermal melanism as a model of phenotypic evolution Heritable changes in body melanisation with possible fitness consequences constitute a suitable model for phenotypic evolution in different insect species.9,14–19 Several studies on moths and butterflies from the temperate regions have exhibited significant positive correlations between melanism and higher latitudes as well as altitudes.9,20 In different insect taxa, there are diverse patterns of body melanisation i.e. (a) several black species of Collembola occur in temperate regions i.e. Pyrenees, Swiss Alps and Himalayas21,22; (b) in D. melanogaster, a cosmopolitan species, extent of melanism varies with geographical location23,24; (c) discrete melanic and non-melanic morphs occur as genetic polymorphism in species of montium species subgroup.25 Such diversity of melanisation patterns might correspond with contrasting changes in thermal and/ or humidity conditions in the temperate vs. tropical regions. In temperate localities, melanics can warm up faster and become active as compared with non-melanics.10,17,26,27 Thus, the increase in the frequency of melanic morph in temperate regions is in agreement with thermal melanism hypothesis (TMH). Several studies have shown fitness consequences of body color variations in ectotherms. Melanism in peppered moth, Biston betularia, has long been a text book case of evolution in action. Industrial melanism in peppered moth protects individuals resting on dark backgrounds i.e. crypsis.28 Wing melanisation has been associated with increased flight ability under colder conditions in Alpine butterflies - Colias species.29,30 In lepidoptera, Pieris species show polyphenism to adapt seasonally varying temperature conditions.31 Melanics display higher mating success as compared with typicals in Adalia bipunctata (coleopteran) and Ephestia kuhniella (lepidopteran).32,33 Melanic morphs in beetles are involved in aposematism (warning of distastefulness to predators).34 Melanism in ladybird beetles (Adalia bipunctata) has been associated with mimicry and sexual selection.35 Thus, based on field observations and laboratory studies, melanics and typicals differ in thermoregulation, mating behavior and camouflage. Such observations suggest possible pleiotropic effects of melanism in diverse insect taxa. In contrast, there are few investigations in Drosophila on the ecological significance of pigmentation polymorphism.36 Melanin synthesis pathways are conserved in diverse insect taxa and Drosophilids. Inspite of the fact that more than three thousand species have been described under Drosophilidae, body melanisation has been analyzed in about a dozen species.36 Diverse Drosophilids vary in their melanin patterns (continuous or interrupted stripes or spots on the abdominal segments) but data on geographical populations and their fitness consequences are limited.17,19,36,37 On the basis of laboratory selection experiments, Dombeck and Jaenike have demonstrated substantial genetic variation for abdominal spot number in Drosophila falleni and showed that the individuals

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without abdominal spots are two times more susceptible to nematode infection as compared with the spotted wild type flies.37 Thus, it is clear from different studies that no single selective force can provide a universal explanation for the evolution of melanisation in diverse insect taxa.

Why melanics occur in the tropics? It is not clear why melanics in diverse insect taxa are found in the tropics.17 Such cases include: (a) occurrence of high frequency of melanics in beetle – Harmonia axyridis in south-Asian localities38; (b) in Adalia bipunctata, the melanic form f. sublunata comprise upto 96% in warmer regions of central Asia39; (c) melanic morphs of two-spot ladybird beetle Coccinella septempunctata, f. confusa occur at a high frequency in south Indian localities.40 Thus, environmental conditions in the tropics can affect melanism in diverse insect taxa. Besides thermal effects, body melanisation can control water balance in ectothermic insect species.41 Role of body melanisation in conferring desiccation resistance has been shown in a generalist species – D. melanogaster,7 in a cold adapted species — D. immigrans42 and a tropical species — D. polymorpha.37 These studies have shown that assorted darker and lighter flies from a given population differ in their desiccation resistance level and show differential rates of cuticular water loss which are negatively correlated with body melanisation.7,42,43 For body melanisation, evolutionary response of thermal effects has been tested experimentally in different insect taxa from temperate regions10,27 but the effects of other climatic variables have not received much attention. In this brief review, we have focused on possible linkages for within population assorted morphs of body melanisation and desiccation resistance in diverse Drosophila species populations from the Indian subcontinent. For both the traits, genetic and phenotypic variability occurring in different Drosophila species at the levels of within populations, between populations and between species have been examined. We have provided data on alternative role of pigmentation in desiccation resistance in several Drosophila species inhabiting the Indian subcontinent. We tested melanism – desiccation hypothesis on the basis of following evidences: ● High vs. low potential of body melanisation and desiccation resistance. ● Phenotypic plasticity as an adaptive strategy (seasonal adaptations). ● Impact of wet – dry conditions on mating propensity.

Mechanistic basis of desiccation resistance (laboratory vs. natural populations) Drosophila species have invaded a wide range of terrestrial habitats and differ greatly in their physiological tolerance limits.44 Thus, wild populations of

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Nature at work: Ongoing saga of evolution

Drosophila species provide good models for study of adaptations to stressful environments. In the laboratory, populations of D. melanogaster respond rapidly to selection and significant higher desiccation resistance levels have been detected within 10–12 generations.45 Selected populations provide a useful comparison to natural populations of Drosophila. Also unselected control populations are maintained for direct comparison with the ancestral condition of populations. Both laboratory and natural populations have shown several adaptations for resistance to desiccation: (i) reduction in the cuticular water loss, (ii) by accumulating more water in hemolymph; (iii) physiological tolerance for greater water loss (Fig. 2; Table 1). The main barriers to cuticular water loss are the epicuticular lipids.46 Some studies have shown geographical divergence in the amount of cuticular lipids which correspond with clinal variation in desiccation resistance.47,48 However, both such cases include non-melanic species i.e. Melanoplus sanguinipes and Zaprionus indianus.47,48 By contrast, in several insect taxa, differences in surface lipids are not sufficient to account for reduced water loss in either laboratory or natural populations.62 According to Gibbs, most insect species produce lipids with higher Tm (> 45 °C due to higher proportion of alkanes) as well as with lower Tm (≤ 25 °C due to alkenes).62 Therefore, there is lack of correlation between Tm of cuticular lipids and cuticular water loss in several insect taxa from diverse habitats. Thus, there is general lack of supporting evidence for the role of cuticular lipids in changing cuticular permeability. If reduction in cuticular water loss can not be explained on the basis of epicuticular lipids, it may be accomplished by changes in other portions of the cuticle. A single study has shown a possible link between body melanisation and desiccation resistance e.g. darker mutant strain (ebony) of

Figure 2. Schematic representation of three different modes of water conservation in ectothermic insects

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287

Table 1 A comparison of the outcome of laboratory vs. natural selection for the mechanistic basis of desiccation resistance in Drosophilids Laboratory selection

Natural Selection

Body water

Accumulation of body water in haemolymph

No variation in water contents

Dehydration tolerance

Lack of differences

Show variation

Cuticular water loss

Negative correlation

Negative correlation

Cuticular lipids

Lack of differences

In some taxa, trait variation impacts desiccation resistance while not in others.

Body size

Lack of differences

Lack of differences (within population)

Body melanisation

Not analyzed

Positive correlation with desiccation

D. melanogaster is desiccation resistant as compared with non-melanic mutant strain (yellow).50 In this brief review, we raised a hypothesis that flies differing significantly in melanisation level (darker vs. lighter) might evidence correlated differences in desiccation resistance and its mechanistic basis i.e. reduction in cuticular water loss.

Melanisation - desiccation hypothesis Melanism seems to have evolved independently through diverse mechanisms in various taxa and different ecological factors could be responsible for selective responses.17 Increased body melanisation at higher altitudes as well as latitudes is generally considered to be adaptive for thermoregulation. Physiological traits such as body melanisation and desiccation resistance have been investigated independently in diverse insect taxa at three levels: within population, between populations and among species.5,9 A substantial number of Drosophila studies have reported clinal variations in both these traits along latitude.5,41 Correlations between these traits remained unexplored in wild and laboratory populations of ectothermic insect taxa including Drosophilids. In insects, there are several investigations on the rate of water loss in laboratory selected strains for desiccation resistance but such analyses have not been considered in context of body melanisation.45,49 Association between pigmentation and desiccation resistance was initially proposed by Kalmus.50 He compared desiccation resistance in wild-type flies to that of yellow (light body color) and ebony (dark body color) mutants in D. melanogaster. Similar desiccation assays were also performed for wild type as well as yellow mutant flies in D. simulans, D. pseudoobscura, and D. subobscura.

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The ebony mutants survived longer as compared with yellow mutants. Fraenkel and Rudall51 and Pryor52 concluded that the darkening and hardening traits of the cuticle are due to the same biochemical processes which may involve cross linking of proteins with melanin. Brisson et al.37 investigated desiccation resistance in D. polymorpha and found that dark flies from open dry environments survived longer in a desiccating environment than lighter flies inhabiting dark humid forest environments. A possible link between melanisation and desiccation can be shown if laboratory selected strains for higher melanisation evidence increase in desiccation resistance; and if within population differences in assorted groups (darker and lighter phenotypes) of body melanisation evidence significant parallel changes in desiccation resistance and its mechanistic basis i.e. rate of water loss/hr. Accordingly, we analyzed darker and lighter phenotypes of body melanisation in six wild Drosophila melanogaster populations from closely located highland and lowland sites at three different latitudinal locations (Table 2). In large population samples, within population variability made it possible to assort non-overlapping phenotypes of body coloration [i.e. lighter (<25%) and darker (> 45%)] for all the populations which were investigated for desiccation resistance and rate of water loss/hr (Table 2). Wild populations of Drosophila melanogaster from diverse geographical locations showed significant associations between

Figure 3. Relationship between % body melanisation and desiccation resistance in wild caught flies assorted into two groups of darker (> 45% melanisation) and lighter phenotypes (< 30% melanisation) for each of the five highland populations of D. melanogaster

11.15

11.21

24.12

24.36

30.44

31.06

--

--

--

Coonoor

Deesa

Mount Abu

Chandigarh

Shimla

mean ± s.e.

s.d.

r values

Lat. (ºN)

Calicut

Pop.

--

--

--

2202

347

1195

136

1747

10

Alt. (m)

--

5.64

21.33±2.30

30.40±2.74

24.00±2.16

22.40±1.97

17.00±1.68

20.50±1.74

14.00±1.14

Light

--

9.69

54.34±3.85

69.02±4.99

60.00±4.02

55.10±4.12

48.00±3.40

52.12±3.02

42.00±2.56

Dark

% Melanisation

0.99±0.08

3.45

12.50±1.41

18.00±1.96

13.00±1.59

14.00±1.76

10.00±1.34

12.00±1.36

8.00±1.24

Light

0.99±0.07

6.18

23.16±2.52

33.00±4.01

26.00±3.60

24.00±3.72

19.00±3.26

22.00±3.14

15.00±2.80

Dark

Desiccation hr.

0.98±0.10

0.42

0.036±0.0017

0.030±0.0005

0.033±0.0007

0.035±0.0004

0.038±0.0005

0.037±0.0002

0.043±0.0003

Light

0.98±0.09

0.79

0.028±0.0030

0.016±0.0006

0.023±0.0007

0.026±0.0007

0.032±0.0008

0.033±0.0005

0.038±0.0006

Dark

CWL (mghr.-1)

Table 2 Data on mean ± s.d. of percent body melanisation, desiccation hours and rate of cuticular water loss/ hr (CWL) in wild caught assorted groups of darker and lighter phenotypes of six populations of Drosophila melanogaster

Testing the melanism-desiccation hypothesis: A case study in Darwinian evolution 289

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Nature at work: Ongoing saga of evolution

darker phenotypes and higher desiccation resistance while lighter phenotypes showed lesser desiccation resistance (Fig. 3). Significant reduction in rate of water loss/hr in darker phenotypes could be indirect evidence in favor of cuticular impermeability due to higher body melanisation.7 In conclusion, analysis of populations of Drosophila melanogaster demonstrates that body melanisation is phenotypically variable and subject to natural selection pressure for increased melanisation under colder and drier conditions. Our data are consistent with the increased desiccation being a consequence of selection on melanisation. Body melanisation may result in cuticular impermeability for reducing water loss under increasing dehydrating conditions.7,42

Evidences for melanisation - desiccation hypothesis High vs. low potential of body melanisation and desiccation stress resistance across species Several investigations have shown rapid response to laboratory selection for desiccation resistance in D. melanogaster.45,53,54 Thus, genetic variation for desiccation resistance appears to be directly or indirectly under selection. However, various studies on the mechanistic basis of desiccation resistance in various Drosophila species have shown that rate of cuticular water loss are negatively correlated with desiccation resistance.7,55,56 For ectothermic insects, more than 90% loss of body water involves cuticular transpiration under desiccation stress.57 If desiccation resistance evolves through changes in cuticular permeability, the target of selection might be cuticular components either cuticular lipids or cuticular melanisation. However, such a possibility has not been investigated in natural populations as well as through laboratory selection experiments. For desiccation resistance, water proofing role of cuticular lipids has been reported for terebrionid beetles, scorpions and grasshoppers. 58,59,47 Cuticular impermeability results due to changes in the amount and/or composition of cuticular lipids.46,60,61 According to Gibbs,62 there is lack of correlation between Tm of cuticular lipids and cuticular water loss in several insect taxa. Thus, the water proofing role of cuticular lipids may not be a universal mechanism for regulating water loss. However, it is not clear whether species specific differences in body melanisation can impact their stress resistance levels. For generalist species, it is generally assumed that populations have abundant genetic variations in quantitative traits for adaptation.63 By contrast, there are limited data on the evolutionary responses of stress related traits in tropical Drosophila species from humid habitats.64 A comparison of geographical variations for body melanisation, desiccation resistance and cuticular water loss per fly in D. melanogaster as well as in

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291

D. ananassae suggested non-overlapping species specific clinical variation (Fig. 4). The most resistant northern populations of D. ananassae are comparable to the least resistant southern populations of D. melanogaster for all the desiccation related traits as well as body melanisation. Thus, the baseline resistance levels are significantly different across these two species. For sympatric populations of these species, selection pressures due to climatic conditions might differ if they vary in their behavioral evasion of desiccation stress under wild conditions. This is possible because both the species are linked with domestic habitats which may offer protection from desiccation stress. In this respect, these species may encounter differential exposure to climatic stress conditions resulting in different slope values for desiccation resistance across species. Alternatively, if there are direct selection pressures on body melanisation which differ significantly across species, we may expect similar response to selection (similar slope values) but with significant differences in intercept or baseline resistance level which correspond to equatorial regions (Fig. 4 A–C). Ectothermic insect taxa may be thermal generalists, or high- or low temperature specialists. Sensitivity to temperature and humidity has the potential to influence the ecology, behavior and adaptive fitness of different insect species. Thus, the extent to which ectotherms can tolerate changes in their ambient thermal environments can be critical in determining their distribution and abundance. On the Indian subcontinent, southern tropical habitats and northern subtropical regions have impacted distribution patterns of D. melanogaster and D. ananassae. However, it is not known which ecophysiological traits are influenced by varying climatic conditions. The contrasting levels of body melanisation correspond with species specific desiccation resistance i.e. body melanisation could be the target of climatic selection. For D. ananassae, inability to colonize high latitudes as well as altitudes may be limited by its quite lower levels of body melanisation. Thus, for these two Drosophila species, variability for body melanisation may be a limiting factor for ecological success under variable climatic conditions. This is supported by distribution of D. ananassae along latitude which has shown a steeper negative slope (b = 2.20 ± 0.21) as compared with D. melanogaster (b= 0.94 ± 0.11; Fig. 4D). Such data suggest that significant reduction in % distribution of D. ananassae along latitude matches with its desiccation sensitivity. However, for D. melanogaster, the changes in % distribution are significantly lower which correspond with its high desiccation resistance level.65 Evidences from seasonal adaptations In montane localities of subtropical regions, winter is the dry season and ectothermic Drosophilids are expected to evolve desiccation resistance to cope with drier climatic conditions. Seasonal variations in quantitative traits are generally assessed by differences in thermal conditions66,67 but evolutionary

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Nature at work: Ongoing saga of evolution

Figure 4. Geographical variation in % body melanisation (A); desiccation resistance (B); and cuticular water loss hr-1 (C) in twelve sympatric populations of D. melanogaster and D. ananassae. The slope values for each trait are quite similar across species but intercept values differ significantly. Regression analysis of changes in % species distribution (D); as a function of RHcv of site of origin of populations

responses due to other environmental factors have received lesser attention.68–71 Seasonal variations have been reported for body size (low heritability) in different Drosophila species72,73; and for desiccation resistance (which show high heritability) in a single population of D. simulans.74 Under seasonally varying environments, wing color patterns in different lepidopterans confer adaptive responses such as crypsis and thermoregulation.75,76 Thus, seasonally varying environments impose strong natural selection and cause rapid phenotypic changes in quantitative traits.70,77,78 However, there are no data on the impact of seasonal climatic variations on desiccation related traits in different Drosophila species from the subtropical regions. Body melanisation shows seasonal plasticity but its role in conferring adaptations to desiccation stress is not clear. Melanic flies are expected to have tighter cross-linkage of the cuticular proteins which would make it less permeable.79,80 If body melanisation is linked with desiccation resistance, seasonal changes in body color due to phenotypic plasticity may impact desiccation related

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traits. Thus, we addressed the following questions: (a) Do quantitative traits (body size, body melanisation and desiccation related traits) vary seasonally in wild populations? (b) Do plastic changes in body size and cuticular lipids correlate with desiccation resistance? (c) Whether plasticity for body melanisation impacts desiccation resistance and cuticular water loss? We examined seasonal changes in body size, body melanisation, cuticular lipids, desiccation resistance and cuticular water loss in six altitudinal populations of D. melanogaster. Wild caught as well as laboratory reared individuals were analyzed to find genetic and plastic effects for these traits. Further, we examined whether seasonal plastic changes in body melanisation are linked with desiccation resistance and cuticular water loss in D. melanogaster. Finally, we considered spatial as well as temporal changes in body melanisation and desiccation related traits which might be in agreement with the co-gradient hypothesis. Our data have evidenced considerable changes in body melanisation and desiccation related traits across two seasons (autumn and winter). However, seasonal plastic effects for body size are not correlated with desiccation resistance. Further, there is lack of changes in cuticular lipids which do not account for variations in desiccation resistance across seasons. To the best of our knowledge, adaptive significance of body color plasticity to cope with seasonally varying desiccation stress has not been previously analyzed. Following are the main findings of our study: Assessment of plastic vs. genetic changes

Insects can cope with seasonal environments through phenotypic plasticity which allows a single genotype to produce different phenotypes. In temperate regions, some studies have compared clines for body size in wild-caught individuals with their laboratory progeny based on common garden experiments.81,82 However, changes in clinal patterns under seasonally varying conditions have not been reported so far. Thus, in order to test genetic as well as plastic effects, we compared slope values for clinal variation in wild vs. laboratory populations for desiccation related traits. Changes in slope values for quantitative traits across seasons are expected due to plastic responses. By contrast, lack of differences in slope values would suggest the absence of plastic effects for quantitative traits. The slope values for desiccation related traits were found to be significantly higher for winter as compared with autumn under wild conditions i.e. plastic effects were 1.64 fold higher for body melanisation as well as desiccation resistance across seasons (Table 3). By contrast, laboratory analysis of seasonal populations found no differences in slope values. Interestingly, the ratios of slope values based on wild data across seasons were identical for body melanisation, desiccation resistance and cuticular water loss. Thus, in montane localities, phenotypic plasticity for body melanisation in D. melanogaster leads to darker flies in winter and lighter

-4.5 ± 0.37 -2.2 ± 0.30 2.04

2.8 ± 0.34 1.43

Desi. h. 4.0 ± 0.39

CWL

5.8 ± 0.37 1.45

8.4 ± 0.48

1.07

% Mel

0.16 ± 0.015

4.84***

2.32**

4.29***

0.48 ns

Wild /Lab. t- test(p)

0.15 ± 0.014

Lab. (L)

WL

Wild (W)

Slope values *10-3 ( Autumn)

-3.0 ± 0.39

7.0 ± 0.74

13.6 ± 1.0

0.20 ± 0.02

Wild

2.50

2.34

1.25

2.01 **

4.88***

7.31 ***

1.50 ns

Wild/Lab. t-test (p)

-2.0 ± 0.31 1.50

2.8 ± 0.45

5.8 ± 0.38

0.16 ± 0.015

Lab.

Slope values*10-3 (Winter )

1.50

1.75

1.61

1.36

1.01

1.01

1.01

1.00

Wild/ Wild Lab/ Lab.

Plasticity across seasons

Table 3. Comparison of elevational slope values *10-3 (b ± S.E. = regression coefficients) for wild and their laboratory progeny; and plastic effects based on their ratios for four quantitative traits in seasonal populations of D. melanogaster. Plastic effects across seasons are also shown. The slope values for different traits in wild vs. laboratory progeny were compared statistically on the basis ‘t’ - test. ** p < 0.01; ***p < 0.001; ns = non-significant

294 Nature at work: Ongoing saga of evolution

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295

Figure 5. Histograms showing changes in % body melanisation of (A) 2nd +3th+4th abdominal segments; (B) 5th +6th +7th segments; and (C) total melanisation per fly in seasonally varying wild - caught individuals from a highland population (Chail - 2226 m a.s.l.) of D. melanogaster

flies during autumn season; and such plastic effects correspond with changes in desiccation resistance across seasons. Plasticity of body melanisation

In D. melanogaster, the developmental specification of the three anterior (2nd, 3rd and 4th) and the three posterior abdominal segments (5th, 6th and 7th) are respectively controlled by the homeotic genes: Abdominal-A and Abdominal-B.83 There is a lack of phenotypic correlations between these two groups of abdominal

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Nature at work: Ongoing saga of evolution

segments i.e. (2nd + 3rd + 4th) vs. 5th +6th + 7th) for all the populations suggesting their independent genetic control (Fig. 5 A–C). For 2nd to 4th abdominal segments, we obtained non-significant differences in slope values for two different seasons. By contrast, the 5th, 6th and 7th segments showed significant differences in slope values for the two seasons. Plastic effects for three posterior abdominal segments (5th +6th +7th) were two fold higher. An interesting observation is that there were no plastic changes for the three anterior abdominal segments (2nd+3rd+4th). However, the magnitude of the genetic effect (ca. 40%) was similar for (2nd+3rd+4th) and (5th + 6th + 7th) segments. Therefore, due to plastic effects, darker flies in winter are more desiccation resistant and show reduced cuticular water loss as compared with the lighter flies of the autumn season. We may infer that plastic changes in (5th +6th + 7th) segments are correlated with plastic changes in desiccation resistance across seasons. In D.melanogaster, plastic effects for body melanisation and desiccation resistance exceed the magnitude of genetic differences along a cline. There are 40% higher trait values (% melanisation, desiccation hours and cuticular water loss) along the altitudinal cline based on either autumn or winter data. By contrast, for each population, trait divergence across seasons was 50% higher for desiccation related traits. Thus, D. melanogaster has evolved developmental plasticity for body melanisation as an adaptive response to seasonally varying environments. In D. melanogaster, spatial and temporal variations for quantitative traits result from genotype-environment effects. We observed variations in desiccation related traits associated with environmental gradient along altitude. Our data indicate that body melanisation is influenced by environmental conditions during different seasons which impact desiccation resistance. We found significant genetic and plastic effects by comparing slope values along a cline for wild vs. laboratory reared progeny for autumn and winter seasons. The adaptive role of plastic changes described here has not been reported previously. The major conclusion is that plasticity for body melanisation confers desiccation resistance under seasonally varying climatic conditions in the montane localities.84 Evolution of behavioral traits through natural selection Behavioral differences between closely related species are common e.g. different species of Drosophila sing different courtship songs. By contrast, when behavioral variation within a species corresponds to variation in environmental conditions, it is an evidence of past evolution. For example, sitter and rover represent foraging behavioral phenotypes in D. melanogaster which are controlled by diallelic variation at a locus i.e. fors and forR. Laboratory studies of Drosophila populations raised in high- and low-density conditions showed clear divergence in behavior linked to foraging gene (for). Significant increase in the frequency of fors in low density populations; and of forR under high-density conditions support the contention that

Testing the melanism-desiccation hypothesis: A case study in Darwinian evolution

297

foraging divergence represents experimental evidence for behavioral evolution in Drosophila.85 Because of the influence of genes on behavior, natural selection can result in the evolution of behavioral traits in populations. Evidences for such evolution are behavioral divergence between as well as within species. In species of Ladybird beetles that harbor melanic polymorphism, dark- colored individuals benefit from increased mating success via thermoregulation (Brakefield, 1984). 32 However, fitness benefits of body color polymorphisms in diverse insect taxa have received lesser attention so far. For body melanisation, evolutionary response of thermal effects has been tested experimentally in different insect taxa from temperate regions10,27 but lesser attention has been paid to other abiotic factor i.e. humidity. We tested this hypothesis in Drosophila jambulina which exhibits color dimorphism controlled by a single locus but its ecological significance is not clear. Dark and light morphs differ significantly in body melanisation, desiccation resistance, rate of water loss, mating activity and fecundity. Interestingly, this species lacks clinal variation for body size, desiccation resistance and life history traits. For body melanisation, lack of geographical variation as well as plastic effects is not in agreement with a thermal melanism hypothesis. However, based on field data, there are seasonal changes in phenotypic frequencies of dark and light body color morphs which correlate significantly with variation in humidity levels. In order to test which of the climatic variables could be a selective agent in the tropics, we raised the following hypotheses: (a) changes in phenotypic frequencies of dark and light morphs may occur through thermal effects and/or humidity changes; (b) humidity changes (dry vs. wet) may impact mating propensity (assortative matings) for different body color morphs; (c) if body melanisation confers desiccation resistance, dark morph is expected to prevail under dry seasons while the reverse may occur for light morph under humid conditions. Under short-term (8 hours) desiccation stress, we observed higher number of assortative matings, longer copulation period and increased fecundity for dark strains as compared with light strains. By contrast, both the morphs when exposed to high humid conditions exhibited higher assortative matings and fecundity for light strains as compared with dark strains. In tropical populations of D. jambulina, body color polymorphism seems to be maintained through humidity changes as opposed to thermal melanism. Thus, seasonal changes in the frequency of body color morphs in this tropical species supports melanism–desiccation hypothesis. Variation in phenotypic frequencies through humidity changes

On the Indian subcontinent, Tave does not vary along latitude as well as longitude. There is lack of correlation between Tave and latitude (r = 0.19 ± 0.44; ns) and also with longitude (r = –0.11 ± 0.70; ns). By contrast, humidity changes are significant along latitude (r = – 0.92 ± 0.10) as well as longitude (r = 0.96 ± 0.20).

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Nature at work: Ongoing saga of evolution

Figure 6. Field data on seasonal changes in the % frequency of dark and light morphs in (A & B) northern vs. southern population; and (C & D) in western vs. eastern population of D. jambulina. For southern and eastern localities, changes in humidity levels are quite low as compared with northern and western localities. The number of wild caught individuals was 200–240 for each season as well as locality

Field data have shown significant seasonal variation in the % frequency of dark and light morphs i.e. greater frequency of melanic morph in autumn as compared to rainy season (Fig. 6 A–C). Longitudinal localities are characterized by similar thermal conditions but higher humidity levels correspond with increased frequency of lighter morph in the eastern locality (Guwahati, 91.35°E; Fig. 6D). Data on varying humidity conditions under laboratory set up has also shown changes in the frequencies of dark and light morphs. Thus, seasonally varying humidity seems to be the principle selective agent for adaptive changes in the frequencies of dark and light body color morphs.

Testing the melanism-desiccation hypothesis: A case study in Darwinian evolution

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Figure 7. Comparison of mating propensity (mated pairs and copulation period) in control vs. flies exposed either to desiccation stress or to high humidity condition. (A) Bar diagrams for mated pairs; (B–D) Changes in copulation period are shown as a function of mating speed for dark and light morphs of D. jambulina. Data are based on analyses of twenty isofemale lines. For all correlation values, p < 0.001

Mating propensity under dry and wet conditions

In order to test whether mating propensity of dark and light morphs in D. jambulina varied with seasons, we attempted no choice mating experiments and the data on observed matings under control and stressful conditions are given in Table 4 and Figure 7A. Mating propensity were estimated on the basis of two components of mating process (mating speed and copulation period) and finally in terms of fecundity differences in four types of observed matings (homospecific and heterospecific reciprocal matings; Table 4). For control experiments, homospecific and heterospecific matings did not differ significantly i.e. under laboratory conditions, matings with no choice method are random between dark and light

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Nature at work: Ongoing saga of evolution

Table 4 Data on the basis of no choice method for frequency of mated pairs (MP), mean ± SD for mating speed (MS), copulation period (CP) and fecundity under control and stressful (dry and wet) conditions for D. jambulina. For each experiment (A–C), there were twenty replicates. *** p < 0.001, ns = nonsignificant. Humidity levels were 68 ± 2 % for control; 5 ± 2 % for desiccation stress; and 85 ± 2 % for high humidity. Experiment

Type of Mated pairs

(A) Control

MP (%)

MS (min) m ± SD

CP (min) m ± SD

Fecundity m ± SD

1.

Light ♀ * Light ♂

26.27

9.38 ± 1.38

6.24 ± 1.31

24.4 ± 2.18

2.

Dark ♀ * Dark ♂

28.20

7.15 ± 1.46

7.50 ± 1.29

28.0 ± 2.07

3.

Light ♀ * Dark ♂

23.03

9.23 ± 1.15

6.00 ± 1.35

24.9 ± 2.61

4.

Dark ♀ * Light ♂

22.50

9.32 ± 1.07

6.19 ± 1.16

21.6 ± 2.44

ns

ns

Contingency χ

ns

ns

(B)

1.

----Light ♀ * Light ♂

20.53 14.0 ± 1.41

4.21 ± 1.43

20.1 ± 2.90

Desiccation

2.

Dark ♀ * Dark ♂

64.47

5.15 ± 1.12

11.4 ± 1.14

44.2 ± 3.45

stress

3.

Light ♀ * Dark ♂

8.70

10.0 ± 1.20

5.08 ± 1.23

23.7 ± 2.84

(8 hours)

4.

Dark ♀ * Light ♂

6.30

10.2 ± 1.33

5.32 ± 1.02

24.5 ± 3.80

2

Contingency χ

-----

2

(C)

1.

Light ♀ * Light ♂

*** 64.44

*** 6.49 ± 1.39

*** 10.0 ± 1.31

*** 35.9 ± 3.25

High humidity

2.

Dark ♀ * Dark ♂

22.22 11.0 ± 1.27

6.13 ± 1.06

22.9 ± 3.11

(120 hours)

3.

Light ♀ * Dark ♂

6.11

10.1 ± 1.34

6.29 ± 1.25

25.4 ± 3.61

4.

Dark ♀ * Light ♂

7.23

8.12 ± 1.28

7.47 ± 1.19

24.1 ± 2.66

Contingency χ

2

-----

***

***

***

***

morphs. However, when dark and light morphs were subjected to short-term desiccation stress, mating propensity between dark ♀ x dark ♂ increased about two fold as compared with control experiments (Table 4). The homospecific matings between light ♀ x light ♂ and reciprocal heterospecific matings decreased significantly (p < 0.001). By contrast, flies exposed to high humidity, under no choice mating conditions, exhibited two fold higher mating propensity for light ♀ x light ♂. However, all other mating combinations (dark ♀ x dark ♂) and reciprocal heterospecific matings showed significant reduction (p < 0.001; Table 4). These significant changes in homospecific mating propensity under desiccation or high humidity conditions as compared with control are illustrated in Figure 7A. Thus, the observed differences in mating propensity are a reflection of the type of humidity conditions experienced by different body color morphs. Regression analyses of data on two important components of mating propensity (mating speed and copulation period) under control and stressful conditions are shown in Figure 7B–D. Population means for mating speed (MS) and copulation period (CP) for all types of mating under no choice method are given in Table 4. For all matings under stressful conditions, mating speed and copulation period were negatively correlated i.e. if the MS was longer, CP was shorter and vice versa.

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Figures 7B to D clearly demonstrate that under desiccation stress, copulation period is longer and mating speed is short for dark morph as compared with light morph (Fig. 7C). Under high humidity conditions, we observed a reverse trend i.e. light morph showed significantly longer copulation period as compared with dark morph (Fig. 7D). Interestingly, under control conditions, there is a significant overlap in mating propensity for dark and light morphs (Fig. 7B). For each experiment, the slope values for both the morphs were quite similar. Thus, regression analysis of copulation period as a function of mating speed helped in discriminating mating propensity under control vs. stressful conditions. In tropical regions, seasonal changes in precipitation cause desiccation stress in autumn. D. jambulina has adapted to wet (rainy season) and dry (autumn) seasons by modifying the frequencies of color morphs through assortative matings. Therefore, humidity rather than temperature is the primary selection agent for body color polymorphism. Field and laboratory data on D. jambulina show evidences in favor of melanism–desiccation hypothesis. Like D. jambulina, other species of montium species subgroup are expected to provide similar evidences. Thus, seasonally varying humidity conditions in the tropics can maintain body color polymorphism and desiccation resistance in D. jambulina.

Conclusions Possible role of body melanisation in conferring desiccation resistance was initially demonstrated for ebony mutant strains of D. melanogaster.50 Subsequently, darker and lighter laboratory strains of D. polymorpha were shown to differ in desiccation resistance.37 For altitudinal populations of D. melanogaster, assorted dark and light flies exhibited significant differences in desiccation resistance and cuticular water loss.24 Furthermore, seasonal data on wild living flies have shown frequency changes of dark and light morphs in D. jambulina which correspond with varying humidity levels. We also observed the impact of short-term desiccation stress or high humidity on mating propensity under no choice experiments. We found a consensus between wild vs. laboratory data on the role of body melanisation in desiccation resistance in this species. Thus, change in humidity level is the principle agent of natural selection which modifies the frequencies of dark and light morphs in D. jambulina. The melanism–desiccation hypothesis finds support from studies on seasonal variations,84 body color mutant strains,50 use of assorted dark and light individuals from a given population43; and on the basis of assortative matings either for dark morph under desiccation stress or light morph under high humidity conditions.8 Finally, laboratory selection experiments on body melanisation and their possible impact on desiccation resistance can form evidence in favor of melanism–desiccation hypothesis but there are no data to support such contentions. However, future investigations are needed to support melanism–desiccation hypothesis across diverse insect taxa.

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Acknowledgement: The help and support of Prof. Dr. K.S. Rai and Prof. Dr. V.P. Sharma is gratefully acknowledged. This work was supported by a research project of UGC, New Delhi.

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19 Origin and evolution of human malaria parasite, P. falciparum and P. vivax Nidhi Datta* and Virender Singh Chauhan** International Center for Genetic, Engineering and Biotechnology, Aruna Asaf Ali Marg, PO Box 10504, New Delhi - 110067, India *[email protected], **[email protected]

Abstract: The global occurrences of malaria make it the major killer among all diseases. Many initiatives are being taken worldwide to control the disease. But, despite all these initiatives, its incidences are increasing constantly. Antimalarial drug resistance and lack of method to assess efficacy of any control initiatives have also worsen the situation. In this scenario phylogenetic study of malaria pathogen, can be effective in helping to develop new drugs and vaccines for the disease as well as to understand host parasite interactions and evolution of drug resistance in the pathogen. Malaria is likely originated in tropical Africa and later spread to other parts of the world due to human movement. Presence of plastid in Plasmodium sp. indicates its affinity with cynobacteria and green algae. Recent phylogenetic studies have indicated that all the four human malaria sp. are remotely related to each other. Close relation between P. falciparum and chimpanzee malaria parasite (P. reichenowi) and P. vivax and malaria parasite species of Asian monkeys has been observed. P.vivax shows higher genomic diversity than P. falciparum which indicates that it is older than the latter. Evolutionary studies suggest origin of P. falciparum and P. vivax in Africa and Asia respectively, and support ancient population expansions for both Plasmodium sp. Resistance in Plasmodium sp. for widely used antimalarial drugs was also developed along with spread of the disease It is Darwin’s fundamental contribution that fired the imagination of scientist’s interest in evolutionary studies, and these studies have already proved of much value in understanding the origin, spread and control measures in malaria. Keywords: Darwin, Malaria, P. falciparum, P. vivax, Phylogeny

Introduction The evolution of organisms is well-established scientific knowledge and has important applications in agriculture, medicine, and other areas. Charles Robert Darwin, known as the originator of the modern theory of evolution, was born V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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on February 12, 1809. In 1859, he published his most important book “On the Origin of Species by Means of Natural Selection.1 In Origin, he gathered the best available evidences for evolution and explained the process of natural selection, the role of hereditary variation, and considered possible objections to his theory. As for most path breaking theories that challenge conventional wisdom, it took sometime for its acceptance, but by the middle of 19th century, naturalists accepted the evolution of organisms. The theory of natural selection is now considered as the central organizing principle of biology. In an address to the American Association of Biology Teachers in 1973, the evolutionist Theodosius Dobzhansky emphasized on the importance of evolution by saying that “Nothing in biology makes sense except in the light of evolution”. Human malaria is an endemic parasitic disease in most tropical and subtropical ecosystems worldwide. Malaria infects more than 600 million people, causing up to two to three million deaths, mostly in sub-Saharan Africa every year.2 However, malaria exhibits few forms of genetic resistance too. Patients of sickle cell anemia show some kind of protection; specifically, a child with the genetic disorder has high chances to survive the acute illness of malaria. Presence of heterozygous HbS provides survival advantages, over people with both the genes for normal hemoglobin.3–4 These genes are able to transmit to next generation and hence spread resistance in a population. People of Africa and their progenies also show resistance against P. vivax. The reason behind this could be the absence of Duffy antigen which is the receptor through which merozoites of P. vivax enter into red blood cell.5 Lack of this receptor does not allow the pathogen to invade the host blood cells and provides him resistance against the disease. Thalassemic patients also show some level of resistance against the disease. However, despite this genetic resistance, cases of malaria infection have increased drastically during the last few years and there is no direct method to check affectivity of malaria control measures. Efficacy of any malaria control measure is generally assessed by its impact on the number of infected individuals, clinical cases registered, treatment failure etc. However, most of the times this process does not give a clear picture of the situation and is also not able to show small changes in pathogen population. In this scenario, study of parasite genetic diversity could be the best way to detect the effect of intervention and any slight changes in the population. Knowledge of evolutionary history of parasite population does not only help to check the affectivity of any disease control measure but also assists to understand the development of genetic resistance in a species which is extremely useful to avoid any disastrous situation to human kind. Study of the genetic basis and evolutionary pattern of resistance is helpful in direct monitoring6 and epidemiology surveillance studies.7 It also encourages to test and use different parasitic antigens as vaccines candidate and can contribute to development of new drugs and vaccine.

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Phylogeny of malaria Phylogenetic analyses provide information on the origins, evolutionary history and relationships between organisms. Serious research work on molecular phylogenetics of malaria parasites began in 1991.8,9 Most early conclusions drawn from such studies were based on Small Subunit (SSU) rRNA, the Circumsporozoite Protein (CSP),8-11 mitochondrial gene for cytochrome13-14 and the gene for the housekeeping enzyme adenylosuccinate lyase.15-16 Plasmodium belongs to the phylum Apicomplexa. Phylogenetic investigators suggested its origin with kingdom of plants, fungi and animals around one billion years ago.17 Presence of plastid in Plasmodium and its genetic similarity with cynobacteria and green alga also indicates close affinity between them.18 Four Plasmodium species viz. P. falciparum, P. malariae, P. ovale and P. vivax are parasitic to humans. Earlier it was thought that all human malaria parasites are closely related, but recent works have shown that all the four human malaria sp. are only remotely related to each other.14 It was conjunctured that Plasmodidae were first appeared in the blood stream and have been transmitted by blood sucking anthropods.19 Some studies suggested that the origin of Plasmodium occurred through host switching from a non-human parasite to human parasite. Close relation of some species of Plasmodium to parasitic species of birds than to species parasitic to human or primates supports this hypothesis.8-9 Through several genetic studies, avian origin of malaria parasite proposed earlier8,9,11,15 was later refuted14 and P. falciparum was placed within the clade of mammalian parasites. The single gene hypothesis determined that ‘Plasmodium’ is paraphyletic relative to genus ‘Haemoproteus’ and ‘Hepatocystis’. Genus Haemoproteus belongs to malaria parasite of amphibian, reptiles and birds while Hepatocystis represents the evolutionary stem of malaria parasite of mammals.19 However, connectivities between mammals, bird and lizard Plasmodium parasites were also found. On the other hand, the four gene hypothesis did not show any paraphyletic relation between Plasmodium and avain Haemoproteus. In the case of malaria parasite of primates, the genus Hepatocystis and quartan group is considered as most ancestral, followed by the tertian group. Subgroup Laverania (P. falciparum and P. reichenowi) is probably the most recent.20 Avian and reptilian malaria parasites also show host shift and the lateral transmission of Plasmodium parasite from monkey host to humans is a common occurrence.21,22 Transmission from humans to monkey has been achieved through experiments22 but it may also occur naturally. The evolution of parasite may or may not always be in sync with the evolution of the host. The pace of evolution of parasite and evolution of the hosts sometimes does not match with each other. Some members of a species of host adapt themselves with the changing environment more easily than the others. Even the effect of environment is also not equal on the host and internal parasites present in the host.23 The difference of time between the change in environment

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for the host and the parasite results in the difference in rate of evolution and ultimately evolution of parasite is slower than the host. One notable example of this inconsistency is of malaria species from a southeast asian bird that groups with lizard malaria parasite.24 It seems that malaria originated in tropical Africa and remained localized in the Paleolithic and Mesolithic periods. In the Neolithic period, human population increased and likewise the malaria infection and its movement spread to Mesopotamia, India, South China, and from Nile Valley to Mediterranean shores and ultimately to other parts of the world.20 Prehistoric man must have been a victim of malaria in ancient time and ultimately developed some resistance to it.25 It seems that the resistance to P. vivax developed earlier than to other human plasmodia. Some recent studies support these observations.26–30 Introduction of new and resistant parasite genotype could be due to population movement. During the course of time, Plasmodium also developed resistance against commonly available drugs. Cloroquine resistance (CR) in P. falciparum was first reported in 1978 in non-immune travelers from Kenya and Tanzania.31–32 Similar kinds of reports of CR were also come from Madagascar,33 Tanzania34 and Kenya.35 It is hypothesized that the CRPF spread to Africa from Asia due to the mobility of man. The genetic similarity between parasites of Africa and Asian origin and difference with South America and Papua New Guinea parasite support this hypothesis.36 Thailand, Kwa Zulun Natal,37 Malawi, Kenya and S. Africa are some of the countries which have replaced CQ as a first line drug. P. vivax resistance against CQ has also been reported from Guyana (S. America),38 Sumatra (Indonesia),39 Myanmar and Mumbai (India)40 and Brazilian Amazon region, and particularly for reappearance of parasite after treatment with CQ from Vietnam.41

Evolutionary history of P. falciparum A study based on CSP and rRNA revealed close relation between P. falciparum and P. reichenowi (a rare parasite of chimpanzees).14,15, 42–44 There are different views about their divergence. Some believed that the two species likely diverged from each other between 5–8 million years ago.10,11 This hypothesis is consistent with the time of divergence between human and Chimpanzee. A very recent study favors hypothesis of evolution of P. falciparum from P. reichenowi, most likely by a single host transfer from Chimpanzee to human between 10,000 to 2–3 MYA. This transfer may have happened due to two genetic mutations in Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene and in EBA -175, respectively.45 CSP and cyt-b tree both shows close relation of P. falciparum with P. reichenowi.46 Close association between both sp. has also been revealed by the analysis of mitochondrial DNA sequence polymorphism. A study of mitochondrial haplotypes isolates of P. falciparum from several endemic areas showed a wave of migration from Africa to southeast Asia and

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South America suggesting origin of P. falciparum in Africa and colonization in Southeast Asia and South America.47 Variation in the mitochondrial DNA sequences showed a sudden increase in the African malaria parasite population that was prominent about 10,000 years ago, and after that it migrated to other regions.48 Study on Microsatellite DNA has suggested, African P. falciparum species as the most ancient. It seems that P. falciparum was derived from one single parasite, a cenancestor, within the last 5,000–50,000 years.49 There is a consistency between the antigenic polymorphisms and the recent origin of the world populations of P. falciparum. Strong natural selection leads to rapid allele substitutions or polymorphisms. Extent of polymorphism at neutral evolving loci indicated towards a common ancestor for P. falciparum sp. which was not recent than 1,50,000–200,000 years ago.50 Balanced polymorphisms at certain antigen encoding loci ruled out the hypothesis that this species underwent a recent extreme bottleneck but occurrence of less extreme bottlenecks could be possible. Such bottlenecks could have occurred at the time of origin of modern humans and again at the origin of West African agriculture.51–52 However, it shows that these bottlenecks were not so severe as to eradicate all of the polymorphism present in P. falciparum, some of which is clearly ancient and selectively maintained. Future phylogenetic analyses may help to identify close relatives of P. falciparum and better understand the evolutionary history of these parasites.

Evolutionary history of P. vivax Till mid-1900s, P. vivax was the most widespread and prevalent of the human malaria parasite. It’s ability to complete its sporogonic cycle at a comparatively minimum lower temperature (16°C) helps it to establish stable foci of transmission in the temperate zones. P. vivax shows extraordinary phenotypic diversity and is found in a broad range of ecotypes. It can survive long in liver through its dormant stages, which ultimately leads to relapses of the infection, weeks after the initial infection.53 The relatively stable GC-rich genome of P. vivax (which has a GC content double to that of P. falciparum) has few di-nucleotide microsatellite markers, and the (TA) and (CA) motifs are rare in P. vivax.54 It also possesses highly polymorphic genome which may present some challenges for malaria control initiatives.55 Most recent studies on origin of P. vivax are based on the mitochondrial genome, nuclear genome and the microsatellite loci. Phylogenetic studies showed origin of P. vivax from a malarial parasite of non-human primates as a result of a host switch.13 Genetic affinity of P. vivax with Asian primates malaria has also been reported14,56 Phylogenetic analysis based on mitochondrial cyt b gene showed close proximity of P. vivax with human and monkey malaria parasite from south east asia.13 There are some phylogenetic evidences that P. vivax derived from Plasmodium found in Southeast Asian macaques.57 There are abundant

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evidences available which shows association of P. vivax to a monophyletic group of Plasmodium species parasitizing old world monkeys (Cercopithecoidea) and suggested spreading of the sp. along with its hosts.10,13,14 It was considered that P. vivax co-evoluted with Africans human population58 probably between 45,000– 81,000 years,56 but a study of sequences of complete mitochondrial genome of isolates of P. vivax, P. malariae, P.ovale, P. knowlesi, P. simium, P. frzagile revealed sequence divergence between P. vivax and P. knowlesi and placed it’s mitochondrial ancestors at around 2–3 million years ago.59 It gives a possibility of transfer of P. vivax from a monkey host to H. erectus in Southeast Asia sometime before 1 million years ago and subsequently spread across southern Asia into Africa through the H. erectus population. It supports findings of a previously given hypothesis on the Asian origin and close relation with Asian monkey’s of P. vivax based on the nuclear-encoded -tubulin and CDC-2 genes of 10 species of Plasmodium.56 Asian populations of P. vivax are considered the oldest. Diversity between Plasmodium sp. Two principal human malaria parasites, P. falciparum and P. vivax, seem to be very different in origin and in phylogenetic resemblance to other species of Plasmodium.

Evolution of Plasmodium parasite as suggested by Richard Carter and Kamini N. Mendis.61

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Further, they both differ not only in the mortality and morbidity they cause but also in the percentage of nucleotide composition in their respective genomes. The African populations of P. falciparum are highly diverse at the DNA level among other populations and in P. vivax the Asian population shows maximum diversity. These studies support African origin of P. falciparum and Asian origin of P. vivax. P. vivax shows higher genomic diversity than P. falciparum which suggests that P. vivax is older than P. falciparum. P. vivax has close genetic affinity with malaria parasite species of Asian monkeys, P. falciparum showed genetic similarity with P. reichenowi and chimpanzee.3 Like other Plasmodium sp., P. falciparum and P. vivax both have 14 haploid chromosome in their genome. The P. vivax genome contains a much lower AT (~55%) content in comparison to the genome of P. falciparum (~80%).60,62 On genetic variation, some reports have suggested a low level of genetic variation in P. falciparum and thus a relatively recent expansion, while other studies revealed a high level of genetic variation, indicating a large population size that has been maintained over hundreds of thousands of years.63 Similar level of diversity between P. falciparum and P. vivax confirmed that both the parasites underwent ancient population expansions. This strengthens the previous hypothesis that these two species were parasites of the hominid lineage before the origin of modern H. sapiens and expanded with population expansion of their host.3 Studies on population structure have provided important indications as to the origin, dispersal and stability of multilocus genotypes and quantify how alleles are spatially dispersed and how the overall genetic diversity is organized. Both P. falciparum and P. vivax use their genetic diversity to fight against anti-malarial drugs and the host immunity. However, the mechanisms of maintenance of such diversities are not known. Thus, study of genetic variations and evolutionary history of Plasmodium parasite genes and populations is critical in understanding several mechanisms in malaria, especially in identifying drug and vaccine targets, understanding virulence pattern, host–parasite interactions for determining source of parasite for imported malaria and for determining genes for drug resistance.7,64,65 On February 12, 2009, world has celebrated 200th birthday of Charles Darwin and 150 years of his magnificent intellectual achievement ‘On the Origin of Species’. He died in 1882, just 2 years after Alphonse Laveran identified Plasmodium, the parasite that caused malaria. Since then, a great deal of development has been achieved in malaria treatment procedures, as well as in issues like drug resistance by malaria parasites, effect of changing human behavior like agriculture and urbanization on the malaria parasite, adaptation by mosquitoes to changing environment and pesticides. Role of natural selection and development of resistant strains of malaria parasite,66 and in insects exposed to pesticides undergo strong natural selection through adaptive mechanisms to survive.67 These

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issues can be resolved by evolutionary studies of the parasite which can help to develop potential remedial measures. Today only because of Darwin’s work and guidance, we have some understanding about the complex evolution of Malaria. On the occasion of two centuries of Darwin, the biologists should acknowledge our gratitude to him and remember his outstanding contributions, which are still so relevant and useful ammunition in the continuing battle to save human lives from infectious diseases.

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20 Evolutionary trends in soil-inhabiting alaimid nematodes Mahlaqa Choudhary* and M. Shamim Jairajpuri** Section of Nematology, Department of Zoology, Aligarh Muslim University, Aligarh - 202002, UP, India *[email protected], **[email protected]

Abstract: In the present article, morphological characteristics with reference to evolutionary patterns of the nematodes inhabiting soil and belonging to the Order Alaimida on the basis of their sensory structures – amphids have been discussed. The alaims are simple nematodes, having a thin slender body and unarmed stoma, more or less gradually widening esophagus single or paired female gonads, single testis, simple and short spicules, few ventromedian supplements and no caudal glands or spinneret. Depending on the type of amphids two different routes of evolution i.e ‘Alaimus group’ and ‘Amphidelus group’ have been traced. A third line that is, ‘Cristamphidelus group’ has diverged mainly on the basis of numerous prominent longitudinal ridges. Possible evolutionary pattern of thirteen genera of alaims has also been discussed. ‘Alaimus group’ having amphidial apertures which are small, pore-like or indistinct and posteriorly placed, diverges into the genus Alaimus and Cosalaimus respectively. In ‘Amphidelus group’ the amphidial apertures are very large, transverse slit – like or oval or even longitudinally oval, placed generally close to the head. Ten different genera coming under this group i.e Amphidelus, Caviputa, Etamphidelus, Laxamphidelus, Megamphidelus, Metamphidelus, Paramphidelus, Postamphidelus, Scleralaimus, Scleramphidelus, have been discussed. These seem to have evolved from one single Amphidelus-like ancestor. Scleramphidelus have large amphids but instead of having a cup-shaped appearance transform to a stirrup-shaped form and also the margins are sclerotized. Probably, later three lines arose such as Paramphidelus slit-like, crescentshaped amphids, Etamphidelus, oval amphids with margins sclerotized and Metamphidelus having small, irregularly oval ones also with the same feature of the amphid margins. The genus Postamphidelus, as the name means posteriorly directed amphids, derive the character of these sensory structures from Paramphidelus. On the other hand, Etamphidelus gave rise to Megamphidelus with very large amphids and Caviputa where only the males have large, oval, kidney-shaped amphids. Lastly Caviputa gave rise to those alaims that have amphids which are oval and bilobed-Scleralaimus and Laxamphidelus with amphids that are kidney-shaped. ‘Cristamphidelus group’ has become a little more advanced in comparison to other alaims in the sense that it has evolved dorylaim characters such as presence of long slender spicules and also a small gubernaculum. Keywords: Alaimida, Nematodes, Evolutionary trends, Amphids and Soil V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Introduction The soil-inhabiting nematodes are, by and large rather, simple and primitive in their body structure and functions. They were discovered only in the late eighteenth century. The nematodes occur in very large numbers and are estimated to consist of about 5,00,000 species of which 30,000 have been described so far. It is said that the nematodes comprise of more than 90% of all metazoan of the world. They have successfully adapted to live, nearly in every ecological niche from marine to freshwater, polar regions to the tropics, as well as the highest to the lowest of elevations. These ubiquitous and in many respects bizarre animals live in marine, freshwaters and terrestrial environments, where they outnumber all other animals, both as individuals and in species count, can also be parasitic, both on plants and animals. The nematodes are placed at a very low level of hierarchial classification of invertebrates, somewhere close to Rotifera, Gastrotricha, Kinorhyncha, Nematomorpha, etc. They are good biological models for various kinds of studies as in genetics, ageing studies, molecular biology, mainly because of their features such as small size, transparent bodies, constant number of cells, shorter life cycles, etc. Their structural, biological and habitat diversities have made them extremely important for a broad spectrum of studies. Their numbers like that of Arthropoda is very large but unlike the later their bodies are not protected by a chitinous exoskeleton and hence cannot conserve water specially when on land. As soon as nematodes encounter atmospheric air, their bodies undergo dehydration. It has to overcome this condition by undergoing cryptobiosis, if it cannot, it may eventually die. This way, these organisms counteract their inability of having a chitinous coat, by hiding themselves. They are always enveloped in a thin film of water as long as they are hiding within the soil or may flourish in aquatic habitats. They represent a wide range of biodiversity. Nematodes also represent an important transitional stage of evolution of animals on earth. Being pseudocoelomates, they form an important link between acoelomates and the true coelomates. Being a variable group, new forms are being added to the number and hence no generalizations can be made regarding their morphology which could satisfactorily apply to all species. Because of the fact that the body is soft and due to the lack of solid shell or skeleton, fossilization is not possible, and consequently there are only a few known but poor fossil records. The oldest impression from Eocene of a Mermithida species, Heydonius antiquus1 which showed that the worm that was wriggling from the abdomen of a beetle was a nematode. In the absence of fossils and also that a huge ecological diversity is displayed by nematodes, it is difficult to correctly trace the phylogeny or geohistory of the group. Their evolution and phylogeny may only be reconstructed from the structure of recent living forms, relationships within and without the group

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and perhaps from their ontogeny. No reliable data is available as far as the phase of their phylogeny is considered, that is whether it is progressive or regressive. Their ancestors also seem to have disappeared entirely without much trace. The evolution which has produced diversity in the form and function and also the interrelationships, can, however, be inferred while studying the contemporary species of nematodes. The richness of species and the variety of their form and habitat suggests that they are comparatively of recent origin and are a dynamically developed group of animals. The alaims or alaimid nematodes that belong to the Order Alaimida2 can be easily recognized in a mixed nematode suspension by having a very thin and slender body and a simple appearance that is without cuticular appendages, absence of setae on head or body, non-annulated cuticle (under light microscope), narrow lip region, pore or slit-like amphids, small unarmed stoma, more or less gradually widening esophagus, single or paired female gonads, single testis, simple and predominantly very short spicules, few ventromedian supplements, no caudal glands or spinneret. The size of the body can range from being small (0.4 mm) to large (4 mm). In this article, stress has been laid on the morphological characteristics of alaim nematodes with reference to their evolution.

Morphological characteristics of alaim nematodes with reference to evolution Due to the lack of fossil remains, there is no concrete proof of when, where and in what possible shape nematodes in general appeared for the first time. Their ancestors, the primitive nematodes, the kind of evolutionary line followed by them, factors that influenced them to follow various lines of evolution. Their unparallel adaptations and advance stage of speciation and also that most ancient nematodes that survive even today are the ones which are supposedly the most advanced representatives of the group, are some of the questions that can be answered through indirect means and that too may be done by profound study of recent forms. The above aspects are very important as far as the evolution is concerned. We can try to put the present forms into an evolutionary series or we can connect them by their relationships. Therefore, in order to do this we can in short discuss the morphological characteristics and thereby evaluate the features phylogenetically as ones that are primary, primitive and also secondary features which are indicative of an advance development of nematodes (Fig. 1). Alaimid nematodes usually have a cuticle that is smooth, whether the smooth cuticle is more primitive than the annulated is difficult to decide. Lip region is somewhat rounded continuous with the body contour, sometimes offset, truncated and conoid in shape. Most of the species have six lips of equal size having a papilla

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on inner and outer margins. Stoma is simple unarmed and triradiate. Cephalic setae are absent, generally these are found in aquatic forms, whether absence of setae is primitive or not is still to be understood (dorylaims are regarded to be more advanced as they are without setae). Amphids and their apertures play an important role in the taxonomy of alaims. They are a pair present, one on each side of the proximal end of body. They may be pore-like, a little posteriorly placed from the anterior end, amphidial ducts between fovea and sensillar pouches is rather short, it is distinctive of Alaimus group. They may also be very prominent, their apertures may be large to very large, transverse, slit-like or oval, close to the head or a little below, amphidial duct between fovea and sensilla is very long. These amphids are not superficial organs, but lie below the cuticle and are said to be modified forms of missing labial papillae.3 If we say that it is a primitive character (originating from papillae) then in case of soil-inhabiting species which are regarded to be more advanced, this character persists and the different genera of alaims are differentiated largely on the basis of this feature. Consequently, it is regarded to be an advanced feature. Pharynx is clearly marked as an anterior part that is narrow, tubular while the posterior region is the swollen part often referred to as a glandularium, enlargement can be about one-fourth to one-sixth of esophageal length. The glandularium also contains three or five uninucleate glands, the glands are not very distinct but their nuclei are very clear, the orifice of the glands open into the lumen of pharynx. The five gland composition may possibly have evolved from the enlargement of dorsal nucleus and to counterbalance this, the subventral nuclei became double. Excretory system is simple and excretory pore occupies a variable position in the oesophageal region. It is also important taxonomically. Alaimids have either a single female reproductive system (monodelphic) or a set of double gonads (didelphic or amphidelphic). If monodelphic, then the reproductive branch can be anteriorly (prodelphic) or posteriorly (opisthodelphic) directed. In monoprodelphic condition vulva is situated post-equatorial. While in mono-opisthodelphic condition, the vulva is pre-equatorial. Amphidelphic condition has both the functional branches. Female reproductive branch is represented by an ovary, oviduct, sometimes spermatheca, uterus and vagina which in turn opens to the outside by vulva. According to Andrássy,3 single branched, unpaired gonad is a primitive character while the paired gonad condition is said to have developed later due to the gradual shifting of the vulva. Male reproductive system usually consists of a testis, vas deferens, ejaculatory duct, cloaca, additional sensory structures, spicules, guiding apparatus and ventromedian supplements. In alaim nematodes, male reproductive system is represented by the presence of a single testis (monorchic). It may also be outstretched and its tip is directed toward the anterior end of body. Spicules in this group are paired, usually equal in size, cuticularized and

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ventrally arcuate. They are guided by a set of protractor and retractor muscles. A variety of types of spicules are seen. Spicules can be straight, robust, cylindroid and provided with rounded ends. Ventromedian supplements vary in number from species to species and it is a good taxonomic character. Gubernaculum, which supports and guides the spicules, is absent in the alaims except in the genus Cristamphidelus where it is present as a small piece (Fig. 2). Length of tail is usually dependent on anal body diameter. Tail is an important feature in the group of alaims. Various sizes and shapes such as elongated, regularly tapering or flagelliform, mucronated, conoid, lanceolate, etc. are found and this character helps in the classification of these nematodes. Sometimes the two sexes may have the same type of tail but there can be differences also.

Figure 1. Alaimus teres (A) Entire female, (B) Entire male, (C) Anterior region (D) Basal region of esophagus, (E) Female gonad, (F) Female tail, (G) Male posterior region

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Scleralaimus (amphids large transversely oval, bilobed)

Megamphidelus (amphids large, longitudinal, oval)

Laxamphidelus (amphid apertures kidney shaped)

Caviputa (males with large, oval, kidney shaped amphids)

Postamphidelus (slit-like amphids, placed far from anterior end) Paramphidelus (slit-like, crescent-shaped amphids)

Cosalaimus (Indistinct amphids)

Alaimus (pore like amphids)

Etamphidelus Metamphidelus (Oval amphids (amphids irregularly with sclerotized margins) oval, small with sclerotized margins)

Scleramphidelus (large, stirrup-shaped amphids, opening slit-like to oval with sclerotized margins)

Amphidelus Cristamphidelus (large amphids, (transverse amphidial aperture, gubernaculum absent) gubernaculum present)

Alaims with pore-like amphids

Alaims with large, prominent amphids

Alaim ancestors Figure2. Evolutionary tree of Alaimida

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Evolutionary aspect De Man4 for the first time proposed the genus Alaimus for those nematodes having long and slender bodies, minute pore-like amphid apertures and a dorylaimoid type of esophagus. Micoletzky5 classified alaims in the family Alaimidae as nematodes with simple appearance, without cuticular appendages, cephalic or somatic setae, having non-annulated cuticle under light microscope, simple rounded head, pore or slit-like amphids, very small unarmed stoma, more or less gradually widening esophagus, simple or paired female gonads, mostly few ventromedian supplements without caudal glands and spinneret. Chitwood6 considered the families Tripylidae, Mononchidae and Alaimidae into an assemblage of unrelated groups and of independent evolutionary lines. Clark7 revised the diagnosis of Alaimina and Dorylaimina placing both Suborders under Enoplida. Gerlash8 and Andrássy5 placed the alaims in the superfamily Oxysomatoidea Filipjev9 of the Order Enoplida, basically on the characters such as absence of cephalic setae and caudal glands. Amphids are extremely small, pore-like or conspicuous, one or two ovaries, gubernaculum lacking, pre-anal supplements, papilliform, etc. The later because of characters such as absence of stomatal armature, distinct ducts connecting the fovea and sensillar pouches, cephalic sensillae in two separate circlets, spermatheca found in females (in some only) and a single outstretched testis, Siddiqi2 suggested that alaims should be placed under a separate Order Alaimida within subclass Enoplia. He rejected the view presented by Maggenti10 which was that Enoplia developed primarily along two lines one marine and the other terrestrial, the later line included dorylaims. Therefore, he was of the opinion that nematodes representing different evolutionary lines independently invaded the niche “land” and successfully occupied it. He, therefore placed only one suborder Alaimina under Alaimida (found on land) and Oxystomina that are sea-dwelling were placed separately. Bongers11 recognized Alaimidae but assigned it to suborder Tripylina under Enoplida. Among alaims, again depending on the type of amphids, two different routes of evolution can be traced, the “Alaimus group” that has amphid apertures which are small, pore-like or indistinct very posteriorly placed (about 1/3–1/2 of esophagus length) on amphidial duct short, includes the genus Alaimus de Man.4 Cosalaimus Siddiqi12, which is also in this group has evolved further by acquiring longitudinal ridges, and at the same time, the amphids have become indistinct. The other, “Amphidelus group” has amphid apertures that are large to very large, transverse, slit-like or oval, or longitudinally oval, generally close to head, about (one-fifth labial widths) from anterior end. Amphidial duct between fovea and sensilla are long to very long. Within this group, 10 different genera have been reported, that is Amphidelus, Caviputa, Etamphidelus, Laxamphidelus, Megamphidelus, Metamphidelus, Paramphidelus, Postamphidelus, Scleralaimus,

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Figure 3. Different types of alaimid amphids. (A) Alaimus, (B) Amphidelus, (C) Cristamphidelus, (D) Cosalaimus, (E) Scleramphidelus, (F) Paramphidelus, (G) Etamphidelus, (H) Metamphidelus, (I) Postamphidelus, (J) Megamphidelus, (K) Caviputa, (L) Scleralaimus, (M) Laxamphidelus

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and Scleramphidelus (Fig. 3). Amphids, being a particular characteristic of alaims is always a pair on each side of the proximal end of the body. The shape and structure varies in all the 10 genera. Very close to the Amphidelus group, a third line that is,Cristamphidelus group has diverged mainly on the basis of numerous (30–60) fine longitudinal ridges, slit-like amphids, characteristic S-shaped vagina, long and slender spicules and the presence of a small gubernaculum. In the earlier two groups, represented by Alaimus and Amphidelus, there is an absence of gubernaculum, however, in the Cristamphidelus group, the presence of gubernaculum shows that this may be close to dorylaims . In Amphidelus group, the amphids are large but the gubernaculum is absent. Scleramphidelus is the genus that has large amphids like Amphidelus but a slight diversion has taken place, where amphids have become stirrup-shaped, the opening is slit-like to oval and their margins are sclerotized. From this, three lines very close to one another, that is, Paramphidelus having slit-like, crescent-shaped amphids, Etamphidelus with oval amphids (sclerotized margins) and Metamphidelus with irregularly oval, small amphids and also with sclerotized margins have diverged out. The slit-like crescent-shaped amphids of Paramphidelus later evolved into those that were pushed behind, far from the anterior end, which is a characteristic feature of Postamphidelus. Etamphidelus later on, gave rise to two types that is, Megamphidelus having amphids that are large and longitudinally oval, while Caviputa in which females have no amphids but the males have large oval, kidneyshaped amphids, though according to Siddiqi, 1993)12 Caviputa comes close to Alaimus de Man (1880)4 but differs from it in having prominent outer labial sensillae and didelphic gonads. Two genera i.e., Scleralaimus and Laxamphidelus have amphids that are large transversely oval, bilobed and amphids that are kidneyshaped respectively having possibly evolved from Caviputa. Acknowledgement: One of us, Prof. M. Shamim Jairajpuri is thankful to INSA, New Delhi for providing financial assistance.

References 1.

Taylor AL (1935) A review of the fossil nematodes. Procf Helmintholog Society, Washington 2:47–49

2.

Siddiqi MR (1983) Phylogenetic relationships of the soil nematode orders Dorylaimida, Mononchida, Triplonchida and Alaimida, with a revised classification of the subclass Enoplia. Pak J Nematol 1(2):79–110

3.

Andrássy I (1976) Evolution as a basis of the systematization of nematodes. London

4.

De Man JG (1880) Die einheimischen, frei in der Erde und im süssen Wasser lebenden Nematoden. Vorläufiger Bericht und descriptiv-systematischer Theil. Tijdschrift der Nederlandsche Dierkundige Vereenigin 5:1–104

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5.

Micoletzky H (1922) Die freilebenden Erd-Nematoden mit besonderer Berücksichtigung der Steiermark und der Bukowina, zugleich mit einer Revision sämtlicher nicht mariner, freilebender Nematoden in Form von Genus-Beschreibungen und Bestimmungsschlüsseln. Archiv für Naturgeschichte, Abteilung A 87:1–650

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Chitwood BG (1937) A revised classification of the Nematoda. Papers Helminth Moscow 67–79

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Clark WC (1961) A revised classification of the Order Enoplida (Nematoda). N Z J Sci 4:123–50

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Gerlach SA (1966) Bemerkungen zur Phylogenie der Nematoden, Mit zwei Anhangenm: 1. Klasification der Oxystominidae, 2. Das Mannche von Onchulus longicaudatus Cobb, 1920. Mitteilungen aus der biologischen Bundesanstatt fur land und Forstwirtschaft, Berlin-Dahlem, 118:35–39

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Filipjev IN (1918) Nématodes libres marins des environs des Sebastopol. Part I (22 parts). Travaux Lab Zool et Stat Biol Sebastopol Ac Sci Russie 2:1–350

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Maggenti AR (1983) Nematode higher classification as influenced by species and family concepts. In: Concepts in Nematode Systematics. Stone AR, Platt HM, Khalil LF (Eds.), Academic Press, London

11.

Bongers T (1988) De Nematoden van Nederland Utrecht

12.

Siddiqi MR (1993) Nematodes of tropical rainforests. 1. Six new genera and eighteen new species of alaims. Afro-Asian J Nematol 3:60–80

21 Evolution of the cerebral cortex in amniotes: Anatomical consideration of neuronal types U.C. Srivastava* and R.C. Maurya** Department of Zoology, University of Allahabad, Allahabad - 211002, India *[email protected], **[email protected]

Abstract: The cerebral cortex is a thin sheet of nervous tissue in the roof of the cerebrum of amniotes. The cerebral cortex of reptiles is divided into four areas viz. medial, dorsomedial, dorsal and lateral cortices having different neuronal components. The areas of the hippocampal complex of birds are subdivided into a dorsal parahippocampal region and a ventral hippocampus. The different neuronal types divide the hippocampus of birds into five fields viz. medial and lateral hippocampus, parahippocampal area, central field of parahippocampal area and crescent field. The cerebral cortex of mammals is a complex structure. The neocortex of mammals shows the typical six layered structure in the frontal, parietal, temporal and occipital lobes having typical pyramidal and nonpyramidal neurons which show phenotypic variations. Neuronal types in cerebral cortex of reptiles, birds and mammals have been compared to determine homologous structures. The possible homologous structures have been mentioned in final comments. Keywords: Cerebral cortex, Hippocampus, Amniotes, Pyramidal, Nonpyramidal neurons

Introduction Cerebral cortex is a structure that constitutes the outermost layer of the cerebrum. The term pallium is used for cerebral cortex coating the surface of the cerebrum (cerebral hemisphere). In the anatomical nomenclature, the cerebral cortex is divided into neocortex (isocortex) and allocortex. The neocortex constitutes 95.6% of the cerebral cortex and is six layered structure. The allocortex is divided into paleocortex and archicortex. The latter is synonym of hippocampus1. The periallocortex or the mesocortex are the terms used to differentiate the transitional zone between the neocortex and allocortex. V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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From the beginning of 19th century, different investigators have differentiated the hippocampus into different regions and used varied terminology/nomenclatures. Cajal2 divided the hippocampus into region superior and region inferior while Rose3 divided the hippocampus into five regions naming H1–H5. Lorente de Nó4 identified four regions using the term CA1–CA4. In fact, he was the first to use the term cornu ammonis for the hippocampus. Of late, Federative committee on anatomical terminology (1998) gave the newest anatomical nomenclature and divided the hippocampus in four regions naming CA1–CA4 which may be written as I–IV. In addition, the term gyrus dentate has been used for fascia dentate. How the cerebral cortex of mammals should be compared to that of reptiles and aves is one of the oldest and most intensely debated questions in the field of comparative neurobiology. In this article the neuronal classes of the cerebral cortex of reptiles, aves and mammals have been compared to show homologous structures for evolutionary interpretations.

Cerebral cortex of reptiles The entire cerebral hemisphere of reptiles from rostral to caudal region is divisible into pallium (includes cortical layers) and subpallium (include septal complex, dorsal ventricular ridge, striatal complex and amygdaloid complex). The cerebral cortex of reptiles is differentiated into four areas having distinct cellular composition. These are named according to their relative mediolateral position as medial cortex (MCx), dorsomedial cortex (DMCx), dorsal cortex (DCx) and lateral cortex (LCx). The cerebral cortex shows three-layered structure consisting of a cell layer (cl) with highly packed neuronal cell bodies sandwiched between cell-sparse outer (opl) and inner plexiform layer (ipl).5-12 The outermost layer-І has only few neuronal somata and also the dendrites ascending from subjacent layers. There are only few somata in layer-I of different reptilian species viz. lizard8,11 and snakes6 whereas in tegu lizard5 neurons are absent in layer-I. Layer-II of the cerebral cortex of reptiles is characterized by the presence of densely packed neuronal somata in P. hispanica,9 M. carinata,11,13 and snakes.6–7 It also contains dendrites descending from outer layer-І and ascending from inner layer-ІІІ. The sheet of somata in the cell layer-ІІ is interrupted by a discontinuity in the different regions of cerebral hemisphere. The cortex of all the reptiles except crocodilian14-15 is characterized by a distinct discontinuity in layer-II.16 Layer-II of the dorsal cortex is placed closer to the ventricle than that of the medial and lateral cortices. In many species layer-II of the dorsal cortex is overlapped by the adjacent cortical layers forming medial and lateral superpositions, whereas in snakes such as Eryx or in turtles15,17 little overlapping is observed. The significance of this discontinuity and overlapping is not clear but the basic trilaminar pattern is replaced by

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five-layered annulus of cortex with dendrites being exchanged between the densely packed somata of overlapping layers.11 The somata are loosely packed in layer-III of the cerebral cortex of reptiles9,11-13,18 and snakes.6–7 It also contains dendrites descending from layer-І & ІІ and ascending processes from ependymal layer. Below the inner plexiform layer an ependymal layer is also observed just above the ventricle (V). Ependymal layer has been observed in all the cortical areas except the lateral cortex.

Neuronal types in reptiles By using classical Golgi-impregnation method19 different investigators described the different number and types of the neuronal classes in all the cortical areas in ophidian and lacertilian groups. In the medial cortex, only one type of neuron in snake genera Natrix and Boa,7 five types in the lizard L. pityusensis,20 P. hispanica9 and M. carinata11, seven types in H. flaviviridis10 have been reported. In case of dorsomedial cortex one type of neuron in the lizard A. agama21 and three types in each layer of snake’s dorsomedial cortex22 have been reported. In the dorsal cortex, four types of neurons in the lizard P. algirus23 and five types in M. carinata11 have been reported. In lateral cortex, four types of neurons are present in all the three layers of snakes,24 whereas three types were observed in the M. carinata11 and four types were present in the H. flaviviridis.25 Of late, different neuronal types described in lacertilian species are pyramidal (may be inverted and bipyramidal), multipolar, monotufted, bitufted, stellate, candelabra like monotufted, monotufted monopolar and monotufted bipolar neurons. Further, they have been classified on the basis of the presence or absence of spines. Different investigators have used different methodology to reveal the morphology of the neurons and neuropil in the cerebral cortex of reptiles.6,7,9,11,13,18,21,26–30 These investigations are important. Various neuronal types described by different investigators in medial, dorsomedial, dorsal and lateral cortex of reptiles have been given in Tables 1 to 4.

Cerebral cortex of birds The boundaries of the avian hippocampus have not been well established39-41, which is very important for understanding the functions of the avian hippocampus. The hippocampal complex (dorsomedial forebrain) of birds, separated from the rest of the cerebral hemisphere by lateral ventricle, is a narrow curved strip of tissue present on the dorsomedial surface of telencephalic hemisphere. Anatomically, the hippocampal complex is subdivided into two main structures, a dorsal parahippocampal area and a ventral hippocampus. The hippocampus is widest dorsally at the junction with the parahippocampal area, and it tapers ventrally

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Table 1 Neuronal types of the medial cortex of various species reported by different investigators S. No. Author (year) 1. Ramón, 189631; Ramón y Cajal, 190432 2. Minelli, 196633 3. 4. 5. 6. 7. 8.

Northcutt, 196734 Regidor et al., 197435 Ulinski, 19777 Wouterlood, 198121 Shen and Kriegstein, 198636 Berbel et al., 198720

Animal Types of neurons C. chamaeleon Pyramidal cells L. viridis, L. muralis I. iguana L. galloti

Very large pyramidal neurons; Vertical bipolar neurons Double pyramidal cells; Small projecting cells m1B, 1C, 1D, 1E

Snakes (Natrix Six types of candelabra cells and Boa) Six types of candelabra cells A. Agama Turtle

Stelllate cells; Pyramidal cells

L. Pityusensis

Sparsely spinous horizontal neurons; Spinous pyramidal neurons; Spinous bitufted neurons; Small, sparsely spinous pyramidal neurons; Spinous multipolar neurons Heavily spiny granular monotufted, medium sized neurons; Heavily spiny bitufted neurons; Spiny bitufted neurons; Sparsely spiny bitufted neurons; Superficial multipolar neurons Monopolar neurons; Monotufted bipolar neurons; Candelabra neurons; Bitufted neurons Aspinous bipolar neurons; Aspinous monotufted monopolar neurons; Aspinous monotufted bipolar neurons; Spinous monotufted monopolar neurons; Spinous monotufted bipolar neurons; Spinous bitufted bipolar neurons; a. Heavily spinous bitufted bipolar neurons; b. Spinous bitufted bipolar neurons; c. Sparsely spinous bitufted bipolar neurons; Spinous multipolar neurons

9.

Luis de la Iglesia P. hispanica and Lopez Garcia, 19979

10.

Srivastava et al., M. carinata 200711

11.

Maurya and Srivastava, 200610; Maurya, 200925

H. flaviviridis

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Table 2 Neuronal types of the dorsomedial cortex of various species reported by different investigators S. No.

Author (year)

Animal

Types of neurons

1.

Wouterlood, 198121

A. Agama

Large pyramidal cells of one types

2.

Srivastava et al., 200711

M. carinata

Pyramidal neurons; Inverted pyramidal neurons; Bipyramidal neurons; Multipolar neurons; Candelabra neurons

3.

Srivastava et al., 200912

H. flaviviridis

Bitufted neurons; Pyramidal neurons; Inverted pyramidal neurons; Bipyramidal neurons; Multipolar neurons; Candelabra like monotufted neurons

Table 3 Neuronal types of the dorsal cortex of various species reported by different investigators S. No. Author (year)

Animal

Types of neurons

1.

Garcia Verdugo et al., 198337 L. galloti

2.

Guirado et al., 198723

P. algirus

Bitufted neurons; Multipolar neurons; Pyramidal neurons

3.

Srivastava et al., 200711

M. carinata

Monotufted neurons; Bitufted neurons; Pyramidal neurons; Bipyramidal neurons; Candelabra neurons

4.

Maurya, 200925

H. flaviviridis Monotufted neurons; Bitufted neurons; Multipolar neurons; Pyramidal neurons

Six types of neuronal soma

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Table 4 Neuronal types of the lateral cortex of various species reported by different investigators S. No.

Author (year)

Animal

Types of neurons

1.

Ulinski and Rainey, 198024

Snakes (C. constrictor, T. sirtalis, N. sipendon)

Bipyramidal neurons; Pyramidal neurons; Polymorphic neurons

2.

Garcia Verdugo et al., 198438 L. galloti

3.

Srivastava et al., 200711

M. carinata

Bipyramidal neurons; Pyramidal neurons; Polymorphic neurons

4.

Maurya, 200925

H. flaviviridis

Monotufted neurons; Bitufted neurons; Pyramidal neurons; Stellate neurons

Four basic types of neuronal soma

with the septum.39,41–45 On the basis of the presence of different neuronal types,39,45 the hippocampal complex have been divided into five different fields viz. medial (HCm) and lateral (HCl) hippocampus, parahippocampal area (APH), central field of parahippocampus (PHc) and crescent field (CF). The adjacent region, corticoid complex (CC) occupies the dorsolateral surface of the telencephalic pallium in zebra finch, Taeniopygia guttata and Strawberry finch, Estrilda amandava.39,45 The corticoid complex is divided into two subfields viz an intermediate corticoid area (CI) and a dorsolateral corticoid area (CDL). The extent of these two areas varies considerably in different species of birds.39,45-46 The intermediate corticoid region and various regions of hippocampal complex progressively disappear in the caudal region at the level of the cerebellum. The avian dorsomedial forebrain or hippocampal complex is involved in memory processes associated with spatial behaviour, food storing.40-41,47-52 The hippocampal volume of the food storing passerine species is larger than non food storing species.49,53-55

Neuronal types in birds On the basis of Golgi study, the neurons of hippocampus of birds have been classified into two main groups. The predominant cell types were projection neurons with spinous dendrites and local circuit neurons having sparsely spinous and aspinous dendrites. In HCm area pyramidal, multipolar, bitufted, monotufted; in PHc and CF region multipolar neurons have been observed. The neurons of

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the corticoid complex are classified into three main cell groups: predominant projection neurons, local circuit neurons and stellate neurons. These neurons are sub classified into pyramidal neurons (located only in CI) and multipolar neurons (located both in CI and CDL). The stellate neurons of the CI have a small round or ovoid cell body that extends 4–6 long thin dendrites. The spines on the dendrites are moderately distributed. The axon of these neurons originates either from cell body or from a dendrite, and ramifies locally. The above mentioned neuronal types and their projections have been completely described in strawberry finch, Estrilda amandava by Srivastava et al.56 The comparative account of different neuronal types of hippocampal complex, corticoid complex and visual wulst in different bird species has been shown in Table 5 to 7. Table 5 Neuronal types of the hippocampal complex of various species reported by different investigators S. No. Author (year)

Animal

Types of neurons

Gallus gallus

Pyramidal neurons/ Bipyramidal; Long axon multipolar neurons; Short axon multipolar neurons; Horizontal neurons

1.

Mollá et al., 1986

2.

Montagnese et al., 199639

Taenopygea guttata

Bitufted neurons (Pyramidal); Multipolar; Local circuit neurons (dense pericellular, basket, radial, netlike)

3.

Tömböl et al., 200041

Gallus domesticus and Columba livia

Pyramidal neurons; Pyramidal-like neurons; Multipolar; Local circuit neurons (small/ medium, large/ medium); Stellate neurons (Columba livia)

4.

Srivastava et al., 200745

Estrilda amandava

Pyramidal neurons; Pyramidal-like neurons; Multipolar; Bitufted neurons; Monotufted neurons; Local circuit neurons

57

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Table 6 Neuronal types of the corticoid complex of various species reported by different investigators S. No. Author (year)

Animal

1.

Montagnese et al., Taenopygea guttata 199639

2.

Srivastava et al., 200956

Estrilda amandava

Types of neurons Bitufted neurons (Pyramidal); Multipolar; Local circuit neurons (radial, horizontal, bipolar/multipolar); Stellate neurons Pyramidal neurons; Multipolar; Horizontal neurons; Local circuit neurons (small, medium sized multipolar); Stellate neurons

Table 7 Neuronal types of the visual wulst of various species reported by different investigators S. No. Author (year)

Animal

Types of neurons

1.

Watanabe et al., 198358.

Coturnix coturnix japonica

Type I; Type II; Type III; Type IV

2.

Tömböl and Maglóczky, 199059.

Gallus domesticus

Type 1d; Type 1b; Type 1a, Type 1c; Local circuit neurons (small, medium, large sized, GABA positive neurons); Stellate

3.

Montagnese et al., 199639.

Taenopygea guttata Multipolar; Local circuit neurons (small and medium sized with few spines); Stellate

4.

Chand P, 200960.

Estrilda amandava

Pyramidal neurons (Moderately spinous, Sparsely spinous); Multipolar (Highly spinous, Moderately spinous, Sparsely spinous) Local circuit neurons (small and medium sized), Aspinous; Stellate, Sparsely spinous

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Cerebral cortex of mammals The cerebral cortex of mammals: a sheet of nervous tissue in the telencephalic roof is commonly referred to as gray matter due to the predominance of cells which appears grayish brown. These neurons are connected to other neurons within the brain by axons located beneath the cortex and within the cortex. The neocortex of mammals shows the typical six layered structure in the frontal, parietal, temporal and occipital lobes, corresponding to the names of skull plates that protect them. The characteristics of six different layers of different fields are different. Among eutherians, Golgi study of the specific sixth layer of cerebral cortex in brains of insectivore, chiroptera, rodentia, lagomorpha, artiodactyla, carnivore and primate revealed differences in cortical thickness, soma types, soma size, dendritic extents and orientation of neurons.61–62 Neuronal morphology and distribution of calcium binding proteins on cortical neurons have been found to be similar in cetacea and artiodactyla but differ considerably from that in primates, carnivores and rodents. This shows several species and order specific patterns which can be used to assess taxonomic affinities among species.63 Pyramidal cells in prefrontal cortex of new world monkey are relatively simple in structure while inferotemporal cells are highly branched and spinous64, although it is generally agreed that the cortical areas have the same complements of neuronal cell types.65 In some cases there are distinct types of neurons which are only found in particular cortical areas or species, such as Meynert and Betz giant pyramidal neurons, spindle neurons and double bouquet cells.66-68 The enlargement and differentiation of the giant pyramidal Betz and Meynert subtype cells can be viewed as a correlate of specialization of functionally distinct regions of the neocortex in the primates.69 In addition, the pyramidal cells sampled from different areas of different primate species, show differences in dendritic arbor size and spine density.64,67,70–74 In addition to this, the pyramidal neurons exhibit a differential distribution among cortical layers and regions, and some of them are differentially represented among species.75 Dendritic spines, a characteristic feature of typical mammalian pyramidal neurons represent important structural specialization of eutherian isocortical neurons and provide most of the postsynaptic sites of axon terminating upon pyramidal neurons.76–77 Differences in pyramidal neuron’s spine density could reflect important functional differences in the isocortex78 and in spine density of pyramidal neurons of visual areas in marmoset monkey.70 These phenotypic differences in pyramidal neurons may reflect some evolutionary trends.

Neuronal types in mammalian cerebral cortex In general, two main types of neurons have been described in mammalian cerebral cortex: pyramidal and nonpyramidal types. The specialization in these neurons

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shows homology and differences not only among the different mammalian species but also in sauropsids (reptiles and birds). The different types of neurons described by different investigators in mammalian species in the cerebral cortex are given in Table 8.

Table 8 Neuronal types of the mammalian cerebral cortex of various species reported by different investigators S.N. Author (year)

Animal/region

Types of neurons

1.

Economo, 192779 Human cortex

2.

Mitra, 195580

Visual, somatosensory and motor cortex of cat

Fusiform neurons

3.

Adrianov and Mering, 195981

Layer II of Broadmann’s area

Small, medium and large pyramidal neurons

4.

Tunturi, 197182

Ectosylvian auditory cortex of Dog

Small, medium and large pyramidal; star; special; double bouquet and fusiform cells

5.

Gilbert and Kelly, Visual cortex of Cat 197583

6.

Lund et al., 1977, Visual cortex of Macaque Pyramidal and stellate cells Monkey 197984-85

7.

Garey and Saini, 198186

Monkey

Multipolar, triangular, bipolar, capsular and neurons with fine axon-like dendrites

8.

Garey et al., 198587

Visual cortex of bottle nose Dolphin

Pyramidal, multipolar/stellate and spindle shaped cells

9.

Ferrer et al., 198661–62

Rat, mouse, hamster, vole, rabbit, dwarf bat, hedgehog, cat, dog, cow, sheep and man; sixth layer of cerebral cortex

Pyramidal neurons (large, medium, small or flattened and atypical or triangular); Multipolar pyramidal; Inverted pyramidal; Fusiform neurons; Horizontal neurons; Fan-shaped neurons; Martinotti cells; Multipolar neurons; Bitufted neurons

Small, medium and large pyramidal neurons

Pyramidal and stellate cells

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...Cont’d from previous page

S.N. Author (year)

Animal/region

Types of neurons

10.

Ferrer, 1986

Mole (Talpa europaea)

Presumptive projection neurons and Local circuit neurons (bipolar, multipolar, double bouquet cells, spiny stellate and star pyramidal cells)

11.

Fitzpatick and Henson, 199489

Auditory cortex, mustached bat

Spiny cells (pyramidal, inverted pyramidal and extraverted pyramidal cells, spiny stellate, multiform cells). Non-spiny cells (multipolar, bitufted and bipolar cells)

12.

Tyler et al., 199890

Dunnart, quakka (both marsupials) and Macaca

13.

Hassiotis and Ashwell, 200378

Australian echidna

14.

Hof and Sherwood, 200591

50 species of 12 mammalian orders –

Pyramidal neurons, spiny stellate neurons, wide arbor basket neurons, medium arbor basket neurons, robust columnar axon cells, double bouquet cells, chandelier neurons, neurons with simple beaded axon arbors, layer 4 type beta 3 neurons. Pyramidal, spinous bipolar, aspinous bipolar, spinous bitufted, aspinous bitufted, spinous multipolar, aspinous multipolar and neurogliaform. Pyramidal neurons, multipolar neurons

88

Primary motor cortex of primates and primary visual cortex of primates Cingulated cortex and insula of hominids

Meynert cells, Betz cells

Spindle cells with vertical, fusiform morphology

Comparison of the neuronal types of the cerebral cortex in reptiles, birds and mammals The medial cortex of reptiles and the hippocampal fascia dentata of mammals have been considered homologous centers on the grounds of their common cytoand chemo-architectonical patterns, their position in cortical circuitry, and their

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late ontogenesis.9,10, 92–93 The neurons of the cell layer-II of the medial cerebral cortex of reptilian species show resemblance with the granule cells present in the hippocampal formation of different mammalian species such as rat,4,94–97 rabbit,98,99 monkey,100 primates101 and human.102 The dentate granule cell layer of mammals is occupied by homogeneous cell population which is very similar in different reptiles,2,4,98 where most morphological differences are of the soma position in the granule layer. Basal dendrites are transitory in immature dentate granule cells of rodents,94,97 frequent in ectopic granule cells,96 and are considered as aberrant characteristic in granule cells under experimental conditions.103–104 Moreover, basal dendrites are rare in mature granule cells of rabbits,2,98 but they are regularly seen in granule cells of other mammalian species105 including primates and humans.2,98,101–102,106 The morphology of dentate granule cells with basal dendrites is very much similar to different types of spiny bitufted bipolar neurons in the cell layer-II of medial cortex of the reptiles.9,10,12 The neurons of the hippocampal complex of the birds share some common features with the neurons present in the homologous structures of the reptilian and mammalian telencephalon. In the hippocampus of birds such as chick and homing pigeon, the dominant projection neurons are the multipolar neurons39, 41,45 unlike the situation in the mammalian hippocampus, where the only projection neurons are pyramidal. In the mammalian hippocampus, the pyramidal neurons are located only in the pyramidal layer while in the hippocampus of the birds the dominant multipolar projection neurons were found in all layers, but the pyramidal and pyramidal-like neurons were located only in the pyramidal layer-II.39,41,45 The spinous and highly branched monotufted neurons present in the lateral hippocampus of birds39,45 and extending their projection towards the pia seems to be similar with those of the candelabra cells,107 granular neurons9 or spiny monotufted neurons10 present in the cell layer-II of the medial cortex in different lizard species. The bitufted neurons present in the lateral and medial hippocampus of the bird39,41–42, 45 also show resemblance with those found in the bitufted and bipyramidal neurons of medial and dorsomedial cortex of the lizards.9–10,21,26 According to Casini et al.108 the hippocampus would correspond to a part of Ammon’s horn, APH to the subiculum, and hyperpallium densocellulare (HD) to the entorhinal cortex. Using Golgi method, Montagnese et al.39 suggested that the medial arm of the V-shaped layer corresponds to Ammon’s horn, the central field of the parahippocampus to the subiculum, and the intermediate corticoid to the entorhinal cortex. They proposed an absence of a dentate gyrus and mossy fibers because of the absence of granule cells in the hippocampus, as did Tömböl et al.41 for the chicken and pigeon. In zinc staining, the hippocampus is stained entirely homogeneously in the chicken and zebra finch,39,109–110 although the mossy fiber bundle from the dentate gyrus to CA3 is very apparent in mammals.

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Similarities between reptilian medial and dorsomedial cortex with avian hippocampus and parahippocampus include the position medial and dorsal to the ventricle, a more or less pronounced three layered organization,26,111–112 neuronal types,9–12, 21, 26, 34 and efferent and afferent projections.26,108,113–114 The dorsomedial cortex of reptiles shows resemblance with the parahippocampal area of birds due to its position dorsal to the ventricle and medial hippocampus, and the presence of multipolar neurons in the parahippocampal area of birds with medium/large cell body and four to six spinous dendritic branches.39,45 The pattern of extra cortical afferences and efferences of the dorsomedial cortex neurons,112,115 and the intra cortical scheme of connections of the lizard cerebral cortex have a clear resemblance to that of the mammalian hippocampus and the entorhinal olfactory cortex.116 The dendritic tree pattern of pyramidal and bipyramidal neurons with their dendritic spines of dorsomedial cerebral cortex of reptiles27 are comparable with the corresponding elements on pyramidal neurons of the CA3 (Cornu ammonis 3) area of the mammalian hippocampus because it emits a prominent commissural-contralateral projection117 and also because it is the main recipient of the zinc-positive ‘lizard mossy fibers’ coming from the medial cortex.27,11 8 Anatomically, reptilian dorsal cortex resembles with the isocortex of mammals in many respect. The three neurons of reptilian dorsal cortex namely bitufted neurons, multipolar neurons and pyramidal neurons have formed the basis for comparing the medial aspect of dorsal cortex being homologous to the mammalian hippocampal formation,119–120 whereas the lateral aspect of the dorsal cortex has been compared to the mammalian isocortex, or at least to part of it.107, 121 Anatomically the dorsal cortex of reptiles differ from the isocortex in being three layered instead of six layered and in lacking columnar organization.122 The dorsal cortex is a structure unique to reptiles, and its relationship to structures in mammalian brain is of great theoretical interest.122–126 The pyramidal neurons of the hyperpallium apicale (HA) of birds39,41,45,58–60, 127 are comparable with the layer V-VI of the mammalian neocortex.128–131 Sparsely spinous pyramidal neurons of the HD are slightly similar with the pyramidal neurons found in the layer-III of the echidna somatosensory cortex and layer-V of bottlenose dolphin visual cortex.78,87 The multipolar neurons of the laminae: interstitial nucleus of the HA, hyperpallium intercalatum and HD of wulst of the birds39,45,58–60,127 show similarities with the sparsely spinous multipolar cells of the layer II-III of the mammalian visual cortex132 and also with the spinous multipolar neurons of the layer III and IV of echidna somatosensory cortex,78 spiny cells without apical dendrites of the rat Sm1 cortex133 and type 7 cells in primate somatosensory cortex.134 The local circuit neurons observed in the HA, HI and HD of birds39,41,59,127 are comparable with the chandelier cells of the somatosensory134–135 and visual cortex of the different mammalian groups.85,136–145 The sparsely spinous

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stellate neurons of the wulst of birds, strawberry finch,60 zebra finch,39 Japanese quail58 show resemblance with the spiny stellate neurons of the layer III, IV and VI of the visual cortex of bottlenose dolphin87 and other mammals.78,146–147 The stellate cells are also referred to as local plexus neurons because of the limited spread of axonal arborization.148 The wulst of birds is comparable to dorsal isocortex comprising occipital, parietal and sensorimotor parts of the frontal cortex.122,125–126,149–150 The visual and somatosensory areas of the wulst resemble the primary visual and somatosensory regions of the occipital and parietal cortex in mammals.150–151 The wulst lamination, however, is inverted as compared to the mammalian isocortex.152 It is inferred that the superficial wulst layer is comparable to deep isocortical layers (V-VI) in that it projects to both striatum and brainstem. The middle layers of wulst are comparable to the middle isocortical layer (IV) in that they receive primary sensory thalamic inputs. The deepest layer of the wulst is comparable to the superficialmost isocortical layers (II-III) in that it projects to the striatum and other telencephalic targets but not to the brainstem.130–131 Wulst of birds includes a somatomotor area,153–154 which could neither be found in lizards112,155–156 nor in turtles157 and shows somatosensory thalamic projections to the cortex. Like the avian wulst,128 the dorsal cortex (DC) of turtles displays a visual area that includes the pallial thickening, which receives a projection from the dorsal lateral geniculate nucleus of the thalamus.157 In lizards, this projection reaches just the pallial thickening.155,158 The reptilian pallium has a three-layered cortex, consisting of a medial, a dorsomedial part (both similar to the mammalian hippocampal formation), a lateral (olfactory) cortex,107 and finally a dorsal cortex (comparable to the wulst of birds) which receives visual projections from the dorsal lateral geniculate nucleus, as well as some somatosensory input.131 The dorsal cortex of reptiles and its avian equivalent, the wulst, are considered to be homologous to both the striate or primary visual cortex and the somatosensory cortex of mammals, as all these structures receive similar sensory projections.131,149,159–160 More precisely, the visual projections from the retina to the thalamic dorsal lateral geniculate nucleus terminate in the posterior dorsal cortex/wulst of sauropsids and in the striate cortex of mammals, respectively. In addition, the somatosensory spinothalamic and the dorsal column-medial lemniscus pathways project to the anterior dorsal cortex/ wulst of sauropsids and to the somatosensory cortex of mammals.131,161 In the corticoid complex (CI) of birds, dominant pyramidal neurons possess spinous apical dendrites that give rise to oblique side branches and several basal dendrites. The axon collaterals of these neurons show connections with their own dendritic spines and other neuronal dendritic spines. Some of these collaterals run laterally towards the CDL and APH regions.39,41,45 The pyramidal cells of mammals have one dominant large apical dendrite that often branches close to

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the soma, whereas the basal dendrites extend extensively in all directions within the deep layers. The apical dendrites and the axon collaterals extend towards the superficial layers, sometimes even reaching the pial surface.162–164 A major afferent pathway to the mammalian hippocampus comes from the entorhinal cortex.165 The projection from the corticoid complex to the HCC in birds108 might originate from CI pyramidal neurons, which can be compared with the projections of the entorhinal cortex to the various parts of hippocampus in mammals.162–164,166–170 The spinous multipolar neurons of the birds are uniformly distributed in the CI and CDL regions and their axons run towards the ventricular wall, whereas the axon collaterals of CDL region run parallel to the entire width of CDL. The CI receives some axon collaterals from the adjacent regions, such as the CDL and APH and vice versa.39,56,127,171–173 The polymorphic multipolar neurons of the entorhinal cortex of mammals have medium-sized somata with spherical to slightly pyramidal shape. These neurons have multipolar spiny dendritic arborizations that extend for a long distance in all directions, instead of having dominant apical dendrites.162–164,169 The horizontal cells of the CDL region of birds39–56 are comparable with the horizontal neurons of entorhinal cortex of mammals characterized by sparsely spiny apical dendrites extending to the pial surface and slightly spiny basal dendritic plexus that extend horizontally, whereas their axon travels up to the angular bundle.162–163,170,174–175 In the entorhinal cortex of mammals, the somata of stellate cells extend dendritic arbor comprising multiple, roughly equal-sized, primary dendrites that branch widely in the mediolateral entorhinal cortex.175–178 The axons of stellate neurons run towards the angular bundle from a primary dendrite or the base of the soma.175 The stellate neurons of the birds39,127, 171–172,179–180 correspond to the stellate neurons of the entorhinal cortex of mammal, having an equal-sized primary spiny dendritic tree. The lateral cortex of lizards may be considered homologous to the mammalian olfactory cortex as it receives the bulk projection from the principal olfactory bulb.181–182 It shows a highly laminated efferent to ipsilateral medial cortex, a projection that can be considered as a ‘lizard perforant path’ in comparison with that of the mammalian perforant path arising from the entorhinal cortex and ending in the hippocampus and outer molecular layer of the dentate gyrus.182 In addition to the pattern of the extracortical afferences and efferences,115 the intracortical scheme of connections of the lizard cerebral cortex also has a clear resemblance to that of the mammalian hippocampus and the entorhinal olfactory cortex.116 The lateral cortex of the lizard forms an intermediate region by which olfactory information is distributed to the other cortical areas,24 showing a similarity with birds in which the CI and CDL regions act as intermediate platforms from which efferent and afferent connections originate and extend to the adjacent areas. The pigeon hippocampus has reciprocal ipsilateral connections with the hippocampus

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and the CDL.183 Thus it can be concluded that the lateral cortex in reptiles serves as an interface between the olfactory bulb and other cortical areas and that the CI and CDL in birds carry out the same work between adjacent regions of hippocampus, whereas the same function is carried out in the mammals by the entorhinal cortex between neocortex and hippocampal formation.184 Thus, all these regions in distant animal groups work as a gateway through which bidirectional information passes.

Final comments The comparison of the morphology and connections of the neuronal classes of the cerebral cortex of reptilian, aves and mammals shows following important outcomes: 1. The medial cortex of reptiles shows homology with the hippocampus of birds and fascia dentata of mammals. 2. The dorsomedial cortex of reptiles shows homology with the parahippocampal area of birds and CA3 region of mammalian hippocampus. 3. The dorsal cortex of reptiles shows homology with visual wulst of birds and mammalian isocortex. 4. The lateral cortex of reptiles shows homology to corticoid complex of birds and to entorhinal olfactory cortex of mammals. These findings may provide a further basis of investigations of homology to establish a more detailed correspondence between subdivisions of hippocampal region of cerebral cortex in amniotes.

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22 Medicinal and aromatic plants: A case example of evolving secondary metabolome and biochemical pathway diversity Suman P.S. Khanuja*, Tripta Jhang** and Ajit Kumar Shasany*** *Former Director, CIMAP (CSIR), C41-42, Double Storey, Ramesh Nagar, New Delhi - 110015, India [email protected], [email protected] **Genetic Resources & Biotechnology Division, Central Institute of Medicinal and Aromatic Plants (CSIR), Lucknow - 226015, India [email protected], ***Senior Scientist, Genetic Resources & Biotechnology Division, Central Institute of Medicinal and Aromatic Plants (CSIR), Lucknow 226015 [email protected]

Abstract: The unique capability of plants, in spite of being immobile in strict sense, to defend and respond precisely to environmental stresses whether biotic or abiotic is relatable to their ability to synthesize an array of phytochemicals as metabolites. We find these plants compounds useful for health care as nutraceuticals, drugs and medicines and even most attractive fragrances and flavors. This huge diversity of low molecular weight compounds is represented by secondary metabolites that confer the power of responding to stimuli in plants. Hence, the network of metabolic pathways in plant species represents the pool of functions and chemical diversity leading to biomolecules such as alkaloids, flavanoids, terpenoids, glycosides, etc. Although the structures of approximately 50,000 such metabolites have already been elucidated, there are probably hundreds of thousands of such compounds which we are not able to detect or decipher within the existing limitation of detection. Only a few of these are part of ‘primary’ metabolic pathways (those common to all organisms). The rest are termed ‘secondary’ metabolites; this term is historical and was initially associated with inessentiality but we know today their necessity in defence to signals and stimuli. In the last decade, the research on plant secondary metabolism has been aiming to understand genetic basis from synthesis to regulation of plant compounds at molecular level. With these gene and genome studies, fascinating insights into the creation of genetic diversity of secondary metabolism have become evident. This has been leading to rising inquisition about the mechanisms of gene recruitment and diversification for novel functions. After more than 100 years of ignorance, biologists have reached a stage to recognize the causal connection between gene diversity and plasticity of secondary metabolism in its indispensable ecological role in the dynamic interactions of the plant V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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kingdom with its continuously changing environment. Nevertheless, the complexities are many folds. The multitude of metabolites found in living organisms and the calculated, unexpected small number of genes identified during genome sequencing projects pose more questions to the biologists. Several processes on the posttranscriptional and posttranslational level lead to the formation of enzyme diversity through structural and functional variant forms explaining partially this surprising situation. Further, lower enzyme specificity may also contribute to metabolome diversity. The bottom-line is the evolution of these genes which may provide multiple forms and thereby leading to metabolic diversity even for any cause including adaptation. The situation of pathway diversity across species and genera breaks the conventional taxonomic barriers and brings in chemo-taxonomic basis as new dimension to explain the ever evolving metabolome and hence vertical to horizontal genomic flow for the natural combinatorial biological chemistry working in plant kingdom as no surprise. Keywords: Medicinal plants, Aromatic plants, Biochemical pathways, Secondary metabolism, Metabolome, Diversity, Evolution, Pathway genes, Metabolic enzymes

Introduction Plants like other living forms in nature tend to conserve the energy in the metabolic activities but are known to produce and accumulate a large number of crucial and structurally very diverse molecules/metabolites. These metabolites directly may not serve as essential molecules of life for not being involved in the primary processes of growth and development. But such molecules are present in almost all plant forms from fungi to higher plants and are termed as secondary metabolites.1 Secondary metabolites, however, are known to have crucial role in plant defence (against herbivores, microbes, viruses or competing plants) and signaling or to attract pollinating or seed dispersing animals. Thus, secondary metabolites represent plant’s adaptive functions essential for its own survival and reproductive fitness aiding in its natural selection during evolution. The huge phytochemicals diversity found in plant kingdom is the result of different selection pressure conditions that plants have been successfully coping with through evolution. Medicinal and aromatic plants have become most attractive category of plants both for researchers as well as industry mainly because of the abundant presence of these prized secondary metabolites which find diverse uses from drugs to industrial and agricultural or environmental applications. Structures of these secondary metabolites appear to have evolved during evolution in such a way that they can mimic the structures of endogenous substrates, hormones, neurotransmitters or other ligands. The distribution pattern of secondary metabolite in a given plant is complex but very dynamic as it precisely links with different tissues/developmental stages. Plant metabolites of this nature can exist as proactive/pro-drug molecules that become activated upon wounding, infection or in

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the body of a herbivore. The biosynthesis of some secondary metabolites is induced upon wounding or infection and is made de novo, for example, phytoalexins.2 This quality of plants represents their capability too of producing enormous pool of molecules dynamically through the metabolic pathways diversity. Perhaps this diversity also is in an ever-evolving state through the continuum of genetic pool recombination, reshuffling and mutations happening and getting accumulated across the Darwinian forces and selection pressures. This state has been taken up here as the case study of evolving genomes in plants and discussed at length.

Evolving plant genomes: Emergence of duplicate genes, their clustering and divergence Understanding the evolutionary process leading to the emergence, distribution, diversification and selection of genes involved in plants’ metabolite biosyntheses is possible by visualising the ancestral forms vis-à-vis present phenotypes whether in primary or secondary metabolism. It is believed that in primary metabolism the new genes/alleles mainly arise by gene duplication followed by divergence.3,4 This equips the organism with one gene that maintains the original function and a second copy that is not restricted by natural selection. This second copy can then accumulate mutations until, rarely, it has acquired a new function and might then become fixed in the population. So gene duplication is assumed to be the major driving force for diversification and gene recruitment.1,5 If a gene that directs an essential function is duplicated, the duplicate is released from the stringent function of the mother gene; it may either be eliminated by inactivation (e.g. pseudogenization) or recruited for modified or new functions. There are at least two routes for duplicate genes to be recruited and functionalized. (1) Continuous modification of its function during plant speciation, as consequence of which large gene families with rich functional diversity arise and (2) the duplicate is recruited for a new stringent function in a novel biochemical environment, as consequence, a new single copy gene originates.6 Further elaborating this rationale are the following examples and cases. Amyrin synthases in oats are entirely distinct from other plants. The gene AsbAS1 seems to have arisen by duplication and divergence of a cycloartenol synthase-like gene, and later clustering with other genes required for distinct steps in avenacin biosynthesis in a region of the genome that is not conserved in other cereals. The components of this gene cluster are required for at least four clearly distinct enzymatic processes (2,3-oxidosqualene cyclization, amyrin oxidation, glycosylation and acylation), it is unlikely that the cluster has evolved as a consequence of duplication of a common ancestor. Although clusters of paralogous genes are common in plants (e.g. gene clusters for rRNA and specific disease resistance), reports of clusters of genes that do not share sequence relatedness and whose products contribute to a single selectable function are rare.7 With a

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series of mutants and their functional characterization, it has been shown that AsbAS1 might have evolved from an ancestral cycloartenol synthase-like gene cycloartenol by duplication and rapid sequence divergence. The close relatedness of AsbAS1 to cycoartenol synthases is interesting because, although cycloartenol synthase and amyrin synthase both use, 2,3-oxidosqualene as a substrate, but the structures of the cyclization products generated are quite different. Although gene clusters for secondary metabolites are not well documented in plants, they are common in fungal biosynthetic components, specific pathway regulators and for auto-resistance to the end-product.8 Transmission of these self-contained ‘gene cassettes’ by horizontal gene transfer has also been suggested as an explanation for the persistence of clustering in fungi although recent phylogenetic analyses underestimate the significance of vertical transmission.9 Clustering facilitates the inheritance of the genes for selective advantage as a single functional unit. Disruption of the gene cluster may lead to failure in producing desired pool of protective chemicals and further could result in the accumulation of deleterious intermediates.7 Synthesis of avenacins, like many other plant secondary metabolites, is highly tissue specific and under strict developmental control. Such situations may also confer undefined selective advantages associated with physical proximity and position effects. Intimate coadaptation of all enzymes of a single pathway is likely to be important as an additional mechanism for strict control and containment of secondary metabolites and their pathway intermediates during synthesis. This coadaptation may extend to physical interactions among pathway components, which would aid the channelling of metabolic intermediates within multienzyme complexes.10 Genes for secondary metabolism may in turn be derived from genes for primary metabolism by gene duplication and divergence or possibly also by allelic polymorphism. Similarly, another group of the plant enzymes, terpene synthases (which collectively mediate production of a diverse class of natural products) are predicted to be derived from genes for primary metabolism by duplication and consequent divergence in structural and functional specialization. The example of the clustering of polyketide synthases (PKSs) genes from different plant species shows them to be into two categories. The two clusters represent chalcone synthases (CHSs) and nonchalcone-synthases of angiosperms. The latter apparently originated from ancient duplication of an ancestral CHS gene. During angiosperm speciation, it seems, one gene retained the essential CHS function while the duplicate underwent functional diversification. Under environmental selection pressure new polyketide synthases might have evolved, producing a rich diversity of polyketides that can be distinguished by their biogenetic starter units, the hierarchy through number of added C2-units and the mechanism involved in cyclization.6

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The other example for the gene duplication worth mentioning is the evolution of benzoxazinone (DIMBOA) and indole.7 Molecular characterization of DIMBOA pathway indicates that non-homologous genes are organized in a gene cluster. The first gene in the sequence (BX1) is believed to have originated from a duplication of the gene encoding the unknown subunit of ubiquitous tryptophan synthase followed by recruitment of the second gene duplicate for the emission of volatile indole. Thus, both gene duplicates acquire completely new functions. The first being involved in the biosynthesis of defence compounds under developmental control of young seedlings, the second, encoding indole-3-glycerol phosphate lyase (IGL) is induced in mature leaves in response to herbivore damage. IGL catalyzes the formation of indole as one of the volatiles in maize emitted as signals in tritrophic defence (the volatile signals allure parasitoids to their prey, i.e. the maize herbivores). Another example relates to homospermidine synthase (HSS), which has already been mentioned as the first pathway-specific enzyme in the biosynthesis of pyrrolizidine alkaloids. HSS evolved by duplication of the gene encoding desoxyhypusine synthase (DHS).11 DHS takes part in the posttranslational activation of the eukaryotic initiation factor 5A (eIF5A). It is evident that HSS retained all kinetic and molecular properties of DHS except the ability to bind the eIF5A precursor protein.12 The ability of synthesizing homospermidine from putrescine and spermidine, a side activity already existent in DHS, ultimately became the core-activity of HSS. Again the duplicate of an essential gene of primary metabolism here was recruited for an entirely divergent function in secondary metabolism. The third example has also been described by Hartmann6 that concerns acyltransferases operating with 1-O-ß-acetalesters (1-O-glucose esters) as acyl donors instead of coenzymeA thio-esters. These enzymes, which play important role in plant phenylpropanoid metabolism, have most likely evolved from serine carboxy peptidases by gene duplication and new functionalization.13,14 Gene duplications are relatively frequent events within genomes and have a high impact on the evolution of new biological functions.15,16 The most likely event after gene duplication is the production of pseudogenes from one of the gene copies by knockoff mutations. However, in rare cases, one copy may acquire a completely new function as a result of beneficial mutations within its regulatory and structural components. According to a model proposed by Hughes, 17 the evolution of functionally distinct daughter genes is preceded by a period in which the ancestral gene is bifunctional. This bifunctionality is accomplished by the deoxypussine synthase (DHS) protein that originally possesses homospermidine synthesizing activity in many separate angiosperm lineages, later becoming the exclusive activity of the other gene copy. The HSS-coding gene copy presumably lost the protein-modifying activity of DHS and escaped the strong selection pressure on this essential function of primary metabolism. Nevertheless, its remaining ability

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to synthesize homospermidine became the object of selection pressure from herbivores enabling some plants to recruit the gene copy to establish the first step in the biosynthesis of defence compounds. There are several examples known in which genes have been independently recruited to a single function within a gene family. For instance, the resistance of insects to specific toxins seems to have been acquired through the independent recruitment of paralogous genes belonging to the cytochrome P450 superfamily.18,19 Plant terpene synthases, such as limonene synthase, have been shown to be repeatedly recruited within their gene family from other terpene synthases20 enabling some plants to recruit the gene copy to establish the first step in the biosynthesis of defence compounds. In case of Solanum habrochaites (formerly Lycopersicon hirsutum) f. typicum LA1777 and cultivated tomato Solanum lycopersicum (formerly Lycopersicon esculentum), it has been observed that the glandular type VI trichomes present on the leaves and stems accumulate monoterpenes in S. lycopersicum21 but sesquiterpenes, as insecticidal carboxylic acid derivatives, are accumulated in LA1777.22 Enzymes related to germacrene C synthase mediate the accumulation of a group of structurally similar compounds termed class I sesquiterpenes (cI-Ss) in LA1777 and S. lycopersicum.23 This represents the existence of a mechanism of secondary metabolites controlling the production of two distinct groups of sesquiterpenes from different precursor pools in the wild species. Such a partition in sesquiterpene biosynthesis could be the result of metabolite channeling, through two distinct farnesyldiphosphate (FPP) synthases in the cytoplasm for the secondary metabolites that are associated either with cI-S synthase or with plastidial transporters and the cII-S synthase.

Case examples of pathway genes diversity for secondary metabolism in plants Secondary metabolites of high structural as well as functional similarities are well known to occur simultaneously across even unrelated families of the plant kingdom. For example, the anti-tumor alkaloid camptothecin (inhibitors DNAtopoisomerase) has been found in the following unrelated orders and families Nothapodytes foetida (Celastrales), Pyrenacantha klaineana (Icacinaceae), Camptotheca acuminate (Cornales: Nyssaceae), Ophiorrhiza mungos, O. pumila, O. filistipula (Rubiales: Rubiaceae), Ervatamia heyneana (Apocynaceae) and Mostuea brunonis (Loganiaceae). 2 Consequently, the co-occurrence of a structural class in two taxa could, but not necessarily, be an indication of a monophyletic relationship. This could be due to convergent evolution or differential gene expression wherein, it is likely that in some cases the genes that encode the enzymes for the production of a given structure or structural skeleton might have evolved early during evolution. These genes were not lost during phylogeny

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but got switched off and on again at some later point.24 An example of lupaine pathway shows that genes evolved early during evolution were turned on in some plants using the alkaloids as chemical defence substances but remained turned off in most instances.25 Wink and Mohamed26 to study the phylogenetics of the Leguminosae by selecting representative taxa covering a broad range of tribes established a large rbcL data set and using secondary metabolites as chemical defence traits, reducing the entire Leguminosae to 95 taxa. Quinolizidine alkaloids are frequently present in all taxa of the subfamily Papilionoideae except the tribe Crotalarieae.27 Crotalaria species sequester pyrrolizidine alkaloids and/or nonprotein amino acids but not Quinolizidine alkaloids. In the genus Lotononis, some taxa produce quinolizidine alkaloids and others produce pyrrolizidine alkaloids. Since Crotalaria and Lotononis have derived from same ancestors, producing quinolizidine alkaloids but not pyrrolizidine alkaloids, the genes encoding biosynthetic enzymes of quinolizidine alkaloid formation must still be present. More likely the quinolizidine alkaloid genes have been turned off in Crotalaria and partially in Lotononis. The formation of pyrrolizidine alkaloids (which are typical secondary metabolite of the Boraginaceae and some Asteraceae) instead appears to be a new acquisition for chemical defence, which probably evolved independently. The protease inhibitors (i.e. trypsin and chymotrypsin inhibitors) distribution pattern is also similar to quinolizidine alkaloids.28 The members of the Caesalpinoideae and many Mimosoideae accumulate protease inhibitors in their seeds, where they serve concomitantly as chemical defence and nitrogen storage compounds. Within the Papilionoideae, protease inhibitors are prominent in the tribes Vicieae, Trifolieae, Cicereae, Abreae, Galegeae, Loteae, Phaseoleae, and Tephrosieae, but are not described in the Mirbelieae. Withanolides represent a group of steroidal lactones with strong insecticidal properties which appear to be restricted to the family Solanaceae.29,30 Withanolide producing genera are typical for the tribe Physaleae, but isolated occurrences have been reported for Brugmansia (Datureae), Hyoscyamus (Hyoscyameae), Lycium (Lycieae), Jaborosa (Jaboroseae), Nicandra (Nicandreae) and Browallia (Browallieae) serving as chemical defence compounds in the plants producing them. Therefore, these compounds constitute important fitness traits and represent adaptive characters with some, but usually have limited value as a taxonomic marker.26 All members of a monophyletic group share a chemical characteristic; favouring their use as a taxonomic marker. In other instances, a particular secondary metabolite may occur in several unrelated clades and/or plant families.26,28 The erratic secondary metabolite distribution can be due to simple convergence, where genes encoding a particular biosynthetic pathway evolved independently in several parts of a phylogeny. There is evidence however for an alternative explanation: In several cases, it is apparent that ancestral members of a group evolved the

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biosynthetic capacity to produce a specific secondary metabolite. The absence of such a trait in phylogenetically derived groups is probably due to differential gene expression, where the corresponding genes are not lost but switched off. Since secondary metabolites play a vital role as defence and signal compounds, their occurrence apparently reflects adaptations and particular life strategies embedded in a particular phylogenetic framework.2 Khanuja et al.31 while assessing the interspecific as well as intraspecific relationships in Mentha at molecular (DNA) level, observed that M. gracilis Sole Cardiaca showed a much higher similarity with M. spicata as well as M. arvensis, which amongst themselves showed rather a greater distance. This indicated that the species might have evolved as a natural hybrid between M. arvensis and M. spicata. The GC and GC/MS of the oil quality for metabolite components of 20 accessions of M. piperita32 showed close relationship of mutant ‘Kukrail’ with Japanese oil, whereas other two accessions were similar to Chinese oil as analysed in the component plot. These genotypes were released as varieties by the names of ‘CIM -Indus’ (high pulegone and menthofuran content) and’ CIMMadhuras’ (peppermint plant having aroma with high acceptability respectively by CIMAP. Similarly, the aromatic grass species Cymbopogon, in the analysis for essential oil biosynthetic components indicated remarkable variation among various species.33 The major essential oil components citral ‘a’ and ‘b’, were detected in Cymbopogon pendulus, C. flexuosus and C. citratus with highest in C. Citratus where as it was not detected in C.winteianus (Jowii) The hierarchical cluster analysis based on essential oil composition placed C. winterianus distantly from all other taxa followed by the hybrid Jamrosa. This hybrid contained high geraniol (68%), low citral (less than 2%) and trace of citronellal (0.5%) in the essential oil composition. The genomic synteny in essential oil compositions along with the differences indicated gene duplication followed by variations to create the metabolite diversity during speciation.

Domain swapping and neo-funtionalization: Another level of functional divergence Domain swapping represents an independent mechanism for the generation of new composite genes with or without prior gene duplications.34 It is theoretically possible for a new allele in one of the plant’s genetic loci to be selected for if it encodes the ability to make a new defence compound, whereas the older alleles still specify the synthesis of another defence compound that however may be no longer effective. Thus, in secondary metabolism, there is a potential for new genes to keep evolving without a prior gene duplication event. In such cases, these orthologous genes in related species might encode proteins with different functions1. It is widely believed that enzymes with more active and specialized

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function evolve divergently from enzymes with promiscuous function. This process is thought to be closely associated with the evolution of metabolic pathways.35–37 It has been observed that that the recruitment of single enzymes from other metabolic pathways might significantly drive the evolution of both enzymes and metabolic pathways.35 It is believed that enzymes with promiscuous functions can be initially shared by two distinct metabolic pathways. Enzymes with promiscuous function might give organism novel metabolic capabilities and, thus, render them adaptable to different environments. When the gene is duplicated, one enzyme is free to abandon the role it had in the previous pathway and thus can specialize its function for the new pathway, and vice versa. This results in divergent molecular evolution of enzymes and a mosaic or patchwork evolution of metabolic pathways. If multiple steps in a metabolic network are catalyzed by a series of promiscuous enzymes, although inefficient, this network would be able to produce a large library of natural products. If any of these products were to be captured by positive selection, the metabolic network could then converge on the pathway through divergent evolution of each component enzyme.38 In a research study, a combination of domain swapping and reciprocal sitedirected mutagenesis was carried out in grand fir between (–)-(4S)–limonene synthase (LS) and (–)-(4S)-limonene/ (–)-1S, 5S–α-pinene synthase (LPS) by Katoh et al.39 Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome. Whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS. This resulted in a 5-fold increase in relative activity. Based on the results of these chimeric studies, reciprocal point mutations were made in each parent monoterpene synthase to provide five single and 33 multiple site mutants of LS, and five single and 12 multiple site mutants of LPS, and were successfully expressed and evaluated. The most effective in altering monoterpene olefin distribution was V384L, a position predicted by modeling to reside in helix D. In this mutant, the proportions of generated α- and β-pinene were reversed and the level of β-phellandrene was found to be nearly doubled. These experiments were conducted, in conjunction with modeling of the two enzymes to know the critical amino acids for product determination. But indirectly this denotes the change in domains among highly homologous genes can lead to change the product distribution itself. In another study, domain swapping experiments between Cl(-)  PINS ( -pinene synthase) and Cl TS ( -terpinene synthase) and between Cl(+)LIMS2 (limonene synthase) and Cl  TS were conducted40 to identify domains within the monoterpene synthase enzymes determining the product specificity. Similarly, a sesquiterpenes cyclase (CASC2) showing 77% amino acid identity with the previously cloned sesquiterpene cyclase CASC1 of Capsicum annuum failed to express in Escherichia coli. However, the chimeric construct of CASC2 in which

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the amino terminal 164 amino acid was substituted by the equivalent portion of either CASC1 or tobacco sesquiterpene, the cyclase was capable of expressing the functional sesquiterpene cyclase activities.41 Relatively closer similarity of GES (geraniol Synthase) and LIS ((R)-linalool synthase protein pairs in Ocimum basilicum indicates that further terpene biosynthetic diversity is continuing to be generated in the basil lineage by gene duplication and divergence.42 This two genes have been found to produce only a single product, either linalool or geraniol, but not both. But the results of domain-swapping experiments by the same group also demonstrated that it is possible to generate a monoterpene synthase that can synthesize both geraniol and linalool. Here, multiple amino acids contribute to such a dual selectivity but such an enzyme is not found in nature till date. Another phenomenon of importance observed relates to the occult biosynthetic capacities of plant either constitutive or induced. This confers them an unlimited potential to produce a large array of different compounds that can be activated when novel substrates become available.43 Through biotechnological route, overexpression of Clarkia breweri floral gene linalool synthase (LIS), an enzyme that catalyzes the formation of (S)-linalool from the monoterpene precursor geranyl diphosphate was attempted in tomato fruit.44 Interestingly, accumulation of (S)-linalool (expected) and 8-hydroxylinalool (unexpected) was observed. In this case, the availability of novel substrate due to expression of a foreign gene enabled endogenous ‘occult’ hydroxylase activity to act on (S)-linalool. When the Clarkia LIS gene was overexpressed in carnation flowers, linalool in the transgenic flowers further metabolized to linalool oxides. Thus, the overexpression of an identical gene in different target tissues and organisms could gave rise to distinct phenotypes, according to the metabolism present or induced in the target organism that might interact with the novel products generated. Similarly, from lemon basil (O. basilicum L. cv. Sweet Dani) geraniol synthase (GES) gene was overexpressed in tomato fruits to modify the aroma and flavor.45 Besides high levels of geraniol, eleven novel additional metabolites sharing a common chemical backbone accumulated. These were derived from geraniol i.e the monoterpene alcohols nerol and citronellol. This way, monoterpene aldehydes geranial, neral and citronellal, the monoterpenol esters geranyl, neryl and citronellyl acetate, geranic and neric acid and rose oxide accumulated in such transgenic tomatoes. In another study,46 the sweet basil type that possesses OMT activities converting methylate chavicol to estragole, was compared with a distinct basil type that accumulates only methyl eugenol (a 3 methoxylated estragole derivative). When chavicol was provided as a substrate it was methylated to estragole in cell-free extracts, although the plant itself does not contain estragole. The “lemon-scented” basil line 197 is also known to contain citral (a mixture of the monoterpene aldehydes geranial and neral) but lacks volatile phenylpropenes in its essential oil. And yet cell-free extracts derived from this lemon-scented line

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could readily convert O-methylate chavicol to estragole, eugenol to methyl eugenol or isoeugenol to methyl-isoeugenol although none of these compounds are present in the plant. This further indicates the presence of ‘silent’ O-methyltransferase activities in basil lines that may readily accept novel substrates if the ability to produce those substrates is acquired by breeding or mutation, easily yielding to novel chemotypes. Biosynthesis of tryptophan, a precursor for indole alkaloids, is well known for the feedback inhibition mechanism to regulate its own production. Hence, its availability can be well understood as a limiting factor in biosynthesis of indole alkaloids. However, the introduction of Arabidopsis thaliana feedback-resistant anthranilate synthase (AtAS) and induction of tryptophan decarboxylase (TDC) in Catharanthus roseus hairy roots did not significantly improve downstream alkaloids even though the levels of early alkaloid precursors tryptamine and tryptophan increased.47 This suggests that the availability of the early amino acid precursor is not limiting for TIA (Terpenoid indole alkaloids) biosynthesis, which confirms the finding that the availability of secologanin was the important ratelimiting step in TIA biosynthesis.48 A superfamily is supposed to be a group of enzymes related by divergent evolution.49 Among these, the plant terpene synthase superfamily is interesting and the most cited example to explain divergent molecular evolution. These enzymes share a strikingly similar active site scaffold comprising several -helices and catalyze the formation of diverse terpenes from several different classes of prenyl diphosphates through wide varieties of carbocation rearrangements. Terpene synthase subfamilies within angiosperms are more closely related to each other than are members in the same subfamily from gymnosperms. In each subfamily, terpene synthases from the same or related species are more closely related to each other than are ones from different species with similar catalytic mechanism based secondary metabolites.20 These observations indicate that divergence in terpene synthase subfamilies evolved after angiosperms and gymnosperms separated and that of terpene synthases within each subfamily arose after a series of subsequent speciation events.38 All terpene synthases so far described show promiscuous function. Among those, -humulene and dselinene synthases are very promiscuous sesquiterpene synthases that are constitutively expressed in Abies grandis, each catalyzing the formation of at least 52 and 36 sesquiterpenes, respectively. In addition, these enzymes can use geranyl diphosphate as a substrate and catalyze the formation of monoterpenes. Although the specific roles of these enzymes have not been identified, it is thought that they might create chemical libraries that are important in general defence against microbial invasion. By contrast, many other terpene synthases have highly specialized functions and are often found to have very specific roles in the formation of bioactive metabolites. For example, (+)-D-cadinene, vetispiradiene and 5-epi-aristolochene synthases catalyze the

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first reaction step of phytoalexin (anti-fungal agents) production in various plant species and yield their respective sesquiterpenes with more than 98, 90 and 70% selectivity, respectively. Thus, it has been suggested that terpene synthases with specific functions have evolved from ones with promiscuous function after having been captured by positive selection.38 Acknowledgments: Prof. Sushil Kumar, former Director, CIMAP who happens to be our teacher showing the path through the pathways in plants and life inspiring this writing attempt.

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23 Conservation of Himalayan bioresources: An ecological, economical and evolutionary perspective Lok Man S. Palni* and Ranbeer S. Rawal** *G. B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora 263 643 (Uttarakhand), India *[email protected], **[email protected]

Abstract: The Himalayan ranges are the youngest and loftiest among the mountain systems of the world. They represent a highly complex and diversified system both in terms of biological and physical attributes. Their vulnerability toward natural and human-induced disturbances is well recognized. On account of richness and uniqueness of biodiversity elements, the region has been recognized as one of the 34 global biodiversity hotspots. It represents 3 sub-centers (west Himalaya, east Himalaya and north east region) of plant origin, which, respectively, contribute 125, 82 and 132 species of wild relatives. The eastern Himalaya and north eastern sub-centers are known for contribution to Musa and Citrus diversity. The prevailing primitive agricultural systems in the region and conscious and unconscious selections by indigenous farming communities have contributed toward enormous enrichment of genetic diversity in the form of land races. Diversity of representative natural ecosystems (grasslands and woodlands) and richness of endemic bioresources have added to the ecological significance of the Himalaya. Particularly, the alpine grasslands and the forests of the region exhibit unique features. Furthermore, medicinal and wild edible plants add substantially to the ecological and economic value of the region. However, Himalayan ecosystems and their components are highly vulnerable both due to geological reasons and on account of stress caused by increased pressure of population. Also, there are growing indications that the ill effects of these factors may be exacerbated on account of the impact of climate change. This would affect the very sustenance of the indigenous communities living in the uplands as well as downstream areas. Therefore, there is an urgent need for making conscious efforts for conserving all representative systems. In this context, the existing conservation area network in the region, which appears to be stronger than the country average, is one welcome initiative. This network, however, requires strengthening to provide adequate coverage to all representative ecosystems, particularly in north east. Need for a major shift in the conservation approach through community support, and by way of promotion of sustainable use concept, has been suggested to ensure conservation of Himalayan bioresources. This is pivotal for maintaining vital stocks of unique, often endemic elements, for the natural evolutionary processes to continue. V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Keywords: Indian Himalayan region, Biodiversity hotspot, Center of origin, Endemics, Community participation, Protected area network

Introduction Charles Robert Darwin, who derived pleasure in inventing a whole new science from the small, commonplace features of the Earth that most of us mortals often ignore; he was able to transform the then prevailing contemporary thinking across diverse spheres of human society. The Origin of Species,1 which established theory of Natural Selection, subsequently popularized as ‘Survival of the Fittest’, marks a watershed in the history of scientific, philosophical, theological ideas. In spite of considerable changes in evolutionary theory over time, the content, arguments and reasoning provided in his great revolutionary work remain relevant even to this date. Among others, the complexity and interdependence of natures’ elements, the entangled bank as he describes in the concluding paragraph, holds the truth of the life’s diversity which manifests itself in its myriad of forms. ‘It is interesting to contemplate an entangled bank, clothed with many plants of many kinds, with birds singing on the bushes, with various insects flitting about, and with worms crawling through the damp earth, and to reflect that these elaborately constructed forms, so different from each other, and dependent on each other in so complex a manner, have all been produced by laws acting around us……………………There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved’.1 Any attempt, small or big, to describe such complexity of nature would be a tribute in itself to the great scientific-visionary. Recognizing this, in subsequent pages, we have tried to explore complexities and interdependence of various elements of the Himalayan Ecosystem. Our attempt is to find out a few workable solutions under changing ecological and economic scenarios in the region which could help in maintaining vital stocks of unique Himalayan biodiversity elements and help in the natural evolutionary processes to continue. The mountains, which cover nearly one quarter of Earths’ land area, host over 12% of global human population. More importantly, >50% of global human population draws benefits directly or indirectly from resources and services emanating from the mountains. These regions, although well recognized for their evolutionary significance and ecological value manifested by ecosystem integrity, adaptability and services, have largely remained marginalized from economic development perspectives. However, in the aftermath of Rio Earth

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Summit and the implementation of its Agenda 21, awareness about the mountains has increased manifold, and the region is being institutionalized on mainstream developmental agenda. In this context, on account of richness (diversity), representativeness and uniqueness, the mountain biodiversity elements have gained considerable attention. The Himalaya: A global biodiversity hotspot While considering the mountains as a special focus, the Himalaya, which represents youngest but complex mountain system on the surface of earth, which is still evolving, has been recognized among the 34 global biodiversity hotspots.2 On account of its being in evolving state, the ecosystem components in the region exhibit great dynamism.This majestic mountain system covers nearly 7.5 lakh km2 area (spanning over 3000 km in length, and rising from < 300 to > 8000 m asl) and stretches from northern Pakistan on the west to northeastern region of India through northwestern states of India, Nepal and Bhutan. The region has a discrete geographic and ecological entity. It produces a distinctive climate of its own and influences the climate of much of Asia.3 However, the variations in topographical features along three-dimensional frame work (i.e. latitudinal: SouthNorth; longitudinal: East-West; altitudinal: Low-High) cause diversity in climate and habitat conditions within the region.4 This leads to overwhelming richness of biodiversity elements and to their uniqueness. The region serves as a rich repository of plant and animal wealth in diverse ecological systems. These ecosystems reflect a mosaic of biotic communities at various spatial and organizational levels. Diversity and uniqueness features with respect to biodiversity of Himalayan hotspot are included (Table 1). The Indian Himalayan Region (IHR), with geographical coverage of over 5.3 lakh km2, constitutes a large proportion of the hotspot and, therefore, contributes greatly to richness and representativeness of its biodiversity components at all levels (i.e. genes, species and ecosystems). Administratively IHR (Fig. 1) covers 10 Table 1 Diversity and uniqueness of biodiversity in the Himalayan biodiversity hotspot [Source: Ref. 2] Taxonomic group Species Percent Endemism Endemic species Plants Mammals Birds Reptiles Amphibians Freshwater fishes

10,000 300 977 176 105 269

3,160 12 15 48 42 33

31.6 4.0 1.5 27.3 40.0 12.3

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states entirely (i.e. Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, Arunachal Pradesh, Nagaland, Manipur, Mizoram, Tripura and Meghalaya), and two states partially (i.e. hilly districts of Assam and West Bengal). The region represents nearly 3.8% of total human population of the country and exhibits diversity of ethnic groups (171 of a total 573 scheduled tribes in India) which inhabit remote inhospitable terrains.5 On account of its contribution (biophysical values) to the Himalayan biodiversity hotspot, IHR holds a significant position.6 While considering maintenance or conservation of its representative biodiversity values (Table 2), the region needs to be examined critically for its evolutionary, ecological and economic contributions.

Himalayan bioresources: Evolutionary and biogeographic contributions The Himalaya, which owes its origin to the collision between two land masses (the Peninsular Indian Plate and the Eurasian Plate) about 60–70 million years

Figure 1. Indian Himalayan region with three centers of plant origin. [Modified after Refs. 6, 22]

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Table 2 Representativeness of Himalayan biodiversity Representation Levels

References

Total number

% of India

Eco-regions Communities

16

-

[60]

Vegetation types Forest types Formation types Species

21 10 11

26 -

[32] [61] [7]

Angiosperms Gymnosperms Pteridophytes Bryophytes Lichens Fungi Mammals Birds Reptiles Amphibians Fishes Specific groups

8000 44 600 1737 1159 6900 241 528 149 74 218

47 81 59 61 59 53 65 43 35 36 17

[62] -do-do-do-do-do[63] -do-do-do-do-

Medicinal Wild edible Trees

1748 675 723

23 67 28

[38] [44] [64]

ago, is the youngest and the tallest mountain system in the world, and is most susceptible to landslips and erosion owing to the presence of residual stresses and highly compressed and tectonized rocks.7 However, the Himalayan orogeny does not relate to a single event, the western part being more recent.8 The temporal and spatial variations caused by diversity in geological orogeny has resulted in to a marked difference in climate and physiography, and consequently in distribution pattern of biotic elements. Also, the spatial position (at the juncture of five major biogeographic regions- Palaearctic, Mediterranean, Indo-Chinese, Indo-Malayan and Peninsular India) and heterogeneous dispersion of biodiversity elements has caused complexity in biogeographical patterns of the region. The erosion of the young Himalaya resulted in production of alluvia that led to filling and drying up of the Tethys Sea in northwest India and also led to the formation of the Indus and Gangetic alluvial plains.9 Considering the wide ranging affinities of the biota, the IHR (Table 3) consists of three biogeographic zones and seven provinces.10,11

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Table 3 Biogeographic divisions of IHR [Source: Refs. 10,11] Biogeographic Biogeographic Geographical Major Biome Zones Provinces area of India Representation (%) Trans Himalaya 1A: Ladakh Mountains 3.3 Tundra 1B: Tibetan Plateau 2.3 Alpine The Himalaya 2A: North west 2.1 Alpine, temperate, Himalaya Subtropical 2B: West Himalaya 1.6 -do2C: Centaral Himalaya 0.2 -do2D: East Himalaya 2.5 -doTropical evergreen forest, very moist Northeast India 9A: Brahamputra Valley 2.0 sal forest, tropical Grasslands Tropical evergreen, tropical moist 9B: Northeast Hills 3.2 deciduous, subtropical, Temperate forests, wetlands

Cradle of flowering plants Considering the evolutionary contribution, the east Himalayan biogeographic province along with the North East Indian biogeographic zone has great significance. This region is rich in biodiversity and harbors largest number of endemics and Schedule I species as compared to any other part of India.12 This region represents a confluence of the Indo-Malayan and Indo-Chinese biogeographical realms. Also, it exhibits intermixing of the Himalayan and Peninsular Indian elements. The region was considered as cradle of flowering plants,13 which represents some of the primitive angiosperm families including Magnoliaceae, Degeneriaceae, Himantandraceae, Eupomatiaceae, Winteraceae, Trochodendraceae, Tetracentraceae and Lardizabaleaceae. The specific primitive genera include Alnus, Aspidocarya, Betula, Decaisnea, Euptelea, Exbucklandia, Haematocarpus, Holboellia, Houttuynia, Magnolia, Magnelietia, Pycnarrhena and Tetracentron.9,14 The region is known for high evolutionary activities which are clearly evident from the cytogeographic studies on selected genera like Rhododendron, Camellia, Magnolia, Budleia, etc.15–20

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Table 4 Distribution of diversity of wild relatives in the Himalaya. [Source 22] Category Distribution in Himalayan Sub-centers West Himalaya Eastern Himalaya North Eastern Region Cereals and millets 29 07 16 Legumes Fruits Vegetables Oilseeds Fibers Spices and condiments Miscellaneous Total spp. Diversity

09 37 25 06 04 10

05 32 12 03 04 09

06 51 27 01 05 13

05 125

10 82

13 132

Centers of Diversity Further, the IHR contributes considerably in the form of wild relatives of several crop plants and domesticated animals. Of the total 8 sub-centers of plant origin, the region represents 3 sub-centers (e.g. Western Himalaya, Eastern Himalaya and North Eastern Region). These sub-centers, respectively, contribute 125, 82, and 132 species of wild relatives (Table 4).21–22 Based on extensive explorations across the world, Vavilov considered the north eastern region of India as ‘Hindusthan Centre of Origin of Cultivated Plants’, which is very important for tropical and subtropical fruits, cereals, etc. This region forms the richest reservoir of genetic variability of many groups of crop plants. The taxonomical and cytogenetic studies have revealed AssamBurma-Siam-Indo-China region as the center of origin of Musa.23 Greatest diversity, with some being endemic, of this genus is known form this region. Banana in north east India grows wild along the hill slopes of Arunachal Pradesh, Meghalaya and Assam. The region is also rich in Citrus wealth with nearly 64 taxa of Citrus growing wild. Also, it is regarded as the center of origin for five species of palms of commerce – coconut, areca nut, palmyra palm, sugar palm and wild date palm.24 In addition, it contributes a whole range of medicinal and aromatic plants with their origin in the region and wild progenitors of a number of ornamentals like Primula, Rhododendron and huge diversity of Orchids. Among wild and domesticated faunal elements, region harbors wild chicken, zebu, mithun, yak, etc.9

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The prevailing primitive agricultural system of raising crops under stress conditions in the region have resulted in much variability, particularly in adaptive traits. Most of the hilly terrain in the eastern Himalaya is under shifting cultivation and several promising, agronomically and physiologically well adapted types/ land races belonging to diverse crop species occur in this region. The specialized habitats in the western part of Himalaya (e.g. cold arid climate) are also responsible for promoting variation in specific traits. In addition, in isolated pockets, various ethnic groups grow their own preferred locally selected cultivars. All these factors have contributed to enormous enrichment of genetic diversity in land races through conscious and unconscious selections by indigenous farming communities in the Himalaya.25 Emerging issues Realizing the above uniqueness, there is a need to place special attention to maintain the evolutionary significance of the region. Apart from inventorization/ documentation of such genetic resources, their evaluation for use and maintenance both as in situ and ex situ gene banks assumes priority. Ecogeographical surveys in areas of diversity will be required to mark out precise pockets for in situ conservation, and integration of such efforts with monitoring of diversity in bioresources is called for. Apart from seed storage in genebanks, in vitro conservation measures assume greater importance, with overall emphasis on complementary conservation strategies.22, 26 This can, however, be achieved only through a holistic understanding of existing ‘man and nature’ relationships. Among others, the practicable solutions for achieving conservation goals with respect to these genetic resources may be drawn from a diverse set of best practices performed by the indigenous communities.

Himalayan bioresources: Ecological contributions The biogeographical diversity (Table 3) has endowed the region with rich and representative biodiversity elements (Table 2), and it serves as a rich repository of plant and animal wealth in diverse ecological systems. These ecosystems reflect a mosaic of biotic communities at various spatial and organizational levels along three spatial transitions: (1) East to West transition: over 2500 km in length, this transition represents cool and moist conditions of east Himalaya (i.e. Arunachal Pradesh) to cold dry conditions of west (i.e. Ladakh). (2) South to North transition: representing tropical/subtropical conditions of southern most Himalaya (i.e. adjoining the Indus-Gangetic plains) to cold desert (i.e. Tibetan Plateau) in the north. This transition results into a width ranging from 240 to 350 km.

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(3) Low to High altitude transition: ranging from tropical and subtropical conditions of low altitudes (<300 m asl) to alpine/arctic conditions of high altitudes (> 8000 m asl). While considering the conservation of bioresources of such a complex mosaic, among various important ecological considerations, representativeness of ecosystems and uniqueness (endemism) of species deserve priority mention.

Ecosystem representation Considering representation of wide structural and functional diversity, the region is broadly grouped in to two major natural terrestrial ecosystems: (1) the Grasslands, and (2) the Woodlands. The alpine meadows represent natural grassland ecosystems in the Himalaya which are widely distributed between two ecotones (tree line and snow line) from extremely dry trans/north west Himalaya to humid east Himalaya. In view of the variations in structure/composition of biodiversity elements in response to climatic conditions two types of alpine grassland ecosystems are discernible in the Himalaya: (1) Alpine Arid Pastures (AAP) – largely represented in cold desert areas of the trans and north west Himalaya these pastures show specialized characters for both plant and animal forms and occur in relatively low densities, often in geographically isolated populations, which are vulnerable to local extinction.27 While these ecosystems are characterized by dominance of xerophytic plant elements they also provide special habitats for certain localized animals such as Tibetan wild ass (Equus kiang kiang), Tibetan wolf (Canis lupes chanco), ibex (Capra ibex sibirica), Ladakh urial (Ovis orientalis), Markhor (Capra falconeri), Tibetan gazelle (Procarpa picticaudata) and widely distributed high-altitude fauna, for example, bharal (Pseudois nayaur), snow leopard (Uncia uncia), Indian wolf (Canis lupus), brown bear (Ursus arctos isabellinus); and (2) Alpine Moist Pastures (AMP) – they represent typical alpine meadows usually extending across the Himalaya occupying the southern slopes, and are known to be the rich habitats with high productivity. Besides, the unique representative assemblages of floral and faunal elements, value of these ecosystems in sustenance of local pastoral communities is well recognized. These ecosystems, on account of higher productivity, support large populations of wild and domestic ungulates. Species such as bharal, Himalayan thar (Hemitragus jemlahicus), Himalayan musk deer (Moschus chrysogaster), brown bear, red fox (Vulpes vulpes) and the Himalayan monal (Lophorus impejanus) depend substantially on these ecosystems.27 For the representative alpine grasslands of the region, annual live shoot production (382–409 g/m2) has been reported high (example central Himalaya) as compared to reported values (13–348g/m2) for alpine grasslands elsewhere.28 These grasslands, despite intense grazing pressure, maintain good vegetation cover, high production and high species diversity, indicating that these meadows

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Nature at work: Ongoing saga of evolution

are grazophil communities, adapted to withstand grazing.29 Also, these alpine meadows have greater productivity and stocking capacity (7.13 sheep/ha) than the values reported for other cold climatic zones (0.4–2.0 sheep/ha).30 Interestingly, variation in height, manner of leaf deployment, such as vertical vs horizontal spread of shoots and periodicity of leafing enables a number of species to occupy same site, by reducing competition for vital resources in these ecosystems.31 Similarly the woodland ecosystems in the region can be grouped into two categories: (1) the forests and (2) the scrubs. Under both the categories various sub categories, in different parts of the Himalaya, are recognizable. While attempting the zonation of vegetation types many researchers have considered the Himalaya as a transition between tropical and temperate regions of Northern Hemisphere, and between dry west and humid east of the Himalaya.32–35 Major shifts in vegetation types are thus discernible across two simultaneously operating environmental gradients (i.e. warm to cool: temperature; and dry to humid: moisture conditions). Considering the close proximity of vegetation boundaries with climatic zones, the Himalayan woodlands can be grouped into following five broad zones (terminology largely follows the one used for northern temperate types35): (1) tropical/sub tropical (represented by three dominant/physiognomic types – Moist evergreen broadleaf forest Tropical Dry Deciduous and Semi evergreen Forests; (2) warm temperate (characterized by two dominant types – dry deciduous forests and needle leaf forests); (3) temperate (represents maximum diversity of dominant types which mainly include broadleaf evergreen forests, broadleaf deciduous forests temperate needleleaf evergreen forests); (4) sub alpine/cool temperate (dominated by Sub alpine conifer forests, Sub alpine broadleaf forests and (5) alpine (represented by alpine scrublands which include two distinct types (Alpine dry scrub and Alpine moist scrub). In view of the spatial geographical position of the Himalaya and uniqueness of its ecosystems, it is pertinent to assess the extent of representativeness in biological components of different ecosystems. Such studies assume significance while native species populations, with their natural interactions in naturally structured communities, are considered as best indicators of biological integrity. Unfortunately, such information is lacking in the region. However, while considering the ecological characteristics of Himalayan forests it is suggested that Himalayan forests differ significantly in the following attributes: (1) tree phenology, chemistry and form; (2) physiological responses; (3) ecosystem processes and (4) vegetation types.3 Uniqueness of biodiversity: Endemism Endemism is yet another important attribute which needs consideration while developing conservation strategies for the Himalayan biodiversity hotspot. Among

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379

floristic elements, besides nearly 32% of species being endemic, the region represents 71 endemic genera and five endemic families (i.e. Tetracentraceae, Hamamelidaceae, Circaeasteraceae, Butomaceae, and Stachyuraceae). A few families, for example Berberidaceae and Saxifragaceae, represent >90% species endemic to the Himalaya. A large number of orchids, many representing neo endemic taxa, have been recently reported from Sikkim and Arunachal Pradesh. Likewise, of the nearly 300 recorded mammal species across the region, 12 are endemic to the Himalaya. The endemics include the golden langur (Trachypithecus geei) – very restricted range in the Eastern Himalaya; the Himalayan tahr (Hemitragus jemlahicus); and the pygmy hog (Sus salvanius) – species restricted to grasslands in the Terai-Duar savannah and grasslands in the Manas National Park. The Namdapha flying squirrel (Biswamoyopterus biswasi) also represents the only endemic genus in the Himalaya, but is a poorly known species described on the basis of a single specimen taken from Namdapha National Park. Of the 979 bird species recorded from the region, 15 are endemics, including one species, the Himalayan quail (Ophrysia superciliosa) representing an endemic genus. Four Endemic Bird Areas (EBAs) overlap entirely or partly with the Himalaya Hotspot. Among other groups, reptiles (177 spp.) represent 49; amphibians (124 spp.) 41; and fishes (269 spp.) 33 species as endemic to this hot spot.2 The studies in IHR have revealed that the high altitude Himalaya (alpine and sub-alpine zone) is rich in plant endemic diversity. This feature is particularly prominent in Trans/Northwest and West Himalaya, suggesting thereby that the high altitude zone of these provinces can be considered as one of the endemic centers. Among endemics, prevalence of shizoendemics is reported from the western part (Kashmir) of the region which points toward their progressiveness.36

Conservation imperatives Realizing that the Himalayan ecosystem is highly vulnerable both due to geological reasons and on account of the stress caused by increased pressure of population, exploitation of natural resources and other related challenges need special attention. These vulnerability aspects may be exacerbated due to the impact of climate change. It is possible that climate change may adversely impact the Himalayan ecosystem through increased temperature, altered precipitation patterns, episodes of drought and biotic influences. This would not only impact the very sustenance of the indigenous communities in uplands but also the life of downstream dwellers across the country and beyond. Therefore, there is an urgent need for giving special attention to sustain the Himalayan Ecosystem.6 This would require conscious efforts for conserving all the representative systems. Further, it needs to be emphasized that the endemics with restricted distribution, and most often with specialized habitat requirements, are among

380

Nature at work: Ongoing saga of evolution

the most vulnerable elements. In this respect the Himalayan biodiversity hotspot, with rich endemic diversity, is vulnerable to climate change. The threats include possible loss of genetic resources and species, habitats and concomitantly a decrease in ecosystem services.37 Therefore, conservation of endemic elements in representative ecosystems/habitats assumes a great significance while drawing conservation plans for the region. Toward achieving the above, we will have to shift toward contemporary conservation approaches, which include a paradigm of landscape-level interconnectivity between protected area systems. The concept advocates a shift from the species-habitat focus to an inclusive focus on expanding the biogeographic range so that natural adjustments to climate change can proceed without being restrictive.37

Himalayan bioresources: Economic contributions Traditionally, indigenous communities in the Himalaya have been dependent on bioresources to meet basic sustenance needs, notably food, fodder, fuel, fertilizer, fiber, shelter, health care, etc. More than 80% of the population in the region is involved in agriculture, animal husbandry, forestry and other biodiversity dependent vocations. Among other bioresources with direct economic value, the Himalayan region is well recognized for diversity of medicinal plants, wild edibles and other nontimber forest produce. Medicinal plants The rich plant diversity of the Himalaya has been a source of medicine for millions of people in the country and elsewhere in the world. IHR supports over 1748 (23.4% of India) plant species of known medicinal value.38 The unique diversity of medicinal plants in the region is manifested by the presence of number of native (31%), endemic (15.5%) and threatened elements (14% of total Red Data Book plant species of IHR). The economic potential of Himalayan medicinal plants and their contribution in providing novel biomolecules is well recognized.39–41 While setting priorities for medicinal plants of IHR, it is reported that41: (1) of the nearly 280 medicinal plants being used in industry 175 are from IHR; (2) more than two third (122: 69.7%) of identified species from IHR are exposed to destructive harvesting, thereby adversely affecting the resource base; (3) industry extensively uses nonnatives particularly in robust life forms (shrubs 96.8%; trees 90.9%); (4) native percentage among exploited herbs is relatively high (20.2%) and most of them are restricted to specialized alpine habitats and (5) contribution of wild-cultivated forms of medicinal plants is poor (20%).

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Wild edible plants Among the economically important bioresources of IHR, the wild edibles have emerged as potential resources for addressing issues of rural development and biodiversity conservation, particularly on account of their nutritional and pharmaceutical potential.42–43 Over 675 species (angiosperms 647, gymnosperms 7; pteridophytes 12; fungi 7 and lichens 2 species) of wild edible species belonging to 384 genera and 149 families are known from IHR.44–45 Across biogeographic provinces, richness of known wild edibles is maximum in West Himalaya (344 spp.; 50.9%); followed by East Himalaya (221 spp.; 32.7%), Central Himalaya (173 spp.; 25.6%) and Trans/North West Himalaya (169 spp.; 25.0%). Of the total reported wild edibles, 39 (5.8%) species were restricted range endemics and 93 (13.8%) near endemics.45 Consumption of wild edibles meets the protein, carbohydrate, fat, vitamin and mineral requirement of poor rural populace in the region.46 Other reports suggest wild edibles can also generate substantial income. A study in Almora district, Indian west Himalaya, reveals that the sale of Myrica esculenta fruits (Kaiphalknown for edible fruits and other by products in sub-Himalayan region) brings each household Rs. 913–3713 (US$ 26–106) per season, which was significant considering low annual per-capita income in the region.43 Likewise, other wild edibles have been identified with income generating potential.42–47 Changing perspectives With respect to medicinal plants in the region, extensive use of wild forms and destructive harvesting by the industry is a serious threat. At the same time, dependence on relatively small proportion of available stock and that too on nonnative elements suggests that currently the pharmaceutical industry in the country is utilizing only a few true Himalayan medicinal plants.41 Further, recognizing the importance of the medicinal plant resources of the region and considering the emerging trends of global market, particularly issues of commercial patenting, there is a need to build the capacity of indigenous people to harness the opportunities. Further, need to integrate traditional knowledge system of health care into the national planning process so as to ensure participation of communities in preserving the resources and perpetuating knowledge for renewed research is being emphasized at different levels. The studies have proved that wild edibles of the region are promising source of nutrients and minerals. This potential coupled with proven medicinal value of some of these species, can be harnessed for promoting their use as health food supplements. The nutritional attributes varied considerably among species implying that potential of different species needs to be harnessed for specific

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attribute. These results have significant bearing on bio-prospecting of wild edibles for rural development in the region wherein some of these can be projected as an important resource for income generation through value addition. Realizing the potential of mainstreaming medicinal and wild plants, there is a need to bring in the much-needed coordination among different players for development of these sectors. Effective measures to support marketing efforts with appropriate fiscal and policy support are urgently needed. However, as elsewhere in the world, there is a need to bring in a paradigm shift in realizing the economic contribution of Himalayan biodiversity. The attention needs to be focused on the value of ecosystem services. Particularly, the globally referred/ recognized concept of ‘Payments for Ecosystem Services’ requires serious efforts for implementation in the region under upcoming international climate frameworks.

Bioresource management: Existing scenario Conservation area network As advocated in the Article 8 of Convention of Biological Diversity promotion of in situ conservation is being considered important for the maintenance of biodiversity. This has led to emerging international commitment for strengthening Protected Area (PA) systems. Responding positively to global commitments to bring representative coverage of biological diversity under in situ conservation through strengthening of Protected Area Network, India has created a network of PAs and other conservation areas which include a total of 700 units (i.e. 99 National Parks, 513 Wildlife Sanctuaries, 43 conservation Reserves, 4 Community Reserves, 25 Ramsar sites and 16 Biosphere Reserves). The area covered under PAs and other conservation sites (Biosphere Reserves, Ramsar sites, etc.) accounts for around 9% of the total geographical area of the country. The Himalaya, a global conservation priority, has increasingly received attention of Government of India under its PA programme (Table 5).The progression of PA network expansion (both numbers and area coverage) in the IHR has been presented (Fig. 2). However, due to poor coordination and sufficient transparency in implementation, there are incidents which suggest that the inhabitants in and around PAs are frequently not aware of PA proclamation, and issues of their rights remain unsettled. This has caused an atmosphere of resentment against PAs in the region. All these issues are deleterious to the overall objectives of preserving PA values.48 The PA coverage in the IHR (7.6%) is higher than the national average (4.75%). The existing figures of PAs is apparently indicative of the satisfactory state of network system for conservation of representative ecosystems in the region. However, a more objective analysis of facts reveals the following:

Grand Total

09 North East

27

12

Total

12

Total

07

2

East

NE Hills

2

Central

05

4

Brahmaputra valley

4

West

3

Total

Northwest

1

Tibetan Plateau

02 Himalaya

2

Ladakh Mtn

01 Trans Himalaya

No. of NPs

Province Name

Zone Name

2.7

1.54

0.63

2.98

3.41

2.74

3.05

6.50

1.92

3.14

2.38

3.67

(% of) Biozone Area

98

36

18

18

58

11

08

14

25

4

3

1

No. of WLS

Table 5 Summary of Existing Protected Area Network in the IHR [Source: Ref. 65]

4.84

2.00

1.65

2.98

6.89

9.08

7.24

5.52

5.26

5.65

7.25

4.56

(% of) Biozone Area

125

48

25

23

70

13

10

18

29

7

4

3

No. NPs + WLS

7.6

3.71

2.29

5.96

10.30

11.82

10.29

12.02

7.18

8.79

9.63

8.22

(% of) Biozone Area

Conservation of Himalayan bioresources: An ecological, economical and evolutionary perspective 383

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Nature at work: Ongoing saga of evolution



 

        

    

    

        

   

  

 



 





   

  

Figure 2. Progression of PA Network in IHR [(A)Numbers and (B) Area]

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● Broadly the random distribution of PAs and representative coverage well above the national average (4.75%) in each province, barring NE Hills [Ladakh Mtn: 3 units (8.22% of area); Tibetan Plateau: 4 (9.63%); North west: 29 (7.18%); West: 18 (12.02%); Central: 10 (10.29%), East: 13 (11.82%); Brahmaputra Valley: 23(5.96%) and NE Hills: 25 (2.29%)], supposedly takes care of representative habitats, ecosystems and biota along longitudinal-east to west gradient of the region. The representativeness of these elements along horizontal (latitudinal-South to North) and vertical (low to high elevation) gradients needs to be looked into for individual provinces. In this regard bigger PAs with adequate vertical and horizontal expanse emerge as better candidates. The analysis of size of existing PAs in IHR, however, reveals that: (1) PAs in Trans Himalaya are relatively large (average size 2321.1 km2, n = 7); (2) in Himalayan zone the average size (310.0 km2; n = 70) is small. Within this zone, East Himalayan province (average size 761.3 km2, n = 13) has relatively large PAs, followed by west (347.77 km, n = 18), North west (171.8 km2, n = 29) and Central Himalayan province (56.1 km2, n = 10); (3) PAs in NE biogeographic zone are invariably smaller in size–average size 132.3 km2 (Brahmaputra Valley – 171.8 km2, n = 23; NE Hills – 96.0 km2, n = 25) and (4) over 50% of PAs in Himalayan biogeographic zone are below 100 km2. These figures need to be viewed for wider conservation implications, particularly considering the fact that small PAs are inadequate to (1) preserve large contiguous tracts of representative ecosystems/habitats, (2) viable populations of large key stone species and (3) safeguard overall ecological value of ecosystems. ● As reflected, the PA coverage in NE biogeographic zone, particularly in NE hill province, is poor. Whereas, from the biodiversity point of view, considering in particular the evolutionary importance as one of the centers of plant origin, this region holds great conservation significance. Therefore, it would require immediate attention for bringing in adequate and representative areas under PA network to ensure natural evolutionary processes to continue through strict in situ protection of habitats and ecosystems of the region. Recently, recognizing the need for holistic management of biodiversity at the landscape level ‘Biosphere Reserve’ concept is being promoted in the region. The concept considers conservation of landscapes having immense biodiversity value by maintaining cultural heritage and fostering economic and human development which is culturally and ecologically sustainable. Between 1988 till date a total of seven Biosphere Reserves, which represent 43.8% of total units in India, have been designated in different states of IHR [Cold desert – Himachal Pradesh (Trans Himalaya); Nanda Devi – West Himalaya (Uttarakhand); Kangechnjunga – Sikkim (Central Himalaya); Manas and Dibru-Saikhowa – Assam (Bahmaputra Valley); Nokrek-Meghalaya (NE Hills); and Dehang Debang – Arunachal Pradesh (East Himalaya)]. Considering the ecological, economic and evolutionary significance

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of such landscapes, there is a strong need to further strengthen Biosphere Reserve network in the Himalaya.6 In spite of all these initiatives with respect to conservation area network in the Himalaya, the indications are such that community involvement in Government sponsored or otherwise implemented management programmes in the region is decreasing. This calls for reorientation of strategies and action plans.

Bioresource management: Toward contemporary thinking Finding solutions through integration of traditional and cultural values The concept of ‘natural cultural landscape’ which sees cultural and biological diversity as mutually supportive49 appears most appropriate to ensure community support for maintaining and conserving the uniqueness of biodiversity (both wild and domesticated). This calls for proper integration of cultural values and Traditional Ecological Knowledge (TEK) system with modern approaches of management. In this context, one needs to find out tangible dimensions of TEK derived from ‘intangibles’ (Box 1). It would, however, also require putting in place adequate reward systems.6 Management: Synchronizing with sustainable use As indicated in the beginning, the Himalaya has emerged as a mega biodiversity center of the world with its biodiversity components (i.e. genetic, species and ecosystems) having global importance. However, the region is always referred to as economically marginal and as a home of the worlds’ poorest whose livelihoods depend heavily on natural resources, and any change in the status of these resources affects them severely.50 In this context, the Himalayan mountains being sensitive

Box 1: Tangible dimensions of TEK derived from intangibles [Source: Ref. 49] Economic: traditional ethnobiology (lesser-known plants and animals harvested from the wild and used for food and medicine) Socioecological: the ways in which traditional societies conserve and use biodiversity to manage soil fertility, nutrient cycling and soil moisture regimes Sociocultural: the cultural, spiritual and religious belief systems of mountain people which are centered around ‘sacred species’, ‘sacred groves’ and ‘sacred landscapes’

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ecosystems for both human induced and natural perturbations have exhibited rapid changes in its resource base and thereby affecting the sustenance of its people. As a result, the indigenous communities in the region have, by and large, begun to show disinterest toward management of natural resources including the bioresources. Specifically, they feel alienated on account of current policies and programmes of conservation which consider nonuse and existence value of biodiversity as points of prime focus. In the context of above, while initiatives for strengthening of conservation areas is essentially required, there is a need for reorienting existing norms and management practices by harnessing the income generating potential of such areas for local communities so as to promote their interest and participation in conservation.6 Biodiversity conservation: Linking with livelihoods There is a strong need to promote sustainable use concept which attempts to establish linkages between conservation and economic growth, and recognizes that the bioresources are a source for sustainable income. Experiences from the region itself have revealed that people become interested in biodiversity conservation when they realize that it has immediate utility value (i.e. subsistence or income generating opportunity) attached and they can harness benefits from it. A few examples include: (1) Developing biodiversity based enterprise while conserving the resources (e.g. Oak-silk in Garhwal (Uttarakhand); traditional local paper from loktak – Daphne spp. in Nepal and Sikkim, high value caterpillar plant ‘yarsha gumbu – Cordyceps sinensis and matsutake mushroom – Tricholoma matustake in Bhutan); (2) Niche product marketing from shifting cultivation (e.g. ‘kholari’ bean from the shifting cultivation areas of Nagaland); (3) Nature valuation for accrued benefits (e.g. valuation of Kangechnjunga National Park and Khecheopalri Sacred lake in Sikkim) and (4) Ecotourism and biodiversity conservation related enterprises (e.g. conservation of snow-leopard through community based tourism in Ladakh, biodiversity ecotourism initiatives in Sikkim, expansion of Chitwan National Park through community forestry and ecotourism development in Nepal).50 Yet another strategy, which has emerged as promising, is to maximize economic benefits by adding value to biodiversity components available in immediate vicinity of indigenous communities. An example can be cited from Uttarakhand, wherein Institute of Himalayan Environment and Education (INHERE), an NGO, focuses on promotion of agrodiversity of the mountain villages by converting this diversity into high-value products for niche markets. Basic to this effort has been the conversion of, largely by default, the organic agriculture of the mountains into certified organic products by adding value.51

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Efforts made by the G.B. Pant Institute of Himalayan Environment and Development (GBPIHED) in Uttarakhand, Himachal, Sikkim and Arunachal Pradesh for capacity building of inhabitants for effective use of bioresource based rural technologies (e.g. bio-briquettes, protected cultivation, poly-pit, nursery development, medicinal plant cultivation, mushroom cultivation, etc.) have received considerable attention across IHR.52 This has led people to reorient their mindset in a way so as to view bioresources or its products as effective source of income for rural inhabitants. Ecosystem approach: Appropriate option With the growing realization that elements of biodiversity are intricately linked and focusing on management of any one, ignoring the others, cannot be sustainable, there is a felt need for considering holistic or ecosystem based approach of management. It recognizes that humans, with their cultural diversity, are an integral component of ecosystems. The above implies that management of biodiversity components in any ecosystem would require integration of research outputs and human dimensions. One example can be drawn from the role of bees as pollinators to enhance agricultural productivity. The extensive researches have proved that the decline in production of apple in Himachal Pradesh is linked with disturbances in pollination process – a reason largely unknown to many of the growers and even agricultural extension workers.53 With this realization the rental of bee hives is getting popular for pollination among apple growers of Himachal Pradesh. This realization has also led India to participate in the formulation of a global pollinator project, being coordinated by GBPIHED, with targets of management of pollinators for sustainable agriculture through ecosystem approach in the Himalaya. Yet another example of holistic view of conservation can be cited as implementation of Village Environment Action Plan (VEAP) developed by GBPIHED. This includes approach of preparing developmental plan for village ecosystem using integration of its resources (physical, biological and human) and environmental hazard management through participatory approach, of village resource mapping, monitoring and evaluation.54 Likewise the Catchment Area Protection (CAP) and Assisted Natural Regeneration (ANR) initiatives of the Institute focus on ecosystem approach for participatory management of natural water and bioresources.55

Effective valuation: Ensuring transfer of payments While considering ecosystem approach, there is a growing concern to effectively attach monitory value to biodiversity, specifically the ecosystem services and make provisions for transfer of payments (compensations) to their protectors. An

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emerging issue is attaching value to forests. Under the efforts to implement the United Nations Framework Convention on Climate Change (UNFCCC), with a growing global realization that fuller valuation of forests is necessary if forests are to be conserved by local people, progress has been made to develop carbon markets. The trends on initiatives across the world are indicative of the fact that forestry efforts could play an increasingly important role in achieving the emission reduction targets agreed by signatories to the Kyoto Protocol. Experiences from the Uttarakhand (west Himalaya), which represents one of the oldest and largest system of Van Panchayat (introduced in 1930; over 12,000 villages; nearly 30% of total forest area of the State), under ‘Kyoto- Think Global, Act Local’ project has revealed that communities can be trained to use technology to measure carbon sequestration rate in their community forests. Estimates suggest that if trained communities are able to submit proposal through proper mechanisms one Van Panchayat alone could receive up to 1.08 Lakhs per annum for carbon sequestered by their forest. The funds generated through the sale of carbon credits can be used to meet their needs and can encourage people to save their forests as ‘Carbon Sinks’.56 The recently released guidelines by the Ministry of Environment and Forests [6] also emphasize upon the need to pay proper attention for harnessing benefits from the programmes like ‘Reduced Emission from Deforestation and Degradation’ (REDD) under the United Nations Framework Convention on Climate Change (UNFCC). This would require integration and involvement of diverse stakeholder groups for developing effective and feasible mechanisms to conserve carbon within existing forests and through slowing down the rates of deforestation and degradation of forests. Watershed Management: Ultimate solution Having understood the fact that linking with livelihoods, transfer of payments for services and ecosystem approach are among essential components of effective management strategy for Himalayan Biodiversity, the extent of scale of operation remains a major concern. The forested mountain watersheds are now viewed as being vital for the ecological health across the world. In addition to their important protective role, they shelter immense biodiversity, provide food and fodder, and are an important source of fresh water. These benefits are realized not only by the upland watershed communities but also by those who live much beyond its physical boundaries in downstream areas. Considering this value, integrated watershed management approach for mountains has emerged as an effective solution. It is an on-going process which actively involves local people and decision makers – in both upstream and downstream areas– in analyzing the problems and developing appropriate solutions to ensure that the activities undertaken are well adapted to the local situations.57

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In the context of Himalaya, Environmental and Social Management Framework; Guidelines and Implementation and Monitoring manual developed for Watershed Management Directorate (Uttarakhand) sets an example which largely follows the above approach of Integrated Watershed Management.58 Conservation Awareness: A key element Notwithstanding that the above mentioned approaches deserve immediate consideration for the adequate management of biodiversity in Himalayan ecosystem, there is an urgent need for designing and implementing programmes for creating mass awareness among different stakeholders. The approaches need to take note of location specific issues and specificities in the region. While recognizing this requirement, various organizations have initiated activities in this direction. However, a critical assessment of such initiatives most often results in realization that the approach followed lacks a holistic vision. As a result their long term effectiveness remains invisible. We, therefore, feel that it is a high time for introspection by all concerned. We would, however, like to cite two examples from the region. These approaches, in our opinion, possess unique strengths and have potential to serve long term management goals for the Himalayan Bioresources. The growing concern over Environmental Education (EE) programmes calls for developing perspective guidelines for implementation in both formal and informal sectors in order to bring in a perceptible shift in the mindset of communities toward integrating conservation science with societal needs. With regard to IHR, mountain specificities need to be accordingly included in such guidelines and programmes. Approaches developed through successful initiatives in the region; both in formal [e.g. initiative by an environmental NGO – Uttarakhand Seva Nidhi (UKSN), Almora (Uttarakhand)] and informal education [e.g. approach developed and tested by G.B. Pant Institute of Himalayan Environment and Development, Almora and Gauhati based CEE- North East] need to be promoted and considered for replication across the Himalayan states with area/location specific modifications. The concerned State Governments may initiate developing programmes in this direction and ensure implementation in consultation with suitable organizations.6

Conclusion Realizing that evolution is much more than a simple change within individual species, it builds up intricate, intertwined relationships between the earth and its inhabitants, we believe there is a vast scope and need of untangling the complexities of59 Himalayan ecosystem. The representative ecosystems and their unique (endemic) elements are different from the remaining world in many ways.

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The prevailing TEK systems, and potential economic values attached with goods and services emanating from the region, provide enough space to draw plans for long term maintenance of its resources. Such plans, while addressing the issues of livelihood needs of indigenous communities, would also accommodate the emerging needs under the changing climate and economic scenario. In particular, with perceived greater vulnerability of the Himalayan ecosystem to changing climate, it is certain that the ‘struggle for existence’ is going to intensify in the region. Therefore, the adaptation process, which largely follows course of natural selection, under changing climate will determine fate of its bioresources. However, understanding the process of change and its impacts is not an easy task. It would require deep wonderings, like Darwin did, for resolving many of nature’s mysteries. Integrated and participatory efforts, with deeper understanding of intricate linkages, will prove beneficial for conserving unique Himalayan ecosystem. In the above context it is pertinent to end this essay with the following quote from the Origin of Species by Charles Darwin.1 ‘Climate plays an important part in determining the average number of species, and periodical seasons of extreme cold or drought, I believe to be the most effective of all checks……………..The action of climate seems at first sight to be quite independent of the struggle for existence; but in so far as climate chiefly acts in reducing food, it brings on the most severe struggle between the individuals, whether of the same or of distinct species, which subsist on the same kind of food…..’ ‘When a species, owing to highly favourable circumstances, increases inordinately in numbers in a small tract, epidemics……often ensue: and here we have a limiting check independent of the struggle for life’. Acknowledgements: Authors thank various individuals and organizations from where the information has been drawn for building this paper.

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(a) P. Dayanandan: Figure 5, Page 5

(a) V.P. Sharma: Figure 1, Page 23

(b) V.P. Sharma: Figure 2, Page 24

(c) V.P. Sharma: Figure 3, Page 25

(d) V.P. Sharma: Figure 6, Page 28

(e) V.P. Sharma: Figure 7, Page 29

(a) Veena Tandon and Gaurangi Maitra: Figure 1, Page 37

(b) Veena Tandon and Gaurangi Maitra: Figure 2, Page 37

(c) Veena Tandon and Gaurangi Maitra: Figure 4, Page 41

(d) Veena Tandon and Gaurangi Maitra: Figure 5, Page 42

(e) Veena Tandon and Gaurangi Maitra: Figure 6, Page 42

(f) Veena Tandon and Gaurangi Maitra: Figure 8, Page 45

(g) Veena Tandon and Gaurangi Maitra: Figure 9, Page 46

~4.5 billion years

Cyanobacteria

Earth birth

Eukaryotes

Single celled

Scale

Multicellular

Mammals Vertebrate

Time

500 million years 50 million years 5 million years

= represents time required for continuous re-incarnation of a 100 year old human more than 50,000 times

(a) Amit Sharma: Figure 1, Page 61

(b) Amit Sharma: Figure 2, Page 65

(c) Amit Sharma: Figure 4, Page 67

(a) Anupam Varma and Shelly Praveen: Figure 1, Page 77

(d) Amit Sharma: Figure 5, Page 69 (b) Anupam Varma and Shelly Praveen: Figure 2, Page 78

(c) Anupam Varma and Shelly Praveen: Figure 3, Page 79

(e) Anupam Varma and Shelly Praveen: Figure 5, Page 82

(d) Anupam Varma and Shelly Praveen: Figure 4, Page 82

(f) Anupam Varma and Shelly Praveen: Figure 6, Page 84

(g) Anupam Varma and Shelly Praveen: Figure 7, Page 86

(h) Anupam Varma and Shelly Praveen: Figure 8, Page 87

(i) Anupam Varma and Shelly Praveen: Figure 9, Page 88 (j) Anupam Varma and Shelly Praveen: Figure 10, Page 89

(a) Radha Prasanna and B.D. Kaushik: Figure 1, Page 125

(a) V.V. Ramamurthy and Asha Gaur: Figure 4 (a,b,c,d,e,f), Page 186

(a)Rajesh Tandon and H.Y. Mohan Ram: Figure 1(a,b,c,d,e), Page 225

(a) Lok Man S. Palni and Ranbeer S. Rawal: Figure 1, Page 372

(b) Lok Man S. Palni and Ranbeer S. Rawal: Figure 2, Page 384