Karyotypes of Parasitic Hymenoptera

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Karyotypes of Parasitic Hymenoptera

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Vladimir E. Gokhman

Karyotypes of Parasitic Hymenoptera

123

Vladimir E. Gokhman Moscow State University Botanical Garden Moskva Russia 119991 [email protected]

Originally published in 2005 with the Russian publisher KMK Scientific Press Ltd. in Russian language. c KMK Scientific Press Ltd., Russian Edition, 2005

ISBN 978-1-4020-9806-2

e-ISBN 978-1-4020-9807-9

DOI 10.1007/978-1-4020-9807-9 Library of Congress Control Number: 2009920978 c Springer Science+Business Media B.V. 2009 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Cover illustration: Micrograph of chromosome set of Cratichneumon rufifrons (Ichneumonidae). Original photo by Vladimir E. Gokhman Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

Foreword

Not so long ago, karyology was considered a vanguard biological discipline, which could solve nearly all problems of systematics and phylogenetics. We liked to believe in the bright future, in a magician who will appear like a Jack-in-the-box and reveal the truth to us. However, excessive hopes related to the chromosomal study came true only in part. In the meantime, new candidates claimed the place of the magician, i.e. phenetics succeeded by cladistics and now by molecular methods in systematics and phylogeny. Nevertheless, it becomes progressively more obvious nowadays that cladistics is just a bright envelope for the fairly primitive and theoretically vulnerable approach that deprives living organisms and their groups of the traces of integrity and reduces them to the plain sum of characters. Modern molecular techniques look more perceptive and may yield more reliable results, although the details are sometimes embarrassing, and comparison with the fossil record does not necessarily reveal their superiority over cladistics. These methods are accessible by research teams with massive funding and good equipment and this strongly decreases the range and diversity of the material studied. However, classifications are often created by individual systematists with the restricted access to molecular methods. In this context, karyological techniques are in the preferable position, although they certainly do not provide direct and immaculate markers of taxonomic and phylogenetic relationships: chromosomal study is a morphological method with all its advantages and drawbacks. However, karyology operates at the level of cells and organelles that absolutely differs from that of conventional morphology, and, as the present monograph demonstrates, it sometimes reveals differences where routine techniques show deep similarities and vice versa. Because of that, karyotypic data can perform the important function of control and correction of the results obtained by other techniques, along with the evaluation and reassessment of these results. On the other hand, technical progress in this field makes karyological methods accessible to individual systematists and applicable to a wide range of objects. As author’s experience shows, if necessary skills are provided, hundreds of specimens can be karyotypically studied under normal conditions without hindering the performance of the main research tasks. The present monograph is unique because it demonstrates the wide range of karyotypic data for an insect group that seemed hopeless in this respect not so long v

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Foreword

ago. An adaptation of the technique of karyotyping immature stages to adult insects allowed solution of a problem that previously seemed insoluble: to routinely obtain karyograms of acceptable quality from individual insects that nevertheless can be reliably identified. In the case of parasitic Hymenoptera (including Chrysidoidea) this means the possibility of working with adults of the definite sex (more often with females that can be classified more easily). Indeed, the enormous diversity of parasitic wasps makes larvae-restricted studies meaningless because we usually cannot identify these stages. In this case, we can use only laboratory-bred species whose larvae can be identified using conspecific adults. Dr. Gokhman has therefore elaborated the unique and perceptive method that has enabled the unique results accumulated in this book to be obtained. Among more than 400 karyologically studied species of parasitic Hymenoptera representing 24 families, only about 60 species from 13 families had been studied before he started his work. In fact, the author himself has created the whole karyological pattern given in this book, with all the conclusions, sometimes nontrivial ones, as the bimodal distribution of chromosome numbers in the group as well as the asymmetry of pathways of the change in chromosome number, i.e. chromosomal polymerisation through aneuploidy and restoration of even numbers, and oligomerisation through chromosomal tandem fusions, etc. However, I am not going to bore readers with repeating the book content. I would only like to note that the present work is rich with new facts, techniques and ideas, and it will be of use to all those interested in karyology and Hymenoptera. September 2008

Alexandr P. Rasnitsyn

Acknowledgments

I express sincere gratitude to my first tutor in parasitic Hymenoptera research, Prof. A.P. Rasnitsyn (Paleontological Institute, Russian Academy of Sciences, Moscow, Russia). I am grateful to the late Prof. N.N. Vorontsov as well as to Drs. E.A. Lyapunova, I.Y. Bakloushinskaya and other members of the Laboratory of Cytogenetics (Institute of Developmental Biology, Russian Academy of Sciences, Moscow) for valuable advice. I greatly appreciate fruitful discussion as well as identifications of the karyotyped material provided by many specialists of the Zoological Institute (Russian Academy of Sciences, St. Petersburg, Russia), especially by Prof. V.G. Kuznetsova and the personnel of the Hymenoptera Division (Laboratory of Insect Systematics): Prof. V.I. Tobias, Drs. S.A. Belokobylskij, D.R. Kasparyan, V.A. Trjapitzin and the late Prof. M.A. Kozlov. Some parasitic wasps were also identified by Dr. V.V. Kostjukov (All-Russian Research Institute for Biological Plant Protection, Krasnodar, Russia), Prof. M.D. Zerova, Drs. A.V. Gumovsky and V.I. Tolkanitz (Institute of Zoology, National Academy of Sciences, Kiev, Ukraine), Dr. E.N. Yegorenkova (Ulyanovsk Teachers’ Training Institute, Ulyanovsk, Russia), Dr. A.E. Humala (Forest Institute, Karelian Science Centre, Russian Academy of Sciences, Petrozavodsk, Russia), Dr. K.A. Dzhanokmen (Institute of Zoology, National Academy of Sciences, Almaty, Kazakhstan) and Dr. N.G. Ponomarenko (Moscow); the author is also grateful to these specialists. I thank Dr. V.V. Buleza (Institute of Problems of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia), Dr. S.Y. Reznik (Zoological Institute, Russian Academy of Sciences, St. Petersburg), Dr. N.S. Rak (Polar Alpine Botanical Garden and Institute, Russian Academy of Sciences, Kirovsk, Russia) and Ms. L.S. Sabitova (Kosino Greenhouse Complex, Moscow) for providing laboratory stocks of parasitic wasps. The author greatly appreciates cooperation of his foreign colleagues, Prof. D.L.J. Quicke (Imperial College at Silwood Park, Ascot, Berkshire and Natural History Museum, London, UK), Dr. M. Westendorff (Deutsches Entomologisches Institut, Zentrum f¨ur Agrarlandschafts- und Landnutzungsforschung e.V., M¨uncheberg, BRD), Prof. L.W. Beukeboom (Institute for Evolutionary and Ecological Sciences, University of Leiden, Leiden, The Netherlands) and Dr. W. V¨olkl (Department of Animal Ecology, University of Bayreuth, Bayreuth, BRD) as well as Prof. J.H. Werren (Department of Biology, University of Rochester, New York, USA), Dr. K.R. Hopper (Beneficial Insect Introduction Research Unit, Agricultural vii

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Acknowledgments

Research Service, United States Department of Agriculture and University of Delaware, Newark, Delaware, USA), Dr. S.L. Heydon (Bohart Museum of Entomology, University of California, Davis, California, USA), Dr. J.A. Johnson (ARS, USDA, Parlier, California, USA), Dr. D. Nordlund (Biological Control and Mass Rearing Research, ARS, USDA, Mississippi State, Mississippi, USA), Dr. G. Boivin (Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, St-Jean-sur-Richelieu, Qu´ebec, Canada), Prof. B. Lanzrein (Division of Developmental Biology, University of Bern, Bern, Switzerland), Prof. J. Steidle ¨ (Angewandte Zoologie / Okologie der Tiere, Institut f¨ur Zoologie, Freie Universit¨at Berlin, Berlin, BRD), Dr. P. Star´y (Institute of Entomology, Czech Academy ˇ of Sciences, Ceske Bud˘ejovice, Czech Republic), Dr. Z. Bouˇcek (London, UK), Dr. H. Baur (Natural History Museum, Bern, Switzerland), Dr. G. Melika (Pest Diagnostics Laboratory, Plant Protection and Soil Conservation Directorate, Vas County, Tanakajd, Hungary), Dr. M. Wanat (Wrocław University, Wrocław, Poland) and Dr. J. Sawoniewicz (University of Białystok, Białystok, Poland). I appreciate support and assistance of the staff of the Botanical Garden of Moscow State University, including its Director Prof. V.S. Novikov and the personnel of the Plant Protection Group that I lead for about 20 years. A number of staff members, graduate and postgraduate students of the MSU Faculty of Biology (including Drs. A.V. Timokhov and T.Y. Fedina) substantially helped me at various stages of the work. Dr. R.B. Angus (School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, UK) has kindly checked the language of the manuscript. The present study is partly supported by the research grant no. 07-04-00326 from the Russian Foundation for Basic Research.

Contents

1 Chromosomes of Hymenoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Karyotype Structure of Hymenoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Main Genetic Features of Life Cycle . . . . . . . . . . . . . . . . . . . . . 1.1.2 Chromosome Numbers and Ploidy Levels . . . . . . . . . . . . . . . . . 1.1.3 Size of Mitotic Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.4 Centromere Position and Centromere Index . . . . . . . . . . . . . . . 1.1.5 Heterochromatic and Euchromatic Chromosome Segments: C-Banding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.6 Nucleolus Organiser: AgNOR-Banding . . . . . . . . . . . . . . . . . . . 1.1.7 Other Techniques of Differential Chromosome Segmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.8 Details of Meiosis and Structure of Meiotic Chromosomes . . . 1.2 Diversity of Chromosome Sets of Hymenoptera . . . . . . . . . . . . . . . . . . 1.2.1 Karyotypic Features of Various Taxonomic Groups . . . . . . . . . 1.2.2 Types of Chromosomal Rearrangements . . . . . . . . . . . . . . . . . . 1.3 Systematic and Phylogenetic Implications of Chromosomal Characters in Hymenoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1 Taxonomic Significance of Karyotypic Features . . . . . . . . . . . . 1.3.2 Phylogenetic Implications of Karyotypic Characters . . . . . . . .

1 1 1 3 5 5

17 17 22

2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Material Studied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Methods of Obtaining Chromosomal Preparations . . . . . . . . . . . . . . . . 2.3 Methods of Analysing Chromosomal Preparations . . . . . . . . . . . . . . . . 2.4 Sources of Data on Taxonomy and Phylogeny . . . . . . . . . . . . . . . . . . . .

31 31 32 33 33

3 Morphological Features of Karyotypes of Parasitic Hymenoptera . . . . . 3.1 Chromosome Number and Nuclear DNA Content . . . . . . . . . . . . . . . . . 3.2 Size of Mitotic Chromosomes: Centromere Position and Centromere Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Differential Chromosome Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Details of Meiosis and Structure of Meiotic Chromosomes . . . . . . . . .

35 35

6 7 7 9 11 11 13

37 38 39 ix

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Contents

4 Chromosomal Evolution of Parasitic Wasps . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Chromosomal Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1 Changes in Chromosome Structure . . . . . . . . . . . . . . . . . . . . . . . 4.1.2 Changes in Chromosome Number . . . . . . . . . . . . . . . . . . . . . . . 4.2 Microevolutionary and Macroevolutionary Karyotypic Changes . . . . .

41 41 41 42 46

5 Phylogenetic Implications of Karyotypic Characters of Parasitic Hymenoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 5.1 Phylogenetic Analysis of Chromosomal Characters . . . . . . . . . . . . . . . 49 5.2 Main Trends of Karyotype Evolution of Parasitic Hymenoptera . . . . . 65 6 Chromosomal Analysis of Parasitic Wasps at Various Taxonomic Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 General Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Superfamilies, Families and Subfamilies . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Genera and Groups of Genera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Species and Species Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.1 Species Grouping and Classification . . . . . . . . . . . . . . . . . . . . . 6.4.2 Clearly Distinct Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.3 Sibling Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.4 Morphologically Identical Populations . . . . . . . . . . . . . . . . . . . . 6.5 Other Implications of Chromosomal Analysis . . . . . . . . . . . . . . . . . . . .

67 67 67 68 70 70 71 71 76 77

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 A Chromosome Numbers of Parasitic Wasps . . . . . . . . . . . . . . . . . . . . . . . . . 83 B Micrographs and Ideograms of Chromosome Sets of Parasitic Hymenoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Introduction

The parasitic Hymenoptera are one of the largest and most taxonomically complicated groups of insects (Rasnitsyn 1980). Certain estimates (Quicke 1997) demonstrate that species diversity of parasitic wasps is not less than that of Coleoptera, which are traditionally considered as the most speciose order of insects. The former group already included about 50,000 described species in the beginning of the 1990s (LaSalle and Gauld 1991), but this is also the estimate of species richness of the family Braconidae only (Dolphin and Quicke 2001). Parasitic Hymenoptera play a very important role in food chains as parasitoids of the overwhelming majority of insects and other arthropods (Gauld and Bolton 1988, LaSalle and Gauld 1991). This is the reason for the high practical significance of parasitic wasps that attack many pests of agriculture and forestry as well as certain beneficial insects, i.e. predators and primary parasitoids (Viktorov 1976, Rasnitsyn 1980, Godfray 1994). In addition, some secondarily phytophagous Hymenoptera that formally belong to the aforementioned group damage certain cultivated plants (Gauld and Bolton 1988). Despite the large practical importance of parasitic wasps, many problems of their taxonomy remain insufficiently studied (Quicke 1997). This situation can be explained by several reasons. First of all, parasitic Hymenoptera are an intensively evolving group that includes several hundred thousand (or even a million or more) species, and many of them look very similar. Moreover, the morphological diversity of the supraspecific taxa is often formed by various combinations of the same external features. Under these circumstances, separation of the mentioned taxa is even more difficult than the distinction between species (Rasnitsyn 1978). There is also another important factor that decreases the efficiency of taxonomic and phylogenetic research of parasitic Hymenoptera. Specifically, many structural characters of these insects have a comparatively high degree of variation depending on the environmental conditions, especially on the external/internal structure and ecological features of the host as well as on the details of its attack and exploitation by the parasitoid. This situation leads to numerous convergent similarities of parasitic wasps that involve their external/internal morphology (Quicke and van Achterberg 1990, Gokhman 1995). In the situation described, use of the modern research techniques can be very helpful because these methods provide independent data for solving taxonomic and phylogenetic problems in the parasitic Hymenoptera. Chromosomal analysis is one xi

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Introduction

of these techniques that are successfully applied to many animal groups including insects (Blackman 1980, 1985, Kuznetsova 1985, Petitpierre 1996, Stekolnikov et al. 2000, Gokhman and Kuznetsova 2006). Karyotypic research has certain indisputable advantages over other experimental methods used in taxonomic and phylogenetic studies of the Hymenoptera (Gokhman 2007c). First of all, chromosomal characters are essentially morphological, and therefore they can be analysed in approximately the same way as other morphological features. Moreover, some karyotypic characters (such as numbers of chromosomes, chromosome arms, nucleolus organisers and heterochromatic segments) can have only discrete values, and most cases of the intraspecific chromosomal polymorphism as well as of hybridisation between forms with different chromosome numbers therefore become easily recognisable. Finally, modern methods of chromosomal analysis are relatively inexpensive and allow examination of an extensive amount of material within a short period of time. Thus, these perceptive methods can be used for screening both laboratory and natural populations of parasitic wasps (Gokhman 1997a, 2000a, 2006a,b, Gokhman and Kuznetsova 2006). However, despite all advantages of using karyotypic analysis for the purposes of taxonomic and phylogenetic studies of parasitic wasps, chromosomes of these insects remained relatively poorly studied up to recent times. The reason for this situation is that chromosomal studies of the parasitic Hymenoptera are hampered by some technical obstacles (Gokhman 2006b). It is known that a successful karyotaxonomic study of a certain group should simultaneously meet two prerequisites. The first of these conditions is the presence of tissues with relatively high numbers of mitoses and/or meioses. In addition to that, studied species (and every examined specimen if possible) should be reliably identified. Regretfully, chromosomes of the parasitic Hymenoptera were studied on preparations usually obtained from immature stages (prepupae and early pupae) up to the middle of the 1980s. This situation almost exclusively restricted the range of studied forms to laboratorycultivated species, because the precise taxonomic identification was possible only by conspecific adult individuals. Material from natural populations of only few groups of parasitic wasps, such as multiple parasitoids (including polyembryonic ones) as well as certain phytophagous Hymenoptera (gall wasps of the family Cynipidae, etc.), was previously studied (see Sanderson 1932, Gokhman 1995 for reviews). As for the adult individuals, they usually were not considered as potential sources of tissues for obtaining chromosomal preparations, because almost all cell divisions occurred in male immature stages, whereas mitoses and meioses were usually uncommon in the ovaries of intact females. Due to this reason as well as due to other circumstances, chromosome studies covered only about 60 species of parasitic wasps (approximately 0.1% of the described members of this group) until the last 25 years. All aforementioned information demonstrates the importance of large-scale chromosomal studies of parasitic Hymenoptera. In this book, the term “parasitic Hymenoptera” is considered in a broad sense, i.e. it includes not only traditional Parasitica, but also wasps of the superfamily Chrysidoidea. This is done mainly for technical reasons. Specifically, chromosomes of Aculeata are generally studied using

Introduction

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prepupae and early pupae (see above) that are extracted from nests of those insects. It is often possible to collect adults of the species studied together with their immature stages, thus allowing reliable identification. However, wasps of the superfamily Chrysidoidea never construct nests (Gauld and Bolton 1988), and they are closer in their biology to Parasitica than to other Aculeata. For this reason, chromosomes of Chrysidoidea are examined with the same techniques as those used for parasitic Hymenoptera. The main goal of the present work is therefore to study the karyotype structure and chromosomal evolution of parasitic Hymenoptera as well as to interpret the results obtained for systematic and phylogenetic purposes (Gokhman 2003). This is the second edition of the monograph that was first published in Russian a few years ago (Gokhman 2005a). However, the present version of the book is updated with new data, conclusions and references.

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Chapter 1

Chromosomes of Hymenoptera

Abstract Karyotypic features of the order Hymenoptera are reviewed. Main genetic features of the life cycle, i.e. arrhenotoky (sometimes changed to thelytoky) and haplodiploidy, are discussed. Models of sex determination in Hymenoptera are listed. Diversity of chromosome numbers, including ploidy levels, and general chromosomal morphology in the order are briefly reviewed. Various structural features of mitotic chromosomes (chromosome size, centromere position and centromere index, euchromatic and heterochromatic segments, nucleolus organiser, etc.) including different banding techniques, such as C-, AgNOR-, G- and restriction banding, fluorochrome staining and in situ hybridisation (FISH, chromosome painting) are described. Details of meiosis as well as the structure of meiotic chromosomes of Hymenoptera are reviewed. The diversity of chromosome sets in various hymenopteran taxa (including types of chromosomal rearrangements detected in the order) is shown. Taxonomic and phylogenetic implications of karyotypic analysis of Hymenoptera are described. Keywords Chromosomes · Hymenoptera · Karyotype · Phylogeny · Taxonomy

1.1 Karyotype Structure of Hymenoptera 1.1.1 Main Genetic Features of Life Cycle The mechanism of sex determination in Hymenoptera attracted researchers’ attention for a long time. In the middle of the nineteenth century Dzierzon (1845, cited in Crozier 1975) suggested that arrhenotoky, i.e. development of males (in contrast to females) from unfertilised eggs, was characteristic of the honeybee (Apis mellifera Linnaeus). Cytological proofs of this hypothesis, however, were obtained only at the borderline between the nineteenth and twentieth centuries (Paulcke 1899, Petrunkewitsch 1901). Data on arrhenotoky in other Hymenoptera appeared at approximately the same time (Wheeler 1903, 1904, Castle 1904), although acceptance of its widespread occurrence in the order lasted for dozens of years (Sanderson 1932, Heimpel and de Boer 2008). V.E. Gokhman, Karyotypes of Parasitic Hymenoptera, DOI 10.1007/978-1-4020-9807-9 1, C Springer Science+Business Media B.V. 2009

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2

1 Chromosomes of Hymenoptera

The second genetic characteristic of the hymenopteran life cycle that is also tightly bound to arrhenotoky is haplodiploidy, i.e. presence of haploid males and diploid females in the overwhelming majority of those insects. This means that at least the large part of the male’s tissues (including gametes) remains haploid, and therefore an abortive meiosis without decrease in chromosome number takes place there. This fact has been first discovered by Meves (1904, 1907) in A. mellifera and then repeatedly confirmed in other members of the order Hymenoptera (Sanderson 1932, 1988, Torvik-Greb 1935, Koonz 1936, 1939, Schmieder 1938, Mackay 1955, Smith and Peacock 1957, Sharma et al. 1961, etc.). As mentioned above, arrhenotoky is characteristic of many hymenopterans, although thelytoky (i.e. development of the purely female offspring from unfertilised eggs; White 1973) is detected in some insects belonging to this group (Heimpel and de Boer 2008). Thelytoky does not usually occur in all members of certain taxa; on the contrary, it can be found sporadically in many groups, and arrhenotokous and thelytokous populations of the same morphospecies often coexist (Hung et al. 1988, Stouthamer and Kazmer 1994, Belshaw et al. 1999, Jeong and Stouthamer 2005, Lattorff et al. 2005). The only exception from this rule is gall wasps of the family Cynipidae. In many Cynipidae, alteration of the so-called sexual and parthenogenetic generations is observed within the life cycle. The parthenogenetic generation consists of females of two types that lay unfertilised eggs. These two types differ in that females of the first type give rise only to males, whereas those of the second type give rise only to females of the sexual generation. In turn, offspring of the males and females of this generation are females of the parthenogenetic generation. Thelytoky in these forms as well as in some other species is therefore cyclical (Doncaster 1916, Crozier 1975, Quicke 1997). Another modification of the normal life cycle is found in the ants Cataglyphis cursor (Fonscolombe) and Wasmannia auropunctata (Roger) in which workers are produced by sexual reproduction, but new queens are almost exclusively produced by thelytoky. Moreover, males of W. auropunctata also reproduce clonally because the maternal half of their genome is likely to be eliminated from diploid eggs (Pearcy et al. 2004, Fournier et al. 2005). Although reports on the discovery of sex chromosomes in parasitic (Guhl and Dozortseva 1934, Dreyfus and Breuer 1944) and aculeate Hymenoptera (Kerr 1951) appeared up to the middle of the twentieth century, it is now considered proven that these chromosomes are absent from hymenopteran karyotypes (White 1973, Crozier 1975). Nowadays, it is widely accepted that sex determination in Hymenoptera occurs through allelic interactions modulated by ploidy levels of individuals (Snell 1935, Crozier 1971). If relatively rare mutations are excluded (see e.g. Beukeboom et al. 2007), hymenopteran females are usually diploid and their males are haploid, although diploid males were also detected in some species (Whiting and Whiting 1925, Speicher and Speicher 1938, 1940, Woyke 1969, Smith and Wallace 1971, Hung et al. 1972, Kerr 1974, Hedderwick et al. 1985, Tsuchida et al. 2002, Cowan and Stahlhut 2004, etc.). Although the single multiallelic locus, csd, was previously thought to be involved in sex determination in the Hymenoptera (Beye et al. 2003, Evans et al. 2004), this locus was recently found

1.1 Karyotype Structure of Hymenoptera

3

only in honeybees. However, its more widespread putative progenitor, fem, probably implements the csd-controlled switch of the developmental pathways (Hasselmann et al. 2008). For example, in the sawfly Athalia rosae (Linnaeus) sex is determined by the locus with 45–50 alleles (Fujiwara et al. 2004), in the bee Melipona interrupta fasciculata Smith it is determined by the locus with 20 alleles (Kerr 1987), and in the wasp Habrobracon juglandis (?= hebetor)1 it is determined by the locus with 9 alleles (Whiting 1943). The heterozygous organism is therefore a female, and the hemizygous (i.e. haploid) or homozygous one (i.e. usually resulting from inbreeding) is a male (Crozier 1977). The aforementioned model of the single-locus complementary sex determination (sl-CSD) has got certain experimental support (Cook 1993b, Butcher et al. 2000a,b, Tsuchida et al. 2002, Wu et al. 2003, Stahlhut and Cowan 2004, van Wilgenburg et al. 2006, Zhou et al. 2006). For example, strict inbreeding under laboratory conditions produced not only diploid, but also triploid males in the sawfly A. rosae ruficornis Jakovlev and the bumblebee Bombus terrestris Linnaeus (Naito and Suzuki 1991, Ayabe et al. 2004). However, the multilocus CSD (ml-CSD) model was also proposed for a few species (Naito et al. 1999, de Boer et al. 2007). Nevertheless, most species with high natural levels of inbreeding (many braconids, chalcids, bethylid wasps) do not fit in the CSD model (Schmieder 1938, Skinner and Werren 1980, Cook 1993a,b, Beukeboom et al. 2000, Wu et al. 2005, etc.). At least in some of the latter cases, the so-called genetic imprinting is likely to play the key role (Dobson and Tanouye 1998, Trent et al. 2006, but see also Beukeboom and Kamping 2006). Under these circumstances, female development is initiated by the presence of paternal chromosomes in the diploid set (Heimpel and de Boer 2008).

1.1.2 Chromosome Numbers and Ploidy Levels The haploid chromosome number (n) in different species of Hymenoptera can vary from 1 to 60, but all this diversity is created by karyotypes of the only family Formicidae (Imai et al. 1990, 2001, Mariano et al. 2008). Moreover, even in ants of the genus Myrmecia, the range of variation of chromosome numbers (n = 1–42) is pretty close to the previous one (Imai et al. 1988). Despite strong variation of these values in Hymenoptera, two modal chromosome numbers, n = 10 and 16, are clearly discernible in Symphyta and Aculeata (Fig. 1.1). If estimates are made using the “genus-karyotype” concept suggested by Crozier (1975) when every chromosome number is counted only once per genus to lower the effect of uneven study of various taxa, an analogous result, n = 8 and 16, is obtained (Fig. 1.2). Values close to the aforementioned ones prevail in the most speciose and karyotypically well-studied superfamilies, Tenthredinoidea, Formicoidea and Apoidea. According to previously published references (Crozier 1975, etc.), the distribution of parasitic Hymenoptera species by chromosome number is also bimodal, but n values of 5 and 10 are the most frequent in this group (see e.g. Gokhman 2003). 1

Unless otherwise stated, authors of species names of parasitic wasps are given in Appendix A.

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Fig. 1.1 Distribution of Symphyta and Aculeata by chromosome number at the species level (based on data from Gokhman 2003, Westendorff 2006 and other references compiled by the author)

An analysis of the distribution of chromosome numbers of Hymenoptera at lower taxonomic levels demonstrates that the characteristic studied is relatively stable within most genera (Crozier 1975). Nevertheless, differences in chromosome number are found between certain closely related species (Goodpasture 1975a,

Fig. 1.2 Distribution of Symphyta and Aculeata by chromosome number at the genus level (based on data from Gokhman 2003, Westendorff 2006 and other references compiled by the author)

1.1 Karyotype Structure of Hymenoptera

5

Goodpasture and Grissell 1975, Crozier 1977) as well as at the intrapopulation level (Imai et al. 1988, etc.). Data on ploidy levels in Hymenoptera are fairly ambiguous. Most researchers agree that gametes usually retain the same ploidy levels as unfertilised eggs, although gonads often contain certain numbers of polyploid spermatogonia and oogonia (Imai and Yosida 1966). Nerve cells are likely to have the same ploidy levels as gametes (Smith and Peacock 1957, Hauschteck-Jungen 1970). Other tissues include a considerable amount of polyploid cells, although chromosome numbers of cells from female tissues are substantially higher than those from the male ones (Sanderson 1932, Smith and Peacock 1957). On the other hand, earlier works stated that cell nuclei of corresponding tissues of the males and females were close to each other in their DNA content (Merriam and Ris 1954, Rasch et al. 1977), although the latter also depended on the levels of secretory activity of cells as well as (in social insects) on the caste (Mello and Takahashi 1971, Woyke and Krol-Paluch 1981). An analysis of these facts leads to the possibility of polyteny of hymenopteran chromosomes (especially in the males) and/or of amplification of certain segments of the nuclear DNA in ontogeny of these insects. A few direct observations and experimental works are likely to confirm these assumptions (Risler and Romer 1969, Kapralova 1985, Sanderson 1988), although at present this problem is far from full solution. Moreover, recent papers (Aron et al. 2003, 2005, Boivin and Candau 2007, Barcenas et al. 2008) demonstrate that an average ploidy level of female cells is substantially higher than that of the male ones.

1.1.3 Size of Mitotic Chromosomes Length of hymenopteran metaphase chromosomes can range from 0.5–1 to 15– 17 µm, but their average length is 3–5 µm. In the Hymenoptera, as in many other organisms, the number and size of chromosomes are inversely correlated. Specifically, length of the single chromosome of the ant species with n = 1 that belongs to the superspecies Myrmecia pilosula Smith2 is 17 µm. In contrast to that, the largest chromosome of Nothomyrmecia macrops Clark from the same family with n = 47 is 3–4 µm long whereas the smallest one is 1 µm long (Imai et al. 1988, 1990). Chromosomes of most Hymenoptera gradually decrease in size (Crozier 1975, Gokhman 2000a), but substantial size differentiation is characteristic of chromosomes of some species (Costa et al. 1992, 1993, Gomes et al. 1995).

1.1.4 Centromere Position and Centromere Index It is well known that chromosomes of Hymenoptera are monocentric (White 1973, Crozier 1975), i.e. every chromosome has a single centromere. The position of the centromere on the chromosome can be quantitatively characterised by the so-called

2

This species was later described as Myrmecia croslandi Taylor (Taylor 1991).

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centromere index (CI), which is a numerical ratio of the length of the shorter chromosome arm to the whole chromosome length (Macgregor and Varley 1988). All theoretically possible chromosome types can be found within karyotypes of Hymenoptera, namely, metacentrics (CI = 37.5–50%), submetacentrics (CI = 25– 37.5%), subtelocentrics (CI = 12.5–25%) and acrocentrics (CI = 0–12.5%; Levan et al. 1964, Imai et al. 1977). This is also true for many large families of the Hymenoptera. For example, metacentrics predominate in chromosome sets of certain ants, but chromosomes of the reverse type, i.e. acrocentrics, are more numerous in karyotypes of other Formicidae (Imai et al. 1988). In this section, it is also appropriate to consider the problem of symmetry of chromosome sets of Hymenoptera. This term was introduced by Stebbins (1950). According to his definition, chromosomes of the symmetrical karyotype “are essentially similar to each other in size and with median or submedian centromeres” (i.e. they are either meta- or submetacentrics). On the contrary, asymmetrical karyotypes “possess many chromosomes with subterminal centromeres, or great differences in size between the largest and the smallest chromosomes, or both”. Although chromosome sets of Hymenoptera are fairly diverse in their structure, symmetrical karyotypes generally predominate in the group (this is especially true for chromosome size). Nevertheless, asymmetrical chromosome sets are characteristic of a relatively small minority of Hymenoptera. For example, a very asymmetrical karyotype is found in the ant species belonging to the Myrmecia fulvipes Roger complex with 2n = 12. This chromosome set contains two pairs of very large acrocentrics and four pairs of relatively small submetacentrics (Imai et al. 1977).

1.1.5 Heterochromatic and Euchromatic Chromosome Segments: C-Banding Chromosomes of living organisms contain regions of two types: those of the first type are condensed during the whole cell cycle, whereas the other regions become decondensed in the end of mitosis and are weakly stained in interphase nuclei. These segments are, respectively, called heterochromatic and euchromatic (Bostock and Sumner 1978). Moreover, heterochromatin can be further subdivided into two classes: structural (constitutive) and facultative (Prokofyeva-Belgovskaya 1986, Macgregor and Varley 1988). Regions belonging to the first class are constantly visualised on chromosomes of various cell types, those of the second class are seen only in the few types (Bostock and Sumner 1978, Smirnov 1991). Segments of the constitutive heterochromatin can be either pericentromeric, telomeric (i.e. situated at the ends of chromosome arms) or intercalary (i.e. situated between the centromere and telomeres; White 1973, Smirnov 1991, Zhimulev 2003). Pericentromeric and telomeric heterochromatin are the most common on chromosomes of Hymenoptera (Imai 1991, Hoshiba and Imai 1993). For chromosomes with the fully heterochromatic shorter arm and the euchromatic longer one, the term “pseudoacrocentric” has been proposed (Hoshiba and Imai 1993). In some cases, chromosomes more or less entirely consist of heterochromatin (e.g. B chromosomes of many insects; White 1973, Gokhman and Kuznetsova 2006).

1.1 Karyotype Structure of Hymenoptera

7

Pericentromeric and some other segments of the constitutive heterochromatin are usually visualised by the so-called C-banding. This banding is supposed to result from DNA denaturation in less-spiralised chromosome regions under the action of alkaline solutions (Macgregor and Varley 1988); however, it is also suggested that C-banding reveals heterochromatin-specific proteins (Zhimulev 2003). Although the degree of condensation of aforementioned regions and their ability to form C-segments are not strictly correlated, there is a common belief that this banding exclusively visualises heterochromatic regions. Moreover, Hymenoptera and other insects often demonstrate the so-called spontaneous C-banding that appears in the process of making chromosomal preparations without special pretreatment (Imai et al. 1977, 1984a, etc.). The nature of this banding also remains obscure, although the results of the “spontaneous” and regular banding usually coincide. For this reason, no distinction is made between these types of C-banding in the present work. The number of papers on C-banding of hymenopteran chromosomes substantially increased during the last years. Aculeate Hymenoptera (Hoshiba 1984a,b, Palomeque et al. 1987, 1988, 1990c, 1993a,b, Pompolo and Takahashi 1990, Costa et al. 1993, 2004, Hoshiba and Imai 1993, Gomes et al. 1995, 1998, Pompolo and Campos 1995, Rocha and Pompolo 1998, Ara´ujo et al. 2002, Rocha et al. 2002, 2003, Brito et al. 2003, 2005, Scher and Pompolo 2003, Domingues et al. 2005, Lopes et al. 2008) and certain sawflies (Rousselet et al. 1998, Kuznetsova et al. 2001) are the most abundant groups of the order studied in this respect.

1.1.6 Nucleolus Organiser: AgNOR-Banding Nucleolus organiser regions of metaphase chromosomes can often be discerned without any pretreatment, just by the presence of secondary constrictions (Bostock and Sumner 1978). However, these regions are more reliably visualised using the so-called AgNOR-banding (see Macgregor and Varley 1988) that includes treatment of preparations with an argentic salt and further reduction of the latter to the metallic state (Goodpasture and Bloom 1975, Howell and Black 1980). The number of works on silver staining of Hymenoptera chromosomes rapidly increases during the last years. Specifically, karyotypes of a few species of the families Tenthredinidae, Formicidae and Apidae were studied using this technique (Palomeque et al. 1987, 1988, 1990a,c, 1993a, Imai and Taylor 1989, Imai et al. 1992, Lorite et al. 1997, Kuznetsova et al. 2001, Maffei et al. 2001). A single pair of active nucleolus organisers is usually revealed on chromosomes of various hymenopteran species (Palomeque et al. 1990b, etc.).

1.1.7 Other Techniques of Differential Chromosome Segmentation The G-banding method that involves either treatment of chromosomal preparations with proteolytic enzymes or incubation in solutions of various electrolytes is widely used in karyotypic studies of higher vertebrates (Macgregor and Varley 1988). However, application of this technique to the chromosomes of Hymenoptera

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deserves special comments. A view predominated for a long time that G-banding did not reveal well-discernible regions on insect chromosomes (Bailly et al. 1973, Fraccaro et al. 1976). Along with that, a number of papers describing results of G-banding on chromosomes of Orthoptera (Webb and Westerman 1978, Camacho et al. 1991) and Homoptera (Lauritzen 1982, Manicardi et al. 1991) were published from the end of the 1970s. Up to now, studies on G-banding of Hymenoptera karyotypes were performed on a few species belonging to the families Vespidae, Formicidae and Apidae (Tambasco et al. 1979, Hoshiba 1984a, 1985a,b, Hoshiba and Okada 1986, Lorite et al. 1996, Gomes et al. 1998, Stanimirovic et al. 2005). A number of G-positive segments often associated with heterochromatic regions are usually revealed in karyotypes of these insects. Judging from images published in these papers, this banding sometimes facilitates search of paired chromosomes within given karyotypes. However, if chromosome sets of different species are compared, their G-banding patterns differ to the extent that substantially hampers the search of homologous chromosomes. This situation is probably explained by certain structural features of chromosomes of Hymenoptera and other insect orders (Rodionov 1999). Differential chromosome staining can also be obtained with chemicals binding with specific regions of DNA (for example, with those enriched with GC- and AT-bases). These chemicals include quinacrine mustard that produces the so-called Q-banding, as well as 4 ,6-diamidino-2-phenylindole (DAPI), distamycin A (DA) and chromomycin A3 (CMA3 ) (Schmid 1980, Schweizer 1980). Studies of that kind were conducted on certain species of the families Tenthredinidae, Formicidae, Sphecidae and Apidae (Lorite et al. 1996, 1997, Brito-Ribon et al. 1999, Ara´ujo et al. 2000, Kuznetsova et al. 2001, Rocha et al. 2002, 2003, Brito et al. 2003, 2005, Costa et al. 2004, Domingues et al. 2005, The Honeybee genome sequencing consortium 2006, Lopes et al. 2008, Mariano et al. 2008). The so-called in situ hybridisation is a very specific method of chromosomal analysis that includes treatment of chromosomal preparations with DNA fragments marked with radioactive isotopes or other chemicals. Technique of that kind involving use of fluorescent markers is called “fluorescence in situ hybridisation” or FISH (Lemieux et al. 1992, Larin et al. 1994, Speicher and Carter 2005). Using in situ hybridisation, karyotypes of certain sawflies of the families Diprionidae and Tenthredinidae (Rousselet et al. 1998, 1999a,b, 2000, Matsumoto et al. 2002, Sumitani et al. 2005) as well as those of Formicidae (Hirai et al. 1994, Meyne et al. 1995, Lorite et al. 1997, 2002a, de Menten et al. 2003, Mariano et al. 2008), Sphecidae (Ara´ujo et al. 2002) and Apidae (Beye and Moritz 1995, Brito et al. 2005) were studied. Further development of the FISH technique has led to the discovery of the socalled multicolour FISH or chromosome painting (Rubtsov and Karamysheva 1999, Langer et al. 2004) in which fluorochrome-marked DNA sequences characteristic of chromosomes or even of their certain regions are used. After this treatment, every chromosome (or its region) acquires specific colour and therefore can be easily recognised. The only species of the Hymenoptera currently studied with this method is the parasitic wasp Nasonia vitripennis (R¨utten et al. 2004; see below).

1.1 Karyotype Structure of Hymenoptera

9

Important information on nucleotide composition and structure of certain chromosomal regions can be obtained from treatment of chromosomal preparations with specific endonucleases that restrict very specific DNA sequences. Up to date, works of that kind are performed only on the ant Tapinoma nigerrimum (Nylander) (Lorite and Palomeque 1998, Lorite et al. 1999) and the bee Melipona mandacaia Smith (Rocha et al. 2003). In particular, results of these experiments confirm not only that DNA of those species (as well as in the majority of eukaryotes) is enriched with GC-base pairs, but it also includes TTAA-sequences (Lorite and Palomeque 1998). It is interesting to note that treatment of chromosomes with the enzyme-restricting TTAA-sequences has led to the pattern analogous to C-banding, whereas action of most other endonucleases resulted in segmentation similar to G-banding (Lorite et al. 1999, Rocha et al. 2003).

1.1.8 Details of Meiosis and Structure of Meiotic Chromosomes Spermatocytes, i.e. precursors of male gametes, develop in testes as groups of cells resulting from divisions of the only spermatogonium and interconnected by cytoplasmic bridges within the so-called cyst (Meves 1907, Roosen-Runge 1977). The meiosis is abortive in hymenopteran males (Crozier 1975); this virtually means deletion of the reductional division. Sometimes (e.g. in some ants) this division is fully reduced (Palomeque et al. 1990a). Karyokinesis is also absent in other cases, although a small cytoplasmic bud (containing no nuclear material) can pinch out of the cytoplasm of the dividing spermatocyte (Smith and Peacock 1957, Sharma et al. 1961); however, this bud was not observed in some other studies (Sanderson 1932, Mackay 1955, Smith 1941, Kumbkarni 1965). Moreover, ambiguous data on the first meiotic division in males of the same species can coexist (Sanderson and Hall 1948, Wolf 1960, Hoage and Kessel 1968). The two latter authors have also observed “budding” of centrosomes out of the cell together with a small amount of cytoplasm. Homologous chromosomes of diploid males do not conjugate in meiosis (Woyke and Skowronek 1969, Yamauchi et al. 2001), and therefore there is no reduction of the diploid set. The equational division in most Hymenoptera usually has no specific features, and every spermatocyte forms two spermatids. In bees, however, the spermatocyte gives rise to a spermatid and a “nuclear bud” that degenerates later (Wolf 1960, Sharma et al. 1961, Hoage and Kessel 1968). In turn, spermatozoa arise from spermatids through the process called spermiogenesis or spermateleosis (Crozier 1975, Roosen-Runge 1977). Some details of chromosome behaviour during the first meiotic division remain unclear. In most species, stages analogous to the normal prophase and metaphase can be observed (Dodd’s 1938, Smith and Peacock 1957, Crozier 1975). Certain papers (Sanderson 1932, Mackay 1955, Sharma et al. 1961, Kumbkarni 1965) indicate that at the later stage chromosomes clump into a compact mass (this is probably an artefact arising from inadequate fixation). However, other references (Sanderson and Hall 1948, Smith and Peacock 1957) demonstrate that the chromosomes retain their individual shape.

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Development of female gametes (oocytes) takes place in ovarioles of the polytrophic type (Crozier 1975, Aizenshtadt 1984, Ivanova-Kazas 1995). Oogonia, i.e. oocyte precursors, develop in the germarium, the narrowest apical section of the ovariole. The dividing oogonium gives rise to the oocyte and nurse cells interconnected to the former by cytoplasmic bridges (Hogben 1920, Sanderson 1932, Smith 1941). Each cell group of that kind is surrounded by its own epithelium and forms a follicle (Hegner 1915, Mackay 1955, Raven 1961, Aizenshtadt 1984). During its development, the oocyte comes through the so-called growth phase in which an intensive synthesis of nutritive compounds takes place that is necessary for the subsequent embryonic development. At this stage, the first (reductional) division of the oocyte occurs; initial phases of this division usually take place in the nurse cells as well. It is supposed that chromosomes of Hymenoptera are weakly stained (or absolutely invisible) at the growth phase (Hogben 1920, Sanderson 1932, Smith 1941, Mackay 1955). However, since the prophase of the first meiotic division occurs at this particular stage, chromosome observation at the growth phase can provide information on the number and distribution of chiasmata. The prophase of the first meiotic division is subdivided into several stages: leptotene (sometimes preleptotene is also distinguished), zygotene, pachytene, diplotene and diakinesis (Darlington 1965, Kiknadze and Vysotskaya 1975). Chromosomes lack definite orientation at the leptotene, but form the so-called bouquet in the zygotene with their telomere ends assembled at a nuclear pole. During pachytene, chromosome conjugation ends, although the structure of bivalents as well as localisation of chiasmata becomes most clearly visible at the diplotene (Smith 1941, Smith and Peacock 1957). Condensation of the bivalents becomes stronger at the diakinesis followed by the metaphase. At this stage, the first maturation division usually ceases and resumes only after egg-laying (Crozier 1975). In the laid egg both maturation divisions proceed very rapidly, and this process usually takes half an hour. Since in the Hymenoptera, as well as in other insects, all products of maturation divisions are retained in the developing egg, fusion of the male and female pronuclei is unnecessary for forming the diploid zygote, e.g. by thelytoky that can be determined by a single locus (Lattorff et al. 2005). According to the classification provided by Suomalainen et al. (1987), there are two main types of thelytoky, i.e. ameiotic (apomixis) and meiotic (automixis). The latter type can be further subdivided into premeiotic doubling, central fusion (of the products of maturation divisions), terminal fusion and gamete duplication. Earlier known types and instances of thelytoky in Hymenoptera have been reviewed by Crozier (1975); however, a number of them were reassessed later (Stille and D¨avring 1980, Beukeboom et al. 2000). It is well known now that thelytoky in this order is often caused by symbiotic microorganisms mostly belonging to the genus Wolbachia (Stouthamer et al. 1993). The structure of meiotic chromosomes was studied in only a few hymenopteran species (Hogben 1920, Sanderson 1932, Smith 1941, Mackay 1955, Smith and Peacock 1957, etc.). Moreover, most of these papers contain only a general

1.2 Diversity of Chromosome Sets of Hymenoptera

11

description of meiosis without citing numbers of chiasmata and other important information.

1.2 Diversity of Chromosome Sets of Hymenoptera 1.2.1 Karyotypic Features of Various Taxonomic Groups Suborder Symphyta. Chromosome sets of more than 360 species of sawflies and horntails from the superfamilies Xyeloidea, Tenthredinoidea, Pamphilioidea, Siricoidea and Cephoidea have been studied up to now (see Westendorff 2006 for review). The haploid chromosome number in the whole suborder ranges from 5 to 35, and this diversity is created rather by karyotypic differences between genera and species, than by those between families (Gokhman 2003). Symmetrical chromosome sets predominate in this group. Superfamily Xyeloidea. Family Xyelidae. Chromosomes of the only species, Xyela julii (Br´ebisson) with n = 25, were recently examined (Gokhman unpublished data). Superfamily Tenthredinoidea. Family Argidae. Nine species of the genus Arge with n = 8–13 are studied (Maxwell 1958, Westendorff and Taeger 2002, etc.). Family Cimbicidae. Four species having n = 8–16 have been examined (Benson 1950, Westendorff and Taeger 2002, Westendorff 2006). Family Diprionidae. Chromosomes of about 40 species with n = 6–15 have been studied (Smith 1941, Maxwell 1958, Rousselet et al. 1999b, etc.). Family Pergidae. Six species belonging to the genera Perga, Philomastix and Pterygophorus have been examined (Maxwell 1958); all have n = 8. Family Tenthredinidae. This group is the best karyotypically studied among Symphyta. Chromosome numbers (n = 5–22) and other karyotypic features are known now for about 290 species of Tenthredinidae (Naito 1982, Westendorff et al. 1999, etc.). The most frequent chromosome number in this family is n = 10. Chromosome sets of the genus Tenthredo with n = 8–21 are the most diverse (Sanderson 1970, Naito 1982, etc.). Superfamily Pamphilioidea. Family Pamphiliidae. About 12 species of the genera Acantholyda and Cephalcia having n = 11–35 have been examined (Battisti et al. 1998, Boato and Battisti 2001). Family Megalodontidae. The only studied species, Megalodontes thor Taeger, has n = 20 (Westendorff 2006). Superfamily Siricoidea. Family Siricidae. Five species of the genera Sirex and Urocerus with n = 8–18 have been examined (Sanderson 1970). Superfamily Cephoidea. Family Cephidae. Judging from chromosome numbers, this group is apparently very variable with respect to karyotypic features. Despite only three species of the family having been examined up to now, the range of variation of chromosome numbers in this group (n = 9–26) is fairly close to that characteristic of the suborder Symphyta as a whole (Mackay 1955, Crozier and Taschenberg 1972, Westendorff and Taeger 2002).

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Suborder Apocrita. Chromosome numbers and other karyotypic features are known for more than 400 species of parasitic Hymenoptera (see Appendix A). Haploid chromosome numbers in this group can range from 3 to 23 (see Chapter 3). Distribution of species by this character at the species level is bimodal with two distinctive peaks at n = 6 and 11 (Gokhman 2003, 2004d). Aculeate Hymenoptera is the best studied group of the order. Chromosomes of about 700 species of Aculeata belonging to the superfamilies Scolioidea, Pompiloidea, Vespoidea, Formicoidea, Sphecoidea and Apoidea have been examined (Goodpasture 1974, Imai et al. 1988, Hoshiba and Imai 1993, etc.). Superfamily Scolioidea. The only studied species of the family Sapygidae, Sapyga pumila Cresson, has n = 25–26 (Goodpasture 1974). Superfamily Pompiloidea. Two species of the family Pompilidae have been examined; both have n = 15 (Hoshiba and Imai 1993). Superfamily Vespoidea. Nine species of the family Eumenidae (often considered as a subfamily of Vespidae) with n = 5–12 as well as about 40 species of the family Vespidae with n = 7–34 have been studied. The latter family is highly diverse in terms of chromosome numbers and other karyotypic features. For example, various species of the genus Polistes have chromosome numbers ranging from 9 (or 6 according to other data) to 33 (Machida 1934, Misra and Srivastava 1971, Hung et al. 1981). Superfamily Formicoidea, which includes the only family Formicidae, is extremely polymorphic with respect to many details of karyotype structure. Specifically, the range of variation of chromosome number in the family determines this characteristic not only for the Aculeata, but for all Hymenoptera as well (see Section 1.1.2), due to the large number of studied ant species (more than 450, i.e. approximately 2/3 of all aculeate Hymenoptera). It is worth noting that modal chromosome numbers of the Formicidae, n = 10 at the species level and n = 11 at the genus one, are also close or identical to those modal for the Hymenoptera as a whole (Figs. 1.1 and 1.2). This is also true for many other details of chromosomal morphology of ants (Imai et al. 1988, 1990). For instance, low n values in the Formicidae are usually associated with the prevalence of biarmed chromosomes, and high values are associated with that of acrocentrics, although intermediate variations are also present in this group (Imai et al. 1988). Superfamily Sphecoidea. Karyotypes of more than 20 species of the family Sphecidae with n = 3–24 have been studied up to now (Costa et al. 1993, Hoshiba and Imai 1993). The highest range of variation (n = 8–21) is revealed within the genus Psenulus (Hoshiba and Imai 1993). Superfamily Apoidea. In the family Apidae, which is the best studied in this group (more than 100 species), n = 8–26 is found. In addition, the chromosomes of three species of Andrenidae (n = 3–10), about 20 species of Anthophoridae (n = 8–21), the same number of Megachilidae (n = 15–17), 9 species of Colletidae (n = 8–28) and 13 species of Halictidae (n = 8–21) have been studied. The modal number for the superfamily is n = 16 (Hoshiba and Imai 1993). As in the Symphyta, variation in chromosome numbers of Apoidea is apparently created rather by karyotypic differences at the genus and species levels than by those between families.

1.2 Diversity of Chromosome Sets of Hymenoptera

13

1.2.2 Types of Chromosomal Rearrangements All karyotypic diversity of the Hymenoptera, as well as that of other organisms, is created by a few types of chromosomal mutations. These changes can be subdivided into two main groups, i.e. rearrangements changing structure of chromosomes and those changing their number (Swanson et al. 1981, Ayala and Kiger 1984). Changes in chromosome structure. Independent deletions, i.e. losses of chromosomal segments, are uncommon in the Hymenoptera (Imai et al. 1977, 1988). In other words, the emerging fragment usually joins the other chromosome, i.e. a translocation occurs (see below). Even an irreversible loss of a small part of the genome is likely to lower the viability of Hymenoptera, especially after the transition to the homozygous or hemizygous (haploid) state. Some indicative deletions found in Hymenoptera usually affect heterochromatic segments that are supposed to contain a few active genes (Imai et al. 1977, 1988). In contrast with that, duplications are relatively frequent in this group (Hoshiba and Imai 1993). A clear example of those rearrangements in Hymenoptera occurring as the so-called tandem growth of the constitutive heterochromatin is found in the two ant species belonging to the genus Myrmecia (Imai et al. 1977). Acrocentrics predominate in Myrmecia pyriformis Smith with n = 41, as in other Myrmecia species with higher chromosome numbers, whereas in M. brevinoda (Forel) with n = 42 all chromosomes are pseudoacrocentric. These chromosomes are formed through the tandem growth of heterochromatic arms. Despite the fact that “inversions are the most common structural aberrations found in natural populations of higher organisms” (Swanson et al. 1981), routine chromosome staining reveals only pericentric inversions that affect the centromere region and change position of the centromere on the chromosome. Moreover, even this type of inversion can be identified with confidence either in a heterozygous condition or if the chromosome set in question is compared to karyotypes of closely related forms. Nevertheless, those rearrangements were found at least in 17 ant species (Imai et al. 1988). In a member of the superspecies M. pilosula with 2n = 9–10, for example, chromosomes of the first pair are of equal length, and at the same time either they can both be subtelocentric or one of them is a metacentric that resulted from inversion (Imai et al. 1977). Certain data show that the centromere position can change without inversion through the so-called shift of centromeric activity (Hoshiba and Imai 1993). For example, a shift of that kind is revealed in Myrmecia croslandi with 2n = 2–4 (Imai and Taylor 1989). The mechanism of this process is uncertain; however, it can be interpreted as an inactivation of the existing centromere with a simultaneous emergence (more precisely, reactivation) of the other one. In particular, two latter phenomena are found in tandem rearrangements and certain translocations in ants (Imai et al. 1988, Imai and Taylor 1989; see also Zhimulev 2003). Translocations are usually believed to lead to the nondisjunction of meiotic chromosomes and to the emergence of partial monosomics, trisomics, tetrasomics, etc. that have substantially low fertility. For this reason, translocation polymorphism in animal populations is considered pretty rare (White 1973). With this information,

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data on relatively frequent rearrangements of that kind in the karyotypes of Formicidae in which at least ten cases of translocation polymorphism are found seem quite unexpected (Imai et al. 1977, 1984a, 1988). These data lead to the conclusion that aneuploids resulting from nondisjunction of meiotic chromosomes are likely to be viable and even fertile at least in ants and probably also in other Hymenoptera (Imai et al. 1984a). Translocations can be subdivided into the single and reciprocal ones. Those of the first type (i.e. insertions or transpositions) represent a transfer of a chromosomal segment to the same or other chromosome; those of the second type represent a reciprocal exchange of segments between chromosomes (De Robertis et al. 1975, Ayala and Kiger 1984). Translocations can occur between either homologous or nonhomologous chromosomes. Transpositions affecting homologous chromosomes are likely to represent the most significant mechanism of the origin of duplications. In particular, it is highly possible that this process has led to the duplication in the longer arm of the metacentric in Monomorium rothsteini Forel (Formicidae). Chromosomes of this pair are present in populations in all possible versions, i.e. two homozygous states and a heterozygous state (Imai et al. 1977). As shown above, translocations often result in the meiotic nondisjunction of chromosomes and origin of aneuploids. For example, that was the mode of origin of five different chromosomal forms with 2n = 21 and 22 in the ant Monomorium indicum Forel (Imai et al. 1984a). Changes in chromosome number. Chromosomal fusions and fissions are the most important mechanisms of karyotypic change affecting the chromosome number (White 1973). The best known among them are centric, or Robertsonian, fissions and fusions that do not change the arm number (Swanson et al. 1981, Ayala and Kiger 1984). In particular, rearrangements of that kind are found in 22 species of Formicidae (Imai et al. 1988). Robertsonian fusions and fissions are best detected in the heterozygous state as well as many other karyotypic changes. Perhaps the most impressive case of the polymorphism on centric fusions is revealed in the Australian ant Rhytidoponera metallica (Smith) or, more precisely, in its “Western form”. The diploid chromosome number ranges in various individuals of this species from 34 to 46, but the arm number is always 46 (Imai et al. 1977, 1988, Crozier et al. 1986). A similar range of variation of the chromosome number is found in the other member of the genus, Rhytidoponera maniae (Forel). Here 2n values can vary from 39 to 48 with the constant NF = 50 (Imai et al. 1977). The so-called inversion–Robertsonian polymorphism, a combination of those rearrangements, is detected in certain ants. For example, a metacentric of the diploid set of an Indian Camponotus species has undergone a centric fission. In turn, one of the two resulting acrocentric chromosomes turned into the new metacentric by inversion. The described rearrangements have disrupted the normal chromosome disjunction in meiosis that resulted in the emergence of three different karyotypes with 2n = 34 and the fourth one with 2n = 35 (Imai et al. 1984a). Along with centric fusions, an increased attention of researchers is recently attracted to the so-called tandem rearrangements (White 1973, Crozier 1975, Imai and Taylor 1989). In these cases, centromere–telomere or telomere–telomere fusions

1.2 Diversity of Chromosome Sets of Hymenoptera

15

result in the origin of the larger chromosome with one of its two centromeres inactivated. Although the assumption on the presence of tandem fusions can arise from the comparison of characteristics of chromosome sets (i.e. the diploid chromosome number must decrease by two units, and the arm number – by two or four units, respectively), these rearrangements are difficult to recognise using routine chromosome staining. Nevertheless, putative tandem fusions are recorded at least in three groups of Formicidae, i.e. in Linepithema humile (Mayr) (2n = 18, NF = 32) and L. piliferum (Mayr) (2n = 16, NF = 30; Crozier 1975), in M. croslandi with 2n = 2–4, NF = 4–8 (Imai and Taylor, 1989) and Ponera scabra (Wheeler) with n = 3–4, NFn = 6–8 (Imai et al. 1988) and probably also in two other species of the M. pilosula complex (Imai et al. 1988). Polyploidy. In the 1930s, when only several dozen species of Hymenoptera were studied karyologically, an assumption emerged that the majority of species of the order were polyploid (Reed 1934, Greenshields 1936). This hypothesis was based on the fact that many chromosome numbers known at that time were divisible by four (Sanderson 1932), and chromosomes of males of certain Symphyta could be more or less successfully arranged in pairs. Analogous views based on the comparison of chromosome numbers of closely related species of sawflies and bees were later expressed, e.g. by Smith (1941) and Kerr and Silveira (1972). The arguments by Reed and Greenshields, however, were criticised soon after their publication (Speicher 1936), and the subsequent accumulation and refinement of data on the karyotype structure of the Hymenoptera have allowed full rejection of the assumption of the important role of polyploidy in the evolution of Hymenoptera (Crozier 1975). Nevertheless, Naito and Inomata (2006) recently demonstrated that the sawfly Pachyprotasis youngiae Inomata & Naito (Tenthredinidae) is a fully thelytokous species with triploid females. However, a few decades ago Naito (1982) also claimed that two other species belonging to the same family, Dolerus lewisii Cameron and Loderus eversmanni obscurus (Marlatt), were polyploid. This assumption was based on chromosomal analysis of embryos in which development had been artificially initiated. It is quite possible therefore that unfertilised eggs could start to develop in the thelytokous mode under the conditions described and thus could give rise to females, not males. It must be also taken into account, however, that many somatic cells and tissues of various hymenopterans become secondarily polyploid (see Section 1.1.2). Finally, truly polyploid individuals can also occur sporadically in haplodiploid species of the Hymenoptera. For example, specimens of that kind are described in Australian ants of the genera Camponotus and Crematogaster (Imai et al. 1977). Aneuploidy. Changes in chromosome number by aneuploidy are substantially facilitated in Hymenoptera by the relatively high frequency of translocation and inversion–Robertsonian polymorphisms with the characteristic irregular meiotic disjunction of chromosomes in the group (see above). For example, at least five cases of that kind have been recorded in the Formicidae up to now (Imai et al. 1988). Moreover, a few karyotypes with the “pure” trisomy/monosomy (i.e. those not directly originated through the translocation polymorphism) are also found in the ant Pachycondyla astuta Smith and in the M. pilosula complex (Imai et al. 1988).

16

1 Chromosomes of Hymenoptera

The frequency of aneuploidy in the Hymenoptera is therefore substantially higher than that existing in many other animals (White 1973). This is probably due to the fact that individuals of this order with unbalanced karyotypes are fertile enough (Imai et al. 1984a). In turn, it is highly likely that this genomic tolerance can be explained by the sex determination system of those insects that involves male haploidy (Imai et al. 1988). According to the recent data, for example, action of many mutations that stop meiosis in the males and therefore usually become eliminated by natural selection is expressed in survival of gametes carrying aberrant chromosome sets in females (Hunt and Hassold 2002). If it is true, higher frequency of aneuploids in the Hymenoptera can be explained, among other factors, by the lack of the aforementioned checkpoints. Finally, another mechanism that increases variability of chromosome numbers in the Hymenoptera is the variation in B chromosomes whose presence in the karyotype is usually considered facultative (Jones and Rees 1982, Camacho et al. 2000, Zhimulev 2003). Chromosomes of that kind are found in the ants Leptothorax spinosior (Forel) (up to 12 B chromosomes per haploid set), L. muscorum (Nylander), Lasius niger (Linnaeus), Podomyrma adelaidae (Smith), Prenolepis jerdoni Emery, some members of the genera Crematogaster, Pachycondyla and Pseudolasius and probably also in Dolichoderus bituberculatus Mayr, Lasius brunneus (Latreille), Odontomachus rixosus Smith and in a number of other Leptothorax species (Imai 1974, Imai et al. 1977, 1984b, 1985, 1988, Go˜ni et al. 1982, Loiselle et al. 1990, Palomeque et al. 1990c, Lorite et al. 2002b). Chromosome numbers of an Indian species of Tetramorium as well as of a species of the M. pilosula complex can vary between different cells of the same individual due to the presence/absence of small mitotically unstable chromosomes (Imai et al. 1977, 1984a). In addition, B chromosomes are detected in Trypoxylon albitarse Fabricius (Sphecidae) and in the bees Melipona rufiventris Lepeletier, Partamona cupira (Smith) and P. helleri (Friese) from the family Apidae (Costa et al. 1992, Brito et al. 1997, Ara´ujo et al. 2000, Lopes et al. 2008). The same type of chromosomal polymorphism is probably characteristic of the sawfly Tenthredo brevicornis (Konow) with 2n = 18–21 (Sanderson 1970). Since trends of the karyotypic change in the Hymenoptera at the macroevolutionary level can be detected with a sufficient degree of confidence only after the phylogenetic analysis of chromosomal variation in those insects, these trends will be discussed later (see Section 1.3.2.4). As far as microevolutionary changes of chromosome sets are concerned, they can be best judged from the types of population polymorphism known for this group, specifically, polymorphisms involving localisation and size of heterochromatic segments, inversions, translocations, Robertsonian rearrangements (including inversion–Robertsonian polymorphisms) and the number of B chromosomes. Karyotypic diversity substantially increases through the origin of aneuploids that can appear in populations with inversion– Robertsonian and translocation polymorphisms (see above). Perhaps, it is also worth noting that proportions of different chromosomal forms in various populations can vary geographically. Specifically, polymorphism involving heterochromatic segments in the ant Solenopsis geminata (Fabricius) is detected only in eastern India, but populations of this species are monomorphic in the west and south of the

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17

country. The Robertsonian polymorphism in Camponotus crassisquamis Forel is also detected in eastern India, whereas all chromosomes are acrocentric in northern populations of this species (Imai et al. 1984a). Another case of that kind is found in the ant Doronomyrmex kutteri (Buschinger), where populations from Germany, on one hand, and other European countries, on the other hand, are reported to differ in two Robertsonian fissions (Buschinger and Fischer 1991). Those differences may at least partly be explained by adaptation of certain populations to the local environmental conditions, although at present these assumptions are not supported by any convincing data.

1.3 Systematic and Phylogenetic Implications of Chromosomal Characters in Hymenoptera 1.3.1 Taxonomic Significance of Karyotypic Features Any individual mitotic chromosome can be characterised by the following structural features (Gokhman and Kuznetsova 2006): morphological type (monocentric, i.e. carrying a single centromere in the case of Hymenoptera); absolute and relative (in percentage of the total length of all chromosomes in the haploid set) length of chromosomes; presence of satellites or achromatic gaps, which in most cases are nucleolus organisers; centromere index. The features of the karyotypes are chromosome number; chromosomal mechanism of sex determination (see Section 1.1.1); number and morphology of B chromosomes; arm number. Using these data, certain integrative values, e.g. the degree of karyotypic symmetry, can be calculated. As far as meiotic chromosomes are concerned, most of their characteristics correspond to those of the mitotic ones (for example, the number of bivalents usually corresponds to the haploid chromosome number). However, some additional features can be revealed during meiosis, including presence and position of chiasmata. Various methods of differential staining provide information on the size and localisation of chromosomal regions with the specific content and structure (Vorontsov 1958, Gokhman 1997a, Pimenov 2001, Gokhman and Kuznetsova 2006). The number of the main classes of chromosomal characters is therefore fairly limited (Gokhman 2006a). Despite this fact, karyotypic characters are widely used in the taxonomy of insects, especially of those belonging to Diptera and Orthoptera (White 1973, Chubareva and Petrova 1979, Hewitt 1979). This is because structural features of the karyotype which are used as taxonomic characters have several important advantages (Gokhman 1997a, 2006a). First of all, many of these characteristics (chromosome number, etc.) can have only certain values, i.e. they change discretely. Hybrids that result from crossings between populations with different chromosome numbers are therefore easily recognisable by their karyotype structure (White 1973, Fedorova et al. 1991). This also means that chromosomal characters allow detection of isolated populations and morphs which can be hardly recognised by their morphological features. In particular, “similarity in the chromosome number is not a sufficient argument by itself for the unification of species with the

18

1 Chromosomes of Hymenoptera

similar 2n under the same group, although differences [i.e. hiatus] in the diploid number of closely related forms testify in favour of their independence at the species level” (Vorontsov 1958). However, chromosomal characters can be both differential and integrative (in the latter case they unite members of a particular taxon; Vorontsov 1980). The integrative role of karyotypic features is explained by their preferential similarity at the certain taxonomic level. In particular, this is the source of the so-called genus-karyotype concept in the Hymenoptera (Crozier 1975). According to this concept, identical chromosome numbers within the same genus are considered as results of the single evolutionary event (see Section 1.1.2). Implications of chromosomal characters for Hymenoptera systematics at various taxonomic levels. The examples listed below mostly illustrate the appropriate use of chromosome numbers of Hymenoptera as the best studied characteristic of chromosome sets of those insects. Superfamilies and families. The data accumulated show that the opportunities of use of chromosomal characters at this level are rather limited. As it was mentioned in Section 1.2, karyotypic diversity of many groups of Hymenoptera is mainly created by differences between karyotypes of lower level taxa (genera and species). The qualitatively different situation is observed only in parasitic Hymenoptera (see Section 1.2 and Chapter 6). Genera and groups of genera. At this level, karyotypic features of Hymenoptera usually provide information that confirms the existing taxonomic status. Specifically, both karyotypically studied genera of the family Siricidae, Sirex with n = 8 and Urocerus with n = 13–18, differ in chromosome number (Sanderson 1970). Differences in this character are also revealed between morphologically similar genera of Apidae, Bombus (n = 16–23) and Psithyrus3 (n = 25–26; Owen 1983, Hoshiba and Imai 1993, Owen et al. 1995). Species and species groups. Data on chromosomal morphology of Hymenoptera can be used for clarifying taxonomic status and species grouping, as well as for searching and recognising sibling species (see Crozier 1975, 1977, Gokhman and Quicke 1995, Gokhman 1997a, 2000a, 2007b for reviews). For example, two species groups of the genus Athalia (Tenthredinidae) differ from each other not only in their morphology and foodplants, but also in chromosome number (n = 6 and 8). Another two species groups with similar karyotypes (n = 8 and 10) are distinguished within the genus Macrophya that belongs to the same family (Westendorff et al. 1999). Bumblebees of the subgenus Subterraneobombus have n = 16, whereas other Bombus spp. have n = 17–23 (Hoshiba and Imai 1993, Owen et al. 1995). Details of karyotype structure are very important for detecting sibling species, because species of that kind virtually have no other distinct features (Gokhman 1997a). In the present work, however, the term “sibling species” is considered in a broader sense and it embraces not only closely related species that are difficult to distinguish by their external morphology, but also erroneously identified “good” species (Table 1.1). This is because these two categories usually cannot be

3

This group is often considered as subgenus of the genus Bombus.

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19

Table 1.1 Sibling species of Symphyta and Aculeata that differ in chromosomal characters∗ Morphological differences

Family

Species

Chromosome set

Diprionidae

Diprion pini (Linnaeus)

n = 7, 2n = 14; Not studied n = 14, 2n = 28

Diprionidae

Diprion similis (Hartig)

n = 7, 2n = 14; Not studied n = 14, 2n = 28

Diprionidae

Gilpinia hercyniae n = 7, 2n = 14 Weak (Hartig); G. (thelytoky); n = polytoma (Hartig) 6 (arrhenotoky) Neodiprion swainei n = 7; n = 8 Not studied Middleton

Diprionidae

Pamphiliidae

Cephalcia alpina n = 18; n = 26 (Klug); C. annulicornis (Hartig) Pamphiliidae Cephalcia arvensis n = 23; n = 24; Panzer n = 26; n = 29 Tenthredinidae Rhogogaster viridis n = 10; n = 14 Linnaeus

Moderate

Not mentioned† Not studied

Tenthredinidae Tenthredo arcuata Foerster

n = 10; n = 15

Not studied

Tenthredinidae Tenthredo ferruginea Schrank Tenthredinidae Tenthredo notha Klug

n = 10; n = 12

Moderate

n = 18; n = 20

Not studied

Formicidae

Formicidae

Formicidae

Formicidae

Formicidae

Aphaenogaster rudis (Buckley) species complex Camponotus compressus (Fabricius) Camponotus cruentatus (Latreille)

n = 16–18; n = 20; Weak n = 22 n = 10; n = 20, 2n = 40

Not studied

2n = 36; n = 20

Not studied

Dorymyrmex bicolor n = 9; n = 13 Wheeler; D. thoracicus Santschi Formica truncorum n = 26, 2n = 52; Fabricius n = 28

Not mentioned

Not mentioned

References Maxwell (1958), Rousselet et al. (1998) Smith (1941), Maxwell (1958), Rousselet et al. (1998) Smith (1941), Maxwell (1958) Maxwell (1958), Rousselet et al. (1999b) Battisti et al. (1998), Boato and Battisti (2001) Boato and Battisti (2001) Sanderson (1970), Kuznetsova et al. (2001) Sanderson (1970), Kuznetsova et al. (2001) Naito (1982), Westendorff et al. (1999) Sanderson (1970), Westendorff et al. (1999) Crozier (1975, 1977)

Kumbkarni (1965), Hauschteck-Jungen and Jungen (1983) Hauschteck-Jungen and Jungen (1983), Lorite et al. (2002b) Crozier (1970)

Rosengren et al. (1980)

20

1 Chromosomes of Hymenoptera Table 1.1 (continued)

Family

Species

Chromosome set

Formicidae

Leptothorax albipennis (Curtis); L. tuberum (Fabricius) Leptothorax muscorum (Nylander) Myrmecia fulvipes (Roger) species complex Myrmecia piliventris Smith species complex Myrmecia pilosula Smith species complex

n = 8; n = 9, 2n =18

Formicidae

Formicidae

Formicidae

Formicidae

Formicidae

Morphological differences References Weak

n = 16–17; n = Moderate 17–18+0–5B; n = 18 2n = 12; 2n = 60‡ Weak

n = 2, 2n = 4; Weak 2n = 34; n = 32, 2n = 64 n = 1, 2n = 2–4; Weak n = 5, 2n = 9–10; n = 9–13, 2n = 18–27; n = 15–16, 2n =30–32 n = 23; n = 24, Not studied 2n = 48

Hauschteck-Jungen and Jungen (1983), Orledge (1998)

Loiselle et al. (1990)

Imai et al. (1977)

Imai et al. (1988)

Crosland et al. (1988), Imai et al. (1988), Hirai et al. (1994)

Myrmica “rubra” Imai (1969), (Linnaeus) Hauschteck-Jungen species complex; and Jungen (1983) M. laevinodis Nylander, M. sulcinodis Nylander Formicidae Pachycondyla sp. 2n = 30; 2n = 34 Weak Mariano et al. (1999) “inversa Smith”; P. villosa (Fabricius) Formicidae Pheidole pallidula 2n = 20; 2n = 24 Not studied Hauschteck-Jungen (Nylander) and Jungen (1983) Formicidae Rhytidoponera 2n = 22–24; Weak Imai et al. (1977), metallica (Smith) 2n = 34–46 (1988), Crozier species complex et al. (1986) Crozier et al. (1986) Formicidae Rhytidoponera 2n = 30; 2n = 46 Not studied tasmaniensis (Emery) ∗ Since discrepancies in chromosome numbers of the same species can result from misprints in the references, data are included only for the species in which karyotypic heterogeneity was especially emphasised. † Moderate biochemical and ecological differences are found. ‡ Karyotypes greatly differ in chromosome size and morphology.

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21

distinguished using reference data. Nevertheless, chromosomal analysis can be of use even in the latter case if one or more species in a given group appear new for the science. In this situation, chromosomal features can manifest the presence of undescribed taxa. The problem of taxonomic status of morphologically similar populations of Hymenoptera that differ in karyotypic features is very important. Available data show that chromosome numbers of those populations in almost all cases differ by a few units, and forms that are intermediate in respect to this character, usually absent. This obviously means that these forms are reproductively isolated (at least if they are sympatric) and that the interpopulation differences are due to several or even many, but not the only one, chromosomal rearrangements. Morphologically similar forms of Hymenoptera that differ in their karyotypic features therefore usually deserve species status (Gokhman 1990a). There are two main groups of views regarding correlation between the degree of morphological similarity between sibling species that differ in chromosomal characters, on one hand, and the time elapsed from the beginning of divergence of these forms, on the other hand. According to one view, forms that are difficult to distinguish by their external morphology are species “in statu nascendi” (see e.g. Vorontsov 1960), especially if they do not differ greatly in chromosomal characters as well. However, at least in the case of the differences by many dozen chromosomal rearrangements, as in the M. fulvipes species complex (Imai et al. 1977), this explanation cannot be accepted. Other ideas that consider the possibility of morphological stasis of isolated populations together with an intensive divergence in chromosomal characters are perhaps more correct. Zoogeographical and palaeontological data show that the external similarity of those forms can remain virtually unchanged for a few dozen and even hundred million years (Rasnitsyn 1987, 2002a). Moreover, for example, many genera of ants, a very well-studied group in terms of karyology, that contain sibling species are known as fossils from the Eocene (Dlussky and Fedoseeva 1988). Taking this information into account, an assumption on the fairly ancient divergence in many groups of sibling species of the family Formicidae seems very possible (Gokhman 1990a). With the high degree of morphological similarity that is characteristic of sibling species, there is a practical problem that concerns the possibility of their formal description and attribution of Latin names according to the International Code of Zoological Nomenclature (1999). This seems fairly possible if all species of the group are new for the science, and they all have discrete morphological differences. However, the situation becomes more complicated, if, as is often happens, certain components of the species complex are already described. Under these circumstances, the possibility of finding correspondence between the karyologically studied forms and the type material of described species becomes crucial. If this is impossible (for example, in sympatric forms that are virtually undistinguishable by their external morphology), it is necessary to abstain from the formal attribution of names to new species. Indeed, Section 13.1.1 of the International Code of Zoological Nomenclature (1999) postulates that every valid name must “be accompanied by a description or definition that states in words characters that are purported to

22

1 Chromosomes of Hymenoptera

differentiate the taxon”. If external differences between the examined forms do not exist, chromosomal analysis of type material of the previously described species is necessary to precisely meet this condition. However, this analysis is obviously impossible (Gokhman 1993).

1.3.2 Phylogenetic Implications of Karyotypic Characters 1.3.2.1 General Principles of Phylogenetic Analysis Using Karyotypic Information During the last decades, interest in the phylogenetic research of living organisms substantially increased (Maddison 1994, Harvey and Pagel 1995, Avise 2006, etc.). This can be explained by the accumulation of data on comparative morphology, physiology, ecology, behaviour, etc., as well as by the intensive development of computerised methods of phylogenetic analysis, mostly cladistics (Quicke 1993). It is worth noting that the first, now classic, examples of use of the results of chromosomal studies in phylogenetic research appeared already in the 1930s–1950s. These were works on the phylogenetic analysis of inversions of the giant chromosomes of Diptera (Dobzhansky 1941, etc.). However, if organisms with polytene chromosomes are excluded, karyotypic characters are rarely used in the phylogenetic studies of many other animal groups. This can be explained by the fact that the number of chromosomal characters is usually small if compared to the number of features of external morphology (see Section 1.3.1). In addition to that, many karyotypic traits are very labile and subject to parallel evolution. Karyotypic parallelisms appear to be much more frequent than morphological ones (Vorontsov 1958). To formulate it more precisely, it is more difficult to distinguish true synapomorphies from homoplasies when dealing with structural features of chromosome sets (Gokhman 1997a). However, the common occurrence of homoplasies cannot be regarded as the basis for rejecting karyotypic characters in phylogenetic analysis. Indeed, phylogenetically important results can be obtained even e.g. from the analysis of the seemingly labile details of the grooming behaviour of Hymenoptera (Basibuyuk and Quicke 1999). An additional argument for including karyotypic characters in the phylogenetic analysis is the fact that these features, unlike many morphological ones, change rather independently of the environment. This makes the chromosomal characters very important for phylogenetic analysis, especially in those cases when studies of morphological and other features fail to provide an unambiguous solution (Gokhman and Kuznetsova 2006). Since the probability of homoplasy obviously increases together with the size of the group, the phylogenetic value of chromosomal characters usually becomes greater as the taxonomic rank decreases. The so-called character devaluation (see Rasnitsyn 2002a) is also important in this case. Indeed, a few examples of phylogenetic trees of insects and other animals based solely on karyotypic features usually cover lower taxa, such as groups of closely related species (Stegniy 1993, Vysotskaya and Stepanova 1996), even though successful attempts of resolving

1.3 Systematic and Phylogenetic Implications of Chromosomal Characters in Hymenoptera

23

the phylogeny of higher taxa are also known (see e.g. Kuznetsova et al. 2004). Analysis of the karyotypic information at higher taxonomic levels often makes use of already available phylogenetic schemes based on other characters (Crozier 1975, Kuznetsova 1985, Emelyanov and Kirillova 1989, 1991). This approach allows characterising karyotypic variability in particular phylogenetic lineages. The fullscale use of chromosomal data for creating phylogenies of large taxa is apparently possible only in conjunction with morphological, biological and other characters (see e.g. Mamontova 2001). A few papers on Hymenoptera phylogeny prepared in that way appeared only during the last decade (Quicke and Belshaw 1999, Quicke et al. 1999). Together with the analysis of karyotypic data from the phylogenetic point of view, an alternative approach also exists. This approach studies the mode of distribution of chromosomal characters using statistical methods. The necessity of phylogenetic analysis of chromosomal variation can be primarily explained by the fact that this analysis allows identification of karyotypic changes that have an independent evolutionary history (Donoghue 1989, Maddison 1994, Harvey and Pagel 1995). If this approach is neglected, this can lead to flaws in identification of the frequency and direction of those changes. Regrettably, even preliminary versions of phylogenies do not exist for many groups of organisms. Under those circumstances, methods of statistical analysis of chromosomal variation take over the phylogenetic ones (see Section 1.3.2.4). For the more detailed study of the possibilities of use of chromosomal characters in phylogenetic analysis, it is necessary to consider these characters using phylogenetic presumptions, i.e. certain assumptions on which this analysis is based. Rasnitsyn and Dlussky (1988) are likely to be the first who have formulated hypotheses of that kind, which were later modified by Rasnitsyn (1996, 2002a). Results of the preliminary analysis of phylogenetic implications of chromosomal characters using most of these presumptions are given below (except for the statements used only in the palaeontological, embryological, morpho-functional and physiological studies; names and definitions of the presumptions are given according to Rasnitsyn (1996)). Presumption of “knowability” of phylogeny claims that any similarity should be considered as inherited (unless and until the reverse is reasonably proved)4. Rasnitsyn (2002a) shows that this statement is the central presumption of phylogenetics because it postulates that true synapomorphies prevail over homoplasies. Whether this statement is correct where chromosomal characters are concerned much depends on (1) the nature of these characters and (2) volume of the taxon. Indeed, karyotypic features are highly unequal in terms of their ability to display homoplastic evolution. It is obvious that chromosome numbers are the least reliable in that respect because they can coincide in many unrelated species. Other chromosomal characters are also fairly variable and can vary simultaneously in

4

The text given in brackets is omitted below from the definitions of all presumptions for the sake of brevity.

24

1 Chromosomes of Hymenoptera

many phylogenetic lineages (for possible mechanisms of this variation see Section 1.3.2.3). Since the probability of homoplasy increases in the larger taxa, reliability of phylogenetic reconstructions can be enhanced by reconstructing evolution of chromosomal characters at the maximally possible lowest level and then by comparing the results to each other. According to the presumption of analogy, if a transformation series is polarised in a group, the results should be considered as valid for another group. As Rasnitsyn and Dlussky (1988) explain, this presumption is the consequence of the fact that many closely related groups evolve simultaneously and acquire similar character states. It is well known that parallel karyotypic variation is usual in the related taxa (see above). In particular, chromosome numbers often decrease in more advanced groups if compared to the less advanced ones (White 1973). The so-called dislocation hypothesis (Navashin 1957) was even based on the prevalence of those phenomena in plants. According to this hypothesis, fusion of two chromosomes leads to the obligate loss of the redundant centromere. Navashin (1957) supposed that this process, in turn, led to the irreversible reduction of chromosome numbers in specialised groups, because centromeres could not arise de novo. Although those assumptions can be disproved (if polyploidy is excluded, chromosome numbers of all organisms should therefore eventually decrease to n = 1), the very phenomenon does take place. However, this pattern is certainly not universal. Indeed, if the mechanism of decrease in the chromosome number exists, the reverse processes must also occur. For example, certain evidence suggests that chromosomal fissions prevailed in carnivorous mammals (Todd 1970). Moreover, analogous phenomena probably took place in the karyotype evolution of ants and other Hymenoptera (Imai et al. 1977, 1988, Hoshiba and Imai 1993). Outgroup presumption (or presumption of the conserved character distribution) declares that a character state found only within a group should be considered apomorphic in respect of that distributed both within and outside the group. The method of the outgroup comparison is based on this presumption. Numerous deviating values of chromosomal characters (e.g. chromosome numbers) in the specialised forms directly illustrate this principle. For example, the majority of ant species of the genus Monomorium have n = 8–19. The only exception from this rule is Monomorium latinode Mayr with n = 35 (Imai et al. 1984a). The latter value is apparently autapomorphic. Moreover, Rasnitsyn and Dlussky (1988) point out that the more ancient character state must be more widespread than the younger one. The latter statement represents an apparent basis for the treatment of modal (i.e. the most widespread) chromosome numbers as initial ones (see Section 1.3.2.2). Presumption of parsimony states that the most likely cladogram is that implicating the least number of homoplasies. Since chromosomal characters are often fairly variable and subject to reversal, this situation determines the phylogenetic significance of that principle. Under these circumstances (“unless and until the reverse is reasonably proved”, see above), the scenario that minimises the number of those reversions seems more correct. Finally, according to the presumption of weighted similarity, in the case of conflicting similarities those which should be considered as inherited are known to

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25

be more reliable in other cases (especially in closely related groups). Similar to the previous assumption, this presumption helps to make a choice between the true and false synapomorphies. Indeed, the preference in phylogenetic reconstruction should be given e.g. to the combination of chromosome lengths and centromere indices, not just to the chromosome numbers as such, if other conditions are equal. 1.3.2.2 Reconstruction of Initial States of Chromosomal Characters To use karyotypic features in the phylogenetic analysis, initial character states should be determined for the analysed taxa. Possible approaches to the solution of this problem are illustrated below on the example of the identification of plesiomorphic values of chromosome numbers in the Hymenoptera. In the practice of karyotypic studies, modal values of chromosome numbers are usually treated as initial (see e.g. Emelyanov and Kirillova 1989, Gokhman and Quicke 1995, Shaposhnikov et al. 1998). In fact, this means the application of the “common-is-primitive” principle to the chromosomal characters. Although there are different opinions on this subject (Frolich 1987, Quicke 1993), the association given above is highly possible, especially if the outgroup presumption is taken into account (see above). Moreover, it is evident that members of any particular taxon are known only in part. In a strict sense, to draw sample-based evolutionary conclusions for a particular taxon, it must be proven first that this sample is a representative one. In this case, it is only necessary to demonstrate that modal chromosome numbers of the sample and those of the whole taxon coincide. Although proving this fact is generally a difficult, if at all soluble, task, it is obvious that the probability of this coincidence directly depends on the level of chromosomal diversity. Indeed, if all members of a given taxon have the same chromosome number, it is necessary to study a single species to obtain the full information on this character. Alternatively, chaotic variation of chromosome numbers deserves the exhaustive study of the whole taxon, but it is impossible to find more or less pronounced modal values in this case. Of course, absolutely stochastic variation of chromosome numbers never occurs in nature. Nevertheless, the existence of several peaks of the distribution pattern of this character, flatness of these peaks, etc. are often explained by the superposition of different distribution patterns of the lower taxa. Under these circumstances, variation of chromosome numbers in all subordinate groups should be studied. This procedure diminishes the probability of homoplasy (see Section 1.3.2.1) as well as of the distortions caused by the uneven study of subordinate taxa. The aforementioned “genus-karyotype concept” (see Section 1.3.1) is based on this technique. Indeed, study of the pattern of chromosomal variability at the genus level is enough for the confident identification of modal chromosome numbers in the majority of animal taxa including Hymenoptera. Finally, coincidence between the objective and sample patterns can be confirmed by the constancy of characteristics of this distribution during the long period of study (see Imai et al. 1977, 1988, and also Timonin 1990). Many varieties of the chromosome number distribution certainly exist even at the genus level. Among these varieties, the distribution with the coinciding modal and

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median values is fairly common (the median value divides the whole data pool into two parts which are equal by the number of species). The normal distribution is the most often example that satisfies this condition (Lakin 1973, McKillup 2005). This pattern of the observed distribution testifies in favour of the more or less full equilibrium between the processes that increase or decrease the chromosome number (White 1973). If it is true, the modal chromosome number of a given taxon must be considered initial. However, the diversity of chromosome numbers even at the genus level can be large enough (e.g. in certain Aculeata) to make the approximate evaluation of the initial chromosome number impossible. There are two apparent opportunities in this case: (1) the group is either unsatisfactorily studied in terms of karyology or (2) chromosomal rearrangements in the taxon were so intensive that the more detailed information on karyotype structure is needed to resolve chromosomal evolution (see also Section 1.3.2.4). Finally, only a certain range of the initial chromosome number can often be determined. Even this approximate evaluation can apparently be used for studying processes of the phylogenetic change of chromosome numbers, especially if they are compared to chromosome numbers of the sister taxa. The procedure of determination of the initial chromosome number in higher taxa certainly cannot be conducted, e.g. by calculating the arithmetic mean of chromosome numbers of the subordinate taxa. In these cases not only information on karyotype structure of less specialised groups, but the very possibility of detecting those taxa is crucial. Phylogenetic analysis is apparently the most effective mechanism of diagnosing those groups. Nevertheless, if phylogenetic reconstruction of the studied taxon is considered difficult for any reason, a detailed chromosomal study of morphologically less advanced groups is necessary even in this case because of the consequence of the phylogenetic presumption of analogy. According to this consequence, if distribution of the two morphoclines is similar, and polarity of the first one is known, the polarity of the second cline can be determined by analogy with the first one (Rasnitsyn and Dlussky 1988). In other words, it is highly possible that relatively less specialised taxa also have relatively weakly changed chromosome sets. 1.3.2.3 Karyotypic Orthoselection and Its Possible Causes After determining plesiomorphic character states of chromosomal features it may seem that the identification of their apomorphic character states is a significantly easier task. However, the search for true apomorphies is complicated by the important phenomenon of karyotype evolution, namely, karyotypic orthoselection. This term was introduced by White (1973) to specify evolutionary traits that are manifested in different lineages and caused by canalising selection. White suggested that there were two distinct ways in which selection favoured various types of chromosomal rearrangements. First, “changes of the same structural type can have the same kind of physiological [i.e. phenotypic] effect”. Second, similar karyotypic changes can be “caused by the need for chromosomal shapes and sizes to remain harmonious

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with the dimensions of the spindles and the cells in all the tissues of the body” (White 1973). It is beyond doubt that particular features of chromosomal organisation restrict the possible spectrum of karyotypic rearrangements. For example, biarmed chromosomes cannot directly undergo centric fusions, and holokinetic chromosomes are more liable to fragmentations if compared to monokinetic ones (to be more precise, the resulting fragments of the holokinetic chromosomes are better retained within karyotypes; Kuznetsova 1979). Moreover, the chromosome number can even affect distribution of the types of chromosomal polymorphism within the same large taxon. Specifically, translocation polymorphism is detected only in the ants with n = 1–12, and the Robertsonian one is detected only in these insects having n = 12–41 (Imai et al. 1988). This distribution may be explained by the fact that bivalents are fixed at the nuclear membrane by both their ends during pachytene (Solari 1970, Moses 1977). Since chromosome length is inversely correlated to the number of chromosomes within a given karyotype, other conditions being equal (see Section 1.1.3), chromosomal interactions are substantially hampered in species with large chromosome numbers, and reciprocal translocations therefore become less frequent. Moreover, processes of chromosomal fragmentation destroy the traces of a few translocations that occur in karyotypes with higher chromosome numbers (Imai et al. 1984a). However, multidirectional traits of chromosomal evolution cannot be explained only by the differences in various details of chromosome structure. The fact that karyotypic changes can proceed in opposite directions (for example, chromosomal fissions and fusions) in closely related groups represents the clearest evidence in favour of this statement. Situations of that kind are frequent in insects and other animals (White 1973, Emelyanov and Kirillova 1989, Shaposhnikov et al. 1998, Mariano et al. 2003). It is obvious that these cases can be explained only by factors that are external with respect to chromosomal organisation (Gokhman 1990a). However, White (1973) believed that analogous recurrent rearrangements could affect the phenotype in a similar way (see above), but this hypothesis seems purely speculative. On the other hand, Vorontsov (1966) already suggested that the karyotypic orthoselection could be associated with the general type of selection, but not with a particular phenotypic effect: “The evolution of chromosome sets is virtually adaptive. A stochastic increase in the chromosome number favours destabilising selection. The decrease in chromosome number narrows the combinative variation, i.e. favours stabilising selection” (see also Emelyanov and Kirillova 1991). Meiotic processes that increase or decrease the level of recombination can also proceed in similar directions in closely related lineages. For example, the mean number of chiasmata per cell in Acrididae (Orthoptera) decreases simultaneously in many lineages (Vysotskaya 1996). Anyway, it is obvious that karyotypic orthoselection leads to the parallel development of similar character states and hampers phylogenetic reconstruction. Under these circumstances, application of the presumption of “knowability” of phylogeny (see Section 1.3.2.1) appears fairly limited. Of course, detection of chromosomal autapomorphies is not usually difficult, but merging taxa in monophyletic groups

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on the basis of their karyotype structure should be done very carefully. This is extremely important if phylogenetic relationships of those taxa were not previously revealed using independent information. 1.3.2.4 Main Trends and Mechanisms of Phylogenetic Change of Karyotypes Chromosomal rearrangements in Hymenoptera affect virtually all karyotypic features, i.e. (1) chromosome number, (2) chromosome size (including size differentiation), (3) proportions of biarmed chromosomes to acrocentrics (the two latter parameters can be considered as changes in the degree of karyotypic symmetry), (4) size and localisation of segments of the constitutive heterochromatin, (5) localisation and number of nucleolus organisers, (6) number and localisation of meiotic chiasmata. Examples of the increase/decrease in chromosome number of closely related groups of Hymenoptera are very numerous (Crozier 1975, Naito 1982, Imai et al. 1988, etc.). In most cases, these changes are fairly moderate (see Section 1.1.2), but in the members of certain extremely polymorphic genera of aculeate Hymenoptera (Myrmecia, Polistes, etc.) the variation range is very wide (see above). The chromosome number is usually supposed to increase in the evolution of Hymenoptera (Imai et al. 1988, Hoshiba and Imai 1993), but these assumptions lack independent phylogenetic evaluation. Karyotypic rearrangements that change the chromosome number in Hymenoptera are described in Section 1.2.2. As mentioned in Section 1.1.3, the number and size of chromosomes are inversely correlated. The comparison of chromosome sizes within a compact group is therefore very convincing. Specifically, chromosomes of sawflies (Tenthredinidae) with n = 6–10 (falling into the range that covers the chromosome number which can be initial for the family; Westendorff et al. 1999) are notably larger than those of species with substantially higher chromosome numbers (Naito 1982). Possible mechanisms of change in the chromosome size in Hymenoptera are also listed in Section 1.2.2. The problem of the directionality of mutual changes between biarmed chromosomes and acrocentrics has been studied in detail by Imai et al. (1988, 2001) who used the so-called minimum interaction hypothesis. According to this concept, “metacentric chromosomes in a broad sense” (including submeta- and subtelocentrics) preferentially turn into acrocentrics by centric fissions and the tandem growth of the constitutive heterochromatin of the shorter arms of the emerging “telocentrics”. In turn, this growth is reversible to a certain extent. Acrocentric chromosomes can again become biarmed through centric fusions or pericentric inversions (the “fission–fusion” or “fission–inversion” cycle). For example, alteration of fissions and inversions is supposed to play the key role in the karyotype evolution of ants (Imai et al. 1988). Regrettably, this hypothesis, as well as many similar assumptions, is not confirmed by thorough phylogenetic analysis. An example of the directional change of localisation and size of heterochromatic segments regards the origin of pseudoacrocentrics in M. brevinoda (Formicidae; see Section 1.2.2). In addition, a comparative study of the localisation of gene clusters

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responsible for the synthesis of 28S rRNA using FISH has also been done on ants of the M. pilosula complex (Hirai et al. 1994). It has shown that there were two clusters of the ribosomal genes in the genomes of the members of this complex with 2n = 3, 8 and 10 (the presumed initial state), whereas species with 2n = 18 and 27 had six and ten clusters, respectively. The ribosomal DNA of an ancestral species was supposed to have been initially localised in a heterochromatic segment, and then the number of its copies increased through centric fissions. Most of the DNA that has been amplified that way was later eliminated along with the heterochromatic segments, and the remaining copies were distributed among the genomes during other chromosomal rearrangements. This reconstruction, however, strongly depends on the correct detection of directionality of the change in chromosome number and size of this group (see above). Regrettably, it is impossible at present to determine any traits of phylogenetic changes of meiosis in Hymenoptera, because data on the number and localisation of meiotic chiasmata in the group are fragmentary and incomplete. However, it is known that acrocentric chromosomes have more chiasmata than metacentric ones, and the chromosome with large heterochromatic segments bears less chiasmata than the analogous element with the small amount of heterochromatin, etc., if all other conditions are equal (John and Freeman 1975, Vysotskaya et al. 1983). Analogously to other insects (see e.g. Vysotskaya et al. 1983), evolutionary changes in the number and localisation of chiasmata will probably be found in Hymenoptera as well. To conclude this chapter, it is necessary to add that phylogenetic and statistical approaches to the study of karyotype evolution are not mutually exclusive, but rather complementary to each other. Statistical methods are the most important when there is no satisfactory phylogenetic reconstruction for the group studied. During the last decades, at least two useful research methods were developed to study karyotype evolution in the Hymenoptera. The first method implies construction of so-called karyographs (Imai and Crozier 1980), the second one implies reconstruction of chromosomal rearrangement networks (Imai 1991). The karyograph is a diagram of the species distribution that uses coordinates of diploid numbers of chromosomes and chromosome arms. It may be true that this technique visualises the most probable rearrangements in a particular taxonomic group, although it cannot determine their directionality, at least without any additional information. In turn, construction of chromosomal rearrangement networks is an effort-consuming process that includes recognition of several types of biarmed and acrocentric chromosomes with respect to heterochromatin distribution, identification of possible pathways of mutual transition between acrocentrics and metacentrics, as well as the estimate of the relative numbers of these transformations (Hoshiba and Imai 1993). It seems that this method can more or less precisely determine the relative frequency of certain chromosomal rearrangements, but it is also fairly limited. The main restriction of this technique is that it requires the precise data on heterochromatin distribution on every chromosome. In addition, this method, along with other statistical techniques, cannot identify independent evolutionary events among other karyotypic rearrangements (see above).

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Chapter 2

Material and Methods

Abstract More than 4,000 individuals of parasitic Hymenoptera mainly collected by the author in European Russia and adjacent countries were studied in this book. Chromosomal preparations were obtained from ovaries of adult females, cerebral ganglia of prepupae and gonads of early pupae of parasitic wasps using air-drying techniques. Routine chromosome staining (including subsequent morphometric analysis) as well as C-banding was used. Chromosomes were studied using conventional and phase-contrast light microscopy. Mitotic chromosomes were classified into four groups: metacentrics, submetacentrics, subtelocentrics and acrocentrics. Meiotic chromosomes were classified according to the number of chiasmata per bivalent. Karyograms of many studied species were prepared. Modern works were used as data sources on taxonomy and phylogeny of parasitic wasps. Keywords Air-dried preparations · Chromosome staining · Parasitic wasps · Phylogeny · Taxonomy

2.1 Material Studied Parasitic Hymenoptera mainly collected by the author during 1983–2008 in European Russia and adjacent countries as well as those obtained from laboratory stocks originating from Russia, the UK, Germany, the Netherlands, the USA and Canada are used in this work. Mass collecting was done at Ozhigovo (60 km SW Moscow), the Botanical Garden of Moscow State University (Moscow) and near Volgograd (about 1,000 km SE Moscow). Small additional amounts of parasitic wasps were collected in Russia, Central and Western Europe as well as in the USA by A.P. Rasnitsyn and other researchers. Adult parasitic Hymenoptera were mainly collected by beating as well as on various plants including blooming Apiaceae (= Umbelliferae). Females of certain species were found on their hibernation sites, inside hollow plant stems and under the tree bark. Some species were reared from their hosts collected by the author and other specialists. V.E. Gokhman, Karyotypes of Parasitic Hymenoptera, DOI 10.1007/978-1-4020-9807-9 2, C Springer Science+Business Media B.V. 2009

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2 Material and Methods

Virtually all studied material was identified by the author. Most identifications were later checked by the leading taxonomists from Russia and other countries: A.P. Rasnitsyn, V.I. Tobias, S.A. Belokobylskij, D.R. Kasparyan, V.A. Trjapitzin, M.A. Kozlov, M.D. Zerova, V.I. Tolkanitz, K.A. Dzhanokmen, V.V. Kostjukov, N.G. Ponomarenko, O.V. Kovalev, D.L.J. Quicke, Z. Bouˇcek, S. Heydon, etc. The overwhelming majority of the studied specimens are deposited in the Zoological Museum, Moscow State University (Moscow), and small amounts of parasitic wasps are deposited in the Natural History Museum (London, UK), German Entomological Institute (M¨uncheberg, Germany) and the University of Rochester (Rochester, USA).

2.2 Methods of Obtaining Chromosomal Preparations The author has examined more than 4,000 preparations which were obtained from ovaries of adult females, cerebral ganglia of prepupae and gonads of early pupae of parasitic Hymenoptera. To make chromosomal preparations from immature stages, the technique developed by Imai et al. (1988) was used; we (Gokhman and Quicke 1995) have substantially modified this method to study chromosomes in adult wasps. The technique by Imai et al. (1988) includes the following main stages: 1. Isolation of material. Insects were dissected in 1% hypotonic sodium citrate solution containing 0.005% colchicine. Necessary organs were extracted. 2. Hypotonic treatment. The organs were transferred to a fresh portion of hypotonic solution and incubated there for 20 min at room temperature. 3. Suspending and fixation. The material was transferred onto a precleaned microscope slide using Pasteur pipette and then gently flushed with Fixative I (glacial acetic acid:absolute ethanol:distilled water 3:3:4). The tissues were disrupted in an additional drop of Fixative I using dissecting needles. Another drop of Fixative II (glacial acetic acid:absolute ethanol 1:1) was applied to the centre of the area and the more aqueous phase was blotted off the edges of the slide. The same procedure was performed with Fixative III (glacial acetic acid). The preparation was then dried for about half an hour. 4. Staining. The preparation was stained for 3–6 h with freshly prepared 2% Giemsa solution in 0.05 M Sørensen’s phosphate buffer (Na2 HPO4 + KH2 PO4 , pH 6.8). The preparation was then rinsed with distilled water and dried at room temperature. To study the chromosomes of adult females, this technique was modified by Gokhman and Quicke (1995) as follows: 1. The insects were fed with 10% honey syrup containing 0.1% colchicine for about 12–16 h prior to karyotyping. 2. The membranous part of the hypopygium was cut at the base of the ovipositor; ovaries with the paired oviducts were extracted and detached from the female’s body. Operated wasps were scarcely different from the intact ones after the usual mounting.

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Differential C-banding was performed using the modified technique by Sumner (1972) on preparations from the cerebral ganglia of prepupae. Before staining, preparations were kept for 2–4 weeks at 3–5◦ C. Slides were transferred for an hour into 0.1 N hydrochloric acid solution at room temperature. The preparations were then rinsed with water and submerged for 1–2 s in 5% saturated barium hydroxide solution at 55–65◦C. After that, the slide was first rinsed with 0.1 N HCl solution and then with distilled water. The preparation was then incubated in 2 × SSC buffer solution (0.3 M sodium chloride and 0.03 M sodium citrate) at 60◦ C. After that, the slide was rinsed with distilled water and stained with 2% Giemsa solution prepared on the phosphate buffer for an hour.

2.3 Methods of Analysing Chromosomal Preparations Mitotic and meiotic divisions were examined and photographed using MBI-15, Zeiss NU-2 and Axioskop 40 FL light microscopes equipped with the regular and phase-contrast optics. To count chromosome numbers and identify numeric features of chromosomes, not less than ten mitoses from every preparation were usually examined. Metaphase plates with the best chromosomal morphology were used to obtain karyograms. Chromosomes were classified into four groups (metacentrics, submetacentrics, subtelocentrics and acrocentrics) according to the papers by Levan et al. (1964) and Imai et al. (1977). Chromosomes of diploid sets were united in pairs and then arranged by decreasing length within the whole karyotypes or in groups. Meiotic chromosomes were classified according to Darlington (1965) and Kiknadze and Vysotskaya (1975), numbers of chiasmata per bivalent were counted. To obtain images of cell divisions, they were photographed on the “Mikrat 300” 35-mm film. The film was later developed and black and white prints were made. Digital images of chromosomes were acquired using Zeiss AxioCam MRc camera. To obtain karyograms, these images were processed with Adobe Photoshop. Images of mitoses scanned directly from the preparations using the static TV camera connected to the personal computer fitted with the image analysis program ImageExpert were used for morphometric chromosomal analysis. Chromosome measurements were taken using Adobe Photoshop. Statistical analysis of the obtained results was done using STATISTICA.

2.4 Sources of Data on Taxonomy and Phylogeny Taxonomic data used in this work were obtained from the modern books (Handbook for the Identification of Insects of the European Part of the USSR 1978, 1981, 1986a,b, Fitton et al. 1988, Gauld and Bolton 1988, Quicke 1997) and separate papers on parasitic Hymenoptera taxonomy (Selfa and Diller 1994, Ronquist 1999, etc.). For the phylogenetic analysis of chromosomal data, modern works on parasitic Hymenoptera phylogeny were used (Belshaw and Quicke 1997, 2002, Ronquist et al. 1999, Campbell et al. 2000, Gauthier et al. 2000, Quicke et al. 2000, Dowton et al. 2002, etc.).

Chapter 3

Morphological Features of Karyotypes of Parasitic Hymenoptera

Abstract Results of karyotypic studies of more than 400 species of parasitic wasps are reviewed. The haploid chromosome number in the group can vary from 3 to 23. Data on the nuclear DNA show that the average DNA content per chromosome in the superfamily Chalcidoidea is at least four times more than that in the Ichneumonoidea. The largest and the smallest chromosomes of parasitic wasps are 12–15 and 0.5–1 µm, respectively, but most chromosomes are medium-sized (3–5 µm). Parasitic Hymenoptera are generally characterised by symmetrical karyotypes. Segments of pericentromeric and telomeric constitutive heterochromatin are the most usual in this group. Paired nucleolus organisers are localised on both homologous chromosomes. Three patterns of transition to thelytoky, i.e. ameiotic parthenogenesis, gamete duplication and central fusion (the two latter patterns belong to meiotic parthenogenesis) are recorded in parasitic Hymenoptera. Keywords Chromosomes · Differential staining · Hymenoptera · Meiosis · Parasitic wasps

3.1 Chromosome Number and Nuclear DNA Content Chromosomes of more than 400 species of parasitic wasps have been studied up to now; their haploid chromosome numbers can vary from 3 to 23 (see Appendices A and B). The lowest chromosome number, n = 3,1 is found in Aphidius sp. (Braconidae), Encarsia protransvena (Aphelinidae), Brachymeria intermedia (Chalcididae) and Perilampus ruschkai (Perilampidae; Hung 1986, Quicke 1997, Baldanza et al. 1999, Gokhman 2000a, 2005b) and the highest one, n = 23, is found in Fopius arisanus (Braconidae; Kitthawee et al. 2004). The distribution of species by this character is bimodal (Fig. 3.1) with the two distinct peaks at n = 6 and 11 (see Gokhman 2003, 2004d, 2006b). Similar modal values, n = 6 and 10, are recorded if chromosome numbers are counted at the genus level, i.e. using the 1

The chromosome number n = 2 found in Oligosita sp. (Trichogrammatidae; Muramoto 1993) needs to be confirmed.

V.E. Gokhman, Karyotypes of Parasitic Hymenoptera, DOI 10.1007/978-1-4020-9807-9 3, C Springer Science+Business Media B.V. 2009

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3 Morphological Features of Karyotypes of Parasitic Hymenoptera

Fig. 3.1 Distribution of parasitic wasps by chromosome number at the species level (based on data from Appendix A)

“genus-karyotype” method developed by Crozier (1975) (Fig. 3.2; see Gokhman and Quicke 1995, Gokhman 2000a, 2003). Estimates of the levels of diversity of chromosomal characters in parasitic Hymenoptera have substantially changed during the last decades. Until the middle

Fig. 3.2 Distribution of parasitic wasps by chromosome number at the genus level (based on data from Appendix A)

3.2 Size of Mitotic Chromosomes: Centromere Position and Centromere Index

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of the 1970s, most specialists agreed that chromosome numbers of parasitic wasps were very uniform. Specifically, Crozier (1975) cited the only chromosome number, n = 5, for all members of the superfamily Chalcidoidea, and n = 10 for all other parasitic wasps (except for an ichneumonid species with n = 11). However, certain simultaneous publications (Goodpasture 1974, 1975a; Goodpasture and Grissell 1975) have demonstrated that members of different families and even closely related species of Chalcidoidea could differ in chromosome number. Later on, analogous data were also obtained for other parasitic wasps (Gokhman 1990a, etc.). It is widely accepted now that chromosome numbers of parasitic Hymenoptera are relatively stable at the genus level, although the differences in this character are sometimes detected even between morphologically indistinguishable populations of parasitic wasps (Gokhman and Quicke 1995, Gokhman 1997a, 2000a, 2002a). Data on the DNA content in the haploid genome of parasitic Hymenoptera that were obtained by various techniques for Diadromus pulchellus (Ichneumonidae), Habrobracon juglandis, H. serinopae (Braconidae), Eupelmus vuilleti (Crawford) (Eupelmidae), Nasonia vitripennis and Catolaccus grandis (Burks) (Pteromalidae) demonstrate that the DNA content in the chalcid genome (3.0×108 to 10×109 base pairs) is two to ten times more than that found in the Braconidae and Ichneumonidae (1.4×108 to 1–2×109 bp; Rasch et al. 1975, 1977, Bigot et al. 1991, Barcenas et al. 2008). Since the chromosome number of Eupelmus and Nasonia (n = 5) is about twice less than that of Diadromus (n = 11) and Habrobracon (n = 10), the average DNA content per chromosome in the Chalcidoidea can be at least four times more than that in the Ichneumonoidea (Gokhman and Quicke 1995). However, further studies are needed for the more precise identification of genome sizes as well as the differences in this character at the superfamily level (Quicke 1997).

3.2 Size of Mitotic Chromosomes: Centromere Position and Centromere Index The largest and the smallest chromosomes of parasitic wasps are 12–15 and 0.5–1 µm, respectively; however, most chromosomes are medium-sized (3–5 µm) (Gokhman 2003, 2004d). On average, chromosome size and number are inversely correlated in parasitic Hymenoptera as well as in other members of the order (see above). Specifically, chromosomes of parasitic wasps of the family Dryinidae and chalcids that have lower chromosome numbers (n = 4–6) are substantially larger than those of the Ichneumonidae with higher n values. Chromosomes of most parasitic Hymenoptera gradually decrease in size, and the largest and the smallest chromosomes usually differ in length by no more than 2–2.5 times. Nevertheless, drastic differences in size between individual chromosomes are found in some Ichneumonidae, Braconidae and Dryinidae as well as in many chalcids of the families Eulophidae and Torymidae (Gokhman 1987, 2002c, 2004e, Gokhman and Kolesnichenko 1998b,c, Gokhman and Mikhailenko 2007). Biarmed chromosomes prevail in most parasitic wasps; acrocentrics are relatively common in many species of the family Cynipidae (Sanderson 1988) and some

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other groups. Moreover, karyotypes of some Ichneumonidae (Venturia canescens, Hyposoter sp. 1, Pristomerus sp.) and Cynipidae (Dryocosmus kuriphilus) contain only acrocentric chromosomes (Speicher 1937, Abe 1994, Gokhman 2001a). Thus, parasitic Hymenoptera are generally characterised by symmetrical karyotypes (see Section 1.1.4).

3.3 Differential Chromosome Staining Although there are relatively few papers that study heterochromatin distribution in karyotypes of parasitic Hymenoptera (Hoshiba and Imai 1993, Reed 1993, Gokhman 1997b, Baldanza et al. 1999, Gokhman and Westendorff 2000, 2003), the results obtained show that segments of pericentromeric and telomeric heterochromatin are the most characteristic of species of this group. Segments of intercalary heterochromatin are less frequent; they are found e.g. in some Ichneumonidae and Aphelinidae (Gokhman 1997b, Baldanza et al. 1999). Pseudoacrocentrics are relatively common in chromosome sets of parasitic wasps (Gokhman 1985, 1997b, Hoshiba and Imai 1993, Baldanza et al. 1999, Gokhman and Westendorff 2000), and some elements of karyotypes (e.g. the last chromosome pair in Aphidius ervi with 2n = 12) consist entirely of heterochromatin (Gokhman and Westendorff 2003). Large taxa of parasitic wasps show virtually no difference in the mode of heterochromatin distribution (see e.g. Hoshiba and Imai 1993, Gokhman 1997b). On the other hand, C-banded karyotypes also look similar in sibling species of Nasonia (Pteromalidae; Gokhman and Westendorff 2000). Nevertheless, strong interspecific differences in the size and localisation of heterochromatic segments were found in parasitic wasps of the families Ichneumonidae and Aphelinidae (Gokhman 1997b, Baldanza et al. 1999). Moreover, population polymorphism that involved size of C-segments has been revealed in Dirophanes invisor (Ichneumonidae; Gokhman 1990a, 1997b). Polymorphism regarding this character has also been detected in the other member of this family (Hoshiba and Imai 1993). Using AgNOR-banding, chromosome sets of a few chalcid wasps of the families Aphelinidae, Eulophidae and Pteromalidae were studied (Reed 1993, Baldanza et al. 1999, Maffei et al. 2001, Giorgini and Baldanza 2004, Bernardo et al. 2008). In all cases, paired nucleolus organisers were localised on both homologous chromosomes. In closely related species with the same chromosome number, Encarsia formosa, E. luteola and E. meritoria (as well as in the Encarsia sophia complex), this region was located on different chromosomes (Baldanza et al. 1999, Baldanza and Giorgini 2001, Giorgini and Baldanza 2004). There was a report on the karyotypic study of parasitic wasps of the family Aphelinidae, Encarsia citrina and E. leucaspidis, using DAPI staining (Baldanza et al. 1999), although no differential segmentation has been revealed in this case. Up to now, karyotypes of the two species of Aphelinidae, Encarsia berlesei and E. inaron, were studied using G-banding (Odierna et al. 1993, Baldanza et al. 1999).

3.4 Details of Meiosis and Structure of Meiotic Chromosomes

39

Although the results obtained seemed not to allow finding unequivocal correspondence between chromosomes of these species, it was possible to identify all chromosome pairs of each species through G-banding. Using similar technique, the chromosome set of N. vitripennis was also studied (R¨utten et al. 2004). Until recently, identification of the chromosomal localisation of ribosomal DNA as well as of DNA of the symbiotic polydnavirus integrated in the genome of the braconid Cotesia congregata was the only chromosomal study of parasitic Hymenoptera using in situ hybridisation (Belle et al. 2002). It was found that rDNA and the viral genome of this species were localised in the shorter arms of two different subtelocentric chromosomes. In addition, all chromosomes of the karyotype of N. vitripennis were identified using the technique of chromosome painting or multicolour FISH (R¨utten et al. 2004).

3.4 Details of Meiosis and Structure of Meiotic Chromosomes As noted above (see Section 1.1.8), the first meiotic division is abortive in hymenopteran males, i.e. it occurs without chromosome reduction. In parasitic Hymenoptera, as well as in other members of the order, a cytoplasmic bud can pinch off the spermatocyte (Koonz 1939). However, various authors had different opinions regarding the existence of this bud even in the same species of parasitic wasps (see e.g. Doncaster 1910, Dodds 1938). Certain electron microscopy data show that these cytoplasmic bodies can contain centrosomes (Newman and Quicke 1998). Analogously to other Hymenoptera (see Section 1.1.8), chromosomes of diploid males of parasitic wasps do not conjugate during the first meiotic division (Torvik-Greb 1935, Pennypacker 1958, Whiting 1968). In females of parasitic wasps, meiosis occurs in the way similar to other Hymenoptera (Tagami and Miura 2007). In particular, meiotic chromosomes usually form a characteristic “bouquet” in zygotene (Stille and D¨avring 1980, Sanderson 1988). As for thelytokous forms, at least three out of five patterns of the transition to thelytoky are found in various species of parasitic Hymenoptera. Specifically, apomictic thelytoky is detected in Trichogramma cacoeciae, Closterocerus formosus (Eulophidae) and possibly also in Neuroterus quercusbaccarum (Cynipidae; Dodds 1939, Vavre et al. 2004, Adachi-Hagimori et al. 2008), gamete duplication in Trichogramma deion, Leptopilina clavipes (Figitidae), Diplolepis rosae (Cynipidae), Muscidifurax uniraptor (Pteromalidae) and E. formosa (Aphelinidae; Stille and D¨avring 1980, Stouthamer and Kazmer 1994, Gottlieb et al. 2002, Pannebakker et al. 2004, Giorgini et al. 2007) and central fusion in V. canescens (Ichneumonidae), Lysiphlebus fabarum (Braconidae), Encarsia hispida, E. guadeloupae Viggiani and E. pergandiella (Beukeboom et al. 2000, Belshaw and Quicke 2003, Giorgini et al. 2007). Terminal fusion is also supposed in Aphytis mytilaspidis (Aphelinidae; R¨ossler and DeBach 1973). Up to now, morphological diversity of meiotic chromosomes of parasitic Hymenoptera is more or less fragmentarily studied (Dodds 1939, Whiting 1968, Macy and

40

3 Morphological Features of Karyotypes of Parasitic Hymenoptera

Whiting 1969, R¨ossler and DeBach 1973, Stille and D¨avring 1980, Sanderson 1988, Gokhman et al. 1999, 2001a,b, 2002a,c, 2003, 2005b, Gokhman and Mikhailenko 2008a,b). The data obtained show that bivalents bearing the only chiasma often prevail in parasitic wasps with relatively high chromosome numbers (e.g. in gall wasps; Sanderson 1988, Gokhman et al. 1999). In contrast to that, not less than half of the bivalents in the meiotic karyotypes of many parasitic wasps with lower chromosome numbers carry at least two chiasmata. For example, five out of six bivalents of the chromosome set of Pediobius planiventris (Eulophidae) as well as three out of four bivalents in Anteon gaullei (Dryinidae) have at least two chiasmata, and every bivalent of Gonatopus clavipes from the latter family bears two chiasmata (Gokhman 2001b, 2002b,c, 2003). In the forms studied, the number of chiasmata per bivalent can vary from one to three and more, usually ranging from one to two on average. The majority of chiasmata are terminal, but subterminal and even interstitial chiasmata are detected in some cases in parasitic wasps (R¨ossler and DeBach 1973, Gokhman et al. 1999, 2002c, 2003, etc.).

sdfsdf

Chapter 4

Chromosomal Evolution of Parasitic Wasps

Abstract Various types of chromosomal mutations that occur in parasitic Hymenoptera are listed, namely, deletions and duplications, inversions, translocations, centric fusions and fissions, tandem fusions, polyploidy, aneuploidy, changes in the number of B chromosomes. Karyotypic changes in parasitic wasps at the microevolutionary level can be judged from various types of population chromosomal polymorphism, specifically, polymorphisms on the localisation and size of heterochromatic segments, translocations and B chromosomes. The mechanisms of the macroevolution of chromosome numbers in parasitic Hymenoptera are mostly asymmetrical. In the group studied, a decrease in the chromosome number occurred through chromosomal fusions, whereas an increase in this parameter was usually related to the origin of aneuploids followed by restoration of even chromosome numbers. Keywords Chromosomal mutations · Macroevolution · Microevolution · Parasitic wasps · Population polymorphism

4.1 Chromosomal Mutations Until the last few years, types and relative significance of chromosomal rearrangements in parasitic wasps were absolutely insufficiently studied due to the low degree of knowledge of parasitic Hymenoptera karyotypes, especially at the level of closely related species. However, the information accumulated (reviewed in Gokhman 2004d) allows making certain important conclusions that are given below. The classification of chromosomal rearrangements presented in Section 1.2.2 is used.

4.1.1 Changes in Chromosome Structure First of all, changes in chromosome structure of parasitic wasps as well as of other Hymenoptera include deletions and duplications that involve heterochromatic segments. These rearrangements were found e.g. in Dirophanes invisor and D. fulvitarsis (Ichneumonidae) that differ greatly in the amount and localisation of heterochromatin within the karyotypes (Gokhman 1990a, 1997b). Moreover, V.E. Gokhman, Karyotypes of Parasitic Hymenoptera, DOI 10.1007/978-1-4020-9807-9 4, C Springer Science+Business Media B.V. 2009

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4 Chromosomal Evolution of Parasitic Wasps

polymorphism involving heterochromatic segments has also been detected in the former species (see Section 3.3). Analogous rearrangements might take place in closely related species of the genus Encarsia (Aphelinidae; Baldanza et al. 1999). Moreover, tandem growth of the constitutive heterochromatin probably occurred in certain parasitic wasps with higher chromosome numbers, e.g. in Chasmias motatorius from the family Ichneumonidae (Gokhman 1985, 1990a). The presence of deletions and/or duplications probably can also be judged from differences in the relative chromosome length in some closely related forms (for example, in Aphelinus asychis, A. albipodus and A. varipes s.l.; Gokhman 2003). Although inversions are difficult to detect (especially in the homozygous condition, see Section 1.2.2), the frequency of these rearrangements can be preliminarily estimated, e.g. from differences in the number of chromosome arms between closely related species that have the same chromosome number. For example, a homozygous inversion has probably occurred in Tycherus osculator (Ichneumonidae) with 2n = 22 and NF = 44, because T. dilleri, a closely related species, has the same chromosome number and NF = 42 (Gokhman 1989, 1990a). However, there is a possibility that these changes in the arm number can also occur through the interchanges of acrocentrics and pseudoacrocentrics by deletions and tandem growth of the constitutive heterochromatin (Imai et al. 1988). Morphometric analysis of chromosomes can probably detect pericentric inversions within karyotypes of parasitic wasps in a more reliable way. Indeed, chromosome sets of the two strains of the A. varipes complex with 2n = 8 significantly differ from each other only in the value of the centromere index of the second chromosome, which also suggests an inversion in one of those strains. Analogously to other groups of Hymenoptera (see e.g. Imai et al. 1988), translocations were probably involved in the karyotypic rearrangements of many related species that have the same chromosome number but differ in chromosomal morphology. For example, differences in karyotype structure between two species of Ichneumonidae, D. invisor and D. fulvitarsis with 2n = 20, can be explained not only by deletions and duplications, but also by reciprocal chromosome translocations (Gokhman 1997b). The most conclusive evidence for a translocation between nonhomologous chromosomes followed by their meiotic nondisjunction was obtained in Tycherus bellicornis (Ichneumonidae) in which three chromosomal forms with 2n = 20 and 21 were found (Gokhman 1989, 1990a). Apparently, this is also true for Glypta ceratites from the same family with 2n = 12 and 13 (Gokhman 2003). Finally, a strong difference in the length of homologous arms of a certain subtelocentric chromosome of Ichneumon gracilicornis (Ichneumonidae) probably also resulted from translocation. A reciprocal translocation could take place between chromosomes of the first two pairs of Aprostocetus elongatus (Eulophidae; Gokhman 2003).

4.1.2 Changes in Chromosome Number Since biarmed chromosomes predominate in karyotypes of parasitic Hymenoptera, material for centric fusions in these insects is rather limited. However, Robertsonian

4.1 Chromosomal Mutations

43

fusions are likely to have occurred in the few groups of parasitic wasps in which acrocentrics prevail (e.g. in the genus Encarsia from the family Aphelinidae; Baldanza et al. 1999). Apparently, a similar rearrangement has led to the origin of the chromosome set with 2n = 8 in most Aphelinus species. In this case, karyotype with 2n = 10 known for Aphelinus mali and Aphytis mytilaspidis is likely to be the ancestral one (Viggiani 1967, R¨ossler and DeBach 1973). In addition, centric-like chromosomal fusion is supposed to have occurred in Andricus kashiwaphilus and A. targionii (Cynipidae) with 2n = 10 in comparison with the closely related A. mukaigawae that has 2n = 12 (Abe 1998, 2007). Recently, a direct evidence for the accumulation of centric fusions (or similar rearrangements) is also found in the chalcid genus Eurytoma (Eurytomidae), in which the majority of species (e.g. Eu. rosae and Eu. brunniventris from the rosae species group; see Zerova 2007) have 2n = 20 and their karyotypes mostly contain subtelocentrics of similar size, but in Eu. robusta (robusta species group) 2n = 14, and in Eu. serratulae and Eu. compressa (both belong to the tibialis species group) 2n = 12 and 10, respectively (Gokhman and Mikhailenko 2008b). Karyotype structure of these Eurytoma species with lower chromosome numbers suggests that they represent respective results of the three, four and five centric-like fusions. Studies of closely related forms also yield information on possible centric fissions. For example, most species of the subfamily Cheloninae (Braconidae) have 2n = 12 and their karyotypes contain five pairs of relatively large metacentrics and a pair of substantially smaller acrocentric chromosomes (Gokhman and Kolesnichenko 1998c). In Chelonus insularis, however, 2n = 14, and one pair of metacentrics is substituted by two pairs of acrocentrics (Silva-Junior et al. 2000b). Apparently, a centric fission has therefore occurred in the latter species. A similar situation is observed in the majority of members of the family Chalcididae in which karyotypes have five pairs of large metacentric chromosomes, whereas one of these pairs is substituted by two pairs of submetacentrics in the chromosome set of Psilochalcis brevialata with 2n = 12 (Johnson et al. 2001). The latter chromosomes have probably resulted from a centric fission with the subsequent growth of the constitutive heterochromatin on the shorter arms of the emerged acrocentrics. In addition, at least one case of the accumulation of fissions of that kind is known in Ch. motatorius (Ichneumonidae; see above). In this species, virtually all chromosomes are pseudoacrocentrics. This suggests the preceding occurrence of subsequent centric fissions accompanied by the tandem growth of heterochromatin (Gokhman 2001a). Karyotype structure of parasitic wasps therefore demonstrates that tandem fusions are considerably more frequent there than the centric ones (see Section 1.2.2). Several examples of these rearrangements can be found in the well-studied subfamily Ichneumoninae (Gokhman 1990a). For example, karyotype of T. bellicornis with 2n = 20 and NF = 38 has apparently originated by tandem fusion from the chromosome set with 2n = 22 and NF = 42 characteristic e.g. of T. dilleri (Gokhman 1989). Apparently, tandem fusion has occurred during the karyotype evolution of Ichneumon sarcitorius with 2n = 22 and NF = 44, because most other members of this genus have 2n = 24 and NF = 48 (Gokhman 1990a). An analogous

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4 Chromosomal Evolution of Parasitic Wasps

conclusion arises from the study of the chromosome set of Eurylabus torvus with 2n = 20, whereas the modal chromosome number for the subfamily Ichneumoninae is 2n = 22 (Gokhman 1987). At least one tandem fusion has taken place in Tycherus ischiomelinus if compared to T. australogeminus (Gokhman 1990a, 1991). Indeed, the karyotypes of all these species with presumed tandem fusions contain pairs of chromosomes that are distinctly longer than the others (see Section 1.2.2). Analogously to the Ichneumoninae, tandem fusions can also be presumed in other parasitic wasps with the similar karyotype structure, e.g. in Encarsia protransvena (Aphelinidae; Baldanza et al. 1999). Judging from the arm number, all aforementioned examples involve telomere–telomere fusions, but centromere–telomere fusions are likely to have occurred, e.g. in certain members of the family Eulophidae. Specifically, chromosome sets of some Aprostocetus (Aprostocetus) spp. with n = 5 have probably resulted from an acrocentric–metacentric fusion in the chromosome set with n = 6, like that of Aprostocetus (Aprostocetus) sp. 2 (Gokhman 2004e). Analogously, the karyotype of Melittobia chalybii with 2n = 10 (Schmieder 1938) might have originated through fusion of the acrocentric and metacentric chromosomes of the set with 2n = 12 characteristic of Melittobia australica (Maffei et al. 2001, Gokhman 2002c). Finally, a similar rearrangement could take place in the subfamily Megastigminae (Torymidae) with 2n = 10 if compared to the majority of Torymidae with 2n = 12 (Gokhman 2005b, Gokhman and Mikhailenko 2007, etc.). A single polyploid species of parasitic Hymenoptera, the thelytokous gall wasp Diplolepis eglanteriae with 3n = 27, is known (Sanderson 1988). Triploid and tetraploid females are found in Habrobracon hebetor and H. pectinophorae (Braconidae), Pteroptrix orientalis (Aphelinidae) and Nasonia vitripennis (Pteromalidae) (Inaba 1939, Whiting 1960, 1961, Macy and Whiting 1969, Baldanza et al. 1991a). However, self-reproducing polyploid strains were not isolated in any of these species. Aneuploid individuals are relatively often found in parasitic Hymenoptera. For example, a specimen with 2n = 25 has been detected in Ichneumon gracilentus usually having 2n = 24. A detailed analysis of the chromosome set of this parasitic wasp has demonstrated that it was a trisomic for a relatively small chromosome (Gokhman 1993). Judging from chromosome number, a trisomic with 2n = 25 has also been found in Ichneumon extensorius (Gokhman 1990a). In addition to ordinary trisomics, aneuploid parasitic wasps can originate from meiotic nondisjunction of chromosomes due to preceding translocations. In particular, these are chromosomal forms of T. bellicornis with 2n = 21 (Gokhman 1989, 1990a) and probably also of G. ceratites with 2n = 13 (Gokhman 2003). It is important that aneuploid specimens of parasitic Hymenoptera are hyperploids, i.e. these individuals carry redundant chromosomal material. Hypoploids, i.e. females with a partly haploid genome are usually not known in parasitic wasps (but see Beukeboom et al. 2007). Moreover, individuals of that kind are generally very rare in Hymenoptera as a whole; specifically, they were detected in only two populations of Australian ants of the Myrmecia pilosula complex (Imai et al. 1988). It is therefore not surprising that nullisomy (meaning the full loss of a particular segment of the genome) is absent in Hymenoptera because it would be lethal for

4.1 Chromosomal Mutations

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those insects. Finally, every chromosome in the species with a lower chromosome number (i.e. number of linkage groups) carries a bigger proportion of the genetic material than that in the species with the higher n value, and therefore not only the loss, but also an acquisition of chromosomes or their large fragments, strongly decreases the viability of aneuploids in parasitic wasps that have lower numbers of linkage groups. This may explain the fact that aneuploids are found only in parasitic Hymenoptera of the family Ichneumonidae with relatively high chromosome numbers (Gokhman 2003), but they were not detected in any well-studied group with lower n values, e.g. Chalcidoidea. Like chromosome sets of other Hymenoptera, karyotypes of certain parasitic wasps contain facultative B chromosomes. In parasitic Hymenoptera, most chromosomes of that kind carry specific factors that are termed psr (from “paternal sex ratio”). N. vitripennis from the family Pteromalidae was the first parasitic wasp where the B chromosome was found in the end of the 1980s (Nur et al. 1988). A few years prior to this discovery, a paternally transmitted heritable factor that led to the development of haploid males even from fertilised eggs was detected in N. vitripennis (Werren et al. 1987). The preliminary study of the developing eggs has shown that some chromosomes clumped into a dense mass and later became eliminated. An assumption regarding the loss of one of the two parental genomes was therefore put forward. According to the researchers’ opinion, results of the subsequent experiments using phenotypic markers seemed to confirm the full disappearance of the male genome. Since this attribute was transmitted only paternally, conclusion on the extrachromosomal nature of the studied genetic factor was considered the most plausible (Werren et al. 1987). However, this explanation could not be accepted because cytoplasmic factors of animals are transmitted only maternally. Nevertheless, the newly discovered B chromosome has been detected on cytological preparations only after identifying DNA structure of the psr factor that demonstrated its chromosomal nature. This factor has appeared to really eliminate the entire paternal genome (except for the B chromosome where this factor was located) from the diploid zygote (Nur et al. 1988, Werren 1991). The specialists, who detected the above described B chromosome, concluded that it was “the most ‘selfish’ genetic element yet described” (Nur et al. 1988). The origin of the B chromosome in the aforementioned case remains obscure, although there was an assumption that it could arise from hybridisation of N. vitripennis with a closely related species (Reed 1993). Molecular data have demonstrated that this chromosome probably got into the genome of N. vitripennis from that of a parasitic wasp of the genus Trichomalopsis (McAllister and Werren 1997). The way of the transmission of the B chromosome perhaps was more complicated because it has been shown that this chromosome was fully functional in the genomes of other species of Nasonia – N. longicornis and N. giraulti (Dobson and Tanouye 1998). Moreover, the so-called artificial B chromosome was created through experimental introgressive hybridisation of N. vitripennis and N. giraulti (Perfectti and Werren 2001). This chromosome did not contain any specific factor, but was mitotically unstable. In addition, centric fragments that differed in

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4 Chromosomal Evolution of Parasitic Wasps

their mitotic and meiotic stabilities were also induced in N. vitripennis via chemical mutagenesis (Perrot-Minnot and Werren 2001). It is worth noting that another psr-bearing B chromosome was found in the chalcid wasp Trichogramma kaykai during the last years (Stouthamer et al. 2001, Werren and Stouthamer 2003). Finally, it is quite possible that an additional chromosome which was found in some males, but not in females of Encarsia asterobemisiae (Baldanza et al. 1999), also contained a genetic element turning the diploid zygote into the haploid one. Features of the sex determination system and population structure of parasitic wasps are likely to predispose them to the acquisition of genomic factors that substantially change offspring sex ratio (Gokhman 2003). Indeed, the loss of the paternal genome in parasitic wasps is not necessarily due to B chromosomes, e.g. as in Encarsia pergandiella (Hunter et al. 1993). It is interesting that the so-called cytoplasmic msr factor (perhaps a microorganism) that altered the sex ratio in favour of females was also detected in N. vitripennis (Skinner 1982).

4.2 Microevolutionary and Macroevolutionary Karyotypic Changes As in other Hymenoptera, karyotypic changes in parasitic wasps at the microevolutionary level can be judged from various types of population chromosomal polymorphism, namely, polymorphisms on the localisation and size of heterochromatic segments, translocations and B chromosomes. Inversion polymorphism is also likely to exist in parasitic Hymenoptera, but it is still not revealed in the group (this can be explained by certain technical difficulties). Translocation polymorphism that involves nonhomologous chromosomes resulting from meiotic nondisjunction leads to the appearance of aneuploids, which substantially increases the karyotypic diversity of the population. Various examples of population polymorphism in parasitic Hymenoptera are given in the preceding section. As for the traits of chromosomal macroevolution of parasitic wasps, they will be considered here in a very general form (mainly using differences between closely related species), because it will be done in detail after the discussion of phylogenetic data for various groups (see Section 5.2). Obviously, both increases and decreases in chromosome number occurred in karyotype evolution of parasitic Hymenoptera; however, these processes considerably differ both in their frequency and particular kinds of chromosomal rearrangement that underlie them. Indeed, the decrease in chromosome number of parasitic Hymenoptera occurred only through chromosomal fusion, because hypoploids are not found in this group (see above). Moreover, the reduction in chromosome number that took place in closely related forms of parasitic wasps was usually accompanied by the appearance of a pair of large chromosomes that presumably resulted from chromosomal fusion. Finally, judging from the prevalence of biarmed chromosomes in the karyotypes of parasitic Hymenoptera, tandem fusions of the telomere–telomere type mainly occur in these insects, although centric and centromere–telomere fusions can also take place in certain cases (see Section 4.1).

4.2 Microevolutionary and Macroevolutionary Karyotypic Changes

47

However, reconstruction of the mechanisms of the increase in chromosome number in parasitic wasps poses a more complex problem. Except for the single case of triploidy known in D. eglanteriae from the family Cynipidae (see above), the increase in this parameter is likely to result from chromosomal fission. Several cases of that kind have actually been observed in parasitic wasps (see Section 4.1), although, for example, chromosomal fusions took place instead of the presumed fissions in certain chalcids (see Goodpasture and Grissell 1975 and Section 5.1). Comparing n values of closely related species of parasitic Hymenoptera that differ from each other in the single pair of biarmed chromosomes may yield additional information on the ways of increasing chromosome numbers. If the smaller of the two 2n values is modal for the given group (e.g. 2n = 24 in the genus Ichneumon), it may be assumed that aneuploidy has occurred in parasitic wasps with the larger 2n value. In turn, this event was followed by the restoration of an even chromosome number through a complete or, more probably, partial tetrasomy. Evidently, I. extensorius, I. suspiciosus and I. inquinatus have acquired karyotypes with 2n = 26 in this way (Gokhman 1993). The chromosome set of Aethecerus dispar (Ichneumonidae) with 2n = 24 that is closely related to Ae. ranini with the modal 2n = 22 apparently is of the same descent (Gokhman 1991). Individuals of Charmon cruentatus (Braconidae) with 2n = 12 have probably also appeared in the same way within populations with 2n = 10 (Gokhman 2002a). Thus, mechanisms of the evolution of chromosome numbers in parasitic Hymenoptera were mostly asymmetrical (Gokhman 2003). In the group studied, a decrease in the chromosome number occurred through chromosome fusions (mainly, tandem fusions), whereas an increase in this parameter was usually related to the origin of aneuploids followed by the restoration of an even chromosome number. In the last case, duplication of certain segments of the diploid genome actually took place (Gokhman 2003, 2004d). As noted in Section 3.2, the number and size of chromosomes are inversely correlated. This means that all chromosomal fusions and fissions affect size parameters of karyotypes. However, processes and mechanisms of the size differentiation of elements of chromosome sets are the most interesting. It is obvious that the fusion of medium-length chromosomes immediately results in the appearance of elements that considerably differ in size from the remaining chromosomes. At the same time, translocations can also change the relative sizes of chromosomes. Examples of these rearrangements are given in the first section of the present chapter. Regrettably, mechanisms of the increase in the proportion of acrocentrics in the karyotypes of parasitic Hymenoptera are relatively poorly studied, because an analysis of the corresponding chromosomal rearrangements is hampered by the taxonomic separateness of the groups with the described properties. Since acrocentric chromosomes originate through centric fissions, deletions of heterochromatic arms of pseudoacrocentrics and inversions in other Hymenoptera (Imai et al. 1988), this may also be true for parasitic wasps in which all these rearrangements have been observed. The relative significance of different groups of karyotypic rearrangements remains obscure, although they are generally fairly restricted with respect to the scale of their manifestation. Evidently, many features of the main

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cycle of chromosomal evolution suggested by Imai et al. (1988) for ants and other Hymenoptera, including centric fissions of biarmed chromosomes that result in the origin of acrocentrics and their further transformation into new metacentrics (in a broad sense) due to pericentric inversions and the tandem growth of the constitutive heterochromatin, are seldom found in parasitic wasps. As shown above, the increase in size differentiation of chromosomes combined with the increase in the proportion of acrocentrics in the karyotype can be considered as components of an integral process of karyotypic dissymmetrisation. This dissymmetrisation, however, is not only limited in scale but is also reversible in terms of evolution (see Section 5.2). The macroevolutionary changes in the number of chiasmata, recently known in parasitic wasps, are also indirectly related to the differences in chromosome number. Indeed, the number of chiasmata per bivalent in many groups of parasitic Hymenoptera with small chromosome numbers (chalcids, Dryinidae, etc.) is almost twice as high as in parasitic wasps with larger 2n values, such as gall wasps (R¨ossler and DeBach 1973, Gokhman 1999, 2001a, 2002b,c). Obviously, various traits of karyotype evolution of parasitic wasps are determined by a complex of external and internal factors. The external factors of chromosomal evolution will be mentioned during the phylogenetic analysis of karyotypic variation of certain groups of parasitic Hymenoptera (see Chapter 5). The internal factors of the evolution of chromosome sets partly include restrictions imposed by karyotypic organisation (see e.g. Section 4.1). In addition, the mode of sex determination in parasitic wasps is also likely to determine the range of preferential traits of karyotype evolution of this group. Indeed, the relatively high frequency of translocations and aneuploid karyotypes in parasitic wasps may be explained by the lack of meiotic control of these rearrangements (i.e. synapsis of homologues and the reductional division) in haploid males. Moreover, the robustness of parasitic and other Hymenoptera to ploidy changes also makes aneuploid forms of these insects more widespread (Gokhman 2003). Haplodiploidy is the possible reason for the existence of specific B chromosomes in certain species of parasitic wasps. These B chromosomes carry special factors that alter sex ratio of the offspring (see Section 4.1).

Chapter 5

Phylogenetic Implications of Karyotypic Characters of Parasitic Hymenoptera

Abstract Karyotypic data show that n = 14–17 should be considered as the initial chromosome number for parasitic wasps. Apparently, the same values are initial for aculeate Hymenoptera, with the chromosome number being substantially reduced at least in the family Dryinidae. A parallel decrease in chromosome number occurred independently and repeatedly in Ichneumonidae, Braconidae and Microhymenoptera. Low chromosome numbers (n = 9–11) are a synapomorphy of Diaprioidea, Cynipoidea, Platygastroidea, Ceraphronoidea and Chalcidoidea. In addition, chromosome numbers were reduced to n = 5–6 in several groups of Chalcidoidea. Following main trends of evolutionary change of karyotypes of parasitic wasps are found: (1) decrease in the chromosome number through tandem fusions (less frequently, centric ones) and (2) karyotypic dissymmetrisation through an increase in the size differentiation of chromosomes and/or in the proportion of acrocentrics in the karyotype.

Keywords Decrease in chromosome number · Hymenoptera · Initial chromosome number · Karyotypic dissymmetrisation · Parasitic wasps

5.1 Phylogenetic Analysis of Chromosomal Characters Different karyotypic characters of the order Hymenoptera are unequal with respect to their use in the phylogenetic analysis in terms of the presumption of weighted similarity (see Section 1.3.2.1). As in other Hymenoptera, chromosome numbers of parasitic wasps are the less phylogenetically reliable among these characters. Indeed, identical chromosome numbers are known for many unrelated taxa of parasitic wasps. In particular, the lowest chromosome number known for parasitic Hymenoptera, n = 3, was recorded in the four species belonging to different families (see Section 3.1), and the second highest number, n = 21, was also found in two species belonging to different superfamilies, Perithous scurra (Ichneumonidae) and Chrysis viridula (Chrysididae; Quicke and Gokhman 1996, Gokhman and Kolesnichenko 1997). V.E. Gokhman, Karyotypes of Parasitic Hymenoptera, DOI 10.1007/978-1-4020-9807-9 5, C Springer Science+Business Media B.V. 2009

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However, chromosome numbers are the only karyotypic characteristic known for all species studied; therefore they are inevitably used in the phylogenetic analysis of chromosomal variation. It is shown in Section 1.3.2.2 that modal chromosome numbers are the best parameters for this purpose. The distributions of species with respect to chromosome numbers are monomorphic or close to normal in many families of parasitic wasps (Table 5.1); therefore, modal chromosome numbers of these groups may be considered initial, at least as a first approximation. The comparatively small variance of this parameter in parasitic wasps (Table 5.1) is an additional criterion for a relative evolutionary stability of chromosome numbers. It is interesting that the variance of chromosome numbers of some genera belonging to certain families of aculeate Hymenoptera, Formicidae and Vespidae, is more than 40 and 50, respectively (Gokhman 2003). If the modal number does not coincide with the median one or there is more than one modal number, this may mean that it is impossible to reliably determine the initial chromosome number for a particular group. In these cases, the entire variation range of chromosome numbers characteristic of the given taxon should be used in the subsequent analysis (Gokhman 2003). Other karyotypic features, e.g. combinations of chromosomal measurements, can certainly also be used in the phylogenetic analysis. Characters of this kind are Table 5.1 Characteristics of the distributions of species by chromosome number in the families of parasitic wasps

Family

Number of studied species

Ichneumonidae 153 Braconidae 62 Gasteruptiidae 2 Cynipidae 23 Figitidae 4 Diapriidae 4 Scelionidae 2 Megaspilidae 1 Mymaridae 2 Eurytomidae 11 Encyrtidae 13 Aphelinidae 22 Chalcididae 5 Eulophidae 41 Eupelmidae 5 Leucospidae 1 Ormyridae 2 Perilampidae 1 Pteromalidae 15 Torymidae 18 Trichogrammatidae 12 Bethylidae 2 Chrysididae 4 Dryinidae 8

Variation range of n Modal n values values

Median n values

Supposed initial n values

Variation

6–21 3–23 14–16 5–10 5–11 8–11 10 9 9 5–10 8–12 3–11 3–6 5–8 5–6 6 5–6 3 4–7 4–10 5 10–14 19–21 4–7

11 9 15 10 10 10.5 10 9 9 10 11 5 5 6 5 6 5.5 3 5 6 5 12 19 4

11 Unknown Unknown 10 10 Unknown 10 9 9 10 11 Unknown 5 6 5 6 Unknown 3 5 6 5 Unknown 19 4

5.8 22.1 2.0 2.5 7.3 2.0 0 0 0 3.6 1.3 6.6 1.2 0.3 0.3 0 0.5 0 0.4 2.3 0 8.0 1.0 1.1

11 6, 10 Absent 10 10 11 10 9 9 10 11 5, 9 5 6 5 6 Absent 3 5 6 5 Absent 19 4

5.1 Phylogenetic Analysis of Chromosomal Characters

51

usually more reliable due to the presumption of weighted similarity. For example, chromosome sets of many parasitic wasps of the family Eulophidae include five pairs of large biarmed chromosomes and a pair of small subtelo-/acrocentrics (Gokhman 2002c, 2004e). This karyotypic feature is the initial one for the family and probably represents a synapomorphy of Eulophidae. However, the character described is often lost in various groups of the family (see below). An analysis of initial chromosome numbers and comparatively narrow variation ranges of this character shows that these numbers fall within the range of n = 9–11 in most parasitic wasps (Gokhman and Quicke 1995). Exceptions from this rule are, on one hand, many Chalcidoidea with the initial n = 5–6 and, on the other hand, Gasteruptiidae with n = 14–16 (Table 5.1). Thus, haploid chromosome numbers of about ten can be regarded as initial for most parasitic Hymenoptera (Gokhman and Quicke 1995), probably except for the two aforementioned groups. Various assumptions on the chromosome numbers initial for Gasteruptiidae and the whole superfamily Chalcidoidea are given below. As for chalcids, chromosome numbers initial for some families also fall within the range of 9–11, although the initial n = 5–6 is obviously characteristic of the majority of Chalcidoidea. The reduction in chromosome numbers therefore took place in the evolution of this superfamily (Gokhman and Quicke 1995). Moreover, if the outgroup presumption is taken into account, this reduction is likely to have occurred within that superfamily. Regretfully, various families of parasitic wasps from the superfamily Chrysidoidea differ in chromosome numbers to such an extent that it is very difficult to restrict the presumed initial number for this group to any particular range. Before coming to a more detailed phylogenetic analysis of chromosomal variation in parasitic Hymenoptera, it is necessary to comment on the members of this group that have comparatively high chromosome numbers. Indeed, Crozier (1975) noted in this review published more than 30 years ago: “The most puzzling aspect of the overall karyotypic evolution of the Hymenoptera is the occurrence of low chromosome numbers in the Parasitica followed by the reappearance of high numbers in aculeates”. However, groups with n = 14–23 were found during the last 20 years in the families Ichneumonidae, Braconidae, Gasteruptiidae and Chrysididae (Gokhman 1997a, 2002a, 2003, Kitthawee et al. 2004). In addition, all studied parasitic wasps of the families Mymaridae, Encyrtidae and the majority of Eurytomidae (superfamily Chalcidoidea) have relatively high chromosome numbers (n = 8–12; Gokhman 2000a), while Aphelinidae are characterised by wide ranges of variation of this character not only in the entire group, but also in individual genera (Baldanza et al. 1999). Preliminary examination of the aforementioned taxa demonstrates that many of them are traditionally considered as weakly specialised (Gokhman 1997a, 2000a). These are the families Gasteruptiidae, Mymaridae and Eurytomidae as well as the subfamilies Pimplinae (Ichneumonidae) and Doryctinae (Braconidae). Finally, modal numbers of the majority of families of aculeate Hymenoptera fall into one of the two ranges, n = 14–17 or 25–26 (see Section 1.2.1). Data on chromosome numbers of many Symphyta also do not shed any light on the initial chromosome number of parasitic Hymenoptera. In particular, members of Tenthredinoidea have

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5 Phylogenetic Implications of Karyotypic Characters of Parasitic Hymenoptera

n = 5–22, Pamphilioidea – n = 11–35, Cephoidea – n = 9–26 and Siricoidea – n = 8–18 (Westendorff 2006). Thus, even approximate estimates of initial chromosome numbers and other characteristics of the karyotype structure are impossible without an analysis of their phylogeny (see Section 1.3.2.1). For this purpose, phylogenetic reconstructions based on morphological, molecular and other data should be used; it is also desirable that these reconstructions are created using computerised cladistics that allows formalising assumptions on which the aforementioned reconstructions are based. Among the few papers of that kind which appeared during the last decade, the study by Quicke et al. (1999) embraces the largest amount of various systems of morphological and behavioural characters. Regretfully, the phylogenetic tree of hymenopteran families provided in this work and later reproduced by Rasnitsyn (2002a) was partly based on the review of chromosome numbers of the order (provided by the author of the present book), and thus could not be used for the analysis of karyotypic features of Hymenoptera from the phylogenetic viewpoint. Phylogenetic reconstructions created by Ronquist et al. (1999) on the basis of the data provided by Rasnitsyn (1988) were therefore used in this book. The shortest possible tree of modern families of the order Hymenoptera (see Ronquist et al. 1999) was used as the main phylogenetic scheme. To be precise, a number of papers trying to reassess the results described in the latter paper, appeared during the last years (summarised in Sharkey 2007). For example, Sharkey and Roy (2002) have declared that wing venation characters used by Ronquist et al. (1999) should be excluded from tree construction, because their variation is correlated with the decreasing body size in Hymenoptera. However, the phylogenetic tree created by Sharkey and Roy (2002) was so less resolved if compared to that given in the paper by Ronquist et al. (1999) that it probably does not allow serious discussion of evolution of the majority of morphological (including chromosomal), behavioural and other characters of Apocrita at the superfamily level. Among the works on the phylogeny of Apocrita that were published in the 2000s, the paper by Dowton and Austin (2001) perhaps deserves the closest attention, because these authors used a set of molecular characters of the Hymenoptera. Nevertheless, the phylogenetic reconstructions obtained, first, have appeared pretty close to those created by Ronquist et al. (1999), and, second, showed that the topology of the reconstructions produced by Dowton and Austin (2001) was subject to substantial changes due to the assumptions used in the phylogenetic analysis of DNA sequences (see also Castro and Dowton 2006). Until recently, most researchers believed that the phylogenetic tree of Apocrita contained two principal branches. The first lineage included members of the superfamily Ichneumonoidea and aculeate Hymenoptera, the second one all other apocritans (Rasnitsyn 1980, 1988, 2002b, Quicke et al. 1999; see also Sharkey 2007). This reconstruction, however, has been put under doubt in the work by Dowton and Austin (2001) as well as in that by Ronquist et al. (1999). In particular, the shortest trees of contemporary taxa given in the last paper, include Parasitica and Aculeata as sister taxa, although phylogenetic reconstructions of the same groups

5.1 Phylogenetic Analysis of Chromosomal Characters

53

that were produced using a posteriori weighting, testify for the earlier opinion (Rasnitsyn 1980, etc.). Analogous contradictions between the cited papers exist on the position of other taxa, e.g. the superfamily Ceraphronoidea (Rasnitsyn 1988, Ronquist et al. 1999). Taking these contradictions into account, the most detailed analysis of chromosomal variation of parasitic Hymenoptera should be performed. It must be done at the level of families, and even to the level of subfamilies, tribes and certain genera in the case of the two families belonging to Ichneumonoidea, specifically, Ichneumonidae and Braconidae. One of the most recent phylogenetic reconstructions of the family Ichneumonidae that is based on both morphological and molecular characters was performed by Belshaw and Quicke (2002). Up to date, 15 subfamilies of Ichneumonidae, i.e. about a half of the taxa of this level (Table 5.2), are involved in chromosomal studies (Gokhman 2001a, Gokhman and Mikhailenko 2008a). The mentioned parasitic wasps belong to all large groups of subfamilies that are known in the Ichneumonidae, namely, Pimpliformes (Pimplinae, Microleptinae and Orthocentrinae), Ichneumoniformes (Adelognathinae, Cryptinae and Ichneumoninae) and Ophioniformes (Tryphoninae, Anomaloninae, Ctenopelmatinae, Metopiinae, Mesochorinae, Banchinae, Campopleginae and Cremastinae). As for the subfamily Orthopelmatinae, it was placed by Quicke et al. (2000) in the separate group, Orthopelmatiformes.1 The modal chromosome number for the family, n = 11, is also modal for Ichneumoniformes and Ophioniformes, although Orthopelmatiformes and most Pimpliformes have n = 152 and 14–21, respectively (Fig. 5.1). However, parasitic Table 5.2 Chromosome numbers of subfamilies of the family Ichneumonidae Subfamily

Number of studied species

Haploid chromosome numbers (n)

Pimplinae Microleptinae Orthocentrinae Adelognathinae Cryptinae Ichneumoninae Orthopelmatinae Tryphoninae Anomaloninae Banchinae Ctenopelmatinae Campopleginae Cremastinae Mesochorinae Metopiinae

18 1 4 1 9 92 1 3 1 8 2 6 1 1 4

8, 9, 13–18, 20, 21 16 10, 14, 18 10 8, 10, 13, 16 8–14, 17 15 6, 10 8 6, 7, 9, 11 11, 12 8, 11, 12 8 7 8, 11

1

Although this group was later merged with Ophioniformes (Belshaw and Quicke 2002), it is retained in the present analysis.

2 An early record for Orthopelma mediator, n = 11 (Hogben 1920), is not confirmed by our recent study (Gokhman and Mikhailenko 2008a).

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5 Phylogenetic Implications of Karyotypic Characters of Parasitic Hymenoptera

Fig. 5.1 Phylogenetic tree of subfamilial groups of the family Ichneumonidae (simplified from Belshaw and Quicke 2002; subfamilial groups are given according to Quicke et al. 2000) with variation ranges of haploid chromosome numbers superimposed onto it. Modal chromosome numbers are given in parentheses, aberrant n values that differ from those characteristic of certain taxa are given in square brackets

wasps of the tribe Polysphinctini are the exception for the latter group because they have n = 8–13. Taking into account that this tribe is highly specialised in terms of larval morphology and biological features (Fitton et al. 1988), lower chromosome numbers that are characteristic of this taxon, must be considered autapomorphic (Gokhman 2003), and higher n values found in other Pimplinae should be treated as initial for this subfamily (Gokhman and Kolesnichenko 1997). In the Pimpliformes, secondary reduction of chromosome number is also supposed in Plectiscus impuratus (2n = 20) and Theronia atalantae (2n = 24). The latter genus from the family Pimplinae was previously united together with Perithous (2n = 42) in the tribe Delomeristini, but Perithous was later transferred to the tribe of its own, Perithoini (Wahl and Gauld 1998, but see Gauld et al. 2002). Identification of the initial chromosome number for all Ichneumonidae is a more difficult problem. At first glance, it seems that n = 11, the modal chromosome number for many subfamilies, should be considered initial for all Ichneumonidae. However, a few circumstances do not favour this assumption. First of all, this number is actually modal only for Ophioniformes and the subfamily Ichneumoninae. In addition, higher chromosome numbers observed in some Ichneumoninae (e.g. in Chasmias motatorius) are probably secondary (Gokhman 1990b). In most other Ichneumoniformes (i.e. in the subfamily Cryptinae), the distribution of chromosome numbers is characterised by a wide variation range (n = 8–16) and has no distinct maximum. Finally, n = 14–17 in almost all less-advanced parasitic wasps of the family Braconidae as well as in most Pimpliformes (see below). Thus, a haploid chromosome number close to 11 can be considered as initial only for Ophioniformes and the subfamily Ichneumoninae, and n = 14–17 – for Pimpliformes and Orthopelmatiformes. Therefore, each of these numbers could appear initial for the Ichneumonidae. The accumulated data allow drawing certain conclusions on the mode of chromosomal variation of some best studied subfamilies and lower taxa of Ichneumonidae. In particular, most of the examined tribes of the subfamily Pimplinae (Ephialtini,

5.1 Phylogenetic Analysis of Chromosomal Characters

55

Pimplini and Perithoini) are characterised by chromosome numbers that are higher than those found in many other Ichneumonidae (except for the majority of remaining Pimpliformes as well as certain members of Ichneumoninae and Cryptinae; Gokhman 1990b). However, most parasitic wasps of the tribe Polysphinctini have substantially lower chromosome numbers if compared not only to the rest of Pimplinae, but to many other Ichneumonidae that have the modal n = 11. In these cases, lower chromosome numbers must be considered as apomorphies of certain groups (Gokhman 2001a, 2003). Differences in variation ranges of chromosome numbers are observed between all tribes of the subfamily Cryptinae, i.e. Phygadeuontini (n = 13–16), Cryptini (n = 10) and Hemigasterini (n = 8), although the amount of karyotypic data is too small to testify in favour of the real absence of overlapping of these ranges. In addition, karyotypic data suggest that two tribes of the subfamily Tryphoninae, Phytodietini and Tryphonini, also have different chromosome numbers (n = 6 and 10, respectively). In particular, substantial differences in karyotypic patterns between lower taxa are found in the subfamily Ichneumoninae, the best studied group of Ichneumonidae (Gokhman 2001a, etc.). Thus, diversity of chromosome numbers in the tribe Ichneumonini (2n = 16–34) is substantially higher than that in the tribe Alomyini (= Phaeogenini sensu Perkins; 2n = 18–24), although modal numbers of these groups are very close to each other and they even coincide (2n = 22) at the genus level, e.g. if counted according to the “genus-karyotype” concept (Gokhman 1990b, 1993). If common features of karyotypic structure of various groups of Ichneumoninae are considered, most Alomyini (except for specialised forms) have 2n = 22 and NF close to 44 (all or almost all chromosomes are biarmed), the subtribe Cratichneumonina has 2n = 22 or usually more (their karyotypes contain at least one or more pairs of acrocentrics), most Ichneumonina have 2n more than 22, although all chromosomes are usually biarmed (Gokhman 1990b, 1993). Since Alomyini are often considered as a group ancestral for all other Ichneumoninae (Gokhman 1988, 1992, but see Laurenne et al. 2006), karyotypes of this subfamily mainly evolved towards an increase in chromosome number, and this evolution was also accompanied by an increase in the proportion of acrocentrics in the karyotypes of Cratichneumonina. An attempt of explanation of these traits of karyotypic changes is given below. The process of karyotype evolution in the Ichneumoninae may be explained using data on the different modes of phylogenetic change of various groups of these insects. According to this concept, the least specialised members of the tribe Alomyini attack prepupae and pupae of small Lepidoptera in exposed cocoons or cases (Gokhman 1988, 1992). Many ectoparasites of the family Cryptinae, the putative sister group to the Ichneumoninae (Gokhman 1992), are morphologically very close to Alomyini and attack hosts ecologically similar to those of the latter group. The author believes (Gokhman 1990b) that it was the competition with Cryptinae which prevented Alomyini from explosive adaptive radiation; in fact, the latter tribe occupies an adaptive zone that must be considered initial for all Ichneumoninae. Originating from this zone, the advanced Ichneumoninae then switched to larger Lepidoptera and therefore occupied a new adaptive zone where competitors from related groups were absent (Gokhman 1988). In the latter zone, processes of the

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5 Phylogenetic Implications of Karyotypic Characters of Parasitic Hymenoptera

intensive adaptive radiation occurred. In particular, these processes have resulted in the origin of the large amount of hardly discernible species in the subtribe Ichneumonina (Heinrich 1967). Todd (1970) suggested a few assumptions regarding the correlation between the intensity of adaptive radiation and the evolution of chromosome numbers. According to this paper, chromosome number is moderate in the members of ancient taxa which evolution was not markedly divergent. At the same time, diploid numbers usually increase in groups that underwent an intensive adaptive radiation. According to Todd (1970), this is the result of an increase in combinative variation. Some other researchers (e.g. Emelyanov and Kirillova 1991) also have similar opinions on this subject. A comparison of the aforementioned assumptions to the characteristics of chromosome numbers of the Ichneumoninae demonstrates that the idea regarding correlation between the intensity of the adaptive radiation and the chromosome number that has been formulated by Todd (1970) generally applies for this subfamily as well. Indeed, Alomyini did not come through the outburst of the adaptive radiation and have therefore retained their initial chromosome number (2n = 22). This number decreases to 2n = 20 and 18 only in specialised members of the tribe. Unlike Alomyini, chromosome numbers in the tribe Ichneumonini, an intensively radiated group, are usually higher than the initial number and it also agrees well with Todd’s ideas. An increase in the combinative variation in the subtribe Cratichneumonina, along with a certain increase in 2n values, is also explained by an increase in the proportion of acrocentric chromosomes within karyotypes. The general tendency for the increased recombination is accomplished in the phylogeny of various groups of Ichneumonini in two different ways, i.e. in an increase either in the chromosome number or in the proportion of acrocentrics in the chromosome set. It is interesting that while chromosome numbers of Ichneumonini generally increase, the modal number in the tribe remains virtually unchanged if compared to less-advanced groups (Gokhman 2003). Apart from Ichneumonidae, a detailed phylogenetic study of the family Braconidae has been done about 20 years ago (Quicke and van Achterberg 1990) without using molecular data. Regretfully, certain technical flaws were revealed in this work (Wharton et al. 1992), and therefore position of many taxa on the obtained phylogenetic trees seemed dubious. Taking these circumstances into account, a phylogenetic scheme of the family was constructed by Dowton et al. (2002) on the basis of morphological and molecular characters. According to this scheme, karyotypically studied groups of the family are subdivided into two lineages. The first of them includes the following subfamilies: Doryctinae, Opiinae, Alysiinae, Braconinae and Exothecinae, i.e. cyclostome braconids, as well as Aphidiinae, and the second the other groups (the subfamilies Charmontinae, Macrocentrinae, Meteorinae, Miracinae, Microgastrinae, Agathidinae and Cheloninae; Fig. 5.2, Table 5.3) (Gokhman 2004c). Results of the phylogenetic study of the subfamily Aphidiinae published by Smith et al. (1999), Belshaw and Quicke (1997) and Quicke et al. (1999) are pretty similar, although phylogenetic reconstructions given in the two latter papers are

5.1 Phylogenetic Analysis of Chromosomal Characters

57

Fig. 5.2 Phylogenetic tree of karyotypically studied subfamilies of the family Braconidae (simplified from Dowton et al. 2002) with variation ranges of haploid chromosome numbers superimposed onto it. Modal chromosome numbers are given in parentheses, aberrant n values that differ from those characteristic of certain taxa are given in square brackets (modified after Gokhman 2004c)

used in the present work because they are supported not only by molecular and biological information, but also by morphological data as well. If the data on chromosome numbers are superimposed onto the phylogenetic tree of the tribes, n = 7 can be found in all of them except for the Praini with the autapomorphic n = 4 (Gokhman 2003). Since the former value is characteristic of all members of the

Table 5.3 Chromosome numbers of subfamilies of the family Braconidae Subfamily

Number of studied species

Haploid chromosome numbers (n)

Doryctinae 1 17 Exothecinae 1 6 Braconinae 5 10 Opiinae 8 14, 17, 19, 20, 23 6 11, 16, 17 Alysiinae∗ Aphidiinae 20 3–7, 9 Meteorinae 4 8–10 Macrocentrinae 1 14 Charmontinae 1 5, 6 Agathidinae 3 9, 11 Cheloninae 5 6, 7 Microgastrinae 6 9–11 Miracinae 1 10 ∗ Haploid chromosome number of 17 is given for Asobara tabida.

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5 Phylogenetic Implications of Karyotypic Characters of Parasitic Hymenoptera

least advanced tribe Ephedrini, and also because of the outgroup presumption (see above), n = 7 is likely to be initial for the Aphidiinae. However, if the information on chromosome numbers and biological features of other groups of Ichneumonoidea is taken into account, n = 7 certainly cannot be considered as initial for the whole family Braconidae. Under these circumstances, either n = 10–11 or 14–20 can be initial for the family. Chromosome numbers close to 10 or 11 therefore prevail in the superfamily Ichneumonoidea. The following lineages are exceptions to this rule: Pimpliformes (Pimplinae, Microleptinae and Orthocentrinae) with n = 14–21 (except for a few species with lower chromosome numbers), Orthopelmatiformes with n = 15, some (mainly cyclostome) Braconidae (Doryctinae, Opiinae, Alysiinae and Macrocentrinae) with n = 14–23 and Aphidiinae with n = 3–9. Karyotypes of parasitic wasps of the superfamily Chrysidoidea are very diverse (see above). In particular, haploid chromosome numbers of different members of this group vary from 4 to 21 (Gokhman 2000a, 2001b, 2002b). Moreover, variation ranges in this parameter in the studied families of Chrysidoidea, namely, Bethylidae (n = 10–14), Chrysididae (n = 19–21) and Dryinidae (n = 4–7), do not overlap (Fig. 5.3). Even if n = 4 and 19 are considered as modal for the Dryinidae and Chrysididae, respectively, it is very difficult to determine the initial chromosome number for the whole superfamily, regardless of the used phylogenetic reconstruction (Brothers and Carpenter 1993, Brothers 1999, Carpenter 1999, Ronquist et al. 1999). Indeed, n = 10–19 is likely to be initial for both the Bethylidae and the Chrysididae; at the same time, initial n values for the whole superfamily fall within the range of 4–19. Nevertheless, modal numbers of many other aculeate Hymenoptera, for example, Scolioidea, Pompiloidea, Vespoidea as well as of the majority of Formicoidea, Sphecoidea and Apoidea vary from 14 to 26 (Gokhman 2003, 2007c), and therefore the initial chromosome number for all Chrysidoidea falls within the range of 14–19. If it is true, lower chromosome numbers found in Dryinidae and probably also in Bethylidae are apomorphic.

Fig. 5.3 Phylogenetic tree of families of the superfamily Chrysidoidea (after Brothers and Carpenter 1993) with variation ranges of haploid chromosome numbers superimposed onto it. Modal chromosome numbers are given in parentheses (modified after Gokhman 2005a)

5.1 Phylogenetic Analysis of Chromosomal Characters

59

In addition to the aforementioned groups, parasitic Hymenoptera include several superfamilies (e.g. Megalyroidea, Evanioidea, Trigonalyoidea and Stephanoidea) that were assigned to the group Evaniomorpha by Rasnitsyn (1980). Chromosomes have been studied only in two species of the family Gasteruptiidae (superfamily Evanioidea) with n = 14–16, i.e. with relatively high chromosome numbers (Hoshiba and Imai 1993, Quicke and Gokhman 1996). All other karyotypically studied families of parasitic wasps, namely, Diaprioidea, Cynipoidea, Platygastroidea, Chalcidoidea and probably also Ceraphronoidea, are sometimes grouped under the common name Microhymenoptera (Rasnitsyn 1980). In many members of this group, modal chromosome numbers are 9, 10 or 11, except for most chalcids with n = 5–6 and Aphelinidae; the latter group has two modal numbers, n = 5 and 9, which belong to both intervals (Table 5.4). In some families of Chalcidoidea chromosome numbers characteristic of other Microhymenoptera (n = 9–11) are the most frequent; hence, these values are likely to be initial for the group studied (Gokhman 2000b). In most Chalcidoidea, however, the chromosome numbers are close to five or six; therefore, the mean chromosome number has decreased by about twofold in many families of Chalcidoidea (Gokhman and Quicke 1995). If this is correct, the described reduction took place within the superfamily and it

Table 5.4 Chromosome numbers of parasitic wasps of the superfamilies Cynipoidea, Diaprioidea, Platygastroidea, Ceraphronoidea and Chalcidoidea Family

Number of studied species

Haploid chromosome numbers (n)

Cynipoidea Cynipidae Figitidae

23 4

5, 6, 9, 10 5, 10, 11

Diaprioidea Diapriidae

4

8, 10, 11

Platygastroidea Scelionidae

2

10

Ceraphronoidea Megaspilidae

1

9

Chalcidoidea Aphelinidae Chalcididae Encyrtidae Eulophidae Eupelmidae Eurytomidae Leucospidae Mymaridae Ormyridae Perilampidae Pteromalidae Torymidae Trichogrammatidae

22 5 13 41 5 11 1 2 2 1 15 18 12

3–11 3, 5, 6 8–12 5–8 5–6 5–7, 9, 10 6 9 5, 6 3 4–7 4–6, 10 5

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5 Phylogenetic Implications of Karyotypic Characters of Parasitic Hymenoptera

occurred at least three times, i.e. in the common ancestor of the families with n = 5–6 as well as in certain Aphelinidae and Eurytomidae that have lower chromosome numbers (Gokhman and Mikhailenko 2008b, etc., see also Fusu 2008d). Decreases of that kind could certainly independently repeat in various groups of Chalcidoidea (see below). For the further analysis of karyotype evolution of Chalcidoidea, it is necessary to compare data on chromosome sets of these insects to their phylogeny. Regretfully, phylogenetic reconstruction of chalcids at the family level that is based on both morphological and molecular data is not published yet. The most detailed contemporary analysis of the phylogeny of Chalcidoidea (Campbell et al. 2000) is mostly preliminary because it is based on the results of the study of the only DNA sequence. Nevertheless, several important conclusions can be drawn on the basis of the phylogenetic tree presented in this paper. Since the fact that the family Mymaridae is the least advanced group of chalcids is supported not only by molecular data, but also by morphological ones as well (Gibson 1986, Noyes 1990, Gibson et al. 1999), n = 9 (or 10–11) is probably initial for this superfamily (Gokhman 2000b). Two branches come off the base of the chalcid phylogenetic tree almost immediately after Mymaridae. These lineages harbour aphelinids of the subfamily Coccophaginae (including members of the genera Coccophagus and Pteroptrix with n = 11) as well as Encarsia with n = 3–10. According to the molecular data, a few other lineages of Aphelinidae occupy a more distal position. In particular, two tribes of the subfamily Aphelininae, Aphelinini (n = 4–5) and Aphytini (n = 5) form a monophyletic group. The reduction of chromosome numbers in Aphelinidae therefore occurred repeatedly – within the genus Encarsia (perhaps also in a few lineages) and in parasitic wasps of the Aphelinini + Aphytini branch (Gokhman 2003). Taking into account the facts that the haploid set, e.g. of Aphytis includes a metacentric and four acrocentric chromosomes, the karyotype of Aphelinus varipes s.l. and A. albipodus includes two metacentric chromosomes and two acrocentrics, and at the same time Aphelinus mali also has n = 5, a centric fusion that is synapomorphic for Aphelinus species with four chromosomes, has probably occurred there. It is interesting that the monophyly of the family Aphelinidae is not supported either by morphological and molecular data (Gibson et al. 1999, Campbell et al. 2000) or by karyotypic information as well. Moreover, the amount of diversity of chromosome numbers of Aphelinidae almost equals to that of Chalcidoidea as a whole (Baldanza et al. 1999, Gokhman 2000b). According to the analysis by Campbell et al. (2000), two other families with relatively high chromosome numbers, Eurytomidae and Encyrtidae, belong to the same vast lineage that also includes an array of forms with lower numbers. Higher n values (and the pronounced karyotypic asymmetry) represent a putative symplesiomorphy of Encyrtidae and Eurytomidae. It is interesting to note that the widely accepted hypothesis about the close relationship between Eupelmidae (n = 5–6) and Encyrtidae (n = 8–12) (see e.g. Trjapitzin 1978 and Noyes 1990) was not apparently supported by both chromosomal and molecular data (Campbell et al. 2000, see also Gibson 1990). However, certain information (e.g. Fusu 2008a) suggests that Eupelmidae can be substantially more variable in terms of karyotype structure

5.1 Phylogenetic Analysis of Chromosomal Characters

61

(including chromosome number) than it was previously supposed. If this is true, higher n values can be found in this family as well (see Fusu 2008e). The reduction of chromosome numbers from n = 9–11 to n = 5–6 is therefore not a synapomorphy of Chalcidoidea, because it occurred within different groups of this superfamily (Gokhman 2000b, Gokhman and Mikhailenko 2008b). Since the phylogeny of Chalcidoidea is insufficiently studied even at the family level, it is impossible now to precisely detect the number of those events in the evolution of this group. On the other hand, this means that several morphological types of chromosome sets must exist in advanced chalcids with lower chromosome numbers. Indeed, haploid karyotypes of many members of Pteromalidae, Chalcididae, Trichogrammatidae, etc. with n = 5 contain five biarmed chromosomes of similar size and most Eulophidae and Torymidae also have an additional subtelo-/ acrocentric. The origin of these karyotypes remains obscure, although it is not impossible that, for example, the haploid set with six chromosomes could originate through tandem fusions in the karyotype of Mymaridae with n = 9 similar to that of Anaphes (Gokhman 2000b, 2002a). Although species with n = 5 prevail in the superfamily Chalcidoidea, it is difficult to determine which of the two most frequent karyotypes is initial for this group of families. Moreover, it is quite possible that those chromosome sets originated repeatedly. In particular, it is highly likely that karyotypes with lower chromosome numbers could originate through a tandem fusion of the small and one of the large chromosomes in chromosome sets with n = 6. To study this possibility in more detail, it is necessary to analyse the information obtained on the karyotype structure of Eulophidae and Torymidae (Goodpasture 1974, 1975a, Goodpasture and Grissell 1975, Kostjukov and Gokhman 2001, Maffei et al. 2001, Gokhman 2002c, 2004e, 2005b, Caprio and Bernardo 2006, Gokhman and Mikhailenko 2007, Fusu 2008d, Kostjukov et al. 2008). Chromosome sets of Eulophidae are fairly diverse. The haploid chromosome number in various species of this family ranges from 5 to 8. Haploid sets of six chromosomes are the most frequent in the whole Eulophidae as well as in all studied subfamilies of these insects (Gokhman 2002c). As in many other parasitic wasps, meta- and submetacentrics prevail within karyotypes of Eulophidae, although a pair (less frequently two pairs) of short acro-/ subtelocentrics is present in the majority of studied species of this family, especially in those with n = 6. Exceptions from this rule are Elachertus sp., in which acrocentrics are similar in size to other elements of the set, Melittobia chalybii, in which only biarmed chromosomes are found (Schmieder 1938) as well as certain species that belong to the subgenera Aprostocetus and Stepanovia3 of the genus Aprostocetus with the analogous karyotype structure (Gokhman 2004e, Kostjukov et al. 2008). In addition, all chromosomes of Aprostocetus (Hyperteles) elongatus with n = 7 are also biarmed and gradually decrease in size (Gokhman 2003).

3

The latter group is sometimes considered as a separate genus (see e.g. Kostjukov et al. 2008).

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5 Phylogenetic Implications of Karyotypic Characters of Parasitic Hymenoptera

Fig. 5.4 Phylogenetic tree of subfamilies and tribes of the family Eulophidae (simplified from Gauthier et al. 2000) with variation ranges of haploid chromosome numbers superimposed onto it. Modal chromosome numbers are given in parentheses (modified after Gokhman 2005a)

The data obtained also help to detect features of chromosome sets that are initial for all Eulophidae. Indeed, a comparison of karyotypic data for this family with the last results of the phylogenetic analysis of Eulophidae (Gauthier et al. 2000) shows that the most probable initial chromosome number in this group is the modal one, i.e. n = 6 (Fig. 5.4). This assumption is also likely to be correct with respect to chromosome morphology (see above). The haploid set containing five large biarmed chromosomes and a small acro-/ subtelocentric must be therefore presumed initial for the Eulophidae (Gokhman 2002c, 2004e). This karyotype structure could be considered as a potential synapomorphy of this family, although the structure of chromosome sets of many studied species of Eulophidae substantially differs from the aforementioned one, i.e. it is autapomorphic. Thus, 2n = 12 in Euplectrus flavipes and Euplectrus sp., as in many other Eulophidae, but their karyotypes contain two pairs of small chromosomes and four pairs of large elements (Gokhman 2002c, 2004e). Moreover, at least one pair of medium-sized acrocentrics (that are relatively larger than those in the majority of other species) present in the chromosome set of the other member of this genus, Euplectrus bicolor. Finally, karyotypes of A. elongatus with 2n = 14 and Elachertus sp. with 2n = 16 appear the most transformed if compared to the initial one, and they differ from the latter karyotype both in the number and size proportions of chromosomes. Regrettably, the detailed reconstruction of karyotype evolution of Eulophidae seems impossible at present, although certain more or less probable assumptions can be already suggested. For example, the chromosomal set of Eu. bicolor probably differs from those of Eu. flavipes and Euplectrus sp. in the tandem fusion involving two smallest elements in species with 2n = 12. In turn, karyotypes of latter species have probably originated by translocation from the karyotype initial for all Eulophidae. Chromosome sets of Aprostocetus (Aprostocetus) and Aprostocetus (Stepanovia) spp. as well as that of M. chalybii with n = 5 perhaps have also resulted from tandem fusions. Judging from chromosome size, it is quite possible that the increase in chromosome number in A. elongatus and Elachertus sp. occurred not through fissions, but by the origin of aneuploids associated with meiotic nondisjunction of chromosomes.

5.1 Phylogenetic Analysis of Chromosomal Characters

63

Karyotypes of parasitic wasps of the family Torymidae are similar to chromosome sets of Eulophidae. As in members of the latter family, modal chromosome number of Torymidae is n = 6, although their haploid chromosome numbers can vary from 4 to 10 (see Appendix A). Since karyotypes that include five large metacentric chromosomes and a small subtelo/acrocentric are found in many Torymidae (except for the specialised genera Megastigmus and Podagrion; Gokhman 2005b, Fusu 2008d), karyotype structure of this kind is likely to be initial for the family (Gokhman and Mikhailenko 2007). However, Goodpasture (1975a) and Goodpasture and Grissell (1975) suggested that n = 5 could be initial for Torymidae, because those authors believed that this number was the most widespread in the whole superfamily Chalcidoidea. As in the Eulophidae, karyotypes with this chromosome number that were actually found in some Torymidae, probably originated through the tandem fusion of the smallest chromosome with a larger one in the set with n = 6. It certainly cannot be excluded that the similarity of initial chromosome sets of Eulophidae and Torymidae represents a synapomorphy of these families. However, since they belong to different lineages of Chalcidoidea (Campbell et al. 2000), it is more likely that those karyotypes have originated independently (see also Fusu 2008d). It is also possible that the haploid chromosome number of six is synapomorphic for the families Torymidae and Ormyridae. The affinity of these groups was noted, e.g. by Gibson et al. (1999). Thus, superposition of the data on the presumed initial chromosome numbers in parasitic Hymenoptera onto the phylogenetic tree of this group that was constructed by Ronquist et al. (1999) shows that the superfamilies with initial chromosome numbers falling within the range of n = 14–17 or higher (aculeate Hymenoptera, including Chrysidoidea, and the superfamily Evanioidea) are at the base of the tree. The karyotypes of these parasitic wasps are more or less symmetrical. The superfamily Ichneumonoidea, in which the initial chromosome number may be either n = 10–11 or n = 14–17, is intermediate with respect to chromosome number. Finally, Microhymenoptera are characterised by n = 9–11, except for most chalcids with initial values of n = 5–6 (Fig. 5.5). Judging from the scheme presented here, n = 14–17 should be considered the initial chromosome number for parasitic wasps (Gokhman 1997a, 2003). Apparently, the same (or very close) values are initial for aculeate Hymenoptera, with the chromosome number being substantially reduced at least in the family Dryinidae. A parallel decrease in chromosome number occurred independently and repeatedly in Ichneumonidae (in the tribe Polysphinctini, the subfamily Ichneumoninae and the group Ophioniformes), Braconidae (in the subfamilies Braconinae, Exothecinae, Aphidiinae and in the noncyclostome lineage) and Microhymenoptera (Gokhman 2007a). In the last of the cases considered here, low chromosome numbers (n = 9–11) are a synapomorphy of Diaprioidea, Cynipoidea, Platygastroidea, Ceraphronoidea and Chalcidoidea. In addition, chromosome numbers reduced to n = 5–6 in several groups of Chalcidoidea. This reduction took place at least two times in Aphelinidae and is likely to have occurred repeatedly in other Chalcidoidea with similar chromosome numbers. It is certainly theoretically possible that evolution, e.g. of the superfamily Ichneumonoidea, went in the reverse direction, i.e. n = 10–11 must be considered initial for

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5 Phylogenetic Implications of Karyotypic Characters of Parasitic Hymenoptera

Fig. 5.5 Phylogenetic tree of superfamilies of parasitic wasps with the most frequent haploid chromosome numbers superimposed onto it. The presumed initial chromosome numbers are underlined (Gokhman 2006b). Reproduced by permission of Wiley-Blackwell Publishing

the superfamily and higher chromosome numbers found, e.g. in most Pimpliformes and cyclostome Braconidae are secondary. However, results of the study of chromosome sets of Ichneumonoidea with higher n values contradict the aforementioned opinion. In particular, karyotypes of those parasitic wasps mostly contain biarmed chromosomes of the usual type, but not acrocentrics or pseudoacrocentrics that can result from centric fissions. In contrast to that, pseudoacrocentric chromosomes do present in chromosome sets of a few members of Ichneumonoidea in which the secondary increase in chromosome number is highly likely (e.g. in Ch. motatorius). As for the possible increase in chromosome numbers due to aneuploidy (see Section 4.1), even in the case of the notable prevalence of these processes (for example, in the subfamily Ichneumoninae), emerging karyotypes are fairly close to the initial ones in their chromosome numbers and the modal n value almost does not change in the group (Gokhman 1990a). Finally, karyotype evolution of parasitic wasps mostly proceeds towards the decrease in chromosome number due to the presumption of analogy (see Section 1.3.2.1). The parallel reduction of chromosome number in parasitic Hymenoptera must be considered as a tendency related to karyotypic orthoselection that can probably be explained in this case by the decrease in the number of linkage groups in the specialised forms of parasitic wasps (see Section 1.3.2.3).

5.2 Main Trends of Karyotype Evolution of Parasitic Hymenoptera

65

In some cases, chromosomal data can confirm the phylogenetically significant results of the study of DNA sequences of closely related species of parasitic Hymenoptera. In particular, the analysis of the Nasonia species complex that was conducted using molecular data (Campbell et al. 1993) has demonstrated that Nasonia vitripennis is likely to have stemmed off the common lineage first (about 200,000 years ago), and other species, N. longicornis and N. giraulti, have split approximately 100,000 years ago. This is also confirmed by chromosomal data (Gokhman and Westendorff 2000), according to which N. vitripennis differs both from N. giraulti and N. longicornis in the size of the second chromosome and the two latter species differ from each other in the size of the third chromosome (Gokhman 2003).

5.2 Main Trends of Karyotype Evolution of Parasitic Hymenoptera The data accumulated therefore suggest that a symmetrical karyotype with n = 14– 17 is likely to be initial for parasitic wasps (Gokhman 2003, 2004d). Taking the performed analysis into account, it is possible to list the following main trends of evolutionary change of karyotypes of parasitic wasps:

1. Decrease in chromosome number. As noted above, this process has occurred repeatedly and independently in different groups of parasitic Hymenoptera, mainly through tandem fusions (less frequently, centric ones). The aforementioned trend has mainly been observed at the superfamily and family levels (e.g. in Microhymenoptera); however, the reduction of chromosome number may have occurred in lower taxa (e.g. in the tribe Polysphinctini (Ichneumonidae), various Aphelinidae of the genus Encarsia, etc.). A limited increase in chromosome number is characteristic only of a few genera and species (e.g. in Ichneumon species with 2n = 26) and occurs through the origin of aneuploids, or, substantially less frequently, through centric fissions, as in Ch. motatorius (Ichneumonidae). 2. Karyotypic dissymmetrisation, in contrast to the former trend, is considerably more limited; it has occurred only in a few families, subfamilies and tribes of parasitic Hymenoptera. These changes took place through an increase in size differentiation of chromosomes, e.g. in parasitic wasps of the subtribe Barichneumonina (Ichneumonini) and of the subfamily Cheloninae (Braconidae) and/or in the proportion of acrocentrics in the karyotype; for example, in the family Cynipidae or in the subfamilies Campopleginae and Cremastinae (Ichneumonidae). However, the symmetry of chromosome sets may increase in individual species. In particular, this process simultaneously occurred in certain members of the families Eulophidae and Torymidae when their initial six-chromosome karyotypes turned into five-chromosome ones.

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5 Phylogenetic Implications of Karyotypic Characters of Parasitic Hymenoptera

Since chromosomal fusions are widespread in parasitic wasps, synapomorphic character states of the corresponding groups should be selected very carefully. In all these cases, not only the number of chromosomes, but also their morphology must be analysed as well. Parasitic wasps of the superfamily Chrysidoidea also exhibit a trend towards the reduction of chromosome numbers, which is most distinct in Dryinidae. In contrast to that, the chromosome number apparently increased in other members of the superfamily (at least in the family Chrysididae). Judging from the considerable proportion of acrocentrics within karyotypes, centric fission should be considered the main mechanism of this increase. Note that the same trend is also ascribed to other aculeate Hymenoptera (Imai et al. 1988, Hoshiba and Imai 1993), in which the respective increase in chromosome and arm numbers through centric fissions and pericentric inversions is mostly detected. This trend of karyotype evolution therefore prevails in Aculeata, but is very rare in parasitic wasps. It is often observed that chromosome number and body size are correlated to each other in parasitic Hymenoptera (Gokhman 2003). Indeed, chromosome numbers are high in many relatively large parasitic wasps (e.g. Ichneumonidae) and are considerably lower in smaller ones (e.g. most chalcids). This relationship is most easily explained by similar directions of morphological and chromosomal evolution in parasitic Hymenoptera. Indeed, miniaturisation is one of the main evolutionary trends characteristic of parasitic wasps (Rasnitsyn 1980), and the group of superfamilies comprising Chalcidoidea, Cynipoidea, Proctotrupoidea and some other taxa has been informally but aptly termed Microhymenoptera. It is worth noting, however, that the decrease in size also leads to the increase in the level of inbreeding, because the radius of reproductive activity of parasitic wasps narrows. A simultaneous increase in the species number and abundance of multiple parasites as well as of parasitic wasps attacking aggregated hosts is observed in Microhymenoptera and sib-crossing becomes usual in these forms (Askew 1968). Under these conditions, selection can favour the development of mechanisms that decrease the levels of recombinative variation due to the decrease in the number of linkage groups (Gokhman and Quicke 1995). Parasitic wasps of the subfamily Aphidiinae (Braconidae) with n = 3–9, on one hand, and many species of the superfamily Chalcidoidea with n = 3–8, on the other hand, are the most obvious examples of this reduction (Gokhman 2007a). Moreover, Braconidae that are usually inferior to Ichneumonidae in terms of body size, also comprise substantially more taxa with lower n values. However, the correlation between the body size and chromosome number in parasitic wasps is certainly not absolute because these parameters are also determined by many other factors. In some groups of parasitic Hymenoptera, the relationship between the body length and chromosome number is inverse. Thus, chromosome numbers of Mymaridae are higher than in many other Chalcidoidea (Gokhman 2000b), although the smallest parasitic wasps of this superfamily do belong to Mymaridae (Rasnitsyn 1980).

Chapter 6

Chromosomal Analysis of Parasitic Wasps at Various Taxonomic Levels

Abstract Taxonomic significance of chromosomal characters of parasitic wasps was studied at various levels, from superfamilies and families to morphologically identical populations. Although these characters can be of use at all taxonomic levels, they are most effective at the species level. Closely related species and forms of parasitic wasps that differ in karyotypic features can be subdivided into the following categories: (1) clearly distinct species; (2) species with weak morphological differences (sibling species s.str.); (3) morphologically identical populations; (4) intrapopulation forms (elements of population polymorphisms); (5) individuals with spontaneous chromosomal mutations. Chromosomal analysis is also a very important method of determining numbers of linkage groups. Karyotypic features of parasitic wasps can be used for identifying their immature stages. Counting chromosomes in embryonic tissues is an effective technique of studying primary sex ratios. Keywords Closely related forms · Immature stages · Parasitic wasps · Sex ratio · Species-level taxonomy

6.1 General Remarks As was shown in Chapters 3 and 5, parasitic Hymenoptera have relatively smaller variation range of chromosome number and other details of karyotype structure than Aculeata do, and they resemble Symphyta in this respect. This feature allows the identification of the main characteristics of chromosomal diversity of various taxa. On the other hand, these characters are variable enough to provide information needed for distinction of groups of different taxonomic rank, from superfamilies to morphologically undistinguishable populations (Gokhman 2006a).

6.2 Superfamilies, Families and Subfamilies In some cases, taxa of this rank have nonoverlapping ranges of variation of chromosome numbers. This is characteristic e.g. of all the families of Chrysidoidea (species of Bethylidae, Chrysididae and Dryinidae have n = 10–14, 19–21 and 4–7, V.E. Gokhman, Karyotypes of Parasitic Hymenoptera, DOI 10.1007/978-1-4020-9807-9 6, C Springer Science+Business Media B.V. 2009

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6 Chromosomal Analysis of Parasitic Wasps at Various Taxonomic Levels

respectively; Gokhman 2002b). Similarly, parasitic wasps belonging to Evanioidea (n = 14–16) and Microhymenoptera (n = 3–12) also differ by the character discussed (see the previous chapter). Some chalcid families (Mymaridae, Encyrtidae and most Eurytomidae) have higher chromosome numbers (n = 8–12) in comparison with the majority of other Chalcidoidea (n = 3–7), with the exception of a certain clade of Eurytoma with n = 5–7, Elachertus sp. (Eulophidae) with n = 8 and also Aphelinidae and Torymidae with n = 3–11 and 4–10, respectively (Gokhman 2000b, Fusu 2008d, Gokhman and Mikhailenko 2008b). Chromosome numbers of closely related subfamilies of Braconidae also differ between each other. In particular, Macrocentrinae have n = 14, whereas n = 5–6 is known for Charmontinae. Differences in the chromosomal characters were also revealed in cyclostome members of the family Braconidae: n = 17, 10 and 6 were found in Doryctinae, Braconinae and Exothecinae, respectively (Gokhman and Quicke 1995). In some cases, additional information can be obtained from the examination of modal chromosome numbers of different groups or ranges that include these numbers. For example, Evanioidea are characterised by higher n values (14–16) in comparison with modal numbers of other superfamilies and families of parasitic wasps (n = 5–11). Finally, two subfamilies of Chrysididae (Hedychrinae and Chrysidinae) also evidently differ in chromosome number (n = 19 and 21, respectively; Quicke and Gokhman 1996). On the basis of the results obtained, it is also interesting to discuss the problem of the taxonomic rank of the subfamily Aphidiinae, which is treated by some researchers as a separate family (see e.g. Tobias 1986). Although chromosomal data rather provide information on heterogeneity of a supraspecific taxon than on its possible rank, certain conclusions can be drawn even in this case. As noted above, n = 14–17 is assumed to be the initial chromosome number for the superfamily Ichneumonoidea. Hence, if Aphidiinae stem by a separate branch off the common lineage of all Ichneumonoidea, together with Ichneumonidae and Braconidae, and at the same time they are ascribed the family rank, it is necessary to accept the deep reduction in chromosome numbers at the family level (from n = 14–17 to n = 3–9), which is not characteristic of other parasitic wasps. Thus, in my opinion (Gokhman 2004c), it would be appropriate to follow many experts treating Aphidiinae as a subfamily of Braconidae; furthermore, this point of view is partly confirmed by both morphological and molecular data (Dowton et al. 2002).

6.3 Genera and Groups of Genera In some parasitic Hymenoptera, differences between ranges of variation of chromosome numbers also occur, e.g. at the tribe level. For example, these ranges do not overlap in all tribes of the subfamily Cryptinae (Ichneumonidae) (see Section 5.1). Members of the tribe Polysphinctini have lower chromosome numbers (n = 8–13) than many other parasitic wasps of the subfamily Pimplinae (n = 14–21) belonging to the same family (Gokhman 2001a). A similar relationship between

6.3 Genera and Groups of Genera

69

chromosome numbers was found in the two tribes of the subfamily Banchinae (Ichneumonidae), Glyptini (n = 6–9) and Lissonotini (n = 11) (Gokhman and Quicke 1995). Karyotypic features of species of the relatively well-studied subfamily Ichneumoninae can be used for solving taxonomic problems at the genus level. In particular, the subtribe Barichneumonina was erected on the basis of morphological characters (Hilpert 1992), but an asymmetrical karyotype with one or two pairs of very small chromosomes is also characteristic of these parasitic wasps (Gokhman 1990a). However, the genus Baranisobas that is provisionally included in this subtribe has a relatively symmetrical chromosome set and therefore may not belong to Barichneumonina (Gokhman 2003). In the majority of parasitic wasps of the subtribe Ichneumonina 2n = 20–34, but species of two closely related genera, Patrocloides and Pseudoamblyteles, have diploid chromosome numbers of 16 and 18, respectively. This character state is likely to be synapomorphic for these taxa. In contrast to that, Stenichneumon, which is also treated as a genus closely related to the mentioned genera, has 2n = 28 (Gokhman 1993, Gokhman and Quicke 1995). In the Ichneumoninae, chromosomal differences were also revealed at the genus level. In particular, species of the genus Dirophanes usually have lower chromosome numbers (2n = 18–20) than parasitic wasps of the two other genera also included into Phaeogenes s.l., namely, Tycherus and Phaeogenes s.str., in which 2n = 22 was found in the majority of species (Gokhman 2003). Vulgichneumon saturatorius, the only examined species belonging to this genus, has 2n = 18, whereas 2n = 22–34 were found in other Barichneumonina. Similar examples could be found in some other parasitic wasps of the family Ichneumonidae (Gokhman 2003). As for other groups of parasitic Hymenoptera, all studied species of the subfamily Opiinae (Braconidae), i.e. Biosteres (n = 14), Psyttalia (n = 17–19), Diachasmimorpha (n = 20) and Fopius (n = 23) differ in chromosome number (Kitthawee et al. 1999, 2004, Gokhman 2004c). Parasitic wasps of the tribe Praini that belong to the same family differ in this feature from all other Aphidiinae (n = 4 and 3, 5, 7 and 9, respectively; Quicke and Belshaw, 1999). Many gall wasps of the family Cynipidae have n = 10; however, n = 9 has been found in all species of the genus Diplolepis (Sanderson 1988). Chromosomal data can be used for solving taxonomic problems in the family Eulophidae (Chalcidoidea) at the tribe level. Thus, the study by Gauthier et al. (2000) has significantly changed the classification of the subfamily Eulophinae. According to the authors mentioned, the tribes Euplectrini and Elachertini established by Bouˇcek (1988) do not deserve an independent status and must be included into Eulophini. With respect to that, it should be noted that karyotypes of species of the genera Euplectrus and Elachertus have distinct apomorphic features (Gokhman 2002c), whereas no character of that kind was revealed in parasitic wasps of the tribe Eulophini s.str. (e.g. in Sympiesis species). The majority of genera of Aphelinidae (Chalcidoidea) has either relatively high (n = 11 in Coccophagus and Pteroptrix) or rather low chromosome numbers (n = 4–5 in Aphelinus and Aphytis). In addition, differences in this character have also been

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found in the two genera of the subfamily Anteoninae (Dryinidae): Anteon (n = 4–5) and Lonchodryinus (n = 7) (Gokhman 2001b). Strong differences in the morphometric features of karyotypes are often found even in members of different genera of parasitic Hymenoptera with the same n values and similar chromosomal morphology. In particular, the overwhelming majority of Pteromalidae has a chromosome set containing five pairs of metacentrics that are fairly similar in size; however, species of the genus Nasonia, Lariophagus distinguendus and one of the forms of Anisopteromalus calandrae with n = 5, differ in the relative length of chromosomes and also in the centromere indices of some of them (Gokhman 2003).

6.4 Species and Species Groups 6.4.1 Species Grouping and Classification As noted in Section 1.3.1, use of the structural karyotypic features in Hymenoptera taxonomy is the most effective at the species level. In particular, results of chromosomal analysis can testify in favour of the grouping of species with an obscure taxonomic position within certain genera. For example, Ichneumon lugens was sometimes included into the genus Chasmias because of the superficial similarity to other species of this genus. I. lugens, however, has 2n = 24, as well as the majority of species of the genus Ichneumon, whereas only 2n = 34 has been found in Chasmias (Gokhman 1997a); 2n = 24 has also been revealed in the other parasitic wasp, Coelichneumon deliratorius, which is usually treated as an intermediate form between Ichneumon and Coelichneumon (Gokhman 1997a). At the same time, 2n = 26 was found in the two other species of the genus Coelichneumon, C. cyaniventris and C. sugillatorius. This fact probably demonstrates that C. deliratorius is more closely related to Ichneumon; however, since C. cyaniventris and C. sugillatorius both belong to the same species group, further chromosomal studies of the genus Coelichneumon are needed for a more accurate taxonomic decision. The same chromosome number, 2n = 26, was also recently recorded in Lymantrichneumon disparis (Gokhman and Mikhailenko 2008a). This information may indicate that Lymantrichneumon is closer to the tribe Protichneumonini (including Coelichneumon) than to Ichneumonini. Data on karyotype structure can confirm the correctness of merging species (into groups or subgenera) and facilitate separation of those groups. In particular, species of the genus Aprostocetus (Eulophidae) that belong to different subgenera (Stepanovia, Ootetrastichus and Hyperteles), have different chromosome numbers (n = 5, 6 and 7, respectively; Gokhman 2004e, Kostjukov et al. 2008). Another example is Andricus mukaigawae s.l. (Cynipidae) with lower chromosome numbers (n = 5–6), whereas all other members of the genus Andricus have n = 10 (Abe 1998). In addition, chromosome numbers confirm phylogenetic relationships between the studied species of the genus Dirophanes (Ichneumonidae).

6.4 Species and Species Groups

71

In this taxon, D. fulvitarsis and D. invisor with 2n = 20 both belong to the same monophyletic group, whereas D. callopus with 2n = 18 belongs to the other clade (see Sch¨onitzer et al. 2006). Finally, chromosomal analysis can reveal and recognise closely related species and forms of parasitic wasps and also provide additional information on the differences between species and populations (Gokhman 2003). These forms that differ in structural features of the karyotype can be subdivided by the taxonomic level and the degree of morphological isolation into the following categories (Gokhman 2007b): (1) clearly distinct species; (2) species with weak morphological differences (sibling species s.str.); (3) morphologically identical populations; (4) intrapopulation forms (elements of population polymorphisms); (5) individuals with spontaneous chromosomal mutations (examples of the two latter categories are considered in Section 4.1).

6.4.2 Clearly Distinct Species At present, a number of closely related species of parasitic Hymenoptera that differ not only in external features, but also in details of karyotype structure, are known. In particular, examples of situations of that kind can be found among species of the genera Dolichomitus, Pimpla, Stenomacrus, Glypta, Tycherus, Phaeogenes, Dirophanes, Cratichneumon, Virgichneumon, Ichneumon, Coelichneumon (Ichneumonidae), Psyttalia, Meteorus, Microplitis, Cotesia, Chelonus, Aphidius (Braconidae), Gasteruption (Gasteruptiidae), Leptopilina (Figitidae), Andricus, Diplolepis (Cynipidae), Eurytoma (Eurytomidae), Ageniaspis, Anagyrus, Copidosoma (Encyrtidae), Aphelinus, Encarsia (Aphelinidae), Brachymeria (Chalcididae), Aprostocetus, Melittobia, Euplectrus (Eulophidae), Ormyrus (Ormyridae), Monodontomerus, Torymus (Torymidae), Anteon (Dryinidae) (see Appendices A and B). The majority of the genera listed above differ from each other in chromosome number, but in some cases these differences involve chromosome size and centromere position (e.g. in D. invisor and D. fulvitarsis, Psyttalia fletcheri and P. incisi, some chalcids of the genera Aphelinus, Encarsia and Torymus), as well as the size and localisation of segments of the constitutive heterochromatin (species of Dirophanes and Encarsia), and even localisation of the nucleolus organiser (Encarsia formosa and E. meritoria, as well as members of the Encarsia sophia complex; see Chapter 3). As a rule, chromosomal characters only complement the differential diagnosis of the aforementioned species; however, these features can obviously be used for identifying parasitic wasps from laboratory stocks (Gokhman 2003, 2006a).

6.4.3 Sibling Species Species with weak or nearly absent morphological differences are called “sibling species” (Mayr 1970). These differences, however, are stable, and populations of this kind can therefore be described as separate species, as it follows from

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the very term “sibling species”. Despite the high degree of external similarity between the sibling species of parasitic Hymenoptera, they can differ significantly in chromosome number and other karyotypic features (Table 6.1).

Table 6.1 Morphologically similar populations of parasitic wasps that differ in chromosomal characters∗ a

Species

Morphological Chromosome set differences

Reference

Ichneumonidae Aethecerus dispar; 2n = 24; 2n = 22 Moderate A. ranini

Gokhman (1991)

Ichneumonidae Tycherus australo- 2n = 22; 2n = 18 Moderate geminus; T. ischiomelinus

Gokhman (1991)

Ichneumonidae Phaeogenes bacilliger; Ph. spiniger

2n = 20; 2n = 22 Moderate

Gokhman (1990b) and unpublished data

Ichneumonidae Ichneumon extensorius

2n = 24; 2n = 26 Virtually absent

Gokhman (1993)

Ichneumonidae I. suspiciosus

2n = 24; 2n = 26 Absent

Gokhman (1993)

Braconidae

Aphidius ervi

n = 5, 2n = 10; 2n = 12

Absent

Gokhman and Westendorff (2003)

Braconidae

Charmon cruentatus

n = 5, 2n = 10; 2n = 12

Absent

Gokhman (2002a), (2003)

Cynipidae

Andricus 2n = 10; 2n = 12; Absent kashiwaphilus; 2n = 10 A. mukaigawae; A. targionii

Aphelinidae

Aphelinus varipes

2n = 8†

Weak

Gokhman (2003)

Aphelinidae

Encarsia sophia‡

2n = 10§

Weak

Giorgini and Baldanza (2004)

Encyrtidae

Copidosoma floridanum

n = 8, 2n = 16; Not mentioned n = 10, 2n = 20; n = 11, 2n = 22

Eupelmidae

Eupelmus n = 5; n = 6; vesicularis; n=6 Eupelmus sp. 1; Eupelmus sp. 2

Pteromalidae

Anisopteromalus calandrae

Pteromalidae

Nasonia vitripennis n = 5, 2n = 10; Absent n = 6, 2n = 12

Moderate

n = 5, 2n = 10; Moderate n = 7, 2n = 14

Abe (1998, 2007)

Patterson (1917), etc., Hunter and Bartlett (1975), Strand and Ode (1990) Fusu (2008a)

Gokhman et al. (1998) Gershenzon (1968), Whiting (1968), etc., Goodpasture (1974)

6.4 Species and Species Groups

73 Table 6.1 (continued)

a

Species

Morphological Chromosome set differences

Reference

Pteromalidae Nasonia giraulti, N. n = 5, 2n = 10 longicornis, N. vitripennis

Weak

Gokhman and Westendorff (2000)

Pteromalidae Spalangia endius

n = 4, 2n = 8; 2n = 12

Not mentioned

Silva-Junior et al. (2000a), Kitthawee and Vasinpiyamongcol (2002)

Torymidae

n = 4, 2n = 8; Not mentioned n = 6, 2n = 12

Monodontomerus obscurus#

MacDonald and Kruni´c (1971), Goodpasture (1975a)

Goodpasture and Torymus n = 6,∗∗ 2n = 12 Weak Grissell (1975) californicus, T. warreni ∗ Since differences in chromosome number mentioned in the available literature could result from a misprint, only data on groups in which karyotypic heterogeneity was especially emphasised, are included into the table. † Statistically significant morphometric differences were revealed between karyotypes of different populations. ‡ Another group of karyotypically different sibling species was recently found in parvella species group of the genus Encarsia (Pedata et al. 2005). § Strong differences in routine chromosome morphology as well as in the localisation of the nucleolus organiser were revealed between karyotypes of different populations. Statistically significant morphometric differences were revealed between karyotypes of different species. # The species with n = 6 is probably Monodontomerus laticornis. ∗∗ The haploid karyotype of Torymus californicus contains six biarmed chromosomes, whereas the last chromosome of T. warreni is acrocentric; in addition, the second chromosome of the latter species carries a secondary constriction.

Torymidae

I will discuss in some detail certain previously known cases of the detection of sibling species of parasitic Hymenoptera. Chromosomal characters are likely to have been used for the first time in parasitic wasps for diagnostics of the two species of the family Torymidae, Torymus californicus and T. warreni (Goodpasture and Grissell 1975). Both these species were described long before their karyotypic study, although T. warreni was later synonymised with T. californicus, because they externally differ only in the body colour. Nevertheless, chromosomal data have confirmed the species status of both these taxa. The situation is more complicated in the Copidosoma floridanum species complex in which chromosomal forms with 2n = 20 and 22 were possibly similar in their external morphology (Hunter and Bartlett 1975, Strand and Ode 1990). However, a karyotype with 2n = 16 that was ascribed to C. floridanum by certain authors (Patterson 1917, etc.) might belong in fact to other morphospecies, e.g. Copidosoma bakeri (Howard) or Copidosoma truncatellum (Dalman) (J. Noyes, personal communication).

74

6 Chromosomal Analysis of Parasitic Wasps at Various Taxonomic Levels

Two different chromosome numbers, n = 4 and 6, were also reported for Monodontomerus obscurus (Torymidae; MacDonald and Kruni´c 1971, Goodpasture 1975a). MacDonald and Kruni´c (1971) might have provided an incorrect identification of their Monodontomerus species that was probably described later as M. laticornis Grissell and Zerova (Zerova and Grissell 1985). Chromosomes of M. laticornis were not studied, although the haploid karyotype of the closely related M. montivagus contained six large chromosome and a smaller one (Goodpasture 1975a), as in parasitic wasps examined by MacDonald and Kruni´c (1971). The same chromosome numbers, n = 4 and 6, were given in different papers for Spalangia endius, a cosmopolitan parasitic wasp of the family Pteromalidae (Silva-Junior et al. 2000a, Kitthawee and Vasinpiyamongcol 2002). Regretfully, a comparative morphological study of individuals that belonged to different populations was not conducted in this case. Nasonia vitripennis, another cosmopolitan species of Pteromalidae, usually has n = 5 and 2n = 10 (Gershenzon 1968, Whiting 1968, etc.), although n = 6 and 2n = 12 were detected in an American population of this species; morphological differences of parasitic wasps of the latter population from the typical N. vitripennis were not found (Goodpasture 1974). Sibling species were also detected in gall wasps of the A. mukaigawae species complex, i.e. in A. mukaigawae s.str. (n = 6), on one hand, and in A. kashiwaphilus and A. targionii, on the other hand (both have n = 5), that differ in plant hosts and in the shape of galls of the parthenogenetic generation (Abe 1998, 2007). Two populations that belong to the same morphospecies, E. sophia (Aphelinidae), were recently found to differ not only in subtle details of external morphology detected by canonical discriminant analysis, but also in certain karyotypic features as well, i.e. in some details of chromosomal morphometry as well as in the localisation of the nucleolus organiser (Giorgini and Baldanza 2004). I have detected and described a few groups of sibling species of the family Ichneumonidae (Gokhman 1991). In particular, a new species, Tycherus australogeminus with 2n = 22, has been found in Southern Russia and adjacent territories, within the distribution range of the closely related parasitic wasp, T. ischiomelinus with 2n = 18 (Gokhman 1991). The former species that inhabits forest zone of the Palaearctis was described as new for the science. It also has later flight time as well as some morphological differences if compared to T. ischiomelinus (Gokhman 1990a). Analogously, a new species of Ichneumonidae, Aethecerus ranini with 2n = 22 that differs from the widely distributed A. dispar with 2n = 24 in certain features of external morphology, was found in Central Russia and Finland (Gokhman 1991). In addition, 2n = 22 and 20 were, respectively, discovered in Phaeogenes spiniger and the closely related Ph. bacilliger that has been erroneously synonymised with the previous species (Gokhman 1990b and unpublished data; see also Sawoniewicz 2003). The study of chromosome sets of sibling species can provide important information even in those cases when these species are already detected and described by morphological characters. In particular, statistically significant chromosomal

6.4 Species and Species Groups

75

differences found in three species of the Nasonia species complex with n = 5, N. vitripennis, N. longicornis and N. giraulti (Gokhman and Westendorff 2000) confirm the phylogenetic reconstruction for this complex that was created using molecular data (see Section 5.1). Analogous differences between strains of the Aphelinus varipes s.l. species complex can probably be used in the phylogenetic analysis of this group (Gokhman 2003). Perhaps the most interesting example of sibling species of parasitic wasps that differ in chromosomal characters has been found in chalcid wasps of the A. calandrae species complex (Pteromalidae) (Gokhman 2007b). The study of populations of this effective parasitoid of various stored-product pests from Russia, Western Europe and the USA has shown that this complex actually included two reproductively isolated sibling species (Gokhman and Timokhov 2002). In addition to certain morphological differences, these species also differ in chromosome number (n = 5 and 7; Gokhman et al. 1998), details of behaviour as well as in important bionomic features (including host specialisation). Parasitic wasps with n = 5 were found only on Anobiidae whereas those with n = 7 were found on Curculionidae and Bruchidae. Individuals of both species can develop on alternative hosts; although even after cultivation on these Coleoptera they usually prefer to attack initial hosts in choose experiments (Timokhov and Gokhman 2003). We have also found that species of the A. calandrae complex substantially differ in characteristic strategies of the life cycle (Gokhman et al. 1999). Females with 2n = 10 have larger body size and prefer to oviposit on host prepupae and pupae, whereas those with 2n = 14 are smaller and attack all host stages that are accessible under the experimental conditions without any distinct preference. Interspecific differences in egg size are well correlated to female body size of these insects. Wasps with 2n = 14 begin to oviposit immediately after hatching, as opposed to the day following egression in those with 2n = 10. Females of the latter species have lower egg production and strongly female-biased sex ratio of the progeny, whilst wasps with 2n = 14 have much higher fecundity and the sex ratio close to 1:1. The preimaginal development is also substantially quicker in the latter species, though its eggs are always destroyed by other females in the case of superparasitism. Oviposition in the species with 2n = 10 is usually preceded by an incomplete host paralysis and followed by marking of infested grains, whereas wasps with 2n = 14 paralyse attacked hosts more completely and never display kernel-marking behaviour. These differences are best interpreted in terms of the r/K continuum and species with n = 5 and 7 are considered as K and r strategists, respectively (Gokhman et al. 1999). Since the type of A. calandrae is lost (Bouˇcek 1988), it is difficult to assign the aforementioned material to any particular species. However, morphological features of the species with n = 7 fit the original description of A. calandrae better (Howard 1881), and therefore this species should be considered as the original A. calandrae (Gokhman and Timokhov 2002). Because the A. calandrae complex actually harboured two sibling species, all information on its bionomic features needed reconsideration. This fact again demonstrates the significance of chromosomal analysis for studies of parasitic wasp

76

6 Chromosomal Analysis of Parasitic Wasps at Various Taxonomic Levels

species, including those of practical importance. Moreover, the mentioned technique of express analysis should be used in descriptions of laboratory stocks of all widely distributed species (Gokhman 2000a, 2006b). This is especially important for those taxa in which substantial karyotypic variation has already been found, e.g. in the genus Copidosoma (see Table 6.1), but the example of A. calandrae proves that sibling species that strongly differ in chromosomal characters can also be found even in groups that are apparently uniform in this respect, like Pteromalidae. Finally, if the aforementioned data are taken into account, the necessity for including karyotypic data into descriptions of new species becomes obvious (Grissell 1973, Gokhman 1991, Johnson et al. 2001, Kostjukov and Gokhman 2001, Kostjukov et al. 2008).

6.4.4 Morphologically Identical Populations If morphologically identical populations differ in karyotype structure, it is usually impossible to describe them as new species. For example, 2n = 10 was found in two specimens of Charmon cruentatus (Braconidae) from Great Britain and Central Russia; however, a morphologically identical female with 2n = 12 was also found in the Moscow Province. At the same time, no specimens with the intermediate chromosome set (2n = 11) were revealed (Gokhman and Quicke 1995, Gokhman 2002a). However, examination of a greater number of Ch. cruentatus specimens is certainly needed for the more accurate analysis of this situation. Chromosomal investigation of a British population of Aphidius ervi (Braconidae) has revealed n = 5 (Quicke and Belshaw 1999). We also found n = 5 and 2n = 10 in the majority of studied specimens from the laboratory stock obtained from the University of Bayreuth (Germany); however, 2n = 12 was revealed in several females (Gokhman and Westendorff 2003). The morphometric analysis of karyotypes with different diploid chromosome numbers has demonstrated that they also differed in an additional pair of small acrocentrics entirely consisting of heterochromatin and occupying a peripheral position on the metaphase plate during mitosis. Since males with n = 6 (and females with 2n = 11) were not detected in the population, the aforementioned chromosomal pair probably carried a certain factor resulting in the appearance of thelytoky. If it was true, then a new class of chromosomal factors (analogous to some B chromosomes of parasitic wasps, although found in females, not in males) was described in the cited work for the first time. These factors (the so-called sex-ratio distorters) change the sex ratio in offspring of parasitic Hymenoptera (Gokhman 2004c). It is worth noting that the existence of those factors is not discussed even in the recent review on the problem mentioned (Stouthamer 2004). In addition, chromosome sets with 2n = 24 and 26 were revealed in parasitic wasps of the genus Ichneumon (in both I. extensorius and I. suspiciosus). Although discrete morphological differences between these co-habiting forms were not revealed in both species, virtually total absence of individuals with an intermediate karyotype structure proves the existence of the reproductive isolation between

6.5 Other Implications of Chromosomal Analysis

77

populations of I. suspiciosus and I. extensorius with different chromosome numbers (Gokhman 1993). It is interesting to compare the degree of divergence of sibling species of parasitic Hymenoptera by morphological and chromosomal characters. First of all, a large number of well-differentiated parasitic wasp species has no significant differences in karyotype structure (Gokhman 2003). On the other hand, the presence of morphologically similar populations that strongly differ in chromosomal characters could mean the comparatively recent origin of these forms (Vorontsov 1960). Nevertheless, species groups with the most significant differences in karyotype structure (e.g. in chromosome number, as in the T. ischiomelinus and A. calandrae species complexes) reliably differ in their external morphology. This fact testifies to the existence of a certain correlation between levels of the morphological and chromosomal divergence in parasitic wasps, in contrast to Aculeata (Gokhman 2002a)

6.5 Other Implications of Chromosomal Analysis In addition to the use for taxonomic and phylogenetic purposes, chromosomal analysis is a very important method of determining numbers of linkage groups (Gokhman 2003). This method is especially valuable in cases where some chromosomes are small or relatively inert in terms of genetics, as, for example, the B chromosome of N. vitripennis (see Section 4.1). Moreover, control of the number of linkage groups is necessary for the evaluation of this feature by indirect estimates, e.g. during the study of random amplified sequences of the polymorphic DNA (the so-called RAPD marker technique; Kazmer et al. 1995, Laurent et al. 1998, Holloway et al. 2000). Since karyotypes of parasitic Hymenoptera are specific enough, chromosomal features of these insects can be used for identifying their immature stages. In particular, I (Gokhman 1987, 1990b, 1997b) have undertaken a study of that kind involving species of the genus Dirophanes (Ichneumonidae; D. callopus, D. fulvitarsis and D. invisor). In these species, chromosome number and morphology have been studied first, and then prepupae that were extracted from parasitized hosts, were identified by their karyotypes. Immature stages of different species of the subfamily Opiinae (Braconidae) that attack fruit flies have been identified in a similar way (Kitthawee et al. 2004). Finally, since males and females of Hymenoptera differ in ploidy levels, counting chromosomes in embryonic tissues is an effective method of studying primary sex ratio that can later be compared to the secondary one, i.e. to the proportion of adult males to females (Dijkstra 1986, van Dijken 1991, Ueno and Tanaka 1997, Kraaijeveld et al. 1999).

sdfsdf

Conclusions

Chromosomes of more than 400 species of parasitic Hymenoptera have been studied up to now. This is certainly not too much if compared to the number of the known species, although if the situation is considered at higher taxonomic levels, it is obvious that about two-thirds of the subfamilies and half of the families are touched by chromosomal studies (Gokhman 2003). Moreover, chromosomal data on parasitic Hymenoptera that were obtained during the last years (see e.g. Gokhman 2004d, 2006a) just refine the pattern as to which basic features remain virtually stable from the middle of the 1990s (Gokhman and Quicke 1995, Gokhman 1997a, Quicke 1997, etc.). Due to this reason, I hope that the conclusions given below adequately describe the existing chromosomal diversity and the processes of karyotype evolution of parasitic Hymenoptera. Arrhenotokous parthenogenesis is characteristic of the majority of parasitic wasps (thelytoky is less widespread there); females develop from diploid eggs, males from haploid (rarely diploid) ones. Chromosomes of parasitic Hymenoptera are comparatively large (their average size is about 3–5 µm), each of them carries a single centromere. Chromosomes usually decrease in size more or less slowly within karyotypes, and most chromosomes are biarmed, i.e. karyotypes of parasitic wasps are fairly symmetrical. Various parasitic Hymenoptera have haploid chromosome numbers of 3–23. The distribution of species by chromosome number is bimodal with the peaks at n = 6 and 11. Centromeric and telomeric segments of the constitutive heterochromatin are the most frequent in karyotypes of parasitic wasps. Bivalents with one or two and more chiasmata are found in meiosis of parasitic Hymenoptera; bivalents with several chiasmata are more frequent in species with lower chromosome numbers. Following types of chromosomal mutations have been detected in parasitic wasps: deletions and duplications of the constitutive heterochromatin, inversions, translocations, centric and tandem fusions and fissions, polyploidy, aneuploidy and the numerical variation in B chromosomes. Various types of chromosomal polymorphism were found in parasitic Hymenoptera, i.e. those in size of heterochromatic segments, translocations and number of B chromosomes. Karyotypic rearrangements in parasitic wasps are asymmetrical in their mechanisms at the macroevolutionary level. During this process the decrease in chromosome number was possible due to chromosomal fusions (mostly tandem ones). In turn, the 79

80

Conclusions

increase in chromosome number mainly took place due to the production of aneuploids and subsequent restoration of even chromosome numbers or (sufficiently less frequently) due to centric fissions accompanied by the tandem growth of the constitutive heterochromatin and production of pseudoacrocentrics. The symmetrical karyotype with a relatively high chromosome number (n = 14– 17) and the prevalence of biarmed chromosomes must be considered as a ground plan feature of parasitic wasps. An independent reduction of chromosome number took place in different lineages of parasitic Hymenoptera. Specifically, this reduction (from n = 14–17 to n = 10–11 and less) occurred in some lineages of the superfamily Ichneumonoidea (in the Ichneumonidae and Braconidae) as well as in the common ancestor of the Proctotrupoidea sensu lato, Cynipoidea and Chalcidoidea. Further multiple decreases in chromosome number to n = 4–6 and less took place in various groups of the superfamily Chalcidoidea as well as in wasps of the family Dryinidae. Two main trends prevailed in the karyotype evolution of parasitic Hymenoptera: the reduction in chromosome number and (to a lesser extent) karyotypic dissymmetrisation (due to an increase in size differentiation of chromosomes and in the proportion of acrocentrics in a chromosome set). An increase in chromosome numbers and in the degree of karyotypic symmetry also took place, although on a more restricted scale. Differences in chromosomal characters of parasitic Hymenoptera have been found at various taxonomic levels: from superfamilies and families to morphologically indistinguishable populations. Use of karyotypic features for solving taxonomic problems in parasitic wasps is the most effective at the species level. About 20 groups of sibling species that differ in their chromosomal characters are known up to now. Specifically, karyotypic analysis has resulted in description of the two new species of the family Ichneumonidae, Aethecerus ranini and Tycherus australogeminus. It was found that Anisopteromalus calandrae (Pteromalidae), a well-known cosmopolitan parasitoid of various stored-product pests, appeared to be a complex of two closely related species that differ in chromosome number (n = 5 and 7), some morphological characters, details of behaviour and life-history strategies. Significant differences in karyotypic characters were found in chalcidoids of the Nasonia species complex (Pteromalidae) and Aphelinus varipes s.l. (Aphelinidae). Morphologically identical populations with different chromosome numbers were found in the families Ichneumonidae (Ichneumon extensorius, Ichneumon suspiciosus) and Braconidae (Aphidius ervi, Charmon cruentatus). Perspectives of chromosomal research of parasitic Hymenoptera apparently include further karyotypic studies of parasitic wasps from natural populations and laboratory stocks (Gokhman 2003). For example, very interesting results could be obtained from the chromosomal study of the superfamilies Megalyroidea, Trigonalyoidea and Stephanoidea as well as of many groups of Proctotrupoidea s.l. that were not previously examined. It is beyond doubt that studies of the structure of chromosome sets will be widely used for searching and detecting sibling species of parasitic Hymenoptera. Studies of multiple parasitoids as well as those attacking aggregated hosts (many chalcids, braconids of the subfamily Aphidiinae, etc.) are the most promising in this

Conclusions

81

respect because situations of that kind are likely to favour the fixation of various chromosomal rearrangements within populations (Gokhman et al. 1998). The data accumulated allow suggesting that the use of various types of chromosome banding (including hybridisation in situ and other methods of molecular genetics) will be very productive for taxonomic and phylogenetic studies of parasitic Hymenoptera (Gokhman 2000a). In addition, use of karyotypic characters (along with the features of external morphology, molecular data, etc.) in phylogenetic studies will become usual, especially at the family level and lower (Dowton and Austin 2000).

sdfsdf

Appendix A

Chromosome Numbers of Parasitic Wasps

Table A.1 Chromosome numbers of parasitic wasps Chromosome number Species

n

2n

Reference

Superfamily Ichneumonoidea Family Ichneumonidae Acropimpla pictipes (Gravenhorst) Adelognathus brevicornis Holmgren Aethecerus discolor Wesmael Ae. dispar Wesmael Ae. ranini Gokhman Aoplus deletus (Wesmael) A. pulchricornis (Gravenhorst) Aperileptus microspilus Foerster Aptesis puncticollis (Thomson) Baeosemus dentifer Gokhman Baranisobas ridibundus (Gravenhorst) Campoplex sp. Centeterus major Wesmael C. rubiginosus (Gmelin) Coelichneumon cyaniventris (Wesmael) C. deliratorius (Linnaeus) C. sugillatorius (Linnaeus) Colpognathus celerator (Gravenhorst) C. divisus (Thomson) Cratichneumon fabricator (Fabricius) C. rufifrons (Gravenhorst) C. sicarius (Wesmael) C. viator (Scopoli) Crypteffigies lanius (Gravenhorst) Ctenichnemon funereus (Geoffroy) Diadromus prosopius Holmgren D. pulchellus Wesmael D. subtilicornis (Gravenhorst) D. troglodytes (Gravenhorst) D. varicolor (Wesmael) Dicaelotus sp. cf. parvulus (Gravenhorst)

(14) (10) (11) (12) (11) (13) (13) (16) (8) (11) (12) (8) (11) (11) (13) (12) (13) (11) (11) (12) (13) (11) (14) (10) (8) (11) 11 (11) (11) (11) (11)

28 20 22 24 22 26 26 32 16 22 24 16 22 22 26 24 26 22 22 24 26 22 28 20 16 22 22 22 22 22 22

Gokhman unpublished data Gokhman (2003) Gokhman (1985) Gokhman (1991) Gokhman (1991) Gokhman unpublished data Gokhman (1990b) Gokhman (2001a) Gokhman (1990a, 2000a) Gokhman and Quicke (1995) Gokhman (1990b) Gokhman (2003) Gokhman (2007d) Gokhman unpublished data Gokhman (1990a) Gokhman (1997a) Gokhman (1990a) Gokhman and Quicke (1995) Gokhman (2005a) Gokhman (2003) Gokhman (2007d) Gokhman (1990b) Gokhman (1985) Gokhman (2003) Gokhman (2007d) Gokhman (1990b) Hedderwick et al. (1985) Gokhman (1990b) Gokhman (1990a) Gokhman and Quicke (1995) Gokhman (1990a)

83

84

Appendix A Table A.1 (continued) Chromosome number

Species

n

2n

D. pumilus (Gravenhorst) Diphyus latebricola (Wesmael) D. raptorius (Linnaeus) Dirophanes callopus (Wesmael) D. fulvitarsis (Wesmael) D. invisor (Thunberg) D. rusticatus (Wesmael) Dolichomitus agnoscendus (Roman)

(11) (12) (12) 9 10 10 (10) (17)

22 24 24 18 20 20 20 34

D. messor (Gravenhorst) Dyspetes arrogator Heinrich Ephialtes manifestator (Linnaeus) Eriplatys ardeicollis (Wesmael) Eristicus clarigator (Wesmael) Eurylabus torvus Wesmael Gambrus extrematis (Cresson) Gelis sp. 1 Gelis sp. 2 Gen. sp. Glypta ceratites (Gravenhorst) G. lapponica Holmgren Glypta sp. Herpestomus brunnicornis (Gravenhorst) Heterischnus nigricollis Wesmael H. truncator (Fabricius) Homotherus locutor (Thunberg) Hyposoter didymator (Thunberg) H. fugitivus (Say)

(16) (10) (15) (11) (13) (10) 10 (13) (13) 15 (6, 7) (9) (9) (9) (11) (11) (11) 12 12

Hyposoter sp. 1 Hyposoter sp. 2 Ichneumon albiger Wesmael I. amphibolus Kriechbaumer I. bucculentus Wesmael I. confusor Gravenhorst I. crassifemur Thomson I. croceipes Wesmael I. extensorius Linnaeus

(12) (12) (12) (12) (12) (12) (12) (12) (12, 13)

Ichneumon sp. cf. extensorius Linnaeus I. formosus Gravenhorst I. gracilentus Wesmael I. gracilicornis Wesmael I. ingratus Hell´en I. inquinatus Wesmael I. insidiosus Wesmael I. lugens Gravenhorst I. melanotis Wesmael

(11) (11) (12, 13) (12) (12) (13) (12) (12) (12)

Reference

Gokhman and Quicke (1995) Gokhman (1990a) Gokhman (1990a) Gokhman (1987) Gokhman (1990b) Gokhman (1987) Gokhman (2003) Gokhman and Kolesnichenko (1996, 1997), Gokhman (2003) 32 Gokhman (2002a) 20 Gokhman and Quicke (1995) 30 Gokhman and Quicke (1995) 22 Gokhman (2001a) 26 Gokhman (2007d) 20 Gokhman (1987) 20 Koonz (1939) 26 Gokhman and Quicke (1995) 26 Gokhman (2003) 30 Hoshiba and Imai (1993) 12, 13 Gokhman (2003) 18 Gokhman and Quicke (1995) 18 Gokhman (2003) 18 Gokhman (2003) 22 Gokhman (1990b) 22 Gokhman and Quicke (1995) 22 Gokhman (1990b) (24) Rocher et al. (2004) (24) D. Stoltz (personal communication) 24 Gokhman (2003) 24 Gokhman (2005a) 24 Gokhman (1990a) 24 Gokhman (1990b) 24 Gokhman (1993) 24 Gokhman (1985) 24 Gokhman (1985) 24 Gokhman (1990b) 24, 25, Gokhman (1993) 26 22 Gokhman (1990a) 22 Gokhman (1990a) 24, 25 Gokhman (1993) 24 Gokhman (1990a) 24 Gokhman (1990b) 26 Gokhman (1993) 24 Gokhman (1990a) 24 Gokhman and Quicke (1995) 24 Gokhman (1990a)

Appendix A

85 Table A.1 (continued) Chromosome number

Species

n

2n

Reference

I. memorator Wesmael

(12)

24

I. minutorius Desvignes I. molitorius Linnaeus I. nereni Thomson I. sarcitorius Linnaeus I. stramentarius Gravenhorst I. submarginatus Gravenhorst I. subquadratus Thomson I. suspiciosus Wesmael

24 24 22 22 24 20 24 24, 26

I. validicornis Holmgren I. vorax Geoffroy

(12) (12) (11) (11) (12) (10) (12) (12, 13) (12) (11)

Gokhman and Mikhailenko (2008a) Gokhman (1987) Gokhman (1990a) Gokhman (1990a) Gokhman (1990a) Gokhman (1987) Gokhman (1990a) Gokhman (1990b) Gokhman (1993)

Itoplectis naranyae Ashmead Lissonota buccator (Thunberg)

20 (11)

(40) 22

L. catenator (Panzer) L. coracina (Gmelin) Lissonota sp. 1 Lissonota sp. 2 Lymantrichneumon disparis Poda

(11) (11) (11) (11) (13)

22 22 22 22 26

Mastrus smithii (Packard) Mesochorus sp. Mesostenus gracilis Cresson Netelia latungula (Thomson) Oronotus binotatus (Gravenhorst) Orthocentrus sp. Orthopelma mediator (Thunberg)

13 (7) (10) (6) (11) (14) 15a

26 14 20 12 22 28 30

Oxyrrhexis carbonator (Gravenhorst) Paraperithous gnathaulax (Thomson) Patrocloides chalybeatus (Gravenhorst) Perithous scurra (Panzer)

(8) (15) (8) (21)

16 30 16 42

Phaeogenes bacilliger Kriechbaumer Ph. melanogonos (Gmelin) Ph. semivulpinus (Gravenhorst) Ph. spiniger (Gravenhorst) Phygadeuon sp. 1

(10) (11) (9) (11) (16)

20 22 18 22 32

Phygadeuon sp. 2

(16)

32

Phygadeuon sp. 3

(16)

32

Phytodietus polyzonias (Foerster)

(6)

12

24 22

Gokhman and Quicke (1995) Gokhman and Mikhailenko (2008a) Ueno and Tanaka (1997) Gokhman and Mikhailenko (2008a) Gokhman (2001a) Gokhman (2001a) Gokhman and Quicke (1995) Gokhman (2001a) Gokhman and Mikhailenko (2008a) Koonz (1936) Gokhman (2003) Gokhman (2001a) Gokhman (2001a) Gokhman (1987) Gokhman (1990a) Gokhman and Mikhailenko (2008a) Gokhman (2001a) Gokhman (2003) Gokhman (1993) Gokhman and Kolesnichenko (1996, 1997) Gokhman unpublished data Gokhman (1990b) Gokhman and Quicke (1995) Gokhman (1990a) Gokhman and Kolesnichenko (1996), Gokhman (2001a) Gokhman and Kolesnichenko (1996), Gokhman (2001a) Gokhman and Mikhailenko (2008a) Gokhman unpublished data

86

Appendix A Table A.1 (continued) Chromosome number

Species

n

2n

Pimpla contemplator (M¨uller)

(18)

36

P. nipponica (Uchida) P. turionellae (Linnaeus)

16 (15)

Plectiscus impurator Gravenhorst Polysphincta tuberosa Gravenhorst Pristomerus sp. Pseudoamblyteles homocerus (Wesmael) Rhorus extirpatorius Gravenhorst Scambus buolianae Hartig S. detritus (Holmgren)

(10) (9) (8) (9) 11 (14) (14)

S. nigricans (Thomson)

(14)

S. vesicarius (Ratzeburg)

(14)

Stenichneumon culpator (Schrank) Stenomacrus sp. 1 Stenomacrus sp. 2

(14) (14) 18

Stethoncus sulcator Aubert Synodites notatus Gravenhorst

(8) (12)

Syspasis albiguttata (Gravenhorst) S. scutellator (Gravenhorst) Theronia atalantae (Poda)

(11) (11) (12)

Thyrateles camelinus (Wesmael) Trichionotus flexorius (Thunberg)

(11) (8)

Triclistus globulipes (Desvignes) T. pallipes Holmgren T. podagricus (Gravenhorst) Tycherus australogeminus Gokhman T. bellicornis (Wesmael) T. dilleri Ranin T. fuscicornis (Wesmael) T. infimus (Wesmael) T. ischiomelinus (Gravenhorst) T. nigridens (Wesmael) T. ophthalmicus (Wesmael) T. osculator (Thunberg) T. suspicax (Wesmael) Venturia canescens (Gravenhorst)

(11) (11) 11 (11) (10, 11) (11) (11) (11) (9) (11) (11) (11) (11) 11

Virgichneumon digrammus (Gravenhorst) (17) V. faunus (Gravenhorst) (11) Vulgichneumon saturatorius (Linnaeus) (9)

Reference

Gokhman and Mikhailenko (2008a) (32) Ueno and Tanaka (1997) 30 Gokhman and Kolesnichenko (1997) 20 Gokhman unpublished data 18 Gokhman and Quicke (1995) 16 Gokhman (2003) 18 Gokhman and Quicke (1995) (22) Gokhman (2001a) 28 Gokhman (2003) 28 Gokhman and Mikhailenko (2008a) 28 Gokhman and Kolesnichenko (1996, 1997) 28 Gokhman and Mikhailenko (2008a) 28 Gokhman (1985) 28 Gokhman (2001a) 36 Gokhman and Mikhailenko (2008a) 16 Gokhman (2001a) 24 Gokhman and Kolesnichenko (1996) 22 Gokhman (1985) 22 Gokhman and Quicke (1995) 24 Gokhman and Mikhailenko (2008a) 22 Gokhman (1990b) 16 Gokhman and Mikhailenko (2008a) 22 Gokhman (2005a) 22 Gokhman (2005a) 22 Gokhman (2000a) 22 Gokhman (1991) 20, 21 Gokhman (1989) 22 Gokhman (1989) 22 Gokhman (1990b) 22 Gokhman (2007d) 18 Gokhman (1991) 22 Gokhman (1990b) 22 Gokhman (1990a) 22 Gokhman (1989) 22 Gokhman (1987) 22 Speicher (1937), Gokhman (2001a) 34 Gokhman (1990a) 22 Gokhman (1990a) 18 Gokhman (1987)

Appendix A

87 Table A.1 (continued) Chromosome number

Species

n

2n

Reference

Zatypota gracilis Holmgren

(13)

26

Gokhman (2003)

Family Braconidae Alysia manducator Panzer

11

22

Aphaereta tenuicornis Nixon Aphidius ervi Haliday

(17) 5, (6)

A. matricariae Haliday A. rhopalosiphi (De Stefani) Aphidius sp. Asobara tabida Nees Bassus dimidiator Nees B. timidulus (Nees) Biosteres blandus (Haliday) B. carbonarius (Nees) Charmon cruentatus Haliday

(7) (7) (3) ≈16–18 9 (9) (14) (14) 5, (6)

Chelonus cylindrus (Nees) Ch. inanitus (Linnaeus)

(6) 6

Ch. insularis Cresson Ch. scabrator Fabricius Chorebus petiolatus Nees

7 (6) (16)

Cotesia congregata (Say) C. glomerata (Linnaeus) Cotesia sp. Dacnusa sp. Diachasmimorpha dacusii (Cameron) D. longicaudata (Ashmead) Diaeretiella rapae (McIntosh) Dyscritulus planiceps (Marshall) Earinus gloriatorius Panzer

10 10b 11 (17) 20 20 (6) 4 (11)

Ephedrus californicus Baker E. persicae Froggatt E. plagiator (Nees) Ephedrus sp. Falciconus pseudoplatani (Marshall) Fopius arisanus (Sonan) Habrobracon hebetor (Say)

7 7 7 7 7 23 10

Habrobracon sp. aff.hebetor (Say) H. juglandis Ashmead

10 10c

H. pectinophorae Watanabed

10

Gokhman and Kolesnichenko (1998a) 34 Gokhman (2004c) 10, 12 Quicke and Belshaw (1999), Gokhman and Westendorff (2003) 14 Gokhman (2000a) 14 Gokhman and Quicke (1995) 6 Quicke (1997) ≈32–36 Kraaijeveld et al. (1999) 18 Gokhman (2003) 18 Gokhman (2004c) 28 Gokhman (2004c) 28 Gokhman and Quicke (1995) 10, 12 Gokhman and Quicke (1995), Gokhman (2002a) 12 Gokhman (2004c) 12 Gokhman and Kolesnichenko (1998c) 14 Silva-Junior et al. (2000b) 12 Gokhman (2003) 32 Gokhman and Kolesnichenko (1998a) (20) Belle et al. (2002) 20 Zhou et al. (2006) (22) Hoshiba and Imai (1993) 34 Gokhman and Quicke (1995) 40 Kitthawee et al. (2004) 40 Kitthawee et al. (1999, 2004) 12 Gokhman and Quicke (1995) (8) Quicke and Belshaw (1999) 22 Gokhman and Kolesnichenko (1996) (14) Quicke and Belshaw (1999) (14) Quicke and Belshaw (1999) (14) Quicke and Belshaw (1999) (14) Gokhman and Quicke (1995) (14) Quicke and Belshaw (1999) 46 Kitthawee et al. (2004) 20 Speicher and Speicher (1940), Rasch et al. (1975) (20) Holloway et al. (2000) 20 Torvik-Greb (1935), Rasch et al. (1977) 20 Inaba (1939)

88

Appendix A Table A.1 (continued) Chromosome number

Species

n

2n

Reference

H. serinopae Ramakrishna Heterospilus prosopidis Viereck Lysephedrus validus (Haliday) Lysiphlebus confusus Tremblay & Eadye L. fabarum Marshall Macrocentrus thoracicus Nees Meteorus gyrator (Thunberg) M. ictericus (Nees) M. pallipes Wesmael M. versicolor Wesmael Microchelonus gravenhorstii (Nees) Microgaster curvicrus Thomson Microplitis demolitor (Wilkinson)

10 (17) 7 6 6 (14) (10) (9) (10) 8 (6) (9) 10

20 34 (14) (12) 12 28 20 18 20 16 12 18 20

M. ratzeburgi (Ruthe) M. tuberculifer (Wesmael) Mirax sp. Pauesia juniperorum Star´y P. unilachni (Gahan) Phaenocarpa persimilis Papp

(11) (11) (10) 6 6 17

22 22 20 (12) (12) (34)

Praon abjectum Haliday P. dorsale Haliday P. volucre Haliday Psyttalia carinata (Thomson) P. fletcheri (Silvestri) P. incisi (Silvestri) Rhysipolis decorator Haliday Trioxys pallidus Haliday

(4) 4 4 (19) 17 17 (6) 9

8 (8) (8) 38 34 34 12 (18)

Rasch et al. (1977) Gokhman and Quicke (1995) Quicke and Belshaw (1999) Quicke and Belshaw (1999) Belshaw and Quicke (2003) Gokhman (2003) Gokhman and Quicke (1995) Gokhman (2004c) Gokhman and Quicke (1995) Gokhman and Quicke (1995) Gokhman (2003) Gokhman (2004c) M. Strand (personal communication) Gokhman (2003) Gokhman (2003) Gokhman and Quicke (1995) Quicke and Belshaw (1999) Quicke and Belshaw (1999) Prince and Stace unpublished data, cited in Crozier (1977) Gokhman and Quicke (1995) Quicke and Belshaw (1999) Quicke and Belshaw (1999) Gokhman (2004c) Kitthawee et al. (2004) Kitthawee et al. (2004) Gokhman and Quicke (1995) Quicke and Belshaw (1999)

Superfamily Evanioidea Family Gasteruptiidae Gasteruption breviterebrae Watanabe G. jaculator Linnaeus

14 (16)

(28) 32

Hoshiba and Imai (1993)f Quicke and Gokhman (1996)

Superfamily Cynipoidea Family Cynipidae Andricus curvator (Hartig) A. fecundator (Hartig) A. fecundatrix Mayr A. kollari (Hartig)

10 (10) 10 (10)

20 20 20 20

A. kashiwaphilus Abe A. mukaigawae (Mukaigawa) A. quercuscalicis (Burgsdorf) A. targionii (Kieffer) Aulacidea hieracii Bouch´e Biorhiza pallida Olivier Callirhytis palmiformis (Ashmead) Cynips divisa Hartig Diplolepis eglanteriae (Hartig)

(5) (6) (≈10) (5) 10 10 10 10 9

10 12 ≈20 10 20 20 20 20 27 (3n)

Dodds (1938), Sanderson (1988) Sanderson (1988) Dodds (1938) Hogben (1920), Sanderson (1988) Abe (1998) Abe (1998) Sanderson (1988) Abe (2007) Dodds (1938) Dodds (1938), Sanderson (1988) Goodpasture (1975b) Sanderson (1988) Sanderson (1988)

Appendix A

89 Table A.1 (continued) Chromosome number

Species

n

2n

Reference

D. nervosum Curtis D. rosae (Linnaeus)

(9) 9g

18 18

D. spinosissimae Girault Dryocosmus kuriphilus Yasumatsu Neuroterus laeviusculus (Schenck) N. numismalis (Fourcroy) N. quercusbaccarum (Linnaeus)

(9) (10) (10) 10 10

18 20 20 20 20

N. serratae (Ashmead) Trigonaspis megaptera (Panzer) Xestophanes potentillae (Retzius)

(10) 10 10

20 (20) (20)

Sanderson (1988) Henking (1892), Hogben (1920), Stille and D¨avring (1980), Sanderson (1988) Sanderson (1988) Abe (1994) Sanderson (1988) Dodds (1938), Sanderson (1988) Doncaster (1910, 1911, 1916), Dodds (1938, 1939), Sanderson (1988) Abe (2006) Dodds (1938) Dodds (1938)

Family Figitidae Callaspidia defonscolombei Dahlbom Leptopilina clavipes (Hartig) L. heterotoma Thomson

11 5 10

(22) (10) (20)

Phaenoglyphis villosa (Hartig)

(10)

20

Gokhman et al. (1999) Pannebakker et al. (2004) Jungen in litt. cited in Crozier (1975) Gokhman (2004b)

Superfamily Diaprioidea Family Diapriidae Acropiesta flaviventris (Thomson) Belyta depressa Thomson Cinetus lanceolatus Thomson Ismarus flavicornis Thomson

(11) 8 (10) (11)

22 (16) 20 22

Gokhman (2003) Gokhman and Quicke (1995) Gokhman and Quicke (1995) Gokhman (2003)

Superfamily Platygastroidea Family Scelionidae Telenomus chloropus Thomson T. fariai Lima

(10) 10

20 20

Gokhman (2003) Dreyfus and Breuer (1944)

Superfamily Ceraphronoidea Family Megaspilidae Dendrocerus carpenteri (Curtis)

(9)

18

Quicke and Gokhman (1996)

Superfamily Chalcidoidea Family Aphelinidae Aphelinus albipodus Hayat & Fatima A. asychis Walker A. mali (Haldeman) A. varipes (Foerster) Aphytis mytilaspidis (Le Baron) Coccophagus lycimnia (Walker) Encarsia aspidioticola (Mercet) E. asterobemisiae Viggiani & Mazzone E. berlesei Howard E. citrina (Crawford) E. formosa Gahan

(4) (4) 5 (4) 5 (11) (5) 10, 11 (5) (5) 5

8 8 (10) 8 10 22 10 20 10 10 10

Gokhman (2003) Gokhman (2003) Viggiani (1967) Gokhman (2003) R¨ossler and DeBach (1973) Baldanza et al. (1999) Baldanza et al. (1999) Baldanza et al. (1999) Baldanza et al. (1991b, 1999) Baldanza et al. (1999) Baldanza et al. (1994, 1999), Giorgini and Caprio (2003), Gokhman (2003)

90

Appendix A Table A.1 (continued) Chromosome number

Species

n

2n

Reference

E. hispida De Santis E. inaron (Walker) E. lahorensis (Howard) E. leucaspidis (Mercet) E. lutea (Masi) E. luteola Howard E. meritoria Gahan E. pergandiella Howard

(5) (8) (9) (9) (9) (5) (5) 6

10 16 18 18 18 10 10 12

E. protransvena Viggiani E. sophia (Girault & Dodd) E. tricolor (Foerster) Pteroptrix orientalis (Silvestri)

(3) (5) (7) 11

6 10 14 22

Giorgini and Caprio (2003) Baldanza et al. (1994, 1999) Baldanza et al. (1999) Baldanza et al. (1999) Baldanza et al. (1999) Baldanza and Giorgini (2001) Baldanza et al. (1994, 1999) Hunter et al. (1993), Baldanza et al. (1999) Baldanza et al. (1999) Giorgini and Baldanza (2004) Baldanza et al. (1994, 1999) Baldanza et al. (1991a)

Family Chalcididae Brachymeria intermedia (Nees) B. lasus (Walker) B. ovata (Say) Dirhinus himalayanus Westwood Psilochalcis brevialata Grissell & Johnson

3 5 5 5 6

6 10 10 (10) 12

Hung (1986) Hung (1986) Hung (1986) Amalin et al. (1988) Johnson et al. (2001)

11 10

22 20

Anagyrus lopezi (De Santis) A. orbitalis Timberlake Copidosoma buyssoni (Mayr) C. gelechiae (Howard) C. floridanum Ashmead

10 (11) 12 8, (10) 8, 10, 11

20 22 (24) 16, 20 16, 20, 22

C. koehleri (Blanchard) Copidosoma sp. Mira mucora Schellenberg Pseudencyrtus misellus (Dalman) Syrphophagus sosius (Walker) Syrphophagus sp.

11 (11) 9 11 (11) (11)

(22) 22 (18) (22) 22 22

Andrade-Souza et al. (2002) Silvestri (1908), Martin (1914), Gokhman (2004a) van Dijken (1991) Fusu (2008c) Silvestri (1914) Patterson (1921), Leiby (1922) Patterson (1917, 1921), Patterson and Porter (1917), Hunter and Bartlett (1975), Strand and Ode (1990) Guerrieri and Noyes (2005) Gokhman unpublished data Fusu (2008c) Gokhman unpublished data Gokhman (2003) Gokhman (2003)

(5)

10

Gokhman (2004e)

(6)

12

Gokhman (2004e)

5 (6) (7) (6) (5)

(10) 12 14 12 10

Gokhman (2002c) Gokhman (2004e) Gokhman (2003) Gokhman (2002c) Fusu (2008b), Kostjukov et al. (2008)

Family Encyrtidae Ageniaspis citricola Logvinovskaya A. fuscicollis (Dalman)

Family Eulophidae Aprostocetus (Aprostocetus) sp. (epicharmus species group) Aprostocetus (Aprostocetus) sp. (lycidas species group) Aprostocetus (Aprostocetus) sp. 1 Aprostocetus (Aprostocetus) sp. 2 A. (Hyperteles) elongatus (Foerster) A. (Ootetrastichus) crino (Walker) A. (Stepanovia) eurytomae (Nees)

Appendix A

91 Table A.1 (continued) Chromosome number

Species

n

2n

Reference

A. (S.) kubanica (Kostjukov) Baryscapus evonymellae (Bouch´e) B. gigas (Burks) B. megachilidis (Burks) B. orgyiae Kostjukov B. pallidae Graham

(5) (6) 6 6 (6) 6

10 12 (12) 12 12 12

Baryscapus sp. 1 (evonymellae species group) Baryscapus sp. 2 (evonymellae species group) Cirrospilus diallus (Walker) Closterocerus formosus Westwood Colpoclypeus florus (Walker) Elachertus sp. Emersonella sp. Entedon sp. Euplectrus bicolor (Swederus) Eu. flavipes (Fonscolombe) Euplectrus sp. Melittobia australica Girault

(6)

12

Kostjukov et al. (2008) Gokhman (2004e) Goodpasture (1974) Goodpasture (1974) Kostjukov and Gokhman (2001) Gokhman (2003) and unpublished data Gokhman (2004e)

6

(12)

Gokhman (2004e)

(6) 5 6 (8) (6) (6) (5) (6) (6) (6)

12 10 12 16 12 12 10 12 12 12

M. chalybii Ashmead

5

10

M. hawaiiensis Perkins Mestocharis bimaculatus Dalman Oomyzus galerucivorus (Hedqvist) Palmistichus elaeisis Delvare & LaSalle Pediobius cassidae Erd¨os P. planiventris Walker Pnigalio soemius (Walker) Sympiesis acalle (Walker) S. sandanis (Walker) Tetrastichus (Musciformia) dasyops Graham T. (M) atratulus (Nees) Tetrastichus (Tetrastichus) sp. 1 Tetrastichus (Tetrastichus) sp. 2 Thripobius javae (Girault) Trichospilus diatraeae Cherian & Margabandhu

(6) (6) (6) (6) (6) 6 6 (6) (6) (6)

12 12 12 12 12 (12) 12 12 12 12

Gokhman and Quicke (1995) Adachi-Hagimori et al. (2008) Dijkstra (1986) Gokhman (2002c) Silva-Junior et al. (2000a) Gokhman (2004e) Gokhman (2002c) Gokhman (2004e) Gokhman (2002c) Silva-Junior et al. (2000a), Maffei et al. (2001) Schmieder (1938), MacDonald and Kruni´c (1971) Silva-Junior et al. (2000a) Gokhman (2003) Gokhman (2002c) Silva-Junior et al. (2000a) Gokhman (2002c) Gokhman (2003) Bernardo et al. (2008) Gokhman (2002c) Gokhman (2002c) Gokhman (2003)

(6) (6) 6 (6) (7)

12 12 12 12 14

Gokhman (2004e) Gokhman (2003) Gokhman (2004e) Caprio and Bernardo (2006) Silva-Junior et al. (2000a)

Family Eupelmidae Anastatus catalonicus Bolivar Eupelmus urozonus (Dalman) E. vesicularis (Retzius)

(5) (5) (5)

10 10 10

Eupelmus sp. 1 Eupelmus sp. 2

(6) (6)

12 12

Gokhman and Quicke (1995) Gokhman (2002a) Gokhman and Quicke (1995), Fusu (2008a) Fusu (2008a) Fusu (2008a)

92

Appendix A Table A.1 (continued) Chromosome number

Species

n

2n

Reference

Family Eurytomidae Bephratelloides pomorum (Fabricius) Eurytoma aciculata Ratzeburg Eu. brunniventris Ratzeburg Eu. californica Ashmead Eu. compressa (Fabricius)

10 (10) (10) 10 (5)

20 20 20 ≈20 10

Eu. flavimana Boheman Eu. pistaciae Rondani Eu. robusta Mayr

(10) (10) 7

20 20 14

Eu. rosae Nees

(10)

20

Eu. serratulae (Fabricius)

6

12

Sycophila biguttata (Swederus)

(9)

18

Gomes et al. (1996) Gokhman unpublished data Gokhman (2003) Goodpasture (1974) Gokhman and Mikhailenko (2008b) Gokhman (2005b) Gokhman unpublished data Gokhman and Mikhailenko (2008b) Gokhman and Mikhailenko (2008b) Gokhman and Mikhailenko (2008b) Gokhman and Quicke (1995)

Family Leucospidae Leucospis affinis Say

6

12

Goodpasture (1974)

Family Mymaridae Anaphes iole Girault A. listronoti Huber

9 9

18 18

Gokhman (2000b) Gokhman (2002a)

Family Ormyridae Ormyrus gratiosus (Foerster) Ormyrus sp.

(5) (6)

10 12

Gokhman (2005b) Gokhman and Quicke (1995), Gokhman (2005b)

(3)

6

Gokhman (2005b)

(5)

10

Gokhman (2003)

5, 7

10, 14

(5) 5

10 10

Gokhman and Quicke (1995), Gokhman et al. (1998) Gokhman and Quicke (1995) Gershenzon (1968), Gokhman (2003) Silva-Junior et al. (2000a) Goodpasture (1974) Gokhman and Westendorff (2000) Gokhman and Westendorff (2000) Pennypacker (1958), Whiting (1960, 1968), Gershenzon (1968), Macy and Whiting (1969), Goodpasture (1974), Nur et al. (1988), Werren (1991), Reed (1993), Gokhman and Westendorff (2000)

Family Perilampidae Perilampus ruschkai Hell´en Family Pteromalidae Amphidocius schickae Heydon & Bouˇcek Anisopteromalus calandrae (Howard) Coelopisthia extenta (Walker) Lariophagus distinguendus Foerster

Muscidifurax uniraptor Kogan & Legner (5) M. zaraptor Legner 5 Nasonia giraulti Darling 5

10 (10) 10

N. longicornis Darling

5

10

N. vitripennis (Walker)

5+0−1B, 6

10, 12

Appendix A

93 Table A.1 (continued) Chromosome number

Species

n

2n

Reference

Pteromalus puparum (Linnaeus)

5

10

P. venustus Walker Pteromalus sp. Spalangia endius Walker

5 (5) 4, (6)

(10) 10 8, 12

Guhl and Dozortseva (1934), Dozortseva (1936) MacDonald and Kruni´c (1971) Gokhman (2003) Silva-Junior et al. (2000a), Kitthawee and Vasinpiyamongcol (2002)

Family Torymidae Megastigmus pictus (Foerster)

5

10

M. strobilobius Ratzeburg Monodontomerus clementi Grissell

5 6

10 12

M. montivagus Ashmead M. obscurus Westwood

6 4, 6

(12) 8, 12

M. saltuosus Grissell

5

10

Podagrion gibbum Bernard P. pachymerum (Walker) Torymus baccharidis (Huber) T. bedeguaris (Linnaeus)

(10) 10 6 (6)

20 20 12 12

T. californicus (Ashmead) T. capillaceus (Huber) T. chloromerus (Walker)

6 (6) (6)

12 12 12

T. koebelei (Huber) T. occidentalis (Huber) T. tubicola (Osten Sacken) T. umbilicatus (Gahan) T. vesiculi Moser T. warreni (Cockerell)

5 6 6 5 (6) 6

10 12 12 10 12 12

Family Trichogrammatidaeh Trichogramma brasiliense (Ashmead)

5

(10)

T. brassicae Bezdenko T. cacoeciae Marchal T. chilonis Ischii

5 (5) 5

10 10 10

T. deion Pinto & Oatman T. dendrolimi Matsumura T. evanescens Westwood T. japonicum Ashmead

5 5 5 5

(10) 10 10 (10)

T. kaykai Pinto & Stouthamer T. nubilale Ertle & Davis T. pretiosum Riley

5+0−1B (5) (5)

(10) 10 10

Gokhman (2005b) and unpublished data Gokhman (2005b) Grissell (1973), Goodpasture (1975a) Goodpasture (1975a) MacDonald and Kruni´c (1971), Goodpasture (1975a) Grissell (1973), Goodpasture (1975a) Fusu (2008d) Fusu (2008d) Goodpasture and Grissell (1975) Gokhman and Mikhailenko (2007) Goodpasture and Grissell (1975) Goodpasture and Grissell (1975) Gokhman and Mikhailenko (2007) Goodpasture and Grissell (1975) Goodpasture and Grissell (1975) Goodpasture and Grissell (1975) Goodpasture and Grissell (1975) Goodpasture and Grissell (1975) Goodpasture and Grissell (1975) Amutha Murugan and Manickavasagam (2003) Laurent et al. (1998) Vavre et al. (2004) Hung (1982), Amutha Murugan and Manickavasagam (2003) Stouthamer and Kazmer (1994) Liu and Xiong (1988) Hung (1982) Amutha Murugan and Manickavasagam (2003) Stouthamer et al. (2001) Hung (1982) Hung (1982), Stouthamer and Kazmer (1994)

94

Appendix A Table A.1 (continued) Chromosome number

Species

n

2n

Reference

Trichogramma spp.

5

10

Fukada and Takemura (1943)

Superfamily Chrysidoidea Family Bethylidaei Epyris niger Westwood Laelius utilis Cockerell

(14) (10)

28 20

Gokhman and Quicke (1995) Gokhman and Quicke (1995)

Family Chrysididae Chrysis viridula Linnaeus Hedychridium ardens (Latreille) H. roseum (Rossi) Omalus djozanus hondonis (Tsuneki)

(21) (19) (19) (19)

42 38 38 38

Quicke and Gokhman (1996) Quicke and Gokhman (1996) Quicke and Gokhman (1996) Hoshiba and Imai (1993)

Family Dryinidae Anteon ephippiger (Dalman) A. gaullei Kieffer A. jurinearum Latreille

(4) 4 (5)

8 8 10

Gokhman (2001b) Gokhman (2001b) Gokhman and Kolesnichenko (1998b) Gokhman (2001b) Gokhman (2005a) Gokhman (2002b) Gokhman (2005a) Gokhman (2001b)

A. pubicorne (Dalman) 4 8 Dicondylus dichromus (Kieffer) (4) 8 Gonatopus clavipes (Thunberg) 4 8 G. formicarius Ljungh (4) 8 Lonchodryinus ruficornis (Dalman) (7) 14 Values given in brackets are extrapolated from the known ones. a Hogben (1920) has reported n = 11 and 2n ≈ 22 in this species from the UK, but this information is not supported by our recent study (Gokhman and Mikhailenko 2008a). b Hegner (1915) has reported n ≈ 12 in this species. c Whiting (1927) has previously reported n = 11 for this species. d Whiting (1949) believed that this species was synonymous to the preceding one. e Muramoto (1993) has reported 2n = 28 for Lysiphlebus sp., but this record is obviously erroneous. f The species was cited in this paper as “Trychofoenus sp.” (Imai in litt.). g Schleip (1909) has reported 10–12 chromosomes in this species (perhaps as the haploid chromosome number). h Muramoto (1993) has reported 2n = 4 for Oligosita sp., but this record needs to be confirmed. i The chromosome number of Scleroderma sichuanensis Xiao, 2n ≈ 18 (see Zhan et al. 2006), needs to be confirmed.

Appendix B

Micrographs and Ideograms of Chromosome Sets of Parasitic Hymenoptera

Fig. B.1 Karyogram of Perithous scurra (Gokhman and Kolesnichenko 1997). Reproduced by permission of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences

Fig. B.2 Karyogram of Theronia atalantae (Gokhman and Mikhailenko 2008a)

Fig. B.3 Karyogram of Paraperithous gnathaulax (Gokhman 2005a)

95

96

Appendix B

Fig. B.4 Karyogram of Dolichomitus agnoscendus (Gokhman and Kolesnichenko 1997). Reproduced by permission of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences

Fig. B.5 Karyogram of Dolichomitus messor (modified after Gokhman 2002a)

Appendix B Fig. B.6 Karyogram of Ephialtes manifestator; mitotic chromosomes in prometaphase (Gokhman 2005a)

Fig. B.7 Karyogram of Scambus buolianae (Gokhman 2005a)

Fig. B.8 Karyogram of Scambus detritus (Gokhman and Mikhailenko 2008a)

97

98

Fig. B.9 Karyogram of Scambus nigricans (Gokhman 2001a)

Fig. B.10 Karyogram of Scambus vesicarius (Gokhman and Mikhailenko 2008a)

Fig. B.11 Karyogram of Oxyrrhexis carbonator (Gokhman 2001a)

Appendix B

Appendix B

Fig. B.12 Karyogram of Zatypota gracilis (Gokhman 2005a)

Fig. B.13 Karyogram of Pimpla contemplator (Gokhman and Mikhailenko 2008a)

Fig. B.14 Karyogram of Aperileptus microspilus (Gokhman 2001a)

99

100

Appendix B

Fig. B.15 Karyogram of Orthocentrus sp. (Gokhman 2005a)

Fig. B.16 Karyogram of Stenomacrus sp. 2; mitotic chromosomes (Gokhman and Mikhailenko 2008a)

Fig. B.17 Karyogram of Stenomacrus sp. 2; meiotic chromosomes in metaphase I (Gokhman and Mikhailenko 2008a)

Fig. B.18 Karyogram of Orthopelma mediator; mitotic chromosomes (Gokhman and Mikhailenko 2008a)

Fig. B.19 Karyogram of Orthopelma mediator; meiotic chromosomes in diakinesis (Gokhman and Mikhailenko 2008a)

Fig. B.20 Karyogram of Glypta ceratites; form with 2n = 12 and all paired chromosomes (Gokhman 2005a)

Appendix B

101

Fig. B.21 Karyogram of Glypta ceratites; form with 2n = 12 and two unpaired chromosomes (Gokhman 2005a)

Fig. B.22 Karyogram of Glypta ceratites; form with 2n = 13 and one unpaired chromosome (Gokhman 2005a)

Fig. B.23 Karyogram of Glypta sp. (Gokhman 2005a)

Fig. B.24 Karyogram of Lissonota buccator (Gokhman and Mikhailenko 2008a)

Fig. B.25 Karyogram of Lissonota catenator (Gokhman 2001a)

102

Fig. B.26 Karyogram of Lissonota coracina (Gokhman 2001a)

Fig. B.27 Karyogram of Lissonota sp. 2 (Gokhman 2005a)

Fig. B.28 Karyogram of Campoplex sp. (Gokhman 2005a)

Appendix B

Appendix B

103

Fig. B.29 Karyogram of Venturia canescens; routine chromosome staining (Gokhman 2001a)

Fig. B.30 Karyogram of Venturia canescens; C-banding (Gokhman 2001a)

Fig. B.31 Karyogram of Hyposoter sp. 1 (Gokhman 2005a)

Fig. B.32 Karyogram of Pristomerus sp. (Gokhman 2005a)

Fig. B.33 Karyogram (Gokhman 2001a)

Fig. B.34 Karyogram of Mesochorus sp. (Gokhman 2005a)

of

Rhorus

extirpatorius;

meiotic

chromosomes

in

diakinesis

104

Appendix B

Fig. B.35 Karyogram of Stethoncus sulcator (Gokhman 2001a)

Fig. B.36 Karyogram of Triclistus podagricus; mitotic chromosomes (Gokhman 2000a)

Fig. B.37 Karyogram of Triclistus podagricus; meiotic chromosomes in metaphase I (Gokhman 2000a)

Fig. B.38 Karyogram of Netelia latungula (Gokhman 2001a)

Fig. B.39 Karyogram of Adelognathus brevicornis (Gokhman 2005a)

Fig. B.40 Karyogram of Trichionotus flexorius (Gokhman and Mikhailenko 2008a)

Appendix B

Fig. B.41 Karyogram of Gelis sp. 2 (Gokhman 2005a)

Fig. B.42 Karyogram of Phygadeuon sp. 1 (Gokhman 2001a)

Fig. B.43 Karyogram of Phygadeuon sp. 2 (Gokhman 2001a)

Fig. B.44 Karyogram of Phygadeuon sp. 3 (Gokhman and Mikhailenko 2008a)

105

106

Fig. B.45 Karyogram of Aptesis puncticollis (modified after Gokhman 2000a)

Fig. B.46 Karyogram of Mesostenus gracilis (Gokhman 2001a)

Appendix B

Appendix B

Fig. B.47 Karyogram of Heterischnus nigricollis (Gokhman 1990b)

Fig. B.48 Karyogram of Colpognathus celerator (modified after Gokhman 2002a)

Fig. B.49 Karyogram of Dicaelotus sp. cf. parvulus (Gokhman 2005a)

Fig. B.50 Karyogram of Centeterus major (Gokhman 2007d)

107

108

Fig. B.51 Karyogram of Tycherus suspicax (Gokhman 1987)

Fig. B.52 Karyogram of Tycherus fuscicornis (Gokhman 1990b)

Fig. B.53 Karyogram of Tycherus infimus (Gokhman 2007d)

Appendix B

Appendix B

Fig. B.54 Karyogram of Tycherus nigridens (Gokhman 1990b)

Fig. B.55 Karyogram of Tycherus ophthalmicus (Gokhman 2005a)

109

110

Fig. B.56 Karyogram of Tycherus australogeminus (holotype) (Gokhman 1991)

Fig. B.57 Karyogram of Tycherus ischiomelinus (Gokhman 1991)

Appendix B

Appendix B

Fig. B.58 Karyogram of Tycherus bellicornis; form with 2n = 20 (Gokhman 1989)

Fig. B.59 Karyogram of Tycherus bellicornis; first form with 2n = 21 (Gokhman 1989)

111

112

Appendix B

Fig. B.60 Karyogram of Tycherus bellicornis; second form with 2n = 21 (Gokhman 1989)

Fig. B.61 Karyogram of Tycherus osculator (Gokhman 1989)

Appendix B

Fig. B.62 Karyogram of Tycherus dilleri (Gokhman 1989)

Fig. B.63 Karyogram of Phaeogenes melanogonos (Gokhman 1990b)

Fig. B.64 Karyogram of Phaeogenes spiniger (Gokhman 1990b)

113

114 Fig. B.65 Karyogram of Dirophanes invisor; routine chromosome staining (Gokhman 1987)

Fig. B.66 Karyogram of Dirophanes invisor; C-banding, homologous chromosomes of the second pair are similar (Gokhman 1997b)

Appendix B

Appendix B Fig. B.67 Karyogram of Dirophanes invisor; C-banding, homologous chromosomes of the second pair are heteromorphic (Gokhman 1997b)

Fig. B.68 Karyogram of Dirophanes callopus; routine chromosome staining (Gokhman 1987)

115

116

Appendix B

Fig. B.69 Karyogram of Dirophanes callopus; C-banding (Gokhman 1997b)

Fig. B.70 Karyogram of Dirophanes fulvitarsis; routine chromosome staining (Gokhman 1990b)

Appendix B

Fig. B.71 Karyogram of Dirophanes fulvitarsis; male, C-banding (Gokhman 1997b)

Fig. B.72 Karyogram of Dirophanes fulvitarsis; female, C-banding (Gokhman 1997b)

Fig. B.73 Karyogram of Oronotus binotatus (Gokhman 1987)

117

118

Fig. B.74 Karyogram of Diadromus subtilicornis (Gokhman 1990b)

Fig. B.75 Karyogram of Diadromus prosopius (Gokhman 1990b)

Fig. B.76 Karyogram of Diadromus troglodytes (Gokhman 2007d)

Fig. B.77 Karyogram of Diadromus varicolor (Gokhman 2007d)

Appendix B

Appendix B

Fig. B.78 Karyogram of Aethecerus discolor (modified after Gokhman 1985)

Fig. B.79 Karyogram of Aethecerus ranini (holotype) (Gokhman 1991)

119

120

Fig. B.80 Karyogram of Aethecerus dispar (Gokhman 1991)

Fig. B.81 Karyogram of Eurylabus torvus (Gokhman 1987)

Appendix B

Appendix B

Fig. B.82 Karyogram of Virgichneumon digrammus (Gokhman 2005a)

Fig. B.83 Karyogram of Vulgichneumon saturatorius (Gokhman 1987)

121

122

Fig. B.84 Karyogram of Baranisobas ridibundus (Gokhman 1990b)

Fig. B.85 Karyogram of Cratichneumon rufifrons (Gokhman 2007d)

Fig. B.86 Karyogram of Cratichneumon fabricator (Gokhman 2005a)

Appendix B

Appendix B

Fig. B.87 Karyogram of Cratichneumon viator (modified after Gokhman 1985)

Fig. B.88 Karyogram of Cratichneumon sicarius (Gokhman 1990b)

123

124

Fig. B.89 Karyogram of Homotherus locutor (Gokhman 1990b)

Fig. B.90 Karyogram of Aoplus pulchricornis (Gokhman 1990b)

Appendix B

Appendix B

Fig. B.91 Karyogram of Crypteffigies lanius (Gokhman 2005a)

Fig. B.92 Karyogram of Eristicus clarigator (Gokhman 2007d)

Fig. B.93 Karyogram of Syspasis albiguttata (modified after Gokhman 1985)

125

126

Appendix B

Fig. B.94 Karyogram of Patrocloides chalybeatus (Gokhman 1993)

Fig. B.95 Karyogram of Pseudoamblyteles homocerus (Gokhman 2001a)

Fig. B.96 Karyogram of Lymantrichneumon disparis (Gokhman and Mikhailenko 2008a)

Fig. B.97 Karyogram of Ichneumon vorax (Gokhman and Mikhailenko 2008a)

Appendix B

Fig. B.98 Karyogram of Ichneumon bucculentus (Gokhman 1993)

Fig. B.99 Karyogram of Ichneumon sarcitorius (Gokhman 2005a)

Fig. B.100 Karyogram of Ichneumon subquadratus (Gokhman 1990b)

127

128

Appendix B

Fig. B.101 Karyogram of Ichneumon memorator (Gokhman and Mikhailenko 2008a)

Fig. B.102 Karyogram of Ichneumon ingratus (Gokhman 1990b)

Fig. B.103 Karyogram of Ichneumon amphibolus (Gokhman 1990b)

Appendix B

Fig. B.104 Karyogram of Ichneumon suspiciosus; form with 2n = 24 (Gokhman 1993)

Fig. B.105 Karyogram of Ichneumon suspiciosus; form with 2n = 26 (Gokhman 1993)

129

130

Fig. B.106 Karyogram of Ichneumon stramentarius (Gokhman 1987)

Fig. B.107 Karyogram of Ichneumon inquinatus (Gokhman 1990b)

Appendix B

Appendix B

131

Fig. B.108 Karyogram of Ichneumon gracilentus; form with 2n = 24 (modified after Gokhman 1985)

Fig. B.109 Karyogram of Ichneumon gracilentus; form with 2n = 25 (Gokhman 1993)

132

Appendix B

Fig. B.110 Karyogram of Ichneumon extensorius; form with 2n = 24 (Gokhman 1993)

Fig. B.111 Karyogram of Ichneumon extensorius; form with 2n = 26 (Gokhman 1993)

Fig. B.112 Karyogram of Ichneumon crassifemur (modified after Gokhman 1985)

Appendix B

Fig. B.113 Karyogram of Ichneumon confusor (modified after Gokhman 1985)

Fig. B.114 Karyogram of Ichneumon gracilicornis (Gokhman 2005a)

Fig. B.115 Karyogram of Ichneumon croceipes (Gokhman 1990b)

133

134

Fig. B.116 Karyogram of Ichneumon minutorius (Gokhman 1987)

Fig. B.117 Karyogram of Thyrateles camelinus (Gokhman 1990b)

Appendix B

Appendix B

Fig. B.118 Karyogram of Stenichneumon culpator (modified after Gokhman 1985)

Fig. B.119 Karyogram of Chasmias motatorius (modified after Gokhman 1985)

135

136

Fig. B.120 Karyogram of Diphyus latebricola (Gokhman 2005a)

Fig. B.121 Karyogram of Diphyus raptorius (Gokhman 2005a)

Fig. B.122 Karyogram of Ctenichneumon funereus (Gokhman 2007d)

Appendix B

Appendix B

Fig. B.123 Karyogram of Coelichneumon sugillatorius (Gokhman 2005a)

Fig. B.124 Karyogram of Coelichneumon cyaniventris (Gokhman 2005a)

Fig. B.125 Karyogram of Psyttalia carinata (Gokhman 2004c)

137

138

Appendix B

Fig. B.126 Karyogram of Alysia manducator; male (Gokhman and Kolesnichenko 1998a)

Fig. B.127 Karyogram of Alysia manducator; female (Gokhman and Kolesnichenko 1998a)

Fig. B.128 Karyogram of Aphaereta tenuicornis (Gokhman 2004c)

Appendix B

139

Fig. B.129 Karyogram of Chorebus petiolatus (Gokhman and Kolesnichenko 1998a)

Fig. B.130 Karyogram of Charmon cruentatus; form with 2n = 10 (Gokhman 2005a)

Fig. B.131 Karyogram of Charmon cruentatus; form with 2n = 12 (Gokhman 2005a)

Fig. B.132 Karyogram of Charmon cruentatus; form with n = 5, meiotic chromosomes in diplotene (Gokhman 2005a)

Fig. B.133 Karyogram of Macrocentrus thoracicus (Gokhman 2005a)

Fig. B.134 Karyogram of Meteorus versicolor; mitotic chromosomes (Gokhman 2005a)

140

Fig. B.135 Karyogram (Gokhman 2005a)

Appendix B

of Meteorus versicolor;

meiotic chromosomes

in diakinesis

Fig. B.136 Karyogram of Bassus dimidiator; mitotic chromosomes (Gokhman 2005a)

Fig. B.137 Karyogram (Gokhman 2005a)

of

Bassus

dimidiator;

meiotic

chromosomes

in

diplotene

Fig. B.138 Karyogram of Bassus tumidulus (modified after Gokhman 2004c)

Fig. B.139 Karyogram of Chelonus inanitus; male (Gokhman and Kolesnichenko 1998c)

Fig. B.140 Karyogram of Chelonus inanitus; female (Gokhman and Kolesnichenko 1998c)

Appendix B

141

Fig. B.141 Ideogram of Chelonus inanitus; haploid chromosome set (Gokhman and Kolesnichenko 1998c)

Fig. B.142 Karyogram of Chelonus scabrator (Gokhman 2005a)

Fig. B.143 Karyogram of Microchelonus gravenhorstii (Gokhman 2005a)

Fig. B.144 Karyogram of Aphidius ervi; male (Gokhman and Westendorff 2003). Reproduced by permission of the German Entomological Institute

142

Appendix B

Fig. B.145 Karyogram of Aphidius ervi; female with 2n = 10 (Gokhman and Westendorff 2003). Reproduced by permission of the German Entomological Institute

Fig. B.146 Karyogram of Aphidius ervi; female with 2n = 12 (Gokhman and Westendorff 2003). Reproduced by permission of the German Entomological Institute

Fig. B.147 Chromosomes of Aphidius ervi; form with 2n = 12 at prophase (left) and prometaphase stage (right). Arrowheads indicate chromosomes of the last pair (Gokhman and Westendorff 2003). Reproduced by permission of the German Entomological Institute

Fig. B.148 Karyogram of Aphidius matricariae (Gokhman 2000a)

Fig. B.149 Karyogram of Diaeretiella rapae (modified after Gokhman and Quicke 1995). Reproduced by permission of the International Society of Hymenopterists

Appendix B

143

Fig. B.150 Karyogram of Gasteruption jaculator (Quicke and Gokhman 1996). Reproduced by permission of the International Society of Hymenopterists

Fig. B.151 Karyogram of Callaspidia defonscolombei; meiotic chromosomes in diplotene (Gokhman 1999)

Fig. B.152 Karyogram of Phaenoglyphis villosa (Gokhman 2004b)

144

Appendix B

Fig. B.153 Karyogram of Ismarus flavicornis (Gokhman 2005a)

Fig. B.154 Karyogram of Acropiesta flaviventris (Gokhman 2005a)

Fig. B.155 Karyogram of Belyta depressa; meiotic chromosomes in diakinesis (Gokhman 2005a)

Fig. B.156 Karyogram of Dendrocerus carpenteri (Quicke and Gokhman 1996). Reproduced by permission of the International Society of Hymenopterists

Appendix B

145

Fig. B.157 Karyogram of Eurytoma flavimana (Gokhman 2005b). Reproduced by permission of the Japan Mendel Society

Fig. B.158 Karyogram of Eurytoma brunniventris (Gokhman 2005a)

Fig. B.159 Karyogram of Eurytoma rosae (Gokhman and Mikhailenko 2008b). Reproduced by permission of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences

Fig. B.160 Karyogram of Eurytoma robusta; mitotic chromosomes (Gokhman and Mikhailenko 2008b). Reproduced by permission of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences

Fig. B.161 Karyogram of Eurytoma robusta; meiotic chromosomes in diplotene (Gokhman and Mikhailenko 2008b). Reproduced by permission of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences

146

Appendix B

Fig. B.162 Karyogram of Eurytoma serratulae; mitotic chromosomes (Gokhman and Mikhailenko 2008b). Reproduced by permission of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences

Fig. B.163 Karyogram of Eurytoma serratulae; meiotic chromosomes in diplotene (Gokhman and Mikhailenko 2008b). Reproduced by permission of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences

Fig. B.164 Karyogram of Eurytoma compressa (Gokhman and Mikhailenko 2008b). Reproduced by permission of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences

Fig. B.165 Karyogram of Sycophila biguttata (Gokhman and Mikhailenko 2008b). Reproduced by permission of the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences

Fig. B.166 Karyogram of Ageniaspis fuscicollis; mitotic chromosomes (Gokhman 2004a)

Fig. B.167 Karyogram of Ageniaspis fuscicollis; meiotic chromosomes in diakinesis (Gokhman 2004a)

Fig. B.168 Karyogram of Syrphophagus sosius (Gokhman 2005a)

Appendix B

Fig. B.169 Karyogram of Anaphes iole; male (Gokhman 2000b)

Fig. B.170 Karyogram of Anaphes iole; female (Gokhman 2000b)

Fig. B.171 Ideogram of Anaphes iole; haploid chromosome set (Gokhman 2000b)

Fig. B.172 Karyogram of Anaphes listronoti (Gokhman 2002a)

Fig. B.173 Karyogram of Aphelinus albipodus (Gokhman 2005a)

Fig. B.174 Karyogram of Aphelinus asychis (Gokhman 2005a)

147

148

Fig. B.175 Karyogram of Aphelinus varipes; AvRpF strain (Gokhman 2005a)

Fig. B.176 Karyogram of Aphelinus varipes; AvRpI strain (Gokhman 2005a)

Fig. B.177 Karyogram of Aphelinus varipes; AvDnF strain (Gokhman 2005a)

Fig. B.178 Karyogram of Aphelinus varipes; AvDnG strain (Gokhman 2005a)

Fig. B.179 Karyogram of Encarsia formosa (Gokhman 2005a)

Fig. B.180 Karyogram of Perilampus ruschkai (Gokhman 2005b). Reproduced by permission of the Japan Mendel Society

Appendix B

Appendix B

149

Fig. B.181 Karyogram of Psilochalcis brevialata; male (Johnson et al. 2001). Reproduced by permission of the Entomological Society of Washington

Fig. B.182 Karyogram of Psilochalcis brevialata; female (Johnson et al. 2001). Reproduced by permission of the Entomological Society of Washington

Fig. B.183 Ideogram of Psilochalcis brevialata; haploid chromosome set (Johnson et al. 2001). Reproduced by permission of the Entomological Society of Washington

Fig. B.184 Karyogram of Entedon sp. (Gokhman 2004e)

150

Appendix B

Fig. B.185 Karyogram of Pediobius cassidae (Gokhman 2002c)

Fig. B.186 Karyogram of Pediobius planiventris; meiotic chromosomes in diplotene (Gokhman 2005a)

Fig. B.187 Karyogram of Aprostocetus (Aprostocetus) sp. (lycidas group) (Gokhman 2004e)

Fig. B.188 Karyogram of Aprostocetus (Aprostocetus) sp. (epicharmus group) (Gokhman 2004e)

Fig. B.189 Karyogram of Aprostocetus (Aprostocetus) sp. 1; meiotic chromosomes in diplotene (Gokhman 2002c) Fig. B.190 Karyogram of Aprostocetus (Aprostocetus) sp. 2 (Gokhman 2004e)

Fig. B.191 Karyogram of Aprostocetus (Hyperteles) elongatus (Gokhman 2005a)

Appendix B

151

Fig. B.192 Karyogram of Aprostocetus (Ootetrastichus) crino (Gokhman 2002c)

Fig. B.193 Karyogram of Aprostocetus (Stepanovia) eurytomae (Kostjukov et al. 2008)

Fig. B.194 Karyogram of Aprostocetus (Stepanovia) kubanica (holotype) (Kostjukov et al. 2008)

Fig. B.195 Karyogram of Baryscapus evonymellae (Gokhman 2004e)

Fig. B.196 Karyogram of Baryscapus sp. 1 (evonymellae group) (Gokhman 2004e)

Fig. B.197 Karyogram of Baryscapus sp. 2 (evonymellae group); meiotic chromosomes in diplotene (Gokhman 2004e)

Fig. B.198 Karyogram of Baryscapus orgyiae (paratype) (Kostjukov and Gokhman 2001)

152

Appendix B

Fig. B.199 Karyogram of Baryscapus pallidae (Gokhman 2005a)

Fig. B.200 Karyogram of Oomyzus galerucivorus (Gokhman 2002c)

Fig. B.201 Karyogram (Gokhman 2004e)

of

Tetrastichus

(Tetrastichus)

sp.

2;

mitotic

chromosomes

Fig. B.202 Karyogram of Tetrastichus (Tetrastichus) sp. 2; meiotic chromosomes in diplotene (Gokhman 2004e)

Fig. B.203 Karyogram of Tetrastichus (Musciformia) atratulus (Gokhman 2004e)

Appendix B

153

Fig. B.204 Karyogram of Euplectrus bicolor (Gokhman 2002c)

Fig. B.205 Karyogram of Euplectrus flavipes (Gokhman 2004e)

Fig. B.206 Karyogram of Euplectrus sp. (Gokhman 2002c)

Fig. B.207 Karyogram of Elachertus sp. (Gokhman 2002c)

Fig. B.208 Karyogram of Sympiesis sandanis (Gokhman 2002c)

Fig. B.209 Karyogram of Anastatus catalonicus (modified after Gokhman and Quicke 1995). Reproduced by permission of the International Society of Hymenopterists

154

Appendix B

Fig. B.210 Karyogram of Eupelmus urozonus (Gokhman 2002a)

Fig. B.211 Karyogram of Anisopteromalus calandrae; male with n = 5 (Gokhman et al. 1998)

Fig. B.212 Karyogram of Anisopteromalus calandrae; female with 2n = 10 (Gokhman et al. 1998)

Fig. B.213 Karyogram of Anisopteromalus calandrae; male with n = 7 (Gokhman et al. 1998)

Fig. B.214 Karyogram of Anisopteromalus calandrae; female with 2n = 14 (Gokhman et al. 1998)

Appendix B

155

Fig. B.215 Ideogram of Anisopteromalus calandrae; haploid chromosome sets, forms with n = 5 (left) and 7 (right) (Gokhman et al. 1998)

Fig. B.216 Karyogram of Lariophagus distinguendus (Gokhman 2005a)

Fig. B.217 Karyogram of Nasonia vitripennis; routine chromosome staining (Gokhman and Westendorff 2000). Reproduced by permission of the German Entomological Institute

Fig. B.218 Karyogram of Nasonia vitripennis; C-banding (Gokhman and Westendorff 2000). Reproduced by permission of the German Entomological Institute

Fig. B.219 Karyogram of Nasonia vitripennis; male carrying B chromosome (Gokhman 2005a)

156

Appendix B

Fig. B.220 Karyogram of Nasonia giraulti; routine chromosome staining (Gokhman and Westendorff 2000). Reproduced by permission of the German Entomological Institute

Fig. B.221 Karyogram of Nasonia giraulti; C-banding (Gokhman and Westendorff 2000). Reproduced by permission of the German Entomological Institute

Fig. B.222 Karyogram of Nasonia longicornis; routine chromosome staining (Gokhman and Westendorff 2000). Reproduced by permission of the German Entomological Institute

Fig. B.223 Karyogram of Nasonia longicornis; C-banding (Gokhman and Westendorff 2000). Reproduced by permission of the German Entomological Institute

Fig. B.224 Karyogram of Pteromalus sp. (Gokhman 2005a)

Appendix B

157

Fig. B.225 Karyogram of Ormyrus gratiosus (Gokhman 2005b). Reproduced by permission of the Japan Mendel Society

Fig. B.226 Karyogram of Ormyrus sp. (Gokhman 2005b). Reproduced by permission of the Japan Mendel Society

Fig. B.227 Karyogram of Megastigmus pictus (Gokhman 2005b). Reproduced by permission of the Japan Mendel Society

Fig. B.228 Karyogram of Megastigmus strobilobius; mitotic chromosomes (Gokhman 2005b). Reproduced by permission of the Japan Mendel Society

Fig. B.229 Karyogram of Megastigmus strobilobius; meiotic chromosomes in diplotene (Gokhman 2005b). Reproduced by permission of the Japan Mendel Society

Fig. B.230 Karyogram of Torymus bedeguaris (Gokhman and Mikhailenko 2007)

Fig. B.231 Karyogram of Torymus chloromerus (Gokhman and Mikhailenko 2007)

158

Appendix B

Fig. B.232 Karyogram of Laelius utilis (modified after Gokhman and Quicke 1995). Reproduced by permission of the International Society of Hymenopterists

Fig. B.233 Karyogram of Hedychridium roseum (Quicke and Gokhman 1996). Reproduced by permission of the International Society of Hymenopterists

Fig. B.234 Karyogram of Anteon ephippiger (Gokhman 2001b)

Fig. B.235 Karyogram of Anteon gaullei; mitotic chromosomes (Gokhman 2001b)

Appendix B

159

Fig. B.236 Karyogram of Anteon gaullei; meiotic chromosomes in diplotene (Gokhman 2001b)

Fig. B.237 Karyogram of Anteon jurinearum (modified after Gokhman and Kolesnichenko 1998b). Reproduced by permission of the International Society of Hymenopterists

Fig. B.238 Karyogram of Anteon pubicorne; mitotic chromosomes (Gokhman 2001b)

Fig. B.239 Karyogram of Anteon pubicorne; meiotic chromosomes in diplotene (Gokhman 2001b)

160

Appendix B

Fig. B.240 Karyogram of Lonchodryinus ruficornis (Gokhman 2001b)

Fig. B.241 Karyogram of Gonatopus clavipes; mitotic chromosomes (Gokhman 2002b)

Fig. B.242 Karyogram (Gokhman 2002b)

of

Gonatopus

clavipes;

meiotic

chromosomes

in

diplotene

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Index

A Aculeata, 3, 4, 12, 19, 26, 52, 66, 67, 77 Andrenidae, 12 Aneuploidy, 15, 16, 41, 47, 64, 79 Anobiidae, 75 Anthophoridae, 12 Aphelinidae, 35, 38, 39, 42–44, 50, 51, 59, 60, 63, 65, 68, 69, 71, 72, 74, 80, 89 Apidae, 7, 8, 12, 16, 18 Apocrita, 12, 52 Apoidea, 3, 12, 58 Argidae, 11 Arrhenotoky, 1, 2, 19, 79

B Banding AgNOR-, 1, 7, 38 C-, 1, 6, 7, 9, 31, 33, 103, 114–117, 155, 156 G-, 1, 7–9, 38, 39 Q-, 8 restriction, 1 Bethylidae, 50, 58, 67, 94 Braconidae, 35, 37, 39, 43, 44, 47, 49–51, 53, 54, 56–58, 63–66, 68, 69, 71, 72, 76, 77, 80, 87 Bruchidae, 75 C Centromere index, 1, 5, 6, 17, 37, 42 Cephidae, 11 Cephoidea, 11, 52 Ceraphronoidea, 49, 53, 59, 63, 89 Chalcididae, 35, 43, 50, 59, 61, 71, 90 Chalcidoidea, 35, 37, 45, 49, 51, 59–61, 63, 66, 68, 69, 80, 89 Chiasma, 10, 11, 17, 27–29, 31, 33, 40, 48, 79 Chromomycin A3 , 8

Chromosomal fission, 14, 24, 27, 47, 62 centric, 14, 17, 28, 29, 41, 43, 47, 48, 64–66, 79 Robertsonian, see centric Chromosomal fusion, 14, 24, 27, 37, 41, 43, 46, 47, 66, 79 centric, 14, 27, 28, 41–44, 60, 79 Robertsonian, see centric tandem, 15, 41, 43, 44, 46, 47, 49, 61–63, 65, 79 Chromosomal polymorphism, 13–17, 27, 38, 41, 42, 46, 67, 71, 79 Chromosome painting, 1, 8, 39 Chromosomes acrocentric, 6, 12–14, 17, 28, 29, 31, 33, 37, 38, 42–44, 47, 48, 49, 51, 55, 56, 60–66, 73, 76, 80 B, 6, 16, 17, 41, 45, 46, 48, 76, 77, 79, 155 biarmed, 12, 27–29, 37, 42, 46–48, 51, 55, 61, 62, 64, 73, 79, 80 metacentric, 6, 13, 14, 28, 29, 31, 33, 43, 44, 48, 60, 61, 63, 70 pseudoacrocentric, 6, 13, 28, 38, 42, 43, 47, 64, 80 sex, 2 submetacentric, 6, 31, 33, 43, 61 subtelocentric, 6, 28, 13, 28, 31, 33, 39, 42, 43, 51, 61–63 “telocentrics”, 28 Chrysididae, 49–51, 58, 66–68, 94 Chrysidoidea, 51, 58, 63, 66, 67, 94 Cimbicidae, 11 CI, see Centromere index CMA3, see Chromomycin A3 Coleoptera, 75 Colletidae, 12 CSD, see Sex determination, complementary Curculionidae, 75

181

182

Index

Cynipidae, 2, 37–39, 43, 47, 50, 59, 65, 69–72, 88 Cynipoidea, 49, 59, 63, 66, 80, 88

L Lepidoptera, 55 Leucospidae, 50, 59, 92

D 4 ,6-diamidino-2-phenylindole, 8, 38 DAPI, see 4 ,6-diamidino-2-phenylindole DA, see Distamycin A Deletion, 9, 13, 41, 42, 47, 79 Diapriidae, 50, 59, 89 Diaprioidea, 49, 59, 63, 89 Diprionidae, 8, 11, 19 Distamycin A, 8 Dryinidae, 37, 40, 48, 49, 50, 58, 63, 66, 67, 70, 71, 80, 94 Duplication, 13, 14, 41, 42, 47, 79

M Megachilidae, 12 Megalodontidae, 11 Megalyroidea, 59, 80 Megaspilidae, 50, 59, 89 Meiosis, 1, 2, 9, 11, 14, 16, 17, 29, 35, 39, 79 Microhymenoptera, 49, 59, 63, 65, 66, 68 Mitosis, 6, 33, 76 Mymaridae, 50, 51, 59–61, 66, 68, 92

E Encyrtidae, 50, 51, 59, 60, 68, 71, 72, 90 Eulophidae, 37–40, 42, 44, 50, 51, 59, 61–63, 65, 68–71, 90 Eumenidae, 12 Eupelmidae, 37, 50, 59, 60, 72, 91 Eurytomidae, 43, 50, 51, 59, 60, 68, 71, 92 Evanioidea, 59, 63, 68, 88 Evaniomorpha, 59 F Figitidae, 39, 50, 59, 71, 89 FISH, see In situ hybridisation, fluorescence Formicidae, 3, 6–8, 12, 14, 15, 19–21, 28, 50 Formicoidea, 3, 12, 58 G Gasteruptiidae, 50, 51, 59, 71, 88 H Halictidae, 12 Haplodiploidy, 1, 2, 48 Heterochromatin, 6, 7, 28, 29, 35, 38, 41, 71, 76, 79 tandem growth, 13, 28, 42, 43, 48, 80 I Ichneumonidae, 37–39, 41–43, 45, 47, 49–51, 53–56, 63, 65, 66, 68–72, 74, 77, 80, 83 Ichneumonoidea, 35, 37, 52, 53, 58, 63, 64, 68, 80, 83 In situ hybridisation, 1, 8, 39, 81 fluorescence, 1, 8, 29, 39 Inversion, 13–16, 22, 28, 41, 42, 46–48, 66, 79 K Karyotypic dissymmetrisation, 48, 49, 65, 80 Karyotypic orthoselection, 26, 27, 64

N Nucleolus organiser, 1, 7, 17, 28, 35, 38, 71, 73, 74 O Ormyridae, 50, 59, 63, 71, 92 P Pamphiliidae, 11, 19 Pamphilioidea, 11, 52 Parasitica, 51, 52 Pergidae, 11 Perilampidae, 35, 50, 59, 92 Phylogenetic presumptions, 23–27, 49, 51, 58, 64 Platygastroidea, 49, 59, 63, 89 Polyploidy, 15, 24, 41, 79 Pompilidae, 12 Pompiloidea, 12, 58 Proctotrupoidea, 66, 80 Pteromalidae, 37–39, 44, 45, 50, 59, 61, 70, 72–76, 80, 92 S Sapygidae, 12 Scelionidae, 50, 59, 89 Scolioidea, 12, 58 Sex determination, 1, 2, 16, 17, 46, 48 complementary, 3 Sibling species, 18, 19, 21, 38, 67, 71–77, 80 Siricidae, 11, 18 Siricoidea, 11, 52 Sphecidae, 8, 12, 16 Sphecoidea, 12, 58 Stephanoidea, 59, 80 Symphyta, 3, 4, 11, 12, 15, 19, 51, 67 T Tenthredinidae, 7, 8, 11, 15, 18, 19, 28 Tenthredinoidea, 3, 11, 51 Thelytoky, 1, 2, 10, 15, 19, 35, 39, 44, 76, 79

Index Torymidae, 37, 44, 50, 59, 61, 63, 65, 68, 71, 73, 74, 93 Translocation, 13–16, 27, 41, 42, 44, 46–48, 62, 79 Trichogrammatidae, 35, 50, 59, 61, 93 Trigonalyoidea, 59, 80

183 V Vespidae, 8, 12, 50 Vespoidea, 12, 58 X Xyelidae, 11 Xyeloidea, 11