Advances in Genetics Volume 13

ADVANCES IN GENETICS VOLUME 13 Contributors to This Volume A. D. Bradshaw K. R. Dronamraju Donald N. Duvick Morris ...

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ADVANCES

IN GENETICS

VOLUME 13

Contributors to This Volume A. D. Bradshaw K. R. Dronamraju Donald N. Duvick Morris Foster Aloha Hannah-Alava C. N. Law Ralph Riley Anna R. Whiting

ADVANCES IN GENETICS VOLUME 13 Edited by

E. W. CASPARI Biological laboratories The University of Rochester Rochester, New York and

J. M. THODAY Department of Genetics university o f Cambridge Cambridge, England

Editorial Board G. W. BEADLE WILLIAM C. BOYD M. DEMEREC

MERLE T. JENKINS JAY L. LUSH ALFRED MlRS KY

TH. DOBZHANSKY L. C. DUNN

M. M. RHOADES CURT STERN

1965 ACADEMIC PRESS

0

NEW YORK AND LONDON

COPYRIGHT @ 1965,

BY

ACADEMICPRESS INC.

All Rights Reserved

N o part of this book may be reproduced i n any form, by photostat, microfilm, or any other means, without written permission from the publishers. ACADEMIC PRESS INC. 111 FIFTHAVENUE NEWYORK,NEWYORK,10003 United Kindom Edition PUBLISHED BY

ACADEMIC PRESS INC. (LONDON) LTD. BERKELEY SQUARE HOUSE,LONDON, W.1

Library of Congress Catalog Card Number: 47-30313 PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME 13 A. D. BRADSHAW, Department of Agricultural Botany, University College of North Wales, Bangor, Caerns., Wales, G.B., and Department of Agronomy, University of California, Davis, California K. R. DRONAMRAJU,* Johns Hopkins University School of Medicine, Baltimore, Maryland DONALD N. DWICK, Department of Plant Breeding, Pioneer Hi-Bred Corn Company, Johnston, Iowa MORRIS FOSTER, Mammalian Genetics Center, Department of Zoology, The University of Michigan, Ann Arbor, Michigan ALOHAHANNAH-ALAVA, Department of Genetics, University of Turku, Turku, Finland C. N. LAW,Plant Breeding Institute, Cambridge, England

RALPHRILEY,Plant Breeding Institute, Cambridge, England ANNAR. WHITING, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee

* Present address: Medical Genetics Unit, School of Medicine, The State University of New York, Buffalo, New York. V

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CONTENTS Contributors to Volume 13

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v

Cytoplasmic Pollen Sterility in Corn

DONALD N . DUVICK

I . Introduction . . . . . . . . . . . . . . . . . . I1. Early Discoveries of Cytoplasmic Pollen Sterility . . . . . . . I11. Comparison of Texas and USDA Cytoplasms . . . . . . . . IV . Classification of Sterility-Inducing Cytoplasms . . . . . . . . V. Genetics of Fertility Restoration . . . . . . . . . . . . VI . Effect of Texas Cytoplasm on Morphological Characters Other Than Pollen Fertility . . . . . . . . . . . . . . . . VII Pleiotropic Effects of Rf, . . . . . . . . . . . . . . VIII . Origin and Expression of Cytoplasmic Male Sterility in Maize . . . I X . Economic Usefulness of Cytoplasmic Male Sterility and Fertility Restoration in Corn . . . . . . . . . . . . . . . . . X . Summary and Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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2 2 4 9 12

20 26 27

47 50 52

Genetic Variation in Chromosome Pairing

RALPHRILEYA N D C . N . LAW

I. I1. I11. IV . V.

Introduction . . . . . . . . Preliminary Consideration of Functions Quantitative Variation in Pairing . . Genetic Control of Pairing Specificity Conclusions . . . . . . . . References . . . . . . . .

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57 59 65 . 81 . 105 . 107

Evolutionary Significance of Phenotypic Plasticity in Plants

A . D . BRADSHAW

I . Introduction . . . . . . I1. Genetic Control of Plasticity . 111. Fitness, Plasticity, and Selection IV . Conditions Favoring Plasticity V. Conditions Disfavoring Plasticity VI . Mechanisms of Plasticity . . VII . Fixed Phenotypic Variation .

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viii

CONTENTS

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VIII Conclusions . I X Summary . References .

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The Premeiotic Stages of Spermatogenesis ALOHAHANNAH-ALAVA

I. Introduction . . . . . . . . . . . . . . . . I1 Maintenance of the Germ Line and Spermatogonial Multiplication I11. Relating the Temporal and Spatial Patterns of Spermatogenesis . IV Radiosensitivity of Spermatogonia . . . . . . . . . References . . . . . . . . . . . . . . . .

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159 196 211 215

The Function of the Y-Chromosome in Man. Animals. and Plants

K . R . DRONAMRAJU I . Introduction . . . . . . . . . . . . . . I1. Man . . . . . . . . . . . . . . . . . I11. Drosophila . . . . . . . . . . . . . . IV . Role of the Y-Chromosome in Sex Determination in Insects V. Fish . . . . . . . . . . . . . . . . . VI Mouse . . . . . . . . . . . . . . . VII . Cat . . . . . . . . . . . . . . . . VIII . Plants . . . . . . . . . . . . . . . . . I X . General Discussion . . . . . . . . . . . . References . . . . . . . . . . . . . .

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Mammalian Pigment Genetics

MORRISFOSTER

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311 312

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314 319 332 334 335

I Introduction . . . . . . . . . . . . . . . . . . I1. Genes and the Cytological Basis of Melanin Pigmentation . . . . I11 Genes and the Ultrastructural Basis of Melanin Granule (Melanosome) Formation . . . . . . . . . . . . . . . . . . IV. Genes and the Biochemistry of Melanin Formation . . . . . . . V Color Genes and Evolutionary Biology . . . . . . . . . . VI . Concluding Remarks . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . The Complex Locus R in Mormoniella vifripennis (Walker)

ANNA R . WHITINQ

I . Introduction . 11. The R Locus .

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I11. Discussion References Author Index Subject Index

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CYTOPLASMIC POLLEN STERILITY IN CORN

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Donald N Duvick Department of Plant Breeding. Pioneer Hi-Bred Corn Company. Johnston. Iowa

Page I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 I1. Early Discoveries of Cytoplasmic Pollen Sterility . . . . . . . . . . . 2 A . Peruvian Source . . . . . . . . . . . . . . . . . . . . . . . . 2 B . Argentinian Source . . . . . . . . . . . . . . . . . . . . . . . 3 C. Inbred 33-16 . . . . . . . . . . . . . . . . . . . . . . . . . . 3 D . USDASource . . . . . . . . . . . . . . . . . . . . . . . . . 3 E . TexasSource . . . . . . . . . . . . . . . . . . . . . . . . . 3 F. Summary of Early Discoveries . . . . . . . . . . . . . . . . . . 4 I11. Comparison of Texas and USDA Cytoplasms . . . . . . . . . . . . 4 A. Genes for Restoration of Pollen Fertility . . . . . . . . . . . . . 4 B . Fertility of Heterozygous “Restorcd” Plants . . . . . . . . . . . . 5 C. Phenotypic Appearance of Anthers . . . . . . . . . . . . . . . . 6 IV . Classification of Sterility-Inducing Cytoplasms . . . . . . . . . . . . 9 A . Number of Discoveries and Their Origins . . . . . . . . . . . . . 9 B . Classification by Fertility Restoration Requirements . . . . . . . . 10 V. Genetics of Fertility Restoration . . . . . . . . . . . . . . . . . . 12 A. I n USDA Cytoplasm . . . . . . . . . . . . . . . . . . . . . . 12 B . I n Texas Cytoplasm . . . . . . . . . . . . . . . . . . . . . . 12 VI . Effect of Texas Cytoplasm on Morphological Characters Other Than Pollen Fertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 A . Plant Height . . . . . . . . . . . . . . . . . . . . . . . . . 20 B. NumberofLeaves . . . . . . . . . . . . . . . . . . . . . . . 22 C. Grain Yield . . . . . . . . . . . . . . . . . . . . . . . . . . 23 D . Resistance to Helminthosporium maydis (Southern Leaf Blight) . . . 26 E . Other Morphological and Physiological Characteristics . . . . . . . 26 VII . Pleiotropic Effects of Rf,. . . . . . . . . . . . . . . . . . . . . . 26 VIII . Origin and Expression of Cytoplasmic Male Sterility in Maize . . . . . 27 A . Cytological Studies . . . . . . . . . . . . . . . . . . . . . . . 27 B. Chemical Studies . . . . . . . . . . . . . . . . . . . . . . . . 29 C. Stability of Cytoplasmic Male Sterility . . . . . . . . . . . . . . 30 D . Creation of Sterility-Inducing Cytoplasms . . . . . . . . . . . . . 36 E . Evolutionary Usefulness of Cytoplasmic Male Sterility . . . . . . . 43 F. Other Typcs of Cytoplasmic Inheritance in Corn . . . . . . . . . . 45 G . Summary of Possible Modes of Action and Origins of Cytoplasmic Male-Sterile Corn . . . . . . . . . . . . . . . . . . . . . . . 46 I X . Economic Usefulness of Cytoplasmic Malc Sterility and Fertility Restoration in Corn . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 A . Methods of Producing Hybrid Corn without Detasseling . . . . . . 47 1

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DONALD N. DUVICK

B. Breeding Procedures Necessary in Order to Use Cytoplasmic Male Sterility. . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Patent of the Process of Producing Hybrids by Means of Cytoplasmic Male Sterility Plus Fertility Restoration . . . . . . . . . . . . . X. Summary and Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

48 49 50 52

I. Introduction

Since about 1950 cytoplasmically inherited pollen sterility in corn (Zea mays L.) has been used extensively as an aid in the conimercjal pro-

duction of hybrid seed corn. I n the course of this practical application a considerable body of genetic information, experimental and empirical, has been accumulated. Some of it is published; some is common knowledge in the seed production field, but has never been summarized in print. This paper will review that part of the information, both published and unpublished, which is pertinent to the general field of cytoplasmic inheritance of pollen sterility. The definition given by Caspari (1948) for cytoplasmic inheritance will be used throughout this review: “Only when a character proves to be more or less constant in a series of backcrosses to the paternal strain, or when genic transmission is excluded by substitution of all chromosomes in a species, and dauermodifications are unlikely since no decrease in the action of the cytoplasm appears in successive gcnerations” will cytoplasmic inheritance be assumed, “unless another interpretation of the facts can be found.” II. Early Discoveries of Cytoplasmic Pollen Sterility

A. PERWIAN SOURCE “In a planting of seeds from an open pollinated ear of maize collected by R. A. Emerson and F. D. Richey a t Arequipa, Peru, a male-sterile plant was noticed by Professor Emerson. It was crossed to a n unrelated individual grown from seed obtained a t Cura-cautin, Chile. The resulting F1generation of 45 individuals was completely male-sterile, but all of the ears were well filled and showed no signs of female-sterility.” I n these words, Rhoades (1933) introduced his thorough analysis of the first recognized discovery of cytoplasmic pollen sterility in corn. Rhoades concluded that the male sterility was due entirely to factors (presumably cytoplasmic) contributed by the female parent and that the chromosomal genes had no effect on the expression of pollen sterility. Later reviews of his published data indicate, however, that there was a small but definite effect of the genetic constitution on the expression of the cytoplasmically induced sterility (Edwardson, 1956; Duvick, 195913). No further genetic

CYTOPLASMIC P O L L E N STERILITY IN CORN

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studies were made of this Peruvian source of cytoplasmic sterility and it has been lost.

B. ARGENTINIANSOURCE Gini, in Argentina in 1939, reported a second case of cytoplasmic male sterility in a variety of maize known as amargo blanco (see Edwardson, 1956). Meiosis was normal in all individuals. An interaction of nuclear genes with a specific cytoplasm appeared to be necessary to produce the male sterility effect, for Gini found that depending on the male parent, progenies either segregated for pollen sterility or were completely male sterile. The interaction of genotype and cytoplasm was apparently more clear-cut here than in the Peruvian cytoplasmic sterile. This source of male sterility also is apparently not in existence today. 33-16 C. INBRED

Josephson and Jenkins (1948) found that the cytoplasm of the U.S. white corn inbred line 33-16 caused pollen sterility to occur when certain genotypes were placed in it. They were led to this conclusion through their efforts to discover why certain white hybrids had had poor seed set, in the form of kernels scattered over the ear, in the season of 1945. Examination of pedigrees of the hybrids, followed by a series of special crosses, clearly showed that the inbred line 33-16 carried a cytoplasm which caused pollen sterility to be exhibited, providing its own germ plasm was replaced (Josephson, 1955) or combined with certain other genotypes. A corollary interpretation to these conclusions was that the 33-16 genotype caused pollen fertility to be exhibited, when in its own cytoplasm.

D. USDA SOURCE D. F. Jones, starting in 1944, established a cytoplasmic basis for the pollen sterility of two plants in a progeny grown by M. T. Jenkins, then at the United States Department of Agriculture (Jones et al., 1957a). This progeny traced back to a genetic tester stock (iojap X teopod) from E. W. Lindstrom, a t Iowa State College. Jones and co-workers found that this source of cytoplasmic pollen sterility, like those described above, was affected in its expression by the genotype: some genotypes were sterile in this cytoplasm; some were only partly sterile; and some were of apparently normal fertility. E. TEXASSOURCE I n the course of developing inbred lines from the variety Golden June, J. S. Rogers, then a t Texas A. and M. College (in 1944), noticed a male-sterile plant in one of the partially inbred lines. He outcrossed this

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DUVICK

plant, and found it to be another source of cytoplasm which induces pollen sterility (Rogers and Edwardson, 1952). He found that it, too, was affected in expression by the genotype.

F. SUMMARY OF EARLY DISCOVERIES All five of these early discoveries of cytoplasmic male sterility were established as such by developing completely male-sterile progenies through backcrossing to specific pollen-fertile male genotypes. I n all five discoveries the expression of the cytoplasmically induced sterility was influenced by nuclear genes; i.e., there was an interaction between cytoplasm and nuclear genes. 111. Comparison of Texas and USDA Cytoplasms

A. GENES FOR RESTORATION OF POLLEN FERTILITY Jones (1954)) Josephson (1955), and others soon found that the USDA and Texas cytoplasms, although superficially similar, were different in several ways. The most easily recognized difference was that a given genotype did not necessarily have the same degree of pollen sterility in Texas cytoplasm that it had in USDA cytoplasm. If, through repeated backcrossing as male, the genotypes of several specifically selected inbred lines are placed in each of the two sterility-inducing cytoplasms the pattern of differences in pollen fertility shown in the tabulation will be exhibited: Inbred line Cytoplasm

A158

Ky21

K55

CEl

Sterile Sterile

Fertile Fertile

Sterile Fertile

Fertile Sterile

-

USDA Texas

The two differentiating inbred testers are K55 and CE1. It is apparent that K55 must lack a gene or genes for restoration of pollen fertility in USDA cytoplasm which both Ky21 and C E I possess; i.e., it has “plasmon-sensitive’) genes (see Caspari, 1948) which cause pollen sterility when in USDA cytoplasm. On the other hand, K55 must have a gene or genes for rest’orationof pollen fertility in Texas cytoplasm which is lacking in CE1. Based on these data alone, one might postulate that (1) Ky21 could (at the least) have a single gene capable of restoring pollen fertility to either Texas or USDA cytoplasm; ( 2 ) K55 has an allele of this gene capable of restoring only Texas cytoplasm; (3) CE1 has a third

CYTOPLASMIC POLLEN STERILITY I N CORN

5

allele capable of restoring only USDA cytoplasm; and (4) that A158 has a fourth allele which cannot restore fertility in either Texas or USDA cytoplasm; i.e., it allows pollen sterility to be expressed. However, Jones and co-workers (1957a) and Duvick (1954) have found that from hybrids of Ky21 with nonrestorer genotypes like A158 one can extract four kinds of genotypes: like K55, like CE1, like Ky21, or like A158. Therefore, there must be a t least two different loci in Ky21 which are concerned with restoration: (1) a t least one locus that governs restoration in Texas cytoplasm, and (2) another that governs restoration in USDA cytoplasm. Investigation of the genetics of fertility restoration of both the USDA and the Texas types of cytoplasmic sterility has shown that in either case a single dominant gene is usually sufficient to restore pollen fertility to most cytoplasmic male-sterile inbreds (Jones, 1951; Thomas and Johnson, 1956; Duvick, 1956; Buchert, 1961) although there are complications with certain genetic backgrounds and in certain environments. These will be discussed in more detail in a later section of this paper. The major gene for restoration in Texas cytoplasm has been termed Rfl. The major gene for restoration in USDA cytoplasm has been called Rfz by Buchert (1961). However, the superscript position is conventionally used for designating members of an allelic series and a s was shown above the gene which Buchert called Rf2 is not a t the same locus a s Rfl. Therefore it will be called Rf3in this review. A second locus for restoration in Texas cytoplasm, found in nearly all types of corn, has previously been termed Rf2.

B. F E R T I L I T Y O F HETEROZYGOUS “RESTORED” PLANTS A comparison of plants of the F, of A158 in USDA cytoplasm X Ky21 (Al58-S X Ky21) with plants of A158 in Texas cytoplasm X Ky21 (A158-T x Ky21) reveals th at plants of both types of cross shed fertile pollen in copious amounts, but that only about 50% of the pollen grains shed by A158-S x Icy21 plants are fertile; the remainder are shriveled and more or less devoid of starch and protein granules. I n contrast, about 95% of the pollen grains shed by A158-T X Ky21 plants are fertile, which is about the level of fertility of pollen from plants with normal cytoplasm. Further, if plants of each of these F1 crosses are backcrossed as male to A158-S and to A158-T, it will be found that nearly all the plants of the backcross A158-S x (A158-S X Ky21) are pollen fertile, to the extent described above for the F1;whereas the similar backcross A158-T X (A158-T X Ky21) will segregate in a 1:l ratio of fertile to sterile plants. Similarly, in Fz, few or no sterile plants result when A158-S X Ky21 is selfed, whereas a 3 : l ratio of fertile to sterile plants is obtained when A158-T x Ky21 is selfed.

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DONALD N. DUVICK

Investigation of this type of behavior led Buchert (1961) to discover a fundamental difference between the USDA and the Texas cytoplasms. Buchert’s conclusion was that in USDA cytoplasm only those microspores which contain the dominant gene for fertility restoration in USDA Cytoplasm (Rf 3 ) will develop into fertile pollen grains; the microspores containing the recessive allele (rf3) abort. I n Texas cytoplasm, in contrast, all (95-1000/,) pollen grains in a plant are fertile if the plant is a t least heterozygous for the dominant gene(s) necessary for fertility restoration in Texas cytoplasm. Buchert found that essentially all of the pollen grains are fertile in plants in USDA cytoplasm if the restorer gene is homozygous (Bf3Rf3).The net result of this phenomenon is to upset segregation ratios when the source of segregating gametes is pollen from plants in USDA cytoplasm which are heterozygous for the major restorer gene. Only gametes with the dominant allele will be obtained. However, Buchert showed that segregation was normal if such plants were used as female, and it also was normal if Rf3rf3 plants in normal cytoplasm were used as eithcr male or female. Buchert’s basic conclusions have been confirmed by studies we have made with different materials, but as is usually the case, there appear to be some exceptions to the general rule. I n some genotypes a small percentage of fertile pollen grains of rf3 genotype are produced by Rf3rf3 plants in USDA cytoplasm, in addition to the fertile R f 3pollen grains. It is not known whether specific modifying genes, or environmental conditions, or a combination of these factors govern this departure from the general rule.

C. PHENOTYPIC APPEARANCE OF ANTHERS 1. Sterile Plants

Completely sterile plants in Tcxas cytoplasm characteristically do not exsert their anthers. In contrast to this inany genotypes which are conipletely sterile in USDA cytoplasm (using as a definition of sterility th a t no fertile pollen grains are produced or shed) will exsert some or most of the anthers in the tassel. However, these anthers characteristically have a slender, stiff , needlelike appearance and have in them only aborted pollen grains (Fig. 1). 2. Partially Fertile Plants

In plants with Texas cytoplasm, anthers which have from about 10 to 95% aborted pollen grains will usually exsert, but will have a characteristic twisted, misshapen appearance, owing to the presence of fertile pollen grains in some regions of the anther and of sterile grains in other

CYTOPLASMIC P O L L E N STERILITY IN CORN

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FIG.1. Sterile anthers, of nonrcstorer genotype in USDA cytoplasm (from Duvick, 1959131.

FIG.2. Partially fertile anthers, of partial restorer genotype in Texas cytoplasm (from Duvick, 195913).

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DONALD N. D W I C K

regions of the anther (Fig. 2). The anther is plump in the regions which contain fertile grains and is shriveled in the regions with sterile grains. I n plants with USDA cytoplasm and which have from 50 to 95% aborted pollen grains, the anthers are usually exserted also, but they tend not t o be twisted and misshapen; rather they are of uniform dimension throughout their length. The anthers, if arranged in order of increasing percentages of aborted pollen grains, are progressively shorter and

FIG.3. Fertile anthers, of corn in normal cytoplasm (from Duvick, 195913)

slimmer, finally reaching the needlelike appearance described above for anthers of completely sterile plants. 3. Heteroxygous Restored Plants

Plants of Rf 1rf constitution in Texas cytoplasm, with proper modifiers and environmental conditions, are fully fertile. They exsert all their anthers and the anthers have the same appearance as those of fully fertile plants in normal cytoplasm (Fig. 3). Plants of Rfarfs constitution in USDA cytoplasm have 50% of the pollen grains aborted (usually) as noted above. All of the anthers are exserted, however. They superficially look the same a s those of the same genotypes in normal cytoplasm, but careful comparison reveals that they usually are slightly smaller, and they characteristically have a pebbly

CYTOPLASMIC P O L L E N S T E R I L I T Y I N CORN

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appearance over their entire surface. This is due to the random distribution of shriveled, aborted pollen grains and plump fertile pollen grains within the anther. The anther wall has been stretched by the expanding fertile pollen grains. The pebbly appearance is nearly identical to that of anthers of plants which are heteroxygous for a reciprocal chromosome translocation and are therefore also 50% sterile (“semisterile”). One can classify Rfsrfs vs. Rf3Rf3 plants, and also normal vs. semisterile plants with fair accuracy simply b y looking for presence or absence of the pebbly surface of the anther. IV. Classification of Sterility-Inducing Cytoplasms

A. NUMBEROF DISCOVERIES AND THEIRORIGINS

I n addition to the five discoveries of sterility-inducing cytoplasm described above, there are in the literature descriptions of ten other discoveries of cytoplasmic sterility (Rhoades, 1950; Jones et al., 1957a; Briggle, 1956, 1957; Alirnova, 1962). Shaver (1956) has mentioned a collection of thirty additional discoveries, and we have a collection of thirty-nine additional different discoveries of cytoplasmic male sterility. This makes a reported total of eighty-four separate discoveries of cytoplasmic male sterility in corn. [Another type of pollen stcrility found in a cross of a translocation stock by the inbred Kys was erroneously stated to be cytoplasmic (Schwartz, 1950), but further testing showed that the pollen sterility was due only to nuclear genes (Leng and Bauman, 1955), with no cytoplasniic effect whatsoever.] The fifteen described sources and the thirty-nine in our collection can be sorted into the following general groups with regard to type of corn in which they were found:

Type of corn

U.S. Corn Belt open-pollinated varieties 1J.S. Corn Belt hybrids U.S. Corn Belt inbreds U.S. sweet corn U.S. Indian flint European flints Turkish flints Latin American open-pollinated varieties Genetic tester stocks

Number of sources 8

8 2

3 1

1 1 26 4

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This compilation is by no means the result of a n effort t o sample systematically the various races of corn throughout the world for presence of sterility-inducing cytoplasms, but it does give convincing evidence of the widespread existence of such cytoplasms in virtually all types of corn. One significant fact about these discoveries should be mentioned here, although it will be discussed at more length in a later section of this paper (Section VIII). All sources were identified initially by making sibor out-pollinations on:, pollen-sterile plants observed in segregating populations, with the exception of the two sources found in inbred lines. I n these two cases, pollen sterility differences between reciprocal crosses led t o the discovery of the sterility-inducing cytoplasms. No sources were manufactured deliberately by putting corn germ plasm into the cytoplasm of a different species, as has been done several times with wheat, for example (Fukasawa, 1959). No sources arose as ‘Lmutations’’ in pure lines known t o have “normal” cytoplasm.

B. CLASSIFICATION BY FERTILITY RESTORATION REQUIREMENTS It was mentioned in Section 111 that specific tester genotypes enable one t o differentiate USDA from Texas cytoplasms, because some genotypes are sterile in one cytoplasm but not the other, some are sterile in both, and some are pollen fertile in both. This type of test can be applied to all discoveries of cytoplasmic male sterility to see if they resemble either the USDA or the Texas source, or if they have a different spectrum of interactions with genotype. Conceivably, for example, one might find a cytoplasm which sterilizes all tester genotypes; or one which sterilizes some but not all of the genotypes which are fertile in both the USDA and the Texas cytoplasms. Thirty-two of the thirty-nine cytoplasmic sterile sources in my collection have been tested in this way. All sources were converted to the genotype of the inbred WF9 by backcrossing for a t least six generations. WF9 is essentially sterile in both USDA and Texas cytoplasms, although it occasionally is very slightly fertile in USDA cytoplasm. WF9 was sterile in all thirty-two of the new cytoplasms. The thirty-two new cytoplasmic sterile forms of WF9, plus WF9-S (USDA) and WF9-T (Texas) were crossed as female to three different inbred testers: CE1, which restores pollen fertility to USDA, but not to Texas cytoplasmic steriles; F5DD1, which restores fertility to Texas but not to USDA steriles; and BH2, which, like Ky21, has genes for restoration in both cytoplasms. Observation of the resulting test crosses showed that with two exceptions each of the new cytoplasmic sources could be classified as identical in reaction t o the USDA or to the Texas cytoplasm. The data, summarized in Table 1, show that all cytoplasms had more or less fertile tassels when

11

CYTOPLASMIC P O L L E N S T E R I L I T Y I N CORN

TABLE 1 Per Cent Fertile Pollen per Plant in Crosses of Several Sources of Cytoplasmic Pollen Sterility (WF9 Genotype) to Three Tester Inbreds* Type of cytoplasm

USDA source 25 other sources Texas source 5 other sources 1 other source 1 other source

XBH2

XCEl

XF5DDl

50 50 95 95 50 50

50 50 0 0 95 50

0 0 95 95 0 95

* Data from Duvick (1962). crossed t o BH2, but that some of the sources when crossed to BH2 had pollen which was about 95% fertile, like Texas; while the others had pollen which was about 50% fertile, like USDA. The five sources whose cross t o BH2 had 95% fertile pollen were also restored by F5DD1 (and again had pollen th at was about 95% fertile), and were not restored to fertility by CE1. Twenty-five of the sources whose BH2 crosses had 50% fertile pollen were restored by CE1 (and again had 50% fertile pollen) and were not restored by F5DDl. There were two apparently exceptional cytoplasms, however. One source of sterility-inducing cytoplasm had pollen which was about 50% fertile in the cross to BH2, was not restored by F5DD1, and was restored by CE1 (like USDA), but the pollen of the cross t o CEl was much closer to 95% than t o 50% fertile (unlike either USDA or Texas). Also, the WF9 genotype in this source unexpectedly had pollen which was about 50% fertile, in the year the test crosses were grown. The second exception, out of the thirty-two tested sources, was restored by BH2 and by CE1, and had 50% fertile pollen in both crosses (like USDA), but it also was restored by F5DD1, and had 95% fertile pollen in t ha t cross (like Texas). Both exceptions are being examined further at the present time, to see whether the exceptional results are due to genuinely different cytoplasms, to gene mutation, or whether, as is entirely possible, there may have been errors in pollination or seed labeling. I n summary, thirty out of thirty-two separate sources of cytoplasmic sterility have been shown to be identical in reaction (insofar as tested) to USDA or t o Texas cytoplasm. Five sources, four from U.S. dent material and one from a Mexican open pollinate, are of Texas type; the remainder are of USDA type. Two sources may be different from either USDA or Texas cytoplasm but in neither case has this been proven conclusively. It should be emphasized that the method of testing used here can never

12

DONALD N . DUVICK

show positively that two sources of sterility-inducing cytoplasm are identical, for one can always suppose that the genotype tried next might be the one that can differentiate the two cytoplasms. V. Genetics of Fertility Restoration

A. IN USDA CYTOPLASM Except for the data given by Buchert (1961) there is little published information about the genetic control of the expression of cytoplasmic sterility in the USDA cytoplasm. As discussed in Section III,B, Buchert has shown that the presence of a single dominant gene (Rf3) in heterozygous condition will cause plants with USDA cytoplasm to be 50% fertile; that is all anthers will shed pollen, and about 50% of the pollen shed will be fertile. When the dominant gene is hornozygous, essentially 100% of the pollen is fertile. Experience has shown that there are many grades of fertility between complete sterility and complete fertility, but whether these intergrades are due to modifiers which affect the Rf3gene, or whether there are more than two alleles a t the Rfz locus, is not known. The chromosonial location of Rf 3 is also unknown. Genotypes which tend to be incompletely sterile in USDA cytoplasm will be made either more or less sterile by the environment, especially by the presence or absence of hot dry weather just prior to and during the normal time of anthesis. Hot dry weather promotes increased sterility; cool humid weather promotes increased fertility. B. IN TEXASCYTOPLASM 1. Classification of Genotypes by Fertility Grade and Ennironmental Stability

Before discussing in detail the genetics of fertility restoration in Texas cytoplasm it would be well to define the terms which will be used to describe various genotypes with regard to their pollen fertility (1) in Texas and in “normal” cytoplasm, and (2) under various environmental conditions. (“Normal” cytoplasm as used herein will mean merely th a t the cytoplasm is one which does not induce pollen sterility, a t least with genotypes available.) The terms are as follows: Nonrestorer genotypes. No pollen is shed when these genotypes are in Texas cytoplasm; all pollen grains abort; and no anthers are exserted under any environmental condition. Restorer genotypes. Restorer genotypes will shed as much fertile pollen in Texas as in normal cytoplasm, in any environment. This usually means that in nearly all environnients in which corn is normally grown, about

CYTOPLASMIC P O L L E N STERILITY I N C O R N

13

95% of the pollen in each plant will be fertile, i.e., plump, filled with starch and protein granules, and viable when placed on receptive silks. However, some genotypes typically are only partially fertile in normal cytoplasm, especially in some environments; these, if they are of restorer genotype, will also be only partially fertile when in Texas cytoplasm. Therefore a restorer genotype, by this definition, need not always have all fertile pollen when in Texas cytoplasm. Partial restorer genotypes. Under at least some environmental conditions partial restorer genotypes will shed less fertile pollen in Texas than in normal cytoplasm. Typical characteristics are: (1) a delay of 1-10 days in anther exsertion; (2) scattered exsertion of anthers; (3) some or all of the exserted anthers deformed because only the portions of the anther containing fertile pollen expand to normal dimensions. Members of this class grade into the nonrestorer class on the one extreme and into the restorer class on the other extreme, s o that one must arbitrarily decide whether or not to classify some genotypes as partial restorers. The author does not wish to imply that all partial restorer genotypes must have identical genes with regard to restoration, nor do we imply this for the steriles or the full restorers. Rather, we merely intend to indicate that one can put genotypes into three classes according to their degree of pollen fertility when in Texas cytoplasm, as compared to when in normal cytoplasm. 2. Efects of Environment o n Cytoplasmic Pollen Sterility

Little controlled experimentation has been used to determine with precision what environmental components cause variations in fertility of Texas cytoplasmic steriles, and within what limits these components operate. The general environmental effects are well known, however. By definition, only partial restorer genotypes (in Texas cytoplasm) are affected by environment. Because it was noted that partial restorers often were more fertile in southern Florida during the winter than in the central and eastern United States during the summer, it was suggested that daylength might be a controlling factor (Blickenstaff et al., 1958). Long-time experience with well-defined genotypes has shown clearly, however, that the important contribution of the Florida “winter” environment is the cool humid weather that usually prevails during the period just prior to and during flowering (Duvick, 1959a). Partial restorers in Jamaica in the “winter” are usually quite sterile, in the relatively hot dry environment that prevails at that time. In general, hot dry conditions at about the time of flowering cause partial restorers to be more sterile, and cool humid conditions (for corn) at this time cause partial restorers to have a higher percentage of fertile pollen. A limited amount of testing has indicated

14

DONALD N . DUVICK

that heavy nitrogen fertilization tends to reduce the pollen fertility of partial restorers (Duvick, 1959a). Some partial restorer genotypes are a t such a low level of restoration that they will be completely sterile under all but the most propitious (cool, humid) environments. Other partial restorers are a t such a high level of restoration that they are fully as fertile as their genetic equivalents in normal cytoplasm in all but the most severe (hot, dry) environments. Therefore, without a clear idea of the environmental range in which a given genotype in Texas cytoplasm has been tested, one cannot properly say whether it should be classified as a restorer, nonrestorer (sterile), or partial restorer.

3. Genetics of Fertility Restoration

a. Complete Restoration. As was mentioned earlier, two dominant genes, Rfl and Rfz, must both be present in a t least the heterozygous form, for complete pollen fertility in Texas cytoplasm. Additionally, one or more modifying genes, probably dominants, also must be present in all but the most favorable environments (Duvick, 1956, 1959a; Jones et al., 1957a). Rfl has been located on chromosome 3, within a few crossover units of the centromere, probably on the long arm of the chromosome (Blickenstaff et al., 1958; Duvick et al., 1961). A limited amount of data indicates that Rfz is on the short arm of chromosome 9, and confirmatory tests of this provisional placement are under way at the present time (Duvick, 1964). Very few naturally occurring rf Zrfi genotypes have been identified. The number and location of the modifiers of Rfl-Rfz-are not known. b. Modifier Genes and Partial Restoration. By backcrossing proccdures genotype can be transformed (Eckhardt, 1954), inbred lines of rflrflRfzRfz into Rf,Rf 1RfzRjz genotype. These converted lines will differ from the original by only the Rf, locus and close linkages with it. Owing to the mechanics of the backcrossing process the converted lines usually are in Texas cytoplasm, as well. An example of one such converted line (in Texas cytoplasm) would be the inbred Al2-TR which before conversion was Minn. A12. A12-TR, when crossed as female to nonrestorer genotypes, or when crossed as male to nonrestorer genotypes in Texas cytoplasm, gives hybrids which are fully restored (of normal fertility in all environments). However, as a homozygous line, Al2-TR is a typical partial restorer. It is rarely fully fertile even in the most favorable environments, and in some seasons it is almost entirely sterile. Many Corn Belt inbred lines resemble A12, in that when converted to dominant Rfl genotype they are partial restorers (to Texas cyto-

CYTOPLASMIC POLLEN STERILITY IN CORN

15

plasm) as homozygous lines but when crossed to unrelated nonrestorers (rflrflRf2Rf2)the resulting hybrids are full restorers. It is presumed that such inbred lines lack a full complement of modifiers for the Rf, and/or Rfz genes. Some inbred lines, such as SK2, apparently carry the modifiers in full complement, for SK2-TR (converted to RflRflRfpRf2 from rflrflRf2Rf2)is fully restored as a homozygous line as well as giving full restorer crosses. To some extent, it may seem that hybrid vigor, per se (whatever that is), causes the fertility of many restorer hybrids to exceed that of their component restorer inbreds. But since many inbred lines, like SK2, are as fertile as inbred lines as they are in crosses (when of Rflrfl or RflRfl genotype in Texas cytoplasm), it is obvious that hybrid vigor, or maximum heterosis, is not the major controlling element here. Likewise, overdominance, the superiority of the heterozygote over either homozygote, cannot be called on to explain the increased fertility of some hybrids, since in the case of inbreds like SK2, the homozygote RflRfl is fully as fertile (in Texas cytoplasm) as the heterozygote Rflrfl. c. Alleles of Rfl and Partial Restoration. I n contrast to the A12-TR type of partial restorer, which is a partial restorer a s a line but gives full restorer crosses, is a second type of partial restorer typified by the inbred M14. When M14 is backcrossed, with no genetic change, into Texas cytoplasm, the resulting line, termed M14-T, is a typical partial restorer in its reactions, i.e., its fertility is rarely normal and varies with the environment. However, M14-T differs from A12-TR in that crosses of M14-T t o nonrestorer lines are always partial restorer in reaction, rather than restorer. The M14 genotype, then, is partial restorer as a line and also gives partial restorers when crossed to nonrestorers. When the Fl of M14-T X SK2 is backcrossed as female to SK2 (rflrfl genotype) a 1:1 segregation of sterile to partially fertile plants is obtained, under appropriate environmental conditions. When the Fl of M14-T X Ky2l (Ky21 is RflRfl) is crossed as female t o SK2, a 1:l segregation of fertile t o partially fertile plants is obtained, again if under appropriate environmental conditions. M14 can be outcrossed and then backcrossed to convert it t o full restoration (as was A12, see Section V,B,3,b), using the procedure described by Eckhardt (1954) which involves crossing to a source of Rfl, then backcrossing several times to M14, always growing the backcross population in Texas cytoplasm and selecting the fully fertile (Rfl-) plants. At the end of the backcrossing procedure the backcross plants are selfed and homozygous progenies, fully fertile and RflRfl in genotype, are selected. The converted M14, termed M14-TR, will now give fully restored hybrids when crossed with nonrestorer genotypes. When these hybrids are crossed to nonrestorer genotypes they give a 1:1 segregation (in Texas cytoplasm) of fully fertile to sterile

16

DONALD N. D W I C K

plants; in other words, the gene for partial restoration is no longer present in M14-TR. The necessary conclusions from these results are that M14 contains a single gene which causes partial restoration; that this gene is at the rfl locus; and that it is dominant to rfl but recessive to Rfl. This gene provisionally may be termed Rfr. We have tested six other inbred lines which like M14 are partial restorers both as lines and in crosses, and have found that all of them owe their partial restoration to a single gene. Four of the inbred lines (including M14) have been tested with each other and with Rfl for allelism, and in all four the gene is at the Rfl locus (see Table 2, for example) or extremely close to it. There are indications that the four partial TABLE 2 Tests for Allelism of Partial Restoring Gene of MI4 and of C2513 with RfI and with Each Other* Per cent of plants in class Pedigree

Sterile

Partially fertile

Fertile

Number

SK2-T t SK2-T X C2513 SK2-T X MI4 SK2-T X BH2t SK2-T (MI4 X C2513) SK2-T (MI4 X BH2) SK2-T (C2513 X BH2)

I00 3 0 0 0 0

0 97 25 0 86 48 46

0 0 75 100 14 52 53

30 20 20

1

202

78 29 2 80

* Data from Duvick ( 1 9 6 3 ~ ) .

t SK2 is rflrfl; BH2 is RjIRf1.

$ Grown in different season but similar environment.

restoring alleles are not of equal restoring strength and tests are under way t o determine whether or not they do indeed differ from each other. Each allele is being backcrossed into the SK2 genotype so that all alleles can be compared in a uniform genetic background. There seems to be no formal reason why one cannot expect to find a multiple allelic series at the rfl locus just as has been found at various loci in several organisms, including corn (Anderson, 1924). These conclusions regarding the genetically rather simple nature of partial restoration of the M14 type are a t variance with other published reports. Blickenstaff et al. (1958) state that several genes appear t o be responsible for the action of partial restorer inbreds. Stead (1960) presents extensive data which he interprets as evidence that two complementary

17

CYTOPLASMIC P O L L E N STERILITY I N CORN

genes (both of them different from Rfl) are responsible for the partial restoring action of several inbred lines, including M14. The reasons for the differing conclusions are easy to understand, after one has seen samples of a given segregating population grown in several environments. Owing to the high interaction of partial restorer genotypes with the environment, a sharp threshold exists which, if passed, means the partial restorer plant will be more or less pollen fertile; if the threshold is not passed the plant will be sterile. Especially in segregating populations, in which background modifiers are segregating, only a portion of the plants of Rf,"rf genotype may exhibit their partial restorer genotype, a t least in soiiie environments. The remainder will be sterile and will have the same phenotype as a n rflrfl genotype. Thus one often cannot be sure what is the true genotype of a phenotypically sterile plant. There are several ways to overcome this difficulty. The simplest is to grow the plants in a n environment which allows virtually all the partial restorer genotypes to pass the threshold and shed pollen. Florida in the winter usually has such an environment. Table 3 illustrates how a testcross TABLE 3 SK2-T X (BH2 X B25D1): a Testcross for Allelism of Rf,(in BH2) and the Partial Restoring Gene of B25D1* Per cent in class Location Sterile Iowa Florida

14

1

Nearly sterile

Nearly fertile

Fertile

Number

17 8

10 26

58 65

86 106

* Grown in Iowa (warm, dry environment) and in Florida (cool, humid environment). Data from Duvick (1963~). for allelism of a weak partial restoring gene and Rfl showed several steriles in Iowa but virtually none in Florida. The entire population was shifted toward higher fertility in Florida. A second technique that increases the precision of analysis is t o use a nonrestorer tester which has modifiers that will enhance the effect of the partial restoring gene. The modifiers will reinforce the environment in pushing the partial restorer genotypes over the threshold into partial fertility. Table 4 illustrates the differing effects of 2 nonrestorer (rflrfl) testers on a n Fz population involving the M14 gene for partial restoration. When A-T was used as tester, the pollen shedding plants, in Florida, were usually quite fertile although not completely normal. When WF9-T was used, most of the shedding plants were only weakly fertile. Many of the

18

DONALD N. D W I C K

TABLE 4 Per Cent Sterile Plants in Crosses to Two Testers of 27 Fz Plants from a Cross of the Partial Restorer Inbred M14 with a Nonrestoring Inbred*

F2Plant

Tester female Presumed genotype of F2plant A-T WF9-T ~

1441 1443 144-11 145-9 145-10 146-11 145-6 144-10

R.f,"R.f;

Average of 8 progenies 149-9 146-16 145-2 146-4 145-17 144-4 145-25 146-10 1445 1447 144-8 145-16 145-18 146-20

Rf

w,

Average of 14 progenies ~~

146-13 146-5 144-13 145-5 146-3

Average of 5 progenies

rfdl

0 0 0 0 0 0 2 6 1

8 2 6 17 22 10 13 34 15

40 45 46 48 49 52 52 52 53 54 54 59 67 67 53

60 50 40 50 72 71 73 54 49 68 52 47 54 76 58

90 92 96 100 100 97

100 100 100 100 100 100

~~~

* Data from Duvick (1959~). partial restorer plants (Rfi"rfl)in the WF9-T testcrosses did not express their genetic potential for pollen fertility at all, as can be seen by comparing the WF9-T testcrosses of individual RrRjT plants with the A-T testcrosses of the same plants. If WF9-T had been the only tester, the single gene segregation would have been obscured or at least made difficult to perceive.

CYTOPLASMIC POLLEN STERILITY I N CORN

19

A third, and the most difficult, method of analysis for partial restorer genes at the Rfl locus is to grow large numbers of testcross progenies and sort them into two groups. If the backcross being tested was made to the partial restorer parent the testcrosses can be sorted into (1) progenies with a high proportion of shedding plants vs. (2) those with a low proportion of shedding plants. If the backcross being tested was made to the nonrestorer parent the testcrosses can be sorted into (1) progenies with only sterile plants vs. (2) progenies with a t least some shedding plants. Retesting of the backcross plants, or rather of their selfs or of further backcrosses to them will give confirmation of the diagnosis. We have used this latter method, in addition to the others mentioned, to remove the partial restorer gene from several inbreds in the course of practical breeding operations. I n each case, when the results of six to eight generations of backcrossing t o a given inbred were summed up, the backcross progenies did, indeed, sort out into two groups, those that proved to be heterozygous and those which were homozygous. d. rfz Genotypes and Partial Restoration. The recessive gene rfi was originally identified in the inbred WF9. In background genotypes which are largely WF9, Rfl-rfzrfz plants in Texas cytoplasm are sterile in all environments. However, when Rfl--rfrfz genotypes are in a background which is largely that of the inbred SK2, it has been found that they can be highly fertile in cool humid environments, even though they are sterile in warmer and drier environments (Duvick, 1963a, 1964). A third class of partial restorer genotypes therefore exists: those of genotype Rfl--rfZrf2 with certain modifiers. The dominance or recessiveness of the modifiers in SK2 is not yet known. e. Other K i n d s of Restorer Genotypes. It is conceivable that a gene or genes might occur which have a dominant epistatic effect, such that they could cause fertility in the presence of Tflrfl, rfzrfz, or of the double recessive homozygote. Allelism tests of newly discovered restorers have not yet revealed such a gene or genes, however. All restorers tested have been of RflRflRfiRf2 genotype, as far as we know. Another kind of genotype which has never been found, to my knowledge, is a recessive fertility restorer. Such a genotype would not restore pollen fertility when crossed to cytoplasmic male sterile lines, but nevertheless would be pollen fertile when it was homozygous in Texas cytoplasm. Such a line has not been found; all restorer genotypes are completely or partially dominant. This indeed is the reason why they were called “restorers,” for the term connotes a dominant gene action, such that the cross of a cytoplasmic male sterile genotype by a “restorer” genotype gives an Fl which has been “restored” to pollen fertility. Theoretical studies of the evolution of dominance might find in the

20

DONALD N. DUVICK

genetics of fertility restorers a worthwhile field of research, for there exists here a wide range of degree of dominance, the genetic reasons for which are reasonably well worked out. VI. Effect of Texas Cytoplasm on Morphological Characters Other Than Pollen Fertility

A. PLANT HEIGHT Several studies (Jones and Mangelsdorf, 1951; Josephson and Kincer, 1962; Johnston and Snyder, 1962) have shown that nonrestorer inbreds and hybrids tend to be shorter in Texas than in normal cytoplasm. The greatest shortening is in the internode below the tassel, although a t times there is some shortening in the lower portions of the stalk as well. When restorer genotypes are compared in normal and in Texas cytoplasm, only a little or no shortening has been detected. There also appear to be interactions between genotype and cytoplasm, within a restorer or a nonrestorer genotype, so that some inbreds and hybrids show more shortening due to Texas cytoplasm than do others. An illustration of the shortening in Texas cytoplasm, with and without restoration, is given in Table 5, which summarizes measurements (1) TABLE 5 Average Height of Six Three-Way Hybrids a t Johnston, Iowa in 1962* Nonrestorer

Height (cm)

Normal

Texas

% ’ Normal

Normal

To ear Above ear Total

105.4 115.2 220.6

99.8 107.2 206.9

94.7 93.1 93.8

104.6 117.8 222.3

Restorer Texas

%Normal

101.2 115.8 217.0

96.7 98.3 97.6

* Each hybrid made as restorer and nonrestorer; each restorer type made in normal and in Texas cytoplasm. Data from Duvick (1962). from the ground to the node a t which the top ear was borne, and (2) from that point to the base of the tassel. The measurements were made on six three-way hybrids in Iowa in 1962. Each hybrid was made in four ways: (1) normal cytoplasm, with nonrestorer male inbred [the male inbred of the three-way cross: (A X B) X C is inbred “ C ” ] ;(2) Texas cytoplasm, with nonrestorer male inbred (inbred “A” was backcrossed 6 or more times as male into Texas cytoplasm) ; (3) normal cytoplasm, full restorer male inbred (inbred “C” was converted to Rf,Rf,Rf~Rj~ by means of a backcrossing procedure like th at described for inbreds A12 and M14 in Section

CYTOPLASMIC P O L L E N S T E R I L I T Y I N CORN

21

V,B,3) ; (4)Texas cytoplasm, full restorer male inbred. The first type of hybrid has normal cytoplasm and is pollen fertile. Type 2 has Texas cytoplasm and is pollen sterile. Type 3 has normal cytoplasm and is pollen fertile; it is slightly different from the previous two types genetically because of the substitution of a piece of chroinosome carrying Rfl. Type 4 has Texas cytoplasm and is pollen fertile; genetically it should be identical to type 3. The assumption is made that six or more backcrosses have been enough to completely transfer the genotype of inbred A into Texas cytoplasm. Separate experiments have indicated that this assumption usually is valid (Duvick, 1960). Table 5 shows that all hybrids with Texas cytoplasm were on the average shorter than those with normal cytoplasm, both below and above the ear. However, the nonrestorer hybrids were shortened by Texas cytoplasm more than the restored hybrids, especially above the ear. Analysis of variance showed the differences due to cytoplasm were statistically significant. The greater reduction of nonrestorers as compared to restorers was also significant, i.e., there was a statistically significant interaction between cytoplasms and restorer genotypes. I n summary, this example illustrates the general phenonienon that plants are shorter in Texas than in normal cytoplasm, and that pollen sterility (which here happens to have been caused by Texas cytoplasm) makes them even shorter. One inust be careful to distinguish, then, between the effects of cytoplasm, per se, and of secondary effects of cytoplasm, i.e., the effect of pollen sterility which itself was caused by cytoplasm. I n the test just discussed, measurenients of total plant height (height to tip of highest leaf) were also made a t intervals during the growing season. Since they did not differ significantly at this stage of development, data for restored and nonrestored hybrids in each cytoplasm were conibined. The combined data are presented in Table 6. The six hybrids, TABLE 6 Average Height to Tip of Highest Leaf of Six Three-Way Hybrids at Three Stages of Growth at Johnston, Iowa in 1962* Cytoplasm Age

(weeks)

Normal

Texas

% Normal

4 7 8

65.9 148.1 189.6

64.0 144.5 185.5

97.1 97.6 97.8

* Restorer and nonrestorer forms are averaged within cytoplasms. Height is in cm. Data from Duvick (1962).

22

DONALD N. D W I C K

restorer and nonrestorer, averaged about 2-3% shorter in Texas than in normal cytoplasm, at each measuring date. The differences were significant, statistically, and occurred in all hybrids in approximately equal magnitude. A second test, made in 1963, has compared thirty-six restored single cross hybrids (RflrflRfzRfz) in normal and in Texas cytoplasm. I n this test the hybrids averaged 1-2% shorter in Texas than in normal cytoplasm throughout the season. I n the mature plants, Texas cytoplasm caused shortening below the ear by a statistically significant amount, but did not cause any shortening above the ear. All hybrids had essentially the same reaction. The important fact in these summaries of height measurement is that differences between Texas and normal cytoplasms have been shown to be present as soon as 4 weeks after planting. A t this stage, tassel priniordia may be differentiated (Kiesselbach, 1949), but it is certain that meiosis and microspore development have not been started. Thus, Texas cytoplasni has an effect on plant development which is additional to its effect on pollen sterility.

B. NUMBEROF LEAVES As a corollary t o the height measurements, counts of numbers of visible leaves per plant in each of the two experiments were taken a t intervals during the growing season and at maturity of the plants. Table 7 TABLE 7 Average Number of Visible Leaves per Plant of Six Three-Way Hybrids in Two Cytoplasms, a t Johnston, Iowa in 1962* Cytoplasm Age (weeks) Normal Texas 3

5 7 8 Mature

7.90 13.03 17.56 19.22 19.86

7.80 12.86 17.30 18.96 19.58

% Normal

98.7 98.7 98.5 98.6 98.6

* Restorer and nonrestorer versions of each hybrid are combined within each cytoplasmic group. Data from Duvick (1962). illustrates the data for the test of the three-way hybrids. I n this test the effect of Texas cytoplasm again was the same for the nonrestored as for the restored versions, so the data for the two kinds of genotype are combined. The data indicate that Texas cytoplasm reduced the number of

CYTOPLASMIC POLLEN STERILITY I N CORN

23

leaves per plant by 1-2%; the difference was evident froni the time of the first count. A reduction of about the same magnitude also was obtained in the test of thirty-six single cross hybrids. Analyses of variance showed that within each of the two tests the consistent reduction in leaf number was not affected by the type of hybrid. I n the single cross test the effect was not significantly different in the two different locations in which the test was performed. Therefore, in leaf number as well as in plant height Texas cytoplasm affects the plant prior to meiosis and does so independently of its effect on pollen fertility. C. GRAINYIELD The published data relevant to the effects of Texas cytoplasm on yield are conflicting. Jones et al. (1957a) reported that some but not all cytoplasmic sterile hybrids yielded significantly more than their normal cytoplasm counterparts. Duvick (1958) stated that cytoplasmic malesterile hybrids were sometimes different in yield from their normal cytoplasm counterparts, with extent and direction of difference depending on both the hybrid and the environment. A sparing effect of pollen sterility during periods of environmental stress was suggested as one cause of the differences obtained. Everett (1960), Josephson and Kincer (1962), and Johnston and Snyder (1962) concluded that the cytoplasm has little or no effect on yield. Stringfield (1958) postulated that Texas Cytoplasm plus the restorer genotype might raise yields. Noble and Russell (1963) found a reduction in yield for restorer hybrids in Texas cytoplasm, as compared to the same genotypes in normal cytoplasm. They also pointed out th a t some of the tests reported in the literature had insufficient replication to test for the sinall but real differences (often about 2-4%) that can exist. Some of the reasons for these conflicting conclusions are probably as follows: I n grain yield, as in plant height, there are differences owing to cytoplasm, but the presence of pollen sterility, per se, also has a n effect, so that one must distinguish between (1) the effect of Texas cytoplasm as such and (2) its secondary effect, of causing pollen sterility in the nonrestorer genotypes. Chinwuba et al. (1961) have shown clearly that pollen sterility as such relieves some of the stress on the plant a t time of flowering, resulting in higher yields for a t least some pollen sterile hybrids a s compared to their pollen fertile counterparts whenever the hybrids have been under reasonably severe stress a t time of flowering. Cytoplasmic male sterility and male sterility caused by detasseling are both effective. High plant populations, drought, and/or hot weather a t the time of flowering are the types of stress which give a n advantage to male steriles.

24

DONALD N. D W I C K

Chinwuba et al. (1961) also predicted that restored hybrids in Texas cytoplasm would not respond to stress conditions in the same way as nonrestored hybrids in Texas cytoplasm. The results of experiments like that summarized in Table 8 bear out their prediction. I n this example the nonrestorer hybrid, C103 X Hy, and its reciprocal, Hy x C103, yield increasingly better in Texas than in normal cytoplasm as plant populations are increased, but when Rfl is inserted into either of the nonrestorer inbreds (as Hy-TR or as C103-TR) the advantage under stress of the Texas cytoplasm forms disappears completely. TABLE 8 Yields a t Three Rates of Planting of Four Variations of C103 X Hy in Texas Cytoplasm, Each Expressed as Per Cent of Its Normal Cytoplasm Counterpart* Plants per acre Pedigree

8,000

16,000

24,000

C103 X Hy Hy X C103 Average

l06t 112t -

12st 136t 132t

163t 1421 1521

98 99 98

101

102

97 -

92 t -

99

97

C103 X Hy-TR Hy X C103-TR Average

lost -

* Data from Duvick (1962). t Significantly different from loo%, at 1 % level. When a large number of comparisons are made, it becomes apparent that in yield, as well as in height and leaf number, Texas cytoplasm forms (if of restorer genotype) yield about 2 % less than their genetic equivalents in normal cytoplasm (Duvick, 1963b). Table 9 shows average yields of sixty-seven three-way hybrids, each made in four forms: (1) normal cytoplasm, nonrestorer; (2) Texas cytoplasm, nonrestorer; (3) normal cytoplasm, restorer; and (4) Texas cytoplasm, restorer. These were made as described for the three-way hybrids in Section VI,A, but they are not the same hybrids. The average yields of the Texas cytoplasm nonrestorer hybrids were the same as those of their normal cytoplasm counterparts, but the average yields of the Texas cytoplasm restorer hybrids were about 2 % less than those of their normal cytoplasm counterparts. These data imply, therefore, that pollen sterility raises yields on the average about 2 % but that Texas cytoplasm on the average reduces yields by about 2%. The gains and losses cancel each other in the comparison of cytoplasms within the nonrestorer types; but in the restorer types, where

25

CYTOPLASMIC POLLEN S T E R I L I T Y I N CORN

TABLE 9 Averages of Yields in Bushels per Acre of Several Three-Way Hybrids, Each Made in Four Forms* Nonrestorer genotype Normal Year Number of hybrids cytoplasm 1960 1961 1962

12 12 43

Weighted average

96.0 109.1 115.0 109.7

Restorer genotype

Texas cytoplasm

Normal cytoplasm

Texas cytoplasm

96.5 109.4 114.0 109.3

96.5 108.8 114.5 109.5

94.4t 105.5t 112.4f 107.13

~

* Tests conducted throughout the U.S. Corn Belt during a 3-year period. Data from Duvick (1962). t Significantly different from normal cytoplasm counterpart at 5 % level, as tested with error mean square. 3 Significantly different from normal cytoplasm counterpart at 1% level, as tested with error mean square. pollen sterility is not a factor, the reduction in yield owing to Texas cytoplasm is revealed. Analysis of variance of the data summarized in Table 9 has shown that although, on the average, yields of non-restored hybrids in Texas cytoplasm equal those of their genetic equivalents in normal cytoplasm, some nonrestorer hybrids in some environments will yield genuinely more in Texas than in normal cytoplasm (as the example showed in Table S), and conversely, some nonrestorer hybrids in some environments will yield genuinely less in Texas than in normal cytoplasm. I n other words, in the nonrestorer hybrids there is a statistically significant interaction between hybrids, environments, and cytoplasms with respect to yield. On the average, with the hybrids and testing conditions employed, the pluses and minuses tend to cancel each other. But if, for example, one divides all yield comparisons of nonrestorer hybrids into two groups-those in which the individual plants were under comparatively severe stress vs. those in which they were not under severe stress (using grain yield per plant as a measure of stress)-the Texas cytoplasm forms will tend to outyield their normal Cytoplasm counterparts in the tests which were under relatively high stress, while they will tend to yield less than their normal counterparts in the tests which were under comparatively less stress (Duvick, 1958, 1962). In restorer genotypes there is little or no effect of hybrids or of environments on the small reduction in yield caused by Texas cytoplasm (Duvick, 196313, 1964). This would seem to indicate that the sparing effect of pollen sterility, per se, is more affected by hybrids and by environments than is the more fundamental effect of Texas cytoplasm, per se.

26

DONALD N. D W I C K

I n summary, it appears that Texas cytoplasm consistently reduces yields by a small amount but that since pollen sterility, per se, on the average raises yields by a like amount, only restored genotypes (and not nonrestored genotypes) will be reduced in yield, on the average. The small amount of yield reduction due to Texas cytoplasm, per se, makes it imperative to have large numbers of replications for detecting and establishing the statistical significance of the reduction. The high interaction of the sparing effect of pollen sterility, per se, with hybrids and with environments, can cause wide variation in results of individual tests comparing yields of nonrestorer hybrids in Texas vs. in normal cytoplasm.

D. RESISTANCE TO Helminthosporium maydis (SOUTHERN LEAF BLIGHT) Mercado and Lantican (1961) and Lantican et al. (1963) have reported that genotypes in Texas cytoplasm are much more susceptible to Helminthosporium maydis than are their normal cytoplasm counterparts. This effect is seen in both restorer and nonrestorer genotypes. Their observations have been made in the Philippine Islands, under conditions of severe infection of H . maydis. To our knowledge, no differences of this sort have been reported or noted in the United States. It may be that the increased susceptibility in the Philippines is a secondary effect of reduced plant vigor, accentuated by the Philippine environment, but there are no data at hand either for or against such a speculation.

E. OTHERMORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS Normal and Texas cytoplasm hybrids have been compared by many investigators with regard to several other agronomically important characteristics. These include moisture content of mature grain, resistance of plants to stalk and root lodging, general appearance of plants and of mature ears, tendency to tiller, and resistance to infection with Helminthosporium turcicum (northern leaf blight). I n no case have any consistent differences owing t o cytoplasm alone been reported (Everett, 1960; Josephson and Kincer, 1962; Jones et al., 1955; Noble and Russell, 1963). We have summarized annually the results of hundreds of such comparisons and invariably have found that the mean values for hybrids in Texas cytoplasm are virtually identical to the values for the same hybrids in normal cytoplasm. VII. Pleiotropic Effects of

Rfl

Since Rfl occurs occasionally in Corn Belt corn and when found is usually hi normal cytoplasm, one wonders whether it has some effect additional to that of restoring pollen fertility to Texas cytoplasm.

CYTOPLASMIC POLLEN STERILITY IN CORN

27

Brooks (1961) has presented data which indicate that mass selection for desirable agronomic characteristics within open-pollinated varieties increased the percentage of genotypes which restore Texas cytoplasm. Whether or not the varieties themselves carried any Texas cytoplasm was not indicated. The restorer genotypes were not rigorously identified and the data do not distinguish between pleiotropic effects and linkages with the restorer gene(s). The data presented in Section V1,C of this paper show that 67 nonrestorer (rfIrf three-way hybrids in normal cytoplasm yielded 109.7 bushels per acre, as compared to 109.5 bushels per acre for their restorer (Rflrfl) counterparts, also in normal cytoplasm. The Rfl genes in these hybrids came from seven different naturally restoring genotypes; analysis of variance indicated that the source of Rfl gene had no significant effect on yield. However, analysis of variance did show that linkages with Rfl, irrespective of the Rfl source, had a significant effect on yield; that is, in some hybrids the Rfl form outyielded the rfl form, whereas in other hybrids the reverse was true. Since (1) the mean yields were the same for the Rflrfl as for the rflrfl forms when yields for all hybrids were averaged, (2) the source of Rfl gene had no over-all effect on yields, and (3) the only differences associated with Rfl vs. rfl were those which were probably due to specific linkages, it seenis probable that there is no pleiotropic effect of any Rfl gene with regard to yield of grain in normal cytoplasm. Similar conclusions as to the effect of Rfl on maturity were indicated by analyses of grain moisture measurements for these hybrids. No other phenotypic associations with Rfl, which could not be ascribed to linkage, have ever been observed to our knowledge. The effect of Rfl on yield when the plants are in Texas cytoplasm is more complicated, due to the side effects of pollen sterility per se. This was discussed in detail in Section V1,C. VIII. Origin and Expression of Cytoplasmic M a l e Sterility in Ma i z e

A. CYTOLOGICAL STUDIES

I. Light Microscope Observations The Peruvian cytoplasmic sterile was reported by Rhoades (1933) to have normal microsporogenesis. He noted differences between the Peruvian and normal cytoplasm types in cytoplasmic inclusions of the microspores. The inclusions of the microspores in sterile cytoplasm were rounded; those of microspores in normal cytoplasm were rod-shaped.

28

DONALD N. DUVICK

Rhoades suggested that the differences were a consequence, rather than a cause, of the male sterility. No other differences were noted. Degeneration of the microspores of cytoplasmic sterile plants began at about the time of first vegetative division. The Peruvian cytoplasm, as noted before, has been lost. All reports of examinations of USDA and Texas cytoplasms state that the cytoplasmic inclusions of microspores in both USDA and Texas cytoplasmic steriles could not be distinguished from those of microspores in the normal cytoplasm equivalents (Chang, 1954 ;Edwardson, 1955 ;Khoo and Stinson, 1957). However, in both types of cytoplasmic sterile, degeneration of microspores began at about the time of the first vegetative division, as had been reported by Roades for the Peruvian cytoplasm. All reports also noted that, like the Peruvian cytoplasmic sterile, microsporogenesis was normal in both USDA and Texas cytoplasms, with no meiotic abnormalities. Chang (1954) reported that microsporocytes of the steriles appeared to be somewhat smaller, but that Feulgen stain techniques revealed no differences between cytoplasmic male-sterile and normal microsporocytes. He stated that the tapetal cells of the cytoplasmic steriles (both Texas and USDA) showed hypertrophy and prolonged persistence, plus slightly greater endomitotic activity. He hypothesized that the difference in the tapetum might be a consequence, rather than a cause, of the pollen abortion. Khoo and Stinson (1957) stated that development of the microspore wall and germ pore were inhibited in ctyoplasmic male steriles. The aborted pollen grains were, a t the time of maturity for normal fertile pollen, small, more or less transparent, and devoid of starch. Studies we have made of pollen grains from partial restorer genotypes have shown a complete range of pollen grain types, from those devoid of either starch or protein granules [air-dry pollen has about 20-30% protein (Vinson, 1927)], to those in which a few scattered granules can be seen, up to pollen grains which are completely packed with starch and protein granules. Thus, there is no all-or-none reaction which determines whether or not a microspore will be completely aborted or completely normal. We have noted no particular differences between pollen grains of partially fertile plants in USDA cytoplasm and those of partially fertile plants in Texas cytoplasm. I n summary, there is no cytological evidence (in the light microscope range) for presence of aberrant cytoplasmic inclusions or for presence of additional kinds of inclusions in the two major kinds of cytoplasmic sterility presently known in corn. Degenerative symptoms may appear in microspores of cytoplasmic male steriles at various developmental stages, from just prior to the first vegetative division up to the final stages of filling with starch and protein granules. I n every case the

CYTOPLASMIC POLLEN STERILITY IN CORN

29

symptoms appear to be those of starvation, i.e., a simple cessation of growth. Both starch and protein deposition are retarded or else never begin.

2. Electron Microscopy Observations Edwardson (1962) has reported that differences in inclusions in two separate parts of the plant can be seen with the electron microscope, when Texas and normal cytoplasm plants are compared. He compared the cytoplasmic male-sterile inbred A158 with its normal cytoplasm counSome root-tip cells terpart. A158 is of nonrestorer genotype (rflrflRfzRf~). of the sterile plants contained small inclusions (ca. 50-60 mp in diameter) in areas of “dense cytoplasm.” Neither the inclusions nor the dense cytoplasm were seen in root-tip cells of the normal cytoplasm plants. Tapetal cells of sterile plants contained inclusions of about 58-64 mp in diameter; tapetal cells of normal cytoplasm plants contained similar but smaller inclusions (ca. 46-52 mp diameter) which were always enclosed by membranes. The inclusions of the sterile plants were not enclosed by membranes, with a few exceptions. All inclusions appeared to contain RNA. Edwardson stated that there are in the literature no descriptions of inclusions similar to these, either in corn or in other plant cells. He also stated that the functions of the inclusions and their relationship to each other and to cytoplasmic male sterility remained to be determined.

B. CHEMICAL STUDIES Edwardson (1955) used paper chromatography to study water-soluble and ether-soluble compounds in tassels of normal and Texas cytoplasm plants (nonrestorer genotype). Tassels were examined at about the time of meiosis. He found no difference between normal and Texas cytoplasms when comparisons were made between fractions containing mostly mitochondria, nor when fractions contained mostly plastids. However, in ether extracts of whole tassels (collected after division I of meiosis) the chromatograms showed a ninhydrin-positive substance, and occasionally a fluorescent substance, in the cytoplasmic steriles but not in the normal cytoplasm plants. The substances were not identified. Proline was present in smaller amounts in the anthers of Texas cytoplasm plants than in the anthers of normal cytoplasm plants at the time of meiosis and also after the vegetative divisions of the pollen microspores, according to Fukasawa (1954). He used paper chromatography to study the free amino acids of anthers. He also reported that asparagine was present in larger amounts in the cytoplasmic sterile anthers than in those from normal cytoplasm plants at the tetrad stage and also after the vegetative divisions.

30

DONALD N. D W I C K

Khoo and Stinson (1957) confirmed the observations of Fukasawa with regard to proline and asparagine. They further noted that alanine was present in anthers of cytoplasmic steriles (Texas) in much larger amounts than in those of normal cytoplasm plants, from about the quartet stage to within 4-5 days of anthesis. At that time the anthers of normal plants also began to accumulate large amounts of alanine. This difference between Texas cytoplasmic steriles and normals appears to be one of timing. Anthers of restored plants (restorer genotype in Texas cytoplasm) were chromatographically identical to those of normal cytoplasm plants (Jones et al., 1957b). The USDA type of cytoplasm did not show the characteristic early accumulation of alanine. The data from these chemical studies may indicate that the abortive microspores are unable to make use of alanine and asparagine (both of which are active in transamination reactions in other plant tissues) and that for this reason the two compounds accumulate in the anthers (in tapetal cells?). The relatively low amount of proline in anthers of cytoplasmic steriles could conceivably be a direct cause of pollen abortion, if it were an essential component of one or more of the storage proteins or of one or more of the enzymes of developing pollen grains. Proline is thought to be uniquely important in determination of the over-all shape of protein molecules (Fowden, 1963), and as such its absence or scarcity may be critical. OF CYTOPLASMIC MALESTERILITY C. STABILITY

1. Empirical Observations

Cytoplasmic male sterility in corn, because of its practical usefulness, has been tested on a very large scale. Each year in the United States approximately three billion plants (a rough but conservative estimate) which carry the Texas cytoplasm plus the nonrestorer genotype are grown and observed. These plants are in crossing fields to be used as pollen sterile, non-detasseled seed parents and they are carefully inspected to be sure that they are sterile. This practice (with fewer plants per year at first) has been in use for about 10 years. To our knowledge, there is no recognized case of any permanent or temporary change in the sterilizing capabilities of any culture of the Texas cytoplasm. There are many instances in which the presence or absence of complete sterility fluctuates with the environment, but these are always cases in which partially restoring genotypes are involved. I n some instances, the same cytoplasmic male-sterile genotype has been planted on a field scale for 10 or 12 years in succession, with seed for it being renewed every 2 or 3 years, at least. If any change in the cytoplasm with regard to pollen sterility were taking

CYTOPLASMIC POLLEN STERILITY I N CORN

31

place, in any culture or subculture, it would almost surely be noted (with alarm), and investigated experimentally. The USDA cytoplasm has also been used commercially for as many years as the Texas, although probably with only 1 to 5 per cent as many plants. But it, too, has been stable and predictable in its reactions, within recognized limits of variation owing to genotype and environment. Both cytoplasms also have been extensively studied experimentally, by geneticists and plant breeders. Here again, with the possible exception of two cases in USDA cytoplasm (Jones, 1956a) no changes have been reported. 2. Effect of Restorer Genes

Since, superficially, the restorer genotypes seem to correct the change brought about by sterility-inducing cytoplasm (although not with respect to size and yield differences) it seems reasonable to think that long association of Texas cytoplasm and Rfl-Rfz-genotype might change the cytoplasm to a more nearly “normal” type (Jones, 1956a). This seems not to be the case, however. Jones et al. (1957a) report that restorer genotypes in Texas cytoplasm have been self-pollinated for several generations, then outcrossed as female to nonrestorer genotypes and from these crosses typical cytoplasmic pollen sterile individuals were obtained. I n our own breeding work we have backcrossed restorer genotypes, as male, into Texas cytoplasm for as many as sixteen generations, and then, on crossing and backcrossing to nonrestorer genotypes, have obtained pollen sterile progenies with all the typical sterility reactions of nonrestorers in Texas cytoplasm. The same kind of results, on a less extensive time scale, has been obtained with USDA cytoplasm (Jones, 1956a). 3. Eflects of Heat Treatments, Chemicals, and Ionizing Irradiation

Acting on the not unreasonable assumption that cytoplasmic male sterility in corn may be due to a virus or viruslike particle, Brawn (1963) has tested the effects of various treatments which have been known to inactivate viruses. I n a series of carefully controlled experiments he tested the effects of (1) heat shock; ( 2 ) acriflavine; (3) streptomycin; and (4) dl-parafluorophenylalanine. All treatments were made on kernels in the early stages of germination. Both USDA and Texas cytoplasms were given the heat treatment; only the Texas cytoplasm stocks were treated with the chemicals. Brawn reported that no effect was produced by any of the treatments, i.e., no changes in pollen sterility could be attributed to treatments. Advanced generations were not grown. Tests to demonstrate penetration of the treatment chemicals into cells of treated kernels were not reported, but it seems probable that penetration did occur since

32

DONALD N. DUVICK

numerous experiments have demonstrated a rapid uptake of water and dissolved chemicals by dry corn kernels in the early imbibition stages of germination. A small experiment (conducted by Dr. William Murdy and the author) to test the effects of ionizing irradiation has produced negative results also. Plants of a nonrestorer genotype in Texas cytoplasm were placed in a cobalt-60 y-radiation field at about the time of megasporogenesis. They were divided into four groups, spatially separated so that after approximately 7 days they had received: 42,74,166, and 667 roentgens of irradiation. They were then removed from the field and pollinated with nonrestorer, normal cytoplasm sibs. The following season the resulting seed was planted, giving rise to the following approximate numbers of plants in each of the four groups (in order of the above listed irradiation amounts) : 200, 175, 250, and 20. All plants in all groups were entirely male sterile, as were the control plantings of untreated seed, indicating that the radiation had not nullified the sterilizing capability of Texas cytoplasm. Advanced generations were not grown.

4. Segregation of Xterility-Inducing Particles Gabelman (1949) suggested that a particulate male-sterile factor, quite similar to chromosomes in its reproduction and distribution, was the cause of one type of cytoplasmic pollen sterility. The postulated particles divided and were distributed in equal numbers to daughter cells a t mitosis but at meiosis they did not divide and were distributed to microspores at random. The presence of one or more of the particles in each aborted microspore was thought to be the cause of its abortion. Fertile pollen grains were said to result when microspores had no particle. He interpreted his data to indicate averages of 1, 2, or 4 particles per somatic cell or $6, 1, or 2 per microspore. Data on which the hypothesis was based were gathered from studies of plants in USDA cytoplasm (Rhoades, 1955), although the report itself mistakenly says they were in the Peruvian cytoplasm studied by Rhoades (1933). Gabelman’s hypothesis is now known to be untenable, in the light of present day knowledge of the genetic and environmental interactions with the pollen sterility induced by USDA cytoplasm. Some of the reasons why it cannot be correct are: (1) Random segregation of sterility-inducing particles at megasporogenesis would mean that the sterility-inducing potential of the cytoplasm would vary from plant to plant and should even disappear in some individuals. As noted above, the USDA (as well as the Texas) cytoplasm is extremely stable, hereditarily, in its sterility-inducing properties.

CYTOPLASMIC POLLEN STERILITY IN CORN

33

(2) The hypothesis cannot explain the presence of completely sterile genotypes or of completely fertile genotypes in USDA cytoplasm, without abandoning the stipulation that sterility-inducing particles segregate at random at microsporogenesis. (3) The effect of environment in increasing or decreasing fertility of given partial-restorer genotypes in USDA cytoplasm without changing the cytoplasm in a hereditary sense would require that particle number be temporarily changed or else that some kind of enhancing effect at a distance from the particle in a pollen grain be invoked. (4) The Poisson distribution, the assumed presence of which was one of the bases for development of the particle hypothesis, cannot apply to Gabelman’s data under the tenets of his hypothesis. The hypothesis says each particle in a pollen grain arose from random distribution of particles in pollen mother cells. This limits the maximum number of particles in any pollen grain to the number in its pollen mother cell (maximum of 4, as the data were interpreted) ; in other words there is a truncated distribution. I n order to have a Poisson distribution there must be a probability (even though small) of getting a very large number of particles (1, 2, 3, . . . , X) in any given pollen grain. Therefore one cannot validly assume that the Poisson distribution describes the distribution of the postulated particles. It is of course true that even having put aside this specific particulate hypothesis, one still is faced with the problem of explaining the seemingly random occurrence of fertile pollen grains in tassels of partial restorer genotypes, whether in USDA or in Texas cytoplasm. I n studying the percentages of fertile pollen of individual anthers, looking at every anther in every floret of an entire tassel, or of specified segments of a tassel, we have been impressed with the way in which “islands” of fertility appear, in tassels which are on the whole quite sterile (Table 10); or, at the other extreme, “islands” of sterility appear in tassels which are quite highly fertile. I n other words, if one anther in a floret has a fairly high percentage of sterile pollen, one or both of the other anthers in the floret also tend to be quite sterile; if a floret tends to be sterile, its companion floret tends also to be sterile, etc. Islands of sterility (or fertility) may range in size from 2-3 anthers, to several florets, several spikelets, or all of a tassel branch or of a primary axis. If one calculates, for per cent sterile pollen, the intraclass correlation coefficients (Q) among anthers within: (1) tassel branches, (2) pairs of spikelets, (3) spikelets, and (4) florets in a spikelet, a typical set of such correlation coefficients (all positive) is 0.411,0.385,0.733,and 0.875 for the four respective classes. I n other words, the degree of similarity between anthers, in per cent of sterile pollen, increases with their proximity to each

34

DONALD N . DUVICK

TABLE 10 Per Cent Fertile Pollen in Individual Anthers of Section of Tassel Branch of a Partially Fertile Plant in Texas Cytoplasm* Spikelet rank Spikelet pairt

Pedicellate

Sessile

Floret

(93) 0 41 0 0 0 (69) (87) 0 0 0 (93) (94) (94)

Outer Inner Outer Inner Outer Inner Outer Inner Outer Inner Outer Inner

0 15

0

(79) 0 0 0 0 0 0 (74) 0 0 0

0 21 0 9 0 0 0

* Florets arranged in order as found on the tassel, with the three anthers in each floret arbitrarily arrayed in linear order. t Spikelet pairs numbered arbitrarily, going from base to tip of tassel branch. $ Anthers with 50% or more fertile pollen grains are enclosed in parentheses to emphasize “islands of fertility.” other, i.e., sterile pollen grains are not randomly distributed throughout the tassel. The tassel in this example was taken from a heteroxygous partially restored plant in Texas cytoplasm. The anthers examined averaged 43% sterile pollen grains. Superficially, a spatial distribution of this sort looks very much like the result of somatic segregation of sterility-inducing particles. We must immediately say that the particles cannot segregate in future megasporogenetic tissue, however, for, as noted above, no case of instability in inheritance of either USDA or Texas cytoplasm has ever been definitely established. Therefore, a chimera hypothesis is hard to build. The notion of “most favored orgnns” seems hopeful, until the data are examined. It seems that one might expect that given a limited amount of “pollen nutrient” the inner floret, for example, might tend always to have more “nutrient” than the outer floret, in an analogy with the way in which upper leaves tend to die last under conditions of nitrogen shortage. There is evidence (Duvick, 1957) that in tassels of some plants the outer florets indeed are significantly more sterile than the inner ones; but in other plants of the same genotypes the reverse (also significant) can also be found. And in many tassels the distribution of sterility percentages

35

CYTOPLASMIC P O L L E N STERILITY I N CORN

among florets in a spikelet is random. Thus, although there may be “favored organs” there seems to be no general rule for predicting which they may be. Often most of the plants of a given partially restored genotype tend to be sterile in one general section of the tassel, say, the primary axis. I n these cases, one usually finds that the sterile regions of the tassel are the ones that might have been in a stage just after meiosis at a time of hot, dry weather. But this is only a generalization from empirical observations. We know of no precise data on the question. And, a t any rate, the small islands, of one or two fertile anthers or florets, cannot be explained by any such diffuse environmental change. Probably a study of partially sterile plants with normal cytoplasm will provide the best explanation. Certain inbred lines are notoriously poor pollen producers, even though it can be demonstrated that they have normal (nonsterility-inducing) cytoplasm. They, like partial restorers, are more sterile in hot dry weather. And most important, they, too, tend to have “islands of sterility” (see Table 11) which can grow so large under extremely adverse conditions that the tassel has only “islands of fertility.” This, to the author, indicates that the variable expression of sterility on TABLE 11 Per Cent Fertile Pollen in Individual Anthers of Section of Tassel Branch of a Partially Fertile Plant in Normal Cytoplasm* Spikelet rank Spikelet pairt

Pedicellate

Sessile 69 80 (0)

69 84 (15) 88 (0) 94 66 87 77

80 81 80 66 (0) (5) 72 74 80 79 (0) (7) 90 84 67 84 84 82 50 (28) 82 95 64 79

Floret Outer Inner Outer Inner Outer Inner Outer Inner Outer Inner Outer Inner

* Florets arranged in order as found on the tassel, with the three anthers in each floret arbitrarily arrayed in linear order. t Spikelet pairs numbered arbitrarily, going from base to tip of tassel branch. 1Anthers with less than 50% fertile pollen grains are enclosed in parentheses to emphasize “islands of sterility.”

36

DONALD N. D W I C K

the tassels of partial restorers in Texas cytoplasm has nothing to do directly with the phenomenon of cytoplasmic male sterility. It is simply the plant’s way of focusing a limited amount of pollen producing potential in at least a few functional entities, instead of spreading it out evenly and inadequately over the whole tassel. We hasten to add that it is presumed that Darwinian selection, rather than any teleological impulse on the plant’s part, has been the real cause for this tendency. It is pertinent to note here that plants in USDA cytoplasm, heterozygous for the dominant restorer gene, with 50% of the pollen grains fertile, do have a random distribution of fertile grains, within anthers and between anthers, so that any anther sampled will give about 50% fertile grains, if that is the general level of the plant as a whole. One other phenomenon, sometimes incorrectly thought of as a chimera, also needs comment. Often, the tassel of a tiller will be sterile although the tassel of the main stalk will be more or less fertile. Conversely, the tiller may have a partially fertile tassel but the tassel of the main stalk will be sterile, or, there will be partial to full fertility on one of the tassels and a different level of fertility on the other. I n all the occurrences of this sort that we have studied, known partial restorer genotypes were involved; in every case the direction of change in the weather (from hot dry to cool moist, or vice versa) correlated with whether the tiller tassel (which always develops several days after the main stalk tassel) was more fertile or less fertile than the tassel of the main stalk. Therefore, it seems obvious that this is an example of the effect of varying environmental conditions on two parts of a single clone. It is not at all different from the effect obtained when two plants of a given genotype are planted side by side a t two different times, at an interval of, say, a week. D. CREATION OF STERILITY-INDUCING CYTOPLASMS 1. Genetic Induction

Rhoades (1950) has described an example in which homozygous iojap ( i j ) plants were crossed with normal cytoplasm, green, male-fertile individuals. Normal fertile, green plants resulted when the iojap plants were used as male. When the iojap plants were used as female, some malesterile individuals occasionally resulted. When these male-sterile plants were pollinated by unrelated normal fertile individuals, some completely male-sterile backcross progenies were produced. Rhoades concluded that the iojap gene had induced a cytoplasmic mutation to a sterility-inducing condition, more or less in the same way that iojap induces plastid mutation (Rhoades, 1943) although the two events are not required to occur simultaneously.

CYTOPLASMIC POLLEN STERILITY IN CORN

37

The description as given in Rhoades’ report is precisely that outlined by Josephson and Jenkins (1948) and Josephson (1955), in which 33-16 was crossed as female with K6, giving 15% of sterile plants, and then crossed and backcrossed to Ky27, giving a cytoplasmic male-sterile form of Ky27. The sterile cytoplasm from iojup and the sterile cytoplasm from 33-16 are both indistinguishable from USDA cytoplasm, and from each other on the basis of their fertility restoration requirements (Stinson, 1962). It is not at all unusual for a more or less fully restored USDA genotype to act as an incomplete recessive when crossed to certain nonrestorer genotypes. A case described by Briggle (1956) also parallels closely the results described for iojup and 33-16. Therefore, it seems possible that the strain of iojup used by Rhoades had a USDA-type cytoplasm plus restoring genes, and that a fortuitous cross plus, perhaps, fortuitous weather conditions revealed the latent ability of the cytoplasm to induce pollen sterility. This argument could be demolished immediately, of course, if there were evidence that the iojup gene had been transferred as male to a proven nonsterility-inducing cytoplasm and that from this stock there then was extracted cytoplasmic male sterility. We know of no such data, however, and have attempted such an experiment twice, without success. One must of course admit that just because a thing has not been done, one has not proved it cannot be done. 2. Transmission Through Pollen

One wonders if it is not possible to transmit (at least in small degree) the sterility agent(s) in either USDA or Texas cytoplasm through the pollen as well as through the egg. Rhoades (1933) reported negative results when he attempted this with the Peruvian cytoplasmic sterile. We have been conducting a small test of this possibility for about 10 years, in which a partial restorer genotype in USDA cytoplasm, and another partial restorer genotype in Texas cytoplasm, have been continually backcrossed as male to a stock which had as original female a source of normal cytoplasm. To date no hint of reduction of fertility has been noticed in any of the backcross individuals. A further step in this experiment might be to replace the partial restorer genotype of the backcross lines with a definitely nonrestorer genotype, to see if it might reveal small differences in sterilityinducing potential. Goodsell (1961) has demonstrated with an ingenious experiment that the reverse phenomenon-transfer of “normal” cytoplasm into “sterile” cytoplasm-does not occur. He has recovered “monoploid” individuals which are the result of development of a pollen sperm nucleus in the cytoplasm of an unrelated plant with Texas cytoplasm. The pollen

38

DONALD N. D W I C K

parent, actually the only parent, had normal cytoplasm. The mother (perhaps “nurse” would be a better term) plant contributed no chromosomes. When the monoploid individuals were cross-pollinated as female to the pollen parent inbred line they produced a few kernels of diploid constitution. Plants grown from these kernels proved to be indistinguishable in pollen sterility characteristics as well as in other phenotypic characteristics from their inbred parent (the pollen donor) when it was in Texas cytoplasm. Thus, even when the sperm cell was the sole source of nuclear genes there was no detectable cytoplasmic contribution from the sperm cell. Of course, on an empirical scale, the fact that repeated backcrossing of cytoplasmic sterile lines to their normal cytoplasm counterparts for as many as twenty generations has produced no visible change is also good evidence that the cytoplasm of the pollen from normal cytoplasm plants produces no heritable change in the cytoplasm of plants with Texas or USDA cytoplasm. 3. Infection-Type Transmission

If cytoplasmic sterility were due t o a viruslike particle it might be possible to transmit the condition to plants with normal cytoplasm by some type of injection, or with insect vectors. Efforts to do so have given negative results (Rhoades, 1933; Chang, 1954) although a really exhaustive series of attempts has not been made. Normally occurring insects would seem to be unable to act as vectors, else the many years’ experience of growing normal and cytoplasmic sterile lines in close proximity in breeding nurseries should have resulted in at least some cases of transmission which would mean that cytoplasmic sterile plants would turn up in known normal cytoplasm cultures every now and then. Such has not been the case. Grafting and dodder-transmission are nearly impossible to use with corn so that these methods, often used to transmit virus infections in other plants, hold little promise as an investigative tool in this problem.

4. Cytoplasms of Foreign Species Most of the cases of cytoplasmic pollen sterility described in Edwardson’s excellent review (1956) are derived from intergeneric, interspecific, or interracial crosses, such that a genome of one taxon was placed in the cytoplasm of another recognizably different taxon. It seems reasonable to hypothesize, therefore, that the two types of sterility-inducing cytoplasm found in corn may be visible evidence of interspecific or intergeneric crosses at some time in the past. The immediate difficulty in testing this hypothesis is the fact that until

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recently it has proved virtually impossible t o hybridize Zea mays with any of its closely related taxa except teosinte (Zea mexicana Schrad. comb. nov.). We have crossed a few individuals of the “florida” strain of teosinte as males to both USDA and Texas cytoplasmic male-sterile plants. I n both types of cytoplasm, teosinte proved to be of sterilizable (nonrestorer) genotype. This would seem to preclude the possibility that the strain we tested could be a source of either of the two types of sterility-inducing cytoplasm, for it then would be male sterile in its own cytoplasm. A largescale test of many individuals from several different races of teosinte should be made, however, to give a more exact picture of its genotype with regard t o fertility restoration. We also have backcrossed corn genotypes into the cytoplasm of a clone of florida teosinte. I n this case, one inbred, WF9, proved to be more or less male sterile. However, it and all other genotypes tested in the teosinte cytoplasm were extremely depauperate, weak, and late maturing, and it seems likely that the cytoplasmically induced depauperate condition rather than a cytoplasmic sterility like either the USDA or the Texas type was the cause of the pollen sterility. WF9 in its own cytoplasm is often partially sterile. Hybrids of the partly sterile WF9 (in teosinte cytoplasm) with other lines of corn, all of which were nonrestorer or weak partial restorer genotypes with regard to both USDA and Texas cytoplasm, had completely fertile pollen, which also precludes any similarity of this teosinte cytoplasm to the USDA and Texas forms, with regard to cytoplasmic pollen sterility. Again, testing of the cytoplasm of many teosinte races would be desirable. Farquharson (1957) successfully hybridized Tripsacurn dactyloides L. and maize, using a triploid Tripsacum plant as female. The maize was a Peruvian variety, Puno. The hybrid was highly male and female sterile, presumably owing to the imbalance of its chromosomal constitution. However, it is also perennial, and such a chance hybrid in nature could perhaps persist and multiply long enough for a viable backcross t o corn to be made, if chance segregation were to put into a n egg cell enough corn chromosomes to make a viable gamete. Or, it seems reasonable to think that a paternal monoploid could occur, resulting in corn genotype in Tripsacum cytoplasm in one generation. The resulting monoploid plant would of course need to have a sector of diploid tissue in the ear in order to move back to a more vigorous and fertile diploid type of corn, but as Goodsell (1961) has shown, the entire process is possible (in corn, a t least), and within the range of odds needed by geologic time. To date, it is not known whether or not the particular clone studied by Farquharson will cause corn genotypes to be male sterile, since no viable seeds of any genotype have been produced by it.

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Another species of Tripsacum ( Tripsacum Jloridanum Porter) crosses readily with corn, at least as a male, giving fertile hybrids (Galinat, 1962). If this species will cross as female also, its cytoplasm can be tested as an inducer of pollen sterility to corn genotypes. New techniques, and perhaps more important, a renewal of interest and increases in attempts to hybridize corn and its relatives are showing that occasional crosses can be made to several different species, even though such crosses are extremely rare. If in geological t h e natural interspecific crosses happened twice, in such a way that the noncorn species contributed the cytoplasm, the origin of USDA and of Texas cytoplasms might be accounted for, at least with reference to Zea mays. The cause of the original differentiation of three distinct cytoplasms from a single cytoplasm of a putative common ancestor of the three species would still need to be accounted for, however. One can think of genotype and cytoplasm as evolving synchronously, so that an original interbreeding population which can be thought of as the ancestor of Zea mays and of at least two other unknown species may have developed gradually into three populations which eventually no longer had effective interbreeding capabilities. Intermediates would have disappeared, and three distinct cytoplasmic types, as well as three distinct types of nuclear germ plasm, might have coexisted. Recent hybridization of the direct ancestor of maize, as male, with each of the other two species, followed by repeated backcrossing of maize as male t o these crosses could then have brought into maize two distinctly different cytoplasms. If one assumes that hundreds of thousands of years were required for the development of the differentiated cytoplasms, then any differentiation now occurring at the same rate would appear to be nil, and any existent differences within a particular cytoplasmic type might go undetected. Recent archeological studies (Mangelsdorf et al., 1964) indicate that a t least one hydridization of corn with a related species (such as Tripsacum or teosinte), followed by backcrossing to corn, occurred in Mexico as recently as 5000 years ago. There seems to be no reason why such a hybridization could not have occurred with corn as male. If this hypothesis were correct, one would expect the restorer genes, Rf,, Rfi, and Rf3, to have come from the foreign species. They probably should still be associated with linkage groups which are concerned with determination of phenotypic characters that differentiate the putative “pure” corn from introgressed types. It is interesting to note that in many of the tests we have made (see Section VI), Rfl has been linked with genes for plant height, for tillering, and for maturity in terms of days to bloom and grain moisture at harvest time. Mangelsdorf et al.

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(1964) characterized wild corn, prior to the postulated hybridization with the foreign species, as lacking tillers, being very short, and extremely early in maturity. However, since genes for tillering, for maturity, and for height seem to be numerous and are probably on several of corn’s ten chromosomes, this coincidence may be less significant than it seems a t first thought. The Darwinian notion that gradual divergence with regard to many small characteristics is responsible for speciation, when applied to the development of cytoplasmic species differences, is rather hard to reconcile with the fact that one or two genes plus modifiers are sufficient to overcome the pollen sterility caused by the “foreign” (USDA or Texas) cytoplasm. However, a type of cytoplasmic pollen sterility in flax has been reported (Gajewski, 1937) to be due to the interaction of a single homozygous recessive gene from one species with the cytoplasm of a second species. I n wheat, the cytoplasmic male sterility resulting when a common hexaploid wheat, Triticum aestivum L. “Bison,” is put into the cytoplasm of T. timopheevi Zhuk. appears to be governed by two factors, more or less like the situation in corn (Livers, 1964). Therefore, it may be that sharp differences with regard to pollen sterility may also exist between the cytoplasms of corn and some of its close relatives. A hint as to where, geographically, a putative cross might have occurred, giving rise to Texas cytoplasm plus some introgressed restorer genes, is given by the distribution of naturally occurring restorer genotypes. Edwardson (1955) reported that 60% of the Latin American varieties tested by him contained genes for fertility restoration, compared to an average of about 11% for the frequency of restoration among United States inbreds, as reported by other workers. We have found that 5% or twenty inbred lines out of 436 U.S. Corn Belt inbred lines tested genotype. Pedigrees of sixteen of these twenty restorer are RflRflRfiRfi inbreds trace their ancestry back to open-pollinated corn froni the southern United States, which in turn (Brown and Anderson, 1948) has a large percentage of its ancestry from either Mexican or Caribbean corns. U.S. Corn Belt corn is derived, in general, from original hybridization of varieties from what is now New England with varieties from the southeastern United States. Shaver (1956) in a thorough sampling of large numbers of indigenous maize genotypes from all over the world found that varieties of maize from southwest and southeast United States, and from Mexico, Central America, and the Caribbean Islands had full restorer genes in approximately 20% of their gametes, as compared to a figure of approximately 5% for varieties tracing back to central and northern United States. A small test we made of Longfellow Flint, a New England open-

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pollinated variety representative of native Indian corns from that area, showed no plants with any restorer gene for Texas cytoplasm. Present day sweet corn is morphologically very close to the New England Flints and represents a relatively undiluted sample of New England flint germ plasm. It has about as low a frequency (or lower) of restorer genes as that found in U.S. Corn Belt germ plasm (Buchert, 1959). All of the evidence, therefore, indicates that restorer genes for Texas cytoplasm have their highest frequency in Latin America more or less in the neighborhood of Mexico. Thus, one might guess that the Texas cytoplasm entered Zea mays somewhere in northern Latin America. Somewhere in this region is also the probable place of origin of Zea mays itself (Mangelsdorf et al., 1964). It should perhaps be mentioned that virtually all genotypes examined appear to contain Rfi. This means that for all practical purposes only one gene for fertility restoration of Texas cytoplasm, Rf is segregating in most types of corn. However, Rfl may have several alleles, in a multipleallelic series, as noted in Section V,B,3. Data for distribution of the restorer gene(s) for USDA cytoplasm are not as plentiful as those for Texas cytoplasm. I n our tests, five of 108 Corn Belt inbreds tested contained the dominant restorer gene described by Buchert (1961). Four of the five inbreds traced back to germ plasm from the southern United States; the fifth was of northern United States Corn Belt ancestry. Shaver (1956), testing the varieties referred to above against USDA cytoplasm, found that varieties from the Caribbean region had about 15% of gametes which “fully” restored USDA cytoplasm, as compared to 0-5% for varieties from all other regions. The Caribbean corns then, are outstanding in having genes for restoration of both Texas and USDA cytoplasm; corns of the northern United States are outstanding for having few or no restoring genes for either type of cytoplasm. Based on these limited data for frequency of restorer genes, one may speculate that USDA cytoplasm also entered Zea mays somewhere in Latin America, perhaps in the Caribbean area. The distribution of sterility-inducing cytoplasms should, like restorer genes, show highest concentrations in the centers of their putative entry into maize. Unfortunately, however, the discoveries of sterility-inducing cytoplasms known to date have not been based on random samplings of the maize varieties of the world. It seems more likely that the distribution of the sources now known is a reflection of the kinds of corn that have been searched the most thoroughly. Latin American varieties, and Mexican varieties in particular, do, however, contain both USDA and Texas types of cytoplasm (see Section IV,A).

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6. Spontaneous Origin

The seemingly small difference which exists between the sterilityinducing and normal types of cytoplasm makes it seem reasonable to suppose that some sort of a mutationlike effect might be the immediate cause of the difference, and that this might occur again. However, no cases of spontaneous occurrence of a cytoplasm which can induce pollen sterility have been recorded. I n the case of the Texas cytoplasm, in which two major genes are required simultaneously to correct the deficiency with regard to pollen sterility, one would suppose that at least two cytoplasmic changes must have occurred. If the odds are very low that either of these could occur singly in some heritable cytoplasmic entity, the odds that both changes would occur in the same egg cell are the product of the two single probabilities and would thus be extremely small. If one further assumes that the cytoplasmic entities were very numerous in each cell, and that random segregation had to be depended upon t o give t o an egg cell only mutant entities (of both types), or some certain preponderance of them, the odds of such occurrence seem to be more than extremely small. As noted in the previous section, geologic time might be required for such sorting out.

6. Summury I n summary, there seems to be no conclusive evidence at the present time of any directed or observed de novo creation of sterility-inducing cytoplasm in corn. However, none of the possible methods: genetic induction, transmission through pollen, infection-type transmission, introduction of foreign cytoplasm, or a spontaneous, mutationlike origin, have been definitely eliminated as possibilities.

E. EVOLUTIONARY USEFULNESS OF CYTOPLASMIC MALESTERILITY One may well ask the question: has evolution had anything to do with conserving, and/or spreading cytoplasmic male sterility in corn? Zea mays is naturally cross-pollinated, with wind-blown pollen, and superficially would seem to need no aid such as male sterility to ensure obligatory cross-pollination. I n many situations, however, the time of emergence of silks (stigmas) and of pollen shedding coincide on a given plant, and in such cases some self-pollination can occur. Corn has virtually no self-sterility mechanisms such as genes for self-sterility. Perhaps the progeny of cytoplasmic male-sterile plants would be on the average at a slightly higher level of hybrid vigor than progeny of male-fertile plants, due t o being from entire cross-pollinated seed. Such a phenomenon

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might tend to perpetuate both the cytoplasm and the recessive gene causing sterility in that cytoplasm. Watson and Caspari (1960), in an interesting theoretical analysis, have concluded that in situations analogous to that in corn (for example, when one might have normal and Texas cytoplasms, plus Rfl and rfl genes) the initial frequency of the two types of cytoplasm would tend to be perpetuated, but the nonrestorer gene would tend to be eliminated in both cytoplasms, unless it continued to arise by mutation. For this case they assumed that there was no effect on viability or fertility of females. I n the light of this theoretical conclusion, the widespread occurrence of Texas cytoplasm must mean either that it arose many times or that it was introduced a t a time when Zea mays had a small area and population or else that some selective advantage has spread it through the species. Watson and Caspari have also calculated that relative frequencies of the two types of cytoplasm will vary with their effects on viability and fertility of the females. Thus, if progeny of plants with Texas cytoplasm were more viable, the Texas cytoplasm would increase in frequency. Here, however, the evidence for Texas cytoplasm becomes contradictory. As noted in Section VI,C, average yields of grain in Texas cytoplasm tend to be about 2% less, when plants are restored, but average yields of grain tend to remain equal in Texas and normal cytoplasm when plants are sterile. Whether grain yields are equated with numbers of kernels per plant, or whether they are in the form of larger kernels, the part yield plays in selection pressure on an entirely domesticated plant like corn is difficult to say, but one might imagine that on the average the Indian cultivators of primitive maize tended to save the better ears for seed (i.e., those from the higher yielding plants). The plants which had come from seed produced on male-sterile plants would tend to have more heteroxygous loci and because of this would perhaps tend to be higher yielding; they also would be more likely to themselves be male sterile. The tendency then would be to select toward Texas cytoplasm and rj1. At the same time, the tendency of rfl to be eliminated (according to the model of Watson and Caspari) might be supposed to be operating, so that Rjl would tend to persist in the population. But, because plants of Rfl-genotype in Texas cytoplasm tend to be slightly lower yielding, they would be selected against, which would operate against increase of both Texas cytoplasm and the Rfl gene. We end up, then, with an intricate system of selective and counterselective forces. Without knowing more about the real frequencies of either USDA or Texas cytoplasms in any specified race of maize, or the frequencies of the restorer genes in the same races, it would be well to stop such speculation at this point.

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F. OTHER TYPESOF CYTOPLASMIC

INHERITANCE I N

CORN

It is probably well to say something about other types of cytoplasmic inheritance that have been demonstrated in corn. The cases of maternal inheritance of chlorophyll abnormalities (Anderson, 1923; Demerec, 1927) are well known. Mazoti (1954, 1958) has described an interesting case in which corn genotypes in teosinte cytoplasm are much reduced in vigor, and also seem to inhibit the expression of some Mendelian genes such as iojap. We seem to have duplicated his results, a t least with regard to vigor, when we backcrossed corn genotypes into a different selection of teosinte, as described above in Section VIII,D,4. In our experiments, some genotypes are more vigorous than others, in the teosinte cytoplasm. Brown (1961) has reported a maternally inherited abnormality, found in a strain of popcorn, which causes a peculiar kind of light green leaf streaking (not like any of the Mendelian genes causing chlorophyll abnormalities), and which also causes an extreme loss of vigor. There is evidence that the vigor effects are modified by genotype. Several workers have found a condition similar to this, which appears now and then in the widely used inbred WF9 (Stinson, 1960). It, too, is affected in its expression by genotype, much as restorer genes affect cytoplasmic sterility (Duvick, 1961, 1964). It appears to be quantitatively different from that described by Brown, since given genotypes are more reduced in vigor in the popcorn cytoplasm than in the defective WF9 cytoplasm, but in other respects the two conditions look much the same. This defective cytoplasm seems to arise spontaneously in WF9, and several independent discoveries of it have been maintained. It has even occurred in cultures of WF9 in USDA and in Texas cytoplasms. Fleming et al. (1960) have described differences in maturity, height, and yield which appear to be dependent on the cytoplasm. They studied reciprocal crosses of double cross hybrids, made of inbreds of southern United States material. Further breeding to develop isogenic stocks in the putatively different cytoplasms has not yet been done, so that this evidence for cytoplasmic inheritance is suggestive but not conclusive. I n summary, it seems clear that several kinds of cytoplasmic inheritance in addition to cytoplasmic pollen sterility exist in corn and are often regulated in degree of expression by the genotype. None of the cytoplasmic effects have yet been found to have such simple genetic controls as cytoplasmic male sterility, but none of them have been investigated as thoroughly. They often seem to be extremely erratic in expression and unstable in inheritance, although until one understands the role of genotype and environment the same things can be said of the action of

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partial restorers in cytoplasmic male steriles. There seems to be no causal connection between cytoplasmic male sterility, either of USDA or Texas type, and any of the other kinds of cytoplasmic inheritance discussed in this section; in fact, cytoplasmic male sterility and the WF9type defective cytoplasm seem to be transmitted independently through the same female.

.

G. SUMMARY OF POSSIBLE MODESOF ACTIONAND ORIGINSOF CYTOPLASMIC MALE-STERILE CORN When all is said and done, little really is known about just how USDA or Texas cytoplasms cause pollen sterility or where they came from. The most important difference between the two cytoplasms is that the corrective action of restorer genes in the USDA cytoplasm is primarily in the gamete (since in heterozygous restored plants only the pollen grains with the dominant restorer gene are fertile, with some exceptions) ; whereas the action of restorer genotypes in Texas cytoplasm must be in the sporophyte, since all pollen grains of a plant heterozygous for restorer genes are fertile. The cytological and chemical data indicate that the aborted pollen grains in either cytoplasm are starved to death, but with the possible exception of the low supplies of proline in anthers of Texas steriles, the data a t hand do not indicate why they are starved. The discovery that Rjl restores the normal free amino acid balance, in addition to restoring pollen fertility, in anthers of plants in Texas cytoplasm is revealing, however; for this indicates that a block in utilization of the free amino acids by microsporocytes in Texas cytoplasm has been removed. But since plants of Rjl-genotype in Texas cytoplasm, although pollen fertile, are nevertheless smaller than the same genotypes in normal cytoplasm by about 2%, as were also rjlrjl genotypes (Section VI), it seems that Rjl merely provides an alternative pathway to pollen fertility and does not act on the cause of the basic difference between Texas and normal cytoplasms. Or perhaps Rfl acts on one of several differences between the two cytoplasms. The electron microscopy data are interesting but do not give any clear indication as to what the different kinds of pictured particles might have to do with pollen sterility. Both types of cytoplasm are very stable, hereditarily, which makes any kind of an explanation based on viruslike particles difficult of application. The most attractive hypothesis explaining the origins of the cytoplasms, primarily because it has the least data for or against it, perhaps, is that outcrossing of corn to two different species may have allowed their foreign (and sterility-inducing) cytoplasms to become incorporated into the species Zea mays, along with the genes needed for restoration of

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the pollen fertility. And this explanation, of course, merely puts back and does not really answer the question of what, cytologically and chemically, differentiates Texas, USDA, and “normal” cytoplasms with respect to pollen fertility and sterility. IX. Economic Usefulness of Cytoplasmic M a l e Sterility and Fertility Restoraiion in Corn

A. METHODS OF PRODUCING HYBRIDCORNWITHOUT DETASSELING The primary reason for the large amount of interest in cytoplasmic pollen sterility in corn has been that it has enabled producers of hybrid seed to eliminate, a t least in part, the annual detasseling of thousands of acres of corn. The fact that corn bears its male and female flowers in different parts of the plant was what originally allowed it to be hybridized on a large scale, for by simply pulling out the immature tassel, which bears the male flowers, plants are completely emasculated and all seed they produce is of necessity cross-pollinated, with whatever other corn plants are growing nearby. By planting blocks of one kind of corn alternating with blocks of another kind, and then removing the tassels of the first kind, one can be sure that virtually all seed produced on plants of the detasseled block is hybrid, a cross of the detasseled and the nondetasseled kinds of corn. Most hybrid corn produced today is of the so-called double cross type; that is, four inbred lines of corn are hybridized in the following fashion: (A X B) X (C X D). Inbreds A and C are detasseled when being crossed on a field scale to inbreds B and D, respectively. Then the single cross A X B is detasseled when being crossed to C X D. However, if inbreds A and B are of nonrestorer genotype (rj1rfl) and if inbred A has also been put into Texas cytoplasm by repeated backcrossing as male, one can plant blocks of pollen sterile inbred A-T alternating with blocks of inbred B and produce completely cross-pollinated seed on inbred A-T, without detasseling. The Fl or single cross (A-T X B) will also be pollen sterile, since inbreds A and B are both of nonrestorer genotype, and it can be planted in alternating blocks with the single cross C X D, to produce double cross seed without detasseling. Now, if inbreds C and D were also of nonrestorer genotype, the double cross hybrid, which is to be planted by the farmer to raise a crop of grain, will also be pollen sterile. This is undesirable (to put it mildly!), so one of two things can be done: (1) A portion of the double cross seed, say one-third, can be produced

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by the hand-detasseling method. This seed can be mixed with the seed produced by the cytoplasmic male sterile method, and will give a population in the farmer’s field in which one-third of the plants shed pollen. This will be sufficient to pollinate all plants in the field. (2) If inbred D had been of restorer genotype (RflRfl) the male single cross, C X D, would be heterozygous for this gene. Single factor segregation would produce two types of plants in the double cross as planted by the farmer: rflrfl and Rflrfl. All plants would be in Texas cytoplasm, and therefore half the plants would be male sterile, half would be pollen fertile. If inbreds C and D were both of restorer genotype all plants in the double cross would be heterozygotes and all would shed pollen. If inbred C were nonrestorer and also were in Texas cytoplasm (C-T) it could be used as the female parent to make the cross C-T X D and would not need to be detasseled. The single cross would shed pollen (if inbred D was Rf,Rfl) when used as male to make the double cross. The 50 % restored double cross now would have been produced without any detasseling at all. Briefly, this is the commercial application of cytoplasmic male sterility to production of hybrid seed corn. Duvick (1959b) has reviewed in detail the publications relative to this application. B. BREEDING PROCEDURES NECESSARY IN ORDER TO USE CYTOPLASMIC MALESTERILITY 1 . Making Cytoplasmic Male-Sterile Lines

As mentioned above, most Corn Belt inbred lines are of nonrestorer genotype. I n our experience, about 75% of the inbred lines tested are rflrfl. Therefore, to make such lines pollen sterile it is only necessary to backcross them repeatedly as males (about seven times) into Texas cytoplasm. (Only about 40% of the inbred lines tested have been completely sterile in USDA cytoplasm and for this reason it is generally not used.) I n order to “sterilize” the partial restorer or restorer inbred lines one must outcross to a source of rfl, and then backcross to the partial restorer line repeatedly, testcrossing backcross plants to a Texas cytoplasm rfl tester in each generation, in order to identify the plants which carry rfl. The backcrossing must be done in normal cytoplasm, for at the end of the backcrossing (say, seven or more generations) the progenies must be selfed to homozygosity for rfL.These homozygous individuals are identified by testcrossing to Texas cytoplasm nonrestorer lines, and then can be put into Texas cytoplasm by backcrossing, to give a cytoplasmic sterile line.

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2. Making Fertility Restorer Lines

Since only 5-10% of U.S. inbred lines are of homozygous Rfl genotype (as mentioned before, nearly all inbreds are homozygous Rf2 genotype), most of the “D” inbreds in double cross hybrids must be transformed, by backcrossing, from nonrestorer to restorer genotype. This backcrossing is in principle the same as that outlined above for removing restorer genes, but since one is inserting a dominant instead of removing it, the process can be done without testcrossing. All backcross plants are grown in Texas cytoplasm, in which the dominants will be pollen fertile. The fertile plants are selected for further backcrossing, and after sufficient backcrosses have been made, they are selfed and homozygous progeny are selected. The converted line will now be in Texas cytoplasm, but this usually makes no difference in its utilization. An important exception to this, however, is any case in which the converted inbred lacks modifiers for Rfl such that the line, although having the dominant form of Rfl, is only partially fertile in‘ Texas.’cytoplasm. Such a line is satisfactory as a source of Rfl to a double cross hybrid, if the other three inbreds in the hybrid contain sufficient modifiers, but the line may be most unsatisfactory in itself if the year in which it is to furnish pollen for the sterile form of inbred “C” is so hot and dry that the converted inbred becomes virtually pollen sterile. I n such cases, it may be well to put the converted inbred into normal cytoplasm, by backcrossing, or else to detassel it and use it as the seed parent to make the reciprocal cross, D X C.

C. PATENTOF THE PROCESS OF PRODUCING HYBRIDS BY MEANSOF CYTOPLASMIC MALESTERILITY PLUSFERTILITY RESTORATION Unique in the history of genetics, either with or without reference to cytoplasmic inheritance, is the fact that a patent (Jones, 1956b) has been granted on the process of using fertility restorer lines to produce a pollenfertile “cytoplasmic sterile” hybrid corn. The patent was granted to one of the early researchers in the field, D. F. Jones, who assigned it to a patent management corporation. This is not a patent granted under the plant patent laws (which exclude plants sexually reproduced) nor is it intended to cover the sale or use of a particular kind of germ plasm (or cytoplasm). Rather, it is a “method” patent, in the sense that patents of chemical processes are “method” patents. The patent does not apply if hybrids are produced without fertility restoration, ie., making part of the seed with the detasseling method and the remainder using cytoplasmic sterility to eliminate detasseling, with this latter portion producing pollen-

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sterile plants. It applies only if one produces seed on cytoplasmic malesterile plants pollinated by pollen derived from “at least one inbred strain capable of restoring pollen fertility to cytoplasmic male-sterile corn.” X. Summary and Concluding Remarks

Cytoplasmic male sterility in corn is characterized by having two and (apparently) only two distinct forms, the USDA and the Texas. They are differentiated by (1) genic requirements for fertility restoration (or conversely, by genes for plasmon sensitivity), (2) the degree of fertility of heterozygous restored plants, and (3) the morphology of the anthers of partially sterile plants. Both types are completely stable, hereditarily, insofar as extensive experience and testing have shown them to be so. Both interact with the nucleus to the extent that their sterility-inducing potential is revealed only by certain genes; and the number of the genes is relatively few for each cytoplasm. Both types express pollen sterility primarily as a gradual starvation of the microspores beginning at about the time they would be filling with starch and protein granules, and both types have intermediate forms of sterility, determined genetically but modified environmentally. Neither type has yet been altered or transmitted to “normal” cytoplasm by artificial or natural means. Neither type has been found indisputably to (1) arise de novo from cultures previously shown to have normal cytoplasm, nor to (2) revert back to “normal.” Both types seem, on the basis of indirect evidence, to have as the center of their origin some place in northern Latin America. Both types have been rediscovered several times, in corn varieties from many parts of the world. The Texas type has been shown to affect, to a small degree, the rate of growth and final size of the corn plant, independently of its effect on pollen sterility. However, in other respects plants appear completely normal in both USDA and Texas cytoplasm. The major gene, Rfl, needed to restore pollen fertility to the majority of maize genotypes in Texas cytoplasm, is probably one of an allelic series at this locus. It seems to have no pleiotropic effects, in or out of Texas cytoplasm. Just what any of the Rfl alleles do in normal cytoplasm is not at all clear. It is hard to imagine a gene with nothing to do! Conversely, other typical Mendelian recessive marker genes react and segregate in entirely normal fashion in Texas and in USDA cytoplasm. The exact cytological and chemical causes of pollen sterility in either Texas or USDA cytoplasm are still unknown, although some interesting chemical and cytological differences have been found. The apparent stability and lack of infectivity of the two sterility-

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inducing cytoplasms makes any viruslike cause seem unlikely, although the recent success in asexual transmission of cytoplasmic pollen sterility in petunia (Frankel, 1956, 1962; Edwardson and Corbett, 1961) may indicate that the same can be done with corn some day. Hypotheses of mutation of self-reproducing cytoplasmic entities, or of segregation of a mixture of them (Michaelis, 1954), are also hard to reconcile with the proven stability of and absence of intermediates between the two sterility-inducing cytoplasms. However, if a process of mutation and sorting-out took place over archeological time (hundreds of thousands of years), such that at least three distinct species with three distinct cytoplasms emerged, intermediates would have disappeared. A relatively recent hybridization of corn, as male, to each of the two foreign species might have introduced their cytoplasms plus some restoring genes into the interbreeding population of Zea mays. To be consistent with this hypothesis, one would have to grant that cytoplasmic differentiation was still going on in “normal” and in the two sterility-inducing cytoplasms. But the amount of such differentiation might appear to be nil in view of the short time scale of present experience. Careful examination may some day reveal differences between cytoplasmic cultures that presently seem to be identical. The future course of investigation of cytoplasmic male sterility in maize probably should be in the direction of study of fine structure of the cytoplasm and elucidating enzymological and other physiological causes of the pollen abortion. The evidence seems indisputable that a self-reproducing quality of the cytoplasm is responsible for differentiating the three known kinds of cytoplasm. This quality is completely independent of the nucleus in heredity, although not in the expression of its effect. Current thinking in molecular genetics holds nucleic acid entities (preferably DNA, although RNA is given some hope of being autonomous in rare cases) responsible for heredity and initiation of expression of genetic potential. These entities are thought of as particulate, meaning they can be divided and reassorted without losing a certain identity. One therefore naturally tends to think of the genetic quality of the cytoplasm as particulate, also. One must admit, however, that for cytoplasmic male sterility in maize there is a t present no evidence that this is so. There is no evidence for any kind of cytoplasmic segregation or mixing. For all we know, the quality of the cytoplasm that determines whether it be USDA, Texas, or “normal” (meaning only not cytoplasmic male sterile) may reside in an indivisible whole structure of the cytoplasm of the individual cell, which can be reproduced only as an entire unit. However, evidence of cytoplasmic mixing and sorting-out from other

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organisms, such as the fungi (Jinks, 1958) makes it seem probable that if techniques can only be devised one can mix and/or sort out maize cytoplasm in a way to demonstrate some kind of particulate basis for cytoplasmic male sterility. On the basis of what is presently known, one can speculate that (1) entities not present in “normal” cytoplasm are present in USDA and in Texas cytoplasm, either as (a) two forms of the same class of entities, or (6) as two qualitatively different classes of entity; or that (2) entities present in “normal” cytoplasm are (a) absent or ( b ) altered in function in USDA and in Texas cytoplasm. Speculations (la) and ( l b ) are essentially restatements of a viruslike cause of cytoplasmic male sterility; speculations (2a) and (2b) would more nearly apply to some type of evolutionary change in cytoplasm, although (la) and ( l b ) are not entirely incompatible with notions of evolutionary change. It seems reasonable to suppose that any such hypothetical entities would have some type of nucleic acid as the basis for their genetic capabilities. An obvious place to look cytologically and chemically, therefore, would be in the microsomal fraction of the cytoplasm, specifically, at the ribosomes. Although these particulate sites of protein synthesis are thought, generally, to be under complete nuclear (which is to say, deoxyribonuclear) direction, it may be that they have at least a degree of autonomy (Hoagland, 1961). Cytoplasmic pollen sterility in maize may help to provide an answer to this question.*

* Recent research results (see Reports of Symposiums: Cytoplasmic Units of Inheritance, in Science 147, 911-913, 1965) have indicated that DNA with coding capabilities and of non-nuclear origin is present in plastids and mitochondria of plants. It may well be that an orthodox explanation for cytoplasmic pollen sterility of corn can be arrived at before many more years go by; this explanation would involve mutation of plastid or mitochondria1 self-reproducing DNA, sorting out (randomly) of the mutated plastids or mitochondria, and selection of complementary nuclear gene mutations (restorer genes and their modifiers). This would correspond to the hypothesis presented in Section VIII, D, 5.

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REFERENCES Alimova, G. K. 1962. Tsitokhimicheskoe izuchenie razvitiya pyl 'tsevogo zerna u rastenii kukuruzy s tekhasskim i moldavskim tipom tsitoplazmaticheskoimuzhskoi steril'nosti. (A cytochemical study of pollen grain development in maize plants with the Texas and Moldavian types of cytoplasmic male sterility). Botan. Zh. 47( lo), 1522-1527 (abstr.). Anderson, E. G. 1923. Maternal inheritance of chlorophyll in maize. Botan. Gaz. 76, 411-418. Anderson, E. G. 1924. Pericarp studies in maize. 11. The allelomorphism of a series of factors for pericarp color. Genetics 9, 442-453. Blickenstaff, J., Thompson, D. L., and Harvey, P. H. 1958. Inheritance and linkage of pollen fertility restoration in cytoplasmic male-sterile crosses of corn. Agron. J . 60(8), 430-434. Brawn, R. I. 1963. Maize Genet. Coop. News Letter 37, 86-89. Briggle, L. W. 1956. Interaction of cytoplasm and genes in male-sterile corn crosses involving two inbred lines. Agron. J . 48, 569-573. Briggle, L. W. 1957. Interactions of cytoplasm and genes in a group of male sterile corn types. Agron. J . 49, 543-547. Brooks, J. S. 1961. Cytoplasmic male-sterile fertility-restoring gametes in varieties of corn in the Oklahoma collection. Crop Sci. 1(3), 224-226. Brown, W. L. 1961. A cytoplasmically inherited abnormality in maize. Proc. Iowa Acad. Sci. 68, 90-94. Brown, W. L., and Anderson, E. 1948. The southern dent corns. Ann. Missouri Botan. Garden 36, 255-268. Buchert, J. G. 1959. Chromogenic control of cytoplasmic male sterility in sweet corn. Proc. Penn. Acad. Sci. 33, 50-54. Buchert, J. G. 1961. The stage of the genome-plasmon interaction in t h e restoration of fertility to cytoplasmically pollen-sterile maize. Proc. Natl. Acad. Sci. U.S.47(9), 1436-1440. Caspari, E. 1948. Cytoplasmic inheritance. Advan. Genet. 2 , 1-68. Chang, T.-T. 1954. Pollen sterility in maize. M. S. thesis, Cornell Univ., Ithaca, New York. Chinwuba, P. M., Grogan, C. O., and Zuber, M. S. 1961. Interaction of detasseling, sterility, and spacing on yields of maize hybrids. Crop Sci. 1, 279-280. Demerec, M. 1927. A second case of maternal inheritance of chlorophyll in maize. Botan. Gaz. 84, 139-155. Duvick, D. N. 1954. Unpublished data. Duvick, D. N. 1956. Allelism and comparative genetics of fertility restoration of cytoplasmically pollen sterile maize. Genetics 41(4), 544-565. Duvick, D. N. 1957. Unpublished data. Duvick, D. N. 1958. Yields and other agronomic characteristics of cytoplasmically pollen sterile corn hybrids, compared to their normal counterparts. Agron. J . 60, 121-125. Duvick, D. N. 1959a. Genetic and environmental interactions with cytoplasmic pollen sterility of corn. Proc. 14th Ann. Hybrid Corn Ind.-Res. Cong. 1969 pp. 42-52. Am. Seed Trade ASSOC.,Chicago, Illinois. Duvick, D. N. 1959b. The use of cytoplasmic male-sterility in hybrid seed production. Econ. Botany 13, 167-195. Duvick, D. N. 1959c. Unpublished data.

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Duvick, D. N. 1960. Unpublished data. Duvick, D. N. 1961. Maize Genet. Coop. News Letter 36, 115-116. Duvick, D. N. 1962. Unpublished data. Duvick, D. N. 1963a. Maize Genet. Coop. News Letter 37, 127-128. Duvick, D. N. 1963b. Performance of restored and non-restored hybrids, in normal and in Texas sterile cytoplasm. 55th Ann. Meeting, Am. SOC.Agron., Denver, Colorado, 1963 Agron. Abstr. p. 78. Duvick, D. N. 1963c. Unpublished data. Duvick, D. N. 1964. Unpublished data. Duvick, D. N., Snyder, R. J., and Anderson, E. G. 1961. The chromosomal location of Rf,, a restorer gene for cytoplasmic pollen sterile maize. Genetics 46(10), 1245-1252. Eckhardt, R. C. 1954. Techniques in using male sterile cytoplasm and a restorer gene in corn. 46th Ann. Meeting, Am. SOC.Agron., St. Paul, Minnesota, 1954. Agron. Abstr. p. 67. Edwardson, J. R. 1955. The restoration of fertility to cytoplasmic male-sterile corn. Agron. J . 47, 457-461. Edwardson, J. R. 1956. Cytoplasmic male-sterility. Botan. Rev. 22( lo), 696-738. Edwardson, J. R. 1962. Cytoplasmic differences in T-type cytoplasmic male-sterile corn and its maintainer. Am. J . Botany 49(2), 184-187. Edwardson, J. R., and Corbett, M. K. 1961. Asexual transmission of cytoplasmic male sterility. Proc. Natl. Acad. Sci. U.S. 47(3), 390-396. Everett, H. L. 1960. Effect of cytoplasms and an Rf gene in maize. Agron. J . 62, 215-216. Farquharson, L. I. 1957. Hybridization of Tripsacum and Zea. J . Heredity 48,295-299. Fleming, A. A., Kozelnickey, G. M., and Browne, E. B. 1960. Cytoplasmic effects on agronomic characters in a double cross maize hybrid. Agron. J . 63, 112-115. Fowden, L. 1963. Amino-acid analogues and the growth of seedlings. J . Exptl. Botany 14, 387-398. Frankel, R. 1956. Graft-induced transmission to progeny of cytoplasmic male sterility in petunia. Science 124, 684-685. Frankel, R. 1962. Further evidence on graft induced transmission to progeny of cytoplasmic male sterility in petunia. Genetics 47( 6), 641-646. Fukasawa, H. 1954. On the free amino acids in anthers of male sterile wheat and maize. Japan. J . Genet. 29, 135-137. Fukasawa, H. 1959. Nucleus substitution of restoration by means of successive backcrosses in wheat and its related genus Aegilops. Japan. J . Botany 17( I), 55-91. Gabelman, W. H. 1949. Reproduction and distribution of the cytoplasmic factor for male sterility in maize. Proc. Natl. Acad. Sci. U.S. 36, 634-640. Gajewski, W. 1937. A contribution to the knowledge of the cytoplasmic influence on the effect of nuclear factors in Linum. Acta SOC.Botan. Polon. 14, 205-214. Galinat, W. C. 1962. Maize Genet. Coop. News Letter 36, 18. Goodsell, S. F. 1961. Male sterility in corn by androgenesis. Crop Sci. 1, 227-228. Hoagland, M. 1961. Some factors influencing protein synthetic activity in a cell-free mammalian system. Cold Spring Harbor Symp. Quant. Biol. 26, 153-158. Jinks, J. L. 1958. Cytoplasmic differentiation in fungi. Proc. Roy. SOC.B148, 314-321. Johnston, G. S., and Snyder, R. J. 1962. The utilization of cytoplasmic male sterility for sweet corn hybrid seed production. Proc. Am. SOC.Hort. Sci. 81, 415-420. Jones, D. F. 1951. The cytoplasmic separation of species. Proc. Natl. Acad. Sci. U.S. 37(7), 408-410.

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Jones, D. F. 1954. Gene and cytoplasm interaction in species separation. Atti. Congr. Intern. Genetica, go. Caryologia Suppl., pp. 1225-1227. Jones, D. F. 195th. Genic and cytoplasmic control of pollen abortion in maize. I n Genetics in Plant Breeding. Brookhaven Sym. B i d . 9, 101-112. Jones, D. F. 195613. U.S. Patent 2,753,663. Jones, D. F., and Mangelsdorf, P. C. 1951. The production of hybrid corn seed without detasseling. Conn. Univ. Storrs Agr. Expt. Sta. Bull. 660, 1-21. Jones, D. F., Stinson, H. T., Jr., Munson, A. P., and Renshaw, C. C. 1955. Field corn and sweet corn report for 1954. Report of Progress-G1, Connecticut Agricultural Experiment Station, Storrs, Connecticut. Jones, D. F., Stinson, H. T., Jr., and Khoo, U. 1957a. Pollen restoring genes. Conn. Univ. Storrs Agr. Expt. Sta. Bull. 610, 1-43. .Jones, D. F., Stinson, H. T., Jr., and Khoo, U. 195713. Transmissible variations in the cytoplasm within species of higher plants. Proc. Natl. Acad. Sci. U.S. 43(7), 598-602. Josephson, L. M. 1955. The use of cytoplasmic male sterility in the production of hybrid maize seed. Empire J. Exptl. Agr. 23(89), 1-10. Josephson, L. M., and Jenkins, M. T. 1948. Male sterility in corn hybrids. J . Am. Soc. Agron. 40, 267-274. Josephson, L. M., and Kincer, H. C. 1962. Effects of male-sterile cytoplasm on yields and other agronomic characteristics of corn inbreds and hybrids. Crop Sci. 2, 41-43. Khoo, U., and Stinson, H. T., Jr. 1957. Free amino acid differences between cytoplasmic male sterile and normal fertile anthers. Proc. Natl. Acad. Sci. U.S. 43(7), 603-607. Kiesselbach, T. A. 1949. The structure and reproduction of corn. Nebraska Univ., Agr. Expt. Sta. Res. Bull. 161. Lantican, R. M., Villareal, R. L., and Mercado, A. C., Jr., 1963. Maize Genet. Coop. News Letter 37, 120-123. Leng, E. R., and Bauman, L. F. 1955. Expression of the “Kys” type of male sterility in strains of corn with normal cytoplasm. Agron. J . 47, 189-191. Livers, R. W. 1964. Fertility restoration and its inheritance in cytoplasmic malcsterile wheat. Science 144, 420. Mangelsdorf, P. C., MacNeish, R. S., and Galinat, W. C. 1964. Domestication of corn. Science 143, 538-545. Mazoti, L. B. 1954. Caracteres citoplasmhticos heredables derivados del hibrido de Euchlaena por Zea. Rev. Invest. Agr. (Buenos Aires) 8(2), 175-183. Mazoti, L. B. 1958. Estudio sobre diferencias citoplasmaticas heredablea entre “Zea Mays” y “Euchlaena Mexicana.” Rev. Arg. Agron. 26, 12-44. Mercado, A. C., Jr., and Lantican, R. M. 1961. The susceptibility of cytoplasmic male sterile lines of corn to Helminlhosporium maydis. Philippine Agriculturist 46(5) 235-243. hIichaelis, P. 1954. Cytoplasmic inheritance in Epilobium and its theoretical significance. Advan. Genet. 6, 287-401. Noble, S. W., and Russell, W. A. 1963. Effects of male-sterile cytoplasm and pollen fertility restorer genes on performance of hybrid corn. Crop Sci. 3, 92-96. Rhoades, M. M. 1933. The cytoplasmic inheritance of male sterility in Zea mays. J . Genet. 27, 71-95. Rhoades, M. M. 1943. Genic induction of an inherited cytoplasmic difference. Proc. Natl. Acad. Sci. U.S. 29( l l ) , 327-329.

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Rhoades, M. M. 1950. Gene induced mutation of a heritable cytoplasmic factor producing male sterility in maize. Proc. Natl. Acad. Sci. U.S. 36, 634-635. Rhoades, M. M. 1955. Interaction of genic and nongenic hereditary units and t h e physiology of nongenic inheritance. I n “Encyclopedia of Plant Physiology” (W. Ruhland, ed.), Vol. 1, pp. 19-57. Springer-Berlin. Rogers, J. S., and Edwardson, J. R. 1952. The utilization of cytoplasmic male-sterile inbreds in the production of corn hybrids. Agron. J . 44( I), 8-13. Schwartz, D. 1950. The analysis of a case of cross-sterility in maize. Proc. Natl. Acad. Sci. U.S. 26, 719-724. Shaver, D. L. 1956. Maize Genet. Coop. News Letter SO, 155-162. Stead, B. 1960. The gene action involved in the fertility restoration of Texas type cytoplasmic male sterile maize and its performance compared with normal cytoplasm. Ph. D. thesis, Univ. Wisconsin, Madison, Wisconsin. Stinson, K.T., Jr. 1960. Maize Genet. Coop. News Letter 34,15-18. Stinson, H. T., Jr. 1962. Unpublished data. Stringfield, G. H. 1958. Fertility restoration and yields in maize. Agron. J . 60,215-218. Thomas, W. I., and Johnson, I. J. 1956. Inheritance of pollen restoration and transmission of cytoplasmic sterility in popcorn. Agron. J . 48,472-474. Vinson, C. G. 1927. Some nitrogenous constituents of corn pollen. J . Agr. Res. 36, 261-279. Watson, G. S., and Caspari, E. 1960. The behavior of cytoplasmic pollen sterility in populations. Evolution 14, 56-63.

GENETIC VARIATION IN CHROMOSOME PAIRING Ralph Riley and C. N. Law Plant Breeding Institute, Cambridge, England

Page I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 11. Preliminary Consideration of Functions . . . . . . . . . . . . . . . . 59 111. Quantitative Variation in Pairing. . . . . . . . . . . . . . . . . . . 65 A. MajorGenes . . . . . . . . . . . . . . . . . . . . . . . . . . 65 B. Complex Systems of Pairing Control. . . . . . . . . . . . . . . . 69 C. The Relationship of the Specificity of Synapsis to the Over-All Regulation of the Extent of Pairing . . . . . . . . . . . . . . . . . . . 79 IV. Genetic Control of Pairing Specificity. . . . . . . . . . . . . . . . . 81 A.Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . 81 B. T r i l i c u m . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 C. Pairing Specificity in Other Allopolyploids . . . . . . . . . . . . . 97 D. The Imposition of Bivalent Formation in Species Other Than Allopolyploids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 E. Secondary Association of Bivalents. . . . . . . . . . . . . . . . . 103 V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

1. Introduction

Among the range of organisms and tissues whose nuclear cytology has been studied, several different modes of pairing between chromosomes occur. I n order to specify those phenomena with which we shall be concerned in the present account it is necessary at the outset to classify these different pairing behaviors. I n doing so, a first separation can be made into pairing that takes place between homologous partner chromosomes and into nonspecific pairing between chromosome regions that have no apparent similarities in their genetic activities. 1. Homologous pairing can be separated into four types defined as follows: (a) Meiotic synapsis-involving the intimate association of homologs, in the manner readily recognized during first prophase and leading to bivalent or multivalent formation. (b) Somatic pairing-involving the close juxtaposition and co-orientation of homologous chromosomes as seen, for example, during the prophase and metaphase of somatic division in Drosophila. 57

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(c) Salivary gland pairing-the extremely intimate association of homologous polytene chromosomes in the salivary glands and other tissues of species in the Diptera. This may be a special expression of the pairing seen in other somatic cells of these species. (d) Secondary association of bivalents-in some polyploid species, a t first metaphase of meiosis, bivalents are not randomly distributed but occur in pairs or groups (Darlington and Moffet, 1930; Lawrence, 1931). It has recently been demonstrated that such secondary association involves bivalents comprised of chromosomes that are related genetically and evolutionarily (Riley, 1960a; Keinpanna and Riley, 1964). 2. Nonspecific pairing can be separated into three types defined as follows: (a) Heterochromatic fusions-these are the associations that lead to the formation of chromocenters, for example in Drosophila, or to the random association of heterochromatjc regions in some plants (McLeish, 1953). (b) The terminal nonspecific association of univalent chromosomes at first metaphase of meiosis (Ribbands, 1937; Person, 1955; Riley and Chapman, 1957a), which Kostoff (1938) and Riley and Chapman (1957a) have also ascribed to heterochromatic fusion. (c) Nonspecific pairing a t prophase of meiosis-such as that reported to occur over small nonhomologous regions when homologous synapsis is interrupted by structural heterozygosity. Darlington (1937) considered this to be due to torsional effects extending from homologously into nonhomologously paired regions. This category also includes the nonhomologous pairing that takes place in some monoploid individuals when homologous partner chromosomes are absent (McClintock, 1933; Rieger, 1957; Kimber and Riley, 1963a). Heterochromatic fusion clearly presents problems of a different nature from all the others listed, and may reasonably be considered to involve events different in kind from these, so that it will be given no further consideration. Evidence on nonspecific pairing during meiosis is rather restricted and, although with more detailed study it may ultimately illuminate the normal processes of synapsis, little can be said about it at this time. Finally, it is apparent that, although all types of homologous pairing may depend upon similar causes, it is only as a consequence of synapsis at meiosis that the process can be described in any detail and variation due to genotypic differences established. We shall therefore be almost

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exclusively concerned with synapsis at meiosis and only passing references will be made t o somatic pairing and to the secondary association of bivalents. Alterations in the behavior of chromosomes a t meiosis are known to result from genetic variation as well as from differences in several environmental factors (for references see Rees, 1961). Indeed it has been argued that the operation of selection on genetically controlled variation in the chromosomal phenotype has been responsible for the development of meiotic mechanisms adapted to the requirements of the breeding structures of species and of their populations (Darlington, 1946; Lewis and John, 1963). The fertility of a n organism and consequently the fitness of the lineage t o which it belongs are intimately related, in sexual forms and in some apomicts, to the efficiency of the over-all meiotic process. The prime function, upon which the efficacy of the process depends, is the segregation of homologous chromosomes. Since the mechanics of this segregation require the initial association of homologs, in dealing with the genetic control of pairing we shall be concerned with those events upon which sexuality hinges. However, it is not only for its function in the mechanical segregation of homologous chromosomes that meiotic pairing is important, for it also provides the opportunities for recombination between homologs. Indeed, the positioning of the points at which recombination occurs, and the frequency of recombination events, are related to the course of the synaptic process. If, as many consider, patterns of recombination are adjusted to regulate the release or conservation of variation in a manner adapted to the demands of the genetic systems of species and of their populations, then one means by which this could be attained is through the selection of genotypes determining either the extent of synapsis or its localization. I n considering the genotypic control of meiotic chromosome pairing we shall therefore be considering processes of some significance in the balance of the over-all genetic system of all sexual species. II. Preliminary Consideration of Functions

Since our primary concern is with meiotic pairing it may be useful first to discuss one point of general importance, namely the timing of synapsis. It has for long been widely accepted by cytologists, on observational evidence from certain peculiarly amenable plant and insect species, that chromosomes enter the meiotic sequence in a single or unpaired state. Early in prophase, during the zygotene period, homologs are held to make contact with each other at one or more points from which synapsis extends into adjacent regions. Synapsis reaches its maximum, when it may or

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may not be complete, during the pachytene period, after which the homologs fall apart except where they are held by the chiasmata, thought to have been formed during their most intimate association. I n order to provide a model that would account for the occurrence of negative interference and for the high (frequency of recombination in trisomics jn Neurospora, and at the same time would be compatible with the then-popular copy-choice theory of recombination which required the association of homologs during deoxyribonucleic acid (DNA) replication, Pritchard (1960a,b) and Pontecorvo (1959) proposed that some form of synapsis takes place before zygotene, either at leptotene or in the preceding interphase. On this view prezygotene association, possibly b y chance, of effective pairing regions results in recombination, and meiotic pairing has no function in recombination but is merely responsible for the numerically equal division of the chromosome complement (Pontecorvo, 1959). However, there is some evidence against this theory of prezygotene synapsis. First, in individuals with homologous chromosomes of different sizes or morphology, bivalents are always formed between chromosomes of contrasted structure, and never between chromosomes each comprised of contrasted chromatids. Consequently, the recombinational events, whenever they occur, cannot be followed by the separation of the recombinant products. I n addition, although it must be admitted th a t the evidence is not entirely one-sided (Haga, 1944; Steinitz-Sears and Sears, 1953), this is generally consistent with the view that the formation of chiasmata can be equated with the occurrence of recombination (Brown and Zohary, 1955; Jain and Basak, 1963; other reFerences in these papers). Since recombination cannot be followed by separation, if it takes place in effectively paired segments during DNA replication, then homologs should be joined by chiasmata before zygotene. While it would be hard to prove that chiasmata are not present a t this time they have never been observed. Further, there is little evidence of the interlocking of bivalents that would be expected if chiasmata were formed when homologs were only locally paired and in a state of pronounced elongation. Other evidence counter to the view of prepachytene chiasma formation has been provided by Lawrence (1961a,b) who showed that radiation treatments, that are known to alter the frequencies of chiasmata, m-ere only effective if applied to cells close to pachytene. Finally, the mechanics of positive interference are hard to visualize on the basis of a prezygotene scheme. However, one of the pieces of evidence adduced in favor of its prezygotene occurrence, the higher recombination in trisomics, may perhaps be explicable in terms of reduced positive interference. I n view of these conditions, although we shall return to the problem in the discussion of certain work on

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asynaptic mutants, it seems reasonable to accept the classical view th a t synapsis is initiated at zygotene. Evidence on the functional basis of chromosome pairing at meiosisor indeed in somatic cells-is completely lacking. Indeed this is probably the only biological process, of universal occurrence in higher organisms, whose causal processes are completely unknown. This ignorance, which is due t o the experimental inaccessibility of meiosis, has led to theproposal of a number of ingenious hypotheses intended to explain the mechanism responsible for synaptic attractions. Before outlining these proposals, however, it is probably wise to indicate that a number of requirements must be met by any hypothesis, for although these conditions are widely appreciated they merit frequent reiteration and reconsideration. First, any postulated system must be capable of bringing together chromosomes that may initially be several microns apart. While this is probably the case in most organisms, White (1954) has suggested that the rarity of interlocking bivalents may indicate some presynaptic co-orientation of homologs-a condition which is almost certainly true for the Diptera in which homologous chromosomes apparently lie adjacent, and with similar linear orientation, prior to synapsis (for references see Smith, 1942). Nevertheless, in the majority of species the relative presynaptic orientation of homologs is apparently randoma condition which is self-evidently true in those with zygotic meiosis-so that pairing affinity is a long range phenomenon. Any physical force proposed would thus need to be effective from one side of the nucleus to the other, and a chemically mediated system, for example analogous to transfer ribonucleic acid (RNA), would have to be capable of selecting and associating two unique chromosomal sites over the same distance. This brings us to the second requirement, namely that the system must have the capacity to produce the locus-to-locus, or at least the chromomere-to-chromomere, specificity of synapsis that characterizes the normal meiotic process. I n general, in organisms in which homologous partners are present, pairing is highly specific. Exceptions t o this specificity of pairing occur where structural heterozygosity restricts the opportunity for the association of homologous regions and enables a form of nonspecific affinity to associate small, adjacent, nonhomologous segments (McClintock, 1933). I n addition, work on maize (McClintock, 1933) and Antirrhinum (Rieger, 1957) nionoploids and on Gossypium hybrids (Brown, 1959) suggests th at nonspecific pairing can also take place when each chromosome has no homologs. This may or may not be of the same nature as pairing between homologs, although it does not lead to chiasma formation except in rare instances which may be related t o the presence of duplicated segments (Alexander, 1964). However, this may

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merely be due to the absence of juxtaposed sites th a t react similarly to whatever is responsible for breakage in recombination-such as the hypothetic recombinases of current theories. I n general, nevertheless, the pairing phenomenon results in the synapsis of homologs only, and this is achieved, moreover, with remarkably detailed longitudinal specificity. This longitudinal congruence of homologs could of course be derived from a n affinity expressed between a relatively few pairing sites followed by the zipper-wise conipletion of synapsis in the hitherto unpaired adjoining segments, in the manner suggested by Darlington (1935). However, that this is always the sequence of events must be doubted, for a third requirement that any hypothetical mechanism must satisfy is that there must be numerous sites, each capable of independent synapsis with its homolog. The evidence from this comes from the completion of detailed patterns of pairing in individuals heterozygous for translocations or inversions or for intercalary deletions, and from the complex multivalents formed in some species by polyploid or polysomic individuals. White (1954), in considering this behavior, visualized not merely a general attraction between homologous chromosomes, but many thousands of different forces of attraction, presumably as many as there are pairs of genes. If this view were expressed in chemical terms it would be necessary to imagine that each pairing site has its individual synapsis-mediating substance. The longitudinal specificity of pairing, which is unimpeded by the rearrangement or deletion of segments, presents a major obstacle to the construction of a plausible model to explain the process. The final requirement for any suggested mechanism is that the system concerned with synapsis in any region must be saturated when the twoby-two association of honiologs has been completed. This is demanded by the behavior of polyploid and polysomic individuals in which-for any region-more than two chromosomes never synapse. There are some forms of pairing in the explanation of which one or the other of these requirements may have to be varied to meet the particular sequence of events observed. For example in the touch-and-go pairing observed a t meiosis in certain species of the Heteroptera and Homoptera (Wilson, 1925; Schrader, 1940) a multiplicity of pairing sites need not be visualized, but the system required to cause the local contacts need not differ from that responsible for the more usual type of synapsis-although it should be added that in White’s (1954) view, pairing is quite normal in these organisms, the differences arising merely because it relapses earlier than usual. Another discrepancy occurs in the pairing of chroniosomes in the salivary glands of Drosophila. No direct coniparison has been possible,

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but pairing in salivary glands is usually assumed to resemble that resulting from meiotic synapsis, although-because of the greater time involved-it may be more complete and intimate. However, there is one significant distinction from every known meiosis, namely that when salivary gland chromosomes are represented more than twice all the homologs pair in a multiple manner throughout their lengths, instead of in two-by-two configurations. Cooper (1938) has ascribed this to the multiplicity of pairing faces exposed by polytene chromosomes. Despite the exceptions in some fungi, in which contracted chromosomes pair (McClintock, 1945; Olive, 1949; Singleton, 1953), many authors have pointed out that generally chromosomes that undergo synapsis are in a state of greater longitudinal extension than at any other time during cell division, with no spiral structure apparent. Indeed Manton (1939) observed that in Osmunda regalis the chromosomes at leptotene were of the order of 50% longer than a t any stage of mitosis. It may be, consequently, that it is only when in this relaxed condition that the detailed alignments necessary for synapsis become possible. If this is so the problem is further compounded for we must then ask not only how chromosomes pair but also how their transient nonspiralized condition is attained during division. Among the hypotheses that have been advanced to account for chromosome synapsis the one that has probably received the most attention was elaborated in its greatest detail by Faberg6 (1942), following an original suggestion by Lamb (1907) and a rather similar proposal by Muller (1941). Faberg6 suggested the possibility that chromosomes were attracted by long-range pairing forces derived from Guyot-Bjerknes effects and that this was followed by events having a different cause that led to detailed synapsis. The Guyot-Bjerknes effect depends upon the physical principle that when diff erent molecular bodies pulsate in phase, in a liquid medium, they are attracted together. Bodies with different vibrational frequencies are unaffected by each other. Faberg6’s notion was that each chromosomal pairing site has a distinctive vibrational frequency, and that homologous sites have the same frequency and are in phase with each other, so that they are caused to approach each other. While this hypothesis is conceptually pleasing it leaves several unanswered questions, such as how pairing is saturated by two-by-two synapsis, although the postulate that detailed synapsis results from a separate phenomenon evades this difficulty. Another problem is the method by which the forces can be switched on and off at the beginning and end of the period of attraction. Another hypothesis is that upon which Darlington’s (1937) precocity theory of meiosis depends, namely that the chromosomes that enter

64

RALPH RILEY AND C. N. LAW

meiosis, because of their single-stranded, unreplicated structure are in an electrostatically unsatisfied state. This state is normally satisfied in somatic cells by the association of sister chromatids. However, at the prophase of meiosis homologs must pair to produce the satisfied condition until the separation into chromatids takes place at pachytene. Objections to this notion have been based on observations that leptotene chromosomes may be detectably double-stranded (Swanson, 1957). Others have proposed that dipole moments, or that attractions resulting from the summed effects of van der Waals’ forces created along homologous pairing segments, may be responsible for synapsis (Rhoades, 1961). Alternative hypotheses have utilized ideas based on relationships between homologous regions like those responsible for immunological specificity (Hinton, 1945), although the difficulty must be recognized that pairing segments are structurally similar, unlike antibodies and their specific antigens. Delbriick (1941) considered that chromosomes might be brought together by chance contacts and synapsis could then result from the reduction of peptide bonds and the formation of resonance bonds between pairing peptides. A change in the redox status of the system could then cause the relapse of resonance bond pairing and be accompanied by the synthesis of new polypeptides along each chromosome. Delbriick’s hypothesis predates the recognition of the significance of DNA in inheritance and, as Rhoades (1961) has indicated, any hypothesis giving no consideration to the role of DNA should now be treated with caution. Indeed, this idea that synapsis might be associated with replication is not consistent with the timing of DNA replication (Moses and Taylor, 1955; Swift, 1953; De, 1961) and with the concomitant replication of histone (Bloch and Goodman, 1955; Gall, 1959; Ansley, 1957). The one feature that all the theories about the causes of synapsis, discussed above, have in common, is that they are untested and indeed are scientifically untestable at the present time. While the elegance of the conceptual design often employed in their formulation must be admired there is no doubt that a direct experimental approach would be more valuable were it possible. For this the demand is for biological material which shows differing synaptic behaviors. Apparently the only attempt to analyze material of this type so far is that of Ansley (1957). I n this work cytophotometric comparisons were made between cells of the harlequin lobe of the testis of Loxa jlavicolis, in which there is no pairing a t prophase of meiosis, and cells of other parts of the testis in which pairing is normal. Ansley was able to show that, so far as could be shown by specific staining, the histone :DNA ratio was 1 :1 in synaptic cells, but was 3: 2 in the asynaptic harlequin cells. These observations paralleled earlier results from asynaptic cells of the house centipede, Scutigera

GENETIC VARIATION I N CHROMOSOME PAIRING

65

forceps (Ansley, 1954). Subsequently, Ansley (1958) was able to show that the histone:DNA ratio in the harlequin cells of Loxa was similar to that in somatic cells and that there was a greater proportion of lysine to arginine, available to bind stain, in cells with normal synapsis compared with asynaptic cells. Ansley considered that these differences in the relative amounts of DNA and histone, and in the apparent composition of the histone, were associated with the different patterns of pairing. However, the results need not imply more than th a t there are changes in the association of histone with other proteins or with DNA, or changes in the responses of histone to the particular fixation procedures employed. Nevertheless, it is apparent th at in this work the first biochemical distinction associated with chromosome pairing has been revealed. Clearly it is along these lines that the functional analysis of the pairing phenomenon should be pursued, but material offering the advantages of Loxa flavicolis is extremely rare. Alternative sources of experimental material may, however, be seen in the comparatively numerous examples of genetic variation in meiotic pairing. I n order t o draw attention to these, and t o indicate their potential value, we have in the following account attempted to review the presently available evidence on the genetic control of chromosome pairing. I n so doing we have considered formal genetic positions, and made whatever assessments of functions the evidence allows, in addition t o discussing those evolutionary considerations that permit a functional interpretation. 111. Quantitative Variation i n Pairing

A. MAJORGENES Genetic variation in the extent of pairing of homologous chromosomes, during the prophase of meiosis, is common. Major genes have been described, in a range of organisms, that disrupt the association of homologs prior to first metaphase, and in all instances the reduced pairing th a t results at first metaphase has been ascribed to the homozygosity of recessive alleles (Table 1). Duplicate loci, th at give rise to reduced pairing only in individuals homozygous for both recessive alleles, have also been reported in a cross between two tetraploid species of Gossypium (Menzel and Brown, 1955). Attempts have been made to classify major genic variation in meiotic pairing into two categories. These depend upon the assumption th a t a meaningful distinction can be made between the complete failure of chromosomes to synapse and the precocious separation of chromosomes between which synapsis has taken place. Genes th a t influence the initial contact of chromosomes are referred to as asynaptic, those th a t influence

66

RALPH RILEY AND C. N . LAW

TABLE 1 Genetic Situations Affecting Synapsis in Which Major Genes May Be Responsible Organism

Description

Gene symbol

Source

Pachytene pairing absent in Huskins and Hearne certain nullisomic plants (1933) Drosophila Recessive gene causing failure Gowen (1933) of chromosome pairing melanogaster Low chiasma frequency Gossypium spp. al and a2 Meneel and Brown ascribed to interaction of two hybrids (1955) recessive genes Hordeum vulgare Complete lack of chromosome Person (1959) pairing Pachytene pairing, b u t univads Enns and Larter lents formed a t metaphase I. (1960) Recessive gene Locusta High univalent frequency at Rees (1957) migratoria first metaphase Five mutant genes which cause asl to asp Soost (1950) L ycopersicon esculentum variable pachytene pairing. All nonallelic and recessive Nicotiana spp. Spontaneous mutation. Failure Swaminathan and hybrids of pachytene pairing for Murty (1959) some chromosomes Pachytene pairing, but univaOryza sativa ds Chao et al. (1960) lents frequent. Recessive gene Reduction in pachytene pairing John and Naylor Schistocerca gregaria (1961) Secale cereale Pachytene pairing, but a varias Prakken (1943) able number of univalents formed. Recessive gene Triticum aestivum Pachytene pairing with low ds Li et al. (1945) chiasma frequency at metaphase. Recessive gene. Deficiency of chromosome 3B Sears (1954) (Table 3) Trifolium Complete failure of chromoWhittington (1958) some pairing Zea maus Pachytene pairing, but with inas Beadle (1930, 1933) creased univalence. Recessive gene Variable pairing-from comMiller (1963) as plete failure to partial pairing

Avena sativa

GENETIC VARIATION IN CHROMOSOME PAIRING

67

the maintenance of pairing between synapsed chromosomes as desynaptic. The distinction between them is often hard to draw in practice, because early prophase stages at meiosis are often indistinct and unresolvable. Asynaptic behavior is consequently difficult to establish, since it is the absence of chromosome pairing before and during early pachytene which enables this condition to be recognized. Some examples of chromosome behavior have, however, been reported in which asynapsis was held to be responsible for the presence of univalents a t first metaphase. An instance of this behavior is provided by the meiosis of the asynaptic mutant as3 of Sorghum described by Ross and associates (1960). These authors claimed that the occurrence of unpaired strands a t zygotene and pachytene, as well as the separation of homologs by other chromosomes a t these stages, provided evidence showing that abnormalities at the time of synapsis were responsible for the univalents seen a t first metaphase. Swaminathan and Murty (1959) similarly found that there was no pairing a t pachytene in two plants of Nicotiuna in which there were no bivalents at diakinesis. The most fully described case of asynapsis determined by a major gene occurred in Zea mays and was ascribed to homozygosity for the recessive allele, as. This condition was initially reported as asynapsis by Beadle (1930) because as homozygotes showed very little pairing throughout all the prophase stages. At first metaphase only univalents were observed in some cells. This description clearly conforms to the condition of asynapsis, in which the chromosomes fail to pair throughout the normal period of synapsis. I n later studies, however, a more variable behavior was observed to result from the homozygosity of the recessive allele. Thus, Beadle (1933, p. 283) described a partial but not complete failure of first metaphase association as, “not due to a failure of initial synapsis but to an abnormal early separation of synapsed homologues.” I n other words, the gene behaved as a desynaptic. More recent observations (Miller, 1963) have also confirnied the variable response found in a s homozygotes of Zeu. Thus, a complete failure of pachytene pairing occurred in some plants, whereas in others pairing was only partially completed. Significantly, however, in those plants with incomplete pairing, the amount of synapsis at early prophase was correlated with that occurring at late prophase. In this case, therefore, it was argued, the reduced pairing at pachyteiie seemed to result from a lack of pairing a t an earlier stage, rather than from the subsequent separation of already synapsed regions. The production of univalents was consequently due to asynapsis. These apparent reversals in classification illuminate the difficulties of recognition very clearly. Moreover, the extreme difficulties of interpreta-

68

RALPH RILEY AND C. N. LAW

tion when only a partial as opposed to a complete failure of pairing is found, is demonstrated by the asynaptic and desynaptic classifications obtained for the same gene by the two different workers. When only partial pairing occurs, then a distinction as to whether an unpaired region has been in this condition throughout meiosis or has undergone desynapsis after initially pairing must, indeed, be extremely difficult. The recognition of desynapsis, because the later stages of prophase are more readily analyzed, is often easier than the recognition of the alternative condition of asynapsis. For this reason, it is perhaps not surprising that most of the genes affecting chromosome pairing are described as desynaptic. Thus, clear descriptions of desynaptic behavior have been reported for a number of plants. For example Li et al. (1945)examined the effects of a recessive gene, ds, in Triticum aestivum in which homozygous recessive individuals were indistinguishable from normal plants during early prophase. During pachytene, however, a variable response was found. In some homozygous recessive plants a close association of chromosomes was maintained throughout pachytene until diplotene, while in others, chromosomes fell apart a t early pachytene. A similar, clear occurrence of desynapsis has also been observed in Hordeum vulgare ( E m s and Larter, 1960). Here, the variable response would appear t o be absent, since complete chromosome association was found in all cases up to diplotene. The synapsed chromosomes then separated and there were no bivalents at all in many cells. Again a recessive gene, ds, was found t o be responsible. Further examples of desynapsis are given in Table 1 which shows that although the gene symbol may represent asynapsis, in a number of cases the descriptions are essentially associated with desynapsis. In contrast to asynaptic behavior, complications arise when the possible causes of desynapsis are considered. For asynapsis the decision is unequivocal and only one cause is possible, namely, a direct failure of chromosomes to pair. Desynapsis may, however, arise from a number of alternative causes. For example, it has often been suggested that a cause of desynapsis is the failure of chiasma formation and that, where the effect is genetic, the occurrence of univalents at first metaphase results from the activities of genes that directly control the frequencies or positions of chiasmata. However, the evidence that desynapsis is due entirely to variation in the regulation of chiasma formation is somewhat equivocal. An alternative cause could be the relaxation of pairing between normally synapsed chromosomes before crossing-over takes place. Indeed, a distinction between these two causes is possible only if the stages which precede the crossover condition are recognizable. Given this, then a distinction between pairing failure which occurs before crossing-over and

GENETIC VARIATION I N CHROMOSOME PAIRING

69

a failure of crossing-over which precedes a relaxation in chromosonie pairing may be possible. On the other hand, it is more than probable that the pre-crossover condition can only develop when normal synapsis takes place. If this is so, a true separation of these two causes may well be indeterminate and the failure of chiasmata mean no more than the failure of chromosonie pairing. A further possible cause of desynapsis must also be mentioned; this depends upon the notion, that has been useful in the past to both geneticists and cytogeneticists, of effective and ineffective pairing. This is based on the assumption that the pairing of chromosomes may appear normal when in fact important departures from normality are present. Under these circumstances the necessary conditions for crossing-over may be absent. The “torsional” pairing proposed by McClintock (1933) may be cited as an example of this kind of pairing. The relevance of these notions to the evaluation of desynaptic behavior cannot be overlooked. In this situation, it is indeed the inability to form chiasmata that gives rise to univalents a t first metaphase. However, the absence, or reduced frequency, of chiasmata arises not from a direct variation in their genetic control but froin modifications in the nature of pairing that indirectly influences chiasina formation. The accounts of asynaptic and desynaptic genes have shown that difficulties arise in clearly distinguishing one from the other in practice. Functional distinctions would appear also very difficult to establish. Asynapsis can only result from a failure of chromosome pairing. Desynapsis, it has been proposed, may have a number of causes some of which, in the final analysis, also arise from initial abnormalities in synapsis. Suggestions, therefore, that clear-cut functional distinctions underlie the separation may mislead rather than inform. Indeed the difficulties of cytological recognition and explanation suggest the desirability of discarding the use of the terms “asynaptic” and “desynaptic.” The alternative term “synaptic” could be used to describe the activities of major genes that influence the extent of meiotic pairing until more exact methods of classification become available.

B. COMPLEX SYSTEMSOF PAIRING CONTROL The study of the relatively simple genetic situations found in individuals carrying mutants of major genes affecting synapsis has shown that the most accessible measure of pairing failure can be obtained from the study of either chiasma or univalent frequencies. The interpretation of this information in terms of pairing only is, of course, dependent on how important are the genes that directly control crossing-over compared with those that are responsible for pairing. It appears, from the study of

70

RALPH RILEY AND C . N. LAW

the synaptic genes, t h a t the largest component of the observed variation results from modification in chromosome pairing. Given that this is SO, the quantitative genetics of chromosome pairing can be studied using the frequency of either chiasmata or of univalents as the appropriate metrics. Moreover, such studies, it may be presumed, provide a greater insight into the detailed genetic control of the character, than can be obtained from the consideration of major genes alone. The effects on gametic viability of the mutant forms of major genes are usually so profound that such variants can rarely have played a significant role under natural conditions. It is from the consideration of genes with influences that are not so drastic, and that are concerned with minor variations around the norm, that a detailed picture of the genetic determination of the character can be delineated. This is not, of course, to deny the significance of the normal alleles of major synaptic genes in the balanced regulation of pairing nor to underestimate the potential value of their mutants in the physiological or biochemical investigation of the synaptic process. The genetic alteration of chiasma frequency has been a n object of study for some time. The most striking and informative studies have been concerned with the sudden disruption of established mating groups and with the influence of this on meiotic chromosome behavior. Such disruptions arise whenever a sudden expansion or contraction in the genetic population occurs. Experimentally these conditions have been brought about either by within-species crosses, that go counter to the normal breeding behavior, or by interspecific crosses. To demonstrate the former, two examples, one from a n outbreeding organism, Secale cereale (2n = 14), and the other from an inbreeding organism, Triticum aestivum (2n = 42), will be described. The disruption of the normal outbreeding habit of Secale cereale by inbreeding, to create a sudden restriction in size of the mating group, has drastic effects on chiasma frequency. Lamm (1936) and Muntzing and Akdik (1948) first showed that a decline in chiasma frequency resulted from inbreeding. More recently, however, Rees (1955a, b), and Rees and Thompson (1956) have investigated the genetic system responsible for the maintenance of the higher chiasma frequencies found in outbred populations of S. cereale. The inbred lines used in this study, without exception, exhibited a reduction in chiasma frequency compared with the level in the original population. On intercrossing these lines, however, the chiasma frequencies of the F1generation were uniformly greater than those of either of the participating parents (Fig. 1). I n other words, a positive heterosis was produced, immediately outcrossing was undertaken, which tended to re-establish the high chiasma frequencies prevalent

GENETIC VARIATION I N CHROMOSOME PAIRING

71

in normal populations. Thus a return to the normal outbred genetic condition had immediate effects on the control of chiasma frequency. A diallel crossing program, carried out among the inbred lines, permitted a n investigation of the genetic system behind the positive heterotic effect. Provided th at certain biological and biometrical prerequisites are satisfied, the results obtained from such diallel crosses can be analyzed by Inbred lines P3

R : 1.854

A v : 0 . 0 0 0 8

P6

E: 1.710

V:0.0048

PI2

R : 1.701

P 13

I: 1.725

4

V:0.0042 V: 0.0167

Fl

3 x 6

1:2.033

v : 0.0001

3 x 12

E:2.072

v:0.0012

3 x 13

R:2.086

v:0.0001

6 x 12

I:2.044

V:0.0007

6 x I3

R:2.036

V:0.0006

12 x 13

I:2.069

V:0.0005

f :,

1.5

1.6

1.7

1.8

Mean XA.

I .

1.9 2.0

2.1

frequency

FIG.1. The distribution of chiasma frequencies in inbred lines of Secale cereale and hybrids between them (Rees and Thompson, 1956).

the covariance-variance method of Jinks (1954). This allows the detection of nonadditive gene action whether due to dominance or nonallelic interaction. By this technique the high chiasma frequencies in hybrids between inbred lines of S. cereale were shown to result from nonadditive gene effects that were, in part, of a nonallelic nature. I n addition, the variation between the parental inbred lines was SO large that it was reasoned th at the cause of inbreeding depression was

72

RALPH RILEY AND C. N. LAW

unlikely to be homozygosity alone, since all the lines were equally inbred and, therefore, presumably similarly homozygous. Consequently, the depression in chiasma frequency was presumed to depend upon the segregation of particular homozygous combinations rather than upon some special demerit of homozygotes. The recent discovery of genetic heterozygosity in similar inbred lines of S. cereale (Muntzing, 1963) may indicate, however, that this argument may not be wholly valid since differential selection for heterozygosity may have occurred between lines. It is clear, however, from these studies, that the genetic system, revealed in S. cereale, is one which is conditioned to operate most efficiently when heterozygous combinations of genes are frequent. Furthermore, because nonallelic interactions are important in the control of chiasma formation, the occurrence of high frequencies of chiasmata cannot be simply a result of the presence of heterozygous loci. Instead it must derive from the operation and interdependence of many loci. It is convenient to refer to such an adjusted system in terms of a genetic balance. Similar effects on chiasma frequency, resulting from inbreeding a normally outbreeding species, have also been reported in Zea mays by Blanco (1948) and ZeEevic (1961). Consequently, the operation of polygenic systems, with considerable amounts of nonallelic interaction, seems likely to be associated with major gene effects in the regulation of chromosome pairing in many species. An analogous effect to that obtaining in S. cereale is found when the normally inbreeding hexaploid wheat, Triticum aesiivum, is outcrossed. Univalents are often found a t first nietaphase in the pollen mother cells of varietal lines of T . aestivum, but their frequency is usually low (Riley and Kimber, 1961). However, in F1 hybrids between varieties, the frequencies of univalents are higher than in either parent (Thompson and Robertson, 1930). Again heterosis occurs just as in S. cereale, but it is a negative heterosis so far as chromosome pairing is concerned. Thus, an expansion of the normal mating group of T. aestivum produces the same effect as a contraction in S. cereale. Each species demonstrates, therefore, that a departure from the normal breeding behavior results in the breakdown of a balanced genetic regulation of meiotic pairing. The similarities do not end there, however, for while in varietal lines of T . aestivum the percentage of cells with univalents is very small, there is some evidence that varieties differ in the frequency of meiotic pairing failure. Thus, in five wheat varieties examined by Hollingshead (1932) the percentage of cells with univalents ranged from 2.9 t o 9.7, and there was a significant heterogeneity chi-square for differences between varieties. More recent studies have shown a similar variation among varieties (Riley and Kimber, 1961 ; Watanabe, 1962). This suggests that the con-

GENETIC VARIATION I N CHROMOSOME PAIRING

73

trol of normal levels of chromosome pairing is a function of particular genotypes rather than simply the product of homozygosity, since most wheat varieties can be presumed to be siniilarly homozygous, although this argument again is not immune from the criticism that selection for different levels of heterozygosity may have resulted in different degrees of homozygosity in separate varieties. The emphasis on the precise genotype in T . aestivum, rather than on the degree of homozygosity or heterozygosity, is analogous to the genetic system postulated for the control of chiasma frequency in S. cereale. These genetic systems have been imposed upon the species both as a result of particular breeding habits and in response to past and present adaptive requirements. I n T . aestivum, obligatory inbreeding has ensured that homozygous rather than heterozygous combinations of genes are more frequently exposed to selection. Homozygotes are consequently better adapted and present a better genetic balance than the individuals with heterozygous complexes. Outbreeders such as S. cereale, however, have the reverse situation, and it is heterozygous combinations that display better balance. The maintenance of pairing a t the optimal adaptive level by genetic balance, in T . aestivum, rather than by some intrinsic merit of homozygosity over heterozygosity, may be illustrated further by consideration of two other pieces of evidence. The first concerns the results of an intervarietal-backcross program in which univalent frequency, as a measure of pairing failure, was examined and recorded over a number of generations (Person, 1956). There were high univalent frequencies in the hybrid and first backcross generations, but after this the return to more normal pairing was very gradual and approximately linear (Fig. 2). Of interest, however, is the fact that some discrepancy occurred between the observed return to normal meiotic behavior and the expected return if homozygosity alone had been responsible for stable pairing. The difference again suggests that meiotic stability is not a direct function of homozygosity. A stronger inference than this cannot be drawn since there was no confirmation that the return to homozygosity was at the expected theoretical rate. Although in the absence of selection such an assumption would seem reasonable, there is some evidence that heterozygosity can be maintained in highly inbred material (Jain and Allard, 1960; Jain and Jain, 1962; Allard and Jain, 1962; Muntzing, 1963) so that the true weight of this evidence cannot be accurately assessed. More exact information is, however, obtainable from a second piece of evidence. This concerns the results obtained from a set of diallel crosses between eight varieties of T . aestivum (Watanabe, 1962) (Table 2). Once again, consistent negative heterosis for chromosome pairing was displayed

74

RALPH RILEY AND C. N. LAW

by the hybrids. The diallel nature of the crossing program, however, enables the genetics of the situation to be examined more precisely by means of the covariance-variance type of analysis already used successfully in S. cereale (Rees and Thompson, 1956). The analysis of Watanabe’s data by this method demonstrates that the lower chiasma frequencies of the hybrids were largely due to nonallelic interactions. Furthermore, to account for the negative heterosis, the interactions must on average have been negative also. Nonallelic interactions, therefore, play an important role in the control of chiasma frequency and chromosome pairing in T. aestivurn, just

F,

I

2

3

4

5

6

7

8

9

10

Bockcross generation

FIQ.2. The percentage of first metaphase cells with every chromosome paired in bivalents in the F1and backcross generations derived from crosses between varieties of Triticum aestivum. Solid circles the observed values, open circles the theoretical return to homozygosity, dashed line the percentage of cells with every chromosome in bivalents in the parental varieties (Person, 1956).

as in S. cereale. Apparently the interaction of many genes-genes which may be said to form balanced complexes-is responsible for the high chiasma frequencies observed in normal populations of these two species. Disturbance of this balance, by sudden alterations in the size of the mating group, breaks down the genetic systems concerned, so that negative interactions, counter to the positive interactions brought about by selection, are exposed. In T. aestivum diallel crossing between varieties brought about such a breakdown and enabled negative nonallelic interaction to be recognized. In S. cereale, diallel crossing between unbalanced inbred lines reinstated the normal breeding habit and genotypic balance, so that positive interactions appeared. The occurrence of positive nonallelic interactions between genes that control chiasma frequency and chromosome pairing has also been demon-

0

M

2

TABLE 2 Mean Percentages of Cells with Univalents at First Metaphase of Meiosis in Eight Varieties of Triticum aestivum and Hybrids between Them *

2

d

4

P

Variety

Kanred

Fulty No. 1

NanbuKomugi

AobaKomugi

K6nosu No. 25

Norin No. 3 Sapporo-Harukomugi No. 1 Saitama No. 27 K6nosu No. 25 Aoba-Komugi Nanbu-Komugi Fulty No. 1 Kanred

21.56 4.40 28.40 37.20 40.40 20.80 21.50 0.80

16.65 19.76 17.00 32.67 34.67 21.30 3.60 -

32.80 23.12 17.28 72.80 34.40 2.00

40.80 52.00 46.80 50.00 4.80 -

38.00 33.20 36.91 2.80 -

* From Watanabe (1962).

-

-

-

-

SapporoSaitama Harukomugi No. 27 No. 1 13.00 38.40 4.80 -

25.03

Norin No. 3 11.25

5.60 -

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

E? 2

’ 8 2 d

m

T1z

8 Bw

2 E? z

rs

76

RALPH RILEY AND C. N . LAW

strated in T . aestivum by another method (Kempanna, 1963). Use was made of nullisomic individuals in which, in turn, each of the 21 pairs of chromosomes of the normal complement was separately deficient. Such nullisomic series enable the effect of each chromosome on any phenotypic character to be determined. Furthermore, if certain assumptions are made, particularly that the effects of all the pairs are additive, then the summation of all the differences of each nullisomic from the euploid value should be equal to the euploid value. By contrast, if the assumption of additivity is false, the calculated euploid value will depart from the score observed in the euploid. The extent and direction of the departure will then provide some measure of the average between-chromosome interaction and indicate whether the interactive effect is positive or negative. This technique was applied by Kempanna to the study of the number of chiasmata produced per bivalent in T. aestivum and the values obtained are shown in Table 3. Nullisomics for five different chromosomes were shown to give chiasma frequencies that were lower than that of the euploid to a statistically significant degree, and the test was probably too insensitive to reveal the participation in the control of the character of other chromosomes giving real though less pronounced effects. Apparently therefore there is a multiple-site regulation of chiasma formation in this species like that in S. cereale. The sum of the differences of the observed chiasma frequency per bivalent in euploids, less the score for each nullisomic separately, produced a value that was more than twice the observed euploid frequency. Consequently, between-chromosome interactions must affect this character, and, as the calculated is greater than the observed frequency, the higher levels of chiasma formation in the euploid must result from interactions that are positive on average. Further, the interactions must be nonallelic since the analysis only detects those that occur between chromosomes. Consequently, the analysis of chiasma frequencies in nullisomics amply confirmed the conclusions drawn from the occurrence of univalents in intervarietal hybrids in T . aestivum. Similarly, complex interactive genetic systems are therefore involved in the regulation of chromosome pairing and chiasma frequency in both 8.cereale and T . aestivum. This similarity may stem from the close phylogenetic relationships of the two species, which intercross freely, although several thousands of generations may be presumed to have elapsed since their divergence from a common ancestor. Nevertheless there are important distinctions between the general genetic systems of these species that are associated with their different breeding behaviors. I n the inbreeder, T . aestivum, nonallelic interactions must be based on loci that are largely homoaygous, so that they are of the homozygous x homozy-

GENETIC VARIATION I N CHROMOSOME PAIRING

77

TABLE 3 Mean Chiasma Frequencies per Bivalent in Euploid Forms of Triticum aestivum and in the Nullisomics Deficient for Each Chromosome Pair in Turn* Chromosome deficient 1A 1B ID 2A 2B 2D 3A 3B 3D

4A 4B 4D 5A 5B 5D 6A 6B 6D 7A 7B 7D

Chiasma/bivalent

Difference (euploid-nullisomic)

1.9818 1.9865 1.8250 2 ,0065 1,8500 1,9200 1.6115 1.2875 1.7428 2 ,0085 1.8400 1,9865 1.9615 1,8825 1.8015 1.9300 2.0215 1,9750 2.0215 1.9668 1.9765

0.1027 0.0980 0.2595 0.0760 0.2345 0.1645 0.4730 0.7970 0.3417 0.0760 0.2445 0.1160 0.1230 0.2020 0.2830 0.1545 0.0630 0.1095 0.0595 0.0877 0.1080

2.0845 -

4.17321

~~

Euploid Total

* From

Kempanna (1963). t o n the assumption of additivity of gene action this value should equal the observed euploid value.

gous type (Hayman and Mather, 1955). By contrast, in the outbreeder, S. cereale, heterozygous loci are likely to be common, so that heterozygous X heterozygous interaction must be frequent. It is the collapse of the balanced interactions of both types that results in reduced chromosome pairing when the breeding system of either species is reversed. It is of interest to determine the outcome of the combination of these two systems that have evolved under different selection pressures t o give distinctive forms of genetic balance. This has been achieved by the production of the synthetic allo-octoploid, ( T . aestivum X S. cereale) (2n = 56), the so-called Triticale, in which it has been widely reported univalents are common (Muntzing, 1939; O’Mara, 1953; Riley and Chapman, 1957b). By the method of its production both the Triticum

78

RALPH RILEY AND C. N. LAW

and Secale components of the Triticale genotype must be initially homozygous, so that pairing failure at meiosis could be due to the effects of inbreeding depression on the Secale component. However, when heterozygosity was introduced into the Secale Component by intercrossing between Triticale lines, the heterozygous F1 hybrids were still unstable; indeed some displayed greater pairing failure than their parents (Muntzing, 1939). The occurrence of bivalent failure in Triticale forms is therefore not apparently due to the breakdown of the genetic system of the Secale or Triticum component of the genotype alone, but more probably derives from the interaction of two mutually unadjusted systems for the control of pairing. This is confirmed by the influence displayed by individual S. cereale chromosomes added to the normal homozygous chromosome complement of T . aestivum. Riley (1960b) demonstrated that the second and third chromosome of S. cereale increased the frequency of univalents in the T . aestivum complement to four times the number occurring in plants with the normal chromosome complement. Single chromosomes of S. cereale can, therefore, influence the genetic system regulating meiotic pairing in T . aestivum. The converse effect of the T . aestivum genotype on the pairing of S. cereale chromosomes has also been recognized in material in which single pairs of S. cereale chromosomes were added to the normal homozygous complement of T . aestivum. The S. cereale honiologs failed to form a bivalent in a large number of cells (Riley, 1960b), thus the genetic system of T. aestivum interfered with their pairing. It is likely that such influences also operate when the full complements of both species are combined. The combination of the T . aestivum and S. cereale genotypes thus causes the breakdown of the balanced genetic regulation of chromosome pairing. This result is not unexpected in view of the distinctive genetic architectures of the character in these species. The homozygous genetic balance found in T. aestivum cannot accommodate the adjustment to extreme heterozygosity of the S. cereale genotype, for reasons that are similar t o those that disturb its integration in intervarietal hybrids. Nor can the heterozygous balance of S. cereale be maintained in combination with the totally different balance of T . aestivum. The conclusion to be drawn from this treatment of the genetic control of the extent of meiotic chromosome pairing can be simply stated. It is apparent that the amount of pairing is regulated by the integrated operation of numerous genes, each producing a n individually small effect, and of major genes with more profound manifestations. Moreover there are pronounced nonallelic interactions between the separate components of

GENETIC VARIATION IN CHROMOSOME PAIRING

79

the system and indeed it is upon such interactions that the balanced adjustment of the character depends to a considerable degree. Thus the regulation of the amount of meiotic pairing is exercised by genetic systems closely similar in construction to those functioning in the control of many more obvious aspects of the phenotype. It is impossible to ascertain from the present information what parts of the genetic systems concerned with the amount of pairing between chromosomes are directly involved in the processes th a t lead to synapsis. For it is likely that some facets of the system are merely indirect participants, being primarily involved in other metabolic processes peripheral to the course of pairing. I n addition the genetic evidence presently available provides no grounds for a choice among the range of hypotheses th a t have been advanced to explain the synaptic phenomenon. Nevertheless, the wealth of genetic material that can be provided for causal analyses is apparent. By the use of this it should ultimately be possible to determine in biochemical terms a number of stages in the synthetic sequence, or sequences, t ha t mediate synapsis.

C. THERELATIONSHIP OF THE SPECIFICITY OF SYNAPSIS TO THE OVER-ALLREGULATION OF THE EXTENT OF PAIRING As Darlington (1932) pointed out, the behavior of chromosomes is subject to two modes of control in any cellular system. The first of these depends upon the internal organization of the chromosomes which, although it is potentially permanent, can be changed by mutation. Second, the behavior depends upon the operation of those genetic systems that, through the intervention of the products of gene activity, modify the purely autonomous determination of chromosomal characters. The ultimate expression of any chromosoinal character is thus the product of a dual control and of the interaction of the operative systems. It is reasonable t o suppose that the expression of meiotic chromosome pairing is unexceptional in also being determined both by the relative internal structures of the potential pairing partners and by the genotype. Hitherto, during consideration of the regulation of the extent of meiotic pairing, attention has been confined t o the influence of the genotype. Indeed, some emphasis has been placed upon the absence of evidence, from within species, that the homozygosity-heterozygosity levels of homologous chroniosonies influence the probability of their pairing. This is not to imply th at homologs cannot attain a degree of distinctiveness a t which pairing is impeded, even in the absence of heterozygosity, for structural differences. However, consideration of this possibility is difficult because it is concerned with that ambiguous extent of evolution-

80

RALPH RILEY AND C. N. LAW

ary divergence between homologs at which genic and chromosomestructural differences merge. Nevertheless, it is apparent that much pairing failure between chromosomes with no obvious structural differences, in hybrids between evolutionarily divergent species (Sax, 1935 ; Stebbins, 1945, 1950), may result simply from the occurrence of differences in gene content-that is primarily from differences in nucleotide sequences. That pairing failure in such interspecific hybrids need not result from an imbalance in genotypic control has frequently been demonstrated by the resumption of normal pairing in the synthetic amphiploids derived from them. An example of this situation can be taken from the work of Sears (1941) who produced a range of hybrids between diploid species in the Aegilops-Triticum group. Certain hybrids had some meiotic cells with pronounced pairing failure, while in other cells pairing was almost normal, so that homologs were still capable of pairing but did so with reduced frequency. There was no evidence that the principal cause of pairing failure was structural heterozygosity. In addition pairing was almost regular in the amphiploids derived from the hybrids, so that the lower pairing in the hybrids was not the result of unbalanced genotypes. Consequently the diminished probability of pairing between homologs derived from different species is most readily ascribable to reduced homology. This can be assumed to have resulted from internal differences in the gene composition of homologs and it implies that some forms of heterozygosity can reduce the likelihood of pairing. Therefore the possibility must be recognized that differences between homologs, that can most satisfactorily be ascribed to the presence of alternative alleles at numerous loci, may give rise to pairing failure. Further, accepting lessons learned from evolutionary studies of many other characters, if such differences influence chromosome pairing in hybrids between species they are likely also to have some-though probably less pronounced-effects within species. Presumably the effects of differences in homozygosity-heterozygosity levels on pairing would be hard to detect within species, because of the relatively small distinctions between homologs and because of the selective adjustment of the genetic regulation of the pairing process to the tolerance of levels of heterozygosity automatically generated by the breeding system (see Section IV,B,B). Certainly, in seeking a cause for the specificity of meiotic pairing, it would be rash to dismiss too freely the possible impact of the extent of genetic equivalence-that is of similarities of nucleotide sequences-of homologs. If specificity is determined in this way then the degree of equivalence, in these terms, of homologs must influence the probability of their synapsis.

GENETIC VARIATION I N CHROMOSOME PAIRING

81

IV. Genetic Control of Pairing Specificity

A. INTRODUCTION The remarkable longitudinal specificity of meiotic synapsis and the restriction of pairing t o homologous partners has alreidy been mentioned. Since the only detailed longitudinal variation in chromosome structure for which there is any evidence relates to the sequence of DNA base pairs, it is natural t o attempt to equate pairing specificity with this. However, it seems inevitable for sequences of base pairs, or of codons, to be repeated a t more than one site in a genome; consequently the problem is posed of how synapsis is confined, in diploids, to strictly homologous sites on partner chromosomes. This difficulty is even more apparent in allopolyploid organisms which, while often maintaining a n entirely bivalentforming meiotic regime, carry a good deal of genetic material duplicated in their constituent genomes. It is not therefore surprising that all the information a t present available from which it can be concluded th a t the specificity of pairing is under any form of genetic control, in the normal sense, comes from allopolyploid organisms. Similar effects would be less easily detected in diploids where organisms in which they appeared would be described as variants with quantitative niodifications in the level of pairing or even as synaptic mutants. I n addition to its influence on theories of the causes of pairing, evidence of the genetic regulation of pairing specificity is of interest in revealing something of the evolutionary adjustment of meiosis in allopolyploids. The disruption of the reproductive system that polyploidy causes is the antithesis of the advantages that the new genotype may afford through fixed hybridity or through the extension of genetic variation and diversity. Even so polyploidy has played a prominent role in the evolution of many higher plant groups and perhaps of some animals. Consideration of the evidence will show th at meiotic balance can be attained in allopolyploids, and the reproductive barrier of polyploidy surmounted, by genetic modification of meiotic pairing specificity.

B. Triticum 1. Formal Genetics

Work on Triticum aestivum (an = 6x = 42), the common wheat of agriculture, has resulted in the production of more information on the genetic control of pairing specificity than is available for any other organism. To explain this work it is necessary first to discuss the cytogenetic structure of the species. T . aestivum is a n allohexaploid which,

82

RALPH RILEY AND C. N . LAW

through the normal processes of allopolyploid evolution, contains within its chromosome complement the full chromosome sets, virtually unaltered structurally, of three distinct 14-chromosome diploid species (for reference see Riley, 1965). These three species-Triticum monococcum, Aegilops speltoides, and Aegilops squarrosa-contributed the A, B, and D genomes, respectively. I n work in which they have evolved beautifully detailed architectural concepts of the cytogenetics of T . aestivum, Sears (1954, 1958) and Okamoto (1962) have shown that all its chromosoines can be classified according to the genome in which they occur and also in terms of their relative genetic activities. The phenotypic defects caused by nullisoniy for each chromosome in turn can be removed by extra dosage of certain other chromosomes. There are seven groups each of three chromosomes within which genetic compensation occurs in any deficiency-excess combination, but there is little, if any, compensation between these groups (Sears, 1964). One chromosome in each group is in each genome, and each genome has one chromosome in each group. Chromosoines in the same group are described as homoeologous, following Huskins (1931) , and their compensatory capacities are clearly indicative of their genetic similarities. Presumably, therefore, homoeologous chromosomes have been derived from corresponding nienibers of the complenients of the original diploid ancestors of the hexaploid, and their genetic equivalence stems ultimately from their coinmon origin froni the same chromosome of the prototype diploid progenitor of the entire Triticum-Aegilops group. A paradoxical situation was created by the recognition of hoinoeologous relationships, because T . aestivum has only bivalents at meiosis, and has disoinic inheritance, so that each chroniosoine pairs a t meiosis only with its fully homologous partner and there is no homoeologous pairing. Moreover, in 21-chromosonie haploid individuals, in which each chroniosome has no full honiolog with which to pair, most chroniosonies are univalents, so that the absence of homoeologous pairing at the hexaploid level cannot be ascribed to what Darlington (1937) called differential affinity-although the infrequent pairing and recoinbination th a t does take place in haploids is predominantly between homoeologs (Okamoto and Sears, 1962). The anomaly is niade inore striking by the high pairing, with means of between three or four bivaleiits per cell and with some cells with seven bivalents, in the 14-chromosome hybrids resulting from crosses of any two of the three diploid ancestors of the hexaploid (Riley and Chapman, 1958; Kiinber and Riley, 1963b), since this implies that honioeologs were originally capable of pairing. That their failure to do so in the hexaploid is not due to major structural changes can be shown by the regular behavior of the chroinosomes of the A or the

GENETIC VARIATION I N CHROMOSOME PAIRING

83

D genomes in hybrids between T . aestivum and either T . monococcum or A . squarrosa (Riley, 1965). Consequently, although homoeologous chromosomes have retained their genetic equivalences, their capacity to pair at meiosis has been either lost or suppressed. The anomalies were resolved by work th a t employed derivatives of some of the numerous aneuploid stocks available in T . aestivum. Sears and Okamoto (1959) showed that when a particular chromosome, number 5B, was absent from hybrids between T. aestivum and T. monococcum, there was much more meiotic pairing than normally occurred, and from this they concluded that this chromosome might have a n effect in the suppression of homoeologous pairing. Simultaneously, Riley and Chapman (1958) demonstrated th at 20-chromosome haploids of T . aestivum deficient for a single chromosome of the haploid set, that subsequently proved to be chromosome 5B (Riley et al., 1959), had a pattern of meiotic pairing very different from that of 21-chromosome euhaploids (Table 4). Whereas there was little pairing in the euhaploids, those deficient for chromosomes 5B had numerous bivalents and trivalents. An activity of this chromosome was therefore clearly responsible for the suppression of some form of nonhomologous pairing and the high frequency of trivalents and rarity of quadrivalents suggested th at this took place between homoeologs. The formation of multivalents in 40-chromosome plants nullisomic for chromosome 5B demonstrated that chromosome 5B restricts pairing in a similar manner in normal hexaploid individuals. T o determine the relationships between the chromosomes th a t pair nonhomologously in the absence of chromosome 5B, Riley and Kempanna (1963) exploited the principle that nonhomologous recombination produces translocations relative to the original chromosome structure. Families heterozygous for translocation differences, %hathad arisen by nonhomologous recombination, were produced from-hybrids between a plant nullisomic for chromosome 5B and a euploid individual. Lines in which each chromosome of the complement was marked, in turn, by being telocentric were crossed with the translocation heterozygotes and the resulting hybrids, th at were again heterozygous, were examined for the participation of the marked chromosome in the translocation multivalent. I n this way the chromosomes involved in a t least nine distinct translocations were determined. However, since all except one of the ten families studied carried more than one translocation it was not immediately apparent between which precise chromosomes there had been exchanges. Nevertheless, when the chromosomes involved were classified according to their homoeologous group, they fell in two’s in the same number of groups as there were translocations heterozygous in the family concerned. From this it could reasonably be inferred tha t the translocations were

TABLE 4 Mean Pairing a t First Metaphase of Meiosis in Haploids of Triticum aestivum with Various Conditions of Chromosome 5B

Haploid type

Chromosome number

Chromosome 5B Long arm

Short arm

Cells

Euhaploid Long arm 5B isochromosome Nulli-5B Short arm 5B telocentric

21 21 20 21

Present Present Absent Absent

Present Absent Absent Present

750 100 2 18

50

Univalents Bivalents Trivalents Quadrivalents 1.05 0.90 3.72 3.82

0.02 1.62 2.22

*

i

8

Mean pairing

18.84 19.20 7.42 6.62

w

M 4 ~

-

d

-

9

0.07 0.02

P H

GENETIC VARIATION I N CHROMOSOME PAIRING

85

between honioeologs and a further test gave unequivocal evidence th a t this was so for three translocations in one family. Consequently, it can be concluded t ha t pairing and recombination between homoeologous chromosomes is normally suppressed in T . aestivum by the activity of chromosome 5B, and that the absence of this chromosome causes the relapse of the suppression. I n a range of hybrids between T. aestivum and Secale cereale (2n = 14), deficient in turn for each chroinosome of the Triticum complement, only hybrids deficient for chromosome 5B had a level of meiotic pairing strikingly higher than th at in euploid hybrids (Riley et al., 1960). I n addition, no 40-chromosome nullisomic, other than that deficient for the same chromosome, is multivalent-forming (Kempanna, 1963). It can therefore be concluded that the genetic systein of chromosome 5B, th a t prevents homoeologous pairing, is not duplicated in any other linkage group of T . aestivum. Further, by comparison of the meiosis of haploid plants with the full gametic set of 21 chromosomes, or deficient either for the complete chromosome 5B, or for its long or short arm separately, the effect can be shown to be confined to the long arm (Table 4) (Riley, 1960c; Riley and Chapman, 1964a). It is not yet known whether a particular region of the long arm is responsible for the effect, but mutants have been obtained in the heterozygous condition by Okamoto (1963), in which one chromosome 5B was apparently changed so that it no longer inhibited hoinoeologous pairing. It seems likely, therefore, that the activity is confined t o a restricted region and probably to a single locus. Indeed, since this is the simplest hypothesis for the control of the inhibitory behavior it will be adopted in our further comments. Chromosome 5B was contributed to Triticum aestivum, and to all the other polyploid species of Triticum, by A . speitoides by the usual processes of allopolyploid evolution. Using crosses between plants of T. aestivum, monosomic 5B, and either A . speltoides (2n = 14) or, for example, A . longissima (2n = 14) comparisons were made between the effects of chromosome 5B and the genotypes of the two Aegilops parents (Riley et al., 1961). Here it must be interpolated that A . speltoides and A . longissima are closely related diploid species, hybrids between them having full pairing at meiosis. Meiosis was compared in the 28- and 27-chromosome hybrids, between T . aestivum and the alternative diploids, with and without chromosome 5B. There was high pairing with inany trivalents and quadrivalents, presumably involving the association of homoeologs, whether chromosome 5B was present or not, in the hybrids between T. aestivum and A. speltoides. By contrast, hybrids involving A . longissima have low levels of pairing in the presence, but high levelscomparable with those in the A . speltoides hybrids-in the absence, of

86

RALPH RILEY AND C. N. LAW

chromosome 5B. From this it was concluded that the genotype of A . speltoides suppresses the inhibition of homoeologous pairing resulting from the activity of chromosome 5B, whereas the genotype of A . longissima does not suppress this effect, nor can it take over the activity by conipensating for the absence of the chromosome. By marking two chromosomes simultaneously in hybrids between T. aestivum and A . speltoides Riley and Chapman (1964b) showed subsequently that pairing takes place between homoeologs, confirming that the activity of chromosome 5B is inhibited. All other diploid species, when combined with T . aestivum in the presence or absence of chromosome 5B, produced hybrids that behave like those of either A . speltoides or A . longissima (Table 5 ) (Riley, 1963), so that there is no species with a genotype that, like chromosome 5B, prevents homoeologous pairing. Probably, therefore, the capacity to do this arose by mutation after chromosome 5B had been incorporated in a polyploid Triticum; a less likely alternative is that the hybrid, from which the polyploids developed, was produced by a mutant gamete of A . speltoides. Certainly, if the mutation occurred subsequent to the origin of polyploidy, it must have taken place early in the polyploid history of the genus, at the tetraploid level, since all polyploid species of Triticum apparently have alleles equivalent in function to that on chromosome 5B of T . aestivum. Three distinct genetic activities affecting homoeologous pairing can thus be recognized. These are, first, that due t o chromosonie 5I3, second, that produced by the genotypes of two diploid species ( A . speltoides and A . rnutica) by which the 5B effect is suppressed, and, third, that produced by A. longissima, and all the other diploids, which is of null effect in terms of the tests described. If all these activities are due to the functioning of different alleles a t a single locus on chroniosoines homoeologous with 5B of T . aestivum, as is required by the simplest hypothesis, then the A . speltoides condition is dominant to the 5B condition which in its turn is dominant to the A . longissima condition. Riley (1965) has symbolized these states as P, in A . speltoides, p 5 in ~ T . aestivum and pl in A . longissima. I n view of the other evidence indicating that chromosome 5B was derived from A . speltoides (Sarkar and Stebbins, 1956; Riley et al., 1958) it is likely that P5B arose from P,, but there is no internal evidence against its having originated from pl. Clearly p5B is an active allele since honioeologous pairing, which is the primitive behavior, only takes place in its absence. However, whether the mutation occurred a t this locus, or whether its activity was exposed by the mutation of a suppressor elsewhere, cannot yet be answered. I n all the hybrids so far examined between T . aestivum and species of Aegilops, Secale, and Agropyron there is a marked increase in pairing in

TABLE 5 Mean Chromosome Pairing at First Metaphme of Meiosis in FI Hybrids from Crosses between Triticum aestivum and Various Species of Aegilops and Secale, with and without 5B of T.aesttdum

Alternative parent

Hybrid chro- Chromosome mosome number 5B Cells

Proportion of complement paired

Mean pairing Univ.

Biv. Triv. Quad. Quin.

A. mutica (2n = 14) A. mutica A. speltoides (2n = 14) A. speltoides

28 27 28 27

Present Absent Present Absent

50 50 50 30

5.32 5.94 6.04 6.13

A. A. A. A. A. A. A. A.

28 27 28 27 28

100 100 50 50 30 30 100 100

24.04 7.50 22.44 8.20 24.50 9.44 24.12 6.77

1.96 7.58 2.46 5.10 1.60 4.98 1.82 5.77

5.14 2.06 1.52 5.24 1.56 1.48 6.64 1.88 0.76 5.23 1.76 1.23

-

Sex. -

-

-

8.cereale (2n = 14) S. cereale S. montanum (2n = 14) S. muntanum

28 27 28 27

Present Absent Present Absent Present Absent Present Absent Present Absent Present Absent

100 100 50 50

25.26 18.00 25.84 12.40

1.37 3.18 0.70 0.11 0.02 1.10 5.64 0.96 0.12 -

A. ovata (2n = 28)

A. wata A. triuncialis (2n = 28) A. triuncialis A. cylindrica* (2n = 28) A. cylindrica

35 34 35 34 35 34

Present 70 Absent 100 Present 30 Absent 30 Present 50 Absent 50

29.67 15.40 26.97 12.33 20.82 13.20

2.62 5.60 3.77 6.27 6.74 6.92

A. turcomanica (2n A. turcomanica

42 41

Present Absent

29.02 4.38 0.98 0.32 19.00 4.28 2.12 0.80 0.52 0.14**

longissima (2n = 14) longissima comosa (2n = 14) comosa umbellulata (2n = 14) umbellulata cauduta (2n = 14) caudata

0.01 0.70 0.06 2.04 0.10 1.76 0.08 2.09

0.03 1.90 0.17 2.33 0.18 1.56

0.56 0.62 0.50 0.06

-

-

-

-

0.55 0.02 0.02

0.36

-

0.50 0.20 0.56

0.09 -

-

-

-

Mean

*

0.81 0.017 0.78 f 0.017 0.79 k 0.013 0.77 rfr 0.017 0.14 f 0.010 0.72 + 0.013

50 50

* A. cylindrica has one genome in common with T.aestivum. ** 0.04 = septavalent, 0.02 = octavalent.

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88

RALPH RILEY AND C. N. LAW

the absence of chromosome 5B (Table 5) (Riley et al., 1959; Muramatsu, 1959; Riley, 1960c, 1963; Cauderon, 1963; Upadhya and Swaminathan, 1963). It is reasonable to regard this as being due to homoeologous pairing within the T . aestivum complement and also between the chromosomes of T. aestivum and those of the alternative parent, although allosydetic pairing occurred only rarely in most crosses when 5B was present. The only exceptions, with no change, were the hybrids involving A . speltoides or A . mutica which, through the suppression of the 5B effect, have homoeologous pairing even in euploid hybrids. With the exception of S. cereale hybrids, the increase in pairing in the absence of 5B was greater to a pronounced degree when the alternative parent was a diploid species than when it was polyploid. Since all the alternative polyploid parents are bivalent-forming allopolyploids, derived from closely related diploids (Lilienfeld, 1951), this contrasting behavior may be indicative of their possession of a system restricting hoinoeologous association, like the 5B effect in Triticum. If this were so the occurrence of an increase in pairing, even though it was less than when the alternative parent was diploid, would seem to imply that these systems are functionally distinct from that of Triticum. This interpretation should be treated cautiously however, since the smaller increase in pairing might alternatively have been the product of greater synaptic competition in hybrids with more hoinoeologs attempting to pair. I n an extension of the examination of initial hybrids displaying homoeologous pairing, Riley and Chapman (1963) produced synthetic amphiploids combining A . longissima and T . aestivum with and without chromosome 5B. Although in the presence of chromosome 5B multivalents were very rare in such amphiploids, in its absence they are large and numerous (Table 6) indicating homoeologous pairing within the T . aestivum complement and between T . aestivum and Aegilops chromosomes. Chiasma frequencies per chromosome were substantially lower in the multivalent-forming lines and this has been interpreted as being due to greater competition a t synapsis. An affinity between homoeologs can be visualized as reducing the probability of homologous synapsis without guaranteeing homoeologous synapsis. The correlation of lower chiasina frequencies with homoeologous pairing must also imply that the association of homoeologs does not depend upon an increased chiasma frequency. It also refutes the notion that homoeologs pair, even in the presence of chromosome 5B, but are prevented from forming chiasmata. The exceptional hybrids, between T . aestivum and a diploid species, that did not have a very pronounced increase in pairing when chromosome 5B was absent, involved rye, Secale cereale (2n = 14) (Table 5). Apparently this diploid introduced a quite distinctive genetic regulation of

0 M

Z

TABLE 6 Mean Chromosome Pairing a t First Metaphase of Meiosis in Amphiploids, with and without Chromosome 5B, from the Cross Triticum aestivum X Aegilops longissimu Mean pairing Chromosome Chromosome number 5B 56 54

Present Absent

Univ.

Biv.

Triv.

1.25 4.10

27.13 18.83

0.03 1.27

Quad. Quin. 0.10 1.67

Oct.

Sex.

Sept.

-

-

-

-

0.20

0.10

-

0.03

Chiasmata per cell

Chiasmata per paired chromosome

50.93 0.89 0.92 3 ~ 0 . 0 2 41.67 zk 0.72 0.83 k 0.07

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8

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RALPH RILEY AND C. N . LAW

chromosome pairing since the level in these hybrids was lower than that in 20-chromosome haploids of T . aestivum deficient for chromosome 5B. Consequently the S. cereale genotype is responsible for a reduction in the amount of homoeologous pairing between Triticum chromosomes. The significance of this observation that the control of pairing in Secale is in some way distinct from that in Triticum is not clear (see Section 111,B). 2. Function

The effects, already described, of the presence or absence of the 5B locus are all based on the observation of first metaphase of meiosis, so that the differences could have arisen from alterations either in the specificity of synapsis or in the formation of chiasmata. There are two kinds of evidence that indicate that the differences were not the product of changes in chiasma formation. First, although there were technical difficulties, Kimber (1962) concluded from observations of pachytene that much greater lengths of chromosome synapsed in 20-chromosome haploids, deficient for chromosome 5B, than in euhaploids. I n addition, a t prophase of meiosis in normal 42-chromosome individuals he was unable t o detect the complex associations that are formed in 5B nullisomics. Second, if there is normally some synapsis between homoeologs, that is not detected a t first metaphase because no chiasmata are found in the regions so paired due to a n inhibitory action of the 5B locus, then higher chiasma frequencies could be expected to accompany homoeologous pairing in the absence of the locus. However, there are actually fewer chiasmata formed per chromosome in 5B nullisomics than in euhaploid individuals (Riley, 1960c; Kimber, 1962; Kempanna, 1963). Similarly, synthetic amphiploids, derived from T . aestivum and Aegilops longissima, had lower chiasma frequencies per chromosome in the absence than in the presence of chromosome 5B, despite the considerab1e';amounts of homoeologous pairing in the former condition (Table 6) (Riley and Chapman, 1963). Apparently, therefore, homoeologs do not synapse in standard plants of T . aestivum, but there is no evidence available on the causal basis of this isolation although knowledge of it must influence the attitudes taken to some of the hypotheses proposed to account for all synaptic phenomena. Thus it seems to be incompatible with the operation of any purely physical force; for example it is difficult to visualize GuyotBjerknes effects being changed by gene action to preclude the synapsis of homoeologs. Alternatively the selective synapsis of homologs might be interpreted to imply that pairing is chemically mediated, in which case the 5B gene could either function directly in the process, modifying a pertinent synthetic pathway, or it could act as a n operator, switching

GENETIC VARIATION I N CHROMOSOME PAIRING

91

off genes positioned on other chromosomes, each perhaps responsible for synapsis in its own region. I n attempting to visualize the operation of the 5B system it is useful to consider the probable relationships of the homologous and homoeologous chromosomes between which it distinguishes in regulating pairing specificity. Hornoeologs may be conceived as having been derived from the same chromosome of some remote diploid progenitor of the entire Triticum-Aeyilopsgroup. However, they were separated from each other when the comnion ancestor split into the divergent groups that evolved into distinct, reproductively isolated, diploid species. This evolutionary divergence was presumably accomplished by mutation and by the selection of different adaptive gene complexes on chromosomes of common origins. Isolated from the equalizing effects of recombination and carrying arrays of genes assembled in response to different forces of selection, homoeologs can be visualized as differing at many loci. There may also have been minor changes in structure although there is no evidence of cytologically detectable alterations. Subsequently, when brought together in the allopolyploid species of Triticum, homoeologous chromosomes were still prevented from recombination by the activity of the 5B locus, so t h a t their heterozygosity was fixed and may indeed have been further exaggerated by the accumulation of additional mutations. The loci at which homoeologs are heterozygous are presumably distributed at random along their lengths. Nevertheless, despite their divergence, homoeologs continue to retain many common functions as is displayed by their capacity to compensate for each other in deficiency-excess combinations. By contrast, homologs have not been isolated in the same way. Their common origin from a single chromosome is much more recent-making the reasonable assumption of a monophyletic origin of the polyploid Triticum species-and they have been subjected to the equalizing effects of recombination. Thus, inevitably, there must be less heterozygosity between homologs than between homoeologs. Indeed, since T. aestivum is inbreeding the level of heterozygosity must be very low in most plants. Clearly, a model to describe the general action of the 5B locus can be based on this reasoning. For if the prime contrast between homoeologs and homologs resides in differences in the sum and continuity of the regions that are genetically distinct, then very likely it is upon this that their different synaptic behaviors depend. I n these terms the 5B locus would narrow synaptic specificity so that hoinoeologs lie beyond the threshold of homozygosity or genetic equivalence required for pairing (Fig. 3). The absence of the 5B locus can be depicted as shifting the threshold so that homoeologs are sufficiently similar to pair.

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These notions have some implication for meiotic pairing in general. for if pairing specificity in Triticum is related to the sum and continuity of the genetically equivalent regions that chromosomes have in common, then the restriction of synapsis to homologs may be similarly founded even in diploids. For the presumed duplication of some sequences of nucleotide triplets in nonhomologous chromosomes would probably not be great enough in extent to cause synapsis. We can now attempt to fit this model to the differences, in their reactions to the 5B system, displayed by the diploid species related to T .

/

Non-hornoeologoud Hornoeologous

I

Homologous

P

Similarity o f chromosomes

FIG.3. A diagram to show a possible model for the activity of chromosome 5B in regulating the specificity of pairing in Triticum aestivum.

aestivum. The genotypes of two species-A. speltoides and A. muticasuppress the effect of the 5B locus in hybrids with T. aestivum. By contrast the genotypes of all other related diploids, with which T. aestivum can be hybridized, do not influence the 5B effect. They do not suppress it, nor can those that have been tested (Table 5) take over the function in the absence of the chromosome. There is a striking correlation between these two behaviors and the breeding systems of the species concerned. A . speltoides and A . mutica are both open-pollinating species and can be regarded as outbreeders, although this behavior is not obligatory (Zohary and Imber, 1963). By contrast all the other diploids are cleistogamous inbreeders (Riley and Chapman, 1957b). However, there is no obvious difference in the meiotic pairing, or the chiasma frequencies, of

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any of the diploids-whether inbreeding or outbreeding. It is tempting to postulate that the existence of a genetic difference in the controI of pairing specificity in the diploids, that can only be detected in their hybrids with polyploid Triticum, relates to their breeding behaviors. If this is so, the outbreeders are adjusted to regulate the pairing of somewhat dissimilar (heterozygous) chromosomes, like those they permit to synapse in T . aestivum. However, the inbreeding diploids have not evolved, or have not retained, this niechanism in controlling the synapsis of their largely homozygous partner chromosomes. This may therefore be a n adaptive adjustment of the meiotic process to the over-all genetic systems of the outbreeding species. Moreover, if genetic variation in this character is selectively maintained a t the diploid level it is easy t o visualize how, by its adjustment, a diploidlike meiotic behavior could be developed by the polyploid species of Triticum. Secale species do not match the behavior of the diploid species of Aegilops for although they are outbreeding species their genotypes do not suppress the 5B effect. However, as has already been indicated, Xecale cereale appears to have a form of genetic regulation of meiosis quite different from that of T . aestivum in that its genotype partially limits pairing between homoeologs even in the absence of chromosome 5B. A unifying hypothesis can thus be developed from the notion that the mechanism of pairing specificity depends upon the level of genetic equivalence of the chromosome involved. This does not provide a functional explanation but it clarifies the position in preparation for a causal analysis. A clue as to causes is suggested by Ansley’s demonstration that there may be a difference between the ratios of DNA: histone in synaptic and asynaptic cells in Loxa jlavicolis. This may imply that for synapsis to occur the DNA must be differently masked by histone in meiotic compared with somatic cells. This should be considered in conjunction with the observations of Boniier et al. (1963) indicating that differentiation may depend upon the masking by histone of DNA that is not active in particular tissues. For if differentiation is so dependent, and differentiation does not follow similar paths in related diploid species, then we can hazard a guess, and it should be placed no higher, that there may be contrasts in the distribution of histones on homoeologous chromosomes. The operation of the 5B locus may therefore be related to this differential distribution, and this may indeed simply be the notion of its dependence upon the genetic dissimilarities of homoeologs expressed in different terms. However, whatever ultimately proves to be the mode of its operation, the 5B effect in Triticum obviously affords peculiar advantages in certain aspects of the causal analysis of synapsis.

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3. Evolution The synthetic allotetraploids derived from the hybrids between the putative parents of the tetraploid species of Triticum, A . speltoides, and T. monococcum, form frequent quadrivalents a t meiosis (Riley, 1965). There are as many as four quadrivalents in some cells and the mean frequency is about one per cell. These quadrivalents are the product of pairing between chromosomes of the different parental sets as well as between fully homologous chromosomes derived from the same parent. There is thus the equivalent of homoeologous pairing in the natural polyploid species of Triticum. The synthetic allotetraploids are of low fertility and are entirely constant from generation to generation, displaying none of the segregation that is to be expected from the occurrence of intergenome pairing and recombination. Presumably therefore those gametes, or zygotes, that carry the products of intergenoinic recombination are inviable and this is the cause of low fertility. If this were the situation in the first tetraploid forms of Triticum there would obviously have been enormous selective advantage to any mutation that precluded intergenomic pairing since it would have offered an immediate escape from the meiotic causes of low fertility. Mutation of the 5B locus, on a chromosome derived from A . speltoides, must thus have had a profound impact and its rapid selection to fixation can be readily envisaged. The first tetraploid was the progenitor of all the other tetraploid and hexaploid species of Triticum and in consequence the mutant 5B allele is found throughout the polyploid species. At the hexaploid level it is as effective in the meiotic isolation of the chromosomes derived from A . squarrosa, in the D genome, as in its initial isolation of the chromosomes of the A and B genomes of the tetraploid. Indeed, without the prior presence of the mutant, a t the tetraploid level, hexaploidy might never have been attained. Classical genome analysis has shown that there has been very little interchange of material between chromosome of the sets derived from the diploid ancestors of T. aestivum (for references see Riley, 1965). The distinction between sets is now largely stabilized by the prevention of homoeologous pairing. I n addition, the elimination of the products of intergenomic recombination in the raw tetraploid illustrates how the integrity of the genomes was maintained during the first generations of polyploidy until the 5B effect became operative. Indeed, unless the distinctions between homoeologs had been so maintained there would have been no basis for synaptic discrimination when the appropriate mutant arose.

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4. Relationships between EIlfects An activity of chromosome 3B is responsible, in part, for the maintenance of regular bivalent formation in T . aestivum, and the absence of this chromosome results in the occurrence of univalents a t first metaphase (Table 3). In order to determine the relationships between the maintenance of regular pairing by this chromosome and the prevention of homoeologous pairing by chromosome 5B, Kempanna and Riley (1962) extracted plants deficient for both chromosomes. Plants simultaneously monosomic for both chromosome 3B and 5 B were pollinated with the rye species Secale montanum (2n = 14) and hybrids were produced deficient for neither chromosome, or for each separately, or for both. I n hybrids lacking chromosome 3B, 99% of meiotic cells had no chromosomes paired, whereas in those lacking chromosome 5B there was no cell without any pairing and a mean of 14.6 chromosomes per cell participated in bivalents, trivalents, or quadrivalents. I n contrast to both these patterns of pairing there was a bimodal distribution of cells in the hybrids deficient for both 3B and 5B (Fig. 4). There was no pairing in 16% of cells which consequently resembled the 3B-deficient pattern, while in the remaining cells there was considerable pairing resembling that in hybrids lacking 5B alone. Thus apparently the effects of both deficiencies were separately expressed in different cells of the same individuals. Bimodality in the level of pairing was shown only in individuals deficient for both chromosomes so th at they displayed greater imbalance of synapsis than those lacking either singly. This may be because the deficiency of chromosome 5 B exaggerates the effect of the absence of chromosome 3B in the reduction of synapsis. Alternatively the failure of synapsis may be more abrupt and drastic when pairing is attempted between homoeologous chromosomes than when the potential synaptic partners are full homologous, as in 40-chromosome 3B-nullisomic individuals of T. aestivum. Chromosomes 3A and 3D of T. aestivum are homoeologous with 3B and they are also active in maintaining meiotic pairing, indicating the possibility of triplicate loci (Table 3) (Kempanna, 1963). Indeed these three chromosomes produce more profound effects on the level of meiotic pairing than any other. However, the greatest contribution t o the character is made by chromosome 3B. This tempts speculation on the possible evolutionary relationships of the activities of chromosomes 3B and 5B in the stabilization of meiosis. Thus it might be envisaged th a t a mutant

RALPH RILEY AND C. N . IJAW

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0

Deficient 35

F

Eal 40

e a"

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2o

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Deficient 38 and 5 8

5

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25

Number of chromosomes paired

FIG.4. Histograms showing the frequency distribution of cells with various numbers of chromosomes paired a t first metaphase of meiosis in hybrids derived from the cross Triticum aestivum monosomic-3B and -5B (2n = 42 - 2) X Secale montanum (2n = 14). Euploid = 28-chromosome hybrids; chromosomes 3B and 5B present. Deficient 3B = 27-chromosome hybrids; chromosome 3B absent. Deficient 5B = 27-chromosome hybrids; chromosome 5B absent. Deficient 3B and 5B = 26-chromosome hybrids; chromosomes 3B and 5B absent (Kempanna and Riley, 1962).

allele arose, a t a synaptic locus on chromosome 3B, that enhanced the amount of pairing in a manner that compensated for a general reduction in synapsis that derived from the narrowed specificity caused by the earlier mutation of the pairing locus on chromosome 5B. If the increased pairing due to the 3B mutant caused no further modification in specificity it might well have offered a marked selective advantage. Attempts to test this hypothesis have so far been inconclusive (Riley, 1963).

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C. PAIRING SPECIFICITY IN OTHERALLOPOLYPLOIDS The evolution from a multivalent-forming condition with intergenomic pairing a t meiosis, to a n entirely bivalent-forming-diploidlike-meiotic regime, can be inferred in a number of allotetraploid species by the use of two kinds of evidence. The first of these is the occurrence of less pairing in natural haploids of tetraploid species than takes place in “raw” unadjusted haploids produced by hybridization between the putative diploid parents. The second is the occurrence of multivalents in the “raw” synthetic tetraploid, derived from the hybrid between its putative diploid parents, even though the natural tetraploid is entirely bivalentforming (for examples see Riley, 1 9 6 0 ~ ).Only tetraploids can be compared in these ways because, in considering higher polyploids, the causes of a pre-existing diploidlike meiosis of a polyploid parent may still be effective at higher chromosome levels in “raw” haploids or synthetic polyploids. There is no conclusive evidence that the change to a n entirely bivalentforming pattern in any allopolyploid species, other than those of Triticum, has had a genetic cause. Indeed it has been proposed that such modifications of meiosis might develop by the selection and accumulation of many changes in chromosome structure until the originally equivalent chromosomes of the component genomes diverged to the extent of being no longer capable of synapsis (Darlington, 1937; Stebbins, 1950). The differential pairing of particular homologous chromosomes in autotetraploids has been produced by the presence of different structural conditions in the duplex state, and in triploids by structural heterozygosity, a s is required by this proposal (Rhoades, 1957; Grell, 1961; Doyle, 1963). However, there are no verified examples of the attainment of a diploidlike status by this process which, if it occurs, must be slow and impose many generations of low fertility, genetic instability, and uncertainty on the “raw” allopolyploid species. By contrast the selection of the localized product of a single mutational step, such as seems likely to have happened in Triticum, is very much simpler to envisage and clearly would be a more rapid and evolutionarily more efficient process. While there are no other unequivocal instances of genetically determined diploidization, nevertheless the cytogenetic literature contains hints t ha t in two genera4ossypium and Nicotiana-analogous systems may operate. I n addition Johnson (1962, 1963) has presented evidence indicating t ha t bivalent formation is genetically imposed in the allotetraploid Stipa nevadensis (2n = 68). However, even if a genetic explanation of the diploidlike meiosis in these cases is accepted, it should be stated at the outset that there is no evidence whether it is functionally depend-

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ent upon altered patterns of chiasma formation or altered synaptic specificity. The natural tetraploid species of cotton, Gossypium hirsutum, G . barbadense, and G . tomentosum (all 2 n = 42 = 52) are allotetraploids which combine the chromosome sets of diploid Asiatic and diploid American cottons (Beasley, 1942). At meiosis they are bivalent-forming and have disomic inheritance although a number of loci are duplicated with representatives in each genome (Rhyne, 1957). Kimber (1961) has inferred that the diploidlike meiotic behavior is genetically determined. The evidence adduced for this is that in the triploid hybrids, derived from crosses between the tetraploids and either of their diploid ancestors, there is good pairing at meiosis between chromosomes of the genomes common to the diploid and tetraploid parents. Consequently, although there are some translocation differences, there has apparently been no major accumulation of structural modifications in the tetraploids such as might impede pairing. I n addition there is reasonably good pairing in hybrids between the two diploid ancestors, although there is virtually no pairing in haploids derived parthenogenetically from the tetraploids. The infrequency of structural changes together with the incongruence of the cycle of meiotic comparisons lead to the conclusion that the absence of intergenomic pairing in the tetraploids and their haploid derivatives is genetically caused. Similar arguments can be developed about the meiotic behavior of Nicotiana tabacum ( 2 n = 4x = 48), which is a bivalent-forming allotetraploid. In this species also the chromosomes derived from the two diploid parents have undergone few structural alterations as is shown by the regular pairing of those in the common genomes in triploid hybrids resulting from crosses between the tetraploid and either of its diploid parents (Goodspeed, 1934). However, there is much more pairing in hybrids between N. sylvestris and N . tomentosiformis, close relatives of its diploid progenitors, than occurs in haploid forms of N . tabacum (Clausen, 1941 ; Goodspeed, 1954; Lammerts, 1934; Talcenaka and Tanalra, 1956; Sficas and Gerstel, 1962). Since the different behaviors of the haploids and diploid hybrids are apparently not primarily due to the structural divergence of chromosomes of the basic genomes, here again the absence of homoeologous pairing may have a genetic cause. Gerstel (1963) has argued against this interpretation of the contrasts between haploids and diploid hybrids in Gossypium and Nicotiana. He pointed out that in synthetic tetraploids, derived from hybrids between the putative diploid parents, multivalents are infrequent presumably due to the differential affinity of chromosomes of the parental sets. Thus he claimed the selective premium favoring genetic systems that eliminated

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multivalents would have been slight in the initial tetraploids. I n addition, from consideration of the meiotic behavior of hybrids between the polyploids and distantly related diploids, he concludes that there is no limitation of pairing between chromosomes long isolated in evolution, in contrast to the behavior of Triticum (Riley et al., 1958). Finally, Gerstel indicated certain genetic evidence that in part suggests that some chromosomes of one genome in both tetraploid Gossypium and in tetraploid Nicotiana may not pair freely with the equivalent chromosomes of the diploid species from which they were derived. The implication of this is that the structural divergence of one genome may have further exaggerated differential affinity in the tetraploids to the point a t which intergenome pairing was eliminated. All the previous discussion of meiotic behavior in Gossypium and Nicotiana has been based on observations of first metaphase, but when prophase pairing is considered new factors emerge. That there has in fact been no structural elimination of intergenomic affinity in Gossypium is indicated from a discussion of the situation by Endrizzi (1962) who drew attention to Brown's (1961) observations that there is an average of 7-9 paired chromosomes per pachytene cell in haploids of G . hirsutum. Thus, the much lower pairing a t first metaphase in haploids of tetraploid Gossypium (Beasley, 1942; Endrizzi, 1959; Sastry and Swaminathan, 1960) than in diploid interspecific hybrids cannot be ascribed to the loss of intergenomic affinities. Endrizzi proposed that differences in the degrees of coiling of chromosomes paired intergenomically in the haploids might be responsible for the rarity of chiasma formation. However, this proposal appears to neglect the occurrence of similar difference in coiling and contraction between the chromosomes contributed by the different species in hybrids, between the putative diploid parents of the tetraploids, in which chiasma formation does take place. Nevertheless, these observations seem to imply that the absence of intergenomic pairing a t first metaphase may be ascribable either to a genetic restriction of chiasma formation or of effectivepairing and that a restriction of synaptic specificity, like that in Triticum, does not occur. No evidence is available on the nature of this presumed genetic regulation. The evidence, from pachytene behavior, that structural divergence has not been responsible for the meiotic isolation of the genomes in Gossypium, throws doubt on the parallel arguments advanced to explain the diploidlike behavior of N . tabacum in terms of structural change. A further suggestion that here also a genetic system may be operative can be adduced from the meiotic behavior of a haploid plant derived from the mutant, coral'by Lammerts (1934). The parental plant was heterozygous for a translocation difference and the haploid apparently arose

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from a gamete with a duplication-deficiency segregation of the chromosomes involved. There was much more pairing at meiosis in this individual than normally occurs in haploid plants of N . tabacum, and more indeed than could be ascribed solely to the presence of the duplicated segment. It is tempting t o infer th at the excess pairing was due to the absence, on the deficient segment, of genes normally responsible for the suppression of intergenomic pairing. It is apparent that some uncertainty exists about the basis of the bivalent-forming behavior of these tetraploids. Nevertheless there are suggestions that it may be genetically imposed, the major objection arising from the low frequency of multivalents in synthesized tetraploids. Sears has suggested that this may result from the greater structural divergence of the chromosomes of contemporary diploid species than of those of the diploids from which the tetraploids were derived (in Gerstel, 1963). Had this been so, higher multivalent frequencies would have occurred in the original natural tetraploid than in contemporary synthetic tetraploids, with a correspondingly greater selective advantage to genotypes that prevented intergenomic pairing. However, it is clear th a t among a great deal of speculation, if the case for genetic systems is to be proved, there is a need for firm evidence of the type th a t settled the situation in Triticum beyond dispute.

D. THE IMPOSITION OF BIVALENT FORMATION I N SPECIES OTHERTHANALLOPOLYPLOIDS 1. Autopolyploids

It is important to differentiate the purely bivalent-forming meiotic mechanisms of polyploids like Triticum, Gossypium, and Nicotiana, in which there is disomic inheritance, from the systems found in species with polysoniic inheritance. The latter species are essentially autopolyploids in which four and more partner chromosomes conjugate a t random in pairs. A well-documented example of this behavior is found in Phleum pratense (2n = 42) which is a hexaploid that displays hexasomic inheritance (Nordenskiold, 1953, 1957). There is, however, a genetic restraint of pairing in the forms studied by the Swedish workers since multivalents are formed very rarely in 42-chromosome plants, and 21-chromosome haploids usually make seven bivaleiits and seven univalents (Nordenskiold, 1941; Levan, 1941), while a 63-chromosome (‘triploid” individual described by Muntzing and Prakken (1940) usually formed twenty-eight bivalents and seven univalents. The true autopolyploid nature of the species was indicated by the meiotic behavior of American material examined by Myers (1944) ;in contrast to the Swedish

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material this had numerous yuadrivalents and some sexivalents. The nature of the almost complete restriction of pairing to the formation of bivalents, in the Swedish stock, is not clear, but a limit is not imposed by low frequencies of chiasmata per paired chromosome (Miintzing and Prakken, 1940). However, results of this type could arise from the operation of only a single small region of synaptic contact on each chromosome, and the implication would follow from the occurrence of different behaviors in different stocks, that a limitation of the regions of contact is genetically imposed. Lotus corniculatus ( 2 n = 24) is another bivalent-forming polyploid, in this case with tetrasomic inheritance (Dawson, 1941 ; Buzzell and Wilsie, 1961 ; Poostchi and MacDonald, 1961). The four honiologs, representing each member of the haploid chromosome complenient, usually associate a t random in pairs and this restricted pairing is again not the result of a limitation in the numbers of chiasmata to one to each pair of chromosomes. I n other instances the autopolyploidy of species that form no multivalents has been inferred on cytological evidence. For example when Fragaria elatior (2n = 6x = 42) is crossed with certain 14-chromosome diploid relatives the resulting tetraploid hybrids regularly form fourteen bivalents at meiosis, displaying homologies within the F . elatior coniplement that are unrevealed a t the hexaploid level (Lilienfeld, 1933, 1936; Schiemann, 1937, 1943). Similarly, in the 42chromosome hexaploid hybrid, between the bivalent-forming decaploid Papaver striatocarpum ( 2 n = 70) and the diploid Papaver nudicaule (an = 14), twenty-one bivalents are formed a t meiosis-so th a t in the decaploid hitherto undetected internal homologies are revealed (Ljungdahl, 1924). Other examples of autopolyploids without multivalents apparently occur in the genus Chryanthemum (Shimotamai, 1933 ; Dowrick, 1952, 1953) and in the hexaploid Solanum nigrum (Jorgensen, 1928). I n these examples, unlike that concerning Phleum pratense, there is no evidence of a genetically imposed pattern of pairing in bivalents. However, if such genetic systems are operative, and not dependent upon over-all restrictions in the frequencies of chiasmata, it is hard to imagine any device, other than competition for limited synaptic regions, capable of achieving the observed regularity. Whatever the mechanism, however, the diploid progenitors of the polyploids must be considered in order to ascertain whether it was selected, because of the improved fertility to which it gave rise, after the origin of polyploidy or whether it already existed a t the diploid level. For example, Nordenskiold (1941, 1945) considers that Phleum pratense is a n autohexaploid of Phleum nodosum, yet newly synthesized autotetraploids of P. nodosum have high multi-

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valent frequencies. Thus it is likely that the essentially bivalent-forming pattern of meiosis in the hexaploid originated subsequent to polyploidy in the Swedish material and this seems to be confirmed by the description of American forms with multivalents (Myers, 1944). Naturally the genetic and evolutionary consequences of bivalent formation with random choice of partners, in autopolyploids, are quite different from those arising out of the strict pairing of full homologs to the exclusion of homoeologs, in allopolyploids. While both no doubt stabilize fertility at higher levels than would otherwise occur, in the latter, genes a t duplicate loci on homoeologous chromosomes are sheltered from selective competition with each other. Thus, the original function of a mutant gene may be continued at a duplicate locus and the way opened for greater evolutionary exploration than is possible with polysomic inheritance when one or other allele may be selected to fixation. I n addition, the functional basis of the systems involved in restricting pairing must differ, since in the allopolyploid forms the process involves a genetic enhancement of a pre-existing differential affinity. By contrast, in the autopolyploids, there is no initial differentiation of genomes capable of giving rise to discriminatory pairing, and without this pre-adaptation the only two-by-two pairing patterns that can be imposed genetically are of the nonspecific nature of those described in Phleum pratense and similar species. Therefore the two conditions are similar only in th a t they represent adaptive adjustments of meiosis for the production of balanced reductions in chromosome numbers. The genetic and evolutionary process t o which they give rise, and their mode of operation, are quite different.

2. Bothriochloa Bothriochloa intermedia (2n = 42 = 40) is a segmental allotetraploid grass in which the chromosome complements of two distinct 20-chrornosome diploid species of Bothriochloa and Dicanthium are combined, although perhaps in a somewhat modified form. Indeed, the largely apomictic nature of Bothriochloa and two neighboring genera, by reducing the significance of sexual reproduction, has apparently allowed the retention of some considerable distinctions even between superficially homologous chromosomes. Yet most plants of B. intermedia have quite regular bivalent formation at meiosis, and the occurrence of trivalents or quadrivalents through intergenomic pairing is rare. Nevert'heless, Chheda and Harlan (1962) recorded one plant with a mean of more than two multivalents per cell and another with a maximum of four quadrivalents. However, in a particular plant studied by these authors only 3% of the cells contained a multivalent, despite the pronounced intergenome

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homologies revealed by the regular formation of ten bivalents in a 20chromosome haploid t o which it gave rise. Using the plant th at gave rise to the haploid offspring a s a female parent, Chheda and Harlan produced a wide range of intraspecific, interspecific, and intergeneric hybrids, among which there were striking differences between sibs in the levels of meiotic pairing. Some of the intraspecific hybrids had means of more than twenty univalents per cell, while in others the mean univalent frequency was less than ten. Analogous differences occurred among the interspecific and intergeneric hybrids. Multivalents were much more common in the plants with fewer univalents. The summed data from the selfed derivatives of this plant, and from its hybrid progenies, fit the hypothesis that segregation a t a single locus is responsible for differences in the levels of pairing. The female parent, and all the male parents producing segregating progenies, must have been heterozygous at this locus, a t which the allele giving high levels of pairing is dominant. Apparently, when the dominant is present the partially distinct homologs pair to form bivalents and even homoeologs pair to a small extent, to give some multivalents. When the recessive is homozygous only the closely similar homologs pair. Chheda and Harlan appear to consider that a n anomaly is created by the infrequency of niultivalents involving homoeologs, in the presence of the dominant. However, on the Triticum model, this could be explained by the imposition of a threshold of similarity, required for pairing, th a t included the partially distinct homologs but that largely excluded homoeologs. However, it should be interpolated, this is entirely speculation since there is no evidence concerning the operation of this genetic control of two-by-two pairing in Bothriochloa. As was indicated earlier, the distinctions between homologs are probably maintained because of the largely apomictic breeding behavior of the group. However, the gene imposing bivalent formation has presumably permitted the facultative apomicts to maintain some sexuality, by ensuring the production of balanced gametes, while also ensuring that functional pollen is produced by the obligate apomicts. I n effect this system has allowed the development of meiotic regularity in the equivalent of a hybrid resulting from a wide cross.

E. SECONDARY ASSOCIATION O F BIVALENTS The occurrence of bivalents in pairs or groups, on the first metaphase plate, in polyploid species has been described by several authors, and bivalents so distributed are said to be secondarily associated (Darlington and Moffet, 1930; Lawrence, 1931). Darlington and Moffet as well as

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Lawrence ascribed the phenomenon to the occurrence of residual attractions between bivalents comprised of genetically and structurally equivalent chromosomes, analogous to the attractions that give rise to primary prophase synapsis. There was no rigorous investigation of the phenomenon, however, until Riley (1960a) and Kempanna and Riley (1964) tested the relationships between secondarily associated bivalents in T . aestivum. Two bivalents were marked simultaneously by the presence in each of one normal and one telocentric chromosome. The relative positions

2oL

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Non-homoeologws

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Homoeologous

-al

u)

lJ u0

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4

0

5

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Number of intervening bivolents

FIG.5. The distributions of fist metaphase cells of Triticum aestivum with from zero to nineteen unmarked bivalents intervening between two marked bivalents. The two marked bivalents were either related homoeologs or unrelated and nonhomoeologous (Kempanna and Riley, 1964).

of the two marked bivalents were scored by recording the number of unmarked bivalents that intervened between them on linear first metaphase plates. The relative spacing of two marked homoeologous bivalents was significantly different from that of two marked nonhomoeologous bivalents, primarily because greater numbers of homoeologs were immediately adjacent to each other (Fig. 5). Thus secondary association does genuinely take place between bivalents composed of genetically equivalent chromosomes. It is not dependent, as some have considered (Heilborn, 1936), on the sorting out of bivalents by sizes due to nonspecific forces of repulsion, since homoeol-

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ogous bivalents in T . aestivum are not more alike in size than nonhomoeologous bivalents. Secondary association may be due t o the attraction together of all honioeologous chromosomes, a t prophase, and to their often remaining adjacent as bivalents, following primary synapsis between full homologs, through to first metaphase. On this view secondary association is a relic of prophase attractions. Alternatively quite separate forces of attraction may operate during the nietaphase congression of bivalents, although this is a less acceptable hypothesis since it requires the assumption of previously undescribed pairing processes. While there is no known genetic variation in the process of secondary association, i t nevertheless illustrates that in. T . aestivum some sort of pairing affinity persists between homoeologs even in the presence of the 5B IOCUS. Indeed it may imply that homoeologous affinity still occurs over long distances and that it is only the realization of intimate synapsis that is inhibited by the 5B system. Interestingly this model of pairing matches FabergB’s (1942) proposal th at quite distinct pairing forces might be involved in the long-range attraction of chromosomes for each other and in their detailed pachytene pairing. I n addition, secondary association may be of considerable value in the investigation of chromosome pairing. The response of chiasma frequencies to environmental and genetic variables has been extensively studied but it is usually impossible to determine to what extent the effects detected on the character have their origins in altered pairing attractions between chromosomes or in altered rates of chiasma formation. Since secondary association is dependent upon pairing attractions alone the system is simplified and will allow directed measurements of the influence of environmental factors on pairing. Moreover, as the work with T . aestivum shows, it is amenable to quantitative analysis. V. Conclusions

Two distinct aspects of the process of meiotic chroniosome pairing are therefore under genetic control. The extent to which synapsis is realized within the chromosome complement is determined by the activities of major genes and polygenes, and by interactions within the genetic system comprised of relevant genes of both kinds. The entire process can be disorganized so that no pairing whatsoever occurs, by some mutants of major synaptic genes, while quantitative modifications of a wide range of magnitudes can result from other genetic conditions. There is also good evidence t ha t the specificity of synapsis can be widened or narrowed by gene action to permit the pairing of chromosomes distantly or closely related genetically and evolutionarily. I n addition some results, from bivalent-forming autopolyploids, lead to the speculation th a t the number

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or size of the regions between which contact between chromosomes is made initially, during synapsis, might be varied genetically. It is reasonable to ask whether, from the available genetic information, any hints can be discerned of the mechanism of synapsis, or whether any choice can be made between the hypotheses outlined in Section 11. Perhaps most help can be gained from the observation that the specificity of synapsis can differ according to the genotype in some species. This observation is incompatible with the notion that pairing takes place because of the electrostatically unsatisfied state of prophase chromosomes, just as it cannot be equated with the idea that a relationship analogous to that between specific antibodies-antigens exists between homologous pairing sites. In neither case is it possible to visualize how, for example in T.aestiuum, an alteration in the genotype could alter the relationships between chromosomes in the manner needed to modify specificity. It is also difficult to equate genetic changes in specificity with the operation of Guyot-Bjerknes effects (Faberg6, 1942) since it must be presumed that the postulated molecular pulsations would be invariable characteristics of each pairing site. Indeed comparable difficulties arise with all hypotheses involving physical forces. Nevertheless, if GuyotBjerknes effects or other physical forces resulted in long distance attractions between all genetically related chromosomes, a second mechanism, of variable specificity, might be postulated as being responsible for true synapsis. This notion of the functional separation of long distance attraction from detailed synapsis, which was originally postulated by Faberge, fits one theory of the origin of the secondary association of bivalents, as was pointed out earlier. The proposal of a double-action mechanism of pairing might be regarded as an unnecessary complication of the hypothesis but judgement should be deferred until further evidence, such as could well be produced from the investigation of secondary association, becomes available. The view that synapsis is chemically mediated is perhaps made more attractive by knowledge of its varied specificity. The genes that operate in the system may, on this suggestion, be regarded as operators that initiate or repress the activities of pairing sites with different specificity ranges. If the observations of Ansley (1957, 1958) are interpreted as meaning that the unmasking of DNA by histone is in some way involved in synapsis, then such genes might affect this process. Although this suggestion is obviously highly speculative, it may at least have the virtue of indicating the direction of future experimentation. The occurrence of pronounced quantitative genetic variation in the amount of pairing also suggests the operation of a biochemically mediated system. Moreover it seems to imply a dependence upon numerous syn-

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thetic processes, as indeed might be imagined for the control of the coinplex series of events involved in the attraction together, and synapsis of, chromosomes and in the maintenance of pairing until the formation of chiasmata. The genetic complexity of the control may also be taken as an indication that the genetic systems responsible operate not only directly on the pairing process, but also upon a range of activities that change a number of components of the cellular and nuclear environment of the chromosomes. While no firm positive evidence about the mechanism of meiotic chromosome pairing is made available by this consideration of its genetic control, the occurrence of the many genetic variants discussed nevertheless provides the tools for its investigation. Moreover they are tools that have yet to be employed in experimental work. If the present discussion has done no more than to attract some attention to these, and to indicate their potentialities in causal analyses, it will have served its purpose.

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Thompson, W., and Robertson, H. T. 1930. Cytological irregularities between species of wheat with the same chromosome numbers. Cytologia (Tokyo) 1,252-262. Upadhya, M. D., and Swaminathan, M. S. 1963. Chromosome pairing and morphological characters in 27- and 28-chromosome hybrids between rye and monosomics of homoeologous group V of bread wheat. Indian J. Genet. 23,225-231. Watanabe, Y. 1962. Meiotic irregularities in intervarietal hybrids of common wheat. Wheat Inform. Sew. 14, 5-7. White, M. J. D. 1954. “Animal Cytology and Evolution,” 2nd ed., 454 pp. Cambridge Univ. Press, London and New York. Whittington, W. J. 1958. Asynapsis in red clover. J. Heredity 49, 202. Wilson, E. B. 1925. “The Cell in Development and Heredity,” 3rd ed., 1232 pp. Macmillan, New York. Zezevic, L. M. 1961. Cytogenetic study of inbred lines of maize (Zea mays L.). 11. Chiasma frequency a t diakinesis. Inst. Biol. Beograd. Rec. Trav. 6, 1 4 3 . Zohary, D., and Imber, D. 1963. Genetic dimorphism in fruit types in Aegilops speltoides. Heredity 18, 223-231.

EVOLUTIONARY SIGNIFICANCE OF PHENOTYPIC PLASTICITY IN PLANTS A. D. Bradshaw Department of Agricultural Botany, Uriversity College of North Wales, Bangor, Wales and Department of Agronomy, University of California, Davis, California

I. Introduction . . . . . . . . . . . . . . . . . . . . . 11. Genetic Control of Plasticity . . . . . . . . . . . . . . 111. Fitness, Plasticity, and Selection . . . . . . . . . . . IV. Conditions Favoring Plasticity . . . . . . . . . . . . A. Disruptive Selection . . . . . . . . . . . . . . . . B. Directional Selection . . . . . . . . . . . . . . . . C. Stabilizing Selection . . . . . . . . . . . . . . . . V. Conditions Disfavoring Plasticity . . . . . . . . . . . VI. Mechanisms of Plasticity . . . . . . . . . . . . . . . VII. Fixed Phenotypic Variation . . . . . . . . . . . . . . VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . A. General Characteristics of Plasticity . . . . . . . . B. Interrelationship of Plasticities of Different Characters C. Open Problems . . . . . . . . . . . . . . . . . . D. Plasticity in Crop Plants . . . . . . . . . . . . . . IX. Summary . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . .

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140 144 . . . . . 145 . . . . . . 145 . . . . . . 146 . . . . . 148 . . . . . 149 . . . . . 149 . . . . . 151

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1. Introduction One must take into account the organism’s capacity for adaptive plasticity. I n this regard the question which will undoubtedly receive especial attention in the future is to what extent different species or forms show different degrees of individual adaptive modification. (Nilsson-Ehle, 1914, p. 549, T~ans. S. A . Cook.)

We are becoming increasingly aware that the individual cannot be considered out of the context of its environment. The way in which it reacts to different environments is as much part of its characteristics as its appearance and qualities in a single environment. At the present time there is a great deal of interest in the way in which an individual can maintain stability in the face of varying environmental influences. A considerable amount of evidence has shown that this stability is under genetic control. Much of the evidence has taken the viewpoint that stability and adaptation are correlated ‘and that lack of stability indicates lack of adaptation. But as Nilsson-Ehle implies, it seems that plas115

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ticity, or lack of stability, can be of positive adaptive value in many circumstances. This essay seeks to explore this viewpoint further. An individual genotype assumes particular characteristics in a given environment. I n a second environment it may remain the same, or it may be different. The amount by which the expressions of individual characteristics of a genotype are changed by different environments is a measure of the plasticity of these characters. Plasticity is therefore shown by a genotype when its expression is able to be altered by environmental influences. The change that occurs can be termed the response. Since all changes in the characters of an organism which are not genetic are environmental, plasticity is applicable to all intragenotypic variability. If plasticity is given this definition then it is necessary to remember that it can have two manifestations, ( a ) morphological and (b) physiological. All changes are physiological in origin, so fundamentally all plasticity is physiological. Where physiological changes have predominantly morphological end effects however, we can talk about morphological plasticity. Such changes will occur during the course of development : they are likely to be permanent for the organ involved. Purely physiological changes, by contrast, can occur at any time, even in mature organs: these may be reversible and not permanent. These two sorts of plasticity will be closely interrelated, often reciprocally, since morphological stability may result from physiological plasticity. Because physiological changes are less easy to observe than morphological changes it is inevitable that most of the evidence about plasticity is concerned with the latter. Plasticity in the sense defined here does not include variation which is directly genetic in origin. It is therefore being used more narrowly than by Salisbury (1940) who included both genetically and environmentally determined variation in his definition. His usage is ambiguous, however, since his subsequent examples were all of environmentally determined modifications. The concept of plasticity does not also have any implications concerning the adaptive value of the changes occurring, although many types of plasticity may have important adaptive effects. Plasticity is therefore not equivalent to phenotypic Jlexibility as used by Thoday (1953), since the latter term is the capacity of an organism to function in a range of environments, and may include plastic and stable responses. Lack of stability or instability, in the sense of Mather (1953a) and others, is the term used to describe variation which is not genetic in origin and which has no observed environmental cause. Since the cause is unknown the variation appears to be random in direction. It is sometimes considered to be due to developmental errors [or developmental noise (Waddington, 1957)] arising from random changes during development,

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unconnected with any environmental influences. As such it can be considered to be a different phenomenon from plasticity, where specific modifications are induced by definite environmental effects. Nevertheless, this distinction seems difficult to maintain. It is clear that in many cases the modifications included under lack of stability have distinct environmental causes, but the observations made preclude the possibility of their determination. It is difficult to believe that any such changes do not ultimately have definite environmental causes. This is supported by the experiments of Went (1953). For this reason lack of stability is considered here to be part of the general phenomenon of plasticity, and evidence concerning it will be considered. For convenience, however, the term may be retained to describe nongenetic changes where the cause is not apparent, and the changes therefore appear random. Similarly, stability can be used to indicate a condition where such changes do not occur. It can also be used generally to indicate any condition where there is lack of plasticity. Currently the term homeostasis is widely used in many contexts. I n the sense of Cannon (1932) it is the tendency for the characteristics of a physiological or morphological system to be held constant. Plasticity of a character can therefore be equated with lack of morphological (or physiological) homeostasis of that character, although plasticity of certain characters may lead to homeostasis of others. The use of the term, however, has recently been confused, and its use will therefore be avoided. More lengthy discussions of the semantic problems involved have been given by Waddington (1957, 1961) and Lewontin (1957). II. Genetic Control of Plasticity

In order to understand the significance of environmentally induced changes, it is perhaps logical to turn to what is known of the basic causes of such changes and details of the mechanisms involved. It has been argued elsewhere (Allard and Bradshaw, 1964) that at the present, owing to the complexities of the developmental pathways concerned, the interactions between pathways and environment will be so complex that it is unlikely that much progress in understanding can be made until more detailed work on the pathways has been carried out. Considerations of basic causes, however, have led to the argument that the degree of plasticity shown by a character can be related to the basic pattern of its developmental pathway. Such a viewpoint has been taken by many authors, such as Klebs (1909). Stebbins (1950) has argued that characters formed by long periods of meristematic activity (such as over-all size, leaf number, etc.) will be more subject to environmental influences and are likely to be more plastic than characters formed rapidly (such as reproduc-

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tive structures) or than characters whose pattern is impressed on primordia at an early stage of development (such as bud scales, leaves, etc.). This argument can be supported by evidence of the differences in plasticity shown by different characters in Achillea and Potentilla in the experiments of Clausen and associates (1940, 1948). The plasticity shown by the characters of these species can be related to their general pattern of development, i.e., its duration, complexity, the number of interacting processes involved, etc., as shown in the tabulation: Plastic

Not plastic

Size of vegetative parts Number of shoots, leaves, and flowers Elongation of stems Hairiness

Pinnate leaf shape Leaf margin serration Shape of inflorescence Floral characters

The contrast of the manner in which plants of determinate and indeterminate growth react to density provides further evidence. Species of indeterminate growth such as Vicia faba tend to respond to density by the number of parts formed, whereas species of determinate growth such as Helianthus annuus tend to respond by changes in the size of the parts (Harper, 1961). While basic developmental pathways are important, it does not seem possible for them to provide an explanation of all observable differences in plasticity. In an experiment where the annual mediterranean grass, Polypogon monspeliensis was grown under low and high fertility conditions, a hundredfold variation occurred in the numbers of spikelets per panicle, while glume and seed size varied by only 10% (Bradshaw, 1958). Variation in density of plants of linseed (Linum usitatissimum) caused the following changes in different characters (Khan, 1963) : Linseed Character

High density

Low density

L.S.D. at 5 %

Capsules/plant Seeds/capsule Seed weight/100 seeds

5.6 8.1 6 .O

77.5 9.3 5.9

22.6 0.86 0.34

I n both of these cases it is difficult to see, using arguments based on development, why seed number should change so enormously with density, and seed size remain so constant. Examination of other investigations, e.g., on barley (Ariyanayagam, 196l), on Vicia faba (Hodgson and Blackman, 1956), on Agrostemma githago (Harper and Gajic, 1961), or on

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subterranean clover or others reviewed by Donald (1963) leads to similar conclusions. This problem can be examined in a different manner. If it was assumed that the degree of plasticity shown by a character was the outcome of the basic pattern of its developmental pathway, certain deductions would follow. First, since general pathways of characters cannot be changed readily, it should not be possible for plasticity to change readily. Second, since the same organ, e.g., leaf, usually has the same basic developmental pathway in different species, the organ should show the same plasticity in different species. That such arguments are not true can be seen from a comparison of the plasticity of a single character in a number of species. A conspicuous type of plasticity is the heterophylly shown by certain water plants. Such heterophylly is rarely characteristic of a whole genus; more commonly it is found only in particular species. Closely related species may differ markedly in the degree of heterophylly they show, as can be seen from the tabulated examples : Heterophyllous

Not heterophyllous ~

Potamogeton natans Ranunculus peltatus Sparganium erectum Juncus heterophyllus

P. lucens R. hederaceus S. minimum J . obtusijlorus

The occurrence of such differences was pointed out 70 years ago by Kerner von Marilaun and Oliver (1895). A detailed consideration of this type of ‘evidence will be made later in this essay. Marked differences can similarly be found in the amount of plasticity shown by varieties within species. Linum usitatissimum, in common with many annual plants, shows considerable plasticity in seed production in relation to variation in density. Khan (1963) has shown that linseed (oil flax) possesses a much greater capacity to respond in seed number to changes in density than flax (fiber flax) (Fig. 1). Indications of similar differences of plasticity in relation to density have been reported in other crops, e.g., in soybeans (Hinson and Hanson, 1962), in cereals (Engledow, 1925), and in sorghum (Karper, 1929). Differences in plasticity within spccies in relation to other environmental factors are to be found throughout agricultural literature, e.g., capsule number of different linseed varieties- in relation to nitrogen (Blackman and Bunting, 1954). Such differences are difficult to explain unless it is assumed that the plasticity of a character is an independent property of that character and is under its own specific genetic control.

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The stability, as opposed to the plasticity, of genotypes has recently received considerable attention. Here again there is evidence that the stability of a character can vary from one genotype to another and is genetically determined. Evidence that the stability of the omnibus character of yield can vary from one genotype to another has been reviewed by Simmonds (1962) under the term “general genotypic adaptation,” and the significance of this to plant breeding discussed by Allard and Bradshaw

70011

600

500

Redwina

/ /

200

I00

0 Densitykquare inches per plant)(log scale)

FIG.1. T h e response of four varieties of Linum usitatissimum t o changes in density (Khan, 1963).

(1964). In tomatoes Williams (1960) has shown that different inbred lines can differ markedly in their stability for a number of characters and that this stability is transmitted to the F1hybrids. An examination of his data also shows that stability levels are specific for individual characters within a single genotype and are not common for all characters of a single genotype. If the four inbred lines and three hybrids are ranked in order of their standard deviations determined in experiment 1, this is very clear (Table 1). It is confirmed by correlation analysis. None of the correlation coefficients determined between mean standard deviation values for all

TABLE 1 Mean Standard Deviations and Their Standard Deviations of Ten Replicates of Four Inbred Lines and Three F, Hybrids of Tomato Ranked in Order of Magnitude* Genotypes Character Fruit number Fruit weight Weight per plant Flower number Flowering date

59

XT/1

42

XT/2

5

XT/27

18

2.11 f 0.25 1 0.39 f 0.04 4 7.63 f 0.83 3 1.81 5 0.16 2 0.66 f 0.06 1

3.04 f 0.15 4 0.46 f 0.01 6 7.08 f 0.67 1 2.61 f 0.21 6 0.70 f 0.04 2

3.97 f 0.37 7 0.28 f 0.01 3 7.68 f 0.88 4 1.76 f 0.12 1 2.16 f 0.21 7

3.37 f 0.33 5 0.24 f 0.02 1 8.54 f 1.27 5 2.40 f 0.20 5 1.05 f 0.05 5

2.71 f 0.26

3.49 f 0.22 6 0.40 k 0.03 5 9.46 f 0.91 7 2.24 f 0.24

2.83 f 0.21 3 0.48 5 0.03 7 7.16 f 0 . 5 8 2 3.05 310.19

4

7

0.82 f 0.07 3

1.13 f 0.09 6

* Data of Williams (1960).

n

6 ‘

0.26 f 0.04 2 9.24 f 0.96 6 2.02 f 0.22 3 0.90 f 0.13 4

z

M 1: 0

32 d

r c u,

2

2

* c3

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A. D. BRADSHAW

possible pairs of characters approached significanceat the 10 % probability level. Ariyanayagam (1961) has shown similar differences in the stability of different characteristics of pure lines of barley in response to various environmental conditions. Evidence of a different nature is given by the studies of chiasma frequency in rye by Rees and Thompson (1956), who showed that control of stability of chiasma frequency between plants, between cells, and between bivalents is not the same at these three levels. Very extensive evidence is given by Clausen et al. (1940) in a series of experiments involving the growth of contrasting populations of a number of species under sun and shade, and wet and dry, conditions. Examination of their data shows that different characters, e.g., stem height, stem number, and flowering time, showed markedly different levels of plasticity. Moreover, their data shows that while contrasting populations differed in their plasticity for particular characters, such variation between populations in the plasticity of one character was not necessarily correlated with equivalent variation in the plasticity of another character. Further evidence is given by Paxman (1956) on stability of various characters in Nicotiana rustica. He demonstrated that, for various leaf and floral characters, different genotypes differed in their stability. Such differences were not the property of the whole genotype but were specific for individual characters. Different characters of a single organ (parts of the flower) did possess related stabilities; this might be expected in characters very closely grouped developmentally. Unrelated characters (leaves and flowers), however, had unrelated stabilities. By comparing the stability of characters in relation to both local fluctuations and gross environmental changes, he showed that these were not necessarily related. While it could be considered difficult to distinguish between these two types of environmental variation, this observation is of considerable significance. It implies that plasticity must be considered in relation to specific environmental effects a t specific stages of development. Langridge (1963) takes the same viewpoint. He has shown that stability due to heterosis in Arabidopsis under different conditions of stress is specific for particular factors. It was shown very positively in relation to high temperature, but not shown at all in relation to optimal temperature, low mineral nutrients, or other factors. It is further confirmed by the early work of Crowther (1934) on cotton. He showed that whereas internode number of the main axis was linked with nitrogen level, mean length was positively correlated with availability of water. Plasticity is therefore a property specific to individual characters in relation to specific environmental influences. This is only an extension of the arguments of Haldane (1946) who pointed out that genotype/environment interactions are ultimately entirely specific.

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In more general investigations where single characters have been examined in relation to over-all, often random, environmental influences, a relationship has been found in both plants and animals between stability and degree of heterozygosity (Dobzhansky and Wallace, 1953; Lerner, 1954; Lewontin, 1957; and others). I n crop plants the evidence similarly suggests that stability of performance can be achieved by heterozygosity (Allard and Bradshaw, 1964). It is clear, however, that there is an equal amount of evidence that stability may be determined by gene systems unrelated to heterozygosity as such. This is apparent from the evidence already presented. Observations on stability of certain characters in pure lines and hybrids of the inbreeding Nicotiana rustica showed that stability was quite unrelated to the occurrence of heterozygosity in the material (Jinks and Mather, 1955). The same situation was found in tomatoes (Williams, 1960). In maize it is clear from the work of Adams and Shank (1959) that while heterozygosity has considerable effects on stability it is not in itself a sufficient explanation of stability; hybrids belonging t o the same level of heterozygosity often differed -significantly in their stability. Evidence of direct genetic control of stability in wheat has been given recently (Frankel and Munday, 1963). Flower morphogenesis is very stable in normal types of Triticum aestivum but is unstable in speltoid mutants where Q is deleted. Experiments which are particularly critical are those where it has been possible to modify the level of stability by selection without any alteration in heterozygosity. I n Drosophila the degree of stability of bristle pattern, determined by the degree of asymmetry, has been changed by selection and shown to be under genetic control (Mather, 1953a; Thoday, 1955; Reeve, 1960). Similarly, selection for low variance of scutellar bristles in scute flies has been effective and has incidentally caused decreased sensitivity to temperature changes (Rendel and Sheldon, 1960). Stability of development time has been altered by disruptive selection in a manner implying an increase in the environmental components of variance (Prout, 1962). Waddington-( 1960) has been able to increase markedly the stability of the bar phenotype in Drosophila in relation to temperature variation. In this investigation it was also possible to show that the gene system determining the canalization of the character in question was specific to that character and did not influence the degree of asymmetry of facet number. The influence that selection may have on the stability or plasticity of a character is perhaps most elegantly demonstrated in the investigations on genetic assimilation by Waddington and others (Waddington, 1961). I n Drosophila the ease of production of venation phenocopies by high temperature shock was radically altered by about a dozen generations of selection for ease of production (Waddington, 1953; Bateman, 1959).

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Similarly the ease of production of bithorax by ether treatment was radically altered by selection. In a character not involving a threshold effect Waddington (1959) was able to improve, by selection, the capacity of a strain of Drosophila to react to increased salt content in the medium by the development of large papillae. This is a particularly significant experiment since in this case the changes appear to be of adaptive significance. The altered strains show increased survival in high salt media. I n all these experiments the environmentally induced effect eventually became assimilated and determined genetically. Nevertheless, the significance of the results to the present consideration is that the plasticity of the genotype in one specific respect and direction could be radically altered by selection in a few generations. 111. Fitness, Plasticity, and Selection

The evidence presented so far suggests that the plasticity of a character can be ( a ) specific for that character, (b) specific in relation to particular environmental influences, (c) specific in direction, (d) under genetic control not necessarily related to heterozygosity, and (e) radically altered by selection. Up to this point no attempt has been made to attach any particular adaptive significance to the ability (or otherwise) of a character to be altered by environmental influences. In many cases wide fluctuations in a character owing to environmental influences can be considered to indicate that there is a lack of adaptation and that the genotype concerned is inadequately buffered against the environment. This is particularly true in animals. Wide deviations in characteristics of animals owing to environmental effects are not usually found because of an organization which buffers the individual against the environment. Deviations can usually be considered as indications of lack of adaptation. The classic data of Bumpus (1899) on sparrows substantiate this. The viewpoint colors much of the current investigations on stability, homeostasis, etc., particularly in Drosophila. It can also be true in plants. A plant at the point of death owing to unfavorable circumstances may be very reduced and show considerable distortion. This can readily be seen in the altitudinal transplant experiments of Clausen et al. (1940, 1948). Plants grown in inappropriate environments in their experiments show a great reduction and upset of growth. Such instability can be correlated with lack of adaptation to those environments. In all organisms, however, maximum fitness does not require the same degree of stability in all characters. As a result of natural selection those characters in which stability is paramount for survival are likely to show greater stability than those in which some plasticity is not a disadvantage.

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This is apparent from the observed coefficient of variation ascribable to asymmetry in wing length of Drosophila, 0.48%, compared with that for sternopleural chaeta number, 4.84 % (Mather, 19534. The argument may be taken further. In some organisms plasticity in some characters may not be neutral in adaptive value, but of positive selective advantage, as has been argued by Baur (1930), Schmalhausen (1949), and Mather (1955). This will be particularly true in those types of organisms in which highly complex buffering processes are not present, where the organism grows in accordance with the opportunities provided it by the environment. Plants are essentially such organisms. It has been pointed out that in studies of environmentally induced variation, it is not sufficient to know that a character is of some significance in determining fitness; the effect of its variation is also pertinent (Lewontin, 1957). In plants more is known of the effects of such variation than in other organisms. Plants therefore provide excellent material in which we may seek to discover the adaptive value of plasticity in evolution. Not all plasticity is adaptive, but some is. The conditions under which plasticity is likely to be selected for, and the nature of the plasticity, must therefore be considered. IV. Conditions Favoring Plasticity

It is convenient to consider plasticity in plants in relation to the various types of selection that may operate at one time or another on plant populations, using the terminology of Mather (195313). It will become clear that many different conditions may favor plasticity. If this seems to result in contradictions, it only illustrates the complexity of evolutionary processes in plants. Two essential general points must be made at the outset. Because of their system of nutrition, plants are essentially static, fixed organisms, incapable of movement except during reproductive processes and then normally only by a rather passive scattering of propagules. The new generation falls to the ground and thereafter must endure the particular environment in which it finds itself, including any fluctuations which may occur subsequently. Because they possess complex behavioral responses and locomotory mechanisms, animals do not of necessity have to do this. The infinity of different behavioral patterns which have evolved in animals relate to the degree animals are often able to move from one environment to another, evading those which are unsatisfactory to them and selecting others (Waddington, 1957). Such behavioral plasticity has little equivalent in plants. It should not, however, be construed that animals are always able to evade adverse conditions and have in contrast no physiological and mor-

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phological plasticity. There are many conditions such as season, chronic shortage of food, etc., which they cannot evade. In relation to these, many different forms of plasticity have been evolved, e.g., in coat color, litter size, etc. Plasticity, however, appears to play a more important part in adaptation of plants. Plants, except those that are not holophytic, must also live in environments which provide the necessary raw materials for their growth. In other words, they must live in situations where they are likely to be strongly exposed to extremes of climate and other influences. A t the same time the holophytic type of nutrition can occur efficiently only in a structure which in many important respects will be more attenuated and physically weaker than that of animals; a structure in which elaborate mechanisms buffering the organization of the individual against the environment cannot easily be developed. The only exceptions to this are plants such as cacti: but these achieve stability only at the expense of rapid growth. A plant is therefore both vulnerable in organization and unable to move away from an environment which is unsuitable to it. Lacking behavioral plasticity, other types of plasticity are likely to be favored.

A. DISRUPTIVE SELECTION Disruptive selection can be due to recurrent variation in selection in either time or space. If it acts on populations without reproductive isolation, it can cause either (a) genetic polymorphism or (b) plasticity. The part played by disruptive selection in causing genetic polymorphism has been discussed by Mather (1955), Huxley (1955), Thoday (1959), and others. The occurrence of genetic polymorphisms in the strict sense has been reported in plants, but much less frequently than in animals. This is difficult to understand, since the occurrence of disruptive patterns of selection on plant populations must be no less frequent than on animal populations. It is possible that the scarcity of evidence is due to the lack of observation, for little is known about levels of genetic variability in plant populations; recently, several investigators have recorded the existence of genetic variability in populations not previously suspected (e.g., Allard, 1963; Zohary and Imber, 1963). Nevertheless, the ease with which plants are able to respond to disruptive selection by plasticity is perhaps itself the reason why genetic polymorphisms are not conspicuous. Theoretical support for this argument has been given by Levins (1963). 1 . Disruptive Selection in Time

If a population is subject to recurrent changes in its environment, whose duration is the same as or less than its generation time, it cannot

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easily respond to the contrasting environments by directly adaptive genetic changes. If the duration of the environmental fluctuation is much less than the generation time, any adaptation that occurs can only take place by plasticity. If the duration of a single environmental condition is more or less equal to the generation time, it is possible for adaptation to take place by genetic polymorphism; the particular morph that is adapted in any one generation becomes the most prevalent, the others being reduced in number. But such a situation leads to considerable elimination of individuals; the progeny of the best adapted plants of one generation tend to be the least well adapted in the next. This also applies when the oscillation of the environment is longer than that of the generation time. A t the same time, any genetic adaptation that occurs is always too late (Kimura, 1955; Crow, 1955). Therefore adaptation by plasticity is more likely. This possibility was envisaged by Darwin; “I speculated whether a species very liable to repeated and great changes of conditions might not assume a fluctuating condition ready to be adapted to either condition” (letter to Karl Semper, 1881). The only situation in which adaptation to recurrent changes in environment of shorter duration than the generation time of the population can be achieved by direct genetic changes is if the change is not random but occurs regularly at a particular phase in the life cycle. Then intrinsically timed developmental changes, which are genetically determined, can be evolved, adapting the population to the particular environmental condition concerned. For example, flowering in many annuals occurs after a certain period of growth and therefore coincides with optimal summer conditions. There is, however, a close relationship between such changes and plasticity which is discussed further in Section VII. If the changes are random and not regular, intrinsically determined changes cannot be successful. Only adaptation by plasticity will be effective. Random changes in environment coincident with, orlshorter than, generation changes are almost inevitable in plants because of the passive and therefore unselective distribution of their seeds. Environmental conditions are neither constant in time, nor are they constant in space. The seeds shed from a plant growing in good conditions will not all land in equally good conditions. The significance of this is nowhere more obvious than in annual weedy and other species. During its life an annual suffers changes in its environment, but from generation to generation the environment in which it finds itself may also vary radically. If it is to survive it must be able to grow and reproduce effectivelyin each condition. There will be selection for the individual which can both survive in a reduced state in difficult conditions and grow vigorously in optimal conditions. The degree of over-all plasticity, particularly of seed production, shown

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by annuals is in great contrast with that of perennials. The annual grass Polypogon monspeliensis has already been discussed : its plasticity of panicle size is in marked contrast with that of the closely related perennial Agrostis tenuis. If we examine, however, whether panicles are produced or not, the plasticity is reversed. Under very adverse conditions P. monspeliensis still produces panicles, although reduced in size; in contrast A. tenuis fails to produce any panicIes at all. I n terms of presence or absence of sexual reproduction, the perennial here is more plastic than the annual. A similar contrast can be niade between Poa annua and Poa pratensis: and between annual and perennial Bromus species (Stebbins, 1964). Everyone is familiar with the plasticity of weeds of cultivation in relation to soil fertility and density. Sonchus oleraceus has been observed to vary in number of capitula from 1 to 1600 (Lewin, 1948) ; this is in great contrast to the related perennial Sonchus arvensis. Chenopodiumalbum shows remarkable plasticity in comparison with C. rubrum, which may explain its success as a weed (Cumining, 1959). Annual weeds in particular are subject to considerable fluctuation in density from one generation to another. Agrostemma githago shows considerable plasticity in relation to density, although in contrast Papaver species respond to density by mortality of some individuals (Harper and Gajic, 1961). The contrast in the plasticity that can be found within a single annual species Linum usitatissimum, between flax and linseed, which have become adapted to being sown at different densities, has already been mentioned (Khan, 1963). An extensive experimental examination of the plasticity of a range of populations within an annual species Capsella bursa-pastoris has been made by SGrensen (1954). The populations were subjected to contrasting environments by being grown in fertile garden soil or in coarse poor sand. The latter condition caused very reduced growth, reduced rate of leaf production, and considerable alteration of flowering time. But the significant fact is that populations differed very markedly in their response to these conditions, not only in amount but also in direction. Thus population 7, collected from a luxuriant cultivated field, was constitutionally early flowering. I ts reaction to growth in sand was to increase the number of leaves and relatively retard flowering. By contrast population 22, COIlected from moist beach sand, was constitutionally late flowering. I n the sand treatment its reaction was to reduce the number of leaves and relatively advance flowering. Other populations showed little or no change in leaf number or in flowering time. Such differences in plasticity could be shown t o be of adaptive significance. Thus, population 22 was perfectly successful in sand, whereas population 7 failed to flower and set seed satisfactorily. These differences can be related to the original habitats of the populations.

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Perennials also may show marked plasticity in particular characters. A species with a long life cycle must of necessity endure variations in environment which cannot be met by genetic changes. Many different types of plasticity may therefore be found in perennials. In contrast with annuals however, perennials do not usually show the same marked plasticity of seed production except in its presence or absence. This can be related to the fact that reproduction by seed does not play such an important part in the survival of a perennial from one year to the next and seed production is spread out over many years. The most obvious environmental changes affecting a perennial are those connected with season. Although perhaps it would not be thought of as a type of plasticity, the deciduous habit must logically be considered a highly developed form of it. A deciduous species undergoes a startling change in shape, involving not only the loss of leaves but also the development of specialized protective structures such as bud scales, by which it becomes adapted to the severity of winter conditions. Closely related species differ in the degree to which they show this form of plasticity, e.g., Quercus robur which loses its leaves over winter, and Quercus ilex which retains them. In California the same contrast is shown by Q . douglasii and Q. turbinella (Tucker, 1952) ; these two species form hybrid populations where they meet, in which individuals with all grades of leaf persistence occur. Herbaceous species may retreat into a variety of underground storage organs. Seasonal variations niay be related to drought. Here again many highly developed types of plasticity have been evolved in adaptation to variation in water supply. For instance, Cercidium floridurn, a desert shrub, produces leaves only after rain and these are shed when dry conditions return. Innumerable other examples of the evolution of plastic responses in relation to seasonal variations are available. Different types of response may be shown not only by closely related species, but also by different populations of a single species. This is apparent from the work of Clausen et al. (1940, 1948) on Potentilla glandulosa and Achillea millefolium. One of the most conspicuous differences between the ecological races of each of these species is in the occurrence of vegetative dormancy. The high altitude populations are all winter dormant, the low altitude populations from dry areas are summer dormant, and the low altitude populations from coastal, moist habitats have no dormant period at all. Such differences can be related readily to the habitats concerned. A second particularly significant set of experiments carried out by these investigators was that already mentioned involving the growth of contrasting populations of a number of species under conditions of sun and shade, and drynessland wetness. They were able to show considerable differences in response

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between populations. Thus, in general, the alpine populations showed little change in general morphology, whereas those from lowland areas showed marked plasticity, e.g., Potentilla glandulosa ssp. nevadensis and ssp. typica, respectively. As has already been pointed out, however, it must be remembered that the plasticity involved only certain characters and not others. The different levels of plasticity between populations and between characters can be related to their adaptative significance in the environment concerned. In Lolium perenne Cooper (1963) has shown that populations from different regions of Europe differ markedly in their rate of leaf expansion with temperature. The northern populations are very sensitive to temperature, showing very reduced rate of expansion at low temperatures. Southern populations are markedly less sensitive showing little reduction in rate of expansion at low temperatures. This difference can be shown to be of adaptive significance; the plasticity of leaf expansion of the northern populations gives higher survival under severe winter conditions. In Solidago virgaurea leaf expansion in response to light intensity has been found to differ in populations from habitats of differing seasonal light regimes, in a manner suggesting adaptive significance (Bjorkman and Holmgren, 1963). The photosynthetic mechanism itself is able to alter in adaptation to variation in environment. Measurements of photosynthesis over a range of environments indicated that both rate and position of optimal photosynthesis can be altered by previous exposure to particular environments. Such changes, termed acclimation, are in the direction that implies increased adaptation to the new environments. Recently, Mooney and West (1964) have shown that five Californian species differ in the degree of acclimation of photosynthesis exhibited to temperature and suggest this may be related to the natural distribution of the species. Bjorkman and Holmgren (1963) have shown that populations of Solidago virgaurea differ markedly in their ability to acclimate photosynthesis to changes in light intensity; the differences appear to be the result of genetic adaptation. Physiological acclimation to drought, frost, etc., is also well known. Both physiological and morphological plasticity therefore occur in adaptation to seasonal fluctuations. Despite the complexity of the responses it seems that such plasticity can be evolved quite readily. Seed dormancy and germination, whether of annuals or perennials, show very clearly the influence of disruptive selection and the development of plasticity in response to it. Seeds are likely to be most successful if they germinate when conditions will continue to be satisfactory for their subsequent development into mature plants. This will happen when the season is appropriate, when they are near enough to the surface to be able

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to reach it without exhausting their reserves, or when there is little competition from other vegetation, etc. If every seed that fell t o the ground germinated in its own inexorable manner without relation to external circumstances, a large proportion would do so in unfavorable situations and fail to survive beyond the seedling stages. This is well shown by the breakdown of dormancy in Papaver hybrids (Harper and McNaughton, 1960). Absence of dormancy in interspecific hybrids permits the seeds to germinate in the field in autumn. These seedlings are then commonly killed by winter frosts. There are a large number of mechanisms of enforced dormancy which prevent or reduce the likelihood of this happening. In many species, e.g., Juncus eflusus and Betula pubescens, only those seeds which are in the presence of light will germinate; in others the only seeds that will germinate are those which have experienced a period of low temperature, especially summer annuals, or a period of high temperature, especially winter annuals. Some require particular oxygen or nitrate concentration. Others, e.g., Stellaria media, have as yet undefined mechanisms, perhaps combinations of the preceding ones, which result in only those seeds germinating which are close to the soil surface. The mechanisms are many. They must all be considered examples of physiological plasticity, because the environment is able t o alter markedly the physiological state of the seed and the occurrence of germination. I n most cases the adaptive significance of such plasticity of germination is very obvious and can be related t o the particular habitat requirements of the species concerned. Closely related species may differ enormously in their germination behavior, as has been described by Crocker and Barton (1957), Harper (1959), and Thurston (1960). An important effect of variation in environment in time concerns reproductive processes, particularly pollination. A common adaptation to difficult conditions is cleistogamy, where flowers are self-fertilized without opening. Some species are permanently cleistogamous, but species are more commonly facultatively cleistogamous (reviewed by Uphof, 1938). The occurrence of cleistogamy in such species is determined by various ecological factors. The flowers of Ranunculus moseleyi, and R. apuatilis (S. A. Cook, 1964) , produced under water are cleistogamous, whereas those above water are chasmogamous. I n Dicliptera assurgens and other species in the West Indies cleistogainic flowers are produced in the dry season and normal flowers a t other times. I n Bromus carinatus cleistogamic flowers are produced in dry years and chasmogamous flowers in wet years (Harlan, 1945). I n Viola odorata cleistogamic flowers are produced under summer conditions. Other factors are important in other species. These species therefore exhibit distinct plasticity in this character.

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I n most cases the occurrence of cleistogamy can be inferred to be of adaptive value, enabling the species to overcome conditions adverse to chasmogamy. Closely related species not subject to such adverse conditions are usually found not to possess this plasticity. I n some species, e.g., Sporobolus subinclusus, cleistogamy is obligate; these are then not plastic. Agricultural situations provide interesting conditions of disruptive selection in time. Variation in crops from one season to another causes variation in environmental conditions for associated species. This is well exemplified by the variation found in Camelina sativa (Zinger, 1909 and Sinskaia and Beztuzheva, 1931, in Stebbins, 1950). This species is a weed of arable fields, and has two subspecies. The typical form is found in many crops, including flax. It has a wide branching growth form which is very plastic. When growing with flax it assumes a taller, less branched form similar to flax. Where flax is grown intensively this form is replaced by subspecies linicola, which is found only as a weed of flax. It is not as plastic and under all conditions possesses the taller, less branched growth form. It appears that the typical form has evolved a plasticity enabling it to survive in a variety of crops, whereas subspecies linicola has evolved specifically in relation to flax and therefore has lost this plasticity. Other species, e.g., Chrysanthemum segetum, Papaver rhoeas, and Chenopodium album, which are weeds of several contrasting crops, possess a high degree of plasticity and like Camelina sativa are able to compete more effectively with the crop in which they are growing. Grazing may be considered a marked environmental effect, the incidence of which varies enormously with time. It is noticeable that species vary considerably in their response to the many different consequences of grazing. Some species tiller strongly at the base and regrow rapidly, e.g., Poa trivialis, whereas others do not, e.g., Poa pratensis. The over-all height of vegetation may fluctuate markedly, and it is interesting that some species possess the ability to adjust the height of their leaves in relation to it. The petiole of Trifolium repens must be a n example of one of the most plastic plant organs known, an idea implied by Kerner von Marilaun and Oliver (1895). Plantago lanceolata achieves the same adjustment by remarkable plasticity in leaf length. Recent work on Trifolium species (Harper, 1961) suggests that species differ in this plasticity, the petiole of Trifolium fragiferum possessing greater plasticity than that of Trifolium repens. Black (1960), working with strains of Trifolium subterm n e u m , has shown recently that these differ remarkably in their ability to elongate petioles. The adaptive significance of this was shown in uncut swards containing a mixture of two strains. The strain with the greatest potentiality for petiole elongation rapidly became dominant. Other factors of the environment which may vary in time are many.

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It would appear that examples of plasticity may be found in relation to all of them, but space does not permit their treatment in this essay. Further examples are given by Kerner von Marilaun and Oliver (1895), Davy de Virville (1927-1928), Salisbury (1940), Clements et al. (1950), and others. 2. Disruptive Selection in Space

The environment of a plant species may not only change in time; it may also change in space. If changes in space occur over reasonable distances the plant species usually adapts by the formation of localized races or ecotypes. The distances necessary for such differentiation are now known t o be as little as 10 meters (Bradshaw, 1963). Changes in environment, however, can occur over much smaller distances than this, distances which may preclude the formation of genetically different populations. The most elegant example of this is provided by areas of shallow water. Here three different environments occur in close proximity, under water, at the water surface, and in the air above the water. Although specialized plants such as Lemma species can grow in one of these environments, the water surface, without growing in the others, species which root in solid material can only grow in the upper environments by also growing in the lower. These plants are therefore subject to a violent and unchanging form of disruptive selection, which can only be met by changes occurring within each individual. For this reason some of the most remarkable types of plasticity are to be found in water plants. This has been argued by Arber (1919). Species of Ranunculus, subgenus Batrachium, show a remarkable degree of heterophylly. Submerged leaves are finely dissected, adapted to the conditions of flowing water, whereas floating leaves are entire or lobed. The species differ in their plasticity, in whether they produce leaves which are of both types. The behavior of the British species (Clapham et al., 1962) is given in Table 2. Experimental studies have shown that these differences in plasticity are permanent and therefore genetic characters of the species. The species that are plastic for leaf shape, th a t are able to produce both sorts of leaf, are all typically species of shallow water; whereas those that can produce only floating leaves are species of mud or very shallow water; and those that can produce only submerged leaves are those of deep or swift flowing water. Similar variation in plasticity is shown by species of Potamogeton (Clapham et al., 1962; Dandy, 1961). All species produce submerged leaves, but some can produce both submerged and floating leaves. Innumerable hybrids are known; their plasticity can be related t o the plasticity of the particular parents and emphasizes its genetic determination. The plasticity, as far as it is known, of the British species and their

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TABLE 2 The Occurrence of Heterophylly in British Species of Ranunculus Subgenus Batrachium * ~~

Leaves Habitat

Species

Floating

-

Submerged

R. hederaceus L. R. omiophyllus Ten. R. tripartitus D.C. 12. fEuitans Lam, R . circinatus Sibth . R. trichophyllus Chaix R. aguatilis L. R. peltatus Shrank

Many Mud or shallow water Many Streams and muddy places Muddy ditches and shallow ponds Some Rapidly flowing rivers and streams Ditches, streams, ponds, and lakes Ponds, ditches, slow streams Some Ponds, streams, ditches, river

Many Many Many Many Many

ssp. peltntus ssp. pseudofluitans R. baudotii Godr.

Lakes, ponds, slow streams Fast-flowing streams Brackish streams, ditches, ponds

Many Rare Some

Many Many Many

* From Clapham et al. (1962). P. NATANS P. POLYGONIFOLIUS

0

P. ALPINUS

P. COLORATUS

P. ACUTlFOLlUS

P. LUCENS

P. PRAELONGUS

P TRICHOIOES

P. PERFOLIATUS

P. EPIHYDRUS

0

//

P. BERCHTOLDII

P. OBTUSIFOLIUS P. FRIES11

@

P. RUTILUS

FIG.2. Heterophylly in British species of Potamogeton subgenus Potamogeton, and in their naturally occurring putative hybrids (Clapham et al., 1962; Dandy, 1961). Circles, species; lines, hybrids: white, heterophylly present; black heterophylly absent.

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hybrids is given in Fig. 2. Further work on this group is, however, necessary. This type of plasticity may be quite complex in some species and involve more than one character. Proserpinaca palustris, for instance, is an aquatic with submerged dissected leaves and entire aerial (not floating) leaves. The dissected leaves are borne on horizontal stems, whereas the entire leaves are borne on erect stems (Burns, 1904). Similar complex responses can be found in Ranunculus species. It is difficult to believe that the character of dissection of leaves and the tropic response of the stems are physiologically connected. It would appear that two characters are involved independently in the one response. Many other aquatic species show similar plasticity, whereas other related species may not. Some examples of this have been given earlier. Further examples are discussed by Gliick (1905-1924). Since it is clear that the character of heterophylly is under genetic control and can be of selective advantage, it would seem likely that variation in this character should be found within species and vary from one population to another. There appear to be no records of this in the literature. In Polygonum amphibium, however, a species which shows marked heterophylly (Massart, 1902), populations have been found which differ markedly in the degree to which they can show heterophylly (Turesson, 1950). Recently, a similar situation has been found in Ranunculus baudotii and R. trichophyllus (C. D. K. Cook, 1962, 1964), and in Ranunculus JEammulaand R. aquatilis (S. A. Cook, 1964). In many other habitats similar extreme variations in environment occur over distances which are too short for adaptation to occur by genetic differentiation. Gregor (1956b) discusses an example of this in Plantago maritima. In an exposed area occupied by a fairly dwarf population, shallow depressions occurred affording some protection from the wind. In these depressions the individuals of Plantago maritima in situ were taller than those of the general area. Upon cultivation, however, in five out of the six samples examined the difference disappeared. Elsewhere in the same locality where the exposure was uniformly less, populations of a genetically taller type did occur. In the mosaic of communities on the exposed summit of Monte Maiella in Italy, Whitehead (1954, 1956) has shown the significance of modifications induced by exposure. He suggests there has been selection for single genotypes with high plasticity, rather than different genotypes adapted to different habitat conditions. He points out that species with low plasticity appear to be limited to one community type. Another apposite example is provided by work on Ranunculus hirtus (Fisher, 1960). This is a plant which occupies the variable habitat condi-

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tions of cool lorest margins in New Zealand. Its leaves show a greater degree of dissection in the pastures outside the forest than within the forest. This appears to adapt it to the relatively dry cool conditions of the pasture. The modification is controlled by temperature. Experimental studies have shown that not all populations possess the same degree of plasticity. Populations from the South Island of New Zealand show the greatest plasticity, those from the North Island less plasticity, and those of a closely related species from southeast Australia, R. plebius, least plasticity (Fisher, 1964). There appears to be an adaptive explanation for this difference in sensitivity, since the most marked variation in climatic conditions occurs in the South Island habitat and the least in the Australian habitat. Under the same forest margin conditions R. hirtus also shows plasticity in hairiness, degree of prostration, and over-all size. These characters are determined by humidity and light intensity, as well as by temperature, and appear to be independent of the plasticity of leaf dissection. It is clear that many other examples are to be found. Some that have already been discussed in relation t o disruptive selection in time are appropriate here. But only in a few cases have they been adequately investigated, and the adaptive significance of the changes been determined.

B. DIRECTIONAL SELECTION If directional selection is very severe and the normal, directly adaptive, genetic variation is limited, further adaptation may be afforded by increased plasticity. Experimental evidence for this in Drosophila comes from the work of Thoday (1955,1958). It is common experience that plant populations collected from severe habitats grow more luxuriantly under garden conditions, although they still retain a considerable degree of distinctness (smaller size, etc.) indicating genetic differences from other populations (Kerner von Marilaun and Oliver, 1895, Turesson, 1922, 1925; Clausen et al., 1940, 1948). This suggests that plasticity is supplementing the genetic adaptation already present. Further evidence comes from the fact that an adaptation observed in the field may be found to be genetically determined in some cases, but environmentally determined in others. Turesson, in particular, has recorded a number of examples (1920, 1922, 1925): Prostrate maritime forms Atriplex latifolium Atriplex patulum Chenopodium album

Lax shade form Dactylis glomerata Dwarf subalpine form Ranunculus acris

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The response t o local variations in habitat by phenotypic change in Plantago maritima (Gregor, 1956b), already discussed, is another example. Plastic response is able to provide adaptation to directional selection in some populations which in others is provided by genetic change. The individuals in the former populations may properly be considered phenocopies of the individuals in the latter populations. From the magnitude of such environmentally determined modifications, it is clear that this type of plasticity may be of considerable significance, although there is no indication how much it may vary from species to species or how much it is present in species not subject to intense directional selection. I n most cases the genetically determined forms, would appear to be relatively simple permanent changes, e.g., in Ranunculus acris and Dactylis glomerata. In the case of the genetically determined prostrate forms of Atriplex latifolium and A. patulum, however, Turesson (1920) showed that this was not so. I n these forms the prostrate habit was not permanent and was lost at low light intensities. The prostration under normal conditions was due to greater sensitivity. These forms therefore retain a certain degree of plasticity which will appear under extreme conditions of low light intensity, for instance under conditions of severe competition, where it will be of adaptive value.

C. STABILIZING SELECTION Not only can plasticity permit a single genotype to assume different phenotypes, it can also enable different genotypes to assume a single phenotype. It can therefore cover up variation in a manner analogous to dominance. Where selection is acting in a stabilizing manner it can permit the population t o assume a uniform phenotype, while retaining a considerable degree of genetic variation. Many of the arguments that apply to the evolutionary significance of dominance are applicable to plasticity. Few clear-cut records of plasticity acting in this manner are available, although common sense suggests th at it could be found commonly if appropriate measurements were made. The most critical records are those of Gregor (1956a). A population of Plantago maritima, growing wild in Iceland, showed less than one-quarter the variation in plant size of a Sample of the same population grown in a n experimental garden. Other populations which were uniform in growth form in their natural habitat also showed considerable variation in growth form on cultivation. Turesson (1922, 1925) records a number of similar cases in which populations which were more or less uniform in their natural habitat showed considerable variability when brought into cultivation. These are listed below:

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Succisa pratensis, maritime dwarf population from Hallands Vadero Centaureajacea, maritime dwarf population from Torekov Atriplex littorale, unspecified maritime population Atriplex sarcophyllum, maritime population from East Coast Sedum maximum, maritime cliff population from Kallen and Varberg Hieracium umbellatum, sand dune population from Vietmolle V. Conditions Disfavoring Plasticity

Not all environmental conditions favor the development of plasticity in plants. Any condition of stabilizing selection, where deviations from the optimum are selected against, will tend to increase canalization and stability, apart from the effects it may have on genetic variance, as has been considered earlier. Thus for some plant characters constancy is of adaptive significance, and deviations lead to reduced fitness. This seems to be true of seed size. It is interesting that in comparison with other characters it shows great phenotypic stability, as has already been shown in Linum and Polypogon. It would seem reasonable to argue that constancy of flower size and shape, in insect-pollinated plants particularly, is of considerable adaptive significance; wide deviations in size could be expected to lead to failure of pollination. General observations show that flowers show much less phenotypic variability in size than other organs. Thus in the hemiparasite Euphrasia presence of a host causes considerable increase in vegetative size (X2.2) but only a very small increase in flower size (Xl.1) (Wilkins, 1963). The significance of stabilizing selection in plants, however, has been discussed by Berg (1959) and will not be considered further here. But there are other conditions where stabilizing selection is not occurring, where plasticity also may not be favored by natural selection. For instance, under some conditions of disruptive selection a system of plasticity might be adaptive in itself but only achieved at too high a cost to thearganism. This is very likely with a character that is permanent, and the only means of modification is by the loss of the organ concerned and the regrowth of a new one. This will be true in perennials when adaptation to seasonal environmental variation can only be produced by many modifications of an individual during its lifetime. In these cases, spectacular plasticity is only achieved when it is accompanied by other processes which minimize its deleterious effects, e.g., in the deciduous habit where shedding does occur, but is accompanied by translocation of important materials out of the leaf before shedding. Waddington (1957) has suggested that it may also be difficult for plasticity to be of value in annuals if the modification is not easily reversible. An early adverse period in the life cycle of an individual might so

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modify it that it was unable to take advantage of succeeding good periods or vice versa. Early waterlogging can cause a plant to have a shallow root system which is ill-adapted to later drought. Such environmental variations are again those of short duration relative to the total life-span. We have already seen that if the variations are of the same length as the lifespan, then spectacular types of plastic response may occur, e.g., in annual weeds. Under these conditions the irreversibility of the plasticity is no embarrassment. It may also be difficult for plasticity to be of adaptive value if the environmental change, to which the plasticity is adaptive, occurs suddenly. The plant may be unable to assume its new state rapidly enough so that it suffers damage before it becomes fully adapted. This must be very frequent in plants, e.g., in relation to frost, storm, drought, etc. In this case, as Turesson (1922) suggested, the species may only be able to adapt by permanent genetic changes, so that it is already in the appropriate state before the critical environmental changes occur. It is difficult to see the validity of the arguments of Underwood (1954) who suggests that such situations are uncommon. Finally, it may be biologically impossible for the plant to achieve the necessary degree of adaptation by a system of plasticity, owing to the mere limitations of the potentialities inherent in the genotypes. In other words, it may be beyond the capacity of the species to evolve an effective system. Such practical considerations go a long way to explain why, inspecies that occupy a range of environments, a single genetic type with infinite plasticity is not found, but rather a myriad of genetically distinct individually adapted populations. Indeed, although adaptation by plasticity plays an important role in many situations, permanent adaptation by genetic change is more common. Most plant species consist of separately adapted ‘genetically distinct populations whose characteristics are determined, not by the optimal conditions of growth, but by the most adverse. This can be seen from an examination of any work on the pattern of differentiation within species (e.g., Clausen et al., 1940; Bradshaw, 1959). Waddington (1957), following Warburton (1955), has suggested that such genetically adapted populations are “sewn into their winter underwear” to stop it from being blown away. Rather it would appear that either they cannot afford to possess other underwear or they are unable t o change it quickly enough. Adaptation by plasticity is exogenous; adaptation by genetic change is endogenous. The latter changes often have effects resembling the former; in most of the examples that have been discussed this is true. In this case such genetic changes can be termed pseudoexogenous (Wadding-

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ton, 1957). Arguments based on adaptive value suggest reasons for this resemblance. If the same factor is involved, even if in one situation adaptation by plasticity is more successful whereas in another situation permanent adaptation by genetic change is more successful, this common factor is good reason why the two types of adaptation should resemble one another, Waddington has produced extensive evidence (review, Waddington, 1961) showing th at such resemblance can arise as a result of genetic assimilation. While it is clear that this is an important phenomenon, it seems logical that the resemblance can also be due to parallel adaptation. VI. Mechanisms of Plasticity

The development of a character can be considered to be determined by a n epigenetic landscape (Waddington, 1957). The stability of a character depends on the degree of canalization. The manner in which a character may be modified by the environment is dependent on the pattern of the epigenetic landscape. The many ways in which plants may react to variations in environment show that the patterns of the epigenetic landscape are many and various. At the one extreme is the character which shows a continuous range of modification dependent on the intensity of the environmental stimulus. This has been termed “dependent morphogenesis” by Schmalhausen (1949). The pathway of development can be considered to be a broad flat delta, so that the individual or character can follow any number of different paths to the final adult state. Such characters as height and seed number follow this sort of pathway. In these characters adjustment can be continuous. This is shown by the remarkable manner in which many species can adjust seed output per plant to density so that the seed output per unit area is constant over a wide range of densities (Harper, 1961). At the other extreme is the character which shows a series of discrete modifications, often only two, with no intermediates. I n this case the pathway can be envisaged as two divergent steep-sided valleys with the environmental stimulus merely acting as switch. This type of plasticity has been termed “autoregulatory dependent morphogenesis” by Schmalhausen (1949). He justifiably argues that it is due to the occurrence of stabilizing selection in each phase of the regularly varying environment. Such a character as leaf form in Ranunculus peltatus follows this pattern of development. The canalization of the two types of leaf shape is considerable; there are no intermediates. Where apparent intermediate leaves do occur, it will be found that these are in fact composites of the two patterns of development and that within the leaf a sudden switch from one t o theother has occurred. This has been described for British

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species of Ranunculus (Cook, 1963). Not all water plants with heterophylly show such a degree of canalization. In Ranunculusflabellaris all gradations of dissection of leaf may be found (Bostrack and Millington, 1962). I n Sagittaria sagittifolia, where failure to form lamina occurs in submerged leaves, all grades of reduction of lamina may be found. Similar gradation can be found in Oenanthe aquatica, Sium latifolium, and Rorippa amphibia. Water plants, however, tend to show a higher degree of canalization of alternate growth forms than most other plants, which can be related to the constancy of the distinctness of the contrasting habitats available. An equivalent high degree of canalization of each of two forms is shown in the deciduous habit, which again can be related to the regularity of the environmental variation concerned. A similar degree of Canalization occurs in facultatively cleistogamous species already discussed (Uphof, 1938). The cleistogamous and chasmogamous forms are usually quite distinct in the degree of exsertion of anthers. It seems reasonable t o believe that this is because intermediate forms are unlikely to be effectively pollinated. Nevertheless, there are all grades of distinctness in associated structures. I n the grass Scleropoa rigida anthers and lodicules of cleistogamous flowers are little different from normal flowers. In Avena scabrivalvis, Bromus carinatus (Harlan, 1945), and other grasses, however, the cleistogamous flowers have very reduced anthers, stigmas, and lodicules. In Mayaca fluviatilis the anthers of the cleistogamous flowers are spoon-shaped and reach over the stigma effecting fertilization easily. I n Viola odorata cleistogamous flowers are geotropic from the beginning, whereas chasmogamous flowers are orthotropic until fertilized. This plasticity may therefore involve a highly complex set of modifications in some species, a point which has been made earlier. A further distinction in mechanism that can be made is whether the plasticity is between plants or within plants. This will depend in particular on the duration of life of the plant. Perennials will inevitably show withinplant plasticity, whereas short-lived annuals are more likely to show between-plant plasticity. It will also depend on the reversibility of the response. Plasticity in irreversible response involving the whole plant can only be expressed between plants. Thus plasticity of seed germination is only expressed between seeds; plasticity of seed number in many annuals tends t o be expressed between plants since seed formation takes place over a short period. B y contrast plasticity of petiole length in Trifolium species is reversible (although it involves the production of new leaves) since a single plant, whether annual or perennial, produces leaves over a long period and the response brought about by one set of conditions can be replaced by that brought about by another. There are, however, only

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conceptual and not fundamental differences between these two types of plasticity. In many cases the same plasticity can be expressed both within and between plants. It would be reasonable to expect that all adaptive modifications are determined by the occurrence of the envjronmental factor to which adaptation occurs. In this manner the intensity of the stimulus determines the degree of response, and the degree of modification can be precisely related to the environment, e.g., in responses to density. Goebel (1893), however, realized that water level was not itself directly responsible for changes in leaf form in some aquatic Ranunculus species. Cook (1963) has shown that change of leaf form of Ranunculus aquatilis is dependent on temperature and photoperiod and not directly on water level. I n Ranuculus Jlabellaris Bostrack and Millington (1962) have shown that the same is true but that there is also a profound effect of water level. Therefore there may be differences between related species in the type of mechanism. The same lack of direct causal connection between the plastic modification and the environment to which it is adaptive is readily seen in the deciduous habit, for leaf fall is determined by an internal rhythm controlled primarily by photoperiod and only secondarily (if at all) by temperature and exposure. Further discussion of mechanisms is given by Sinnott (1960). Such cases, where an adaptive modification is determined by an indirect stimulus, may have various explanations. The first is that of reliability. In many circumstances the response would be most valuable if it occurred with complete reliability, even if the stimulus were feeble. This will be particularly true of adaptations connected with seasonal changes, which are usually regularly repeating oscillations, yet which may not always be clear-cut and reliable. This is perhaps also the explanation of the mechanism in Ranunculus aquatilis and related species. The stimulus provided by the water surface will not be clear-cut, particularly in moving water, where the tips of the plant oscillate in the current. The pattern of growth and plasticity is not directly connected with it. The plant begins growing in spring from its overwintering rootstock and grows for a period producing submerged leaves; during this time it reaches the surface. But it does not change leaf shape necessarily on reaching the surface and may continue extending for a time, submerged just below the surface. Then in response to the stimulus of temperature and photoperiod it suddenly produces floating leaves on the surface. This occurs with considerable reliability. The second possible explanation is that of preadaptation or anticipation. If a plastic response is initiated by a particular stimulus, response to that stimulus cannot occur immediately; it will take place only after

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an appreciable period of time. This is a necessary outcome of the normal mechanisms of growth. The only cases where response can be rapid are in various nastic movements such as those shown by stomata or the leaves of Mimosa pudica and Oxalis acetosella. Such mechanisms are, however, uncommon. Therefore, in many cases, adaptive modifications would be much too late if they were initiated by the environmental change to which they are adaptive. This will be particularly true of environmental changes that begin very suddenly, such as the onset of winter or wet season. It has already been argued that, in this case, adaptation may be possible only by permanent genetic change. If the adaptive modification, however, can be initiated by a minor environmental change occurring regularly in advance of the major change, then the plant can be already fully adapted by the time of onset of the major change. The change will be anticipated. It would appear that exactly this has occurred in a number of cases. The most obvious stimulus of this nature available to the plant is that of photoperiod. It would appear that this is concerned in many cases of adaptive response where the stimulus is secondary in nature. Not enough is known of plasticities of this type to be able to generalize about the mechanisms involved, but it would appear that other factors, e.g., temperature, may also be involved where appropriate. The mechanisms of adaptive modification involving secondary stimuli can be considered to be the most elaborate and advanced of the mechanisms studied. Although they may be elaborate, however, it is impossible to say they are more advanced than other mechanisms. They have, for instance, inefficiency in that since the major environmental change is not the stimulus, the degree of modification cannot be related to its severity. Another inefficiency of this type of mechanism is shown when the pattern of the operative stimulus breaks down. Then the adaptation may not be appropriate. In Ranunculus aquatilis and R. peltatus the plants found growing out of water on mud in midsummer possess dissected leaves and not laminate leaves as would be expected. It has been shown (Cook, 1963) that this occurs because laminate leaf production is normally initiated in stem apices under water and that when these are raised above the water (a condition that would not normally occur) they will only produce dissected leaves. Ranunculus flabellaris, in which the degree of submergence does control leaf shape, does not appear to show this anomalous behavior; the leaves of terrestial plants are not capillary. The more complex mechanism may therefore lead to anomalous behavior when normal environmental patterns are disturbed, whereas the simpler mechanisms will not. But in the absence of disturbance the more complex mechanisms are extremely effective.

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It would therefore appear that no one mechanism can be considered the most advanced. Each has its own advantages and disadvantages. Each appears to be related to the particular disruptive situation involved and gives a fine degree of adaptation to the different environments concerned. VII. Fixed Phenotypic Variation

We have seen that for various reasons an adaptive modification can become determined by an indirect stimulus. For the same reasons it can be argued that an adaptive inodification can be determined autonomously by the stage of growth or some other character of the plant itself. If a seed always germinated under a specific set of conditions which are different from those experienced by the adult, modifications in relation to these could be of adaptive value. These modifications could be directly determined by the effect of the environment on the seedling. But if the particular environment always occurred at the seedling stage of development, the stage of development itself could determine the modification. This would lead to both regularity and anticipation. It would appear that this happens in a number of cases (Goebel, 1900; Sinnott, 1960). Thus Acacia, Adenostoma, and Ulex species develop expanded leaves in juvenile stages which are not found in adults in nature. The seeds of these species develop under moist conditions not necessarily experienced by the adults and an adaptive value can be inferred. Such a type of development, termed heteroblastic, has been discussed extensively by Goebel (1900). In water plants the early development of the seedling is inevitably below water. Correlated with this the juvenile leaves of some species, e.g., of Sagittaria sagittifolia and Alisma plantago-apuatica are always of submerged type, thin and ribbonlike, in contrast to those of the adults. These modifications can be considered t o be fixed and to be related to the stage of development since they occur whether the plants are grown in water or not. In these cases there is an apparent plasticity which is not true plasticity, since the modification occurs independently of the environment. The phenotypic variation is in fact fixed, being endogenous and not exogenous. By definition such fixed phenotypic variation must not be considered plasticity at all: it is ontogenetic differentiation. It has a close connection with true plasticity, however, for the boundary between it and true plasticity is not a clear one. The same response may at some stages be fixed, but a t other stages be determined by environment. Thus, in Campanula rotundifolia adult linear leaved plants can return to the juvenile condition of rounded leaves under conditions of low light intensity.

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The linear leaved juvenile state of Sagittaria sagittifolia and the juvenile states of various other species are siniilarly reversible in the adult (Arber, 1919). Many other cases are known (Goebel, 1900). Such fixed phenotypic variation occurs in characters other than morphology. While some species may be facultatively cleistogamous and others obligately cleistogamous, there are some species which are permanently both cleistogamous and chasmogamous (Uphof, 1938). Panicum clandestinum bears cleistogamous flowers a t the base of side tillers, a s well as normal panicles ; Amphicarpum jloridanum bears cleistogamous flowers a t the ends of rhizomes, as well as normal panicles. Seed dormancy also involves fixed phenotypic variation. Despite the presence of mechanisms involving plasticity of seed behavior already discussed, causing seeds to germinate in conditions likely to be satisfactory for subsequent development, on many occasions these conditions might not be maintained and all the seeds that germinated would die. Therefore a species is likely to be most successful if all its seeds do not germinate at any one time in a particular habitat, however appropriate the immediate conditions may be. It is found that many species show a between-seed variability or polymorphism in dormancy. I n some cases this seems to be due to genetic variation. But in a number of cases there is no genetic component; it is determined developmentally by the parent plant. I n Xanlhium there are two seeds in the fruit, the lower of which germinates readily, the other only after a period of dormancy. In Avena ludoviciana there is a similar difference between the seeds of upper and lower florets of the spikelet. This is not found in the related species Avena fatua except in the subspecies septentrionalis (Thurston, 1960). I n other species, particularly of the Compositae, similar differences between seeds are found. Such variation in seed dormancy is very similar to that already considered as plasticity, but it is permanently fixed and is independent of the external environment. Like the other examples of fixed phenotypic variation it may be of considerable adaptive value. VIII. Conclusions

A. GENERALCHARACTERISTICS OF PLASTICITY The many different sorts of evidence show unequivocally th a t the ability of plants to be modified by the environment is genetically determined. The general pattern of development of a character will exercise some control over the degree of modification possible. But it is clear that a large measure of control is afforded by the detailed developmental

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pathway, the epigenetic landscape, which has its own genetic determination. The degree of control is considerable. There can be rigorous canalization into one pathway, giving stability; canalization into two distinct pathways, giving the possibility of switching between two precisely determined forms and therefore discontinuous plasticity; or broad control with canalization only between wide limits, giving continuous plasticity. This control is not general to the whole genotype, but is specific for individual characters, and usually specific for individual environmental influences. All this is apparent from a study of the modifications that can be induced in the characteristics of plants and animals. Since the degree of plasticity of a character is under genetic control, it must follow that it can be influenced by natural selection. There are many situations where the plasticity of a character can have considerable adaptive significance. As we have seen, this is particularly true in plants, because they lack the animals’ complex response mechanisms of movement and behavior. As a component of evolutionary adaptation in plants, plasticity appears to play a considerable part. It also plays a considerable part in the processes of competition. Although competition may express itself by differential mortality of the interfering components, it more commonly expresses itself by differences in response (Harper, 1961; Donald, 1963).

B. INTERRELATIONSHIP OF PLASTICITIES OF DIFFERENTCHARACTERS The fitness of an organism in a varying environment will be maximized by phenotypic changes if these (a) minimize any deleterious effects of the environment and (b) maximize any advantageous effects. Such fitness, dependent on both vegetative and reproductive characters, is the outcome of the interaction of all the component characters. We have seen that, these characters can each have their own plasticity and response to environmental variation. It must therefore follow that maximal fitness can be obtained in a number of ways, by response in some characters and stability of others. If a number of interacting characters are all plastic, it is possible that maximal fitness can be obtained by adjustment of one character in one species and by adjustment of another character in a second. An analysis of such interrelationships for yield in cereals has been presented by Grafius (1956). Nevertheless, essential attributes and balance of organization must be maintained and limits will be set by particular characters which lack the ability to respond or in which response could only be deleterious. We may therefore find increased vegetative growth that may not be achieved by over-all increase in size of all organs equally because of structural limitations, but by increase in their number. There may there-

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fore be stability of certain characters which is compensated for by plasticity of others. Maximal fitness will also involve interaction between physiological and morphological characters and their plasticities. Physiological plasticity may permit morphological stability. But if plasticity in a particular physiological system is not possible, the fitness of the organism may be maintained by morphological adjustment. I n reproductive growth the same complex interactions will be found. For various reasons constancy of flower and seed size is important. Maximization of fitness therefore usually occurs by variation in the number of flowers. I n some organisms, however, there may be variation in some floral characters, such as numbers of seeds per flower. Interaction between vegetative and reproductive characters will also clearly occur. Maximization of fitness will not necessarily occur by unlimited response of both. After the onset of adverse conditions to an annual plant, further vegetative growth may seriously reduce the possibility of subsequent reproductive growth. Under these circumstances we may find the development of a mechanism diverting vegetative growth into reproductive growth. The occurrence of such interactions is well exemplified by the responses of populations of Capsella bursa-pastoris, studied by Sorensen (1954) , already discussed. Poor growing conditions cause a reduced rate of growth and leaf production which the species cannot overcome apparently by physiological plasticity. As a result flowering, which usually occurs after production of a certain number of leaves, tends to be later. Some populations can, however, under poor conditions, reduce the number of leaves produced before flowering and thereby maintain the date of flowering under poor conditions that they usually show under good conditions. Populations not possessing such plasticity of leaf number are unsuccessful under poor conditions. There is therefore a hierachy of plasticities. I n the evolution of processes maximizing fitness a variety of different solutions may be developed in different plants. This can be seen by comparison of plant species. The essential common character of all such solutions will be that some characters of the plant will for various reasons be held constant, whereas others will be permitted to vary, and therefore show high plasticity. Characters which are held constant can be properly said to show homeostasis (in the sense of Cannon, 1932) or canalization (in the sense of Waddington, 1957). For such constancy can be considered to be at least in part the outcome of the plasticity of the other characters. Which characters are held constant and which vary depend on the exigencies of the structure, physiology, and environment of the species in question.

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C. OPEN PROBLEMS In view of the integral and complex part played by plasticity in adaptation it is remarkable that there are so few critical analyses of specific cases of plasticity in plants, particularly of its genetic control and response to selection. It can be argued that plasticity is under genetic control. An attempt has been made in this article to stress the cases where marked differences in plasticity exist in closely related species or varieties, since this is circumstantial evidence of the ease with which plasticity can be changed by selection. But the direct experimental evidence is meager. In order to fit the concept of plasticity into our framework of evolutionary principles, we need t o know the amount of genetic variability for plasticity that is available in natural populations, its genetic control, and the ease with which it can be selected. The same point has been emphasized recently for animals (Mayr, 1963). It seems also necessary that we should have more primary evidence on the occurrence of different types of plasticity and their underlying mechanisms. The spectacular types of plasticity involving a number of major characters are well documented and are becoming well understood. The simpler types of plasticity are more common, yet have rarely been studied critically except in crop plants. The evolutionary relationship between the two types of mechanisms is not clear. Much of the recent work on plant adaptation has eschewed very carefully any consideration of plasticity. Any modifications induced by the environment during the course of an experiment are usually considered only an embarrassment. In this connection it would be particularly valuable to know the mechanism by which several unrelated characteristics can be associated in one particular plastic response. We are familiar with the fact that in genetic polymorphisms several characteristics can be determined by one switch gene. This can be due either to a common system of canalization so that a number of different processes all take place as the result of a single switch. It can also be due to the switch being in effect a set of ganged switches operating separately on a set of independent characters (Mather, 1955). I t seems reasonable to believe that the same alternative systems exist in mechanisms of plasticity, with the environment and not a gene being the switch. But there is little evidence giving proof of this. The deciduous habit seems to be an example of the first system. The second system seems to occur in Ranunculus hirtus where the characters associated in the one response appear to be controlled by separate environmental factors. An analysis of the plastic response of segregating hybrid populations derived from crosses between individuals with contrasting

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plasticities and a search for mutants affecting single components of a response would be valuable. A third aspect of mechanisms about which we need information is the degree to which the direction of response can be controlled. An essential part of the arguments that have been presented is that the direction of response can be controlled by the organism. The evidence already presented unequivocally shows that responses can be in particular directions, e.g., in the adaptation of Drosophila to salinity (Waddington, 1959) or in the flowering of Capsella bursa-pastoris (Sorensen, 1954). Indeed, without such control of direction, the evolution of adaptive responses would be impossible. But we know very little about this control. Initially, plasticity may be undirected: that which is normally considered lack of stability or developmental noise is perhaps of this nature. But a t some stage in the evolution of a plastic response randomness in direction and in extent must be replaced by fixation of direction and extent. D. PLASTICITY I N CROPPLANTS Finally, it must be realized that an understanding of plasticity is important not only to the framework of evolutionary theory, but also to the practical problems of plant improvement. The environment of any crop plant varies from season to season, from locality to locality, and even from place to place in the field, and much of this variation cannot be controlled by the farmer despite cultivation (Allard and Bradshaw, 1964). The plant breeder is therefore concerned with the stability of final yield. Stability of final yield may be due to inherent stability of the crop; but it may also be due to plasticity of the components of final yield (Grafius, 1956). This is very clear from the reaction of crop plants to density (Donald, 1963). Plasticity is therefore as much an essential part of the adaptation of crop plants as of wild species. IX. Summary

The expression of an individual genotype can be modified by its environment. The amount by which it can be modified can be termed its plasticity. This plasticity can be either morphological or physiological; these are interrelated. I t can be argued that the plasticity of a character is related to the general pattern of its development, and apart from this, that plasticity is a general property of the whole genotype. A review of the evidence suggests that neither of the conclusions is tenable. Plasticity of a character appears to be ( a ) specific for that character, (b) specific in relation to particular environmental influences, (c) specific in direction, (d) under

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genetic control not necessarily related to heterozygosity, and (e) able to be radically altered by selection. Since plants are static organisms, plasticity is of marked adaptive value in a great number of situations: (a) Disruptive selection in time. Where changes in environment are of the same or shorter duration than its generation time, a species cannot adapt by genetic changes, but only by plasticity. ( b ) Disruptive selection in space. Where changes in environment occur over very short distances, adaptation by the formation of genetically differentpopulations may be precluded. I n these conditions very spectacular types of plasticity may be evolved. (c) Directional selection. If selection is very severe and directly adaptive genetic variation is limited, further adaptation may be afforded by plasticity. (d) Stabilizing selection. Plasticity permits different genotypes to assume the same phenotype. Under certain circumstances, this may permit the retention of genetic variation in a population in a manner analogous to dominance. Examples of all these situations in plant species are discussed. They indicate that adaptation by plasticity is a widespread and important phenomenon in plants and has been evolved differently in different species. There can be a considerable interrelationship between the plasticities of different characters; plasticity of one character can allow stability of another. The plasticity of a species must therefore only be considered in terms of the plasticity of its individual characters. The mechanisms involved are varied. At one extreme the character may show a continuous range of modification dependent on the intensity of the environmental stimulus. At the other the character may show only two discrete modifications. The stimulus causing these modifications may be direct, i.e., the environmental factor to which the adaptation occurs, or it may be indirect, i.e., an environmental stimulus not part of that to which adaptation occurs. Under certain circumstances the modifications may be determined by the stage of growth of the plant itself. This is no longer true plasticity, for it is fixed and independent of the external environment: the adaptation is endogenous and not exogenous. The mechanisms found can be related to the particular environmental situation involved. ACKNOWLEDGMENTS I am grateful t o the Trustees of the Leverhulme Research Awards for the award of a Fellowship and to Professor R. W. Allard of the Department of Agronomy, University of California, Davis, for his hospitality and stimulus during the period of the fellowship.

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I would like t o thank the many friends with whom I have discussed this problem on many different occasions, Mr. J. E. Ilandy for the inforination concerning Potamogeton, Dr. S . A. Cook for the quotation of Nilsson-Ehle, and Ihs. C. D. K. Cook, S. A. Cook, F. J. F. Fisher, Professor J. L. Harper, Mi-. J. G. Pusey, and Professor G. L. Stebbins for their critical reading of the manuscript and valuable suggestions. I owe much to their guidance and help. The views expressed in this article must, however, remain my, and not their, responsibility.

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Turesson, G. (1925). The plant species in relation to habitat and climate. Hereditas 6, 147-236. Turesson, G. (1950). Personal communication. Underwood, G. (1 954). Categories of adaptation. Evolution 8, 365-377. Uphof, J. C. T. (1938). Cleistogamic flowers. Botan. Rev. 4, 21-49. Waddington, C. H. (1953). Epigenetics and evolution. Symp. SOC.Exptl. Biol. 7, 186-199. Waddington, C. H. (1957). “The Strategy of Genes.” Allen & Unwin, London. Waddington, C. H. (1959). Canalisation of development and genetic assimilation of acquired characters. Nature 183, 1654-1655. Waddington, C. H. (1960). Experiments on canalising selection. Genet. Res. 1, 140-150. Waddington, C. H. (1961). Genetic assimilation. Advan. Genet. 10,257-293. Warburton, F. E. (1955). Feed back in development and its evolutionary significance. Am. Naturalist 89, 129-140. Went, F. W. (1953). Gene action in relation to growth and development. I. Phenotypic variability. Proc. Natl. Acad. Sci. U.S. 39, 839-848. Whitehead, F. (1954). A study of the relation between growth form and exposure on Monte Maiella, Italy. J. Ecol. 42, 180-186. Whitehead, F. (1956). Preliminary investigations of factors determining the growth form of Cerastium tetrandrum Curt. J . Ecol. 44, 333-340. Williams, W. (1960).Relative variability of inbred lines and F1 hybrids in Lycopersicum esculentum. Genetics 46, 1457-1465. Wilkins, D. A. (1963). Plasticity and establishment in Euphrasia. Ann. Botany (London) [N.S.] 27, 533-552. Zohary, D., and Imber, D. (1963). Genetic dimorphism in fruit types in Aegilops speltoides. Heredity 18,223-231.

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THE PREMElOTlC STAGES OF SPERMATOGENESIS Aloha Hannah-Alava Department of Genetics, University of Turku, Turku, Finland

Page 157 159 161 164 172 196 197

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . IT. Maintenance of the Germ Line and Spermatogonial Multiplication . . . . A. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . B. Methods of Spermetogonial Renewal. . . . . . . . . . . . . . . . C. Spermatogonial Stages . . . . . . . . . . . . . . . . . . . . . . 111. Relating the Temporal and Spatial Patterns of Spermatogenesis . . . . . A. Chronometry of the Stages of Spermatogenesis . . . . . . . . . . . B. The Genetic Criteria for Relating the Temporal and Spatial Patterns of Spermatogenesis. . . . . . . . . . . . . . . . . . . . . . . . IV. Radiosensitivity of Spermatogonia . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The dearth of basic information . . . has proved no hindrance t o the flowering of speculative hypothesis and magnificent generalizations. M . M . Rhoades (1961)

1. Introduction

The years devoted to mutation genetics, following H. J. Muller’s discovery, in 1927, that X-rays were effective in inducing mutations, have resulted in the accumulation of an enormous body of genetic facts on mutagenesis in Drosophila, but surprisingly little corollary and substantiating information on the cytomorphology of the germ cells. Part of the reason for this had been because it was quickly realized that spermatogenesis in Drosophila, as compared to some of the other dipterans and especially as compared to the orthopterans, was particulary refractory to cytological investigat.ions. As early as 1908 Stevens reported that D . melanogaster was exceptionally unfavorable material for chromosome studies, and other cytologists must have been equally unsuccessful inasmuch as it was not until 1914 (Metz, 1914) before the next paper on Drosophila cytology appeared, and over twenty years before the publication of G u y h o t and Naville’s description (1929) of spermatogenesis in D. melanogaster. Today, with the impetus of mutation research on stage-sensitivity, 157

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the geneticist is faced with the problem of interpreting the experimental evidence without the essential cytomorphological information on the stages of spermatogenesis. But cytology has not been the only neglected aspect in the study of Drosophila germ cells. In his preface to Biology of Drosophila, Demerec (1950) stressed the fact that there had never even been a comprehensive and detailed treatment of the anatomy and histology of D. melanogaster in spite of the fact that for three decades it had been one of the most important species for biological research. Consequently the various authors of the volume not only had the problem of evaluating an enormous and often contradictory body of facts, but also the task of making new investigations in order to resolve major discrepancies between descriptive and experimental evidence. It was not realized then, of course, how important a role the elucidation of the informatioil-particularly in the scholarly chapters by Bodenstein, Cooper, Poulson, and Sonnenblickwould play in subsequent studies of mutagenesis in this species. S 1 evertheless, the trend in genetic research in the past decade has been such that there is a real necessity for further factual information before it will be possible to interpret many of the experimental results. When this review was undertaken, its purpose was only to incorporate the most recent information on spermatogonia into the rather large body of evidence on germ-cell maturation in D. melanogaster in order to be able to relate the temporal and spatial patterns of sperniatogeriesis more precisely. However, it soon became apparent that all too frequently the terminology, as well as some of the concepts currently in use, was based upon nomenclature or concepts that have long since been discarded. The resultant ambiguity frequently made it difficult to even identify the spermatogonial stage under consideration. Clarification of some of the controversial points has necessitated a review of the literature on spermatogenesis for a number of other animals. Even this has not been entirely satisfactory, inasmuch as there is surprisingly little information on spermatogonia, even for those species exceptionally favorable for cytological investigation. Historically, the reason for the neglect of the spermatogonial stages in the insect is multifold. There is little doubt that the bitter and lengthy controversy over the function of the apical cell (cf. Carson, 1945) has been one of the contributing factors to the misconceptions, since many of the original concepts of spermatogonial renewal involved the apical cell. Another and perhaps the most important factor was the realization, very early in the history of cytology of the germ cells, that the meiotic stages were the most important in interpreting genetic recombination. Consequently spermatogonia have received oiily cursory treatment, e v w in

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reviews of spermatogenesis (cf. Wilson, 1925; Depdolla, 1928 ; Geitler, 1934; Darlington, 1937; White, 1954; Rhoades, 1961). It may even be that the overgeneralized picture of the premeiotic stages, standard for entomology and cytology texts, has played a considerable role in the development of the terminology and current ideas of the spermatogonia and spermatogonial multiplication. Whatever the cause, the lack of definitive information has had important consequences in the development of the present-day concepts of the fuiiction and fate of the spermatogonia in the insect. I n contrast, almost from the earliest studies of spermatogenesis in the rodent, it was realized (von Ebner, 1888; Regaud, 1901) that spermatogonia were a very important component of the testis. Furthermore, for almost a century before the mouse became a genetic tool, the various tentative interpretations of germ-cell maturation in the rodent were refined or discarded, resulting in a logical concept of spermatogenesis t,hat is compatible with most of the experimental evidence (Roosen-Runge, 1962; Clermont, 1962). II. Maintenance of the Germ Line and Sperrnatogonial Multiplication

One of the consequences of the introduction of cytology and cellular embryology, during the time the concept of the continuity of the germ plasm was being formulated, was the development of the idea that even after the gonad was established, certain of the premeiotic cells had a dual function, just as the germ-line cells had a dual function during embryogenesis. Some of the earlier workers (e.g., Verson, 1889; Munson, 1906; GBrard, 1909) were erroneously of the opinion that the apical cell was responsible for the germ cells; others (e.g., LaVallete St. George, 1897; Griinberg, 1903; Mumon, 1906; Buder, 191Fj) believed, possibly also erroneously, that quasidichotomous divisions of the young spermatogonia resulted in the segregation of the germinal and nongerminal elements of the testis. However, it was clearly obvious to Davis (1908), who could also present substantiating cytomorphological evidence in support of his beliefs, that certain of the spermatogonia were maintained throughout the reproductive life of the individual while others were directly destined to become the spermatozoa. Development of this idea resulted i n the formulation of the concept of a stem-cell method for spermatogonial renewal in the grasshopper (Selson, 1931), Drosophila (Harris, 1929; Tihen, 1946), the rodent (Roosen-Runge, 1952; Clermont and Leblond, 1953), and the silkworm (Kondo, 1961). I n spite of the accumulation of considcrable evidence for maintenance of the germ line through quasidichotomous (stem-cell) divisions of certain of the spermatogonia, due to the fact that the diagrammatic representations of the germ line in animals almost always show the spermatogonial

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divisions as dichotomous (cf., Wilson, 1925; Geitler, 1934), it has been tacitly assumed that spermatogonial multiplication is always dichotomous. Second, even though it was realized very early that different generations of spermatogonia have different cytomorphological properties, the spermatogonial stages have never been clearly identified. Third, and in contrast to the cytoplasmic inclusions with their multifold synonymy in terminology (cf., Tobias, 1956), nomenclature for spermatogonia has been limited, especially in insects, almost exclusively to the two terms “primary” and “secondary” even in reviews (e.g., Depdolla, 1928), when it should have been obvious that entirely different cell types were under consideration. The main reason for the relatively little information in the literature on spermatogonia or spermatogonial stages, however, is probably because of the concentration of investigations on the meiotic stages following Sutton’s (1900, 1902, 1903) demonstration that the behavior of the chromosomes during meiosis offered a mechanical explanation of the Mendelian laws. This resulted in the spermatogonia being relegated to a status of no greater significance, in respect to spermatogenesis, than a somatic cell. This may be the reason why, in reviews dealing with the physicochemical properties of dividing cells, spermatogonia are not even considered, or no distinction is made between spermatogonia and somatic cells in spite of the fact that recent studies using modern cytochemical techniques (e.g., Ansley, 1958) clearly show that there is greater similarity between spermatogonia and meiotic cells than between spermatogonia and somatic cells. Historically, a factor that played a considerable role in the development of the various opinions of spermatogonial multiplication, as well as their nomenclature, was the controversy over the function of the apical cell (cf., for reviews, Davis, 1908; Deegner, 1928; Nelson, 1931; McClung, 1938; Carson, 1945; Sado, 1963). Even though Toyama’s (1894) conclusion that the apical cells in the silkworm, Bombyx mori, did not divide to produce germ cells was quickly substantiated for several species of Lepidoptera (Griinberg, 1903), Diptera (Cholodkowsky, 1905), and Orthoptera (Davis, 1908), the lack of substantiating evidence was no impediment to the speculations of those who were convinced of its germinal nature. It is not too surprising, therefore, with the accumulation of overwhelming evidence in favor of a nutritive and supporting function for the apical cell (Schellenberg, 1913; Mohr, 1914; Lewis and Robertson, 1916; Goldschmidt, 1931; Robertson, 1931; Nelson, 1931; McClung, 1938; Carson, 1945) that all corollary hypotheses were discarded along with the hypothesis that the apical cell was a germinal cell. Inasmuch as one of the corollary hypotheses was that of a stem-cell method for spermatogonial

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renewal-and earlier frequently even called the apical-cell method-it was also rejected, only to be revived in recent years in order to interpret some of the experimental results. During the intervening period the problem of spermatogonial renewal was either left in abeyance or it was assumed that all spermatogonial divisions were dichotomous. Conversely, the assumption that there are no apical cells in some species of Diptera (Cholodkowsky, 1905; Miller, 1950) has added to the misconceptions since the apical cells have then been confused with the younger spermatogonia. There is no question of their occurrence in Drosophila (Geigy, 1931; Gloor, 1943; Aboim, 1945), but because they are a group of 10-12 small cells, about the same size as the youngest spermatogonia (Cooper, 1950), it is not surprising that they have been overlooked, even though their nuclear morphology is distinctly different from that of the spermatogonial cells (Fig. 7A and Hannah-Alava, 1965; Meyer, 1964). I n addition it has yet to be established whether or not the apical cells in the Drosophila testis subsequently degenerate, as they do in the Lepidoptera (Griinberg, 1903; Zich, 1911),Orthoptera (Robertson, 1931), and some of the other Diptera (Friele, 1930). A. TERMINOLOGY One of the greatest impediments to an understanding of the function of the spermatogonia is the vagueness of the terminology. With the exception of a standard nomenclature for the mammalian spermatogonia that has been in common use since 1953-with Clermont and Leblond’s description of Type A, Intermediate, and Type B spermatogonia of the rat-there is no standardized and concise terminology for the premeiotic stages as there is for the meiotic stages. Two or more types of spermatogonia can readily be distinguished in the testes of many insects, but the terminology that has been used by one author may mean something completely different to another author. Thus in some species the terms “primary,” “secondary,” etc., may refer to the major types of spermatogonia, but in others the term “primary” has been used to describe the first generation, the first two or three generations, or all but the last generation of the encysted spermatogonia, with the cells of the remaining generations being called the “secondary” spermatogonia [cf. the terminology in Depdolla’s (1928) review as compared with the terminology used by the original authors]. The terms “primary” and “secondary” have also been used to differentiate between the unencysted and the encysted spermatogonia (Cooper, 1950; Kaufmann and Gay, 1963; Sado, 1965). Finally, owing to a similarity in nuclear morphology, preleptotene spermatocytes frequently have been misclassified as spermatogonia or

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vice versa (Wilson, 1925; McClung, 1927; Oakberg, 1957, 1965; Westergaard, 1964). I n referring to the spermatogonia responsible for the maintenance of the germ line, i.e., the stem-cell spermatogonia, the terms “dormant mother cell” or “dormant A-type spermatogonium” have been rather consistently used for the mammals (cf. Roosen-Runge, 1962). Although the terms “Ursperniatogoiiie” or “primordial spermatogonia,” “apical cell,” “pole cell,” “primary spermatogonia,” and “predefinitive spermatogonia” have been used to describe the spermatogonia functioning as stem cells in the insect, all of them, with the exception of predefinitive spermatogonia, have also been used in a purely descriptive or morphological sense with no implication as to the cell’s function. Finally, the term “apical cell” is characteristically used to describe the supporting or nurse cell or cells in the apical end of the insect testis or ovary (cf. Carson, 1945). The following terminology will be used subsequently in discussion of the spermatogonial stages, u ith the exception of direct quotations and the retention of the terms “A type” and “B type” when the mammalian spermatogonia are under considerat,ion : 1. Apical complex: The group of cells in the anterior end of the insect testis or a follicle of the testis consisting of the apical cell or cells, the spermatogonia imrncdiately surrounding it, and the septal or follicular cells destined to form the cyst membrane (Nelson, 1931). 2. C ~ s tA : group of spermatogonia, spermatocytes, or early spermatids of common origin surrounded by the septal (cyst) membrane (Davis, 1908). Synonymous with gonocyst (Munson, 1906) and spermatocyst. 3. Spermatid bundle or sperm bundle: The group of spermatids or spermatozoa of common origin embedded in a nutritive cell or maintained as a unit during spermatid and sperm differentiation ( G u y h o t and Naville, 1929). 4. Apical cell: The specialized cell or group of cells in the anterior end of a follicle or testis with a trophic or supporting but not a germinal function (Griinberg, 1903; Nelson, 1931). 5. Germ cells: The reproductive cells as distinguished from the somatic cells in general and the apical cell in particular. 6. Primordial germ cells: The reproductive cells during the time of establishment of the gonad, and in certain insects the germ cells in the embryonic gonad as distinguished from the pole cells, which are the reproductive cells prior to the establishment of the gonad (Sonnenblick, 1941, 19.50). Probably synonymous with the terms “primitive germ cell” (Sado, 1965) and “gonocjrte” (Shaver, 1953). 7. Spermatogonia: The youngest reproductive cells after formation of the testis (La Valette St. George, 1876) which through division or differentiation are destined to become spermatocytes. They include:

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a . Predejinitive spermatogonia: The spermatogonia (at the anterior end of the testis and immediately surrounding the apical cell or apical cells in many species of insects) that are destined through quasidichot,omous (stem-cell) divisions to produce cells like themselves as well as the definitive spermatogonia (Tihen, 1946). In a cytogenetic sense, the term “predefinitive spermatogonium” is probably synonymous with the terms “Urspermatogonie” as used by Mohr (1914), the “Ursamenzelle” of Waldeyer (cf. Roosen-Runge and Barlow, 1953), “pole cell” as used by Muller et al. (1954), and probably also “primordial spermatogonium” in the sense of Cretschmar (1928) and Kondo (1961). It is synonymous with the terms “dormant mother cell” or “dormant A-type spermatogonium” in the mammal (cf. Roosen-Runge, 1962). Predefinitive spermatogonia may be the same as or the direct descendants of pole cells, primordial germ cells, or primordial spermatogonia. 6. Indefinitive spermatogonia: The generations of spermatogonia between the stem-cell division and the definitive divisions (Sado, 1965). Synonymous with one meaning of the term “primary spermatogonia” as used by Sad0 (1961, 1963) and Kondo (1961). Possibly synonymous with the term “primary spermatogonia” as used by Cooper (1950), Seidel (1963), and Kaufmann and Gay (1963). c. Definitive spermatogonia: The spermatogonia irrevocably destined, through dichotomous division and differentiation, to become the spermatocytes and eventually the spermatozoa (Tihen, 1946). All generations of definitive spermatogonia are derived from an initial definitive spermatogonium (or initial primary spermatogonium) by dichotomous and synchronous divisions. With the possible exception of the initial cells, the definitive spermatogonia in the insects undergo division within a cyst. The term is synonymous with the term “secondary spermatogonia” as used to describe the encysted spermatogonia in Drosophila (Cooper, 1950; Kaufmann and Gay, 1963), the silkworm (Sado, 1961, 1963), and the dragonfly (Omura, 1955, 1957). The generations of definitive spermatogonia as defined here include the last one (Westergaard, 1964) even though the products of the last division may immediately begin to differentiate as spermatocytes (RlcClung, 1927). Thus the number of cells per cyst after the last definitive division is the same as the number of primary spermatocytes of common origin. I n general three morphological types of definitive spermatogonia will be recognized : i. Primary spermatogonia: Consist of a t least the initial definitive spermatogonia (Mohr, 1914), but may include (depending upon the species being discussed) the products of one to three or more successive divisions beginning with an initial definitive spermatogonium (Robertson,

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1931). By this definition primary spermatogonia can be equated with the dichotomously dividing A-type spermatogonia of the mouse (Monesi, 1962a). ii. Secondary spermatogonia: Depending upon the species under consideration, include the last or the last two to four generations of definitive spermatogonia (Mohr, 1914; Robertson, 1931). By this definition the secondary spermatogonia of the insect and the B-type spermatogonia of the rodent can be equated. iii. Intermediate spermatogonia: Products of one or more of the divisions intermediate between the primary and secondary divisions of the definitive spermatogonia (Clermont and Leblond, 1953). I n the species with progressive nuclear or cellular changes during the course of spermatogonial multiplication, the spermatogonia of the intermediate divisions which cannot be readily identified as either primary or secondary can usually be classified as intermediate. B. METHODSOF SPERMATOGONIAL RENEWAL One of the basic concepts of spermatogenesis, and one substantiated with evidence for many of the higher invertebrates as well as most vertebrates, is that a new generation of germ cells begins to differentiate before the last generation has completed maturation so that there is a more-orless continuous production of mature sperm throughout, the reproductive life of the individual. This means that there not only is a constantly recurring cycle of spermatogenesis in the animals with a continuous production of mature sperm, but also that there must be an integrated mechanism for replacing the cells that are irrevocably lost because they undergo the processes leading to the mature sperm. The significance of this concept, even though recognized by von Ebner in 1871 (cf. von Ebner, 1888) and clearly understood by Duesberg (1908) has not been fully appreciated by either the cytologist or geneticist. Even the students of cell lineage were not able to envision the spermatogonial stages as a part of the highly regulated and integrated spermatogenic cycle. Consequently, until quite recently concise information on spermatogonial renewal was almost totally lacking. By definition, the predefinitive spermatogonia are separated from the indefinitive and definitive spermatogonia because of their dual role, i.e., replacement of the cells that have begun to differentiate and multiply, and the production of new predefinitive cells for maintenance of the germ line (Tihen, 1946). The two types of spermatogonia have rarely been separated morphologically although there has been some attempt to distinguish between the indefinitive and the definitive spermatogonia in the species with both types, or between the early and late generations of the

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definitive spermatogonia. Finally, it has not been generally realized that if there is a stem-cell mechanism for spermatogonial renewal, such as envisioned by Davis (1908), Nelson (1931), and Monesi (1962a), and depicted diagrammatically in Fig. lC, there must be two different two-cell stages, one resulting from the quasidichotomous divisions of predefinitive spermatogonia (Fig. 4A) and one from the dichotomous divisions of the initial definitive spermatogonia (Fig. 4B). There is considerable evidence, for the rodent especially, but also for the grasshopper and the silkworm, that the primordial germ cells multiply during embryogenesis, and it has been presumed that this multiplication is dichotomous and for the purpose of increasing the number of predefinitive spermatogonia. Spermatogonial multiplication also takes place during the pre-adult stages, but certainly after birth (or eclosion) it is the primary means for a continued supply of germ cells. Maintenance of the germ line can be accomplished in three general ways: (1) The primordial (or indefinitive) spermatogonia multiply dichotomously only during the embryonic stages so that the whole supply of initial definitive spermatogonia is present in the gonad a t the time of initiation of meiosis (Fig. 1A). This interpretation, probably first proposed by Boveri, was held by many of the earlier investigators (cf. Wilson, 1925) and is still a common assumption in fecundity studies. (2) Either primordial spermatogonia, or more probably the indefinitive spermatogonia, multiply dichotomously throughout the reproductive life of the individual, during which time cells are singled out, almost a t random, to undergo the definitive spermatogonial divisions (Fig. 1B). This hypothesis is actually only a modification of the first one, and, perhaps, was adopted when it was realized that the space required for the whole supply of germ cells could exceed the size of the adult testis, or because of the observation that the “younger” spermatogonia continued multiplying even late in adult life. Undoubtedly abandoning the idea that the apical cell was the progenitor of the germ cells also contributed to the adoption of this method. (3) The third method is an extension of Weismann’s concept of the dual role played by the embryonic cells destined to participate in the formation of the germ line, and is thus called the stem-cell method of spermatogonial renewal. According to this concept, the two cells resulting from a division of a predefinitive spermatogonium have different fates. One becomes a new predefinitive spermatogonium responsible for the next generation of germ cells, and the other, the definitive spermatogonium, is irrevocably destined, through multiplication and differentiation, to give rise to the mature spermatozoa. Although it was apparently an acceptable hypothesis prior to about 1920, it fell into disfavor when it became evident

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I

1

LEGEND OPredef hit ive(sterncell)spermt~nh Olndefinitive spermtogonio Blnitiol definitive spermotogonium QnemtKns i f definitive spermtogonio

FIG. 1. Models of methods for maintenance of a continued supply of functional spermatozoa throughout the reproductive life of a male. The circles represent the primordial germ cells, predefinitive spermat,ogonia, or indefinitive spermatogonia which are responsible for maintenance of the germ line, and the triangles the n number of dichotomous definitive divisions prior to differentiation of the spermatocytes. The temporal pattern of spermatogenesis is indicated by “stair-stepping” the successive spermatogenic cycles in the models with a stem-cell method for spermatogonial renewal (C-F), or the triangles of definitive spermatogonial divisions in the models in which the initial definitive spermatogonium is singled out almost a t random from a large supply of primordial or indefinitive spermatogonia (A-B). See text for

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that the apical cell was not a germ cell. It has only been revived recently to explain the spermatogenic cycle in the mammals (cf. Roosen-Runge, 1952, 1962; Clermont and Leblond, 1953; Monesi, 1962a), the broodpattern results in Drosophila (cf. Rluller et al., 1954; Puro, 1963, 1964; Hannah-Alava, 1964), and spermatogonial multiplication in the silkworm Bombyx mori (Kondo, 1961). A corollary of the stem-cell hypothesis is that the time of origin of the new predefinitive (stem-cell) spermatogonium can be predicted on the basis of counts of the number of definitive spermatogonia, or spermatocytes, of common origin (Hannah-Alava, 1964). Since the definitive spermatogonia multiply dichotomously, if the new predefinitive spermatodetails. A. The whole supply of primordial germ cells or their direct descendants is present at the time of initiation of the first spermatogenic cycle, with cells being singled out at random, throughout the reproductive life of the male, to become the initial definitive spermatogonia. B. The primordial germ cells undergo continuous dichotomous division, possibly as indefinitive spermatogonia, with the initial definitive spermatogonia singled out at random. Thus successive broods of oftspring could be derived from cells that had undergone a varying number of indefinitive divisions prior to the definitive divisions. C. A stem-cell method for spermatogonial renewal in which each predefinitive spermatogonium divides quasidichotomously, with one of the products becoming the initial definitive spermatogonium and the other cell the predefinitive spermatogonium for the next cycle of spermatogenesis. This type probably occurs in the grasshopper (Nelson, 1931) and the mouse (Monesi, 1962a). 1). A stem-cell method in which one of the products of the quasidichotomous division of a predefinitive spermatogonium is the ncw predefinitive spermatogonium and the other is an indefinitive sperrnatogonium which then undergoes one or more dichotomous divisions prior to the definitive divisions. E. A stem-cell method in which the new predefinitive (stem-cell) spermatogonium is isolated after the second division of the initial definitive spermatogonium. The three remaining definitive spermatogonia continue multiplying, dichotomously, to produce a total of 12 cells after two divisions, or 24 cells after three, or 48 cells after four divisions instead of the 2" number (e.g., 16, 32, or 64) as in Model C. F. A stem-cell method in which the new predefinitive sperrnatogonium is isolated after one or more indefinitive divisions, and the initial definitive spermatogonia are derived from the indefinitive spermatogonia. G. A stem-cell method for spermatogonial renewal such as suggested from studies of the spermatogenic cycle in the rat (Clermont, 1962). According to this method, the stem cell is isolated after the second definitive division, but it divides to form a pair of predefinitive spermatogonia (which in turn become the pair of initial definitive spermatogonia for the next cycle). In each cycle one of the pair divides dichotomously 2%times while the other divides twice before the new predefinitive spermatogonium is isolated, and the remaining definitive spermatogonia continue dividing dichotomously. In the rat, with five definitive divisions, some groups of spermatoeytes of common origin will have 32 cells while other groups will consist of only 24 cells.

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gonium is isolated in the first division (Fig. lC), the number of definitive spermatogonia of common origin will be a geometric multiple of 2 (i.e., 2" = 4, 8, 16, 32, 64, etc., depending upon the total number of definitive divisions). If the stem cell is isolated after the second definitive division (Fig. l E ) , the number of spermatogonia derived from its three sister cells will be a geometric multiple of 3 (i.e., 6, 12, 24, 48, etc), and after the third division a geometric multiple of 7 (i.e., 14, 28, 56, etc.) as shown in the model by Clermont and Leblond (1953). The time of isolation of the new predefinitive spermatogonia can be determined directly in insects by counting the number of cells in a cyst (Otte, 1906; Robertson, 1931; White, 1955; Hannah-Alava, 1964). In animals without encysted spermatogonia it can be estimated from data on the relative frequency of spermatogonia in each stage of the cycle (Roosen-Runge, 1951, 1952; Clermont and Leblond, 1953; Oakberg, 1956a,b). Nevertheless, use of such estimates in predicting the sequence of spermatogonial events has its limitations (Oakberg, 1956b, 1957) as is evidenced by the different models that have been proposed for spermatogonial renewal in the rodent (Roosen-Runge, 1952, 1962; Clermont and Leblond, 1953; Clermont, 1962; Monesi , 1962a). 1. Mammals

As the result of the discovery that spermatogenesis in mammals is cyclic even when there is no seasonal fluctuation in the sperm output, the thesis has been developed that the continued production of sperm is accomplished by means of stem cells (dormant A-type or dormant mother spermatogonia) which are responsible for the replenishment of their kind, as well as for the replacement of cells directly destined to become the sperm. Clermont and Leblond (1953), who established one procedure for investigating stem-cell replenishment, were of the opinion that each stem cell in the rat produces, in two divisions, four A-type daughter cells, three of which continue multiplication to produce the B-type cells, but the fourth cell becomes the new stem cell for the next cycle (Fig. 1E). As the result of five divisions there would be, therefore, twenty-four spermatocytes of common origin. The stem cells, after a period of dormancy, initiate a new cycle. More recently, however, and after taking into consideration degeneration of spermatogonia, Clermont (1962) concluded that each spermatogenic cycle in the rat is initiated by pairs of A-type stem cells in the manner shown in Fig. 1G. One cell of each pair divides dichotomously five times to produce two generations of A-type, one of intermediate, and two of B-type spermatogonia, and thus a total of thirty-two spermatocytes of common origin. The other cell of the pair also divides dichotomously to produce A-type spermatogonia, but after the

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third division one pair of cells becomes dormant A-type spermatogonia, while the remaining (intermediate) type spermatogonia divide twice more, producing the B-type spermatogonia and a total of only twenty-four spermatocytes of common origin. Following a period of dormancy, the new pair of A-type stem cells initiates a new cycle of spermatogenesis. It was first presumed that the stem-cell spermatogonia originated in most rodents after the second division (cf. Roosen-Runge, 1962; Clermont, 1962) but Monesi (19624 found, by use of the tritiated-thymidine labeling technique, that the first division of a n A-type spermatogonium, in the mouse, was the quasidichotomous one, with one cell becoming the new dormant stem-cell spermatogonium and the other undergoing the definitive divisions to produce the primary spermatocytes (Fig. 1C). Since the mouse has five definitive spermatogonial divisions, the number of spermatocytes of common origin, barring degeneration of some of the cells, is thirty-two. The time of origin of the stem-cell spermatogonium may also be different for the ruminants than either model depicted for the rat (cf. Roosen-Runge, 1962; Clermont, 1962).

2. Orthoptera There is no doubt that three of the acridian species of the Orthoptera have a stem-cell method for spermatogonial renewal (Davis, 1908; Nelson, 1931; Bishop, 1942) similar to that described for the mouse by Monesi (1962a). I n these species, the predefiiiitive spermatogonia immediately surrounding the apical cell divide quasidichotomously, with one of the products being retained as a predefinitive spermatogonium and the other becoming associated with a cyst cell and undergoing the encysted dichotomous definitive divisions (Fig. 1C). On the basis of the organization of the testis, it could be presumed that there is also a stem-cell method for spermatogonial renewal in the other acridiaii species of Orthoptera, and if so, the first division should always be the quasidichotomous one since the number of spermatogonia or sperniatocytes in a cyst is always 2" (White, 1955). Nelson's (1931) observation that not all predefinitive divisions are stem-cell divisions-in the sense that the two daughter cells have different fates-has important implications in interpreting genetic data. Since the proximity of a daughter cell to the apical cell determines its fate (Fig. 2A), the chance orientation of the spindle of a dividing predefinitive spermatogonium, or the extent of crowding of cells in the apical complex could result in two stem cells with the same epigenetic constitution, or conversely, the loss of a stem cell through both daughter cells becoming initial definitive spermatogonia. However, since the majority of the pre-

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definitive divisions are stem-cell divisions in the species studied b y Nelson, it could be presumed that this is true also for the other species. 3 . Lepidoptera

There is little cytornorphological evidence for a stem-cell mechanism for germ-cell renewal in the Lepidoptera with the exception of one of the microlepidopterans, Tisheria angusticolella Dup., in which there is continuous production of definitive spermatogonia from a single “Urspermatogonie” a t the apical end of each follicle (Knaben, 1931). The organization of the testis in the silkworm, Bombyx mori, suggests that there is also some means for continued production of the definitive spermatogonia in this species (Nakanishi et al., 1963) in spite of the short reproductive life of the male (Tazirna, 1964), and, according to Sado (196.5), that it would be similar to the method proposed by Omura (1955, 1957) for the dragonfly, i.e., type D or F in Fig. 1. Genetic evidence also points to the probability that there is a stem-cell mechanism for sperniatogoriial renewal in t,he silkworm (Kondo, 1961) but a somewhat different one than in the mouse or grasshopper. The number of stem cells estimated by Korido on the basis of mutation results was only eight or nine. But in order for this number of stem cells to produce the large number of “primary” (type-A) spermatogonia (counted by Sado) he concluded that each stem cell would “develop into type-A gonial cells after four or five divisions.” Because of the indefinitive divisions, between the stem-cell and the definitive (encysted) spermatogonial divisions, the sequence of spermatogonial events in the silkworm is probably similar to the model pictured in Fig. 1D. Nevertheless, a model similar to Fig. 1C cannot be ruled out if the number of stem cells is greater than estimated by Kondo. A considerably higher number is indicated from the counts of primordial spermatogonia by RiIiya (1958, 1959), Kobayashi (1962), and Sado and Otsuki (Sado, 1965). But the question of spermatogonial renewal in the silkworm is largely academic, inasmuch as the reproductive life of the male is very short, frequently not more than a single mating (Tazima, 1964) and the “indefinitive” or “primary” spermatogonia produced later than the fifth instar are ineffective in the production of functional “eupyrene” spermatozoa (Sado, 1965).

4. Drosophila With the exception of Clayton’s (1957) counts on the relative frequency of the different, types of germ cells in the testes of D . virilis males, there is little cytomorphological information on the mechanism for maintenance of the supply of germ cells throughout the reproductive life of a Drosophila male. Nevertheless, it is obvious that there must be a means

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for a continued supply of sperm since a male can produce as many as 4000-10,000 offspring during a period of 20-30 days following eclosion (Duncan, 1930; Hannah-Alava and Puro, 1964). Inasmuch as this is two to three times the length of a spermatogenic cycle (cf. Kaufmann and Gay, 1963; Puro, 1964), either some of the youngest spermatogonia must be held in reserve for a long period of time or there is a means for continued production of the initial definitive spermatogonia. The two currently accepted opinions concerning maintenance of the supply of spermatogonia in Drosophila are: dichotomous multiplication of unencysted “primary” spermatogonia as in Fig. 1A and B from which cells are singled out to become the definitive spermatogonia (cf. Auerbach, 1941; Alexander, 1954, 1960; Savhagen, cited by Ytterborn, 1962; Seidel, 1963) and the stem-cell method (Fig. 1C) in which the predefinitive spermatogonia divide quasidichotomously and the definitive spermatogonia dichotomously (Harris, 1929; Tihen, 1946; llfuller et al., 1954; Puro, 1964; Hannah-Alava, 1964). h/lost of the evidence supporting a stem-cell method for spermatogonial renewal is derived from experimental results, with the greater part of it, being a by-product, of brood-pattern studies. The brood procedure was first used by Harris (1929) who proposed, on the basis of the size and distribution of clusters of identical mutations in the spermatogonial broods, that there is a stem-cell mechanism for spermatogonial renewal in D. melanogaster. According to him “proliferation of germ cells in the testis probably occurs through a system of one or a very few indefinitely reproducing cells functioning like apical cells.” On the basis of fecundity results, Auerbach (1941) concluded that it is unlikely that one or two “apical” cells could account for all of the sperm produced by a single male. Tihen (1946) was also of the opinion th a t production of the sperm by dichotomous divisions of one or two spermatogonia was highly improbable. He considered it more likely th a t about eleven of the germ cells-which he called the predefinitive spermatogonia-behaved as cambial cells in that each produced, a t each division, a cell like itself and a definitive spermatogoniuni destined, through multiplication and differentiation, to become the functional sperm. If there is a stem-cell method for spermatogonial renewal in the testis of D. melanogaster males it would most probably be like the model depicted in Fig. 1C because there are sixteen cells to a cyst (Pontecorvo, 1944). However, since both Cooper (1950) and Kaufmann and Gay (1963) were of the opinion that there are asynchronously dividing unencysted ‘Lprimary” spermatogonia, spermatogonial multiplication similar to the model shown in Fig. 1D cannot be ruled out (e.g., Seidel, 1963). Even though there is good genetic evidence for a stem-cell method for spermatogonial renewal in D. melanogaster (Muller et al., 1954; Puro, 1964)

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there is no supporting cytological evidence. Even the details of what the stem cells are and how they function remain to be clarified. Harris’ (1929) arid Friesen’s (1936, 1937) results had led to the assumption that there were only one or two cells per testis functioning indefinitely as stem cells (cf. Muller et al., 1954), a number questioned by both Auerbach (1941) and Tihen (1946) even before methods were developed for calculating it from genetic evidence. Recent studies suggest that although there may be as many as 15-20 stem cells a t the time of the organization of the testis (Sonnenblick, 1950; Hathaway and Selman, 1961), the number is progressively reduced throughout the life of the male. As many as thirteen pole cells survive treatment with ultraviolet light when the embryos are treated in the polar-cap stage (Muller, et al., 1954), and a minimum of five to ten following treatment of first-iiistar larvae with 900 r of X-rays (Alexander, 1954; Khishin, 1955; cf. Puro, 1964). Only five or six stem cells survive when an adult male is treated with 3000 r of X-rays (Puro, 1963, 1964; Auerbach, 1963), but possibly fewer with higher doses (Friesen, 1936, 1937; Abrahamson and Friedman, 1964). Apparently the number is further reduced, perhaps to as few as two to four cells, in the older males (Harris, 1929; Luers, 1956; Abrahamson and Friedman, 1964; Olivieri arid Olivieri, 1964). This reduction in number of stem cells could account for the progressive reduction of off spring in increasingly older males (Hannah-Alava and Puro, 1964; Hannah-Alava, 1965). Hence senile sterility in D. melanogaster may be due to the failure of the ejaculatory mechanism (Duncan, 1930), or to the lack of seminal fluid for activation of the sperm (Lefevre and Jonssori, 1962), but it can also be due to the lack of germ cells. In the Orthoptera and Lepidoptera, depletion of the spermatogonia begins with the degeneration of the apical cell (Griinberg, 1903; Zich, 1911; Nelson, 1931; Robertson, 1931) and the transformation of the predefinitive spermatogonia, immediately surrounding the apical cell, into definitive spermatogonia which then undergo the divisions leading to the formation of the sperm.

C. SPERMATOGONIAL STAGES Spermatogenesis is a continuous process in which a single spermatogonium gives rise, as the result of cell multiplication and differentiation, to the primary spermatocytes, each of which in turn after a n enormous increase in size and a series of complex nuclear changes, undergoes the first meiotic division (MI) to produce the secondary spermatocytes. These in turn divide (MII) and the resultant cells differentiate first into spermatids and finally into the mature spermatozoa. The meiotic stages, beginning with the differentiation of the young primary spermatocytes, are well known and have most recently been reviewed by Rhoades (1961).

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The most recent review on spermatogonia of the insects was made by Depdolla (1928) , and of the mammals by Roosen-Runge (1962). The sequence of events prior to the meiotic stages in the insect especially, and to some extent also in the mammal, is not very well documented. This not only includes the spermatogonial stages proper, but there is relatively little published information on the exact changes taking place between the time of organization of the testis with its primordial germ cells and the initiation of spermatogenesis. Thus it is not known for most species whether the primordial germ cells undergo both differentiation and multiplication or only differentiation before becoming predefinitive spermatogonia. Furthermore, although there is genetic evidence for a stem-cell mechanism for spermatogonial renewal in some of the insects, the cytological information is so scant,y that it is not known whether there is spermatogonial multiplication between the quasidichotomous predefinitive and the dichotomous definitive divisions. With certain exceptions to be described in detail, there are no intervening “indefinitive” mitoses in most of the species which have been carefully studied; in others the number of so-called primary spermatogonia is so large that multiplication prior to the definitive divisions would be indicated. 1. The Primordial Germ Cells, Primordial Spermatogonia, and

Predejinitive Spermatogonia a. Mammals. The primordial germ cells in the rat and the mouse, and presumably in all mammals, arise extragonadally and migrate into the genital ridges. The testis is established with incorporation of the primordial germ cells, frequently called gonocytes, into the medullary cords and the appearance of an incipient tunica albuginea (cf. Franchi et al., 1962). The changes that occur subsequently have been described in detail for the rat (Hucltins, 1963; Beaumont and Mandl, 1963; Franchi and Mandl, 1964). The testis in this species is differentiated by day 14 of gestation, some 2 days after the primordial germ cells have migrated into the genital ridges, but the A-type spermatogonia are not present until 4 or 5 days after birth. The population of primordial germ cells increases from ca. 20,000 to some 140,000 cells from the time of the establishment of the testis until about 18.5 days; then mitotic activity ceases until 4 or 5 days after birth. During this time the germ cells, although in a prolonged interphase, increase in size, then (on days 4 or 5) they become transformed into a “darker transitional” cell type. The transitional cells then become at,tached to the basement membrane. With the resumption of mitosis, the smaller A-type spermatogonia first appear, presumably as division products of the transitional cells. However, the transitional cells may also produce cells like themselves since their number does not de-

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crease with the increase in number of A-type spermatogonia. I n one experiment (Beaumont and Mandl) the gonocytes decreased from 87,900 to 25,810, the transitional cells increased from 4380 to 69,890, and the A-type spermatogonia from 330 to 62,050 from day 4 to day 6. With the exception of a size difference, the cytomorphological change from the gonocyte-stage to the transitional stage was greater than the change from the transitional stage to the A-type spermatogonia. The changes consisted of polarization of the mitochondria with the beginning of the long interphase and its redistribution just before resumption of mitosis by the transitional cells, attachment of the transitional cells to the basement membrane, and formation of several distinct types of cytoplasmic inclusions in the cells that do not become attached and presumably are destined to degenerate. Since similar changes have been reported for several mammals (cf. Beaumont and RIandl, 1963; Franchi and Mandl, 1964) it could be that the primordial germ cells in many vertebrates undergo complex cytomorphological changes as well as multiplication prior to differentiation of the spermatogonia. In spite of the large number of A-type spermatogonia in the gonad very soon after birth, new A-type spermatogonia are produced throughout the reproductive life of the mammal (cf. Roosen-Runge, 1962). Experimental evidence has indicated that sperniatogonial renewal is accomplished in many mammals by means of a stem-cell method in which the A-type cells are derived from dormant A-type spermatogonia (cf. RoosenRunge, 1962; Rlonesi, 1962a; Clermont, 1962). On the basis of their observations of sperniatogonial multiplication in the rat, Clermont and Leblond (1953) suggested that the dormant mother cell and the A-type sperniatogonium could be produced by a special type of mitosis with a n unequal distribution of chromosomal or cytoplasmic material, but as yet there is no good evidence for a morphological difference between the Atype cells destined to be the stem cells for the next generation of spermatogonia and the A-type spermatogonia destined to divide dichotomously as definitive spermatogonia (Oakberg, 1965). 6. Orthoptera. The most detailed description of the primordial germ cells and their fate during the organization of testis was made by Nelson (1931) for the short-horned grasshopper, Melanoplus d<j'erentialis. About the time of hatching, the primordial germ cells become isolated in groups of one to five, due to the partitioning activity of the differentiating connective tissue cells, to form the follicles. One of the mesodermal cells then moves in between the germ cells in each follicle and differentiates as the apical cell. Simultaneously, the germ cells resume mitotic activity and multiply until there are 8-10 cells surrounding each apical cell. An apical cell with the single layer of germ cells surrounding it and the ac-

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companying small mesodermal septa1 cells is called the apical complex (Fig. 2A). It is usually a t the tip of the follicle but it may be somewhat displaced, by the developing cysts of spermatogonia. When the apical complex is established, Nelson referred to the primordial germ cells as the primary spermatogonia. Although there was already a considerable literature on orthopteran spermatogenesis-including Sutton’s (1900, 1902, 1903) papers on spermatogonial divisions, McClung’s (1902) review paper on the X chromosome, and Otte’s (1906) report that the number of cells in a cyst indicated the order of spermatogonial multiplication, Davis (1908) was the first to observe that some of the spermatogonia immediately surrounding the apical cell had a different fate than others. On the basis of his investigations-primarily on the short-horned grasshopper, Dissosteira Carolina, but also other species of both the short- and long-horned grasshoppers-he concluded that when one of the spermatogonia immediately surrounding the apical cell divided (as shown in Fig. 2A), one of the daughter cells is usually forced out of the layer, becomes intimately associated with one or more connective tissue cells which form an investment around it, and then divides to form a cyst of spermatogonia and eventually spermatocytes. Davis used the terms “primary” and “secondary” to differentiate between the two types of spermatogonia. This sequence of events was confirmed for another short-horned grasshopper, Melanoplus diferentialis, by Nelson (1931) who also found that “the primary spermatogonia remain as such so long as they retain their intimate association with the apical cell.” According to him the fate of the daughter cells depends both upon orientation of the spindle of the dividing “primary” spermatogonium, and upon the extent of crowding of the cells around the apical cell. Experimental evidence (Bishop, 1942) from a third species, Circotettix verruculatus, not only proved that each cyst of spermatocytes was derived from a single cell (which he called a “primordial germ cell”) but that several cysts could be derived from the same cell. Thus in the acridian grasshoppers the spermatogonia immediately surrounding the apical cell are predefinitive spermatogonia in that they behave as stem cells with one of the division products usually being retained as a predefinitive spermatogonium and the other becoming the initial definitive spermatogonium. Morphologically, the spermatogonia surrounding the apical cell appear to be somewhat different from the encysted spermatogonia (Brunelli, 1910) and the X chromosome may not be bipartite during metaphase in those cells closest to the apical cell (Fig. 5A) as it is in the early generations of encysted spermatogonia (Fig. 5B). Even though Davis was the first to be aware of what is now known as

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FIG.2. Organization of the testis in the Orthoptera and Lepidoptera. A, B. Dissosteira carolina (Orthoptera, Acrididae). (A) The apical complex with the apical cell (lower center), three spermatogonia (in interphase, metaphase, and telophase) and four cyst cells. (B) A cyst of spermatogonia surrounded by the cyst wall and its cells. The “horseshoe-shaped structure” connecting all of the cells of a (8- or 16-cell) cyst is derived from the spindle bridges. From Davis (1908). C, D. Bombyx mori (Lepidoptera). Spermatogonial stages in the testis of a thirdinstar larva. (C) The apical complex with the apical cell (apc) surrounded by the primordial (predefinitive) spermatogonia (pgc). (D) Generations of encysted spermatogonia (sg) and the cyst-wall cells (cwc). From Nakanishi et al. (1963). E. Tischeria angusticolella Dup. (Lepidoptera). The testis with the single “Urspermatogonie” (Up) which functions as a stem-cell spermatogonium, the five generations of encysted definitive spermatogonia and a cyst of spermatocytes. From Knaben (1931).

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the stem-cell method for spermatogonial renewal, and Brunelli clearly pictured cyclic heteropycnosis of the X-chromosome in successive generations of the encysted spermatogonia, Schellenberg (1913) and Mohr (1914) were the first to differentiate between the different types of spermatogonia in the orthopteran testis, and surprisingly enough in species without the clear-cut niorphological diff erenccs characteristic of the successive generations of spermatogonia in the acridian grasshoppers (Fig. 5A-L). Schellenberg clearly distinguished between predefinitive and definitive spermatogonia in the long-horned grasshopper, Diestrammena marmorata de Hahn. nlohr identified three types of sperniatogonia, the “Urspermatogonien” and two types of definitive cells, which he called the primary and secondary spermatogonia, in the testis of another long-horned grasshopper, Locusta viridissima (Tettigonia viridissima L., according to White, 1940). But it cannot be deduced from nlohr’s description if he considered the primordial sperrnatogonia to be the progenitors of the primary spermatogonia. Furthermore, since he described and pictured the “primary” spermatogonia as being in cysts surrounding the apical cell, 110 conclusion can be reached about spermatogonial renewal in this species on the basis of n!rOhr’S description. White (1940) pointed out that ‘“Iohr emphasized the distinction between the primordial sperrnatogonia and the different generations of secondary spermatogonia.” Sinre White also reported that the general features of nlohr’s description apply fairly well to all Tettigonidae (long-horned grasshoppers), it may be that the Acrididae and Tettigonidae have somewhat different methods of spermatogonial renewal. c. Lepidoptera. Because of the differences in testicular organization in the various suborders of the Lepidoptera, a difference in the sequence of spermatogenic events might also be expected. However, with the exception of one group, the apical complex and the morphology of the spermatogonial cysts in most species appear to be similar to that described for the silkworm by Nakanishi et al. (1963; Fig. 2C,D). Nevertheless, four types of preineiotic germ cells have been identified in Bombyx mori, the primordial germ cells, the primordial spermatogonia, the primary spermatogonia, and the secondary spermatogonia (Toyama, 1894; Kawaguchi, 1928; Miya, 1959; Sado, 1961, 1963, 1965; Kobayashi, 19G2; Nakanishi et al., 1963; Tazima, 1964), but the descriptions indicate a good deal of synonymy in terminology. “Primary” spermatogonia as defined b y Sado include the stem cells and the dichotomously dividing “indefinitive” spermatogonia, with the encysted spermatogonia being called “secondary” spermatogonia. Carson (1945) described only two types of spermatogonia in the butterflies, Papilio turnus and Colias philodice, the “primary” spermatogonia, with cytoplasmic processes extending into the

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cytoplasm of the apical cell, and the generations of encysted spermatogonia. Three types of spermatogonia have been distinguished in two species of Orgyia (Lymantriidae), the “primordial spermatogonia” surrounding the apical cell, the young spermatogonia before the first definitive division, and the encysted spermatogonia (Cretschmar, 1928). I n contrast to these species, in the microlepidopteran, Tischeria angusticolella Dup., with no apical cell during any stage of the life cycle, the single “Ursperniatogonie” a t the distal end of each follicle divides successively to produce the initial cell for each group of encysted spermatogonia (Knaben, 1931, and Fig. 2E). The germ cells in Bombyx mori, after becoming established in the germ band, differentiate in several of the abdominal segments, but only those in the sixth to the ninth segments are folded into the right and left genital ridges, which then contract to form the gonads (Miya, 1959; cf. Tazima, 1964). Although each testis in the earlier stages consists of a single follicle with a few primordial germ cells, just prior to hatching the gonad is partitioned into four follicles each with an apical cell surrounded by primordial germ cells. The number of primordial cells per gonad, according to Kobayashi (1962; cf. Tazima, 1964) increased from 47.5 a t 2 days before hatching, to 171.4 one day after hatching. The “primary” and “secondary” spermatogonia are differentiated only three days after hatching (Sado, cf. Tazima, 1964). I n the third-instar larvae, the cytoplasm of each “primordial germ cell” is connected to the cytoplasm of the apical cell by a spinelike process, while the spermatogonia are encysted (Nakanishi et al., 1963, and Fig. 2C,D). Since according to these authors even the youngest spermatogonia have a “single crescent-shaped ‘cystwall’ cell” which divides to form the envelope enclosing the groups of multiplying spermatogonia, the difference between the primordial germ cell, the primordial spermatogonia, and the primary spermatogonia functioning as stem cells could be a temporal one rather than a spatial or morphological one. d. Drosophila. The germ cells in Drosophila, as in most of the other Diptera, are first identifiable when they are budded off, as pole cells, at the posterior end of the egg during blastema and blastoderm formation about the second hour after egg deposition (Huettner, 1923; Sonnenblick, 1950). Even a t this time the pole cells in D. melanogaster are considerably larger and morphologically distinct from the blastema or blastoderm cells, but one of the characteristic differences is the presence of polar granules in the cytoplasm of the pole cells (Rabinowitz, 1941; Counce, 1963). After their multiplication and organization into the polar cap, some of the pole cells that enter the body either by interblastodermal migration (Rabinowitz, 1941; Poulson, 1947, 1950; Counce and Ede, 1957; Poulson and

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Waterhouse, 1959; Counce, 1963) or via the midgut (Rabinowitz, 1941; Sonnenblick, 1941, 1950; Hathaway and Selman, 1961) migrate into the mesodermal germ bands on either side of the midgut, become surrounded by small mesodermal cells, and are organized into the two gonads (Poulson, 1947, 1950; Sonnenblick, 1941, 1950). When the definitive testes are formed, the pole cells are called primordial germ cells (Sonnenblick, 1950), but with the exception of the loss or morphological changes in the polar granules (Counce, 1963), there are apparently no pronounced cytological differences between the pole cells and the primordial germ cells. They probably do not undergo any extensive multiplication from the time of the interblastodermic migration until 6 hours after the gonads have been formed, i.e., at 16 hours of development (Sonnenblick, 1941, 1950; Aboim 1945). At the time of organization of the testes, the number of primordial germ cells has been estimated as averaging about 20 but the counts have ranged from 10-25 (Sonnenblick, 1941; Aboim, 1945) or 3-37 (Hathaway and Selman, 1961). On the basis of cell counts, the embryos fall roughly into two groups, those containing 5-7 cells per gonad and those containing 9-13 cells (Sonnenblick, 1941, 1950). Muller et al. (1954) were of the opinion that of the 10-26 cells incorporated into the gonads there were only “about half as many in the male (10-14) as in the female (18-26).” But, since the male gonads are always larger than the female gonads from the time that it is possible to identify their sex (Kerliis, 1931) and the size difference is due to the number of germ cells (Sonnenblick, 1950), it is more probable that during the sexually undifferentiated stage the gonads with the larger number of cells are testes (Bodenstein, 1950). On the basis of genetic evidence-the ratio of a given lethal to all tested chromosomes derived from the same genome of the same treated polar cap-Muller et al. (1954) concluded that about thirteen pole cells survive treatment with ultraviolet light to act as progenitors of the germ line, and pointed out that this figure agrees well with the “10-14 based on cell counts of the primordial testis.” However, the number of cells per embryo, following treatment of the polar-cap stage (2-3 hours) with a microbeam of ultraviolet light, averaged only 2.15 (range 0-31) in a 15hour-old embryo (Hathaway and Selman, 1961). The discrepancy between the morphological and genetic est,imates of the number of germ cells in the newly established testes could be resolved if it were assumed that the male embryo had the larger number of germ cells, i.e., 18-26, and that the pole cells are particularly sensitive to cell-killing by ultraviolet light, resulting in a t least 50% loss with the treatments given b y Muller and his co-workers, and a considerably higher loss following treatment with a microbeam of light.

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Between the sixteenth hour and hatching, germ-cell division is resumed. Sonnenblick (1941) reported that one or two mitoses occur before hatching, a t 20 hours, with some gonads containing 8,10, or 12 and others as many as 36-38 “primordia” in a newly hatched larva. According to Bodenstein (1950) the larva hatches at 25 hours, and a t this time each gonad has 36-38 “primordial germ cells.” Because of the wide range in the number of cells in the 13- to 15-hour gonad, and the lackof distinction between female and male gonads, either one or two synchronous or several asynchronous divisions could have taken place between 16 and 25 hours. It has been assumed (e.g., Auerbach, 1941; Tihen, 1946) that these divisions are dichotomous, resulting in an increase in the number of primordial germ cells, but genetic results (Alexander, 1954; Khishin, 1955; cf. Puro, 1964) do not support this assumption. A newly hatched male larva has well-formed gonads (Kerkis, 1931, 1933; Sonnenblick, 1950). The germ cells are entirely spermatogonia (Huettner, 1930; Khishin, 1955), but presumably they are morphologically similar to the primordial germ cells since Aboim (1945) found that diff ereiztiation of the sperniatogonial types did not, begin until about 36 hours of development, that is 12 hours after hatching. With the exception of Geigy (1931) and Aboim (1945) who reported that there were both spermatogonia and spermatocytes just prior to or during the time of the molt, only spermatogonia have been found in the testis of the first-instar larvae (Kerkis, 1933; Gloor, 1943). Spermatocytes occur for the first time, in the 50-hour-old testis (Bodenstein, 1950), but perhaps even later (Seidel, 1963), possibly as late as 64 hours, since Khishin (1955) reported that “at this time the first signs of differentiation of the testis can be noted. The more advanced spermatogonia have started to arrange themselves into parallel cysts . . . .” On the other hand Kaplan and Sislcen (1960), who fed tritiated thymidine to 16-hour-old larvae and fixed them 4 hours later, concluded that labeling of the posteriorly located nuclei indicated that “very early in spermatocyte development , . . the chromosomes are duplicate.” Thus sperniatocytes can be present as early as 36-40 hours after egg-laying even though they cannot be distinguished from the spermatogonia on the basis of cytomorphological differences. Huettiier (1930) who also was of the opinion that very young larvae have only spermatogonia, stated that in the older larvae and pupae the spermatogonia could be distinguished from the young spermatocytes on the basis of the intensity of staining of the chromatin, especially in Feulgen preparations, as shown in Fig. 7C. Nevertheless, there must be considerable variation among the larvae from experiment to experiment in the time of occurrence of the primary spermatocytes, inasmuch as Kaplan et al. (1964) found that the number of labeled sperm per bundle in sperm

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samples from 1-day-old males, that had been fed tritiated thymidine as 16-hour-old larvae, ranged from 2-32 in one experiment, but from 8-64 in a second. Since all 64 sperm in a bundle would be labeled if incorporation had taken place during spermatocyte development, bundles with fewer than 64 labeled sperm must come from cells that were in spermatogonial stages a t the time of chromosome labeling. I n general the meiotic divisions first occur about the time of pupation (cf. Khishin, 1955; Kaufmann and Gay, 1963; Abro, 1964; Lefevre and Jonsson, 1964). With the exception of some glaring discrepancies, e.g., Gleichauf’s (1936) report of spermatozoa in the late larval testis, most authors found fully formed but nonmotile spermatozoa only in very late pupal testes, but Khishin reported that spermatozoa were present at 174 hours, that is about 24 hours before eclosion. Since there is lack of synchrony between initiation of a specific stage of spermatogenesis and stage of metamorphosis (Abro, 1964), an error as great as 8 hours can be made if the timing of spermatogenesis is based upon stages of metamorphosis (e.g., compare the results of Bodenstein, 1950; Khishin, 1955; Lefevre and Jonsson, 1964). Very little is known about the morphological differences between the different kinds of spermatogonia. The ones called “primary” spermatogonia and characterized as being nonencysted and having asynchronous divisions (cf. Stern, 1941; Cooper, 1950; Kaufmann and Gay, 1963) have only been pictured by Seidel (1963) in studies using classic cytological techniques. Since Cooper also refers to them as the “predefinitive” spermatogonia, they could be the stem-cell spermatogonia, which, on the basis of genetic evidence (cf. Hannah-Alava, 1964) probably divide quasidichotomously to produce the initial definitive spermatogonia as well as the predefinitive cells for the next spermatogenic cycle. Or they could be dichotomously dividing “indefinitive” spermatogonia. Even the definitive spermatogonia cannot always be identified with ease since the Drosophila testis lacks the well-defined cysts characteristic of most other insect testes (Huettner, 1930). I n whole mounts of adult testes, the cells immediately surrounding the apical cells appear to be larger and morphologically somewhat different from the more distal and encysted spermatogonia (Hannah-Alava, 1965). This is in line with Kerkis’ observation (1933) that the younger oogonia in D. melanogaster females are larger than the oogonia derived from them. It is also in agreement with the numerous examples, especially in the Orthoptera, that the spermatogonia immediately surrounding the apical cell are larger than the later generations of encysted spermatogonia. Comparison of electron micrographs of Meyer (1961a,b) and Kaufmaim and Gay (1963) suggest that there may be considerable differences

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FIG.3. Electron micrographs of the “primary” and “secondary” spermatogonia in Drosophila hydei. (A) “Primary” spermatogonia with a spindle bridge (sb) connecting two cells (lower left). (B) Encysted “secondary” spermatogonia with a spindle bridge (sb) connecting three cells (lower left). N, nucleus; I, interstitia or cyst walls. Courtesy of G. F. Meyer (original).

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in the morphology of the “primary” and “secondary” spermatogonia in D. melanogaster. There are distinct differences between the two cell types in D. hydei (Fig. 3A,B; and Meyer, 1964): The nuclei of the “primary” spermatogonia are smaller and considerably more invaginated than the nuclei of the LLsecondary’lspermatogonia, and both have a distinct nucleolus that is morphologically different from the spermatocyte nucleolus. The apical cells do not show invaginations of the nuclear envelope; thus they can be differentiated from the spermatogonia in their immediate vicinity. The sheath of a secondary spermatogonial cyst does not differ from the sheath surrounding a cyst of spermatocytes, but the interstitium surrounding the primary spermatogonia has a more complex organization. Only sister primary spermatogonia a,re connected by intercellular bridges, while the secondary spermatogonia, which are arranged in cysts, show intercellular bridges connecting more than two cells. On the basis of the similarity in electron micrographs it seems reasonable to conclude that the “primary” and ‘(secondary” spermatogonia of D . melanogaster and D . hydei are very similar morphologically. Consequently it should be possible to differentiate between the different kinds of spermatogonia in D. melanogaster. I n many respects the differences between the “primary” and “secondary” spermatogonia of D. hydei are comparable to the differences between the unencysted and encysted spermatogonia of other insects, especially the grasshopper (Fig. 4) : Intercellular bridges connect only sister cells of the unencysted spermatogonia, but connect>several cells of a cyst to frequently form the “horseshoe-shaped” structure (Hannah, 1942). The sheath surrounding cysts of spermatogonia is present, or at least, more conspicuous in the later generations of encysted spermatogonia (Robertson, 1931). The mitochondria of the spermatogonia lying nearest the apical cell are distinctly polarized, and there even may be mitochondria in the interstitia (Carson, 1945). 2. The Indejtnitive and Definitive Spermatogonia

The definitive spermatogonia in general can be distinguished from the other cells of the testis, not only because of their distinct morphology, but also because they divide synchronously, dichotomously, and usually a specific number of times before differentiating into the spermatocytes. Although there is a considerable range in the total number of spermatogonial divisions for each of the different groups of animals, e.g., 4-9 in the grasshoppers (Robertson, 1931; Powers, 1942; White, 1955), 4-5 in the Drosophilidae (cf. Copper, 1950), 3-7 in the mammals (cf. Tobias, 1956; Roosen-Runge, 1962; Monesi, 1962a), 5-6 in the Lepidoptera (cf. Depdolla, 1928; Knaben, 1931; Oksala, 1944), with rare exceptions (e.g.,

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Fig, 4, For descriptive legend see opposite page.

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Janssens 1924; Creighton and Evans, 1941; White, 1955) it is speciesspecific. The dragonflies (Odonata) appear to be a special group in this respect since the divisions of the encysted spermatogonia vary from 12 to 17 both within and between species (Oksala, 1944; Omura, 1955,1957). Although h4cGregor (1899) introduced the terms “primary” and “secondary” to describe diff erelit, spermatogonial types (in Amphiuma), Regaud (1901) was the first who clearly recognized, on the basis of the appearance of the chromatin, two types of definitive spermatogonia. Clermont and Leblond (1953), using Allan’s nomenclature, distinguished three types in the rat, a n A type with lldust,y”chromatin, an intermediate type, and a B type with “crusty” chromatin. This terminology is now in general use for classifying the mammalian spermatogonia (cf. RoosenRunge, 1962; Clermont, 1962). The different generations of definitive spermatogonia should be more easily identifiable in the insect than in the rodent since the definitive spermatogonia are generally encysted (Figs. 2, 4,6A). Consequently the exact generation can be determined b y merely counting the number of cells in a cyst (Otte, 1906), yet with rare exceptions (e.g., Cretschmar, 1928; Robertson, 1931; Nakanishi et al., 1963) this has not been done. Neither has a uniform terminology been adopted for describing the spermatogonial types or generations in the insect, in spite of the fact that the most conmionly used terms are “primary” and “secondary.” Further confusion is added by referring to the spermatogonia surrounding the apical cell as a cyst of “primary” spermatogonia (e.g., illohr, 1914) or considering the “primary” and ‘lsecondary” spermatogonia as distinct from the synchronously dividing spermatogonia (e.g., Schaff er, 1920). a. Mammals. The subject of spermatogonia in the mammal has recently been reviewed in considerable detail by Leblond and Clermont (1952a), Tobias (1956), Roosen-Runge (1952, 1962), and Clermont (1962). Since the process of spermatogenesis in the mammal is characterized by a high degree of correlation between adjacent cells and successive generations (cf. Roosen-Runge, 1962) the sequence of events in spermatogonial multiplication has been established quantitatively by the use of a number of different techniques: A precise classification of the stages of

FIG.4. Spermatogonial stages in Hippiscus rugosis (Orthoptera, Acrididae) following treatment of the testis with special techniques (Altmann’s aniline-fuchsin counterstained with light green following osmic acid fixation) to demonstrate thc spindle bridges. (A) Two-cell stage, probably from a predefinitive division. (B) The two-cell stage of the first definitive division. (C) A cyst of four cells. (D) A cyst of 16 cells with a different configuration of the spindle bridges than the “horseshoe-shaped structure” characteristic of this stage. (E) Four cells from a 32-cell cyst to show details of the spindle bridges. From Hannah (1942).

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spermatogenesis in the cycle of the seminiferous epithelium has been achieved by the use of the acrosonial changes in the spermatid-as revealed by periodic acid-Schiff and hematoxylin staining-as reference points (Leblond and Clermont, 1952a; Oakberg, 1956a). The number of spermatogonial generations has been estimated by determining the number of mitotic peaks in spermatogonial multiplication after treatment of the testis with colchicine (Roosen-Runge, 1951; Leblond and Clermont, 1952b). Autoradiographic studies of DNA synthesis following incorporation of P32(Ortevant, 1954), C14 (Pelc and Howard, 1956; cf. Edwards and Sirlin, 1958), or tritiated thymidine (Monesi, 1962a,b), have made it possible to time the spermatogonial divisions as well as to clarify the sequence of the spermatogonial events. Thus Monesi, in addition to determining the method of spermatogonial renewal in the mouse, established the fact that although the average life span of the spermatogonia ranged only from 27 to 30.5 hours, the time required for each of the stages of the mitotic cycle varied for the different generations. Cytological studies have suggested precocity of the sex chromosomes (cf. Darlington, 1937) and electron micrographs have revealed that cytoplasmic bridges probably connect the B-type cells as well as the early spermatocytes in the rat (Watson, 1952; cf. Roosen-Runge, 1962). The morphological differences between the A-type and B-type spermatogonia have also been studied in detail. Two distinct types of spermatogonia are identifiable in the rat (Leblond and Clermont, 1952a,b; Clermont and Leblond, 1953; Clermont, 1962) and the mouse (Oakberg, 1956a,b) on the basis of the “dusty” or “crusty” type of chromatin in the nuclei. In the male of some other species, the ram for example, (cf. Roosen-Runge, 1962), the nuclei of the A-type cells have large nucleoli rather than the “dusty” chromatin, but the nuclei of the B-type cells are “crusty.” I n general the A-type cells are also larger and have less contracted metaphase chromosomes than the B-type cells. Finally, the changes from one generation to the next, in most mammals, are progressive with the last division, in the gerbil a t least having similarities to a meiotic division (Tobias, 1956). Meiotic-like precocity has also been reported for the last division in some insects (cf. Oksala, 1944) and perhaps is more common than is generally presumed. This may be one of the reasons for the confusion of spermatocytes and spermatogonia. With the exception of the guinea pig, most of the rodents appear to have five definitive spermatogonial divisions (cf. Roosen-Runge, 1962). The first two divisions in the rat produce A-type, the third intermediate, and the last two B-type spermatogonia (Clermont and Leblond, 1953; Clermont, 1962). In the mouse, however, there are three A-type, one intermediate, and one B-type definitive divisions (Monesi, 1962a).

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b. Orthoptera. The lack of uniformity in nomenclature of the encysted spermatogonia is more apparent in this group than in almost any other order of animals. Consequently, the term “primary” has been used for every generation of spermatogonia but the last, and the term “secondary” for every generation of definitive spermatogonia. Nevertheless, owing to the occurrence of cyclic reversibility in X-chromosomal heteropycnosis (White, 1935, 1940), it is possible to derive considerable information from the cytological literature, as shown in Fig. 5. Because of the interest in establishing continuity of the chromosome, it was customary for the earlier investigators to a t least, picture “early” and “late” spermatogonia even though the authors were unaware of the significance of X-chromosoma1 heteropycnosis in identification of the spermatogonial generations (see McClung, 1902, 1914; Wilson, 1925; Depdolla, 1928, for reviews). The change from negative to positive heteropycnosis is progressive in many, if not all, of the short-horned grasshoppers (Acrididae), the crickets (Gryllidae), and some of the species of grouse locusts and pigmy grasshoppers (Tetrigidae) with the X-chromosome being negatively heteropycnotic in the first several spermatogonial divisions, but behaving more like an autosome in the later divisions, so that in the last it is positively heteropycnotic (Robertson, 1930, 1931; White, 1940). The X chromosome, in two acridian species, Locusta migratoria (White, 1935) and Hippiscus rugosis (Hannah, 1942), is negatively heteropycnotic during the first three, and positive in the last three divisions of the encysted spermatogonia. It was the main character used by Hannah to differentiate between the “primary” and ‘Lsecondary”(encysted) spermatogonia. It is entirely possible, as shown in the publications of Davis (1908), Brunelli (1910), arid Wenrich (1916), that the “primary” and “secondary” generations of definitive spermatogonia can be identified in many orthopteran species on the basis of cyclic heteropycnosis of the X chromosome. The two types of encysted spermatogonia can also be separated, a t least in some of the Acrididae, on the basis of several other cytomorphological characteristics, including cell size, length of the chromosomes during metaphase, and nuclear morphology during interphase (Davis, 1908; Brunelli, 1910; Lewis and Robertson, 1916; Wenrich, 1916; McClung, 1927; White, 1935, 1940, 1954; Powers, 1942; Hannah, 1942). Perhaps even the intercellular bridges (Fig. 4) could be used as a diagnostic criterion since they are revealed equally well with certain classic techniques (Hannah, 1942) and the electron microscope (Fawcett el al., 1958; Yasuzumi et al., 1958). However the “horseshoe-shaped structure” (Fig. 2B) derived from the spindle remnants and intercellular bridges, and characteristically found in the 8- and 16-cell cysts, is not a consistent

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Fig. 5. For descriptive legend see opposite page.

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structure (Figs. 2B, 4D) and hence not a completely reliable criterion for separating the primary from the secondary spermatogonia (Hannah, 1942). However, on the basis of all of the cytomorphological characters, it should be possible to distinguish between the early and late generations of encysted spermatogonia without resorting to cell counts. Cyclic reversibility of the X chromosome during the spermatogonial divisions is not characteristic for all the species of Orthoptera. The X chromosome is positively heteropycnotic in all generations of the encysted spermatogonia in the long-horned grasshoppers (Schellenberg, 1913; White, 1940) and probably also in some of the grouse locusts or pigmy grasshoppers (Robertson, 1930, 1931). Consequently this character cannot be used for classification of the different generations of encysted spermatogonia in these species. But a t least early and late generations can be distinguished on the basis of the length of the metaphase chromosomes as well as cell size, and possibly also by differences in nuclear morphology (Mohr, 1914; Robertson, 1930, 1931 ; White, 1940). Mohr, who introduced the terms “primary” and “secondary” to describe the different types of definitive spermatogonia in a long-horned grasshopper (White, 1940), considered only the first generation to be primary spermatogonia. The literature, on the other hand, would suggest that about half of the divisions, if morphological differences are used as a criterion, produce primary spermatogonia and half secondary spermatogonia in the Tettigidae ( = Tetrigidae) at least (Robertson, 1931). However, Robertson’s observation that the products of the first four of the eight definitive divisions are not encysted would bear reinvestigation because of its possible significance in interpreting the unencysted “primary” spermatogonia of Drosophila as described by Cooper (1950), Seidel (1963), and Kaufmann and Gay (1963). Since the spermatogonial stages of the Orthoptera are particularly favorable for cytological investigation using the classic techniques, comparative studies of the structure of the

FIG. 5 . Stages of mitosis in successively later spermatogonial generations of Acridian grasshoppers demonstrating progressive heteropycnosis of the X chromosome and cytomorphological differences of the cells. (A) Polar view of a metaphase plate of a cell near the apical cell; the X chromosome is probably not negatively heteropycnotic. (B) Metaphase plate of an early encysted spermatogonium with the X chromosome negatively heteropycnotic. (C) Prophase of early spermatogonia with the X chromosome (upper left cell) negatively heteropycnotic. (D) Prophase of late spermatogonia with the X chromosome (the lowest chromosome in both cells) positively heteropycnotic. (E and F) Anaphase from early and late generations of spermatogonia. (G-I) Telophases from early, late, and the last spermatogonial generation. (J-L) Very late telophases or interphases from early, late, and the last spermatogonial generations. [A, C, D, F from Hippiseus rugosis (original); B, E, G, H from Phrynotettiz magnus (Wenrich, 1916); I-L from Dissosteira Carolina (Davis, 1908).]

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cyst walls, as revealed by the electron and light microscopes, would be of inestimable help in differentiating between the unencysted, indefinitive and encysted, definitive spermatogonia as well as between the different types or generations of definitive cells. Finally, if the definitive spermatogonia of the Orthoptera are separated into three categories, i.e., primary, intermediate, and secondary, it should be feasible, in the majority of cases, to clearly distinguish between the primary and secondary spermatogonia on the basis of morphological differences. If so, comparisons could then be made of the similarity or difference in physiological properties between the A- and B-type spermatogonia of the rodent, and the primary and secondary spermatogonia of the insect. c. Lepidoptera. On the basis of the organization of the testis in Bombyx mori with a typical apical complex in the proximal end of each follicle, and successive generations of encysted spermatogonia more posteriorly removed (Toyama, 1894; Griinberg, 1903; Kawaguchi, 1928; Nakanishi et al., 1963), it would be presumed that the spermatogonial stages in this species fit the general pattern for the insects, i.e., the apical cell immediately surrounded by predefinitive spermatogonia and the definitive cells more posterior and surrounded by cyst walls. Although the morphological studies of the testes of third-instar larvae (Nakanishi et at., 1963) suggest that there are only two types of spermatogonia, the “primordial” cells and the encysted spermatogonia (Fig. 2C,D), according to Kondo (1961) and Sado (1961, 1963, 1965)) there is also a series of “indefinitive” divisions between the primordial or stem-cell spermatogonia and the encysted LLsecondaryll spermatogonia. The cells of the indefinitive divisions as well as the primordial germ cells have been called “primary” spermatogonia by Sado, who considered them similar to the A-type spermatogonia of the mouse. Kondo wasof the opinion, however, that there were four or five divisions between the stem cells and the A-type (= “primary”) spermatogonia. Sado differentiated only between the “early” and “late” generations of the encysted spermatogonia. Nakanishi et al. were able, on the basis of cell counts, to count five generations (1-cell-32-cell) in the third-instar larva. They also pointed out that the spermatogonia within a cyst are joined together by intercellular bridges. Detailed cytological studies of the definitive spermatogonia of B. mori have not been published, and since the male is the homogametic sex, it is not possible to say whether the different generations of encysted spermatogonia could be distinguished cytomorphologically . d . Drosophila. Even less is known about the spermatogonia of Drosophila than of any of the previously described species. Ever since Stevens (1908) reported that the chromosomes of D. melanogaster were psrticu-

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FIG.6. Organization of the anterior end of the testis of Lhosophila melanogaster. (A) A section from a testis of a late second or early third instar larva that had been fed on tritiated thymidine-labeled food for 8 hours prior to fixation. Photographed a t grain level. The zone of incorporation is clearly restricted t o the anterior region of spermatogonia and young primary spermatorytes (W. 1). Kaplan, original). The photograph also clearly shows the “rows” of encysted cells characteristic of the Drosophila testis. (B) Photograph of the anterior end of a whole mount of a testis from a 3-day-old male fed colchicine 4 hours prior to fixation. Feulgen and phase contrast (original). ( C ) Photograph of a testis from a 2- to 4-hour-old male fed colchicine 8 hours prior to fixation. From 50-80% of the sperrnatogonia are in C-mitosis. Feulgen and phase contrast (original).

larly refractory to the commonly used cytological stains, it has been assumed that the Diptera are unsuitable for cytological investigation of spermatogenesis (cf. Depdolla’s 1928 review). Nevertheless, Guy6not and Naville’s (1929) observations on spermatogenesis in D. melanogaster were surprisingly detailed, Huettner (1930) both pictured and described

Fig. 7. For descriptive legend see opposite page. 192

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the morphological differences between the spermatogonia and spermatocytes (Fig. 7C), and Metz (1914, 1916, 1926) investigated the pairing of spermatogonial chromosomes. Subsequently, even though the meiotic stages and spermiogenesis have been followed in considerable detail (Zujtin, 1929; League, 1930, 1931 ; Woskressensky and Scheremetjewa, 1930; Frolova, 1931 ; Woskressensky, 1933; Cooper, 1949, 1950, 1952; Gay et al., 1957; Seidel, 1963; Wbro, 1964), only G u y h o t and Naville, Huettner, and Seidel have really pictured spermatogonia (Fig. 7). Furthermore, some authors were even of the opinion that the definitive spermatogonia were not encysted (Kerkis, 1933; Miller, 1950), and it was not known before 1941 (cf. Pontecorvo, 1944; Tihen, 1946) that there were four definitive spermatogonial divisions. Even as late as 1950, Cooper felt that gonial cytology in D. melanogaster was still not well understood, and that the best account was by Dobzhansky (1934) for D. pseudoobscura. In this species the anterior portion of the testis of a n adult male is occupied by a solid mass of spermatogonia and primary spermatocytes. The spermatogonia are rather small rounded or slightly polygonal cells with relatively large nuclei, each with one, or more seldom, two spherical nucleoli. They, like the spermatocytes and spermatids, are grouped into nests, “each nest consisting of a definite number of cells of similar age and undergoing all of the transformations simultaneously. The nests are due to a rhythmic succession of divisions and interphases among the progeny of a single primary spermatogonium . . . .” Thus the term primary spermatogonium, as used by Dobzhansky, could mean either a predefitiitive cell or the initial definitive spermatogonium. There is no question but that the spermatogonial cells and chromosomes in D. melanogaster are small (Figs. 6, 7) and that the DNA in certain stages of meiosis and spermiogenesis is refractory to basic dyes (Cooper, 1950; Abro, 1964; Hannah-Alava, 1965). However, some of the technical difficulties can be circumvented by the use of phase contrast FIG. 7. The spermatogonia of Drosophila melanogaster. ( A ) The anterior end of a whole mount of a testis from an 8-day-old male showing the group of apical cells (apc) surrounded by the youngest spermatogonia. Because of a break in the testis wall most of the spermatogonia and youngest spermatocytes are lying free (to the right) (original). (B) A drawing of a section from a testis depicting the smaller and larger cells which G u y h o t and Naville called sperrnatogonia and spermatocytes, but which are more likely apical cells and spermatogonia. From Guyknot and Naville (1929). (C) A section from a testis showing the distinct differences between the last generation of spermatogonia (spg) and the youngest sperrnatocytes (spc) following Feulgen treatment. From Huettner (1930). (D) Cross-section of a testis from a first-instar larva with primary spermatogonia, and (E) Drawing of a spermatogonium showing the nucleolus (N); from material fixed in Os04, embedded in methacrylate, and sectioned a t 1-2 1; phase contrast. D and E from Seidel (1963).

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and possibly also by some of the methods recently developed for human chromosomes. The electron microscope studies by a number of investigators (Yasuzumi et al., 1958; Ito, 1960; Meyer, 1961a,b; Meyer et al., 1961; Kaufmann and Gay, 1963; Tates, 1963) have shown that this technique should prove useful for the spermatogonial stages (Fig. 3A-B). Autoradiography (Fig. 6A; Kaplan and Siskin, 1960; Kaplan et al., 1964) and colchicine treatment (Fig. 6B,C and Hannah-Alava, 1965) should also contribute to a n understanding of the premeiotic stages of spermatogenesis in this species. Nevertheless, interpretation of the electron micrographs and autoradiographs, as well as the genetic results from radiation experiments, depend upon a clear concept of cell types and of the sequence of stages, and a t the present time this information is available only by virtue of the classic cytoinorphological studies. Meyer (1965) was even of the opinion that it would be “. . . very difficult to establish a reliable time sequence of premeiotic stages with t>heelectron niicroscope” although this technique would help in answering some of the questions still open. In their report on spermatogenesis of D . melanogaster G u y h o t and Naville (1929) described the syermatogonia as very small cells with a diameter of 4-5 p , with a nucleus perhaps 3 p in diameter. Similar sizes for spermatogonial cells and nuclei were reported by RIiller (1950), but according to Cooper (1950) the longest diameter of the “secondary” spermatogonia averaged from 7-10 p , with the nearly spherical nuclei being from 5.6 to 7.1 p in diameter. On the other hand, Woskressensky and Scheremetjewa (1930) concluded that the spermatogonia had diameters of 16-21 p and the nuclei were up to 12 p in diameter. G u y h o t and Naville’s drawing (Fig. 7B) suggests that the cells they called spermatogonia and spermatocytes, respectively, are more likely apical cells and spermatogonia (Fig. 7A); if so, the spermalogonial size is probably closer to that estimated by Cooper, rather than to that estimated by Wosltressensky and Scheremetjewa. Huettner (1930) also found the spermalogonia to be “. . . rather small as compared with spermatocytes, but they are conspicuous in that they stain heavily with all chromatin stains . . . the chromatin appears to be coarser when stained with hematoxylin and much finer and more delicate when the Feulgen reaction is applied.” But Huettner pictured only late definitive spermatogonia (Fig. 7C). Tihen (1946) used the terms “definitive” and “predefinitive” to describe two types of spermatogonia, but Cooper (19,50) was of the opinion that the cells Tihen called predefinitive spermatogonia could have been apical cells. Nevertheless, and after a careful and detailed study, Cooper concluded th at: “As with many insects, two sorts of spermatogonia occur in the apex of the testis of D. melanogaster. One

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type of spermatogoiiial cell [the ‘primary’ or ‘predefinitive’ spermatogonia] is characterized by not occurring in groups and b y undergoing asynchronous niitoses . . . . Contrasting with the above are the ‘secondary’ or ‘definitive’ spermatogonia . . . . They occur (excepting the initial one) in fairly well-defined cysts, all members of each being derived from the same original secondary spermatogonium . . . . The cell bodies are somewhat polygonal in shape . . . . The interphase or resting stage of the living spermatogonial cells has a nucleolus (more rarely two smaller nucleoli) nearly a third the diameter of the nucleus in size. The rest of the nucleus seems an empty void under the ordinary light-field microscope. I n the cytoplasm, generally to one side and appressed against the nucleus, there occur numerous granules which are probably of mitochondria1 nature . . . . A smaller number of such granules are scattered through the cytoplasm. When such resting-state cells are fixed, the nucleus shows a reticulum of varying coarseness and staining density dependent upon the fixative and dye employed.” Thus there is no evidence from Cooper’s description for a size difference among the different generations of encysted spermatogonia. Abro (1964) likewise made no distinction between different generations or types of spermatogonia. Electron microscope as well as light microscope studies of Kaufmann and Gay (1963) revealed that: “. . . The primary and secondary gonia lying near the apex of the testis are actively proliferating cells with relatively small nuclei . . . . Primary spermatogonia undergo mitosis independently of one another so that they do not produce clusters of synchronously dividing cells . . . . A secondary spermatogonium, on the other hand is characterized by its ability to produce two cells that divide in concert; their progeny and theirs in turn maintain a pattern of synchronous divisions until cysts of 16 cells are created . . . . Nuclei of secondary spermatogonia contain large nucleoli during interphase and early prophase. Their cytoplasm carries numerous mitochondria . . . [and] secondary sperniatogonia are connected by intercellular bridges.” Seidel (1963) both described and pictured “primary” spermatogonia. She found only primary spermatogoiiia in the testes of first-instar larvae (Fig. 7D). “Secondary” spermatogonia occurred for the first time in the second insfar, and the primary spermatocytes in the third-instar testes. Comparison of the electron micrographs of the spermatogonia of D. melanogaster (Rleyer, 196lb; Kaufmann and Gay, 1963) and D. hydei (Rleyer, 1964) suggests that there are morphological differences between the early and late generations. Of particular interest in this respect are the intercellular bridges (especially bridges connecting more than two cells) and the septa between the cysts of cells. Both are clearly revealed arid easy to identify in electron micrographs (Fig. 3A,B). Use of these two

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characters for identification of the different generations of spermatogonia should resolve the question of the difference between “primary” spermatogonia as used by Cooper, Seidel, and Kaufmann and Gay, and “primary” spermatogonia as defined in this review. A study of whole mounts of gonads from young adult D. melanogaster males that had been fed colchicine 2-8 hours before fixation (Fig. 6B,C) suggests that the larger cysts of synchronously dividing spermatogonia occupy the greater part of the tip of the testis with the 1-, 2-, and 4-cell stages being present only in the immediate vicinity of the group of apical cells; however, this must be confirmed with sectioned material before it can be settled whether there are intervening generations of “indefinitive” spermatogonia in this species. Clayton (1957) in a study of the frequencies of spermatogenic stages in larval, pupal, and adult testes of D. virilis males found that the absolute and relative frequency of spermatogonia decreased progressively throughout life. The spermatogonia, according to Clayton, are small cells with a centrally located nucleus containing a large nucleolus. On the basis of genetic evidence, Alexander and Stone (1955) concluded that there are two types of spermatogonia in D. virilis, which because of similarity in susceptibility to radiation injury could be homologized with the A- and B-type spermatogonia of the mouse (Alexander, 1959). “Primary” and “secondary” spermatogonia of D. pavani have been pictured and described by Donoso (1961). Although he reported that there was no fundamental difference in spermatogenesis between this species and D. subobscura (and D . melanogaster) as described by Philip (1944), it is of interest to note that only the older spermatocytes in D. pavani are depicted as being encysted. An observation made by Philip, and of considerable interest to the radiation geneticist, was that meiosis subsides in D. subobscura during periods of differentiation and stress; consequently she found no divisions in males up to 2 days after emergence. The spermatogonial divisions in D. melanogaster males, however, apparently are not affected, because males less than 12 hours old may have many C-mitoses following colchicine feeding (Fig. 6C, and HannahAlava, 1965). 111. Relating the Temporal and Spatial Patterns of Spermatogenesis

The extent of organization of the testis and accessory organs as well as the maturity of the germ cells a t the time of birth or eclosion varies considerably from species to species. This factor as well as the length of the reproductive life of the adult male is of considerable significance in experimental work, especially in investigations concerned with the effect of mutagenic agents upon the spermatogonial stages. I n view of the fact

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that there are indications from the Drosophila work of a difference in recovery of mutations, if not a difference in sensitivity, of germ cells in the same physiological stage but derived from different times in the life cycle (Moore, 1934; Ives, 1960; Auerbach and Sonbati, 1960; Lefevre and Jonsson, 1964; Alderson and Pelecanos, 1964), the origin of the material must be kept in mind in interpreting mutation results. The early stages of spermatogenesis occur in some insects primarily during the larval or nymphal stages, with only the latest stages of meiosis or spermateliosis taking place after sexual maturity. I n others most of the sperm is derived from cells that had begun spermatogonial multiplication after embryogenesis. Thus, in Bombyx mori, with the reproduct,ive life of the male being a t the most only a few days (Tazima, 1964), the greater part of the functional sperm will come from cells that have undergone the spermatogonial divisions prior to the fifth larval instar (Sado, 1965). In Drosophila melanogaster, with a reproductive life on the average twice as long as the time required for a single spermatogenic cycle, a large part of the sperm is produced after eclosion of the male. Consequently, sensitivity studies of the premeiotic stages in B. mori would of necessity involve treatment only of the larval stages, whereas comparable studies could be made with D . melanogaster from treated larvae, pupae, and adults. A.

CHRONOMETRY O F THE STAGES O F SPERMATOGENESIS

With the interest, in the past few years in differential sensitivity of the germ cells, a number of methods have been developed for assay of the mutations and more refined criteria employed for relating the brood pattern of offspring to the temporal pattern of spermatogenesis. But paradoxically, the more refined the techniques have become for the sampling of mutations-for example, testing a single offspring from each male (Abrahamson and Friedman, 1964; Oftedal, 1964a,b)-the less useful the data are for relating the temporal and spatial patterns of spermatogenesis. The brood procedure was first used in Drosophila experiments with adult males by Harris (1929) and Hanson and Heys (1929) for comparing radiation-induced sensitivity of the spermatogonia and the more mature germ cells. Interpretation of the brood data is based upon the reasoning that “when irradiated males are mated to a succession of virgin females and separate broods are reared from successive mating periods the spatial pattern of spermatogenesis becomes translated into a temporal pattern of genetic effects in successive broods. With effects known to be limited to certain stages this brood-pattern can be re-translated into the underlying sensitivity pattern of spermatogenesis” (Auerbach, 1954). I n addition to the tedious technical details, the brood procedure has the drawback that

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ALOHA HANNAH-ALAVA

if successive stages occur in a relatively short period of time, they cannot be separated in successive broods b y means of the commonly used procedures, e.g., 3-4 females per male a t 3-4 day intervals (cf. Olivieri and Olivieri, 1964; Fahmy and Fahmy, 1964). More recently daily broods have been used, especially in determining spermatogonial mutation rates, in order to separale, insofar as possible, broods derived from young sperniatocytes, young spermatogonia, older spermatogonia, and predefinitive spermatogonia, since all of these stages occur within a period of 2-3 days in an adult D . melanogaster male (Olivieri and Olivieri, 1964; Puro, 1964). The type of brood procedure used for the larval and pupal stages in Drosophila (Auerbach, 1949; Khishin, 1955) and by necessity in the silkworm (Sado, 1961, 1963; Tazima, 1964) is based upon the reasoning that if treatment with a mutagenic agent is given just a t the time of initiation of a stage in the larva or pupa, the products of that stage will be the first sperm to be sampled after eclosion of the male, with successively earlier stages sampled in successively later broods. But because synchrony between the stage of metamorphosis and stage of spermatogenesis is not absolute, a t least in Drosophila (League, 1931, cited by Parker, 1948; Gleichauf, 1936; i%bro,1984), chronology based upon time of initiation of larval and pupal stage may be quite unreliable. The chronology of spermatogenic events in the larval and pupal testes of Drosophila and Bombyx is primarily from morphological studies (cf. Bodenstein, 1930; Khishin, 1955; Seidel, 1963; Sado, 1961, 1963; Tazima, 1964). I n the mouse, and also in Bombyx, it has been measured by the progressive disappearance and reappearance of cell types or cell generations following treatment with irradiation (Hertwig, 1938; Oakberg, 1955a,b, 19ri9; Sado, 1961, 1983). Genetic evidence, such as “dominant lethality” in certain broods or the presence of clusters of identical mutations following treatment with a mutagenic agent is more commonly used as the criterion for timing spermaltogenesis in the adult (Friesen, 1937; Luning, 1952; Auerbach, 1954; Alexander and Stone, 1955), but it is also used for the larval and pupal stages in the insects (Khishin, 1955; Tazima, 1984). More recently autoradiography has been employed in a n effort to make more precise determinations for both the adult and embryonic stages in Drosophila (Chandley and Bateman, 1962; Kaplan and Sisken, 1960; Kaplan et al., 1964; Kaufmann and Gay, 1963) and the rodent (cf. Oakberg, 1957; Edwards and Sirlin, 1958; JIonesi, 1962a,b). The accuracy of stage identification is dependent not only on the reliability of the criteria used to relate the temporal and spatial patterns of spermatogenesis (cf. Oakberg, 1957; Auerbach, 1958; Hannah-Alava, 1964), but also upon the chronological stability of the spermatogenic

THE PREMEIOTIC STAGES OF SPERMATOGENESIS

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stages within an individual or between individuals (Oakberg, 1957; Edwards and Sirlin, 1958; Kaufmann and Gay, 1963). Another factor that must be taken into consideration in estimating stage chronometry is that the radiation or other treatment used for the timing may alter the chronological sequence of one or more stages of spermatogenesis (Shaver, 1953; Oakberg, 1956a,b, 1957; Clark, 1961; Kaufmann and Gay, 1963). Finally, an important source of bias is the result of misclassification of the stages, which in the mouse is probably mainly due to confusing spermatocytes with spermatogonia (Oakberg, 1957), but in the insect is more probably the consequence of the lack of information on the exact sequence of the premeiotic stages. 1. The Mouse

The duration of one sperinatogenic cycle in the mouse has been estimated to be about 34.5 days with the intervals between the time of treatment (with 100 r of X-rays) and the release of the mature sperm from the tubule ranging from 27.6-28.8 days for the B-type, 28.8-30.8 for the intermediate, and 33.5-35.5 days for the A-type spermatogonia (Oakberg, 195613, 1957). The tracer results, however, are not in complete agreement, but according to Oakberg, the estimates of Pelc and Howard (1956)-based upon autoradiographic rcsults following labeling of adenine with carbon-14-would be, if the proper correction is made for identification of the cells initially labeled. Monesi (1962b) concluded, from studies using tritiated-thymidine labeling, that there was as much as a 3.5-hour delay in mitosis for all types of sperrnatogonia after 100 r of X-rays, but previously Oakberg (1956b) and Edwards and Sirlin (1958) had reported that there was no delay in spermatogenesis following either 100 or 200 r of X-irradiation. However, Moncsi’s (1962a) results not only were in agreement with Oakberg’s (1957) in that the average life-span for the intermediate and B-type cells was 26-28 and 29-30 hours, respectively, but he was also able to show (by the tracer studies) that the lifespan of the A-type spermatogonia was also 26-31 hours. Finally, because the significant difference, among the different cell types, was in the duration of the stages of the cell cycle, Monesi concluded that the difference in X-ray sensitivity of the different generations of spermatogonia could be related to the longer (radiation-sensitive) interphases and shorter (radiation-resistant) prophases of the more sensitive intermediate and B-type spermatogonia. 2. Bombyx mori

The duration of spermatogenesis in the silkworm as determined from histological observations and the time required for regeneration of the

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ALOHA HANNAH-ALAVA

germ cells following acute X-irradiation (Sado, 1961, 1963) is about 20 days. The spermatogonial stages last a few days, the spermatocyctes 10-12 days, spermatids 5-6 days, and the spermatozoa 4-15 days, part of which time they may be stored. 3. Drosophila melanogaster

The length of time required for the spermatogenic cycle before eclosion has been estimated to be 9-10 days (Bodenstein, 1950; Khishin, 1955; Kaufmann and Gay, 1963; Kaplan et al., 1964). Tracer results, following labeling of DNA with tritiated thymidine, suggest that about 10-11 days are required for the youngest spermatocytes to become mature sperm in the adult male (Chandley and Bateman, 1962; Kaplan et al., 1964), but genetic results indicate that, following irradiation, definitive spermatogonia are already sampled in the 10- to ll-day broods, and predefinitive spermatogonia as early as the day 12 (Fritz-Niggli, 1958; Schacht, 1958; Olivieri and Olivieri, 1963, 1964; Puro, 1964). Since there is a minimum of five mitotic divisions between an initial predefinitive cell and the sixteen spermatocytes derived from it, each of which is from 8-15 hours in duration (Olivieri and Olivieri, 1964; Hannah-Alava, 1965), extensive radiation-induced mitotic delay is not indicated even after doses as great as 3000 r of X-rays. Conversely, Kaufmann and Gay (1963) reported, on the basis of tracer studies, that spermatogenesis is retarded as much as 24 hours following treatment of male pupae with 800 r, or adults with 1000 r of ionizing radiation. Because of the difference of opinion in respect to the time of initiation of the spermatogenic stages in the larva and pupa (Bodenstein, 1950; Khishin, 1955, Kaufmann and Gay, 1963; Seidel, 1963; Lefevre and Jonsson, 1964) and the difference in interpretation of the mutational brood pattern (see Olivieri and Olivieri, 1964; Puro, 1964; Hannah-Alava, 1964 for reviews of the literature), stage chronometry in D . melanogaster is as yet only a rough estimate. Part of the variance in the experimental results is due to the numerous environmental and genetic factors which may affect the time of sampling of the sperm derived from a specific stage, or even the time required for completion of one or more of the stages of spermatogenesis (Demerec and Kaufmann, 1941 ; Auerbach, 1954, 1958; Mossige, 1955; Kaufmann a.nd Gay, 1963; McCarthy and Nafei, 1963; Wedvik and Stromnaes, 1963; Fahmy and Fahmy, 1964; Hannah-Alava, 1964). Another factor that may play a very important role in the preciseness of chronometry in Drosophila is that of chronological instability of spermatogenesis (Miller, 1950; Lefevre and Jonsson, 1964) resulting in the mixing of sperm of different ages (Fahmy and Fahmy, 1960, 1964; Ives, 1960). Kaufmann and Gay (1963) concluded

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that there may be differences in the rate of germ-cell maturation because, in sections of the mature testis of D . melanogaster, some bundles of tagged spermatozoa were found to be near the testicular duct and others were more anterior, with many unlabeled bundles lying in between. Unless this could be explained either as incorporational differences, or as a normally expected anterior movement of the bundles during spermiogenesis as in the hemipteran, Leptocoris (Payne, 1934), a precise system of chronometry would not be possible in Drosophila. B. THEGENETICCRITERIA FOR RELATING THE TEMPORAL AND SPATIAL PATTERNS OF SPERMATOGENESIS The two most commonly used criteria for relating the brood pattern of mutational results with the spatial organization of the testis are: (1) a period of excessive sterility followed by resumption of fecundity, and (2) the distribution of clusters of identical mutations or crossovers in the progeny of males that have been treated with a mutagenic agent (Friesen, 1937; Russell, 1951; Auerbach, 1954; Puro, 1964). The first criterion has been used for all three of the species, Drosophila melanogaster, Bombyx mori, and the mouse, commonly employed for mutation studies, but the use of clusters as a criterion has been restricted, until rather recently, to Drosophila because its effectiveness as a criterion is related to the size and number of broods available from each male (Hannah-Alava, 1964). 1. Excessive Sterility

The occurrence of a sterility period subsequent to treatment of the male with irradiation is well established for Drosophila (cf. Sobels, 1963b Geerts, 1965), Bombyx (Sado, 1961, 1963), and the mouse (Russell, 1951; Russell et al., 1958; Oaltberg, 1955a,b, 1959). But, while the sterility period in the silkworm is due to elimination of meiotic stages, and in the mouse to killing of the spermatogonia, in Drosophila it has been attributed to loss of spermatids, spermatocytes, or spermatogonia. Furthermore, its usefulness as a criterion is limited, in Drosophila at least, since the extent of sterility is apparently somewhat dose-dependent (Ives, 1960; Abrahamson and Friedman, 19G4), and it can be obscured if there has not been exhaustion of the sperm (Sobels, 1963a). After treatment with ionizing radiation, the period of excessive sterility of an adult D . melanogaster male occurs between days 7 and 10, depending upon the dose and the brood procedure: Ives’ results (1960), with doses ranging from 500 to 1000 r of y-rays, suggest that the period of lowest fecundity is during the day 9 and day 10. But Sobels and Tates (1961) using two-day broods, found that fecundity was only slightly lowered in brood 4 after 788 r of X-rays, almost complete sterility en-

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ALOHA HANNAH-ALAVA

sued in the third brood after 1928 r, but after 2010 r both the third and fourth broods were sterile. Day 8 was the most sterile period, following treatment with 3000 r of X-rays (Hannah-Alava, 1964), but there was also considerable sterility both prior to and following day 8. Kvelland’s (1962) results, from daily broods following 1000-2200 r of X- or y-rays, show that there is variation from experiment to experiment, with the sterile period ranging from day 7 to day 9 even with the same dose. Finally, with lower doses the sterile period may result from killing of a single cell type, but with very high doses, i.e., 6000-12,000 r, all of the postspermatogonial cells could be killed (Abrahamson and Friedman, 1964). Second, there is a considerable difference in opinion as to what cell type is killed by the irradiation. Both Friesen (1937) and Welshons and Russell (1957) concluded, from histological studies of the testes of irradiated adult Drosophila males, that the late spermatogonia and early spermatocytes were the most sensitive cells to the killing effects of X-irradiation. On the basis of the earlier genetic results, loss of specific cell types was attributed either to “dominant lethality” of the spermatids (Luning, 1952) or the later meiotic stages (Demerec and Kaufmann, 1941; Bateman, 1956). More recently Sobels and Tates (1961) concluded th a t since the highest mutation rates were in the most sterile broods, these broods represent sampling of spermatocytes and possibly very early spermatids, an opinion supported by Abrahamson and Friedman (1964). Conversely, Chandley and Bateman (1960) were of the opinion that the spermatocytes were sampled prior to the sterile period, and Auerbach (1963) that they were sampled during the sterile period. Finally, and on the basis of the size and distribution of crossover cluslcrs, Olivieri and Olivieri (1964) and Puro (1964) concluded that early spermatocytes could be sampled after the sterile period. Thus, there is no doubt th a t the sterile period is a valid criterion for a stage in Drosophila spermatogenesis, but as a criterion for exactly what stage has yet to be clarified. The autoradiographic studies (Kaplan et al., 1964) would appear to substantiate the thesis that very young spermatocytes can be sampled after the sterile period since the first tagged sperm are in the day 10 brood. But if each mitotic cycle requires 15 hours (Olivieri and Olivieri, 1964), sperm derived from predefinitive spermatogonia should not be sampled before day 13 if spermatogonial multiplication is similar to the model shown in Fig. 1C and even later if there are indefinitive divisions between the definitive and predefinitive stages (Fig. 1D). Experimental evidence suggests th a t predefinitive spermatogonia are usually first sampled on day 12 (Olivieri and Olivieri, 1963; Puro, 1963, 1964; Auerbach, 1963). Furthermore, if there is a brood-pattern shift with increasing doses of radiation, as found

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203

for translocations (Clark, 1961) and recessive lethals (Traut, 1963), there is no a priori reason for assuming that the first brood following the sterile periods is from early spermatocytes, especially in experiments with the higher doses of acute irradiation. The sterile period has also been used for separating meiotic and premeiotic stages in the mouse (Russell, 1951; Russell et al., 1958), b u t in the silkworm pronounced sterility following irradiation occurs as the result of damage to one or more of the later meiotic stages. Kogure and Nakajima (1959) concluded, from the histological picture of the testis, that the early prophase of the primary spermatocytes was the most sensitive stage, with the damaged cells being transformed into apyrene sperm, but other experiments (Sado, 1961, 1963) suggest that the later primary spermatocytes are even more sensitive to radiation killing. Furthermore, both genetic and histological results indicate that the spermatogonia in the silkworm are in reality the most sensitive cells t o the killing effects of X-rays, but that this is not observable in the fertility pattern, owing to the fact that there is replacement of the spermatogonial cells (Sado, 1961, 1963; Tazima and Kondo, 1963). The histological studies of Oakberg (1955a,b, 1956a,b, 1957, 1959) and the tracer studies of Monesi (1962a,b) have shown that the mouse spermatogonia are extraordinarily sensitive to irradiation killing, with the intermediate and B-type cells being the most sensitive and the A-type cells heterogeneous in sensitivity. Sad0 also reported that the encysted spermatogonia of the silkworm were the most sensitive cells, and equated them to the B-type spermatogonia of the mouse.

2. Clusters of Crossover Recombinants or Mutations a. Crossing-Over Recombinants. It has long been known th a t crossingover in the germ cells of Drosophila males is a very rare event, but that it can be increased by various genetic means or induced by a number of mutagenic agents and radiomimetic chemicals (cf. Friesen, 1933; Akhundov, 1937; Whittinghill, 1955; Sobels, 1956; Sobels and van Steenis, 1957; Ives, 1960; Auerbach and Sonbati, 1960; Fahmy and Fahmy, 1960). Considerable evidence has now been accumulated (cf. Friesen, 1936, 1937; Schacht, 1958; Savhagen, 1961, 1964; Thompson, 1962; Olivieri and Olivieri, 1963, 1964; Fahmy and Fahmy, 1964; Puro, 1964; HannahAlava, 1964) that after treatment of an adult male with a mutagenic agent, crossing-over recombinants are recovered from the sterile a n d immediately poststerile broods as singles, but beginning during 11-13 days, large clusters of complementary types are more frequently found than singles or small clusters.

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ALOHA HANNAH-ALAVA

During the course of experiments to determine the cause for a highly nonrandom occurrence of X-ray-induced recombinants in the progeny of D. m e h o g a s t e r males, Friesen (1932-1937, cf. 1936, 1937) concluded, and was supported by Auerbach (1954), that the recombinants first recovered were induced in meiotic cells, whereas those in the later broods, when fertility was again on the increase, were the result of induced spermatogonial crossing-over. On the other hand, Parker (1948) was of the opinion that “pseudo-clusters” could occur in Drosophila as the result of the induction of identical mutations in each cell of a cyst of primary spermatocytes. This has not been proved experimentally, but if identical mutations could be induced in all of the cells of a cyst, large clusters could be recovered from broods as late as the first meiotic metaphase. The induction of spermatogonial crossing-over has been substantiated by the recovery of recombinants from Drosophilu males that had been treated as young larvae (Patterson and Suche, 1934; Parker, 1948; see Whittinghill, 1955; Savhagen and Kemmer, 1964) when the gonads probably consisted entirely of spermatogonia (Bodenstein, 1950; Alexander, 1954 ; Khishin, 1955). Characteristically, the larger crossover clusters in the spermatogonial broods are complementary types (Friesen, 1936, 1937; Auerbach, 1954) and frequently they are too large to have come even from a definitive spermatogonium. For example, one of the larger clusters reported by Friesen (1936), consisted of 190 and 146 flies, respectively, of complementary crossover types, and was recovered from three consecutive broods extending over a period of 12 days. The recovery of very large and continuing crossing-over clusters from premeiotic broods of irradiated adult males has been confirmed by Olivieri and Olivieri (1963) and Puro (1964). On the basis of size and distribution of clusters of crossingover recombinants and mutations, Puro (1963; see also Auerbach, 1963; Puro, 1964) concluded that large clusters, beginning about day 12 after irradiation, and continuing for several subsequent broods, are evidence for a stem-cell mechanism for spermatogonial renewal, and thus a means for distinguishing between the broods derived from definitive and predefinitive spermatogonia. A summary of the relevant data, part of which is shown in Table 1, supports this concept. Large and continuing clusters usually begin 12-13 days after treatment of an adult male with ionizing radiation, and continue for 1-10 or more days. Their size and distribution suggests that they could not have been derived from definitive spermatogonia unless there had been such a degree of synchronization of the cells between cysts, as well as within a cyst, that all cells responded in the same way to the mutagenic agent. This is not completely inconceivable in view of the fact that synchronization of mitosis in a cyst of spermato-

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gonia is so great, in D. pseudoobscura, that even the metaphasic pattern of the chromosomes is similar in all cells (Dobzhansky, 1936). Another way to account for large clusters is by the method proposed by Savhagen (as cited by Ytterborn, 1962) to explain the variance in size and distribution of clusters of crossovers. According to her, “primary spermatogonia a t division are not always the source of one primary and one secondary spermatogonial cell, as suggested by Tihen, but may be divided into two primary or into two secondary spermatogonia.” Even though there is no conclusive evidence supporting this method of spermatogonial multiplication in Drosophila, it cannot be discounted since it has been observed to occur in the grasshopper (Nelson, 1931). Continuing clusters should offer critical information for clarification of this point; however, the data on continuing clusters are particularly meager because of the prevalent use of a 3- to 4-day mating interval, or sampling selected broods, and because of the way of presenting the spermatogonial data. The results of Friesen (1936), Olivieri and Olivieri (1963), and HannahAlava (1964) do not indicate th at there is any pronounced change in the frequency of crossover off spring, relative to total off spring, in successively later broods from the males with continuing clusters as would be expected if two functionally identical cells resulted from the division of a stem-cell spermatogonium. On the contrary, as shown by the continuing clusters in Table 1, the relative size of a cluster more frequently increases progressively in successively later broods, just as would be expected if there was random loss of “mutant” and “nonmutant” stem cells with increasing age of the male (Muller et al., 1954). A third factor responsible for variability in the recovery of recombinants from crossovers induced in the predefinitive sperrnatogonia,‘is the pattern of distribution of chromosomes to the daughter cells, as had been so elegantly demonstrated for somatic crossing-over by Stern (1936). If crossing-over occurs in the two-strand stage, both daughter cells should be identical in their chromosomes and the recombinants in successive broods should be the same. But if crossing-over occurs during the fourstrand stage (as suggested by Friesen, 1937; Shapiro, 1945; Olivieri and Olivieri, 1964), distribution of the chromosomes should be reflected in the brood pattern of recovery of the reciprocal types, in other words by “twin-spots’’ in time rather than in space (Table 2). Critical proof for four-strand crossing-over would be a brood pattern in which one of the complementary types was recovered in one brood and the other type in later broods (Tables 1, No. 3). This type could also occur in the singlebrood clusters, and would be evidenced as a higher frequency of one of the two complementary types, but inasmuch as continuing clusters frequently differ in number of offspring of the complementary types (Table 3) a

TABLE 1 Continuing Clusters of Crossover Recombinants in Drosophila Males Following X-Irradiation* Broods (days after mating) Males

2.

Progeny

Crossover Ratio: F,/CO

3.

10

11

12

73 (6& 2.0

13

14

Total F, Crossover

Crossover Ratio: Fl/CO Notch mutation Ratio: F1/Mut

16

58 65 2.5

-

Ratio: F1/CO

4.

15

3.3 7 20.0

3.8

17

1s

19

20

21

4

1.6

z

Total Fl Crossover No. A.

--66---

---57--

---94--

--133--

--170--

--140--

--132--

(1

Crossover No. B.

5. Ratio: Fl/CO Crossover No. C. Ratio: FI/CO

---22--

1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 12-12433.6

(

3 L - 2 L - 2 3 6 1-15 $-20$---15 111 81---2 5.2 3.0 3.8 3.7 6.9 2 - - - - - - - - - - - 6-5+613 10+2$" 15.6 11.0

(

---st_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

) 2 1) 5.5

e

i!

5,

z

4.4

k

* Flies heterozygous for mutant-marked second- or third-chromosomes; dosage 3000-4000 r of X-rays. - - - = number of days per brood; -and - - = continuing clusters; ratio = relative frequency of crossover or mutant flies to total F, progeny, and therefore usually the number of stem cells proliferating to form the sperm. t Symbols: S = male parent sterile; I> = male parent dead. 3 Crossover flies are also Minute. Males Nos. 1 and 2. Reciprocal recombinant types (from a crossover in the st-cu region of chromosome 3) to show the increase in the relative ratio of recombinants to noncrossovers in successive broods, and the difference in number of stem cells between males from the same experiment (from Friesen, 1936, Table 2). Male No. 3. One of the reciprocal types in one brood, followed by the other type as a continuing cluster indicating crossing*ver (in the en-vg region of chromosome 2) during the four-strand stage (Olivieri and Olivieri, 1963, Table 3). Male No. 4. Reciprocal types (from a crossover in the ri-p region of chromosome 3) in two broods, and a X-chromosomal mutation cluster in a single brood (from Hannah-Alava, 1964, Table 7). Male No. 6. Three crossovers in the progeny of another male in the same experiment as Male No. 4: Crossover No. A. Reciprocal types from a crossover in region p-bx, but recovered as three individuals in widely separated broods. Crossover No. B. Continuing cluster of reciprocal types probably from an unequal crossover in region e-ca since both types are homosygous lethal and one is also Minute. Crossover No. C. Reciprocal types from a crossover in region h-th as well as a mutation cluster from an independent, Minute,mutation (in chromosome 2 or 4) in the same stem cell (Hannah-Alava, 1964, Table 7).

M

E

2 0 0

q

--

TABLE 2 Distribution of Reciprocal Crossover Types in Successive Broods of Offspring from a Male with a Crossover Induced in a Predefinitive Spermatogonium* First generation from the predefinitive spermatogonium Kinds of exchange and genotype Chromosomal

(++++---(;ff'---++++ ++++

Definitive

++++-------

++++

Reciprocal CO

Predefinitive

Following generations Predefinitive

Definitive

3equence of reciprocal crossover types in successive broods of ffspring from the male ~

++++---++++---- ++++---- - - - ++++ - - _ _ ++++ - - - - ++++ -

Reciprocal crossover types in all broods

++++++++ ------ -

Reciprocal types only in the first brood

++++---- - - - ++++

-

Reciprocal CO

-

Reciprocal CO

Noncrossover

++++++++ --- -----

++++---- - - - ++++

Noncrossover

Reciprocal CO

++++++++ - _ - - ++++

++++----------

One type of CO

-

Other type CO

++++---++++++++ + + + + + + + + ++++++++ - - - - - - - - - - - - ++++ - - - - ++++ - - - - ++++ 3ne type of CO Other type CO

Reciprocal types beginning the second brood Reciprocal crossover types: one type in the first brood, and the other in the rest of the broods

* Assuming t h a t there is a stem-cell mechanism for the predefinitive divisions and that there is segregation of homologous centroand - - - - - - - - are the two normal chromosomes and ---meres during the mitotic divisions, and - - - are the two recombinant chromosomes. CO = Crossover.

+++ +

+ ++++ + + +

++++

THE PREMEIOTIC STAffES O F SPERMATOGENESIS

209

TABLE 3 Distribution of Reciprocal Crossing-Over Recombinants in the Poststerile Broods of Drosophila melanogasler Males Treated with X-Irradiation* Cluster in only one brood t

Crossing-over recombinants

Continuing clusterst a b c d e f g Total __

A. Reciprocal types: 1. In equal frequency 2. One, then the other 3. Unequal in frequency a. I n the same broods b. One, then both c. Both, then one 4. Special B. Only one of the reciprocal types: 1. Rz-type (Crossover) 2. R,-type (Crossover, mutant, or deficiency)

3 4 - 3 - 1 1 __ 1--1-

12 2

1 6 1---1 - 1 1 - 1 2 I---1 I---- 1

9 2 5 2

- 4

9

-_

- 3 1 - 1

2---3 * The reciprocal types were considered to be unequal in frequency if they differed by more than 10%. t Summarized from: (a) 4000 r of X-rays; Broods: 12-15, 16-19 days (Friesen, 1936, Table 1 ) . (b) 4000 r of X-rays; Broods: 10-13, 14-17, 18-21 days (Friesen, 1936, Table 2). (c) 4000 r of X-rays; Broods: 12-14,15-17,18-20,21-23 days (Friesen, 1937, Table 7). (d) 4000 r 4000 r of X-rays; Broods: 12-14, 15-17 days (Friesen, 1937, Table 8). (e) 1000 r of X-rays; Broods: 12-13,14-15, 16-18,19-21 days (Olivieri and Olivieri, 1963, Table 3). (f) 4000 r of X-rays; Broods: 12-13, 14-15, 16-18, 19-21 days (Olivieri and Olivieri, 1963, Table 3). ( g ) 3000 r of X-rays; daily broods, 12-24 days (Puro, 1964; Hannah-Alava, 1964, 1965). - 1

+

difference in their frequency is not proof of four-strand crossing-over. At present the data do not furnish extensive evidence for induction of crossing-over in the chromatid stage. This might be expected, however, since ionizing radiation produces premutational lesions of preexisting chromosomal strands, not an effect on the duplication process (Muller et al., 1961 ; Kimball, 1964). Nevertheless, the distribution of identical chromosomal aberrations in cysts of young spermatocytes derived from irradiated primordial germ cells of the grasshopper strongly suggests that the breaks leading to chromosomal aberrations can be induced by ionizing radiation in either the two- or four-strand stage (Helwig, 1933). Crossovers recovered immediately after the sterile period are either from early meiotic or late spermatogonial stages (Schacht, 1958; Chandley and Bateman, 1960; Olivieri and Olivieri, 1964; Puro, 1964). It may even

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be possible to differentiate between the two stages on the basis of the frequency of crossover events since, according to Olivieri and Olivieri, the late spermatogonia are more sensitive to induction of crossovers than the spermatocytes. However, proof of this would require a large material, and at the very minimum, daily broods. I t should be possible also to distinguish between the early (primary) and late (secondary) generations of definitive spermatogonia on the basis of goodness-of-fit to a Poisson distribution (Olivieri and Olivieri) if daily matings are used. Nevertheless, even this brood procedure may not have short enough intervals since indirect estimates indicate that each spermatogonial cell cycle is only 14-15 hours (Olivieri and Olivieri) and may even be somewhat shorter, inasmuch as more than 50% of the encysted spermatogonia are in C-mitosis 8 hours after colchicine treatment (Fig. 6C). Finally, with such a short mitotic cycle, if cell division is affected by the mutagenic agent, there may be considerable mixing of the spermatogonial stages even in daily broods. b. Clusters of Identical Mutations. Because of the interest in stagesensitivity, the brood pattern for a number of mutational types is well documented (cf. Sobels, 1963b). Mutant clusters have also been used to relate the spatial and temporal patterns of spermatogenesis, but the information on the size and distribution of mutation clusters in Drosophila, at least, is neither as extensive nor as reliable as the crossover clusters (cf. Hannah-Alava, 1964). Nevertheless, in animals without suitable genetic markers for crossing-over studies, mutant clusters may be the only means of distinguishing between induction of mutations in spermatocytes and spermatogonia, or between definitive and predefinitive spermatogonia. Conversely, mutant clusters have the advantage over crossing-over clusters in that “pseudo-clusters” can be more readily identified, with the exception of the X-chromosomal lethals and possibly homozygous viable mutants, by means of cross-testing. Large (and sometimes continuing) clusters of recessive autosomal lethals have been recovered in spermatogonial broods from D. melanogaster males treated in almost any stage of the life cycle and with a variety of ionizing radiations, as well as with ultraviolet light and some of the chemical mutagens (e.g., Berman, 1939; Auerbach, 1954, 1963; Auerbach and Sonbati, 1960; Muller et al., 1954; Fahmy and Fahmy, 1960; Puro, 1963, 1964; Abrahamson and Friedman, 1964). Continuing clusters of specific-locus mutations have been reported following treatment of 24-hour-old larvae with 900 r of X-rays (Alexander, 1954), but with few exceptions, not after exposure of adult males to X- or y-rays (Kvelland, 1962; Hannah-Alava, 1964; see also Friesen, 1936, 1937, for rl-type recombinants which could have been mutations). The lack of

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clusters of visible mutations may be due, in part, to the very low mutation rates of specific loci, but it may also be because of the loss of the predefinitive spermatogonia after one or two divisions since such clusters are rarely continuing clusters (Hannah-Alava, 1964). Clusters of specific-locus mutations have also been recovered following irradiation of the larval stages of the silkworm (Tazima, 1964). Kondo (1961), on the basis of theoretical considerations, confirmed Tazima’s conclusion that when treatment is given in a very early larval stage, the number of mutations per male deviates appreciably from a Poisson distribution. This would suggest that the clusters were derived either from very early indefinitive spermatogonia or from the spermatogonia functioning as stem cells. However, since there are six definitive divisions (Kawaguchi, 1928) and possibly four more indefinitive divisions between the “primary” spermatogonia and the stem cell (Kondo, 1961), the lack of a Poisson distribution for the clusters does not necessarily indicate that the large clusters, in Bombyx mori, are derived from mutated stem cells. IV. Radiosensitivity of Spermatogonia

The apparent agreement of the experimental evidence on radiationinduced mutations in the germ cells of Drosophila, the silkworm, and the mouse resulted in the establishment of a set of principles of mutagenesis which were presumed to apply equally well to all animals under all conditions. One of the most fundamental of the concepts to emerge from the Drosophila work was that of the striking quantitative difference in the response of germ cells in different stages of maturation a t the time of treatment with a mutagenic agent. Thus, following Harris’ demonstration (1929) of a five- to tenfold difference in recovery of X-ray-induced sex-linked lethals from mature sperm and spermatogonia, it was generally accepted that spermatogonia were considerably less sensitive to mutagens than the more mature germ cells even though subsequent estimates of the relative ratios varied from 2 : 1 to 18: 1 (cf. Schultz, 1936; TimofheffRessovsky and Zimmer, 1947; Muller, 1954; Oftedal, 1964a, for reviews of the literature). But with the discoveries that the mouse spermatogonia may be as much as fifteen times more sensitive to induction of mutations than Drosophila spermatogonia (Russell, 1951, 1956), that chemical mutagens may be more effective in inducing spermatogonial mutations than ionizing radiation (Auerbach, 1949; Fahmy and Fahmy, 1956; Auerbach and Sonbati, 1960), that there is a differential in the rate of induced mutations or cell-killing not only between the more mature germ cells and the spermatogonia, but also between different spermatogonial stages (Auerbach, 1954; Oakberg, 1955a), that the relative sensitivity of the spermatogonial stages may be mutagen-dependent (Watson, 1962), and

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that the induction of spermatogonial mutations in the mouse is not independent of radiation intensity (Russell et al., 1958), it becomes imperative to reinvestigate the whole problem of spermatogonial mutagenesis. A survey of the enormous body of literature that has accumulated in the past decade (e.g. proceedings edited by Ebert and Howard, 1963; Sobels, 196313; Geerts, 1965) makes it obvious that there are many differences in fact, or opinion, yet to be resolved before it will be possible to establish a logical concept of the spermatogonial stages and their response to treahment with mutagenic agents. Even though there is considerable evidence for similarity in patterns of sensitivity for comparable meiotic stages in a number of different species (Kimball, 1963), the development of reasonably clear concepts of spermatogonial sensitivity on the basis of the diverse and often contradictory facts has met with many difficulties. This may be one of the reasons why, in spite of the refined methods of assay of mutations and the concern with technical aspects of the brood procedure, the recent estimates of the relative radiosensitivity of spermatogonia as compared to that of the mature sperm vary as much as the earlier estimates (cf. Oftedal, 1964a). One of the greatest difficulties in the development of a logical concept of spermatogonial sensitivity is the lack of critical cytomorphological information on the sequence of the spermatogonial stages, as well as a uniform nomenclature for comparable stages in the different species used for mutation studies. At present, with the lack of morphological information on the methods of spermatogonial renewal and spermatogonial multiplication, comparison of species in respect to stage-sensitivity may be quite meaningless. Moreover, if it turns out that there are several generations of indefinitive spermatogonia between the definitive and predefinitive stages in Drosophila or Bombyx, the question can be raised whether it is legitimate to homologize the encysted definitive and unencysted indefinitive spermatogonia of the insect with the unencysted B- and A-type spermatogonia of the rodent, or to compare species in which the initial definitive spermatogonium is derived directly from a predefinitive (stem-cell) spermatogonium with a species in which there are several intervening generations of indefinitive spermatogonia. Second, even though the experimental results strongly suggest th a t the latest generations of spermatogonia are more sensitive than the earlier generations to X-ray induction of centromeric crossing-over in Drosophila (Olivieri and Olivieri, 1964) or cell-killing in the silkworm (Sado, 1961, 1963), the grasshopper (Cocchi and Uggeri, 1944), the mouse (Oakberg, 1955a,b, 1959), and probably also in Drosophila (Abrahamson and Friedman, 1964; Oftedal, 1964a,b), it is frequently not clear whether the “early” spermatogonia refer only to the cells dividing dichotomously, or

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whether the predefinitive spermatogonia have also been sampled. Inasmuch as the cells that repopulate the testis are particularly resistant to radiation-killing (Oakberg, 1955a, 1959; Tazima and Kondo, 1963; see also Auerbach, 1963; Abrahamson and Friedman, 1964), samples of “early” spermatogonia could include both radiation-sensitive and radiation-resistant cells. Heterogeneity in sensitivity to irradiation of the A-type spermatogonia in the mouse may be due to variation in the lengths of time required for the sensitive and resistant periods during the mitotic cycle (Rilonesi, 1962b). But the difference between early and late spermatogonia to induction of crossing-over in Drosophila could be a reflection of the extent of chromosomal pairing, which, in some dipteran species at least, is greater in the later than the earlier generations (Keuneke, 1924; Bauer, 1931). Third, even though the brood-procedure is the most common method for detecting the sensitivity pattern, the way of assessing the spermatogonial mutations has not been standardized. Consequently there are reports in the literature varying from complete counts of daily broods, or selected broods, to a single offspring per male (cf. R4cCarthy and Nafei, 1963; Abrahamson and Friedman, 1964; Olivieri and Olivieri, 1964; Puro, 1964; for different methods recently used in the Drosophila work). The daily broods of individual males give the most information on the variance in the brood and mutational patterns, but the tedious mating procedures and technical details frequently preclude scoring of sufficient off spring to be able to demonstrate significant differences between broods (e.g., Hannah-Alava, 1964). The use of brood periods of longer than one day limits detection of stage-sensitivity, since the early spermatocytes and late spermatogonia, from adult D. melanogaster males, are sampled during days 9 and 10, early spermatogonia in the 10- to 12-day broods and the predefinitive spermatogonia beginning on day 12 or day 13 (Friesen, 1936, 1937; Olivieri and Olivieri, 1963, 1964; Puro, 1964). Detecting differences between successive generations of spermatogonia, or even between early and late spermatogonia, consequently would require even shorter than daily broods since the cell cycle for spermatogonia is probably not more than 15 hours (Olivieri and Olivieri, 1964). Finally, sampling of spermatogonia from broods later than the day 13 may not even be an estimate of the initial mutation rate, but only a measure of the funct>ionalactivity (survival) of the stem cells with mutations relative to the functional activity of the stem cells without mutations (R4uller et al., 1954; Puro, 1964). Estimating mutation rates for the predefinitive (stem-cell) spermatogonia presents further problems, and problems yet to be resolved. According to Puro (1963; see also Auerbach, 1963) clusters of mutations,

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beginning in the day-12 and day-13 broods of irradiated adult males, and continuing for one or more subsequent broods (Table l ) , are evidence for a stem-cell method of spermatogonial renewal in Drosophila. The number of stem cells-i.e., the ratio of mutations to all tested chromosomes of the same genome (Muller et al., 1954)-in a young adult male is about ten, but the number probably decreases with increasing age of the male (cf. Puro, 1964; Hannah-Alava, 1964). Random loss of stem cells in the broods subsequent to day 12 or day 13 should not result, in any change in the relative frequency of progeny from mutant and nonmutant stem cells. But if only the males with continuing clusters are considered, as in Table 1, the frequency of mutations relative to the total offspring increases in successive broods (Muller et al., 1954). On the other hand, selective loss of the stem cells with mutations results in a decrease (in successively later broods) of both the absolute and relative frequencies of mutations (Hannah-Alava, 1964). Finally, the greatest difficulty in ascertaining spermatogonial mutation rates, however, is that they are not based upon the initial mutations, but upon the relative frequency of mutants to nonmutants after repeated cell divisions. Consequently one of the problems has been the development of suitable methods for reliably estimating the initial mutation rates (Alexander, 1954; Muller et al., 1954; Kimball, 1956; Welshons and Russell, 1957; Kondo, 1961; McSheehy, 1964; Olivieri and Olivieri, 1964.) Presentation of the spermatogonial data also varies from author to author. Spermatogonial rates are usually presented as the relative frequency of mutations to total offspring per broods, with the standard error based on the variance in the cluster size (Muller et al., 1954). But the data have also been tabulated as the number of independent events per male (Hannah-Alava, 1963), or the frequency of males with mutations and the number per male (Olivieri and Olivieri, 1964; Savhagen, 1964; Savhagen and Kemmer, 1964), or the number of independent events per chromosome at the time of treatment (Puro, 1963, 1964; McSheehy, 1964). Another method coming into favor is the sampling of a single offspring from each male (Abrahamson and Friedman, 1964; Oftedal, 1964a,b). Although this method bypasses the statistical problem of calculating the error due to the cluster, it has the disadvantage that it gives no information on the brood pattern, and this information may be very essential in determining stage-sensitivity (Hannah-Alava, 1964). At any rate, it is obvious that it is frequently impossible to compare the results of different investigators when the data have been presented in a variety of ways. Until adequate methods for estimating spermatogonial mutation rates have been developed, perhaps the best one was that used

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by Friesen (1936, 1937) who tabulated the performance of each individual male.

ACKNOWLEDGMENTS Because a considerable portion of the literature for this review was available only through interlibrary loan, the author is greatly indebted to members of the Library staff-particularly Miss K. Rautaniemi, Mr. P. Tiainen, and Mr. A. Lehtonen-of the University of Turku for their help in obtaining the publications. She also wishes to express her appreciation to Prof. Esko Suomalainen, of the University of Helsinki, for making the Federley library available for her use. Of the many who have read and made valuable comments and suggestions for the manuscript, special acknowledgment is made to: Drs. T. Sad0 and Y. H. Nakanishi a t the Japanese National Institutes of Genetics and Radiology, Drs. G. F. Meyer and Sigrid Seidel of the Max-Planck Institut fur Biologie a t Tubingen, and Dr. W. D. Kaplan of the City of Hope Medical Center for use of original photographs and unpublished experimental results. Finally, the author especially wishes to thank Miss Pirkko Heinonen, Mrs. Sirpa Roman, and Miss Leila Seppalii without whose help, in the morphological studies of the Drosophila testis, and during the preparation of the manuscript, this review could not have been written. Figures 2A,B, and 5B,E,G-L, courtesy of the Museum of Comparative Zoology of Harvard; Figs. 2E and 7C, courtesy of Springer-Verlag and Fig. 7B reprinted from L a Cellule. This work was supported by the International Atomic Energy Agency and the United States Atomic Energy Commission (IAEA Research Contract No. 31/US-AT (30) 2960).

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THE FUNCTION OF THE Y-CHROMOSOME IN MAN. ANIMALS. AND PLANTS*

K . R. Dronamrajut Johns Hopkinr University School of Medicine. Baltimore. Maryland

I . Introduction . . . . . . . . . . . . . . . . . . . . . . 11. Man . . . . . . . . . . . . . . . . . . . . . . . . . A . Y-Linkage . . . . . . . . . . . . . . . . . . . . . B . Cytogenetics of the Y-Chromosome . . . . . . . . . I11. Drosophilu . . . . . . . . . . . . . . . . . . . . . . . A . The X-Chromosome . . . . . . . . . . . . . . . . . B . Fertility . . . . . . . . . . . . . . . . . . . . . . C. Bobbed . . . . . . . . . . . . . . . . . . . . . . . D . Heterochromatin . . . . . . . . . . . . . . . . . . E . Heterochromatin and Polygenes . . . . . . . . . . . F. Heterochromatin and Variegation . . . . . . . . . . G . Sex and Antigenic Differences . . . . . . . . . . . . H . Other Effects of the Y-Chromosome . . . . . . . . . I V. Role of Y-Chromosome in Sex Determination in Insects . . A . Lymantria dispar . . . . . . . . . . . . . . . . . . B . Bombyx mori . . . . . . . . . . . . . . . . . . . . C . Other Insects . . . . . . . . . . . . . . . . . . . V . Fish . . . . . . . . . . . . . . . . . . . . . . . . . VI . Mouse . . . . . . . . . . . . . . . . . . . . . . . . . V I I . Cat . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Plants . . . . . . . . . . . . . . . . . . . . . . . . . A . Angiosperms . . . . . . . . . . . . . . . . . . . . B . Bryophytes . . . . . . . . . . . . . . . . . . . . . I X . General Discussion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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1 Introduction

The first function of the Y-chromosome to be discovered was in Drosophila melanogaster, by Bridges (1913, 1914, 1916) who showed th a t it was necessary for fertility in males . For although flies with XO and XY constitution were both males, the former were entirely sterile . As was * This paper is dedicated to the memory of my guru, Professor J . B . S. Haldane, who died in India on December 1, 1964. t Present address: The State University of New York, Buffalo General Hospital, Buffalo, New York . 227

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shown by Safir (1920) the sperms of XO males were nonmotile and remained in the cystic bundles. The first, Y-linked gene in any species was discovered by Johannes Schmidt in the year 1920 for a black pigment spot in the fish Lebistes reticulatus. A Y-linked color gene was independently reported in the fish Aplocheilus latipes by Aida (1921) and, as Castle (1921) pointed out, Aida’s observations go beyond those of Schmilt (1920) in the important respect of showing the occurrence of crossing-over between the X- and the Y-chromosomes. The discovery of the existence of sex chromosomes was made much earlier, however. Henking (1891), studying spermatocyte divisions in the bug, Pyrrhocoris, observed an unpaired element which led to the formation of two kinds of spermatozoa, one carrying eleven chromosomes and the accessory chromosome, the other carrying only the eleven chromosomes. This observation was confirmed by other investigators but none of them recognized its significance. It was McClung (1902) who first suggested that the unpaired or “accessory” chromosome was the male sex determiner. Later work showed that this “accessory” chromosome was present twice in the female (XX) and only once in the male (XO). Soon after, Wilson, working on Hemiptera, and Stevens, working on Coleoptera, independently discovered that the X-chromosome of the male was accompanied by a mate of different type, often much smaller than the X (Stevens, 1905; Wilson, 1905a,b). The sex-chromosome terminology as it is used today was proposed by Wilson in 1908 in his address as the Vice-president and Chairman of section F-Zoology, of the American Association for the Advancement of Science, Baltimore (Wilson, 1909a). As this address is of considerable historic interest the following sentences are quoted: “. . . The spermatozoa are in fact of two classes, equal in number, that differ in respect to one or more of the chromosomes that enter into the formation of their nuclei; and the facts clearly demonstrate that fertilization of the eggs by one class produces males, by the other class females . . . . “In all the species half the spermatozoa are characterized by the presence of a special nuclear element which I shall call the “X-element,” while the other half fail to receive this element. I n the simplest and clearest case (which was that first discovered) the X-element is a single chromosome, now generally known by the name of the “accessory chromosome” given to it by McClung, but it is also called the “odd” or “het,erotropic” chromosome, the “monosome,” or the “unpaired idiochromosome.” I will here employ McClung’s more familiar term. As a single accessory chromosome the X-element has been found in

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many representatives of the Hemiptera, Orthoptera and Coleoptera, and in certain Odonata, Myriapoda and Arachnida . . . . I n many cases, however, the X-element (otherwise identical with an accessory chromosome) appears as a “large idiochromosome” which has a synaptic mate known as the “small idiochromosome.” This latter chromosome, or its homologue, I shall designate as the “Y-element.” I n a few cases the X-element consists of two chromosomes (Thyanta, Fitchia), of three (Prionidus, Sinea), or even of four (Gelastocoris), accompanied in each of these cases by a single Y-element . . . . I n all cases the spermatozoa are formed in pairs, and the chromosomes are so distributed in the maturation-divisions that one member of each pair receives the X-element (whether it consists of one, two or more chromosomes), the other member the Y-element if it be present . . . . Half of all the spermatozoa thus receive the X-element, while the other half may receive a Y-element in its place, though this may be absent. L‘Comparisonof the male and female somatic chromosomegroups proves, indirectly, but conclusively, that the two classes of spermatozoa thus formed are, respectively, female-producing and male-producing (I do not say female- or male-determining), as already stated. In both sexes the somatic groups are identical save in respect to the X- and Y- elements; and the difference can only be a result of fertilization by the two respective classes of spermatozoa. This is a t once proved in species having a Y-element by the fact that this chromosome is found only in the male . . . . In some cases, however, the Y-element is as large as the X-elemcnt ( N i z a m , Oncopeltus) and no visible difference between the sexes appears to the eye.” Interested readers are referred to the series of papers by Wilson and Stevens, and also Castle, for a discussion of some of the earliest theories of sex determination (Wilson, 1905a,b, 1906, 1907, 1909a,b, 1915, 1925; Castle, 1909; Stevens, 1905, 1906). Bridges (1916), in his classic work on nondisjunction, demonstrated that while Drosophila flies without a Y-chromosome could survive, those without a n X could not. YY and YO zygotes are inviable. Males with an extra Y in addition to their normal X Y complement also showed no peculiarities. Thus it was suspected very early in genetic research that the X-chromosome carried genes vital for life while the Y had no important genes. One exception to this presumed inertness of the Y-chromosome in Drosophila was the suggestion of Sturtevant (later proved by Stern, 1926, 1927) that both X and Y carry a t least one bobbed locus. The

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Y-chromosomes of other organisms also proved inert. The hypothesis of Y-chromosome inertness soon became established and accepted by most geneticists of that time. This will be discussed in detail in a later section. During the past thirty years, much work has been done on the function of the Y-chromosome in various organisms including Drosophila, mouse, man, etc., and it is beginning to be widely recognized that the Y-chromosome is not as inert as it was believed to be and that it may even bear some functional units of great importance. This review is an attempt to bring this evidence, observed in man, animals, and plants, together, and to evaluate the importance of the function of the Y-chromosome. Genetics and cytogenetics of the Y-chromosome in man will be considered first. II. Man

While considerable progress has been made in the past fifty years in understanding the location and behavior of sex-linked genes in man there has been no corresponding progress in cytogenetic research. This was mainly due to the lack of refined techniques, especially in fixation and spreading of the chromosomes in human cells. These techniques have only become available since the mid-’fifties. The historical perspective of cytogenetic research in man, with special reference to the Y-chromosome, will be discussed in Section 11, B.

A. Y-LINKAGE In theory at least, Y-linkage in man may be either complete or incomplete. In complete Y-linkage the gene is solely confined ho a locus in the Y-chromosome and does not exchange genetic material with an X-chromosoma1 allele. Y-linked transmission in organisms with heterogametic male sex, as in man, is characterized by (a) presence of the trait in males only; (b) its reappearance in all sons of affected males (assuming full penetrance); and (c) the daughters of affected men being not only phenotypically normal, but in addition, not transmitting the trait to their offspring (see Fig. 1). Enriques (1922) and Castle (1922) were the first to suggest the existence of Y-linked inheritance in man on the basis of a pedigree of webbed toes published by Schofield (1921). This pedigree, which was of Schofield’s own family, contained fourteen affected males and no affected females in four generations. Schofield himself made no mention of Y-linkage, suggesting that the trait was inherited as a secondary sex character. Enriques coined the word “holandric” (Greek holos = entire, an&, andros = man or male) to describe a type of inheritance in which a trait is inherited by all the sons of an affected male; daughters are neither affected nor

Y-CHROMOSOME

I

FUNCTION

23 1

=-r

mo

m i

r5

FIG. 1 . A hypothetical pedigree of a corriplctcly Y-linked trait.

do they transmit the gene to their sons. Prior to this work, however, a pedigree of hypcrtrichosis of the pirma of the ear was published by Tominasi (1907a,b), which provided evidence in support of complete Y-linltage (Cockayrie, 1933; Gates, 1946). 1. Partial Sex-Linkage

In incomplete Y-linkage (or partial sex-linkage) crossing-over between alleles in the homologous regions of X- atid Y-chromosomes can take place. Such regions are known as the pairing segments of the sex chromosomes, as opposed to diflerential segments which are dissimilar and do not pair. Partially sex-linked genes were first discovered by Aida (1921, 19.10) in the fish Aplocheilus latipes. The suggestion of partial sex-linkage in man was first made by Haldane (1936) who assumed that the cytology and genetics of X- arid Y-chromosomes in man were essentially like those in the rat, (Koller arid Darlington, 1934). The conditions irivestigated by Haldane are given below in the order of the weight which he attached to the evidence of their linkage: 1. Xerodernia pigmentosum : recessive. 2. Achromatopsia (complete color blindness) : recessive. 3. Retiriitis pigmeritosa: recessive form riot associated with deafness, arid riot completely sex-linked. 4. Retiriitis pigmerit,osa : dominant form, in some pedigrees only. 5 . Oguchi’s disease: recessive. 6. Epidermolysis bullosa dystrophic*a: recessive form.

At the time of Haldane’s work the evidence i n support of partial sexlinkage in man seemed very strong, but later gerietical and cytological research lent no support to this hypothesis (Makino, 1941; Macklin, 1944, 19.52; Matthey, 1951; Sachs, 1954; Ford and Hamerton, 1956; Morton, 1957). On the other hand, Darlington arid Haque (1955) arid Kodani (personal communication to Taiiaka, 1962) reported that they

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have observed partial pairing between X- and Y-chromosomes in man, but these observations need further confirmation. While the existence of partially sex-linked genes in man is now considered highly doubtful, final judgment has to be postponed until decisive cytogenetic evidence is available. The occurrence of such genes may be more likely in certain mammals such as Apodemus mystacinus and A . sylvaticus in which Wahrman and Ritte (1963) reported the formation of heteroniorphic X-Y dyads. 2. Complete Y-Linkage

a. General. I n his compendium on Human Genetics, Gates (1946) listed fourteen (apparently) completely Y-linked traits (Gates, 1946, pp. 82, 83). Most of these, however, were excluded in a later list prepared by him (Gates, 1954). Only the following four were left: (1) Ichthyosis hystrix; (2) black hairs in the ears; (3) webbed toes; and (4) radio-ulnar synostosis; a fifth one, “(probably) foot ulcers,” was added. It is interesting th a t although Gates (1946) did not include “foot ulcers” in the list of “genes in the Y” on page 82, he included this trait and four others (webbed toes, hairy ears, ichthyosis hystrix and radio-ulnar synostosis) in “holandric genes” in Fig. 23 of his book (Gates, 1946, p. 81). In the Handbook of Biological Data (19Fi6) radio-ulnar synostosis and foot ulcers were excluded from these five but keratoma dissipatum, originally included by Gates (1946), and a new trait, color vision anomaly, reported by Reed et al. (1951), were added. Another trait entitled “abnormality of the external ear” was considered by Stern (1957). The seventeen traits listed in Table 1, which were considered to be Y-linked a t one time or another, are based largely on Gates (1946). The evidence in favor of Y-linkage of most of these traits was shown to be inadequate and at best ambiguous (Stern, 1957, 1958). Penrose and Stern’s (1958) re-examination of the Lambert pedigree of ichthyosis hystrix gravior demolished the support for Y-linkage of this trait. Another family of ichthyosis hystrix of less spectacular type was previously studied and affected members of both sexes were found (Curth and Macklin, 1954). It is highly probable, as concluded by Penrose and Stern (1958), that a n autosomal dominant gene was responsible for the ichthyosis in the Lambert pedigree. For some of the traits listed above, the probabilities for the assumptions of sex-limited autosomal inheritance were small and further studies should be attempted (Stern, 1957). One such trait is “black hairs in the ears.” b. Hypertrichosis Pinnae Auris. As mentioned earlier a pedigree of hypertrichosis of the ears was published by Tommasi (1907a,b). Addi-

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TABLE 1 List of Traits Claimed to be Y-Linked in Man and References to the First Reports on Pedigree Data Trait

Reference

-~

1. 2. 3. 4. 5. 6. 7.

Ichthyosis hystrix Black hairs in the ears Webbed toes Coloboma iridis (?) Sedgewick’s case Cataract (?) : Harman’s case Keratoma dissipatum (3 families) Peroneal atrophy, 1 case of cross-over,

Cockayne (1933) Tommasi (1907a,b) Schofield (1921) Streatfield (1858), Sedgewick (1861) Dyer (1846) Brauer (1913) Herringham (1889), Gates (1946)

x-Y 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Epidermolysis bullosa simplex (Yaffe) Radio-ulnar synostosis (1 pedigree) Hyperextensibility of thumbs? (1 pedigree) Hypermobility of joints (1 pedigree)? Blue sclera and brittle bones (1 pedigree) Adherent tongue (possible) Camptodactyly (1 pedigree)? (Probably) foot ulcers Color vision anomaly Abnormality of the external ear

Yaffe (1942) Davenport e l al. (1924) Key (1927) Single pedigree? (Stern, 1957) Weiss (1929) Fantham (1924) Smith (1934) Reed et al. (1951) Crow (1956)

tional pedigrees were published in recent years by Gates (1957), Dronamraju (1960a,b, 1964)’ Gates and Bhaduri (1961), Sarkar et al. (1961), and Gates et al. (1962). i. Phenotype. The phenotype is characterized in most affected individuals by the presence of long hairs growing closely together on the helix region of the pinna (Fig. 2). It should be noted that hypertrichosis of other regions of the ear such as the external auditory meatus does not concern us here. I n order to emphasizk this point and to refer to the trait by an appropriate scientific term the name hypertrichosis pinnae auris and the gene symbol He were suggested by Dronamraju (1960b). Th’IS was necessary in view of the vague descriptions “black hairs in the ears” and “hypertrichosis of the ears” that were in vogue prior to Dronamraju’s paper. The hairs may grow along the whole length of the helix or they may be localized on its lower part only. In many Israelis, for instance, the hairs appear on top of the helix (Slatis and Apelbaum, 1963). The number and texture of hairs varies greatly. In milder cases there may only be one or two hairs on one or both ears. But the expression is never unilateral when the growth is prominent. Sarkar et al. (1961) as well as Gates et al. (1962) attempted to classify individuals on the basis of degree of hairiness, ranging from very scanty

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FIG.2. Right (a) and left (b) ears of a 28-year-old Brahmin male from Andhra Pradesh. Note the long hairs over most of the helix region including the apex.

to very marked. However, prior to their work a preliminary attempt a t such classification was begun in Dronamraju’s paper (1960b) where affected men with fewer hairs were grouped in the mild category. The present classifications of degree of growth (five in Sarkar et al. arid six in Gates et al.) are a t best ambiguous and further refinement in the scale is needed before such classifications can be used in population studies. The youngest affected male which I observed was 17 years old, but the age of onset varies considerably within and between populations (Dronamraju, 196la,b). My own studies were largely done on Indian and Ceylonese populations, whereas Slatis and Apelbaum studied Israeli populations. No female has been observed to show this trait. In a brief report on hairy pinnae in Malta, Gates and Vella (1962) reported a 12-year-old girl in Gozo who had “short fine black hairs on the ear lobes and lower parts of the pinnae.” Sarkar and Ghosh (1963) reported some cases of girls “with hairy ear rims” but no photographs of these cases were published. Vella (1963) later, in a letter t,o the Lancet, pointed out that “this infantile condition seems more likely to be a hormonic effect than a genic variation, but the hormone may be genetically controlled. It differs from the adult variety in that (1) it occurs in both sexes, (2) it disappears within a few months, arid (3) the hair is fine and extends over most of the

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edge of the pinna.” The photograph, of the ear of a 3-month-old girl, published by Vella (1963) shows fine hairs on the helix which, according to her mother, were also seen at birth on the ears of another of her three daughters, but later disappeared a t the age of 6 months. ii. Tommasi’s pedigree. In the Italian pedigree published by Tommasi (1907a,b) all ten male descendants in the male line from an affected male ancestor and none of the seven female descendants had hairy ears. The pedigree spanned five generations. Two unaffected boys, who were said to have been too young to show the trait, and who had one older affected brother, were also reported. Three older sisters in the same sibship were reported as unaffected. The pedigree shows good agreement with the hypothesis of complete Y-linkage. However the accuracy of evidence on which this pedigree was based is of considerable doubt because the propositus and his wife were the only individuals seen by Tommasi and all other information was obtained through them. Furthermore, the propositus was 81 years old when questioned and an inmate of a mental institution (Tommasi, 1907a,b). The hearsay method of obtaining evidence for this trait is, in my opinion, highly unreliable and this point has been repeatedly emphasized by Dronamraju (1961a, 1964) and Dronamraju and Haldane (1962). iii. Recent work. Gates (1957) published three small Indian pedigrees which are compatible with, but do not give strong support to, the hypothesis of complete Y-linkage. Stern (1957) pointed out that Gates’ pedigrees were fragmentary. It should be emphasized that Tommasi (1907a,b) was the first l o publish a pedigree of hypertrichosis pinnae auris (though he did not discuss it in terms of the Y-chromosome) and that Gates (19Fj7) was the first to report the occurrence of the trait in Indians. However, no offspring of normal female sibs of affected males were recorded in their pedigrees. Such offspring were recorded for the first time in a pedigree of Dronamraju (1960b) which satisfied aZZ the conditions of complete Y-linkage. It should be added that the brief note published by Gates (1960), though claiming the collection of over twenty pedigrees from India, contained no pedigrees or numerical data. Dronamraju (1960b) published three pedigrees from the state of Andhra Pradesh in southeastern India. Pedigree A of this group is comprised of the author’s own relatives and the propositus was the author himself (i.e., the present writer). The two other pedigrees are of unrelated families. Pedigree A was the largest of the three and also of all pedigrees published by other authors a t that time. This pedigree of seven generations contained 51 males and 56 females of over 17 years of age, and 40 males and 39 females of below 17 years of age. Thirty-three of the 51 adult males

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were seen by the author personally. In the three pedigrees all the sons and none of the daughters of affected men were affected. Two unaffected men of ages 26 and 58 with unaffected fathers, and a n affected maternal grandfather in each case, were recorded. All the male sibs of their mothers (i.e., mothers of the two men aged 26 and 58 years) were also affected. One of these was a male, aged twenty-nine years at that time, who was unavailable for examination, but was subsequently examined and found to show a clear manifestation of hypertrichosis of the helix of both ears (Dronamraju, 1964). The pedigree is thus complete. The information concerning dead individuals was obtained through two or more independent sources and all these individuals were listed in the 1960 paper (Dronamraju, 1960b). Direct questioning for information was found to be unnecessary as all the members of the pedigree were always favorably disposed to discussing the trait. Once the subject was raised in a conversation various individuals would readily name affected individuals (some dead) familiar to them. Information thus volunteered by different individuals at different places was compared and the condition of the dead individuals was recorded. All the living members of the pedigree were personally examined by the author. The subject of hairy ears is of general interest to the people of Andhra Pradesh because it is popularly believed to be positively correlated with longevity. Pedigree B is the smallest of the three showing transmission from a father to his son. Two unaffected sons, aged 15 and 12 years, of a n affected man were recorded. Pedigree C is larger than B, showing male-t,omale transmission in three generations. Five unaffected daughters of affected men were recorded. While pedigrees B and C are of little value in themselves, they provide important confirmatory evidence. iv. Discussion of the mode of inheritance. The three pedigrees from Andhra Pradesh are completely compatible with the hypothesis of complete Y-linkage, satisfying all three important conditions, i.e., the trait is confined to males, present in all the sons of affected males, and not transmitted to their daughters’ sons. In order to exclude possible alternative modes of inheritance, two other hypotheses were considered (Dronamraju, 1960b). The first one was that the gene for hypertrichosis pinnae auris is a dominant only manifested in males but that, like some genes described by Winge and Ditlevsen (1947) in Lebistes reticulatus, i t can occur on the Y- or on the X-chromosome, whether its occurrence on the X is due t o crossing-over or to mutation. High frequencies of the gene ruled out the possibility of recent mutation (Dronamraju, 1961b, 1963~)If . it crosses over, even with a frequency of 1% or so, (or even if i t has arisen by repeated independent mutations), we should expect the gene to be as common on the X as on the Y. If a man carried the gene on

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his X-chromosome, he would not transmit it to any of his sons except by crossing-over from X to Y,which is rare. There are five “independent” sources of the gene in the three pedigrees arid all these five men transmitted it to their sons without exception. I n view of this evidence the hypothesis of facultative X-linkage can be clearly ruled out. The second hypothesis is that the gene is an autosomal dominant, but only manifested in males, like premature baldness (Harris, 1946). The pedigrees are compatible with this hypothesis but, as shown in my paper (Dronamraju, 1960b), it is extremely improbable that it is true. For the sake of simplicity in the calculations it was first assumed that the gene is so rare that one can ignore the possibility that any of the wives of affected males may have carried it. The combined probability of the three pediThis probability increases to grees was found to be less than G.2 X on the assumption that the gene is not rare. It is thus clear 4.1 X that the hypothesis of autosomal dominance may be decisively rejected for these pedigrees. It was added, however, that “It is not of course impossible that an indistinguishable, hut autosomally determined character may be found in another family.” v. Later work Gat,es and Bhaduri (l961), and Gates et al. (1962) subsequently published additional Y-linked pedigrees from India. Sarkar et al. (1961) published seven small pedigrees on the inheritance of this trait. One of these is fully compatible with the hypothesis of complete Y-linkage but the others contained some exceptions. For instance, they recorded one affected and two unaffected sons of over 30 years of age from an affected father. Droriamraju arid Haldane (1962), commenting on the pedigrees of Sarkar et al. (1961), suggested that such exceptions could be due t o incomplete penetrance, “and it is therefore necessary, in favour of the hypothesis of autosomal dominance, to look for affected men whose fathers are unaffected but whose maternal grandfathers are affected.” However, no such cases were reported by Sarkar et al. (1961, 1962). They further pointed out that in the work of Sarkar et al. (1961) only one person in each of three pedigrees and two in another were actually seen by the authors. These pedigrees, like the one published by Tonimasi (1907a,b), were largely prepared on hearsay evidence. Hearsay evidence for this trait could be unreliable for at least three reasons: (a) mild cases may be overlooked and reported as negative; (b) a thick bushy hair growth near the external auditory meatus might be misreported by an informant as posil ive, and (c) considerable manifestations may be regarded as negative by individuals who are accustomed to seeing extreme manifestations (Drouamraju, 1961a, 1964). Slatis and Apelbaum (1963) studied Israeli males and commented a s follows: “The late age of onset of this trait has made it difficult to deter-

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mine its mode of inheritance. This study tested the simple assumption that hairy pinna is Y-linked, and observations were limited to men carrying the same Y chromosome as an affected propositus. If one takes into account the reduced penetrance before the age of 70, there is exceptionally good agreement between the observations and the expectations on the basis of Y-linkage. An assumption of autosomal inheritance either as a dominant or a recessive sex-limited trait gives unsatisfactory results with far too many affected relatives of affected men. Thus, it would appear that hairy pinna is Y-linked and has a high degree of penetrance at, advanced ages.l 1 vi. Frequencies in India and Ceylon. The ears of over 1700 men in Ceylon, and in the states of Andhra Pradesh, West Bengal, and Orissa in India were examined (Dronamraju, 1961b, 1963a,b,c). The over-all incidence in West Bengal was found to be l6.0%, in Orissa 14.5%, and in Andhra Pradesh 6.1%. The 414 men examined in Ceylon included outpatient,s of the General Hospital in Colombo, workers of the Coconut Research Station at Lunuwila, and the staff of the Royal Botanic Gardens, Peradeniya. The over-all incidence in Ceylon was found to be 36.9%! There was no significant difference between the frequencies among Buddhists and Christians (Dronamraju, 1965). vii. Frequencies in diferent age groups. The classification of men on the basis of age suggests that the frequencies in West Bengal, Orissa, and Ceylon increase with age and that this increase is significant in men aged 45 years or over in Orissa, and nearly so in Ceylon (Table 2). It is reasonable to assume that this increase is due to the phenotype appearing at the late age of 45 years or over in some cases. “It appears probable that several genes may be involved in controlling the expression of the phenotype and that the gene or genes responsible in the case of the Dronamraju pedigree caused early onset in all sons of affected men” (Dronamraju, 1963b). Slatis and Apelbaum (1963) reported a similar increase in incidence among men aged up to 60 years or so. They postulated an allele that causes early onset of the trait in Indians. Among 373 Israeli men of the 18-29 years of age group examined by them, only 1.1% had hairy pinnae. In contrast to this low frequency, the incidence in a younger group (18-20 years) in Orissa, India was 5.4%! An over-all incidence of 19.6% was reported by Abbie and Rao (1965) in the Australian aborigines. Much regional variation in the incidence was recorded. The trait appeared to be commoner in men over 40 years of age. There may be different alleles for the early and late ages of onset whose incidences may vary significantly between populations. There is, however, no specific evidence supporting this particular hypothesis, and certain alternative explanations may also be given. For instance, as sug-

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TABLE 2 Frequenries of Hairy Pinnae in Different Age Groups in Orissa, West Bengal, and Ceylon* Age group (in years)

Percentage with hairy pinnae

India Orissa: 15-17 21-23 30-32 45 West Bengal: 15-23 24-29 30 Andhra Pradesh: all ages Orissa tribals: all ages Ceylon : 18-20 24-26 30-32 36-38 45

0 17.0 34.0 58.3 0. 10.8 26.0 6.1 12.9 7.7 20.8 33.9 56.5 59.8

+

+

+

* Total frequencies for Andhra Pradesh and tribals of Orissa are also given. gested by I’rofessor H. ,J. Jluller, “cases of such a n apparent or phenotypic difference could be explained by modifying genes in other loci, which would surely influence the result, just as age also influences the result. In order to conclude that the alleles were different there would have to be data of a special kind, rather difficult to obtain” (Muller, 1964). The possibility of genetic heterogeneity also exists, as it is Imown for so many other human traits such as muscular dystrophy (Rlorton arid Chung, 1959) ; and deaf-mutism associated with arid without albinism (Stevenson and Cheesman, 195.5; llargolis, 1962; Tietz, 1963). In a recent paper Stern et al. (1964) c+onfiriiiedDronamraju’s (1963a,b) observation 011 the increase in inciderice with age in Berigal and South India. I n their study, “The observed frequency of the trait among relatives in the inale line of affected propositi is higher than expected by chance. Expectations are derived for nuniber of affected according to models of a Y-linked, an autosomal dominant, and a recessive allele for hairy pinnae. If an affected person is defined as one who has at least one long hair on one pinna . . . the data do not permit discrimination between the three genetic hypotheses. If an affected person is defined as one who has at least . . . a minimum of about (5 hairs per pinna, . . . expectations according to both types of autosomal inheritance are compatible with the observations, while the data deviate a t the .035 per cent

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level from Y-linkage expectations.” Stern et al. (1964), agreed, however, that the large Indian pedigree published by Dronamraju and the later work of Slatis and Apelbaum in Israel are strong support for Y-linkage. Thus, in view of the overwhelming evidence in favor of the hypothesis of Y-linkage acxuinulated in recent years one may interpret the results of Stern et al. a s rather exceptional; their inability to discriminate between the Y-linked, autosonial dominant and recessive models could possibly be due to inadequate data. It should be firmly emphasized that their recent work did not give any evidence agai*nst Y-linkage. B. CYTOGENETICS OF

THE

Y-CHROMOSOME

Tjio and Levan (1956) made the fundamental discovery that the somatic chromosome number of man is 46 and not 48 as was widely believed until then. The reports of early investigators are of considerable historic interest. In 1891, von Hansemann made the first successful counts of chromosomes in human cells and found 18, 24, and 40 in three cells. Later Sutton (1903) and Boveri (1903) pointed out the hereditary significance of t#he chromosomes. De Winiwarter (1912) observed the chromosome number as 46 autosomes 1 sex chromosome and no Y-chromosome, and this was later confirmed by De Winiwarter and Oguma (1926). Painter (1921, 1923) was the first to report the Y-chromosome in human males but he observed that the total diploid number was 48: 46 autosomes X and Y in males. This number was confirmed by various later researchers including Evans and Swezy (1929), Shiwago and Andres (1932), Minouchi and Ohta (1934), Andres and Navashin (1936), Koller (1937), Hsu (1952), Mittwoch (1952), and Darlirigton and Haque (1955). The presence of the Y-chromosome in males and the XX-XY type of chromosome constitution in man was clearly observed by Tjio and Levan (1956) and Ford and Hamerton (1956), who used improved techniques and established the correct diploid number as 46. For a recent review of cytogenetic techniques see Ferguson-Smith (1964a). The development of modern techniques was soon followed by an avalanche of publications on normal and abnormal human karyotypes and a standard system of criteria for the identification and classification of human chromosomes has been evolved (Robinson, 1960; Anonymous, 1963). I n most males the Y-chromosome can be recognized as the largest of the small acrocentric chromosomes. The centric constriction is usually indistinct. The short arms are shorter than those of chromosomes 21 and 22, and do not have satellites or secondary constrictions. The long arms, on the other hand, may have a secondary constriction. They tend to lie close together, not wide apart as in the other chromosomes. The Y-chromosome is late replicating in comparison to others (Hsu, 1964).

+

+

Y-CHROMOSOME FUNCTIOK

24 1

Its special characteristic is its heterochromatic nature and “fuzzy” appearance after special fixation. 1. Variation in Length

Several authors, led by Bender and Gooch (196l), reported Y-chromosomes of abnormal length (see Fig. 3). The normal Y-chromosome corresponds in size to chromosome 21 or is slightly larger and was classified in Group 21-22 a t the Denver Conference (see Robinson, 1960). The abnormally long chromosomes are reported to be equal in length to chromosomes of group 16-18. Bender and Gooch (1961) reported a long Y-chromosome in a normal man. Kallen and Levan (1962) reported three male patients with Marfan’s syndrome who had Y-chromosomes of normal size. Van V\-ijck et al. (1962) studied three males with oligospermia or azoospermia and found that one had a short Y, another a long Y, and the third a mosaic with some cells having a normal Y and others a long one. Makino et al. (1963; Makino and Sofuni, 1963), and Makino and AIuramoto (1964) also reported long Y-chromosomes. Makino et al. (1963), in a survey of chromosomes in normal individuals and in abnormal ones with congenital disorders and neoplasia, discovered two Japanese families with Y-chromosomes of unusual size. Four such individuals were found, two of whom were a phenotypically normal father and a paternal grandfather of a girl with Down’s syndrome. The other two were a 3-month-old male infant and his father, normal in phenotype. The infant had multiple malformations but external genitalia were normal. The malformations included low-set, left ear, lagophthalmic left eye, microphthalmic right eye, absence of the right optic nerve, bilateral nystagmus, a high arched palate, and a n umbilical hernia. Gripenberg (1964) reported four individuals with long Y-chromosomes in a survey of seventeen normal and abnormal males. Two of the males with long Y-chromosomes were normal, but the other two were mentally retarded, one showing signs of mongolism. In total, eighty-four metaphase plates obtained from leucocyte cultures were studied. The author stated that the occurrence of two secondary constrictions in some of the large Y-chromosomes and the nonperipheral location of the large Y contradicted the view that the increase in length was caused b y a delayed spiralization as was earlier suggested by Bishop et al. (1962). Cohen’s (1964) preliminary work suggests that the variation in length of the human Y-chromosome may significantly differ between different ethnic groups. On calculating the “Y index,” total length of the Y chromosome average length of long arms group 21-22

242 K. R. DRONAMRAJU

FIG.3a. I, Metaphase plates from peripheral blood cultures of a normal male having a Y-chromosome of the usual length (arrow); 11, from a Ibyear-old boy with webbing of the neck, pulmonic stenosis, and lymphadema of the legs, having a long Y-chromosome (arrow). Courtesy of Dr. Catherine S. N. Lee.

N

+

w

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K . R. DRONAMRAJU

FIG.3b, I. Karyotypes from the corresponding cells shown in Fig. 3a, I. Courtesy of Dr. Peter Bowen.

in a group of healthy - Jewish and non-Jewish males he found that the group means were XJews= 1.73; range 1.56-2.00 and Xnon-~ewe = 1.49; range 1.34-1.69. Further work along these lines including other ethnic groups and populations will be awaited with much interest. Inheritance of long Y-chromosomes was reported by Bishop et al. (1962), Makino et al. (1963), and de la Chapelle and Hortling (1963). Deleted Y-chromosomes were also reported by some authors (Rhldal and Ockey, 1961; Conen et al., 1961; Vaharu et al., 1961; Klevit et al., 1963). Muldal and Ockey (1961) reported deletions comprising a third to half of the longer arm of the Y-chromosome in four males, three of whom showed hypospadias with a mild degree of manifestation. An extremely small Y was observed by Conen et al. (1961) in a true hermaphrodite with probable XO/XY mosaicism. Vaharu et al. (1961) found 45 chromosomes plus a fragment which seemed to be a portion of the Y in a female with gonadal dysgenesis and an enlarged clitoris. Klevit et al. (1963) reported triple

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FIG.3b, 11. Karyotypes from the corresponding cells shown in Fig. 3a,II. Courtesy of Dr. Peter Bowen.

chromosomal mosaicism [45/XO, 46/X-isochromosome long arm of Y (or partially deleted Y), 47/X-isochromosome long arm of Y (or partially deleted Y) - Y (long arm partially deleted) karyotype] in a male pseudc hermaphrodite with asymmetrical gonadal differentiation. 2. Role in Sex-Determination

In man, individuals without a - Y-chromosome are phenotypically female and those with a Y a r e normally male. Thus female sex differentiation occurs in XO, XX, XXX, and X X X X individuals, and male sex differentiation occurs in XY, XXY, XXXY, and X X X X Y individuals (Table 3, Fig. 4). Those with two Y-chromosomes, such as XYY, XXYY, and XXXYY, are also male. The main function of the human Y-chromosome is probably to ensure that the fetal gonad develops into a testis rather than an ovary. The first sex chromosome anomalies were discovered in the year 1959 by Ford et al. (1959a,b) and by Jacobs and

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K. R . DROSAMRAJU

TABLE 3 Phenotypic Sex and Sex Chromosome Constitution in Man Sex chromosomes in: Male phenotype Female phenotype

XY XYY XXY XXYY XXXY XXXYY XXXXY

xx/xxv

XY/XXY S Y /XXXY XO/XY/XXY -/XXY/XX XXXY/XXXXY Xxxx/XXxxY XXXY/XXXXY / X X X X X Y

XO XX

xxx xxxx XXXXX SO j X X

xo/xxx xo/xx/xxx XX/XXX XOjXYY XXX/XXXX XX/XXX/XXXX

FIG4. XXXY phenotype. Courtesy of Dr. M. A. Ferguson-Smith.

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Strong (1959). I t was previously established that normal males were of XY constitution and normal females of XX. The XXY constitution was discovered in persons suffering from Klinefelter’s syndrome. Though phenotypically nearly normal males, such individuals have small testes with usually complete azoospermia. Gynaecomastia is also frequent. Mental retardation may also occur in some cases. Those with XO constitution suffer from Turner’s syndrome. These patients, though outwardly female, have immature external genitalia, and very small uterus. They are further characterized by total absence of gonads and also webbed neck, cubitus valgus, and mental defect. Several new types of chromosomal anomalies have been reported in recent years (see, for instance, FergusonSmith, 1961a’b; Miller, 1964), and this subject has been exhaustively reviewed by Polani (1962), McKusick (1964), Miller (1964), and Court Brown et al. (1964), among others. Mention must be made of XYY males who cannot be recognized by any specific clinical features. Hauschka et al. (1962) reported a clinically normal XYY male who had a chromatin-positive X X daughter with primary amenorrhea and total absence of internal sex organs, a 21-trisomic mongo1 daughter, and a “blue baby,” one of fraternal twins. Eight other cases have been reported since but no consistent features have been noticed among them with the possible exception of hypogonadism (Dent et al., 1963; Hustinx and von Olphen, 1963; Ricci and Malacarne, 1964; Sandberg et al., 1961, 1963). Chromatin-positive individuals with an XXYY constitution can be distinguished from the usual cases of Klinefelter’s syndrome by the presence of 48 chromosomes per cell with an additional X and Y (Rhldal and Ockey, 1960; Carr et al., 1961a; Vague et al., 1961; Muldal et al., 1962; Davies, 1963; Laurence et al., 1963; Stimson et al., 1963). Such individuals are eunuchoid with a rather tall stature; slight tendency to gigantism, and enlarged frontal sinuses are also common (Laurence et al., 1963). Varicose veins and ulcerations of the lower extremities are frequently present in older cases (Carr et al., 1961c; Ellis et al., 1961; Stinison et al., 1963). Ellis et al. (1961) reported small immature testes and germinal aplasia in an adult case of XXYY. Cases of mosaic karyotypes XXXY/XXXXY (Lamy et al., 1963); XXXX/XXXXY (Stinison et al., 1963); and XXXY/XXXXY/ XXXXXY (Anders et al., 1960) were reported, in all of which:the phenotype is male. 3. T h e “Inertness” of the H u m a n Y-Chromosome

The presence of an additional Y-chromosome in apparently normal individuals (XYY), the variation in the length of the Y-chromosome

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K. R. DRONAMRAJU

without accompanying drastic effects on the phenotype, and the viability of zygotes without a Y-chromosome (developing into Turner’s syndrome) as opposed to the inviability of YO or YY zygotes-all these facts seem to indicate that the Y-chromosome may be lacking in important genes, a t least in comparison to the X-chromosome. In view of the experience from Drosophila genetics one has to be cautious in believing that the Y-chromosome is genetically “inert.” From the foregoing discussion it is clear th a t the Y-chromosome plays an important role in sex det)ermination. In comparing the functions of Y- and X-chromosomes the difference in their sizes should also be taken into account. Regarding the variation in the length of Y-chromosome, the recent experiments of Wennstrom and de la Chapelle (1963) are of special interest. Their studies showed no significant differences in DNA content between Y-chromosomes of normal and abnormal length. Thus if such variation is caused by contortion or elongation of chromosomes the reports on normal individuals with Y-chromosomes of abnormal size do not necessarily support the hypothesis of “inertness.” The inheritance of long Y-chromosomes could be due to the fact that such behavioral peculiarities of specific chromosomes may be inherited in some families. Further investigations along the line of Wennstrom and de la Chapelle’s work are needed. It is of course possible that long Y-chromosomes may also be due to duplication of part of the chromosome, or translocation in the zygote of chromosomal material from other chromosomes. Since some of the individuals with long Ychromosomes show some signs of Klinefelter’s syndrome it is possible, though unlikely, that a translocation of genetic material from X onto the Y occurred in these cases. The problem of variat,ion in the size of Y-chromosome in relation to “inertness” will be discussed in greater detail later on.

4. Other Y-Linked Genes? The Y-chromosome in man may bear histocompatibility genes as it does in the mouse. However, as emphasized by McKusick (1964), the possibilities of testing for such genes in man are very limited since two genetically identical individuals differing only in the presence or absence of Y-chromosome are required. The work of Lejeune and Turpin (1961) and also of Turpin et al. (1961) on skin grafting between X O (Turner’s syndrome) and X Y (normal male) identical twins suggested no histocompatibility genes on the Y-chromosome as reciprocal grafts in each case were satisfactorily accepted. However, this report does not exclude histocompatibility genes altogether because interfetal circulatory connections in these twins might have led to induction of immune tolerance. Renkonen et al. (1962) suggested that the immunization of a small

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number of mothers to Y-chromosome-determined antigens might be responsible for the decline in the sex ratio observed with birth order. Further evidence is required to support this hypothesis because certain alternative hypotheses are equally likely (see, for instance, Szilard, 1960). Further, the Y-borne factors in man may influence fertility as in Drosophila males (Bridges, 1916). Investigations of fertility and family size in polygamous societies should yield some interesting information on this point. Some Y-linked genes may be responsible for the sex dimorphism in the rate of skeletal development and physical maturation (Tanner et al., 1959)) but little is known yet concerning the genetic control of hormonal differences between the two sexes. Further genetical and endocrinological research is needed in understanding the function of the Y-chromosome. The function of the Y-chromosome in general and in specific organisms, and the possible “inertness” of the Y-chromosome in man, animals, and plants will be discussed at, the end of t,he review. 111. Drosophila

The short arm of the Y-chromosome (Ys) ranges in length from half to two-thirds of the long arm (Y”). The girth of the Y-chromosome, in the long arm a t least, is similar to the X of its set. According to Cooper (1959), “If allowance is made for the tendency of the short arm to stretch in its proximal region, the length of the condensed Y ranges from approximately 1.7 microns in smaller cells to somewhat over 4.5 microns in the largest) neuroblasts and spermatocytes I have studied. The average length a t first metaphase of meiosis is somewhat less than that of X, being about 2.3 microns, although not every Y-chromosome was shorter than the X with which it was paired.” The length of the Y-chromosome varies greatly within species. I n D. pseudoobscura, for instance, a t least seven different types of Y have been reported occurring in nature (Dobzhansky, 1937). They differ in the relative proportions of the two arnis. In the repleta group the Y varies greatly in size and shape. It may be large or small, metacentric or acrocentric, or may even be absent altogether as in the case of D. annulimana and in some strains of D. mercatorum (see Bauer,ll936a,b). This was also reported in some other insects. In the neuropteran Chrysopa vulgaris, Naville and de Beaumont (1933) have shown that the Y varies in size from one individual t o another. In several families of Heteroptera such variation is known from genus to genus, the Y sometimes being larger than X, or smaller in ’some genera, and may even be absent altogether (Wilson, 1905a,b, 1906, 1907, 1911). Males of Metapodius femoratus

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K. R. DRONAMRAJU

(Coreidae) from certain localities lack the Y, while it is usually present in other males (Wilson, 1910). The mitotic Y-chromosome in Drosophila is entirely composed of heterochromatin (Heitz, 1933). Both arms are composed of a number of distinct segments. The Y-chromosome in early prophase of large neuroblasts appears t o be divided into as many as eight or ten separate heteropycnotic elements whose structural details are still largely unknown. Cooper (1959) gives a description of the larger features and cautions that the major components may be aggregates of smaller segments. The distal end of Ys appears to be a little thicker than its proximal segment (Dobzhansky, 1932a). It is separated from the main body of Y by a clear gap (Heitz, 1933; Kaufmann, 1933, 1934). A secondary constriction is commonly seen slightly distal to the middle of YL (Bridges, 1927; Dobzhansky, 1932a; Kaufmann, 1933, 1934). Another secondary constriction may also be seen more proximally, and the main length of YL is normally subdivisible into at least three approximately equal parts (Cooper, 1959). A subterminal constriction, roughly one-sixth the arm length from the distal end, was described by Prokefyeva (1935). Cooper (1959) reports subdivision of the distal end of YL and also of the proximal and medial thirds but observes that such regions with very marked subdivision do not show a consistent pattern. Following his classification, the chief heterochromatic parts of the short arm (Ys) may be represented by SA and SB and those of YL by LA, LB, and LC. The nucleolus organizer of Ys is conspicuously situated proximally on a beadshaped body which was regarded by Heitz (1933) to be the bearer of the kinetochore forming the proximal end of YL and lying between the nucleolus organizer and kinetochore. On the other hand, Kaufmann (1933, 1934) and Cooper (1959) believe this element to be the proximal component of Ys, and that the kinetochore lies in the constriction separating this element from the main body of YL (see Fig. 25 in Cooper, 1959).

A. THE X-CHROMOSOME Approximately the proximal half of the X-chromosome, designated X h by Cooper (1959), is heteropycnotic (Heitz, 1934; Kaufmann, 1934). The kinetochore is subterminal (Kaufmann, 1933, 1934; ProkefyevaBelgovskaia, 1937a,b,c, 1935). More than halfway between the kinetochore and the isopycnotic-heteropycnotic junction there is a prominent constriction, which was shown to be a nucleolus organizer by Kaufmann (1933, 1934), and Heitz (1933). Two secondary constrictions are present, each about the middle of the heterochromatic regions, on either side of the nucleolus organizer. The four large heterochromatic segments

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thus formed are designated, from genetic right to left, as “hA,” “hB,” ‘%C,” and “hD” and it is probable that each of these segments is a composite entity (Cooper, 1959).

B. FERTILITY In the course of his classic studies on nondisjunction of the X-chromosomes of Drosophila melanogaster Bridges (1913, 1914, 1916) discovered that the Y-chromosome is necessary for fertility in males because flies of XO constitution, though normal in somatic appearance, were found to be absolutely sterile. Bridges (1916) tested fifteen primary exceptions and found them all to be sterile. Courtship and copulation were normal in such sterile males (Sturtevant, 1915). The females that were mated with XO males laid eggs which did not develop. Safir’s (1920) investigations of such males are of particular interest. An examination of the spermathecae of females mated to XO males showed that no sperm was present. An examination of the external genitalia revealed no differences between XO and XY males. The testes in XO males, though normal in appearance, contained compact bundles of sperm which when artificially separated produced nonmotile sperm. It was also found that considerably less sperm was produced by the XO male than normal. Shen (1932) showed that spermatogenesis in males without a Y-chromosome is perfectly normal but results in the production of nonmotile sperm. Schultz (1947), however, stated that Safir and Shen erred in concluding that the spermatogenesis of the XO male is normal. According to him, “. . . the sperm nuclei do not go through the final stages of the elongation of the sperm head. The head remains in these abnormal males as a round mass, strongly staining in acetic-orcein, and never attains the slender filamentous form of the mature Drosophila sperm. Similar pictures are found in certain of the rearrangements with sterile males; differences of degree are present, the elongation of the sperm head being stopped apparently a t different stages. There is in all these cases, therefore, an effect upon the behavior of the chromosomes themselves, in their final differentiation in the nucleus of the sperm head.” Unlike D . melanogaster, the XO males of D. hydei are phenotypically different from their XY brothers. The testes of XO males in D . hydei do not contain any spermatids or spermatozoa. Developmental stages older than first spermatocytes have never been found, indicating that spermatogenesis is blocked by the lack of the Y-chromosome in the early growth stages of the spermatocytes (Hess and Meyer, 1963). Later work showed that the sterility of XO, x^Ys/O, and f Y L / O males is brought about by deficiencies for fertility factors present in the Y-chromosome, a set from each arm of Y ( k l in the long arm and k~ in the

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K. R. DRONAMRAJU

short arm) being necessary for male fertility (Stern, 1929a). Further studies on fertility were made by Neuhaus (1936a,b, 1937, 1938, 1939), who induccd a series of Y-4 translocations that produced only sterile males in the absence of another normal Y. He tested the fertility of males that carried the proximal portion of one translocation and the distal portion of another, assuming that the translocation carried a mutant fertility gene at the site of the translocation. Neuhaus concluded that a t least five genes acting complementarily on spermatogenesis are located in the short arm of the Y and a t least four such genes, as well as another one having a normal allele near the bobbed locus of the X-chromosome, are located in the long arm. He further suggested that the proximal regions are genetically inert, because his studies using the position effect of the cubitus interruptus gene showed that breakages in the Y occurred in the distal ends only. Brosseau’s (1960) recent work suggests that there are a t least two fertility genes on Ys and a t least five on YL. Since a Y lacking in fertility factors would be eliminated, in his experiments Brosseau used attached-XY chroinosomes, which carried (attached to a single centromere) all the sex chromosome material necessary for male fertility and viability. By means of very impressive complementation tests he showed that the fertility complex can be best pictured as a linear array of particulate units, i.e., in a manner similar to euchromatin. His work suggests that two of the genes, K1-4 and K1-5, and also probably another pair Kl-3 and KZ-4 are less widely separated than the rest. Brosseau (1960) considers that the slight difference in the number of genes on YL found by Neuhaus and himself (four vs. five) is probably due to the small number of YL-4 translocations available to Neuhaus. The difference for Ys (five vs. two) is considerable and may have been due to the different methods used by them. I n Drosophila busclci the right arm of the Y-chromosome is euchromatic and contains viability factors; XO males of this species are not normally viable but such males appear when the Y-chromosome is substituted by the short, proximal euchromatic element of the X-chromosome. It appears, therefore, that the male viability factors are not solely confined t o the Y-chromosome but have homologs or allelomorphs in the proximal part of the X-chromosome (Krivshenko, 1939, 1941a,b, 1950, 1952, 1955).

C. BOBBED As A. H. Sturtevant first suggested, and as Stern (1926, 1927) later proved, both X- and Y-chromosomes carry at least one bobbed locus. Bobbed has bristles that are shorter and finer than those of wild type and frequently has abdominal bands which are broken and less heavily pig-

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mented than in wild type (Sturtevant, 1929). The effect in D. melanogaster is more extreme on the bristles and relatively less on the abdomen, in comparison t o D. simulans. The effect in females of D. simulans is generally less extreme than in males, but the appearance in males is variable. Sturtevant studied the effects of Y’s from different sources by crossing bobbed females to males from various wild stocks. Several of them were found to have Y’s that intensified the bobbed character in males. The Y of D. melanogaster normally suppresses bobbed rather than intensifying it as does that of D. simulans (Stern, 1927). As a result the sex dimorphism in D. melanogaster is in the reverse direction from that found in D. simulans. Males appear wild type, whereas females show the character. Bobbed is completely fertile and recessive in nature. Cooper’s finding that the bobbed+ locus is located near the nucleolus organizer (Cooper, 1958) and probably in hB, is in agreement with Dobzhansky’s studies of Xh duplications which showed that the bobbed+ locus is a t a physical distance not less than 0.5 times, nor more than 1.5 times, the length of the fourth chromosome from the right end of X (Dobzhansky, 1932b), which places it proximal to the nucleolus organizer and roughly in hB. It is not known if crossing-over occurs between bobbed+ and the spindle fiber locus in females with normal X-chromosomes. Even if such crossing-over occurs it cannot bc measured, because no genetic marker is known to exist in the kinetochore region. Stern (1926, 192913) and others have shown that a normal X exchanges, though rarely, with a normal Y in the male, but studies of these events have so far failed to give much insight as to which arm of Y carries bobbed+ and if any homologous interchanges in fact have occurred. If we assume that bobbed+ is located in only one arm, proximal to the K-set of that arm, then, as Cooper showed, a t least twelve XYArmproducts have to be considered. I n view of the availability of only fertility factors and bobbed or bobbed+ classes and the rarity of exchanges (2-8 X Cooper, 1944, 1959) the problem of location of bobbed+ cannot be solved by simple experiments. Neuhaus (1936a,b) mated l?YSX?s 9 9 with YXTY d 3 and obtained 33,220 flies of which two 9 9 were XX and four 3 3 carried a short-armed Y. He claimed that cytological analysis showed the newly formcd Y-chromosome to consist of the two short arms of the Y-chromosome. He concluded that crossing-over occurs between the short arm of the Y- and the X-chromosomes. On the basis of some crossing experiments he suggested that the bobbed+ gene is located in the short arm of the Y-chromosome and that the long arm of the Y-chromosome contains a very extreme allele of bobbed. His later work (Neuhaus, 1937) suggested

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that X and Y interchanged to the right of bobbed in X in a few cases whether exchange is homologous or not. This was based on the analysis of progenies from the cross c7 y bb/Y X Q x^X/Ys; sixsYL/Ys males were obtained out of 26,557 tested, and two of the XYL chromosomes tested were bobbed and the other four bobbed+. However, from the work of Lamy and Slizynska (1950) and Cooper (1952) it is known that X R (the small segment to the right of the kinetochore in the X-chromosome) may exchange, and the results of Neuhaus could be accounted for by a YL exchange in the X R to either side of a bobbed+ locus located proximally in YL. On those grounds Cooper (1959) criticized Neuhaus’ experiments and concluded that they do not offer adequate and conclusive evidence on the location of bobbed+ in Y. Experiments involving attached Xchromosomes or derived Y-chromosomes that possess parts of X are ambiguous and cannot be relied upon for information concerning the homology and the sites of exchange between X and Y (Cooper, 1959; also see L. V. Morgan, 1938; Muller and Herskovitz, 1954; Lindsley, 1955; Cooper, 1956). Philip (1935) reported double crossing-over between t,he X- and the Y-chromosomes in males of D. melanogasier. She regarded YL as carrying the normal allele of bobbed because most X, b a L behaved like bobbed, while Q y / b a s homozygotes had the bobbed phenotype. However, Cooper (1959) pointed out that Philip’s conclusion is not warranted as other explanations are equally likely. Others like Gershenson (1940) and Bridges and Brehme (1944) generally believed that a normal allele of bobbed exists in YS and a weak one in YL. Because of the uncertain history of the ‘(YL” chromosome in experiments involving attached X-chromosomes Cooper rejects Neuhaus’ ( 1936a,b, 1937) claim that a (‘weak” allele of bobbed exists in YL.The possibility that only YL carries bobbed+ seems unlikely because then X R and X h must each have at least one point of exchange, and exchange with X h must be asymmetrical (distal to bobbed in YL,and proximal in Xh), resulting in deficiencies and duplications for bobbed. No such asymmetry is required if bobbed+ is localized in Ys. Thus it seems more likely that Y carries a single bobbed+ locus, probably a compound one, which is situated in Ys. Some support to such an assignment comes from three directions (Cooper, 1959); (1) the ring Y (Muller, 1948, 1949; Lindsley, 1955) having a complete set of K1 fertility factors, and at least three widely spaced constrictions, but lacking in the bobbed+ locus, some short-arm fertility factors, and probably the nucleolus organizer as well; (2) the relative frequencies of the presence or loss of the bobbed+ locus when either y+ or bw+ was deleted in irradiation experiments from the sc8 . Y : bw+ chromosome, suggesting that bobbed+ is proximal to Kz (Baker, 1955, 1957); and (3) the appearance

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of bobbed-deficient, in certain stocks of Schultz, which retains KI and K:! but shows deletion of the short arm to about one-third the length of a normal Ys. This evidence, however, is not conclusive, as was pointed out by Cooper (1959), because the Y b b - of Muller’s stock h2 showed no structural differences from a normal Y.

D. HETEROCHROMATIN The term heterochromatin was introduced by Heitz (1928) to describe certain chromosomal regions which differ from the others b y their staining and retain a dense and compact structure during interphase. Thus, the original meaning was purely cytological. I n a series of brilliant papers Heitz (1928, 1933, 1934, 1935) produced the classic concept of heterochromatin. He was able to show th at the heterochromatic regions differed in the degree to which they changed in the telophase from the compact and dense appearance of the metaphase, to the diffusely stained uncoiled interphase. On the basis of his studies on mitotic and salivary gland chromosomes he recognized two types of heterochromatin: a,which does not uncoil, and p, which uncoils and exhibits diffuse structure. However, chromosomal regions that are heterochromatic at one stage, or in one kind of cell, or in one sex, may not remain so under different conditions. Darlington and La Cour (1940) suggested that different chromosomal regions show staining properties according to the DNA content in the chromomeres. Heterochromatin has been defined as any chromosomal region which becomes heteropycnotic at some stage of its cycle. Schultz (1947), on the other hand, considers them as the chromosomal regions which form massive blocks of positive material in intermitotic nuclei. Cooper (1956) prefers to use the words “chromocentral region” instead of “heterochromatin” because “heterochromatin today is what a n investigator chooses to make it or imagine it . . . .” Soon after Heitz’s cytological work it was realized that the so-called genetically inert regions of Drosophila chromosomes are heterochromatic (Muller and Painter, 1932). I n D . melanogaster the proximal third of the X-chromosome and the entire Y are totally heterochromatic. Studies of the heterochromatic regions have suggested th at heterochromatin has many functions, some of them remarkable. This subject has been reviewed in the past by Schultz (1941, 1944, 1947, 1952), Pontecorvo (1944), Resende (1945), Darlington (1947), Vanderlyn (1949), Goldschmidt (1949, 1955), Barigozzi (1950), Hannah (1951), Sharma and Sharma (1958) , Cooper (1956, 1959), and also Prokofyeva-Belgovskaia (1935, 1937a,b,c, 1939a,b,c, 1946, 1947). Some of the functions attributed to heterochromatin are to control mutation; to modify specific gene action; to regulate

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crossing-over and chiasma localization; to cause variegation and control variegation brought about by genes located in other chromosomes; to control cell size; to act as a region of transfer of energy and/or substances a t the nucleolar and nuclear membranes; to provide neutral anchorage for rearrangements and translocations, etc., in speciation; to take part in syntheses of nucleic acids, proteins, nucleolar material, etc.; and to regulate growth and differentiation probably by playing a role in sex determination ! The length of the major heterochromatic regions in a salivary gland chromosome of Drosophila is relatively much shorter than in the somatic chromosomes (Dobzhansky, 1944; Heitz, 1934, 1935; Pavan, 1946). The Y-chromosome, which is longer than the X in mitotic chromosomes, is reduced to about nine discs in the salivaries (Prokofyeva-Belgovskaia, 1937a,b,c). For an account of the structure of heterochromatin see the early papers by Painter (1934), Painter and Griffin (1937), and Metz (1941). With the discovery of crossing-over between the X and Y (Kaufmann, 1933; Philip, 1935; Neuhaus, 1936a,b) it became clear that the so-called “inert” region cannot be full of degenerate genes as was suggested previously (Muller, 1914a,b, 1918). Muller recently stated that “In 1914 the presence of the normal allele of bobbed bristles in the Y chromosomes as well as the X had not been found by Sturtevant, but neither he nor I ever took the position that the evidence showed the Y to be completely inert; in fact, we realized that the sterilit,y of XO males found by Bridges proved the presence of some genes necessary for sperm motility in the Y of Drosophila” (RIuller, 1964). Muller and Gershenson (1935) showed that inert regions are essentially of norigenic material and function in elaborating accessory substances, which form the blocks of stainable material. Later these regions were discussed in terms of the substances responsible for the staining, the riucleoproteins or the nucleic acids (Schultz, 1936a). Schultz (1936a, 1947) during the course of his investigations on variegation, compared the behavior of the normal salivary gland chromosomes with different balance of heterochromatin in the nucleus. It was found that the structure of the salivary gland chromosomes changed as the heterochromatin in the nucleus was varied. They became plumper and stained more intensely with extra Y-chromosomes. In the males with no Y-chromosome (XO) they became flaccid and pale. Schultz (1947) confirmed these earlier results. In later work the shape of the chromosomal ends was found to be a more sensitive index. These are mainly of three kinds in the giant chromosoiiies : straight ends, fan-shaped ends, or bulbous nipplelike ends (see Hinton, 1945; Kodani, 1947). The fanshaped ends increase in frequency as we proceed from XY to XO males,

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whereas the nipples appear chiefly in the XYY male and gradually decrease in the order XYY -+X Y -+ XO. The straight ends appear to be most frequent in X Y males and decrease in the order X Y -+ X O + XYY. Schultz (1947) concluded that the number of Y-chromosomes present in the nucleus exerts a determining effect on the structure of all the chromosomes. It was also found that the bands stair1 lightly in the absence of a normal Y complement. The bands appeared more dispersed and nucleoprotein staining fibers were found between them, so it was considered that the nucleoprotein components were affected in amount as well as in arrangement. Studies with the phase microscope showed that these differences were present in vivo also. The X O nucleus looked more watery, the nucleolus not so compact, and the chromosomes less substantial. It was concluded that some common precursor substance was lacking and it was this that prevented the synthetic system in the chromosomes from working normally. It was explained that the morphologically normal appearance of such individuals may be due to the fact that there are substances in the chromosomes either present in excess for normal function of the genes, or substances which do not have to do with the specific functions of the individual genes. It was previously shown that in X X Y females the Y-chromosome influences the nucleic acid content of the egg cytoplasm (for details of the early work see Schultz, 1941, 1947). Schultz (1956) reported that the Y-chromosonie in D. melanogaster plays a prominent role in the metabolism of thc riucleic acids during the formation of the egg. It synthesizes more DNA in the nucleus of the nurse cell than one should expect from its cytological appearance. The RNA of the mature egg cytoplasm changes in base constitution, and the amount of thymidirie is raised considerably, but the total amount of RNA or nucleoside precursors is unaltered. The X-chromosome suppresses variegation (in the iniagirial tissue)-"a phenomenon which is shown by the reversal of the aniethopterin effects upon it, to contain a process involving the synthesis of thymidine." I n later work on the nature and the amount of the ribonucleic acids in two types of unfertilized D. melanogastei. eggs, X X and X r P 8 it was found that the RKA in the latter type contains a significantly higher proportion of adenine than the RNA in the X X type. The quantity of RNA per egg is substantially equivalent in the X X and the X %!Yysc8 types. The content of purine compounds in the extracts from X%!Ysc8 eggs is more than double that of the X X type. Cytosine was found in only small traces in the X gV8type while it was in considerable amounts in the X X , and the converse was found to be the case for thymine. The conclusion was reached that the Y-chromosome affects the composition of RNA in the

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egg cytoplasm and the synthesis of the nucleic acid bases (Levenbrook et al., 1958; Travaglini et al., 1958).

E. HETEROCHROMATIN AND POLYGENES

It has been suggested that the heterochromatin in the Y-chromosome carries a system of polygenes (Mather, 1941, 1944). These studies were based on the variation in the number of sternopleural chaetae and wing hairs. Mather used the following six stocks, which supposedly differed in the Y-chromosomes of their males : Basic Oregon, Amherst, Ockley, Rothamstead, Samarkhand, and Wellington. The females were used to detect variation due to the presence of unreplaced autosonies and t o the effects of environmental differences between cultures. The six Y-chromosomes gave the following order of chaetae-producing power when compared with the X O r chromosome, common to all lines:

The greatest difference, between Y O k and YAm,was found to be 0.593- (-0.588) or 1.181 chaetae. This is about 6.1% of 19.4 chaetae, the general mean number in all males. Other genetic effects of Y-chromosomes were observed between stocks. For instance, the YOk chromosome seemed to make its male carriers less ready to mate than others. On the basis of such experiments Mather (1944) concluded that the variation in phenotype to which these genes give rise is continuous. The Y-chromosome in Drosophila like the supernumeraries of Zea, Sorghum, etc., has all the properties of a polygenic combination, as does the heterochromatin of the X. These experiments and also those of Barigozzi (1952), and Barigozzi and di Pasquale (1953) have been subjected to much criticism, especially by Cooper (1945, 1959) and Schultz (1947). Later Goldschmidt and others (see Goldschmidt et al., 1951; GoldSchmidt, 1955) described certain phenotypes, designated as podoptera, which are characterized by a very low and variable penetrance. Modifying effects were found for the Y-chromosome and other regions. It was concluded that heterochromatic genes act as a series of multiple factors which act in a general unspecified manner resulting in interaction of a pseudoallelic type bet'ween loci.

F. HETEROCHROMATIN AND VARIEGATION It was found, thirty-two years ago, that an extra Y-chromosome tends to suppress the mottling brought about by rearrangements, and restores the phenotype to normal or nearly normal (Gowen and Gay, 1933, 1934).

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However, later work showed that when there are two extra Y-chromosomes as in XXBY females and X3Y males they cause a mottling of the self-colored eye and sterilization of the male (Cooper, 1956). I n females with attached X-chromosomes also (as in a Ys~YL/Ysx^YL)mottled eyes of varying degrees of variegation are observed. Supernumerary Y-chromosomes also seem to affect the uniformity of the coloration of the eye. A few mottled males of X2Y type were reported but Cooper considers these to be mosaics of X3Y and X > 3Y tissues. The eye color of a strongly mottled female fly of the constitution apr v/apr+v may have a n over-all orange cast and in ordinary light the blotches of mottling appear as lemon-yellow washes of pale color a t the posterior margin of each eye. Sometimes the blotch in each eye may appear triangular, or the wash of color may look like a narrow light streak from the eye's center to the mid posterior region. The two eyes are generally not symmetrically affected but the extent of mottling may be similar. Darkening of the eye in older flies may emphasize the color contrast but in others with certain genotypes detection in older flies becomes increasingly difficult. Certain other somatic effects of hyperploidy for Y-chromosomes are also noticed in XXBY females and X3Y males, and such effects are often correlated with more extreme degrees of mottling. Some of these are shortening and thickening of the posterior and middle legs, spatulate end of the tibia, and coarse bristles (Cooper, 1956). Extremely affected females, sometimes resembling superfemales (XXX) , often have reduced viability and fertility or may even be sterile. There is no evidence at present to indicate that the deformity and sterility in these females are primary effects of supernumerary Y-chromosomes. T ha t supernumerary Y-chromosomes in males cause sterility was first reported by Schultz (Morgan et al., 1934). Cooper (1956) studied the matings of several such males with successive females for a period of at least 7 days. The males were chosen on the basis of the mottling of their eyes and a few of them were cytologically tested. I n the series of sc, dl-49, v, BM1males, 12% were found to be fertile and 3 out of 4 of the fertile variegated males actually turned out to be cytologically X2Y of sc, dl-49 v, BM1X-chromosome constitution. Only one fertile male was found in the 195 other males tested and Cooper calculated that the 95% confidence interval for fertility in X3Y males is roughly from 0.05% to 2.9%. The discovery of sterility in flies with supernumerary Y-chromosomes came as a surprise because some years ago Bridges (1916) had demonstrated that XO males are sterile, too. The sterility in both kinds of males appears to be similar in that the testes and allied genital structures are normal, spermatogenesis is regular, and bundles of spermatozoa are produced. The spermatozoa are nonmotile and generally the bundles

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degenerate a t the proximal end of the testis. Free spermatozoa are occasionally seen in the testicular ducts of X3Y males but they are also immotile. It is not known whether X4Y males are viable. In D. hydei, Hess and Meyer (1963) reported that the number of spermatozoa is reduced in XYY males which are, however, fertile. It was also found that>the total length of spermatozoa is considerably greater in such males than in normal males. Cooper (1956) attempted to answer the very interesting question: Do the traiisformed “males” (extreme female intersexes with male phenotype, discovered by Sturtevant, 1945) mottle with two Y-chromosomes as would be expected on the basis of their genotype, or would they require three Y-chromosomes as in true males? It was found th a t the XXSY, tia/tra females variegated like others with XX2Y constitution and contained rudimentary testes with no spermatocytes or spermatozoa, thus confirming that the genetic response of the transformed “male” is that of a feiiialc (Sturtevant, 1945; Goldschmidt, 1953). Experiments with fractional hyperploids showed that something less than X2Y chromosomes in addition to normal complement is enough to cause mottling in some individuals and sterility in some males. It was further shown that tlie factors causing sterility in hyperploid males are dependent, to a certain extent, on those causing variegation, and th a t sterilization may involve certain elements outside the K1 and Kz complexes (Cooper, 1956). As mentioned earlier, Gowen and Gay (1933, 1934) have shown th a t an extra Y, i.e., XXY in female and X2Y in male, suppresses variegation of the white gene. Since then the ability of an extra Y to suppress variegation has been demonstrated for many R(+)euchromatic loci. Several investigators have tried to determine the effect of an extra Y on the R(ci+)position effect but no satisfactory answer has been obtained. The supernumerary Y-chromosomes suppress (or modify) the characteristic phenotypes of dominant and recessive V-type position effects which cause mottling. (The effect of a gene may be dependent upon its position with regard t o neighboring genes.) The term “Dubinin effect” is applied to position effects of the cubitus interruptus gene, first discovered by Dubinin and Sidorov 1935) who found th at ten among nineteen translocations involving the fourth chromosoiiie showed a weakened dominance of ci+ when they were tested against a chroniosome bearing the recessive (ci) mutant. Grell (1959) coinpared two groups, which were genotypically equivalent except for the presence or absence of an extra Y-chroinosome, for the expression of R(ci+)/ci and found a highly significant difference; the effect of the extra Y in the two rearrangements studied was t o partially suppress the Dubinin effect. In another experi-

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ment the group with two extra Y-chromosomes showed a greater suppression of the Dubiriin effect. The fertility of the females varied inversely with hyperploidy for extra Y-chromosonies. For early work on the phenomenon of position effect see Sturtevarit (1925), Dobzhansky (1936), Catcheside (1947), Lewis (1950), and Baker (1953, 1954). The V-type appears t o be detectable whenever a euchroniatic gene is brought adjacent t o heterochroniatin under certain conditions. Cooper (1956) tested the assumption th at complementation of the workings of the position effect and the supernumerary Y-chroniosonies could occur, resulting in the elimination of each of their phenotypic effects on the eye, and concluded that the dominant V-type position effect A l o k and the recessive I n ( l ) w m 4suppresses the action of the Y-chroniosomal elements that cause variegation. It has been emphasized that variegation is a consequence of “heterochromatinization,” i.e., somatic inactivation of euchromatic genes placed in close continuity with chromocentral material (Schultz, 1936a and b, 1941, 1944, 1947, 1952; Noujdin, 1938, 1939, 1946a,b; ProkofyevaBelgovskaia, 1939a,b,c, 1946, 1947). Schultz suggested that the suppression of variegation in the presence of additional Y-chromosomes is due to the heightening of the general pool of nucleic acids. Cooper (1956) regards this as an inadequate explanation to account for the mottling caused by Y-chromosome hyperploidy (also see Prokofyeva-Belgovskaia, 1939a,b,c ; Cole and Sutton, 1941; Callan, 1948; Gersh, 1949; Schultz and Caspersson, 1949). Chromocentral loci have been regarded as forming a balanced system with euchromatic loci (Schultz, 1936a and b, 1941, 1944, 1947, 1952). Ordinary fluctuations in the genotype may change the euchromaticheterochromatic balance. The suppression of variegation in flies with a n additional Y-chromosome and the variegation in hyperploids with two extra Y-chromosomes above normal could be explained in terms of changes in such a balance (see Cooper, 1956). A similar explanation could account for the sterilization of males with supernumerary Y-chromosomes (Schultz, 1941; Morgan et al., 1934, 1935). The phenotypic effect of the two different groups of fertility factors on the Y-chromosome may be the result of a delicate balance maintained between them and the opposing action of genes elsewhere. Infertility may result when the balance is upset by the presence of too many Y-chromosomes. Baker and Spofford (1959) have shown that a t least fifteen different segments of the Y-chromosome differ in their effects on variegation arid that the differences often are not related to the size of the segment, thus indicating that the Y-chromosome probably has linearly differentiated factors which could modify the variegated phenotype.

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G. SEX AND ANTIGENICDIFFERENCES I n 1936 Dr. S. G. Levit and co-workers a t the Medico-Genetical Institute in Moscow obtained evidence that the Y-chromosome of Drosophila could be distinguished immunologically. It was shown that this was not just a male-female difference, since comparison of females with and without a Y-chromosome confirmed this observation (Levit, et al. 1936). Fox (1958) and Fox and Yoon (1939, using techniques modified from Ouchterlony and Bjorklund, investigated antigenic differences between males and females in Drosophila. Two coisogenic stocks have been used. I n one, known as Oregon R, the females have two X-chromosomes (X/X), while males have one X and a Y (X/Y). I n the other stock, known as YA, the males are X/Y as in Oregon R, but the females have attached X-chromosomes (x^X/Y). Since the stocks are coisogenic, antigenic differences between the two types of females from the two types of males could be attributed to the X-chromosome dosage. It was also thought th a t antigenic differences distinguishing Oregon R females on the one hand, and Oregon R males, females, and males of YA stock on the other would be at,tributable to the presence of the Y-chromosome in the latter genotypes. Their technique disclosed two antigenic components specific to Oregon R and YA females ( 9 -1 and 9 -2), and two components specific to Oregon R and YA males (9-1and 9-2). The two female-specific components are inhibited by extracts from either female genotype but neither male genotype. The two male-specific components are inhibited by extracts of either male genotype but neither female genotype. According to Fox (1958), “since these differences are not associated with the presence or absence of the Y-chromosome they must be attributed to the difference between the two types of males and the two types of females with respect to X-chromosomal dosage. I n the presence of two X-chromosomes the female components are produced, while the male components are produced in the presence of one X-chromosome . . . . It is not unlikely that the mechanism responsible . . . involves the balance of X-chromosomes with respect to autosomes, as in sex determination.” Fox, however, observed another antigen, named Y-1, which showed different properties in the two stocks. The antigen in the Oregon R stock is capable of inducing antibody formation and capable of uniting to form visible precipitate; here the antigen is complete. I n the YA stock, on the other hand, the Y-1 specific antigen induces antibody formation and will unite with antibody to produce inhibition without the formation of precipitate; the term “incomplete” antigen is used to describe these properties. He suggested that this could be attributed to either a case of cytoplasmic inheritance or to an effect of the Y-chromosome in the

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oocyte of the mother on her offspring. Further tests were carried out to distinguish between these two possibilities and it was concluded that the observed phenomenon is not due to the presence or absence of the Ychromosome in the genotype of an individual, but to the presence or absence of the Y in the oocyte of a female which determines the type of Y-1 to be carried by her offspring. Fox suggested that since the antigenic specificity of complete and incomplete Y-1 is the same, the difference could be due to the gross physical attributes of the molecule to which the specificity is attached. He considers it probable that the specificity of Y-1 is the product of one or more euchromatic loci on the X-chromosomes or autosomes. I n their recent paper Fox et al. (1962) reported that the modification of Y-1 occurs regardless of the presence or absence of the Ychromosome in the progeny and is independent of the source of cytoplasm transmitted through the egg. They commented that a mechanism might exist which derives its information from the genetic material but is capable of self-replication after that material is removed and that such a mechanism could consist either of an enzymatic system for the synthesis of RNA, utilizing preformed RNA as “primer,” or of a chromosomal element on the autosomes which is altered by the presence of the Y-chromosome in the oocyte. I n either event the mechanism would be subject to decay in the germ line and would be supplementary to ‘(messenger” RNA. Y-linked antigens will be discussed again in the section dealing with mice.

H. OTHEREFFECTSOF THE Y-CHROMOSOME It has been suggested by Meyer et al. (1961) that the intranuclear structures (recognizable only in the electron microscope), occurring in spermatocyte nuclei of D . melanogaster during certain developmental stages, are morphological indications of phase-specific chromosomal activity. They demonstrated that the Y-chromosome is involved in their formation and that certain parts of the Y-chromosome, probably YL, are more effective than others in producing these structures. The morphological appearance of such structures is different in different species of Drosophila. Meyer and Hess (1962) found highly complicated structures in the spermatocyte nuclei of the closely related D. hydei and D. neohydei. The chromosomal functional structures of these two species consist of at least four loop-shaped components which are considered similar to the loops of lampbrush chromosomes in oocytes of Amphibia (Gall, 1954; Callan and Lloyd, 1960). They are paired and polarized. In D . hydei one pair of loops consists of two highly refractive threads originating at the nucleolar region and becoming diffuse distally. The compact parts of these threads are embedded in tubular material. A

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second pair of loops consists of a spongy matrix appearing in the form of two clubs which are covered with large hollow granules 0.5-0.8 p in average diameter. A third pair, called the “pseudonucleolus” (Meyer and Hess, 1962), is built up by the fusion of two subunits. The fine structure of the “pseudonucleolus” is quite distinct from th a t of the nucleolus. The former has a spongelike structure with tunnels while the latter has a granular structure without such tunnels. I n addition to these, loose ribbons of tubular material similar to that described by Meyer (1961) and Meyer et a1. (1961) in D . melanogaster were also found. I n D. neohydei one pair of loops consists of two threads embedded in tubular material The second pair of loops (“clubs”) consists of two elongated elements with large granules 0.5-0.8 p in diameter arranged in a single line. There is no pseudonucleolus but there are two clumps of loose ribbons (see Fig. 1 in Hess and Meyer, 1963). 1. XO-Males

Meyer et al. (1961) found that the intranuclear structures in spermatocytes of D. melanogaster depend upon the presence of a complete Ychromosome. In D. hydei and D. neohydei, since no females with attached X-chromosomes are available, XO males can only be obtained as a result of primary nondisjunction, which can be detected with suitable recessive markers in D. hydei but not in D. neohydei (Spencer, 1930, 1949). The spermatocyte nuclei of XO males in D. hydei appear relatively empty as the paired loops, which are very striking in normal spermatocyte nuclei, are absent. There are only a few big granules, which are composed of smaller granules of varying size and shape, forming clumps or hollow spheres. Some tubular material is seen occasionally under the electron microscope, but it is considerably larger than t h a t observed in the tubuli of XY spermatocyte nuclei. In contrast to this empty appearance, crystal needles are found in XO spermatocytes of D. melanogaster which also contain tubular material in small amounts. 2. X Y Y Males The first pair of loops, which has normally two highly refractive threads, always contains four distinct compact threads in XYY males. The duplication of the club-shaped loops is often seen but is not openly manifest in all cells as a result of fusion of the clubs with each other. About one-third of the nuclei contain two distinct pseudonucleoli instead of one, each with a pair of protrusions. The remaining two-thirds have only one pseudonucleolus which can be recognized as a duplication by its size and by the presence of four protrusions. The duplication of the diffuse ribbons of tubular material is difficult to see because they are coiled and

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folded up tightly. The number of spermatozoa is reduced in the X Y Y males, but they are fertile. The average length of their spermatozoa is 12-14 mm while in normals it is 6.6 mm. A correlation between the presence of the Y-chromosome and sperm length is also established: 1.1 mm in XO males, 1.75 mm in X Y males, and 3.7 mm in XYY males. The investigations of Hess and Meyer (1963) have shown th a t the paired structures found in spermatocyte nuclei are structural modifications of parts of the Y-chromosome. On studying the spermatocyte nuclei of hybrids from D . hydei X D. neohydei crosses they found that these spermatocytes consist of intranuclear formations which are characteristic for the species from which the Y-chromosome was introduced into the cross. They concluded “that the aspect of species specificity of the paired structures depends entirely on the constitution of the Y-chromosome.” It is considered possible that the species specificity of the looplike structures is based on the synthesis of specific proteins whose polypeptide sequence might be coded on the Y-chromosome itself. Fox and associates (1962) have recently demonstrated the involvement of the heterochromatin of the Y-chromosome in protein synthesis. Another possibility is that the information for the synthesis of proteins required for the development of the loops might be Iocalized elsewhere and that the Y-chromosome selects certain “suitable” proteins for the development of the looplike structures. It is obvious from the results already obtained by Meyer, Hess, Beermann, and their co-workers that a most exciting new field has been opened up which requires much further work in order to appreciate the role played by heterochromatin in protein synthesis. It would be of considerable interest to investigate other species of Drosophila also from this point of view. Seiger’s recent work on the genetics of the phototactic responses in crosses between Drosophila pseudoobscura and D. persirnilis is also of considerable interest. Flies were tested by introducing one individual at a time into a Y tube, one arm of which was exposed to daylight from a window and the other shaded. The fly walked down the tube to the bifurcating point and chose between the light and dark arms. Each fly was run twice, and twenty flies of each sex were used. Thus, for a given strain or cross there were forty male and forty female tests. The results of several reciprocal crosses as well as back crosses to persirnilis and pseudoobscura males may be summarized as follows (quoted from M. Seiger’s personal communication, 1965) : 1. Partial dominance of photopositive (persirnilis) chromosomes over photonegative (pseudoobscura) chromosomes. 2. Maternal effect (F, progeny).

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3. Y chromosome modifies phototactic response dependent on whether it came from the pseudoobscura or the persirnilis father (F1and backcross males). 4. Effect of dosage of chromosomes. This is evident from reciprocal backcrosses. Either F1 female crossed t o a persirnilis male produces highly photopositive progeny. The backcross t o pseudoobscura, on the other hand, yields progeny having a greater ratio of photonegative (pseudoobscura) chromosomes to photopositive (persirnilis) chromosomes and consequently show only a moderate photopositive response.

IV. Role of the Y-Chromosome in Sex Determination in Insects

I n Drosophila, the Y-chromosome is not primarily concerned in sex determination as in man, but is required for fertility in males. X X Y or XXYY flies are female, but X O flies are sterile males (Bridges, 1916; Stern, 1929b). Bridges’ (1932) balance theory proposes that sex is determined by a balanced influence between X-chromosomes and autosomes. The X-chromosomes influence development toward femaleness, and the autosomes influence toward maleness. The system is so balanced that a ratio of one set of autosomes to one X-chromosome results in femaleness, while two sets of autosomes to one X gives maleness. The Y-chromosome has no influence. A. Lymantria dispar Goldschmidt’s classic work on sex determination and diploid intersexes in the gypsy moth, Lymantria dispar, is a n important contribution to the understanding of sex-determination mechanism (Goldschmidt, 1934, 1955). This moth occurs naturally in Europe, North Africa, and northern Asia. There is a striking sexual dimorphism in this species. A number of geographic races are known and crosses within each race normally result in male and female offspring. However, crosses between individuals from different geographic regions, e.g., Europe and Japan, produce some intersexes in addition to normal males and females. As in birds and most other moths, but unlike man and Drosophila, Lymantria has heterogametic female sex. Goldschmidt (1920, 1931, 1934) classified the intersexes into male intersex, which has an XX constitution and exhibits female as well as male characters, and female intersex, which has an X Y constitution but exhibits both male and female characters. The intersex is a sex mosaic with varying degrees of maleness and femaleness and sex-reversal i.e., an X Y functional male. Goldschmidt proposed that sex in Lymantria is determined by the interaction of factors for maleness carried in the X-chromosome and factors for femaleness in the Y or in the cytoplasm (also see Caspari, 1948). The “strength” of these factors is said to vary. In any given race, a system has evolved (through natural selection) in which two doses of

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maleness (carried in two X-chromosomes) overcome the femaleness and produce a normal male, while one dose of maleness, in one X-chromosome, is dominated by the femaleness and gives rise to a normal female. The sex factors in the Japanese race of Lymantria are “strong” and in the European race “weak.” Female intersexuality is produced in an F1when a weak female is crossed with a strong male. Male intersexuality results in a proportion of males in the Fz of the cross, strong female X weak male. I t is concluded that the system of sex determination has the nature of a genic balance, sex being dependent upon the balance or lack of balance of certain determiners. On the basis of various crossing experiments GoldSchmidt suggested that sex is determined by a relational balance between a Y-borne maternally inherited female determiner, designated F , and an X-borne male determiner M which is present in single dose in the female and double dose in the male. Intersex is the result when in a hybrid combination M and F are not properly balanced. It was further proposed that the intersexes begin their development as individuals of one sex and continue to do so up to a certain critical point, called “turning-point,” after which their development is of the alternative sexual type. The intersexes are thus explained as due to the switching from the developmental course of one sex to that of the alternative one. This hypothesis has met with objections and cannot be regarded as established [see, for instance, Baltzer’s (1937) work on the marine worm Bonelliu viridis; and Seiler (1937)]. The relative “strength” and “weakness” of the sex-determining genes can be indicated in terms of numerical values assigned to M and F , e.g., M s is much stronger than M a . The “strength” or “valency” of the sex factors differs between races but is fixed within each race. It is suggested that this fixity is quantitative, depending upon definite amounts of the sex-determining material present in any case, different degrees of strength and weakness revealing the existence of a series of multiple allelomorphs. The expression of male intersexuality is influenced by autosomal allelomorphic modifying genes.

B. Bombyx mori In the silkworm, Bombyz mori, Tazima (1943) found that the female is XY (ZW) and that the female determiners, which are located in the Y-chromosome, are so strong that any egg with a Y will be female regardless of the number of X-chromosomes which contain the male determiners. However, as Goldschmidt (1955) explained, since X X individuals are males, the strong Y which all eggs possess before fertilization cannot have a predeterminative influence, because if this were so all males would be intersexual. Goldschmidt (1955) further stated that, “. . . the actions

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controlling female determination must have taken place in the unfertilized egg by way of cytoplasmic predetermination or conditioning, which remains active in the once determined way throughout all the development, since it controls (in balance with the X-chromosomal male factors) all the processes of sexual differentiation.” The diploid chromosome number is 56 in both sexes. Extensive investigations have revealed no morphological traits determined by genes in the W (or Y) chromosome (see Tazima, 1952; Yokoyama, 1959). A full Z (or X ) chromosome is essential for viability. Hypoploids deficient for different amounts of the Z-chromosome and a normal W all died regardless of the portion deleted. Hyperploids for the Z-chromosome, in the presence of a W, all lived and showed no sexual anomalies.

C. OTHER INSECTS The heterogametic sex in crane flies (Tipulidae) is the male, which has a n X Y sex chromosome constitution as demonstrated by the sexlimited inheritance of Y-autosome translocations in Tipula oleracea, Pales crocata, and P. ferruginea. Ullerich and associates (1964) reported three pairs of large autosomes and a small pair of heteromorphic X Y chromosomes. Triploid individuals with normal spermatogenesis have been found in Pales lunulicornis, and P. ferruginea. Their sex chromosomes were found to be XXY. In P. ferruginea, female offspring with XX constitution, and male offspring with XY, XYY, and X X Y occur in crosses of XYY males with normal X X females. All these offspring are fertile. The authors conclude that, “Their occurrence proves the existence of an epistatic male determining factor (or factors) in the Y. Presence or absence of Y thus is responsible for sex realisation.” I n several species of the midge Chironomus the Y-chromosome has been shown to be concerned with sex determination (Beermann, 1955). The role of the Y-chromosome has been suggested in the sex determination of the fly Aphiochaeta zanthina (Tolrunaga, 1955, 1958). Partial sex-linkage of several genes was also reported in this species (Ondraschek, 1953). I n Culez molestus, Gilchrist and Haldane (1947) have shown that a recessive gene for white eye is partially sex-linked and that “maleness appears to be due to a single dominant gene in the same chromosome as that for white eye. Crossing over is unusually variable. Three gynandromorphs, two being mosaic for eye color, are described. These latter cannot be due to chromosomal elimination, and may be due to somatic crossing over.” I n the beetle, Phytodecta variabilis, Galtin (1931) found a n unequal pair of chromosomes in the male, and partial sex-linkage occurs (see

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de Zulueta, 1925; Gilchrist and Haldane, 1947). The same sets of genes for natural polymorphism have been claimed to be found both in the X and Y chromosomes with very little crossing-over. A compound sex-chromosome mechanism has been encountered in Tenodera, Paratenodera, Mantis, Stagmomantis, and in Hierodula, the male sex chromosomcs being XIXzY and the female’s X I X I X ~ X(Oguma, ~ 1921; King, 1931 ; Asana, 1934) ; the males of many genera of the praying mantis produce XO males (White, 1941; Hughes-Schrader, 1950). See White (1954) for an extensive account of the sex chromosomes in insects as well as other animals. Gowen (1961) recently reviewed the subject of sex determination in some detail, and Beat,ty (1964) dealt especially with vertebrates. V. Fish

The first organism in which a Y-linked inheritance was demonstrated was the guppy, Lebistes reticulatus (Schmidt, 1920; Winge, 1921). Two different color patterns were found to be transmitted from father to son, son to grandson, and so on in the male line and the trait never passed through the mother. Later research revealed several Y-linked and partially sex-linked genes. Partial sex-linkage was, however, first reported in another fish Aplocheilus latipes by Aida (1921) who also reported on a Y-linked color gene in this organism, quite independent of Schmidt’s discovery in Lebistes [see postscript in Aida (1921) and footnote by Castle (1921) a t the end of Aida’s paper]. Aida (1930) later reported that, in Aplocheilus latipes, there is a large preponderance of the crossing-over from Y to X rather than from X to Y. The gene R (for the formation of yellow pigment) was shown to cross over from Y to X with a frequency of 1:300, but from X to Y with a frequency of 1:1200. The nondisjunctional form X X Y is male and fertile, and produces mainly female offspring when mated with a normal female. Though genetic evidence indicates that the male is heterogametic in Lebistes the X- and Y-chromosomes are not distinguishable cytologically (Vaupel, 1929). In fact, all 23 chromosome pairs are of the same size. The X and Y are homologous for most of their length, the differential region being quite short. The genes are dominant in the male, and no color pattern is normally exhibited in the female even when it possesses a homozygous genotype. More than twenty color genes have been reported in this species (Winge, 1922, 1927; Blacher, 1927, 1928; Kirpichnikov, 1935). Crossing-over between X and Y has been reported in several cases (Winge, 1923b, 1927, 1934). Genes (probably allelomorphs) showing complete Y-linkage are M a , I r , Pa or A r (Maculatus, Iridescens, Pauper, Armatus). Some partially sex-linked genes are Ti, Lu, Co, V i , El (Ti-

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grinus, Luteus, Coccineus, Vitellinus, Elongatus). The maximum percentage of cross-over was found to be only 10 (Winge and Ditlevsen, 1947). Genes concerned with other traits and also certain color patterns were found in the autosomes (Winge, 1927; Haskins and Druzba, 1938; Goodrich et al., 1944). Winge and Ditlevsen (1938, 1947) discovered a recessive lethal gene in the Y-chromosome. I n inbreeding work it was found that even though the YMa YPa males were fully viable and fertile the YMa YMa type was lethal. This lethal gene, which is situated close to the Ma gene, was believed to contribute in the maintenance of normal sex determination within the YMa race (Winge and Ditlevsen, 1938). The completely Y-linked gene Maculatus always appeared as a unit in Winge’s experiments. In certain selection experiments males with a small black spot on the dorsal fin were selected against, and finally some Xo YMa males were obtained which had no black spot. However, the same male from this group gave spotted and spotless sons when mated to different females. Winge ascribes this result to the interaction of minor factors affecting the spots and not to possible changes in the Maculatus gene itself. Blacher (1928), on the other hand, suggested that Ma is composed of at least two genes one of which produces a black spot in the dorsal fin and the other a red side-spot on the body. Sex determination. According to Winge (1932, 1934), and Winge and Ditlevsen (1947) some interracial crosses gave rise to X X males and XY males. Normally the Y-chromosome contains a strong male-determining gene closely linked to, or identical with, the completely Y-linked Maculatus gene. The X-chromosome may similarly possess a corresponding female-determining gene. However, sex-determining genes of varying degrees of potency, some pulling in a male direction, and others in a female direction, have also been postulated. It was suggested that XX males arose as a result of the accumulation of so many male-determining genes in the autosomes that the male sex developed even in the absence of a Y-chromosome. Through repeated back-crossings of the daughters to XX males equal numbers of colored X X males and uncolored X X females were obtained. As Winge and Ditlevsen explained, “as the inheritance of the X-linked genes is now no longer sex-linked but merely Mendelian, we may reasonably assume that now a pair of autosomes has become decisive in sex-determination. The new sex balance was less firmly established than the normal.” Similarly XY females arose occasionally as a result of the accumulation of female-determining genes in the autosomes that could overcome the strong sex-determining gene in the Y-chromosome. Unlike the situation in man and Drosophila, YY individuals are viable and fertile, thus

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confirming that the genetic difference between X and Y is slight. This interpretation of sex determination was criticized by Goldschmidt (1937). But later work by Winge and Ditlevsen (1947) confirmed the original explanation (Winge, 1934). Similar exceptional individuals with regard to sex chromosomes have been found in nature. Gordon (1946, 1947) observed XY males and X X females in the wild stocks of the platyfish, Platypoecilus maculatus from Mexico. Those from rivers in British Honduras, on the other hand, were WZ females and ZZ males. The chromosomes W and Y have several common characteristics. Such exceptional chromosomal types have also been found in experimental stocks of the platyfish (Breider, 1942; Bellamy and Queal, 1951). In Aplocheilus latipes, Aida (1921, 1936) reported XY females and X X as well as YY males. In the medaka, Oryxias latipes the diploid chromosome number is 48 with a Q X X - 8 X Y system of sex determination. There is no significant morphological difference between the X- and Y-chromosomes. It has been possible to obtain XY females and X X males in breeding stocks. It is considered that the primary sexual characteristics are determined by genes located throughout the autosomes and that these genes are activated by stimulating genes whose quantity determined the sex. Sexreversal has been demonstrated by incorporating hormonelike substances in the diet (Yamamoto, 1953, 1959a,b). The occurrences of such sex chromosomal exceptions are considered by some as an indication of a primitive system of sex determination. Differentiation of sex chromosomes is unknown in most Amphibia and fishes while it is clearly seen in the higher vertebrates. No sex chromosomes are recognizable in the males of the elasmobranch fishes (Matthey, 1937; Makino, 1937) or in other teleosts (Makino, 1934; Prokofyeva, 1934). Earlier accounts of morphologically distinguishable X- and Ychromosomes in the Anura (Witschi, 1924,1933) are considered erroneous (see Makino, 1932; Saez et al., 1935, 1936). VI. Mouse

+

The mouse has a diploid number of 38 XY chromosomes in males and 38 X X in females. The sex-determination pattern is similar to man, the Y being the male determiner (Welshons and Russell, 1959). It has been shown that XO mice are females and fertile (Welshons and Russell, 1959; McLaren, 1960; Cattanach, 1961a and b; Kindred, 1961). Their modal chromosome number is 39 as found in the bone marrow cells. The existence of sex chromosome constitutions XO, XX, and XY is further confirmed in experiments using X-linked genes (Russell et al., 1959).

+

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K . R . DRONAMRAJU

Cattanach (196la,b) made several crosses of XO females with X Y males. Four classes of offspring were expected theoretically in equal numbers, XX, XY, XO, and OY, out of which X X and X Y actually occurred in equal numbers, XO was represented by only about one-third of the expected number, and OY is presumed inviable. The low frequency of the XO class was explained as being due to a preferential loss of chromosome sets lacking an X to the polar bodies during the meiotic divisions of the egg (Cattanach, 1961a,b). After birth, however, he found that the XO and X X females had equal viability. Russell (1961, 1962) pointed out that among XO females those having a maternally derived X-chromosome were more frequent. Experiments for the induction of abnormal sex chromosome constitutions in the mouse have been summarized by Russell (1962). X X Y mice are normal males in size and general appearance, but are sterile. Their chromosomal constitution was identified on cytologic and genetic evidence (Russell and Chu, 1961; Cattanach, 1961a,b; Kindred, 1961). RIcLaren (1960), however, reported an X X Y mouse before, but this male, which was stunted, died a t the age of 25 days. X X Y males have normal external genitalia and the testes have tubules but no spermatogenic cells (Cattanach, 196la,b). The Y-chromosome of the mouse is strongly male determining. Female determining factors may be aulosomal and/or X-linked (see review by Beatty, 1964). Histocompatibility antigen. Eichwald and Silmser (1955) postulated a histocompatibility gene located in the Y-chromosome of certain strains of mice which is responsible for the rejection of all male skin grafts by female recipients. However, transplants between two females, or between two males, or from female to male are always accepted. Fox (1956, 1958) pointed out that the antigen production could be a consequence of the difference in the number of X-chromosomes, two in female and one in male, and not to the presence or absence of the Y-chromosome. It may be recalled t ha t Fox (1958) reported some evidence in support of a similar hypothesis in Drosophila melanogaster. In mice, however, further work in recent years has conclusively established that the determination of male antigen is clearly due to the Y-linked histocompatibility system and not to a result of the single X-chromosome in males. Hauschka and Holdridge (1962) obtained immunological as well as cytogenetical evidence in support of the Y-linked hypothesis. I n immunogenetic tests, using female mice previously immunized with male tissue, certain unexpected rejections of female grafts by females occurred. Since technical causes were eliminated in the procedure they postulated that a piece of the antigenic Y-chromosomal material, after translocation to

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either an autosome or an X-chromosome, “contaminated” the female karyotype. I n order to test this hypothesis, such (yellow) females were first classified, by their response to male grafts as being either malecompatible (RTC) or male-incompatible (MI). It was thought that if the male antigen were entirely confined to the Y-chromosome, all female to female grafts within t,hat strain should be successful. On the other hand, if the RlC females coritairied the translocated Y antigen their skin should be rejected by RII feniales because the latter did not have the Y antigen. The experimental results supported the Y-translocation hypothesis. This was further confirmed by the highly successful compatibility shown by FI females (from the cross male compatible C3Hf or DBA/B females X C57BL males) to C57BL male skin grafts as these F1 females inherited the Y translocation from their C3Hf or DBA/2 mothers. Later tests were designed to distinguish between the two possibilities, one, that the translocation involves an X-chromosome, and, the other, that it involves a n autosome of the female. The E’I hybrid males from the cross C 5 7 B L 8 X DBA/2 0 were back-crossed to the C57BL females, all of which carried the X-chromosome of DBA/B grandmother, but only half carried her autosomes. When these daughters were grafted with C57BL 8 skin about half of them (actually 450/,), as would be expected if an autosome were involved, accepted the graft. Thus the data supported the hypothesis (I that antigenic Y substance has contaminated a DBA/2 autosome.” Cytologic and growth comparisons of sarconias were also studied. Two sarcomas were induced bilaterally in the same C57BL male arid then transplanled to isologous mice of both scxes. Sarcoma B P 8 7 B had a stem-cell karyotype with 41 chromosomes including the Y in most of the neoplastic cells and hence this tumor grew very poorly in females. Sarcoma B P 3 7 A had a stem-cell karyotype of 39 chromosomes with no Y. Accordingly sarcoma B P 3 7 A grew equally well in both male and female C57BL mice. Hauschka and Holdridge (1962) thought that a very small translocation which was not cytologically detectable was probably involved. The cells of male-compatible females did not seem to have sufficicnt Y antigen for the experimental induction of male tolerance (see Billingham arid Silvers, 1960). Billinghani and Silvers (1958) demonstrated the absence of allelism in the Y factor by inducing male tolerance in certain females that were injected as infants with adult male tissue from several strains. Regardless of the donor strain used, all Ihe trcated females became tolerant of isologous male skin. Celada and Welshons (1963) studied mice having exceptional chromosomal constitutions, X O and XXY, using a quantitative method based on the detection of transplanted spleen cells in irradiated recipients pre-

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viously sensitized against the donor cells (Celada and Makinodan, 1961). The quantitative assay method is based upon a secondary antibody response by preimmunized recipients. Spleen cells that were presumed to be carrying a male antigen were injected to pretreat recipient females. After 10 days, the recipients were irradiated and injected with spleen cells from isologous male donors. These spleen cells served the purpose of a secondary stimulus to the recipients which may have already been preimmunized. If the recipients had shown an immunological reaction to the primary and secondary stimuli the spleen cell transplant would have failed, and if not, the donor cells would be successfully transplanted into the irradiated hosts. The assessment of the success or failure of transplanted cells was made by their ability to produce antibody against rat erythrocytes t o which they had been sensitized prior to transplantation. Their results from experiments using X O and XX females showed that XO’s behave antigenically like XX females, and that the titer differences were significant between controls and for both X Y and X X Y males which, in turn, did not differ from each other. As anticipated, no difference was found between male and female recipients that did not undergo pretreatment. I n another experiment it, was shown that male antigens cross-react extensively and those of a male with a BL-Y appeared identical to th a t of a male with a C3H-Y. No strain-specific antigens were found. I n order to test whether the determination of male antigen is dependent upon the presence of male germinal or interstitial cells, 10-day-old BL/10 males were orchidectomized and roughly 3 months later used for the preimmunization of isologous females. Tha t the differences in titer between recipients pretreated with normal control males and orchidectomized males are not statistically significant, supports the hypothesis that the determination of Y-linked antigen is not dependent upon the presence of male germinal and interstitial cells (Celada and Welshons, 1963). Haldane (1956) pointed out that a histocompatibility gene located on the X-chromosome could give rise to rejection of z1 skin graft from a mouse of an inbred strain by parts of its male F1 progeny. Eichwald and associates (1958) tested this hypothesis but found no strain-specific X-linked histocompatibility genes. VII. Cat

The diploid chromosome number of the cat is 38 including a n X Y pair in males. The X-chromosome is three or four times the length of the Y (Komai, 1952; Komai and Ishihara, 1956). Komai reported that the X-chromosome has the kinetochore and gene loci in its pairing segment

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and that the differential segment contains female-determining factors. The Y-chromosome is supposed to have one pairing segment (containing the kinetochore) and two differential segments, one carrying factors for maleness corresponding to the differential segment of the X carrying female determiners, and the other (at the other end of the chromosome) containing the male fertility complex. Tortoise-shell male cats are reported to have resulted from a Ychromosome crossing over with the pairing segment of the X, which contains the Orange gene, in such a way th at the segment for maleness is incorporated but the fertility segment is lost. The fertilization of such a gamete by a n egg containing a normal X-chromosome carrying the wild type instead of the 0 gene results in a sterile tortoise-shell male. The probability of this event is, however, small. Only sixty-five tortoise-shell male cats were reported in Japan, out of which three are fertile. Such fertile males are due to the still rarer double crossing-over which incorporates the 0 gene and the fertility complex as well. Sterile tortoise-shell males have small, firm testes and reduced spermatogonial development. Ishihara (1956) reported that their origin cannot have been due to nondisjunction (as for Klinefelter’s syndrome in man) because the karyotype is 2A XY. However, recent work by Thuline and Norby (1961) suggests that the X X Y karyotype might exist among the tortoise-shell cats. Two out of ten tortoise-shell males examined had a modal chromosome number of 39 and their buccal mucosal smears showed the normal female type of sex chromatin. The other ten showed the normal male pattern. One of the two X X Y males had a normal male phallus and normal scrotum containing descended testes, but no spermatids and spermatozoa; and the other also had a normal male phallus but the scrotum was undeveloped and no testes could be palpated or located in the inguinal canals. No gonadal or other internal reproductive structures were found. This report, as stated by the authors, is only preliminary and further confirmation giving more detailed information about the karyotype is required. The organization of fertility complexes and male-determining factors on the Y-chromosome of the cat has a close parallel in the plant Meland r i u m which is discussed along with other plants in the next section.

+

VIII. Plants

Many dioecious plants have no distinct sex chromosomes. There is no a priori reason to suppose that heteromorphic sex chromosomes should be of widespread occurrence among dioecious plants where dioecism is in most cases of recent origin (Westergaard, 1958). Most of these plants have

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very small chromosomes and the recognition of heteromorphic pairs is not always convincing. A. ANGIOSPERMS Sex chromosomes in higher plants were first reported by Blackburn (1923), and Winge (1923a) in AJelandrium, Humulus, etc.; by Kihara and Ono (1923) in Rumex acetosa; and by Santos (1923) in Elodea. Blackburn (1923) investigated the genus Lychnis, one species of which, Melandrium rubrum Garcke (L. dioica L.), was previously studied b y Strasburger. I n the closely related L. alba Mill., she reported that out of the twenty-four somatic chromosomes, two are larger than the rest. I n the female these two appear similar, but in the male the two large chromosomes differ from one another both in size and shape; “the larger one is bent, somewhat in the shape of a hockey stick, with the curved end pointing outwards from the spindle, whilst the smaller somewhat pear shaped one is not more than two-thirds its size . . . we have here a definite case of an XY pair of chromosomes in the male with a corresponding XX in the female. This is the first definite record of sex chromosomes in a Dicotyledon.” Westergaard compiled a list of species in which heteromorphic chromosomes were convincingly reported on the basis of the three following criteria: (a) demonstration of an unequal pair (XY) during the meiotic cycle of the heterogametic sex; (b) the absence of such an unequal pair in the homogametic sex; and (c) the identification of the sex chromosomes in the somatic cells of both sexes. The subject of sex chromosomes and sex determination in flowering plants has been reviewed by many authors (Correns, 1928; Darlington, 1937; Allen, 1940; Iiuhn, 1942; Lewis, 1942; Westergaard, 1958). According to Westergaard (1958) the least convincing are the reports of Billings (1932, 1934) concerning the sex chromosomes of Loranthaceae and Atriplex. Allen (1940) listed the dioecious plants with no heteromorphic sex chromosomes. To this list should be added Sedum rosea (Uhl, 1952), and Empetrum nigrum, and E. hermaphroditum (Westergaard, 1940, 1958). 1 . Y-Linked Genes in Plants

Winge (1927) first reported Chlorina gene in Afelandrium to be Y-linked on the basis of the following points: (a) Chlorina plants were

Y-CHROMOSOME FUNCTION

2T7

always male, and (b) when Chlorina males were backcrossed with their mothers, no Chlorina females were obtained. However, his later work showed that the gene for Chlorina is X-linked, and is lethal in the homozygous state (XX) (Winge, 1931). This explains the absence of Chlorina females. Winge (1931) then suggested that, “It is better, therefore to call the character in question aurea, as it is well known that aurea types are coninionly lethal in double dose” (Winge, 1930). Winge (1931) reported two other genes, one of which is located in the Y-chromosome arid the other found in both X and Y. a. Variegated. All true variegated plants are female because an inhibitor of variegated is carried in the Y-chromosome. Winge reported sex determination in about 1400 variegated plants, evidence which confirms the Y-linked nature of the inhibitor. In a few males it has been possible to detect a very weak variegation. The intensity of variegation varies but, in Winge’s work, there was a gap bctweeii the least variegated females and the most variegated males. Fourteen inales out of a total of 271.5 were included in the variegated group. b. Abnormal. Abnormal (n) is a recessive gene to be found either in the X-chromosome or in the Y, the allelomorph ( N ) giving normal plants. The abnormal type was originally segregated from a cross between M . album and ,II. rubrum, in the 172 generation and also appeared in two backcrosses between l g l plants and a 11.album female. All possible types have been produced, X,X,, X,Y,, X,X,V, X,YN, XNY,, XNXN, and XNYN. The following segregations can be produced in the intercrosses; (1) normal males and abnornial females, (2) abnormal inales arid normal females, (3) all abnormal, and (4) all normal. Abnormal plants are characterized by pale coloring of the upper part of the plant including the calyx, thc stems, and the leaves. The calyx never opens, and the petals are entirely hidden, even when the plants are in full bloom. Abnormal plants are less viable than normal and furthermore, male plants are often in smaller numbers than females. 2. Sex Determination

a. Il4elandtium. The most extensively studied plant for the understanding of the sex determination mechanism is Afelandrium. Comprehensive studies of natural diploid strains were made by the early geneticists, Correns, Baur, G. and P. Hertwig, Shull, and Winge. The first successful results of cihromosome doubling in this species were published by Westergaard (1938), and Orlo (1939a,b, 1940a,b). The diploid hermaphrodites show a great deal of variation in sex expression. Correns proposed the name “androhermaphrodites” for plants which give bisexual flowers first and later pure males, arid “euhermaphro-

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dites," with all the flowers bisexual. Some of the hermaphrodites give female, hermaphrodite, and some male offspring. Others give only females and hermaphrodites. Winge discovered a hermaphrodite type which bred true and a balanced lethal system was proposed to explain this. All the diploid hermaphrodites are considered to be heterogametic. Westergaard (1946a,b) and Warmke (1946a,b) made extensive studies of the polyploid Melandrium. i. Euploid autosome sets. A comparison between Melandrium and Drosophila in this respect was made by Westergaard (1958). I n Drosophila the sex expression is independent of the presence or absence of the Ychromosome. I n Melandrium the influence of one Y-chromosome is strong enough to suppress the female potencies of three X-chromosomes and four autosomal sets. However, the presence of one more X-chromosome (XXXXY) results in bisexual and male flowers (Table 4). They are TABLE 4 The Relation between Chromosome Constitution and Sex in Melandrium and Drosophila*

Chromosome constitution 1. 2. 3. 4. 5. 6. 7.

2A 2A 3A 3A 3A 4A 4A

8.4A 9.4A 10. 11. 12. 13. 14. 15. 16.

2A 2A 2A 2A 3A 3A 3A 17. 4A 18. 4A 19. 4A 20. 4A 21.4A 22.'M 23. 4A

XX XXX X XX XXX XX XXX

xxxx xxxxx

Melandrium

0 0

-

P 0 0 0 0

0

XY XYY XXY XXYY XY XXY XXXY XY XXY XXYY XXXY XXXYY XXXXY XXXXYY

* From Westergaard (1958).

Drosophila

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mostly bisexual in Warmke’s strains and mostly pure males or only slightly hermaphroditic in Westergaard’s strains. Warmke found that two Y-chromosomes (XXXXYY) almost double the male effect. TWO Y-chromosomes balanced the effect of 4 X-chromosomes and gave a majority of male plants. Warmke used the ratio of X /Y chromosome numbers as a scale for measuring changes from complete male to hermaphroditic types. Out of about two hundred plants studied by Warmke, all with a Y (XY, XXY, XXXY) were males and all without the Y (XX, XX X , X X X X ) were females. From these data he concluded th a t the autosomes are unimportant in the mechanism of sex determination. Westergaard’s (1940,1946b) studies with European strains of Melandrium gave results which are in general agreement with those of Warmke, showing that the Y-chromosome plays a decisive role in sex determination while the autosomes are unimportant in the series having X X Y 2, 3, or 4 autosomal sets. However, as already mentioned, the Y-chromosome in the strain used by Westergaard appears to have elements of greater male sex potential than the one used by Warmke. ii. Possible role of autosomes. In crosses involving aneuploid types, Westergaard obtained male, female, and hermaphrodite offspring. I n a cross (3 A X XX) X ( 3 A XXY), he obtained ten hermaphrodite plants, twenty-one females and fifteen males. When the offspring of these hermaphrodites were studied for several generations it became clear that their sex expression is influenced by combinations of X-chromosomes and certain autosomes which counterbalanced the female-suppressing action (to be explained later) of the Y-chromosome. When the X-chromosomes were increased from one to four the hermaphrodites increased from 0 to 100% in the presencc of the Y-chromosome, while in euploids they would all be males. Out of 205 aneuploid (XXXY) plants studied, 133 were hermaphrodites and only 72 were males. This variation can only be ascribed to autosomes. Gowen (1961) quantized the effects of sex chromosomes and autosomes in JIeZandrium. He assigned a value of 1 for the male, 2 for the hermaphrodite, and 3 for the female. When the types are mixed, as in a male with a few blossoms, a certain value of 1.05, or 1.10, depending on the number of blossoms, is assigned. Gowen applied his rule to Westergaard’s (1948) dat,a on aneuploids. Analysis by least square method shows that the sex type may be predicted from the equation:

+

+

Sex type

+

=

1.37 Y

+ 0.10 X + 0.01 A + 2.34

Gowen concludes that the Y-chromosome has a strong effect toward maleness, the X-chromosomes are next in importance, with each X having

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only one-thirteenth the effect of Y, in the direction of femaleness, and the autosomes have one-tenth the effect of X toward femaleness. The equation for Westergaard’s data of 1953 for euploids (assigning the value of 1.5 to 4 A XXXXY):

+

Sex value

=

-1.29 Y

+ 0.10 X - 0.01 (autosome sets) + 2.53

Here the autosomes are treated as sets since they are direct multiples of each other, so the value of the individual autosome is one-eleventh the value given in the equation. The result shows that the Y-chromosome has slightly less effect toward maleness than in the aneuploids; the X-chromosome’s effect has not changed but there has been a slight shift toward autosomal effects on sex. I n additional data from Westergaard’s (1948) paper information on counts made for proportions of aneuploids which are male to those which are hermaphroditic was published. These include plants with one Y and one X (all males), those which have either one, two, or three Y-chromosomes balanced by two X-chromosomes (89% males), some with X X X Y or XXXYY (36% males), and others with X X X X Y or X X X X Y Y (no males). These data are of special interest because the sex differences can be measured on an independent quantitative scale. The equation for these data is Percentage of males = 13.2 Y - 36.4 X 134.4

+

These results show that the Y-chromosome increases the proportion of males, and the X increases the proportion of females. Warmke’s (1946a) data give the numbers of X- and Y-chromosomes found within the plants but none of autosomes (considered as 2, 3, and 4 genomes). For these data, Sex type = -1.05 Y

+ 0.22 X - 0.04 A + 2.25

The A effect is now in terms of the diploid type equaling 2, the triploid 3, and the tetraploid 4. From these studies the following conclusions may be drawn. The Ychromosome is strongly male determining, with slightly greater effect in Westergaard’s data than in Warmke’s. The X-chromosomes in Warmke’s data have nearly twice the female influence of Westergaard’s. The autosomal values are small for both. iii. Plants with fragmented Y-chromosomes. The location of the sex determiners has been studied by both investigators by making use of deleted Y-chromosomes (Warmke, 1946a,b; Westergaard, 1946b). Plants with fragmented Y-chromosomes may arise in diploid strains. Akerlund’s

281

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+

(1927) description of a hermaphrodite with the AA XX constitution was interpreted by Westergaard (1946b) as AA XY, in which part of the Y has been deleted because a fragmented Y-chromosome has the same size as a normal X-chromosome (Westergaard, 1940). Westergaard (1953, 1958) gives an interesting account of fragmented Y-chromosomes, which may be of two kinds. I n the first, designated Y’, the distal part of the differential segment is missing. The Y’-chromosome is slightly longer than the X. The length of the lost fragment measures slightly less than two-thirds of the differential arm. Such plants have both male and female sex organs which are normal and all the flowers are bisexual (named “euhermaphrodites” by Correns). On crossing to normal females or on selfing, such plants gave females and hermaphrodites, but never males. On crossing to normal males, they gave females with normal X-chromosomes, hermaphrodites with the fragmented Y-chromosome (Y1-type) and one or more X-chromosomes, and normal males that had a normal Y-chromosome, one or more X-chromosomes, and probably a fragmented Y-chromosome also. From such evidence, Westergaard concluded that Y ‘-type lacks the segment which suppresses the formation of female sex organs. The second type, designated Y3, lacks the pairing segment and also part of the differential segment. This chromosome is longer than Y’. Plants having Y3 but no normal Y-chromosome are male sterile. The pollen mother cells go through a normal meiosis and then degenerate. Viable pollen is never formed. The first plant obtained by Westergaard was slightly intersexual and could be propagated by crossing to normal males. The same type was reported by Warmke and Davidson (1944) in American strains of Melandrium. It should be noted th a t plants with one or more fragmented Y3-chromosomes are always male sterile but a s the female suppressor is still present, the female potency of the zygote is not released. Three different regions have been identified so far. “If the distal part is absent (the Y’-type), a normal bisexual plant results. Therefore the function of this segment must be to suppress the formation of the female sex organs. If part of the other arm is lost (the Y3-type) a sterile male results. Hence this segment must include genes (or a gene) which control the last stages in anther development. If the whole Y-chromosome is absent (in XX-plants), a female plant is the result. Hence the middle region of the Y-chromosome must include genes (or a gene) which control the initiation of anther formation” (Westergaard, 1958). Thus Westergaard believes that the Y-chromosome in Melandrium determines the sex through complete linkage between female-suppressing genes and genes which initiate and complete anther development.

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K. R . DRONAMRAJU

The sex-determining mechanism in Melandrium may be summarized in the following points: (1) A trigger mechanism is built up by a n absolute (Y-) linkage between a female-suppressor region and at least two blocks of essential male sex genes. (2) The X-chromosomes and certain autosomes have female potencies which can be demonstrated only when they react with the trigger mechanism in polyploids and aneuploids. Whether the female potencies in the autosomes and X-chromosomes are controlled by major sex genes or blocks of modifying genes is not known at present. Westergaard regards the latter possibility as more likely. An interesting observation is that the female plants infected with the smut fungus Ustilago violacea develop anthers! It is probable that the fungus provides some essential sex hormone which is normally controlled by the anther-initiating gene in the Y-chromosome. b. Humulus. Winge (1923a) identified the heteromorphic sex chromosomes in the two species, H . japonicus, and H . lupulus but reported a t that time a simple X-Y mechanism. Later, Kihara (1928, 1929a,b) found an XYlYz mechanism in H . japonicus. H. lupulus has a n X Y mechanism in which the X is definitely longer than Y. In H . japonicus the three sex chromosomes are longer than the autosomes and the Y-chromosomes are heterochromatic for the greater part of their length (Jacobsen, 1957). I n an earlier investigat,ion Dark (1952) failed to observe the sex chromosomes but Jacobsen’s work leaves no doubt as to their existence. According to Jacobsen (1957) the Y-chromosomes of H u m u l u s are heterochromatic and probably partly inert. The data concerning the sexdetermination mechanism are fragmentary. Triploid strains of commercial hops include many bisexual types which also occur in some of the diploid parental strains. I n H . japonicus, Ono (1940~)obtained triploids from crosses between diploid and colchicine-induced tetraploids and these triploids comprise females with twenty-two chromosomes and monoecious forms with twenty-five chromosomes. Based on Ono’s work, Westergaard (1958) suggests that the Y-chromosome in H . japonicus has no significant role in sex determination which may be of Drosophila type. c. Rumex. Meurman (1925) and Kihara (1925, 1927) failed to distinguish the heteromorphic sex chromosomes in hexaploid R u m e x acetosella. Later work by Love (1943) has convincingly demonstrated them in the diploid and tetraploid types. Love and Sarkar (1956) identified the sex chromosomes in the somatic plates of R. paucifolius. The species R u m e x acetosa has a diploid complement of fourteen chromosomes including one pair of X-chromosomes, and six pairs of

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autosomes in the female. The male has one X-chromosome, two Y-chromosomes, and six pairs of autosomes. Intersexes may have two X-chromosomes, two Y-chromosomes, and three sets of autosomes (Kihara and Ono, 1923; Kihara, 1925; Ono, 1930,1935; Kihara and Yamamoto, 1935). Ono (1935) and Yamamoto (1938) studied polyploids and trisomics of R. acetosa. Yamamoto showed that the six pairs of autosomes were not equally balanced in their potency toward male sex. The chromosomes called al, a4, and as, had net effects toward maleness, while pairs az and as had similar effects toward femaleness. Gowen (1961) applied his equation to Yamamoto’s data and found that the X-chromosomes contribute a strong female influence and each Y a less effective male influence. The chromosomes a2,a3, and a5 have their sex genes almost in balance, but al, a4, and as are somewhat more potent toward maleness than the Y-chromosomes. I n R. paucifolius, Love and Sarlcar (1956) studied tetraploids with twenty-eight chromosomes. The sex chromosomes were suggested t o be X X X X and X XXY, the male being heterogametic. The Y was the smallest chromosome in the complement. They concluded th a t the Y in this species has strongly epistatic male determinants. In Rumex subgenus acetosella, Love (1957) identified five species out of which two are diploid, one tetraploid, one hexaploid, and one octoploid. The diploid species have the complements X X Q and X Y 3. The natural tetraploid R. tenuifolius has X X X X and XX X Y in the females and males, respectively. Hexaploids, derived by alloploidy from the diploid R. angiocarpus and the tetraploid R. tenuifolius have 6 X-chromosomes in the female and 5 X Y in the male. The octoploid R. graminifolius (an autotetraploid of R. tenuifolius) has 8 X-chromosomes in the female and 7 X Y in the male. The diploid number is 57 or 58 and slightly intersexual individuals occur in this group. From such studies Love (1957) concluded that the sex mechanism in this group is based on the evolution of a strong male determinant in the Y-chromosome, similar to the Y in Melandrium, but stronger. Thus, within the genus Rumex, some species seem to have the male determinants in the autosomes, and some others in the Y-chromosomes. There is more variation in the male-determining capacity of Y-chromosomes than in the female-determining capacity of X-chromosomes. d . Acnida. Murray (1940a,b) studied crosses between dioecious Acnida and monoecious Amaranthus. There is only one male flower in each flower cluster in A. retro$exus, A. hybridus, A . caudatus, and A. powelii. This group is known as the first type. I n A . spinosus, on the other hand, all the flowers in one cluster are of the same sex, but the clusters of staminate flowers are placed terminally on the main axes and on the

+

+

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K. R. DRONAMRAJU

lateral branches, whereas the female flowers develop only on the axes of the branches and on the base of the terminal inflorescence. This species is known as the second type. Hybrids between Acnida species and “first type” monoecious species agree with the assumption that the dioecious species has male heterogamety. Crosses between Acnida and the “second type” produced almost 100% male plants and a few monoecious plants. This is explained by assuming that the male sex genes of the monoecious parent are epistatic to the female sex genes of the dioecious species, while the male sex genes of the dioecious species are epistatic to the female genes of the monoecious parent. Evidence from polyploids indicates that X X X Y plants are males. In crosses between 4 X female and 4 n XXYY male, 131 Q Q and 1633 88 were obtained (Murray, 1940a,b).In the cross 4 X Q X XXYY 8,a preponderance of X XXY plants were always produced because X Y gametes were formed in excess. The cross 4 A 4X Q X 4A X X X Y 8 gave equal proportions of males and females. These results indicate that the Y-chromosome plays a decisive role in sex determination. e. Bryonia. Correns’ classical work on Bryonia has recently been confirmed and extended by Heilbronn (1949, 1953), and Heilbronn and Bagarman (1942). Crosses between dioecious and monoecious species gave males and females, in ratios which were in agreement with male heteroganiety of the dioecious species. Monoecious B. macrostylis and the dioecious B. multiflora were used. Crosses between two dioecious species (dioica Q X multiJlora 8 )gave monoecious offspring, and crosses between two monoecious species (alba Q X macrostylis 8)gave dioecious offspring. I n Ecballium eluteriurn,the populations consist of a monoecious species (var. monoicum)which occurs only in northern Spain, while dioecious var. dioicum occurs in southern Spain. Intravarietal crosses, intercrosses, and backcrosses have been successfully studied (Galfin, 1951). In Thalictrum, Kuhn (1930) crossed the dioecious T . fendleri with three hermaphrodite species. The results show t,hat the T. fendleri females are homogametic. Subandroecious plants of Thalictrum give females and more-or-less subandroecious males in the ratio 1 Q : 3 8 8.When the males are crossed to normal females, two-thirds of the males give Q Q and 8 8 in equal proportion; one-third, however, give unisexual families of male plants only. This is explained by assuming that the male sex is heterogametic and that both males and subandroecious hermaphrodites have the X Y constitution. On selfing, XY plants give 1 X X , 2 XY, 1 YY. The YY males when crossed to X X females, produce all male offspring, Based on these and other results, Westergaard (1958) suggests th a t the Y-chromosomes in all these dioecious species contain sex-deciding

+

+

285

Y-CHROMOSOME FUNCTION

genes which by some mechanism suppress the manifestation of female sex potentials. Further, the X-chromosome of Bryonia contains sex-deciding genes which prevent the formation of the male organs and, the Ecballium X-chromosome plays no role in sex determination. f. Asparagus. The plants of A . oficinalis are ordinarily staminate or pistillate but occasional rudimentary organs of the opposite sex might appear. The male and female plants occur in approximately equal numbers. Rudimentary organs of the male sex sometimes develop and set seed. The mechanism of sex determination appears to be similar to Thalictrum (see Rick and Hanna, 1943; Sneep, 1953; Westergaard, 1958). g . Mercurialis. Unlike Thalictrum and Asparagus, the YY males in Mercurialis differ from normal X Y males. The former produce very little pollen. Such males give 100% X Y normal male progeny in crosses with females. No male or subandroecious offspring are produced by the subgynoecious plants. Analysis of the progeny of hermaphrodite plants suggests that the male sex is heterogametic and that YY types are viable (see Gabe, 1939; Kuhn, 1939). h. Xilene otites. The early work of Correns (1928) and Sansome (1938) suggested female heterogamety in S. otites. However, Warmke’s (1942) polyploid method showed that the male sex is heterogametic. Using artificially induced tetraploids, 4 A 4 X and 4 A XXYY, Warmke studied 4 X Q X X Y 3 cross which is expected to give equal proportions of XXX females and X X Y males (or hermaphrodites) if the male sex is heterogametic. He obtained 50% 9 Q and 50% 3 3 in actual experiments, showing that the male sex is definitely heterogametic. Westergaard suggests that some species of Silene may have female heterogamety and other closely related species male heterogamety. This possibility was earlier mentioned by Sansome (1938) who considered 8. otites to have female heterogamety, and the closely related S. pseudootites male heterogamety. Further investigations are needed. i. Valerianu dioica. Correns (1928) reported th a t subandroecious plants give only male offspring and that the proportion of males is higher in natural populations. Moewus (1950) showed that the sex ratio may vary greatly between diff went populations but assumes male heterogamety. Further work is needed. j. Cannabis. Hirata (1924, 1929) was the first to describe the sex chromosomes of Cannabis sativa but McPhee (1924) failed to recognize them. According to Westergaard (1958) , Hoffmann’s (1952) photomicrographs are not too convincing. Before him, however, Yamada (1943) clearly identified the X and Y in somatic plates and reported that the Y is larger than X. A great variety of sex forms exists in the cultivated dioecious strains.

+

+

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K. R. DRONAMRAJU

Sex expression is influenced by environment and the sex ratio may vary considerably from one strain to another (see Hoffmann, 1947, 1952; von Sengbusch, 1952; Huhnke et al., 1950; Neuer and von Sengbusch, 1943). According to both von Sengbusch (1952) and Hoffmann (1947,1952), dioecious hemp has normal male heterogamety. However, they differ in their interpretation of monoecious (or “uniform”) varieties which, according to von Sengbusch, have the constitution XX, the variation in sex expression being due to the heterozygosity, of sex genes in the X-chromosomes and in the autosomes. According to Hoffmann, the monoecious strains may be comprised of all the three sex types, XX, XY, and YY, and the function of sex determination is taken over by the autosomes. Hoffmann reported heteromorphic XY-bivalents in both males and monoecious plants of both varieties in cytological preparations. He was not able to identify the XX or X Y types by studying meiosis. He has not studied the somatic chromosomes of the various sex types. Thus the evidence available is insufficient a t the present stage to reach definite conclusions. k. Spinacea. Spinacea oleracea has six chromosome pairs. The X- and Y-chromosomes are cytologically distinguishable. Recent studies on sexdetermining mechanisms have been made by Bemis and Wilson (1953) and Janicli et al. (1959). The Y-chromosome bears a satellite which is absent in the X. Janick and Ellis (1959) located the sex chromosome pair by making use of some primary trisomics. Their work is in general agreement with that of Zoschke (1956) and of Dressler (1958) confirming th a t the morphology of the chromosomes containing the X Y factors varies between races. Janick and Stevenson (1955) found that in polyploids a single Y-chromosome is male determining even when it is opposed by three doses of the X-chromosome. The XX, XXX, and XXXX plants formed pistillate flowers while the XY, XXY, X X X Y , and XX Y Y plants were of the staminate type. The fullest expression of male determiners is seen in plants with two Y-chromosomes as these never set seed while X Y progenies sometimes do. 1. Carica papaya. Much of the breeding work on the papaya was done by Hofmeyr in South Africa and Storey in Hawaii. There are mainly three sex types; males which occasionally produce female or hermaphrodite flowers, stable females, and hermaphrodites which may be female sterile in certain times of the year (Storey, 1953). Meurman (1925), Suguira (1927), Lindsay (1930), Hofmeyr (1938), and Storey (1941) reported that they could discern no heteromorphism in chromosome pairs either among somatic chromosomes or among chromosomes in various stages of meiosis. Kumar et al. (1945) have

Y-CHROMOSOME FUNCTION

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reported that one pair of chroinosoines separates precociously at anaphase I of meiosis in males and hermaphrodites. They observed no such precocious anaphasic separation in meiosis in the female, and have remarked that the difference in behavior is in agreement with what has been observed in other species of plants which have heteromorphic sex chromosomes. They failed, however, to detect morphological differences between disjoining homologs. Storey (1953) confirms this but states that such precocious observation was not observed in every cell at anaphase. The analysis of crosses between different sex types has been reported (Hofmeyr, 1938,1938a,b1 1945, 1953; Storey, 1953). The YY combination is usually lethal and plants without an X are inviable. According to Westergaard (1958) it is not possible to locate the sex-determining genes without further data. Both Hofmeyr and Storey proposed a factorial explanation of the sex-determining mechanism. According to this hypothesis, a female has the constitution mm, a male Mlm, and a hermaphrodite Mzm, the three genes illl, M 2 , and m being allelic. The conibiriations M1M1, M 2 M 2 ,and M l M 2 are inviable. Storey (1953) and Westergaard (1958) have, however, doubted whether such a simple formula can explain how such a dioecious system has originated from bisexual ancestors during the course of evolution. m. Vitis. Very extensive studies on sex determination in grapes have been made by Breider and Scheu (1938), Oberle (1938), Negrul (1936), and Loomis et al. (1954). Most wild species are probably dioecious but hermaphroditic strains have been selected for breeding purposes. There are three types of hermaphrodites: those which breed true to type, others segregating females and hermaphrodites in a 3 : 1 ratio, and some others which give 9 :3 : 4 ratios. In the third type, a t least one pair of autosomal sex genes probably interact with the sex-chromosomal genes. The Y-chromosome must contain the sex-deciding genes but further work on a quantitative basis is needed (Weshergaard, 1958). n. Coccinea indica. Kumar and Vishveshwaraiah (1952) have demonstrated a large Y-chromosome in t,he somatic cells of males but the X-chromosome has not been identified. The Y-chromosome may be male determining but further evidence is required in support of this assumption.

B. BRYOPHYTES I n most Bryophytes, the gametophytes are two kinds of haploids, one female (XA) and the other male (YA). Allen (1917) described the large X and the small Y of Sphaerocarpus donnellii, in which the number of autosomes is seven. Distinguishable sex chromosomes have since been identified in several other Bryophytes (Allen, 1936; see Darlington, 1937).

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Mackay and Allen (1936) found X : 2 A and 2 X : 2 A female gametophytes and 2 Y : 2 A male gametophytes in Sphaerocarpus. But gametophytes with the X Y :2 A constitution were found to be intersexes. Knapp (1936) suggested that the male-determining genes were largely in the autosomes and that the Y was neutral. IX. General Discussion

I n reviewing the importance of the Y-chromosome in various organisms one is struck by the diversity of functions performed by the Ychromosomal material. It is obvious that so many new functions of the Y-chromosome have gradually been discovered in recent years that no apology need be made for having attempted to review this subject. It seemed quite appropriate fifty years ago to suggest th a t the Y-chromosome contained degenerate genes, or no genes a t all (Muller, 1914a,b). It should be noted that even a t that time it was not regarded as completely inert (Muller, 1964), a fact which has unfortunately been overlooked by many later investigators. The idea that the Y-chromosome was largely inert became established for several reasons. I n Drosophila (and in man and other mammals too, as discovered in recent years), individuals without Y-chromosomes (XO) were found to be viable, while those without an X-chromosome (YO or YY) were inviable. The viability of XO flies, which are sterile males, suggested that the Y-chromosome carried genes for male fertility, but no vital genes essential for viability. Further, as Muller (1914a,b) stated, “a female occasionally, owing to a n abnormal reduction division (‘non-disjunction’), receives a Y-chromosome in addition to the two X’s, yet such a female is indistinguishable from the ordinary form, which contains no Y. The Y-chromosome either contains no genes or else only genes that are allelomorphic to those in X, but never dominant to them. This is proved also by the fact that mosaic flies sometimes develop, of which a part of the body is female but another part is male, owing to the accidental loss of one of the X-chromosomes in an embryonic cell-division. These male parts must have an X, but no Y, and yet they are indistinguishable from corresponding parts on real males which contain a Y. Furthermore, Mr. C. B. Bridges has obtained (again by ‘non-disjunction’) males which must have two Y’s, yet these also show no peculiarities.” This idea of inertness was supported by an apparent lack of obviously important Y-linked traits and the relative importance of X-linked traits in many organisms. A parallel argument for inertness of the Y-chromosome in plants, where it was found to vary in size within a species, was made by plant geneticists and cytogeneticists (Darlington, 1932, 1937). Muller further argued that suppression of crossing-over was followed by the accumulation of recessive changes in

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the Y-chromosome. Assuming further that mutations sometimes consist in losses of genes, a degeneration of the Y-chromosome was expected to have occurred. Now if we assume further that recessive losses are more apt to occur than recessive additions of genes, the Y-chromosome will gradually become functionless (Muller, 1914a,b; 1918). The discovery of the gene for bobbed did not appreciably change the idea of inertness. I n subsequent years Stern, followed by Neuhaus, Cooper, and recently Brosseau, analyzed the fertility factors. Studies on the structure and function of heterochromatin by Muller, Heitz, Schultz, Cooper, and others have made rapid strides since the ’thirties. Cooper’s studies on the effects of Y-chromosome hyperploidy, and a t the cellular level, Schultz’s work on nucleic acid metabolism, and that of Hess and Meyer on the possible role of the Y-chromosome in D. hydei and D. neohydei in controlling the synthesis of specific proteins and their polypeptide sequence have emphasized only too well the diverse functions this chromosome (or more precisely, the material it contains) fulfills. Fine analysis began with the work of Brosseau on fertility factors, and of Baker and Spofford on variegation. If we look back now a t the original concept of inertness of the Y it is amazing indeed to realize how far we have advanced from the old degenerate-genes-in-the-inert-chromosome concept to the latest work showing th at the heterochromatic regions of the Y may in fact be organized as discreet linear functional units similar to those in the euchromatin. I n man, Stern’s (1957) review showed th at not a single function could be convincingly attributed to the Y-chromosome. Then came the discovery in 1959 that the Y in man is strongly male determining, unlike the one in Drosophila. I n fact it is so “strongly” male determining that even XX X X Y individuals and mosaics of the type X X X Y /X X X X Y / XX X X X Y are phenotypically male. Preliminary work in other mammals (mouse, cat, etc.) has confirmed that the Y is male determining in those animals also. The evidence in favor of the complete Y-linkage of hypertrichosis of the pinnae of the ears in man is strong a t present. It is reasonable t o assume that such traits as hypertrichosis are probably superficial manifestations of some basic mechanism and studies concerning the genetic control of hormonal substances in different sexes would be of considerable interest. Furthermore, suspected Y-linked characters could be detected by investigating the progeny of different women married to the same man. The same method could be used in the study of pedigrees in populations such as those of Islamic countries which still practice polygamy. The investigation of suspected Y-linked traits in individuals with such abnormal karyotypes as X X X Y and X X X X Y might yield important confirmatory evidence in favor of Y-linkage, provided the

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abnormal endocrinological factors did not influence the trait under study. I would like to repeat here a suggestion which I first made in my 1960 paper. Some economy in the genetic system would be effected if the genes responsible for male epigamic characters were also on the same chromosome as the male determiners. This would be similar to the close linkage observed between different traits in a heterostylic system of plant species like Lythrum salicaria. It is possible that beard growth, for instance, may be controlled as a subsidiary function of the Y-chromosome, and if so, males of the first generation from a cross of two human races which differed considerably in the development of the beard might resemble their fathers, rather than their maternal grandfathers. The Y-chromosome in man is generally regarded as inert because many genes are known on the X-chromosome and the Y appears to be comparativcly lacking in specific genes. It may have general functions which are not detectable with the tools of methodology now available. Also, is it the right question to ask if it contains as many genes as does the X ? It is much smaller than the X-chromosome. The variation in the size of the Y-chromosome has been pointed out by some as an indication of inertness. Recent work, by Wennstrom and de la Chapelle (1963) using autoradiographic methods, suggests that this variation does not necessarily involve differences in DNA content. The Y-chromosome may be subject to such morphological changes as contraction and elongation more readily than other chromosomes and inheritance of ((long” Y-chromosomes may mean that this behavioral peculiarity is heritable. It would be of considerable interest to study individuals having the same surname (and hence Y-chromosomes derived from a common ancestor) in closed populations such as the Amish and other isolates. The only plant species in which the function of the Y-chromosome has been intensively investigated is Melandrium. Here, as in man, the Y-chromosome is strongly male determining. I n most other plant species the heteromorphic pair of sex chromosomes are usually very small and cytological observations are not always convincing. Much of the evidence concerning the role of the Y-chromosome in sex determination in plants comes from studies of polyploids, and crosses involving dioecious, monoecious, bisexual, and subdioecious species. Localization of the female suppressor, anther initiation, and anther development has been very successfully done in Melandrium. Much further research is needed to understand the function of Y-chromosome in various plant species. ACKNOWLEDGMENTS My investigations on Y-linked inheritance were undertaken when I was working under the direction of the late Professor J. B. S. Haldane, D. Sc., F. R. S., first a t the Indian Statistical Institute, Calcutta, and later a t the Genetics and Biometry Labora-

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tory of the Government of Orissa in Bhubaneswar. During this period I benefited very greatly from Professor Haldane’s instruction, advice, and inspiring guidance. H e also financed part of my investigations. I am grateful t o Professor P. C. Mahalanobis, F. R. S., and Professor C. R. Rao for their encouragement. In my field work in India and in many other ways I received help from Mr. M. R. Shastry, Mr. P. S. Rao, Mr. D. B. Raju, and Mr. D . V. Rao. The investigation in Ceylon was made possible by the invitation of Dr. W. R. C. Paul, President of Ceylon Association for the Advancement of Science (1960) to participate in its Sixteenth Annual Session. To him and the following people in Ceylon who assisted in my work I express my thanks: Dr. V. Basnayake, Dr. P. E. P. Deraniyagala, Mr. W. A. K. Fonseka, Dr. W. Pallie, Dr. M. L. M. Salgado, Dr. I. Samaraweera, and Dr. J. Wanigasooriya. It is a pleasure to acknowledge the suggestions, some of which are incorporated in the present review, which I received from Professor H. J. Muller, Dr. Jack Schults, Dr. Curt Stern, and Dr. I. I. Oster in connection with writing an earlier but much shorter paper on the subject. I would also like to acknowledge my debt to Professor V. A. McKusick and Dr. J. F. Crow for their helpful comments in writing the paper, and to Dr. Catherine S. N. Lee, Dr. D. S. Borgaonkar, and Dr. William J. Breen who have drawn my attention to certain publications. It is a pleasure to acknowledge the typing and other assistance of Mrs. Muriel Klarman.

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MAMMALIAN PIGMENT GENETICS* Morris Foster Mammalian Genetics Center, Department of Zoology, The University of Michigan, Ann Arbor, Michigan

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 11. Genes and the Cytological Basis of Melanin Pigmentation.. . . . . . . . . . . . . . . 312 111. Genes and the Ultrastructural Basis of Melanin Granule (Melanosome) Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. A. Introduction. . C. Genetically Oriented Studies D. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Genes and the Biochemistry of Melanin Formation.. . . . . . . . . . . . . . . . . . . . . A. Introductory Remarks.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Biochemical Pathway to Eumelanin.. . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Question of Phaeomelanin.. . . . . . . . . . . . . . . . . . . . . . . . D. Genes and the Structure of Eumelanin.. . . . . . . . . . . . . . . . . E. Color Genes and Assays of Melanogenic Activity.. . . . . . . . . . . . . . . . . . . . F. Biochemical Studies of the Agouti P a t t e r n . . . . . . . . . . . . . . . . . . . . . . . . . . V. Color Genes and Evolutionary Biology.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Comparative Genetics of Coat Color. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Color Polymorphisms in Natural Populations of the House Mouse. . . . . D. Coat Color and Adaptation in Deer Mice.. . . . . . . . . . . . . . . . . . . . . . . . . . VI. Concluding Remarks ............ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

Genetic studies of mammalian melanin pigmentation have long enjoyed the active interest of many types of investigators because of the many different color phenotypes a t various levels of organization which are amenable to analysis. While the mammalian pigmentation geneticist, by providing suitable material for study, has thus aided his colleagues in other fields (or has himself used techniques developed by them), for example, in biochemistry, cytology, embryology, behavior, population biology, and evolution, he has himself profited when the findings obtained

* I n preparing this manuscript and obtaining some recent research findings cited here we have been greatly aided by N. I. H. Research Grants CA-04305 (currently HD-01254) and CA-05559 (currently HD-01259), of the U.S. Public Health Service. 311

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by such studies have enriched areas directly pertinent to genetic concepts or phenomena; i.e., genic expression in terms of protein, enzyme, or other organic biosyntheses; the regulation of genic expression, involving both intra- and intercellular interactions of genes and their products, as well as genotype-environment interaction; genic interactions, involving both allelic and nonallelic genes; ultrastructural, cytological, cytogenetic, morphogenetic, and behavioral aspects of the roles of genes in development; multiple or pleiotropic effects; and, finally, the relationship between genetics and evolutionary biology, involving such aspects as gene distributions in populations, adaptive genotypes, and genetic homologies deduced from comparative genetic studies. I n order to provide a n economical guide to the wealth of literature on these various facets of mammalian pigment genetics, recent reviews of various aspects, each with a n extensive bibliography, will be cited as soon as possible after the introduction of a topic. II. Genes and the Cytological Basis of Melanin Pigmentation

Melanin pigmentation in mammals depends upon the synthesis of dark (eumelanin) or yellow (phaeomelanin) granules by specialized cells (melanocytes). These cells are situated in a variety of locations: a t the basal layer of the superficial epidermis and in the hair bulbs of the skin, responsible, respectively, for skin and hair pigmentation; in the choroid, iris, and retina of the eye; the leptomeninges; the parathyroid, thymus, ovary, nictitans, spleen, and dermis (Silvers, 1961 ; Billingham and Silvers, 1960). Indeed, in one strain of house mouse P E T / M C V , melanocytes are distributed throughout the connective tissues of many organs, being consistently absent only from the connective tissue of the gut mucosa (Nichols and Reams, 1960). With the exception of the pigmented retinal cells, which are derived from the optic cup, all the other cells are believed to originate from the neural crest (Rawles, 1948; Silvers, 1961; Billingham and Silvers, 1960). Absence of melanin pigmentation is due to one of two major causes. I n albinism, amelanotic melanocytes, or “clear cells,” were observed in the hair bulbs of mice and guinea pigs (Silvers, 1956). Thus the absence of pigment’ation is due to failure of melanogenesis, correlated with failure to demonstrate tyrosinase activity in the skin (Foster, 1951, 1956, 1959). On the other hand, the piebald condition, or white spotting, in both house mouse and guinea pig was diagnosed as the absence of amelanotic melanocytes (Silvers, 1956; Mayer and Maltby, 1964). Because many gene loci are associated with white spotting, the etiology of this defect might vary with the specific mutation. For example, such a mutational defect could be attributed to any of the following: (1) defective differenti-

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ation of neural crest cells into precursor melanoblast cells, (2) disturbed melanoblast migration, or (3) failure of melanoblasts to survive and undergo final differentiation in their final locations. These possibilities were tested by transplanting neural crest from fetuses normally destined to become “one big spot” (black-eyed whites) into the anterior eye chamber, a favorable site for differentiation. Where the donor neural crest involved “one big spot” homozygotes at, for example, the W locus (“dominant spotting”) or Miwh locus (“dominant white”), no pigmented melanocytes were obtained from the intraocular grafts. In these cases failure of neural crest differentiation was implicated (Silvers, 1961). However, in another case, belted (btbt), in the house mouse, a hostile hair follicular environment was implicated (Mayer and Maltby, 1964). The decision as to whether a differentiated melanocyte will produce eumelanic or phaeomelanic granules is made by the tissue environment, and then only in the hair. Moreover, transplantation experiments indicated the decisive role played b y alleles a t the agouti ( A ) locus in conditioning the influential role of the hair cells serving as the melanocyte tissue environment. Thus, for example, when house mouse melanoblasts normally destined to produce eumelanic black or brown granules (genotypes aa BB or aa bb, respectively) found themselves among hair bulb cells containing the allele for yellow (Ar)pigmentation, the cells produced phaeomelanin instead. Moreover, it was demonstrated th a t Aua BB or Aga bb nielanoblasts differentiated, respectively, into black or brown melanocytes whenever they occurred outside of hair bulbs (Silvers, 1957, 1958a, 1958b; Silvers and Russell, 1955). Other experimental and biochemical aspects of gene action a t the agouti locus will be discussed in a later section (Section IV,F). I n concluding this section it seems desirable to mention several additional features connected with white spotting. Studies of the pleiotropic effects of spotting factors, as well as of the interactions between spotting and other color genes, lead to the general conclusion that white spotting is often symptomatic of a more deep-seated genetic disturbance of development or function (Gluecksohn-Waelsch, 1963; Mayer and Maltby, 1964; E. S. Russell, 1963; Searle, 1961; Nachtsheim, 1959; Billingham and Silvers, 1960; Silvers, 1961; Wright, 1963). With respect to pleiotropy, homozygotes a t the W (“dominant spotting”) or Sl (“Steel”) locus exhibit similar patterns of lack of pigmentation, anemia, and sterility (E. S. Russell, 1963). Moreover, “The Vienna white rabbit has epileptic fits and the white cat tends to be deaf” (Searle, 1961). Concerning nonallelic genic interaction, mice doubly heterozygous for dominant spotting factors exhibit a greater than additive amount of white coat when compared with the separate spotting effects of heterozy-

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gosity at each locus taken singly (Silvers, 1961). Moreover, Wright (1963), in considering interactions between recessive spotting (ss) and the tortoise-shell pattern (epep) of finely mingled yellow and eumelanic pigmentation in the guinea pig, has pointed out that in the combined mutant (ss epep) “the amount of yellow is increased and there is often a strong tendency to segregation of yellow and eumelanin into a few large areas each,’’ often separated by white streaks. “Sometimes a streak between eumelanic areas is white at one end, yellow at the other, indicating th a t the determination of yellow is related to the process that leads in more extreme cases to white by absence of the pigment cells.” 111. Genes and the Ultrastructural Basis of Melanin Granule (Melanosome) Formation

A. INTRODUCTION 1. Terminology

Following the recent recommendation of Fitzpatrick and Quevedo (1964, personal communication), the term “melanosome” will be used to describe the melanogenic granule of a pigment-producing cell from its earliest recognizable unpigmented stsageto its completely or partially electron-dense definitive pattern of melanization. Such a definition is independent of the amount of melanin polymer present on the protein framework or matrix of the granule, or of the possible growth in size during granule ontogeny. 2. Signijicance of Ultrastructure

B y virtue of its structure and function, the melanosome can be classified as the specialized organelle of a differentiated cell. Such a n organelle, once assembled from its constituent proteins, presumably gene products, could then serve as a device for stabilizing and coordinating the expressions of those genes (often unlinked) which contribute interacting architectural and functional (enzyme) protein components to organelle construction. Such a viewpoint has recently been stated in general terms by Waddington (1962), and has been specifically applied to the melanosome concurrently by Moyer (1963) and Foster (1963a, 1961-1962). Thus the study of mutational alterations of melanosome architecture, function, and ontogeny might prove instructive in analyzing the macromolecular basis of coordinated genic action and interaction.

B. STUDIES WITHOUT IMMEDIATE GENETIC ORIENTATION The application of electron microscopy to studies of melanosome fine structure is progressing rapidly. While most of the published reports

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are not immediately pertinent to genetic consideration of mammalian pigmentation, and hence are beyond the scope of this review, they nevertheless merit citation because of their probable contributions to the perspectives, insights, or techniques of geneticists interested in the ultrastructure of mammalian melanogenesis. From this standpoint studies on melanin formation in goldfish scales of color variants are pertinent (Mishima et al., 1962; Mishima and Loud, 1963; Loud and Mishima, 1963). Studies involving freckles or Langerhans cells are also of interest (Breathnach, 1964; Breathnach et al., 1963). Melanotic tumors from various sources have been exploited in various ways on a n extensive scale. Descriptive studies have recently been reported on human (Zelickson, 1962), hamster (Staubli and Loustalot, 1962; Nakai, 1963; Nakai and Rappaport, 1963a,b; Rappaport et al., 1963a,b), and mouse melanomas (Hirsch and Zelickson, 1964). Wellings and Siege1 (1963) have reported on melanomas from human, mouse, and hamster sources. Electron microscopy following various experimental treatments is also relevant. For example, Hu and Cardell (1964) studied B16 mouse melanoma cells following their propagation in monolayer culture. Electron microscopic autoradiography, using tritium- or C14-labeled substrates, has also been recently reported (Nakai and Shubik, 1964; Zelickson et aE., 1964) involving S-91 and Harding-Passey mouse melanomas. Finally, Drochinans (1963) reviewed studies on a variety of malignant and nonmalignant melanocytes. A most rewarding series of recent studies combining sucrose density gradient ultracentrifugal separation of melanoma cell particles with incorporation of radioactive tracers and electron microscopy are immediately pertinent to our own considerations (Baker et al., 1960; Seiji et al., 1961a,b, 1963; Seiji and Iwashita, 1963, 1964). These studies have provided evidence that melanosomes and mitochrondria are distinct cytoplasmic organelles and that the melanin-producing enzyme, tyrosinase, becomes a firmly bound structural component of the melanosome after the enzyme has been synthesized on ribosomes.

C. GENETICALLY ORIENTEDSTUDIES 1. Studies of Differently Colored H u m a n Hairs

Some of the complications in determining the inheritance of human hair color have been discussed by Barnicot (1956, 1957) and by Hanna (1953, 1956, 1961). It would seem quite certain, however, that such strikingly different hair color phenotypes as very dark brown or black, red, blond, and albino white hair would represent quite distinct color genotypes. Thus the early findings of differences a t the level of fine

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structure among dark, blond, red, and albino melanosomes in human hair melanocytes (Barnicot et al., 1955) provided the encouragement to advance to more refined studies with improved techniques (Birbeck et al., 1956; Barnicot and Birbeck, 1958; Birbeck and Barnicot, 1959; Birbeck, 1963). The early findings indicated that the dark hair melanosomes were longer and more electron-dense than the smaller, rounded melanosomes, with more obvious internal structure, of red hair. Albino melanocytes contained ellipsoidal bodies which could reasonably be interpreted as anielanotic melanosomes. The more recent studies (Birbeck and Barnicot, 1959; Birbeck, 1963) lead to the following summarized observations and views: Concerning melanosome ontogeny, the earliest morphological structures are small vesicles ( 0 . 0 5 ~diameter) of the Golgi zone which b y fusion or growt,h become large, initially spherical, vesicles. These large vesicles then elongate and approximate the size of the mature (melanized) melanosome. Siniultaneously inner membranous structures arise. I n cross section the inner membranes appear to be arranged either concentrically or as a single membrane folded into a n irregular spiral. Consistent with either arrangement is the arrangement of parallel strands appearing in longitudinal sections of the inner membranes. Particulate sites presumed t o represent locations of both melanin synthesis (localized tyrosinase molecules) and deposition are spaced about 80 A apart along the longitudinal section of a n inner membrane. Moreover, these sites occur alternately on either side of the membrane, so that after melanin deposition has begun the alternating nielanization pattern gives the impression of a helical thread structure. As melanization proceeds in dark hair (considered the standard or “wild type”) the melanosome internal fine structure becomes increasingly obscured. Finally, no inner structure can be resolved when the melanosome interior becomes sufficiently melanized to appear uniformly electron-dense. At this stage the mature granule, with its tyrosinase largely or completely inactivated, is transferred via the melanocyte’s dendritic processes into the keratinizing epithelial receiving cell of the growing hair. Some melanosome growth might occur within the hair cell. With respect to probable genotypic differences, albino granules exhibit some internal membranous fine structure, although no deposition of electron-dense material (melanin) occurs. Thus the internal membrane represents the protein matrix also seen in the immature granules of pigmented cells, and the lesion in albinism involves failure of melanin synthesis and deposition. White hair seems to be similar to the whitespotted areas of laboratory animals previously noted, since no hair

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bulb melanocytes could be detected in such subjects. T h e melanosomes of red hair are rounder and smaller than those of dark hair, and they contain a loose aggregate of dense subparticles enclosed by a n often ill-defined and irregular outer membrane. I n the case of blond hair the melanosonies resemble an arrested stage in the formation of dark hair types. They are smaller and less numerous than in dark hair, and melanization does not proceed to the point of obscuring internal fine structure. Thus they resemble a stJage intermediate between albino melanosomes and melanosomes from a dark-haired subject. 2. Color Genes and Retinal Pigmentation in the House Mouse

The first systematic study of melanocytes using modern electron microscopy and exploiting part of the wealth of available genetically defined color variants was performed by Moyer (1961, 1963), who studied inherited variations in the fine structure of melanosomes in the pigment epithelium of house mouse retina. These cells were found to differ from the previously described human hair bulb melanocytes in several respects. First, the retinal cells lack both a well-developed granular endoplasmic reticulum and a Golgi apparatus when melanosome synthesis has gotten well under way. Instead, free-floating clumps of ribosomes (polysomes?) were implicated in the synthesis of melanosome matrix protein. These differences in cytoarchitecture might be related to the different rates of matrix protein synthesis (slower in retina, according to Moyer), or to the difference in final disposition of melanosomes. Retinal cells retain their granules, while hair follicle melanocytes export their melanosomes to the epithelial receiving cells and thus can be considered secretory cells. The second major difference between mouse retina and human hair bulb melanocytes concerns melanosome ontogeny. I n the latter the melanosome protein matrix is formed as one or more concentric or spiral sheets inside a n enclosing membrane. I n the retina, on the other hand, thin protein fibers, a t first not membrane-enclosed, appear to be “spun out” from different ribosomal clumps, then form parallel crosslinked aggregates, and then undergo progressive thickening by melanization. Finally, uniformly dense material, presumably melanin, spreads from one end of the melanosome to the other end, resulting in the mature granule. Such differences between the two different pigment cell types might be ascribed to species differences or to differences in histologic type, irrespective of species source. Moyer investigated t~hepossible roles in nielanosome ontogeny of six gene loci, shown by transplantation studies (Silvers, 1961) to act autonomously within melanocytes ; c (albinism), b (brown), p (pink-eyed dilution), d (maltese dilution), In (leaden), and ru (ruby-eyed dilution).

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Homozygous mutants at three of these loci (b, c, and p ) were found to affect melanosome fine structure. Thus the ovoid brown (bb) melanosomes contained loose, coarsely granular melanin, whereas the rod-shaped black ( B B ) melanosomes contained dense, finely granular melanin. Albino (cc) melanosomes were smaller, less numerous, and unmelanized when compared with melanosomes from pigmented cells. Homozygous pink-eyed dilution ( p p ) resulted in a disorganized array of matrix fibers and a reduction of cross-linking between the fibers. While both dilute (dd) and leaden (Znln) genotypes exhibit clumping in the hair, only the former exhibited clumped melanosomes in the retina. Finally, the smaller and less numerous melanosomes of the ruby-eyed (ruru) mice were attributed to delayed onset of melanization from the usual beginning at 15 days in utero to parturition (about 6 days later). These observations have led Moyer to conclude that the melanosome protein matrix consists of an ordered aggregation of the protein products of at least three gene loci (b,c, and p ) , thereby providing a structural basis for regulatory genic interactions by way of their interacting protein products participating in a common undertaking. 3. Color Genes and Melanosomes in Hair Bulb Melanocytes of the House Mouse

The second modern, systematic, genetically oriented, study of melanosome fine structure was recently performed by Rittenhouse (1962, 1965). She investigated the separate and several combined effects of mutation at the b, c, d , p , and Miwhloci in house mouse hair bulb melanocytes. Some of her observations are summarized below: a. Regarding cytoarchitecture, the mouse hair bulb melanocyte resembles the human hair bulb melanocyte rather than the mouse retinal pigment cell. For example, the mouse hair melanocyte (5 and 8 days, postpartum) contains an extensive Golgi region and granular endoplasmic reticulum. Thus histogenetic similarities transcend species boundaries. b. Melanosomes appear to originate in border areas where Golgi vesicles, free ribosomes, and occasional rough-surfaced membranes occur together. The formation of an external membrane enclosing a rolled internal membrane, described for dark human hair bulb melanosome ontogeny, approximates the origin of intense black mouse ( B B CC DD PP) melanosomes. In general, both Golgi vesicles and free ribosonies appear to be involved in melanosome formation. c. Mutation from black ( B B ) to brown (bb) affects both melanosome shape and structure. The oval intense black granules begin with an internal structure of rolled membranes, whereas the round brown melanosomes contain an internal framework resembling a tangled ball of strands.

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d. Mutation a t the locus for maltese dilution (dd) is associated chiefly with the crowding of melanosoines within the melanocyte, dd inelanosomes being more densely crowded than DD granules. Evidence of possible interaction between the b and d gene loci is suggested by the observations that bb dd granules are slightly larger than bb DD granules, more often oval, and with more frequent off-center pattern of melanization. e. Mutation a t the pink-eyed dilution ( p ) locus affects the size and degree of inelanization. In brown (bb) cells, nielanosoine shape is also affected. Mutant granules are lightly melanized and are smaller than PP granules. In 5-day-old bb p p mice the granules are often oval and elongate, while in 8-day-old mice of the same genotype the melanosomes are more often round. f. Mutation to albinism (cc) allows formation of granule matrix, but prevents melanization. It has not yet been possible to distinguish reliably between BB cc and bb cc granules. g. In “one big spot” ( M z w h l l / l i w h ) mice no amelanotic melanocytes of the albino type have been found. h. One major difference between the observations of this study and of Moyer’s study on retinal cells concerns the disruptive effect of mutation on melanosome fine structure. In retina, substitution of p p for PP causes major disruption, while in the hair bulb inelanocyte it is the substitution of bb for BB that leads to disorderly granule framework.

D. CONCLUDING REMARKS Clearly, much remains to be done in extending studies of inherited ultrastructural variations in melanosomes to a wider variety of tissue and taxonomic sources, to a wider variety of available color genotypes, and to a variety of experimental techniques coupled with electron microscopy. Speculation concerning the possible mechanism of genic interaction by way of interacting gene-produced proteins participating in organelle construction and function has already outrun the painstakingly gained observations. The next few years should witness an exciting period when facts may catch up with, and even surpass, current fancy. IV. Genes and the Biochemistry of Melanin Formation

A. INTRODUCTORY REMARKS By now it is evident that mammalian melanogenesis cannot be considered a relatively siniple sequence from precursor to end product. Knowledge concerning the structure of the melanin polymer end product and its niode of attachment to melanosome deposition sites is still

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incomplete. The attachment of tyrosinase to specific binding sites of the melanosome protein matrix requires this enzyme to “behave simultaneously as both a structural protein and as an active enzyme” (Birbeck, 1963). Moreover, the elegant studies demonstrating structural and regulatory genes affecting soluble tyrosinase synthesis and activity in Neurospora (Horowitz et al., 1961) and Drosophila (Lewis, 1962; Lewis and Lewis, 2963) are not yet directly applicable to the mammalian melanosome system, where, somehow, the kinetic aspects of the solid state must be taken into account. Furthermore, if, as seems reasonable, several color genes contribute “architectural” proteins to the melanosome matrix, it is not unreasonable to expect that mutational alteration of one or another matrix protein might result in a syndrome of secondary consequences such as (1) altered attachment, stability, and specific activity of tyrosinase, (2) altered number or effectiveness of melanin polymer binding sites, and (3) disturbed melanosome architecture (Foster, 1961-1962, 1963a). Thus, such secondary mutational alterations in enzyme properties, such as stability and specific activity, might mimic the effects of mutation in the true structural gene determining the enzyme’s primary structure or amino acid sequence. Finally, in the absence of quantitative extraction procedures for obtaining tyrosinase from rioninaligriarit melanocytes, it is difficult to distinguish between altered amount and altered specific activity of tyrosinase assayed in different color genotypes. Obviously, the rewarding possibilities of studying such a richly challenging system are matched by current difficulties in analyzing it biochemically.

B. THEBIOCHEMICAL PATHWAY TO EUMELANIN The pathway from the colorless precursor, tyrosine, to the dark, insoluble melanin polymer has been well reviewed on numerous occasions (Brunet, 1963; Fitzpatrick and Kukita, 1959; Fitzpatrick and Lerner, 1954; Fitzpatrick et al., 1958; Lerner, 1955; Lerner and Case, 1959). Briefly, the copper-containing oxidase with dual specificity, tyrosinase, catalyzes the oxidation of tyrosine to 3,4-dihydroxyphenylalanine(dopa), and of dopa to dopa quinone. Dopa can serve not only as a substrate but also as an activator or primer in the oxidation of tyrosine catalyzed by tyrosinase. Intramolecular rearrangements and additional oxidations are involved in the rest of the sequence from dopa quinone to leucodopachrome to dopachrome to 5,6-dihydroxyindole to the polymerizing unit, indole-5,6-quinone. These later reactions do not require enzymatic catalysis in vitro, and no additional enzymatic catalysis of any of these later reactions has been demonstrated in vivo. In any event, the polymer of indole-5,6-quinone becomes attached through its quinone linkages

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to amino or sulfhydryl groups of melanosome matrix protein. (See Fitzpatrick et al., 1958.) Four factors regulating melanin synthesis, at least in vitro, have recently been summarized by Fitzpatrick et al. (1958). They are: 1. Availability of free tyrosine, perhaps made available by peptidases incorporated into the melanosome protein matrix. 2. Sufficient level of tyrosinase to oxidize both tyrosine and dopa. 3. Tyrosinase activators, principally dopa. 4. Inhibitors, including competitive inhibitors such as N-acetyltyrosine or phenylalanine, and sulfhydryl compounds, such as glutathione, that combine with the copper of the tyrosinase molecule.

C. THEQUESTIONOF PHAEOMELANIN The absence of both eumelanin and phaeomelanin in albino skin lacking tyrosinase activity, as well as the presence of some tyrosinase activity in phaeonielanic skin capable at least of oxidizingdopa (producing, however, a black pigment), indicates that tyrosinase plays a key role in the formation of both dark and yellow pigments. These observations, together with the discovery of a tryptophan-oxidizing enzyme in mouse skin (Foster, 1951), provided the basis for the following tentative hypothesis put forward by Fitzpatrick et al., (1958) and by Fitzpatrick and Kukita (1959). Phaeomelanin might be derived from tryptophan as the chromogen. An o-aminophenol derived from tryptophan might then be oxidized by dopa quinone to produce a yellow pigment. Attempts to demonstrate directly the formation of phaeomelanin from tryptophan have been unsuccessful. Markert (1955) and Coleman (1962), while obtaining positive results with C14-labeled tyrosine, were unable to show incorporation of CI4-labeled tryptophan into melanin of mouse skin. Moreover, attempts at histochemical demonstration of net phaeoinelanin synthesis by incubating mouse skin sections with tryptophan or its derivatives gave no encouraging positive results (Foster, 1954-1955, unpublished results). More recent studies suggest the possibility that tyrosine might still be the common precursor of both eumelanin and phaeomelanin. FuseauBraesch (1960), using radioactive-labeled tyrosine, was able to demonstrate tyrosine incorporation in cricket cuticle (Gryllus), not only in black areas but also in areas colored by a stable yellow pigment. Moreover, using the N-acetyltyrosine derivative, N-acetyltyramine, as an in vitro substrate for incubating cuticle, she was able to demonstrate the formation of a yellow pigment catalyzed by a copper-containing phenolase in the cuticle. Thus, as pointed out by Brunet (1963), stable yellow

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pigments ultimately derived from tyrosine might account for the yellow pigmentation of both cricket cuticle and mammalian hair. Further investigation is obviously warranted.

D. GENESAND

THE

STRUCTURE OF EUMELANIN

1. Results of Chemical Studies

From extensive analytical studies of in vitro dopa melanin or squid ink (Sepia) it could be concluded that eumelanin is a highly irregular, three-dimensional polymer, and that, in addition to indole-5,6-quinone other intermediates such as dopa quinone, 5,6-dihydroxyindole, and dopachrome take part in the formation of the polymer (Nicolaus, 1962; Swan, 1963; Blois et al., 1964). However, a spatially more highly ordered biosynthesis involving enzymatically catalyzed melanin formation (Mason, 1959), with additional restrictions on randomness due to structural orientation of tyrosinase and melanin binding sites in a mammalian melanosome matrix, might lead to a more orderly polymerization. Indeed, more recent findings of Nicolaus et al. (1964) with a number of natural melanins indicate several differences between squid ink melanin and the melanin obtained from human hair and rat melanoma. Moreover, the high sulphur content they found in human hair melanin might well be due to a “higher content of protein-pigment bonds in the melanin from human hair.” 2. Inherited Variations in Free Radical Contents of H u m a n Hair Melanin

The recently developed technique of electron spin (or paramagnetic) resonance (ESR or EPR) spectroscopy permits the determination of free radical contents of biologically important polymers due to the Paramagnetic properties of unpaired electrons resulting from biological oxidations and subsequent polymerization. (General references: Androes and Calvin, 1962; Isenberg, 1964.) This technique has therefore recently been applied to the study of melanin obtained from various sources (see Blois et aZ., 1964). According to Mason et al. (1960) the free radical property of melanin could be due to the stabilized semiquinonoid form of indole-5,6-quinone, the polymerizing unit. Such stabilization of a semiquinonoid form would depend upon the degree of conjugation, upon chain length, and upon the redox state of the polymer. Thus extensive application of this technical approach to various color genotypes might lead to a better understanding of possible gene-controlled variations in melanin polymer structure. Only two genetically relevant ESR studies have recently been reported, both dealing with human hair. Mason et al. (1960) noted a

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decreasing order of free radical content with black highest, followed in order by brown, red, and “fair” colored hair. Munday and Kerkut (1961-1962) both confirmed and extended the range of human hair color variants by noting the following decreasing order in free radical content: dark brown, mid-brown, light brown, and finally, grey, the last with little or no free radical content observed. In each report the free radical content measurements were given as “free spins” or as “free radicals” per gram of hair. Since darker hair should be expected to contain more melanin per unit weight of hair than lighter samples, it is not yet clear whether these observed differences in free radical content represent only differences in amount of melanin or whether they might, in addition, reflect some differences in melanin polymer structure. In any event, it seems warranted to extend the methods of ESR spectroscopy to material which is more uniform and better defined genetically. Moreover, such material, represented by color stocks and strains of laboratory rodents, permits the study to be extended to phaeomelanic genotypes. It has been reported that strong ultraviolet irradiation results in increased free radical content of dark hair (Mason et al., 1960). It is therefore possible that in some organisms melanin might act as a biological electron exchange polymer, or electron trap, which by virtue of its capacity for oxidation and reduction and also its stable free radical state, could protect a melanin-containing tissue or associated tissue against harmful free radicals produced by irradiation or other factors. In a related type of study Sever et al. (1962) showed that in melanin granules of beef eyes visible light irradiation resulted in rapid generation of free radicals, and free radical decay from the irradiated level to a basal level occurred rapidly in the dark. This reversible generation and decay of free radicals in the presence and then absence of visible light is thought to involve the quinone subunits of the melanin (Cope, 1963). According to these authors, ‘lThe presence of rapid and reversible photoactive free radical-generating melanin granules in the eye in close proximity to the rods and cones suggests that they may play a more important role in the visual process than merely to absorb stray light.” If this is so, then mutations affecting eye pigmentation and, perhaps, visual function as well, could result in various pleiotropic effects, including behavioral modifications, occasioned by such pigmentary visual defects. 3. Genes and Infrared Spectra of Melanins

Spectrophotometric studies involving absorption peaks at specific wavelengths in the visible and ultraviolet range (2000-7000 8)have been useful in identifying individual molecules or component niolecules of

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macromolecules. This approach in this range of wavelengths has, however, proved disappointing in studying melanin preparations, because of the general light absorption observed. Recent confirmations of such data and their evaluation are presented by Nicolaus (1962) and by Blois et al. (1964). Infrared spectrophotometry, in the wave number range 40001300 cm-l (corresponding wavelengths = 2.5-7.7 p ) can be used to identify functional groups with absorption peaks in this range, and thus might provide clues to the structure of melanin polymers in various color genotypes, although little encouraging evidence has yet been published. This approach has thus far been dismissed by Nicolaus et al. (1964), and the observations reported by Blois et al. (1964) indicated similar infrared spectra for natural squid melanin, and in vitro melanins produced by autoxidation of catechol, L-dopa, and hydroquinone. However, the infrared spectra of several invertebrate and mammalian (ox-eye choroid) melanins indicated not only some general similarities but also some detailed differences (Bonner and Duncan, 1962). At this stage in applying this technique to melanins both the sources and the methods of sample preparation might turn out to be extremely important. Very recently, Siu and Foster (1964, unpublished data) have used melanin obtained from alkaline thioglycolate house niouse skin suspensions prepared in the same way as was previously briefly described (Foster, 1959, 1963a,b). The suspended melanin from individual skins was centrifuged, washed with alcohol, dried, niixed with KBr, and pressed into a pellet. I n the KBr pellet form each inelanin sample could be tested repeatedly in a n infrared spectrophotometer. Preliminary results with samples from homozygotes for the four known alleles a t the brown ( b ) locus: black ( B B ) ,light (BttBBlt), cordovan (bcbc),and brown (bb);suggest that generally reliable genotypic identification is possible. The cordovan samples uniquely exhibit a peak at around 2350 cm-1 or 4.2 p, perhaps caused by S-H bonds. The peak at around 2960 cni-l (about 3.4 p ) occurs much more frequently in brown than in black samples. Decisions concerning the regularity and significance of these and other infrared spectrum characteristics must await more information.

4. Conclusion While purely physical methods have as yet contributed little to the comparison of melanins, more significant and genetically relevant contributions might be forthcoming from the study of additional sources and differently prepared samples.

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E. COLORGENESAND ASSAYSOF MELANOGENIC ACTIVITY 1. Introduction

I n attempts to clarify the physiological genetics of mammalian melanin pigmentation, a number of techniques for assaying melanogenic activity have been systematically employed in the comparison of various color genotypes within different species of laboratory mammals. Thus genotypic comparisons of the in situ induced melanization of hair bulb melanocytes upon incubation of frozen skin sections with dopa (the dopa reaction) have been reported for the rabbit (Kroning, 1930), the guinea pig (Kroning, 1930; W. L. Russell, 1939), and the house mouse (L. B. Russell and W. L. Russell, 1948). [The author has not seen Kroning’s paper, and is relying upon the citations and comments of W. L. Russell (1939) and of Wright (1963).] Darkening of skin extracts, with a t least some dopa oxidase activity, has been used for genotypic comparisons in the rabbit (Danneel, 1941) and in the guinea pig (Ginsburg, 1944). Measurements or estimates of C14-labeled tyrosine incorporated into the melanin of house mouse skin (principally in active hair bulb melanocytes) from various genotypes have involved in vitro incubation of skin slices in media containing the labeled tyrosine (Fitzpatrick et al., 1958; Fitzpatrick and Kukita, 1959; Coleman, 1962; Wolfe and Coleman, 1964), or in vivo administration of labeled substrate (Coleman, 1962), followed by radioactivity measurements of extracted melanin (Coleman, 1962; Wolfe and Coleman, 1964) or in situ estimates from autoradiographs of sections (Fitzpatrick et al., 1958; Fitzpatrick and Kukita, 1959). An additional recently developed melanin assay, involving turbidimetric measurements of alkaline thioglycolate skin suspensions, has proved useful for measuring both initially present (in unincubated controls) and newly formed tissuebound melanin resulting from in vitro incubation of lyophilized skin obtained from the house mouse (Foster, 1959, 1963a,b; Foster and Thomson, 1961) and from the deer mouse, Peromyscus (Foster and Barto, 1963). Finally, oxygen consumption nieasurenients providing information on the earlier oxidative phases of melanogenesis, with tyrosine or dopa as substrates, were originally employed as the sole or principal melanogenic assay, using homogenized skin of the house mouse (Foster, 1951) or guinea pig (Foster, 1956), and subsequently as part of a balancesheet approach, involving both oxygen consumption measurements and melanin assays, to the study of both the early and late phases of melanogenesis in the house mouse (Foster, 1959, 1963a,b; Foster and Thomson, 1961) and in Peroinyscus (Foster and Barto, 1963). Finally in our most

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recent studies (Foster, 1963a; Foster and Thomson, 1961 ; Foster and Barto, 1963) we have added to our balance-sheet approach turbidimetric measurements of darkening of incubation media owing to escape of diffusible melanogenic intermediates from incubated skin. (The emphasis on our more recent combination of manometric oxygen consumption and turbidimetric melanin assays is occasioned by some published criticism of my 1959 paper to the effect that that study depended solely upon oxygen consumption data and lacked measurements of actual pigment formed. This criticism of the 1959 paper is, of course, without foundation. Either the criticism was intended for an earlier study, in which case I should be the first to concur, or else there was singular oversight of both the whole matrix of turbidimetric melanin assay data [Foster, 1959, Table 1, pp. 306-3071 in which the oxygen consumption data were embedded and the text itself, which explicitly contrasted t,he manometric and turbidimetric assays performed on the same skin samples.) Strictly speaking, none of the above-mentioned types of assay can be termed simply “tyrosinase” or “dopa oxidase” activity assays, although they are related to these activities. Since these enzyme activities refer only to the first two oxidative reactions in the melanogenic sequence, and since the subsequent oxygen-consuming reactions are not known to be enzymatically catalyzed, one is not strictly justified in calling the oxygen consumption measurements of all the utilized oxygen direct measurements of tyrosinase or dopa oxidase activity. As for the various melanin assays previously summarized, these depend, in the main, upon insoluble melanin deposits formed in incubated skin. Thus diffusible melanogenic intermediates are constantly leaking into the medium and and are washed away at the termination of an incubation experiment. Thus it might be difficult to diagnose those genetic lesions in which high enzymatic activity might happen to be correlated with diminished ability to deposit melanin upon a defective melanosome matrix. In such a case, what in fact should be termed “high tyrosinase activity” could be construed as “low tyrosinase activity” on the basis of faulty melanization. For these reasons the less specific term “melanogenic activity” is used here. Aside from the qualifications mentioned above, some additional comments are in order. First, oxygen consumption measurements with tyrosine or dopa substrates might not be entirely relevant to melanogenic activity; some nonspecific oxidation could occur. Thus additional checks should accompany such assays, for example the nonmelanogenic activity of albino skin toward these compounds. Moreover, the manometric technique is less sensitive than the histochemical dopa reaction in detecting weak react,ions, especially on lightly pigmented backgrounds,

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On the other hand, the visual grading technique probably imposes a lower ceiling on the observer than the manometric method, especially serious when dopa is used as substrate and there is much initially present eumelanin (Foster, 1956; Wright, 1963). The more recently developed assays for newly formed melanin (Foster, 1959; Coleman, 1962) seem to strike an optimal compromise among the desirable features of sensitivity, range, objectivity, quantitative scale, and independence of initial state of melanization. Furthermore, since no single assay procedure can be considered adequate to measure all phases of mammalian melanogenesis, it would be desirable to employ a combination of assays in order to arrive at a more reliable judgment of the likely locus of a genetic lesion, as well as of the possible syndrome of secondary consequences. Finally, the difficulby of distinguishing between gene-controlled alterations in amount and specific activity of enzyme has already been mentioned. 2. Physiological Genetic Interpretations of the Roles of Several Color Loci

When viewed in sufficiently broad perspective many of the sets of observations agree on such matters as order of effect as a function of genotype, or a t least they can be reconciled as different partial samplings of the total complex picture of mammalian melanogenesis. It is therefore instructive and, in any event, desirable to consider how various contributions lead to conclusions concerning the roles of some of the better known and studied color loci. a. The Albinism Locus (c)-The Structural Gene f o r Tyrosinase. From the vantage point of hindsight it is easier to recognize that the early thermolability studies of Danneel (1941) and others implied that the gene locus for albinism is probably the only significant gene (determining primary structure or amino acid sequence) for the tyrosinase molecule. Thus Danneel’s studies showed increasing thermolability of dopa oxidase extracts of skin containing lower albino series allelic combinations in the rabbit, Moreover this conclusion is supported by all reported observations on albino homozygotes, which generally lack melanin and also lack demonstrable melanogenic activity. In addition, intermediate levels of activity between that for homozygotes for full color at this locus (CC) and homozygotes for the lowest allele (cc) could be demonstrated in a number of intermediate allelic homozygotes and compounds. Indeed, the only apparent exception noted involved the inability to demonstrate manometrically any melanogenic activity in intensely colored eumelanic guinea pig genotypes lacking the full color allele C (Foster, 1956). This drastic effect was in all likelihood accentuated both by the low sensitivit.y

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of this method and by the thermolability of the enzyme tyrosinase with respect to preparation and assay procedures. The most recent and convincing demonstrations of both intermediate levels of melanogenic activity for intermediate albino series allelic genotypes and increased thermolability of tyrosinase in these genotypes was provided by Coleman (1962) in the case of the house mouse. Our own preliminary studies with both house mouse and Peronzyscus albino series genotypes confirm Coleman’s observations as to reduced levels of melanogenic activity in intermediate allelic combinations and also numerous instances of incomplete dominance. I n conclusion, then, a variety of studies involving different investigators, techniques, and tested organisms converge on the gene locus for albinism as the structural gene for the tyrosinase molecule. b. The Brown Locus (b). Despite the fact that in various tested organisms black (BB) genotypes produce much more melanin than do brown (bb) genotypes, often about twice as much (E. S. Russell, 1946, 1948; Wright, 1949, 1963; Foster, 1956, 1959, 1963a,b), the common experience with melanogenic assays has been to observe either no corresponding decreased activity or, more often, increased activity of brown skin over black skin when treated with exogenous substrates (W. L. Russell, 1939; Ginsburg, 1944; L. B. Russell and W. L. Russell, 1948; Foster, 1956, 1959, 1963a,b; Coleman, 1962; Wolfe and Coleman, 1964; Fitzpatrick et al., 1958; Fitzpatrick and Kukita, 1959; Wright, 1963). Only our own balance-sheet approach, using a combination of assays of melanogenic activity, indicates the additional occasions where black skin is superior to brown skin-as indeed it must be in vivo (Foster, 1959, 1963a,b; Foster and Thomson, 1961). Thus, for example, when skins are left to their own endogenous resources, i.e., incubation simply in phosphate buffer, the black homozygote, in both house mouse and Peromyscus (Foster and Barto, 1963; also 1964, unpublished data) is superior in its darkening ability to all other homozygotes or heterozygotes for other alleles at the brown locus. In the house mouse, then, this BB superiority is extended to comparisons involving not only the b allele, but also the the alleles “light” ( P )and cordovan ( b c ) (Foster, 1961, unpublished data). From the above considerations it seems that the proper question to ask is not simply ‘(What is the basis for the experimentally observed melanogenic superiority of brown over black skin?” but rather, “What is the genetic damage in the brown genotype which, in comparison with the black genotype, results in less natural melanin produced, less darkening of incubated skin when it must depend upon its own endogenous resources, and greater assayed enzymatic responsiveness to the presence

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of added substrate?” Moreover, it seems pertinent to recall th a t the electron microscopic study by Rittenhouse (1965) indicated that intense brown hair bulb melanosomes suffer from disorderly internal structure when compared with corresponding black nielanosomes in the house mouse. An attempted integration leads to the following tentative explanation (see Fost,er, 1963a; Foster and Thomson, 1961; Foster and Barto, 1963): The brown locus is the structural gene for a protein, other than tyrosinase, serving as an architectural Component of the melanosome matrix. Moreover, mutational alteration of this b protein could, secondarily, cause alteration of one or more of the following: (1) specific activity of tyrosinase, (2) number or binding effectiveness of melanin polymer binding sites, (3) provision of endogenous substrate, and (4) structural organization of the protein matrix. The increased enzymatic responsiveness of brown skin toward added substrate and also its weak in vitro darkening in the absence of added substrate might, perhaps, be attributed to the same cause-disturbed organization of substrate provision in vivo. Based on this viewpoint more enzymatically active centers, not combined with endogenous substrate, would be free to combine with added substrate in brown than in black skin. Even Coleman’s (1962) results with in vivo administration of labeled tyrosine might be explained in this way, especially in view of the tyrosine and dopa injection experiments of Galbraith (1964). Finally, the brown defect could even result in altered melanin polymer formation. This is suggested by the previously mentioned differences in infrared absorption spectrum patterns of melanin preparations from brown, cordovan, and black house mouse skin. It must be emphasized that the explanation proposed above is highly speculative, and it must eventually be tested b y separating, identifying, and recombining the protein components of the melanosome matrix, and thereby restoring a pattern characteristic of a specific genotype. I n any event, the broad strategy of explanation seems reasonable, namely, that an altered single component of a pattern can alter, simultaneously, various observed aspects of that pattern. If these different aspects are considered different unit characters or traits a t the biochemical and ultrastructural levels, then one can readily visualize a syndrome of simultaneous secondary consequences, or pleiotropic effects, resulting from a single gene mutation. Obviously, such a viewpoint also implies a structural basis for genic interaction by way of protein-protein interactions of the gene products participating in the construction and functions of an organelle. c. T h e Pink-Eyed Dilution Locus ( p ) . According to Rittenhouse (1965), the principal effect of pink-eyed dilution ( p p ) in both bb and BB

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house mouse hair bulb melanocytes is to decrease the amount of pigment deposited on the melanosome granule framework. Although earlier studies provided no adequate clue as to the nature of this melanogenic lesion (W. L. Russell, 1939; Ginsburg, 1944; L. B. Russell and W. L. Russell, 1948; Foster, 1956), more recent studies point to defective melanization, rather than defective tyrosinase activity, as the major effect of mutation a t this locus. Thus Fitzpatrick et al. (1958), Fitzpatrick and Kukita (1959) and Coleman (1962) noted diminished incorporation of CI4-labeled tyrosine by p p house mouse skin. Moreover, Foster (1959), contrasting manometric and turbidimetric assays of melanogenic activity in house mouse skin noted that p p genotypes, when contrasted with their PP counterparts, did not in general darken as well as might be expected from their manometrically measured abilities to oxidize tyrosine and dopa, thereby leading to the conclusion that this mutation results “in defects in later parts of the melanogenic sequence, perhaps in polymerization or in the conjugation of a late intermediate with the colorless portion of the melanin-forming granules.’’ Our most recent studies with Peromyscus (Foster and Barto, 1963; also unpublished data) provide additional support for this conclusion. In this mammal the two similar pale mutants, pink-eye ( p p ) and ivory (ii), on a black (BB) background, exhibit far more manometrically measured oxidative activity toward tyrosine and dopa than does intense black skin. This dramatically increased oxidative activity in these mutants is correlated with dramatically increased darkening of the dopa incubation medium, when compared with the darkening of medium produced by intense black skin. Nevertheless, the total quantity of tissue-bound melanin (both initially present and newly formed on incubation with saturating amounts of dopa) in these mutants is less than that observed for intense black skin. Thus it appears that. the increased levels of manometrically measured oxidative activity of these mutants are relevant to melanogenic activity, but the high enzyme levels lead to relatively greater loss by leakage of diffusible melanogenic intermediates from the incubated tissue into the incubation medium, rather than to more insoluble total tissue-bound melanin than is found in intense black skin. All of these results point to a defect in the number or effectiveness of the melanosome binding sites to which the melanin polymer becomes attached. Finally, it seems reasonable to conclude that the locus for pink-eye ( p ) , as well as the ivory (i) locus in Peromyscus, is the structural gene for still another protein component of the melanosome matrix, which either provides melanin binding sites itself, or else contributes indirectly to the melanin binding capacity of the melanosome matrix. Mutational alteration of either protein leads to a defect in melanin binding capacity

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and also, secondarily, to increased tyrosinase activity. Additional, currently unknown, secondary alterations in melanosome function might contribute to the drastically reduced pigment deposition on the melanosome matrix. 3. Conclusion

It seems premature at this time to discuss the chemical or ultrastructural roles of other color genes, including some whose main effects appear to be on granule distribution or melanocyte morphology (Silvers, 1961; Billingham and Silvers, 1960). While the enzymatic and ultrastructural roles of numerous other color genes in various species await study, enough information has already emerged to indicate th a t the structured system of mammalian melanogenesis constitutes an excellent source for analyzing cases of genic interaction and pleiotropy at biochemical and ultrastructural levels, F. BIOCHEMICAL STUDIES OF THE AGOUTIPATTERN The biochemical basis of the wild-type agouti pattern, a yellow subterminal band on an otherwise eumelanic dorsal hair, has been studied intensively in several laboratory mammals, especially during the past fifteen years. For example, in the case of the rabbit, Danneel (1949) viewed the yellow band as a possible consequence either of a temporary inhibition of dopa oxidase activity or of a temporary drastic reduction in the rate of dopa oxidase synthesis. Moreover, Cleffinann (1953) noted that in the rabbit the period of phaeomelanin synthesis was associated with two features: (1) The melanocytes showed increased levels of sulfhydryl conipounds (which might inhibit tyrosinase activity) ; and (2) there was an increased growth rate of the hair and a correlated increased mitotic index in the cells of the hair matrix. These descriptive observations led to Cleff mann’s more extensive combined descriptive and experimental analysis of gene action a t the agouti locus in the house mouse (Cleffmann, 1954, 1963). Foster (1951, 1956), on the basis of oxygen consumption studies with house mouse and guinea pig skin, suggested that yellow band formation might be due to sharp fluctuations in the level of a sulfhydryl tyrosinase inhibitor, presumably emanating from the keratinizing epithelial pigment-receiving cells of the growing hair. The influence of the follicular environment in deciding whether melanocytes would produce eumelanin or phaeomelanin had been conclusively demonstrated by Silvers (1957, 1958a,b) and b y Silvers and Russell (1955) by means of skin transplantation studies in the house mouse. The more recent study by Cleffmann (1963), involving experimental manipulations of house mouse skin of different agouti locus

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genotypes cultivated in vitro, implicates even more strongly the correlated features of increased sulfhydryl levels and increased hair follicle mitotic activity. Moreover, he observed that different agouti locus genotypes exhibited different thresholds of response (phaeomelanin synt’hesis) to glutathione added to the culture medium. This point could be studied because all agouti locus genotypes produced eumelanin under standard culture conditions. Thus ut (black-and-tan) and u (nonagouti) melanocytes were induced to form yellow pigment a t the highest glutathione concentrations used. AY (yellow) cells needed only a very low glutathione concentration to resume phaeomelanin synthesis in vitro. Finally, A (agouti) melanocytes showed a fluctuation in threshold sensitivity, with the period of lowest threshold or greatest sensitivity to glutathione corresponding to the time when the yellow band would normally have been formed. Another set of variables has recently been reported b y Galbraith (1964), who experimentally modified the house mouse agouti pattern by local administration of the mitotic inhibitor, colchicine, and also b y the local infusion of tyrosine and dopa. These treatments resulted in a switch from phaeomelanin production to eumelanin production. Galbraith’s suggested explanation is that during the period of rapid hair growth, substrate (presumably tyrosine) common to both hair growth and melanogenesis could be drained into hair production, “thereby reducing below a critical threshold the amount available for melanogenesis. Consequently, synthesis of eumelanin ceases and is not resumed until the supply of substrate is sufficient to meet the demands of both hair production and melanogensis.” There appears to be no contradiction between the work of Galbraith and of Cleffmann. Their different findings seem rather to complement one another in demonstrating that yellow band formation in each case is associated with a period of rapid hair growth correlated with conditions unfavorable for eumelanin synthesis. V. Color Genes and Evolutionary Biology

A. INTRODUCTION I n this section only a small sampling of information indicating the relevance of mammalian pigment genetics to various aspects of evolutionary biology will be cited. More extensive information is readily available in the reviews and other references to be cited below.

B. COMPARATIVE GENETICSOF COATCOLOR The meaningful relationship between genetics and evolution was clearly pointed out by Dunn (1922) in his comparisons of color inheritance

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in different rodents. Criteria of genetic homology required by Dunn included phenotypic similarity, similarity in mode of inheritance, and siniilarity in linkage relations. I n addition Little (1958) has proposed the criteria of similar pleiotropic effects of genes, similarity of multiple allelic series, and similar morphogenesis and function of pigment cells. Additional considerations are stated by Searle (1961) and Nachtsheim (1959). It might even become possible to homologize specific nucleic acid sequences of genic dimensions (see Hoyer et al., 1964). Several well-known color genes meet at least some of the criteria of genetic homology, for example, the agouti, brown, albinism, and pink-eye loci. Moreover, the criterion of similar linkage relations is met by the gene loci for albinism and pink-eye in three rodents: Mus musculus, Rattus norvegicus, and Peromyscus maniculatus (Little, 1958). IN NATURAL POPULATIONS C. COLORPOLYMORPHISMS OF THE HOUSEMOUSE

Although there have been some recent interesting studies of cat coat color gene frequencies in Singapore (Searle, 1959) and Boston (Todd, 1964), i t is not entirely clear to what extent the cats observed represent samplings of feral populations. I propose, therefore, to consider very briefly some polymorphisms in house mice only. I n conjunction with their study of the distribution of t alleles in feral house mouse populations, Dunn et al. (1960) noted that all mice captured exhibited the black agouti phenotype, except for one extreme whitespotted variant. I n general, their population samples indicated three color polyiiiorphisms involving gray, yellow, and white belly color (alleles A , A L and A W ) ;white spotting, and intensity of over-all color (light and dark agout,i). No gene frequency estimates were given. As part of an extensive survey of polymorphisms in natural populations of the house mouse in the Ann Arbor, Michigan, area, Dr. Michael L. Petras (1962, unpublished data) has tested captured wild mice for polymorphism with respect to a number of color genes. I n confirming Dunn et al. (1960), Petras has also observed polymorphism at the agouti locus (alleles AW and A ) . Moreover, the estimated allelic and genotypic frequencies confirm other data obtained b y Petras indicating a significant numerical deficiency of heterozygotes when tested against HardyWeinberg equilibrium expectations. One possible explanation suggested by Petras for this heterozygote shortage is the structuring of the natural population into small panmictic breeding units (demes) with associated gametic correlation. Additional quite extensive tests of heterozygosity for a number of recessive mutants met with negative results (i.e., b, c, p , s, d, pa, ru).

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Petras’ proposed explanation is that any homozygous mutant arising in the wild would be conspicuous to its usual predators and, hence, would be subject to strong selection. Moreover, for the sample size tested, and assuming reasonable levels of inbreeding and mutation rate, it is not surprising that no heterozygotes at these loci were found. D. COATCOLORAND ADAPTATION IN DEERMICE Dice (1947) devised an experiment in which buff (allele G ) and gray (allele g) deer mice were subjected to owl predation under conditions of alternate exposure on two colors of soil, one type matched one mouse color variety, and the other type of soil the other variety of mouse. Under these experimental conditions the mice that were inconspicuous when viewed against their backgrounds were favored, i.e., less subject to predation. In a population study of the distribution of the same alleles G and g in Peromyscus maniculahs blandus in New Mexico, Blair (1947) noted differentiation in buff -gray frequencies in populations several miles apart and living on differently colored soils. Thus on dark red soil, buff was favored over gray, and on pinkish gray soil the nonadaptive buff was more subject to predation. Thus, at least one of the selective forces molding the complex biochemistry of melanogenesis to produce inconspicuous coloration is predation involving visual cues. VI. Concluding Remarks

I have resisted the strong temptation to discuss additional aspects of mammalian pigment genetics, such as various associated biochemical, physiological, or even behavioral features which might, or might not, represent pleiotropic manifestations of color genes. These and other tempting topics were judged ruthlessly against the question, “What will this tell us about mammalian pigment genetics?” I hope, however, that enough pertinent information has been presented to indicate that this subject enjoys not only a distinguished history but also expectations of a challenging and rewarding future. ACKNOWLEDGMENTS I am happy to acknowledge the courtesy of Dr. Elizabeth W. Rittenhouse and Dr. Michael L. Petras in kindly permitting references to some of their unpublished observations. My thanks also go to Dr. Elizabeth Barto for her guidance to literature concerning Peromyscus. I am also indebted to Mrs. Evelyn M. Peterson and Mrs. Sarah S. Measel for devoted virtuosity in typing the manuscript and to Miss Lucinda Thomson for her painstaking,time-consumingassistance connectedwith proof-reading text, bibliographic references, and research cited.

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Sever, R. J., Cope, F. W., and Polis, B. D. 1962. Generation by visible light of labile free radicals in the melanin granules of the eye. Science 137, 128-129. Silvers, W. K. 1956. Pigment cells: occurrence in hair follicles. J. Morphol. 99, 41-55. Silvers, W. K. 1957. Melanoblast differentiation secured from different mouse genotypes after transplantation to adult mouse spleen or to chick embryo coelom. J . Exptl. 2001.136, 221-237. Silvers, W. K. 1958a. An experimental approach to action of genes a t the agouti locus in the mouse. 11. Transplants of newborn aa ventral skin to ata, A% and aa hosts. J . E ~ p t l2002. . 137, 181-187. Silvers, W. K. 195813. An experimental approach to action of genes at the agouti locus in the mouse. 111. Transplants of newborn Am-, A - , and &skin to Ag-, A w - , Aand aa hosts. J. Exptl. 2001.137, 189-196. Silvers, W. K. 1961. Genes and the pigment cells of mammals. Science 134, 368-373. Silvers, W. K., and Russell, E . S. 1955. An experimental approach to action of genes a t the agouti locus in the mouse. J. Exptl. 2001.130, 199-220. Staubli, W., and Loustalot, P. 1962. Electron microscopy of transplantable melanotic and amelanotic hamster melanomas. Cancer Res. 22, 84-88. Swan, G. A. 1963. Chemical structure of melanins. Ann. N . Y . Acad. Sci. 100, 10051019. Todd, N. B. 1964. Gene frequencies in Boston’s cats. Heredity 19, 47-51. Waddington, C. H. 1962. Specificity of ultrastructure in developing cells and its genetic control. J. Cellular Comp. Physiol. 60, Suppl. 1, 93-105. Wellings, S. R., and Siegel, B. V. 1963. Electron microscopic studies on the subcellular origin and ultrastructure of melanin granules in mammalian melanomas. Ann. N . Y . Acad. Sci. 100, 548-568. Wolfe, H. G., and Coleman, D. L. 1964. Mi-spotted: a mutation in the mouse. Genet. Res. 6 , 432-440. Wright, S. 1949. Estimates of amounts of melanin in the hair of diverse genotypes of the guinea pig, from transformation of empirical grades. Genetics 34, 245-271. Wright, S. 1963. Genic interaction. In “Methodology in Mammalian Genetics” (W. J. Burdette, ed.) pp. 159-192. Holden-Day, San Francisco, California. Zelickson, A. S. 1962. The fine structure of the human melanotic and amelanotic malignant melanoma. J. Invest. Dermatol. 39, 605-613. Zelickson, A. S., Hirsch, H. M., and Hartmann, J. F. 1964. Melanogenesis: a n autoradiographic study a t the ultrastructure level. J. Invest. Dermatol. 43, 327-332.

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THE COMPLEX LOCUS R IN Mormoniella vitripennis (WALKER)" Anna R. Whiting Biology Division, Oak Ridge National laboratory, Oak Ridge, Tennessee

I. Introduction. . . . . 11. The R Locus. . . . . A. Eye-Color Mutants B. U n s t a b l e 0 . . . . C. Deleterious Factors 111. Discussion. . . . . . References . . . . . .

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1. Introduction

Mormoniella vitripennis (Walker) and Nasonia vitripennis (Walker)are two long nanies given to a 2-mm short wasp parasitic on the pupae of blowflies and other large species of muscoid Diptera. This wasp is a giant among its relatives of the Chalcidoidea, the largest superfamily of the order Hymenoptera, which are mostly minute egg parasites. The two generic nanies were used with equal frequency when the genetical work was begun in 1948, a t which time Mormoniella was advised. At present Nasonia is approved, but because all papers on genetics published since 1948 have used Mormoniella, this name is being retained to avoid confusion. Since there is only one known species of the genus, the specific name is omitted in most genetical papers. The sexes can be readily separated, both as pupae and as adults, and large numbers of virgin females can be obtained by breaking open the puparial shells of infected hosts arid sexing the wasp pupae. About 80% of the progeny of mated females are female (diploid), and the remainder are males (haploid). Diploid males are not normally produced. Unmated females oviposit as readily as niated feniales and lay the same numbers of eggs, all of which develop into males. As many as 200 adults can be

* Research on Mormoniella was conducted by P. W. Whiting and associates a t the University of Pennsylvania with support from Contract AT(30-1)-1471 between the U.S. Atomic Energy Commission and the University. The review was prepared a t the Biology Division of the Oak Ridge National Laboratory, operated by Union Carbide Corporation for the U.S. Atomic Energy Commission. 341

342

ANNA R. WHITING

obtained from a single infected pupa of the large blowfly, Sarcophaga bullata Parker, the species used by Whiting for his research. Fifty to seventy-five is the optimum number for obtaining wasps of maximum size. One female may produce 600 progeny if a sufficient supply of hosts is available. A generation at 28°C is about 10 days in length. Exposure of females to X-rays gave the first mutants, eye colors oyster and scarlet, and the exceptional genetical behavior of these was disclosed at once. They, as well as the complex locus which they exposed, were named for their finder, D. T. Ray. The mass of mutations associated with the R locus suggests a veritable opening of Pandora’s box, as Whiting has expressed i t (1956). At the Mormoniella stock-keeping center at Dartmouth College there are sixty-one stocks involving different mutations in this complex locus, and this is a relatively low percentage of the possibilities. Because of the frequency of eye-color mutations and the diverse behavior patterns of many, phenotypically identical, each mutant which has been kept has been given either the initials of the finder, or in later work, a three-digit number combined with a n abbreviation of the color. Purple body color (pu), recessive to wild type, was found in a wild population in Woods Hole, Massachusetts. It is linked with the R locus and gives about 11% recombinations. II. The R Locus

A. EYE-COLOR MUTANTS Wild-type eye color in Mormoniella is a dark reddish brown. Scarlet a minute black fleck, whereas oyster (oy) is a dark pearl gray with no trace of red. These mutant characters are designated st-DR and oy-DR. The females which produced them had been exposed to 6000 r of X-rays. Each mutant stock showed normal fertility and viability. Wild-type eye color was found to be dominant to each, and mutant males were produced by Fl females heterozygous for either color in numbers comparable to wild type. For each mutant the relation to wild type was “orthodox.” Crosses between scarlet and oyster gave wild-type daughters, suggesting that different loci were involved, and i t was expected that haploid males of four genotypes would be produced by the F1 females. However, only scarlet and oyster males appeared. (st) is a brilliant clear red with

x st 3 3 st/st 9 9 x 02/ 3 d oy/st 9 9 unmated OYlOY 9 9

+ 99 +99

1257 oy $ 3 456 2771 st 33 1827 st $ 3 4376 oy 33 4215

COMPLEMENTATION IN

Mormoniella

343

Fortunately, wild type is a color quite distinct from these mutant eye colors, so that no question could be raised about the phenotype of the F, females. Why has the wild type never been produced by these F, females among 25,135 FB males recorded? Morgan, et al. (1925) wrote, “In all cases of multiple allelism it has been found that the genes affect the same character or characters in approximately the same way and when two of the genes, other than the wild-type from which they are derived, are brought together, they do not give the wild-type character but a character that is as a rule more or less intermediate between the two . . . . The two allelomorphic recessive genes do not produce a character in excess of that produced by the more effective gene.” As judged by the F1 results, oyster and scarlet are not alleles; by Fz results, they segregate as a single pair of allelic genes. Thus, with the very first mutations found in Mormoniella, the clear-cut behavior of the R-locus mutants was demonstrated and found to be unorthodox. No recombinations have been found between the “elements” 0 (for oyster) and S (for scarlet). The genetical possibilities, og.+, +.st, and oy.st all exist, but each mutant type has been obtained by mutation of one or the other or both of the factors. Mutations to scarlet and oyster have occurred many times and are between two and three times as frequent as all other eye-color mutations combined, with scarlet from two to three times a s frequent as oyster. If lethal and sterility factors are excluded, mutation rates in S and 0 are approximately equal. Double mutations in these factors are about ten times as frequent as expected on a random basis. Spontaneous eye-color mutation from wild-type stock is probably somewhat less than 0.004 %, X-ray-induced mutation as high as 1.8% (Caspari, 1958). Whiting uses the term “factor” for the 0 and S elements and “gene” for the segregating unit, produced by mutation and characterized by one or more mutant “factors.” Demerec in 1961 wrote, “Mutants resulting from mutations at different sites of the same locus (alleles) or even a t a single site, may differ in several respects. As a rule, however, such mutants have one feature in common, they are changes with regard to one specific function controlled by that particular locus.” Visible mutant changes in the R locus illustrate this whether each represents a site or a factor in the gene. Kayhart later carried out X-ray experiments similar to those of Ray and found a third and a fourth eye-color mutant, garnet, ga, and vermilion, vm (Kayhart and Whiting, 1949). The three “red” eye colors form with wild type a series of decreasingly dark pigmentation. Wild type is reddish brown, whereas garnet is brownish red. Vermilion is a brilliant color with a very prominent black fleck. Scarlet is similar to

+.+,

+

344

ANNA R. WHITING

vermilion except that the red is clearer, appearing more intense, and the fleck is very minute, often invisible. Oyster eyes are dark pearl gray without a trace of red. All pigment appears to be lacking in them. Vermilion was found to be allelic with scarlet, whereas garnet segregated freely from the other colors and was neither a n R factor nor a n R-linked gene. Although the original mutants mentioned above were X-ray-induced, they were accompanied by no deleterious effects. This was fortunate, for it enabled Whiting and his students to draw conclusions which would have been obscured, perhaps, by sterility or lethal factors. Many of the X-ray-induced eye-color changes in the R locus found later were so accompanied. Eye colors are discussed first and the more complex aspects associated with deleterious mutations later. Work on the R locus was continued, and the information obtained up to 1954 is presented in Table 1 (Whiting, 1951, 1954). The R-locus TABLE 1 Phenotypes of the Ten Different Females Possible as Compounds for Five Alleles Formed by Mutation in Two Hypothetical Gene Elements Compound females

Hypothetical gene elements

dahlia-GF vermilion-MK dahlia-GF scarlet-DR dahlia-GF oyxter-DR dahlia-GF oyster-NH vermilion-MK scarlet-DR vermilion-MK oyster-DR vermilion-MK oyster-NH scarlet-DR oyster-DR scarlet-DR oyster-NH oyster-DR oyster-NH

da. +.urn da. -

+

~

+ da. + +.st

+

OY.

+

da. __ oy.st

+.urn +.St

+.urn __ oy.

+

+.urn oy.st

+.st ~

oy.

+

+.st oy.st OY. ~

+

oy.st

Phenotypes

Dominance relationships

Brown

Complete complementation

Brown

Complete complementation

Bright red

Partial dominance

Bright red

Partial dominance

Light vermilion

Blending

Brown

Complete complementation

Light vermilion

Blending

Brown

Complete com plementation

Scarlet

Complete dominance

Oyster-white

Homoaygous oyster

COMPLEMENTATION IN

Mormoniella

345

symbols representing factors within each locus are separated by periods. Note a second eye-color mutant, dahlia, in the 0 factor. Initials of the finders were still being used to designate each stock. Dahlia (du) had been found by Gladys Friedler, and a n oyster white (oy.st) phenotypically like oy-DR but by mutation a double recessive, by Nellie Harris. Symbols are clearly identifiable with colors, but one can foresee that, as studies continue, phenotypes are going to be increasingly difficult to describe. Attempts were made a t first to identify colors very exactly by comparison with color charts. This was soon found to be of little value. Changes of color with aging, angle a t which eyes were observed, etc., made a n objective decision concerning names difficult if not impossible. TABLE 2 Interaction of Eye-Color Genes Garnet (ga) and Tomato (to) with Alleles in the IZ Series

R Alleles

+.+

da.

+

+.vm +.st

+

OY. oyst

++ Brown Dark red Bright red, dark fleck Scarlet Oyster Oyster

9a

+

Dark red Bright red Scarlet Scarlet Oyster Oyster

+to Dull red Dull orange Bright orange Bright orange Oyster Oyster

ga to

Orange Dull orange Bright orange Bright orange Oyster Oyster

Further complications in this respect can be visualized from Table 2 (Whiting, 1954), for non-R eye-color mutants garnet (previously mentioned as found by Kayhart) and tomato had appeared by this time. There are represented in this table ten haploid phenotypes, twenty-four items. Garnet and tomato, both non-R, segregate independently of each other. Multifactor mutants in the R locus were obtained only by mutation, never by recombination. In addition to the 0 and S factor sites, one more was added in 1957 by G. B. Saul. This was described at first as mahogany and the factor designated M . Further study deliionstrated its resemblance to dahlia, and it was called da-845, but the M designation was retained. A fourth factor, P , was identified by a dahlia (du-442) practically identical in phenotype t o the other dahlias. This is very closely linked to the R locus and is considered to be in it, although it very rarely undergoes recombination. Since no recombination within the R region has been found, with the exception of very rare cases between M and P , the order of the elements in i t had to be arbitrarily assigned. 0 was placed a t the extreme left, and S at the right of 0, followed by M and P.

346

ANNA R. WHITING

I n summary, there have been found mutations to eleven allelic states in 0, five in S , one in M , and one in P (see Table 3). Many of these have occurred several times; since they often show minor differences in phenotype or in behavioral patterns, each has been kept separate. I n 0 and in S mutations to oyster and scarlet, respectively, have been relatively frequent. Any one in one factor in combination with any one in a second factor gives wild type, and segregation takes place without recombination. With the eighteen mutant types listed (Table 3), the numbers of possible TABLE 3 Eye-Color Factors and Their Alternative States in the R Locus

0

+

Oyster Dahlia Tinged Orange Scarlet Mahogany Tomato Reddish Apricot Red Peach

M

S

+

1Scarlet Vermilion Mahogany Red Dahlia -

P

+

Dahlia -

Dahlia

-

-

-

-

-

-

-

-

-

-

genotypes can beestimated. It is possible to obtain 288 kinds of ganietesor of haploid males, and gametes of 288 kinds are capable of combining to form 41,616 diploid genotypes or females. Twenty-four of the haploid phenotypes are oyster, since oyster masks other colors. One can deduce from these genetical facts the problem of indicating phenotypes, all of which must lie within the range bounded by oyster and dark brown. It would seem that a n analysis such as th at made by Whiting and his students would have been impossible were it not for haploid males. Of the maximum of 288 haploid eye-color genotypes, fifty-five have been identified. Twenty scarlet stocks have been used (+.st, stst, mh.st, da.st, etc.), and eleven oyster stocks (oy.+, oy.st, etc.). It will be noted that the same name is sometimes given to colors in different factors. This is because of their resemblance to each other phenotypically. Deleterious factors have been mentioned, and one mutant gene, st-474, is the only one in which there is a lethal associated with a spontaneous eye-color mutation. All unfertilized st.Z eggs fail to develop. I n association with oyster, +.st.Z/oy. females will produce oyster

+.+,

COMPLEMENTATION

IN

Mormoniella

347

sons only. When mated to wild-type males, daughters will be wild type their genotypes phenotypically (+.st.E/+.+.+ and oy.+.+/+.+.+), identified by breeding tests.

B. UNSTABLE 0 A female compound for dahlia-GF and scarlet-DR (da.+/+.st) produced a mutant son. His eyes had a slight pinkish tinge, and the trait was described as “tinged” (ti-277) (Whiting, 196Ob). His gene formula was shown to be tist, suggesting that had mutated to ti in the +.st egg froin which he had developed. Seven other pale mutants were subsequently found among 9847 red-eyed sons of da. +/+.st females (da-GF/st-DR). They appeared in different vials and therefore represented different mut,ations. Four of these were found to be oyster in appearance, but three were tinged, resembling ti-277 phenotypically. One of the tinged proved to be in a non-R locus and a second, 23-248.3, to be ti.st like ti-277. Reciprocal crosses were made between Woods Hole wild-type and ti-277 stocks. When females of the former stock were used the cross gave rise to many bright-eyed mutants (scarlet, orange, and peach) among the expected wild-type and tinged Fn males, a t a rate of about one in 1000. Mutations of tinged to oyster (tist to oy.st) were not included, as their classification would have been time consunung a n d uncertain. When the reciprocal cross was made, ti-277 females by WHf males, mutation rate was no higher than that found in pure stocks, less than one among 25,000. Because the tinged-277 niulant male (tist) and his descendants have S in the st condition, no dark eye pigment could develop in his mutant male descendants or in pure stock females. There appeared oy.st, pe.st, or&, and to.st, and these could be identified by phenotypes, oyster, peach, orange, and tomato. All scarlet and indistinguishable are st.st, da.st, mh.st, rdh.st, and +.st. They can be differentiated in females compound for oy-DR (oy.+), as tabulated:

+

Female

Phenotype

+ + mh.st/oy. +

st.st/oy. da.st/oy.

rdh.st/oy. +.st/oy.

+

+

scarlet dahlia mahogany reddish wild type

348

ANNA R. WHITING

Mutant differences always showed linkage with purple body color, pu, normally eleven crossover units distant, suggesting that the change was a t R rather than due to some modifier a t another locus. Moreover, in fraternities from females compound for a stable allele, or oy.st, for example, and a gene descended from ti.& new mutations were associated with purple in the same way that the majority of types from t i s t were associated. After 4 years, continued appearance of 0-factor mutant eye colors was demonstrated from breeding tests. No lethal or sterility effects were associated with the eye-color changes, and the new eye colors have always been double-mutant as previously, remaining st in S. I n summary, gene descendants of a certain mutated gene, ti-277 (ti&) (tinged eye color), are characterized by a n unstable condition of factor 0 giving a range of red eye colors from reddish (rdh) near wild type, through mahogany (mh), dahlia (da), tomato (to), apricot (up), orange (or), peach ( p e ) , to tinged (ti) near oyster white. None of these relatively frequent spontaneous mutations is accompanied by a n y deleterious condition.

+.+

C. DELETERIOUS FACTORS 1. General

Perhaps the best method of demonstrating the complexity of the R locus, which was exposed as research on it continued, is to present Table 4 for comparison with Table 1 and for reference during the following discussion. There are now six factors; the X indicates those for which homologies are uncertain (Whiting, 1962a, b). These R-locus genes were selected from a large number that have been obtained by irradiation. The majority of them, unless indicated by initials of other finders, occurred in experiments carried out by S. B. Caspari (1958). This list includes two with semilethals, two with male steriles, two with near steriles, and ten with female steriles. Phenotypes of diploid compounds may be readily determined from the formulas of their two genes. Thus oy.+.+.+.+.fsb.+/+.st.+. fsa.+.+ is a wild-type female and fertile. Her sons will be oyster or scarlet and fertile, but will contribute sterility factors t o all their daughters. Sterility caused by fsb incapacitates the homozygous females so that they cannot sting the host and therefore cannot feed. T h a t caused by f s a prevents egg laying but does not affect other reproductive activities. Of the deleterious factors, only the semilethals, female steriles, and near steriles can be transmitted by haploid males; with the near steriles

COMPLEMENTATION IN

Mormoniella

349

TABLE 4 Designations of Some R-Locus Genes and Their Formulas Designations

Wild type Apricot-837 Dahlia-GF da-442 da-817 da-838 da-845, 846 Mahogany-441 rnh-819 mh-835 Orange-336 Oyster-DR OY-NH OY-250 oy-423 oy-800-804 OY-805 OY-816 OY-840 Peach-333 Scarlet-DR st-474 st-689 st-808, 810 st-811 ~t-812 ~t-813 st-814 st-821 ~t-823 st-824 st-829 st-830 st-831 ~t-834 ~t-841 ~t-855,856 Tinged-277 Vermilion-MK urn-809

s

0

M

P

+ . + . + . + . + . + . + . + . + . + . + . + . + + .. da+ . + . d a . + . + . + . fsa + rdh . + .. dd aa .. ++ .. + + + . + mh . + . + . + . + rnh . + . + . + . + + . mh . + . + . fsa or . st . + . + . + . + . + . + . + . . + . + . + . st . + . + . + . st . + . + . fsa . + . + . + . + . st . + . + . + . + . + . + . + . + . + . + . + . st . + . + . + Pe + . st . + . + . + + . st . + . + . + + . st . + . + . fsa + . st . + . + . + + . st . + . + . + + .. stst . + . + . fsa . + . + . + + + . st . + . + . + + .. stst . + . + . + . + . + . fsa + + .. stst . + . + . + . + . + . fsa + + .. stst . + . + . + . + . + . fsa + + . st . + . + . + + .. stst .. ++ .. ++ .. ++ + . ti . + . + . + + . vm . + . + . + + . urn . + . + . +

. + . + . + . + . + . +

aP da

OY

OY

st

OY

OY

OY OY

OY

OY

st

x

B

A

. .

. . . . . . . . . .

. +

fsb + .

l

+ + + + + . + + + + +

. . . . l . . . . .

X

+ + + + x + + + + +

. + . I s . + . l x . . msx . . nsx

+ +

. + . + . + . + . + . 1 x . + .. slx+ . ? . + . 1 . + . l . + . 1

. .

+

.

x x x msx

+

.

P

.

?

. +

. 1 x . 1 x

. + . +

. + . z x .

+

.

1

.

+

.

z

. .

+ . + .

x slx nsx x

. + . +

. + . + .

fsb

.

1x

350

ANNA R. WHITING

transmission is so difficult that it is more convenient to treat them as lethals or male steriles. The nonhomologous factors fsa and fsb have been mentioned. A balanced stock with mahogany435 contains them both. Its formula is +.+.+.da.+.fsb.+/+.mh.+.+.fsa.+.Zx. The symbol lx represents a lethal of which the homology has not been determined. These females produce dahlia sons only, .da. .fsb. the mahogany being associated with Zx. Fertile daughters will be like their mothers, fsblfsb being sterile. Thus a n alternation of types occurs at each generation. Diploid males have not been discussed, and a brief description of polyploidy as i t acts in Mormoniella should be given (Whiting, 1960a). It arose by mutation in a laboratory culture, with increase of the chromosome numbers from 5 to 10 in males and from 10 to 15 in females. Diploid males, like their haploid brothers, have no reduction in spermatogenesis. They are fully fertile and sire triploid daughters which, when bred unmated, produce a few sons, haploid and diploid. Haploid or diploid males always develop from unfertilized eggs, diploid or triploid females from fertilized. I n testing homologies between lethals, work on the haploid-diploid level is not adequate because these factors cannot be transmitted by haploid males (Whiting, 196213). Diploid males heterozygous for one lethal are crossed to females heterozygous for one. Daughters are triploid, and all carry the lethal borne by the father; in addition, one-half carry that borne by the mother.

+.+.+ + +,

Cross: +.st.+.+/oy.+.la.+ 9 X pe.st.+.+/+.st.+.Zb Daughters: +.st.+.+/pe.st.+.+/+.st.+.Zb oy.+.la.+/pe.st.+.+/+.st.+.Eb

3 scarlet wild type

The wild-type daughters carry both lethals. Their haploid sons will the all be peach. If wild-type sons are present, oy.+.Za.+/+.st.+.Zb, nonhomology of la and Eb will be demonstrated. Since triploid females produce few progeny, a fair number of them will have to be set to secure sufficient data. On the diplo-triploid level, 270 of 325 possible combinations of 26 R alleles carrying lethals, near steriles, or male steriles were made. I n only 17 of these was it shown that the lethals were homologous or identical, and these fell into one or the other of two homologous series. This is further evidence for the great complexity of the R locus. Whiting (1958) notes that the distinction between lethals and male steriles is somewhat arbitrary. Scarlet lethal-474 acts before hatching; oyster lethal-803 kills the half-grown larvae; scarlet lethal413 kills the pupae; vermilion lethal-809 acts at eclosion, the adults unable to walk

COMPLEMENTATION IN

Mormoniella

351

about, feed, or mate; scarlet-814 mates but produces no offspring; oyster840 rarely produces females, which are, in turn, near sterile. Ingenious methods of demonstrating the behavior of these deleterious factors have been devised by Whiting and his students, and some of these will now be described. 2. Stock Maintenance

The mutant stocks of Mormoniella may be refrigerated as diapause larvae for several months or up to 2 years. True breeding stocks without deleterious factors may be kept in active condition, slowed down by lowering of the temperature, and renewed every generation by setting females after they have emerged from host puparia and mated with their brothers. Genes with lethal or male-sterile factors must be run with “normal” mutant genes (without deleterious factors) or balanced against genes with female-sterile factors. Female-sterile-a (fsa) genes niay be balanced against nonhomologous female-sterile-b (fsb). Thus, scarlet-lethal-474 may be run with oyster-DR, in which case the compound feiiiales, +.st.lx/oy.+. are wild type and the homozygous oy-DR females must be discarded every generation; or scarlet-474 may be run with oyster-NH, in which case the compound females are scarlet, homo+.st.lx/oy.st. zygous st. If scarlet-474 is run against oy-423, there is a balanced stock, +.st. +.Zx/oy.st.fsa. with the lethal-bearing females scarlet and fertile, the oyster females sterile, homozygous fsa. This stock may be kept in mass culture without loss or decrease of the lethal-bearing gene. An fsa gene may be run against an fsb gene such as oy-423/da-442 (oy.st.+.+.fsa.+/+.+.+.da.+.fsb), which may be kept in mass culture with females of three types, the homozygous oyster and dahlia being sterile, the wild-type compound fertile and producing oyster males with fsa, dahlia males with fsb. Dahlia-442 may similarly be carried with a scarlet fsu gene, such as st-829 (+.st. .fsa. +). A male-sterile gene may be balanced against a female-sterile gene, such as st-814 against oy-423 (+.st.+.msx/oy.st.fsa.+), or oy-816 against st-829 (oy.+.+.msx/ +.st.fsu.+). I n the former case the scarlet males are sterile, the oyster males fertile. In the latter case the oyster males are sterile, the scarlet fertile. The fertile females are scarlet in the former case, wild type in the latter, and heterozygous for all four factors involved. Table 5 shows eight alternations of generations, or “Merry-GoRounds” as Whiting calls them (Whiting and Caspari, 1957), each involving three R-locus alleles: one lethal or male sterile and two either normal or fsa-bearing. For each Merry-Go-Round, the former are shown in bold type and, as the formulas show, carry an 1x or a n msx factor.

+, +,

+,

+.+

w cn

TABLE 5 Alternation of Generations

R Alleles Designations st-474 1 OV-DR

tQ

Odd generations 9 9

Formulas

Retained

9 9 Discarded

Even generations

33

9 9 Retained

9 9

Discarded

33

+.st.lx OY.+.+

OY-NH oy.st.

+

Scarlet

(Oyster)

Oyster

Wild type (Oyster)

Oyster

Dahlia

(Wild type)

Scarlet

Scarlet

(Wild type)

Dahlia

Wild type

Scarlet

Oyster (larvae)

Oyster

(Scarlet)

Scarlet (larvae)

Scarlet

(Apricot)

Apricot (scarlet pupae)

Wild type (Apricot)

OY-806 0 y . s t . h 2 st-DR +.st.+ ~ u - G F du.+.+

OY-803

OY.

+.+.h +

3 oy-423 ~t-689

oy.st.fsa.+ +.st.fsa.

St-813 4 UP-837

UP.+.+

+.st.h

OY-NH oy.st.

+

Oyster (scarlet pupae)

vm-809 +.vm.lx 5 or-336 mh-819

or.st.+ mh.

oy-816 6 pe-333 st-DR

oy.+.msx pe.st.4+.st.+

+.+

Wild type (Mahogany) Orange (vrn2)

Scarlet

(Mahogany) Wild type (vml)

Wild type

Tinged

(Scarlet)

(Scarlet)

Peach (oyster)

Scarlet (oyster)

0 0

TABLE 5 (Continued)

R Alleles Designations

Formulas

Odd generations 9 9

9 9

Retained

Discarded

+

st-814 .s~.~sz 7 pe-333 pest.+ Wild type OY-DR OY.+.+ da-817 8 up-837 st-858

+.da.fsa.lx up.+.+.+ +.st.fsa. 4-

Dahlia

5 M 5

l?

Even generations

$8

9 9 Retained

9 9 Discarded

(Tinged)

Peach (oyster)

Scarlet

(Tinged)

(Wild type)

Apricot

Wild type (Wild type)

$3

2

Oyster (scarlet)

Scarlet

0

3 * m* F c1

354

ANNA R. WHITING

These genes are present in every generation in the females retained. Both of the other two genes characterize the females discarded every generation, and they alternate every other generation between the females retained and the functional males. The mothers of the odd-numbered generations, F1, Fa, etc., have the first and second genes designated, the fathers have the third. The mothers of the even-numbered generations, F,, F4, etc., have the first and third, the fathers have the second. Phenotypes are given for each genotype in successive generations. Phenotypes of females discarded are given in parentheses, as are also those of the nonfunctional males. M I . This shows phenotypic alternation in females retained, which are scarlet in odd generations and wild type in even, the latter due to coniplenientary allelism. The oyster males of the former are oy-DR, of the latter, oy-NH. M d . This was devised by Mrs. Caspari in order to maintain oy-805, a triple-factor mutant from wild type. This gene cannot be run conveniently with scarlet-DR because females to be retained and females to be discarded are both scarlet and nonseparable. It cannot be run conveniently with dahlia-GF because the homozygous females to be discarded overlap in phenotype with the oyster-dahlia females to be retained. This alternation of generations solved the problem. The wildtype females to be discarded are easily distinguished from the red to be retained. The third eye-color factor, dahlia-M mutation, da-846 (Saul, 1957), now makes it possible to maintain oy-805 in combination with this gene alone, oy.st. .Zx/+. .da. The dahlia females are discarded, the wild-type retained for the stock. MS. This is a n oyster lethal run with two fsa genes giving sterile scarlet discard females. The lethal is oy-803 and, unlike the egg lethals of the first two, has its incidence in the larval stage. Half-grown larvae, which will live for many days after their viable adult brothers have emerged, are to be found in the puparia. A striking demonstration may be made by setting the females unmated; counts show these larvae to be almost equal in numbers to the adult males. If large numbers of the scarlet females are set with a few host pupae, a few eggs will be found because f s a sterility is not complete. These eggs develop normally, resulting in viable adults. M4. This contains a scarlet lethal gene with incidence in the pupal stage. Scarlet-eyed dead male pupae are found and may be obtained in large numbers from unmated mothers. M 5 . This lethal has incidence at time of eclosion. Many of the vermilion haploid males are able to eclose but are unable to walk about, feed, or mate. Their eyes appear rather small and are very dark red, much

+

+ +.

COMPLEMENTATION

IN

Mormoniella

355

darker than the color characteristic of vermilion-MK. These haploid inviable males, designated vml (vermilion lethal) in the records, are a characteristic type. Females compound for orange-336 or other genes, pale or oyster in 0, scarlet in X, such as peach-333, ‘tinged-277, oy-NH, may best be designated scarlet, but minute clumps of black pigment are scattered through the eye tissue, appearing very different from the eyes of other scarlet such as st-DR. The designation “vermilion” is here a coinpromise term characteristic of a theoretical intermediate between the dark red of the haploid male and the scarlet of the compound females. It is very different from the verinilion of om-MK, but nevertheless a mutant state of the same factor, X. I n combination with a (non-R) gene for black eye-color such as bk-424, the compound “scarlet” females have very pale lavender eyes. The very dark red eyes of the haploid inviable males become deep purple. Another compromise term appears in M5 in connection with mahogany-819. This “nornial” mutant gene not only lacks any deleterious factors, but it determines wild-type eye color in the haploid males (see even-generation males) and in the honioaygous stock females. The mahogany effect, actually somewhat brighter than some other 0-factor mahoganies such as mh-441, appears only in females compound for pale or oyster 0-factor mutations (see discard females). It was on this basis that the mutation to gene mh-819 was detected, the original mutant (pe.st pu/mh.+ +) having been found by S. B. Caspari among the progeny of an X-rayed (3000 r) wild-type male crossed with a peach purple female. The treatment presumably produced not only the change in factor 0 but also a crossover reducer, “very closely linked with purple, demonstrating only about 1.0% recombination rather than the expected 11%” (Caspari, 1958). M 6 and M7 contain an oyster male-sterile and a scarlet male-sterile gene, respectively. These are, therefore, the colors of the sterile males in each generation. Females compound for peach and oyster have tinged eyes, closely resembling eye color of tinged-277 stock. Tinged feinales are therefore retained in the even generations of M6, because here pe-333 combines with the oyster male-sterile gene. In M7, tinged females are the discards every generation because the two normal genes are tinged and oyster. The deleterious genes of 1-7 illustrate increasing viability in later lethal incidence from egg lethals to male steriles. Scarlet-474 and oyster805 are egg lethals. Oyster-803 is a larval lethal, but the inviabIe halfgrown larvae normally live longer than their viable adult brothers. The scarlet-eyed-813 pupae appear to die and dry before their viable brothers. The eclosed adult “vermilion”-809 males might be called sterile, but

356

ANNA R. WHITING

none are able to move about, feed, or mate, and many are unable to eclose. The actively mating males of oyster-816 and scarlet-814 sire no offspring and may truly be called viable but sterile. Many other conditions have been found involving viability and sterility. Among these may be mentioned the near steriles, oy-840 (oy. .nsx) and st-841 (+.st.nss). The males with these genes are normally viable, active, and aggressive in mating attempts, but very rarely accomplish a mating. When this occurs, several daughters may be sired. These females are for the most part sterile, but occasionally a few offspring are produced. Scarlet-841 has a factor suppressing crossing-over with purple and is run as a balanced stock with oy-423 purple, +.st. +.nsx +/oy.st.fsa. +pu. No purple scarlet-841 have been reported. Females homozygous for factor fsa are not completely sterile but occasionally lay a n egg, and such eggs develop normally to adults. M 8 has a lethal-bearing gene, dahlia-817, masking the presence of female sterility, fsa. When this is combined with a normal mutant allele and a n allele carrying fsa such as st-858, normally fecund females bearing the lethal alternate as mothers with the near sterile. A few wild-type females produce the odd generations from which a large number of dahlia females are set to produce the even generations. I n the even generations the females retained and the females discarded are of the same wild phenotype. By setting a few of these singly and selecting the dahlia daughters after they have mated with their apricot brothers, adequate near-sterile females are obtained to produce the next sparse generation.

+

Ill. Discussion

Pontecorvo (1958) has reviewed the research on the genes, recombination, allelism and conzplenzentation up to 1956. He notes that examples of close (or complete ?) linkage between complementary genes with similar or related phenotypic effects have been found in Drosophila melanogaster, Aspergillus nidulans, Neurospora crassa, Salmonella, Escherichia coli, phage T4, the mouse, silkworm, and cotton. Catcheside (1964) has discussed present developments in this field. He defines complementation as the ability of one defective mutant to compensate for the defectiveness of another mutant. He notes that, “Originally, it was supposed that no pair of allelic genes would complement, while every pair of non-allelic genes would do so,” and that complementation probably occurs in all living organisms. Demerec in 1961 discussed the effect of these studies on the theory of the gene. I n 1934 a type of mosaic male found in another hymenopteran species, Habrobracon juglandis Ashmead, was described in which “physiological” complenientation was observed (Whiting et al., 1934). The boundary

COMPLEMENTATION

IN

Mormoniella

357

line between two genetically different haploid areas passed through the external genitalia, and on one side of this line feminization of genitalia occurred. Since many of these niosaics were produced by unmated mothers, their haploidy could not be questioned. Whiting deduced from these that each of the two cells giving rise to a female had a different allele and that femaleness arose from complementation. All tests gave results which were consistent with this concept; and with the discovery of fused, a gene closely linked with the sex locus, he was able to show that femaleness is associated with heteroxygosity of alleles at the sex locus, maleness with azygosity or homozygosity. Nine alleles were ultimately identified at the sex locus, and no exception has been found to make it necessary to modify the original concept. It appears that sex in a wasp is determined by the balance of two agents that have a short diffusion distance and that each must be produced by a different allele a t one genetic locus. A complete summary of these results was published by Whiting in 1943. The complementary action of the sex alleles in Habrobracon has been compared to complementary alleles a t the R locus of Mormoniella by Whiting (1951). The era of-allelic complementation in microorganisms was opened by Calef in 1956 with his demonstration of intragenic complementation?n the adenine-9 locus of Aspergillus nidulans. This was quickly confirmed by Fincham and Pateman (19.57a, b) and Giles et al. (1957). The biochemical era of complementation of gene products was begun when Woodward (1959) combined in vitro crude extracts froin stains with different mutant alleles and made a n active enzyme. The rate of progress in this field becomes obvious as one scans the list of references in the Pontecorvo, Catcheside, and Demerec reviews, all too long for further discussion in a review such as this one.

ACKNOWLEDGMENT The author wishes to express her appreciation of the help given by the reviewers of this manuscript, Drs. R. F. Grell, F. J. de Serres, and R. C. von Borstel. Their suggestions have been most helpful.

REFERENCES Calef, E. 1956. Functional relationships between three adenineless mutants in Aspergillus nidulans. Heredity 10, 279. Caspari, S. B. 1958. An X-ray sperm-dose-action curve for mutations at a single locus in Mormoniella. Radiation Res. 8, 273-283. Catcheside, I). G. 1964. Interallelk complementation. Brookhaven Symp. Biol. 17, 1-14. Demerec, M. 1961. “Theory of the Gene,” 13 pp. Brookhaven Lecture Series, No. 10.

358

ANNA R. WHITING

Fincham, J. R. S., and Pateman, J. A. 1957a. A new allele at the AM locus in Neurospora crassa. J . Genet. 66, 456-466. Fincham, J. R . S., and Pateman, J. A. 1957b. Formation of an enzyme through complementary action of mutant ‘alleles’ in separate nuclei in a heterocaryon. Nature 179, 741-742. Giles, N. H., Partridge, C. W. H., and Nelson, N. J. 1957. The genetic control of adenylosuccinase in Neurospora crassa. Proc. Natl. Acad. Sci. U.S. 43, 305-317. Kayhart, M. E., and Whiting, P. W. 1949. X-ray mutations and fecundity of Mormoniella. Biol. Bull. 97, 344. Morgan, T. H., Bridges, C. B., and Sturtevant, A. H. 1925. The genetics of Drosophila. Bibliog. Genet. 2, 1-258. Pontecorvo, G. 1958. “Trends in Genetic Analysis,” 145 pp. Columbia Univ. Press, New York. Saul, G. B. 1957. A new element at the R locus in Mormoniella. Genetics 42, 393. Whiting, P. W. 1943. Multiple alleles in complementary sex determination of Habrobracon. Genetics 28, 365-382. Whiting, P. W. 1951. Multiple complementary alleles in Habrobracon and Mormoniella. J . Genet. 50, 206-214. Whiting, P. W. 1954. Comparable mutant eye colors in Mormoniella and Pachycrepoideus (Hymenoptera: Pteromalidae). Evolution 8, 135-147. Whiting, P. W. 1956. Spontaneous eye-color mutation in Mormoniella (The opening of Pandora’s box). Proc. Penn. Acad. Sci. 30, 226-246. Whiting, P. W. 1958. Mormoniella and the nature of the gene: Mormoniella vitripennis (Walker) (Hymen0ptera:Pteromalidae). Proc. 10th Intern. Congr. Entomol., 1956 Vol. 2, pp. 857-866. Mortimer Limited, Ottawa, Canada. Whiting, P. W. 1960a. Polyploidy in Mormoniella. Genetics 46, 949-970. Whiting, P. W. 1960b. Unstable-0 R-locus mutations in Mormoniella. Proc. Penn. Acad. Sci. 34, 243-249. Whiting, P. W. 1962a. R-locus deleterious factors in Mormoniella. Genetics 47, 115-129. Whiting, P. W. 196213. R-locus factor homologies in Mormoniella. Genetics 47, 921-936. Whiting, P. W., and Caspari, S. B. 1957. Mormoniella Merry-Go-Rounds. J. Heredity 48, 31-35. Whiting, P. W., Greb, R. J., and Speicher, B. R. 1934. A new type of sex-intergrade. Biol. Bull. 66, 152-165. Woodward, D. 0. 1959. Enzyme complementation in vitro between adenylosuccinaseless mutants of Neurospora crassa. Proc. Natl. Acad. Sci. U.S. 46, 846-850.

AUTHOR INDEX Numbers in italics refer to pages on which the complete referciices are listed

A

B

Abbie, A. A., 238, 291 Bailey, J. D., 244, 294 Aboim, A. N., 161, 179, 180, 215 Baker, R. V., 315, 336 Abraham, A., 286, 300 Baker, W. K., 254, 261, 291, 292 Abrahamson, S., 172, 197, 201, 202, 210, Balteer, F., 267, 292 212, 213, 214, 216 Banerjee, A. R., 233, 237, 306 Abro, A., 181, 193, 195, 198, 216 Barigoezi, C., 255, 258, 292 Adams, N. W., 123, 161 Barlow, F. D., 163, 223 Aida, T., 228, 231, 269, 271, 291 Barnicot, N. A., 315, 316, 336 i$kdik, S., 70, 110 Barr, M. L., 247, 293 Akerlund, E., 280, 291 Barto, E., 325, 326, 328, 329, 330, 336 Akhundov, N. A., 203, 215 Barton, L. V., 131, 162 Alderson, T., 197, 216 Basak, S. L., 60, 109 Alexander, D. E., 61, 107 Bagarman, M., 284, 298 Alexander, M. L., 171, 172, 180, 196, Baternan, A. J., 198, 200, 202, 209, 216 198, 204, 210, 214, 215 Bateman, K. G., 123, 161 Alimova, G. K., 9, 53 Bauer, H., 213, 216, 249, 268, 292, 308 Allard, R. W., 73, 107, 109, 117, 120, 123, Bauman, L. F., 9, 66 126, 149, 161 Baur, E., 125, 161 Allemang, W. H., 244, 294 Beadle, G. W., 66, 67, 107 Allen, B., 322, 323, 337 Beasley, A. B., 333, 336 Allen, C. E., 276, 287, 288, 291, SO1 Beasley, J. O., 98, 99, 107 Altenburg, J., 163, 167, 171, 172, 179, Beatty, R. A., 269, 272, 292 205, 210, 213, 214, 222 Beaumont, H. M., 173, 174, 216 Altenburg, L. S., 163, 167, 171, 172, 179, Beermann, W., 194, 221, 263, 264, 268, 205, 210, 213, 214, 222 292, 302 Anders, G., 247, 291 Bellahch, J., 247, 300 Anderson, E., 41, 63 Bellamy, A. W., 271, 292 Anderson, E. G., 14, 16, 45, 63, 64 Bemis, W. P., 286, 292 Andres, A. H., 240, 291, 306 Bender, M. A,, 241, 292 Androes, G. M., 322, 335 Berg, R. L., 138, 161 Ansley, H. R., 64, 65, 106, 107, 160, 216 Berman, Z. I., 210, 216 Apelbaum, A., 233, 237, 238, 306 Bhaduri, P. N., 233, 237, 296 Applen, J. E., 232, 233, 306 Bhattacharjee, P., 233, 237, 306 Arber, A., 133, 145, 151 Billingham, R. E., 273, 292, 312, 313, Ariyanayagam, D. V., 118, 122, 161 330, 336 Asana, J. J., 269, 291 Billings, F. H., 276, 291 Auerbach, C., 171, 172, 180,197,198,200, Birbeck, M. S. C., 315, 316, 320, 336,998 201, 202, 203, 204, 210, 211, 213, Bishop, A., 241, 244, 292 216, 116 Bishop, D. W., 169, 175, 216 359

360

AUTHOR INDEX

Bjorkman, O., 130, 161 Blacher, L. J., 269, 270, 292 Black, J. N., 132, 161 Blackburn, K. B., 276, 292 Blackman, G. E., 118, 119, 161, 163 Blair, W. F., 334, 356 Blanco, J. L., 72, 107 Blank, C. E., 241, 244, 292 Blaschko, H., 315, 356 Blickenstaff, J., 13, 14, 16, 63 Bloch, I).P., 64, 107 Blois, M. S., 322, 324, 356 Blume, R., 247, 307 Bodenstein, D., 179, 180, 181, 198, 200, 204, 216 Bolton, E. T . , 333, 537 Bonner, J., 93, 107 Bonner, T. G., 324, 356 Bostrack, J. M., 141, 142, 161 Boveri, T., 240, 292 Bradshaw, A. D., 117, 118, 120, 123, 133, 139, 149, 161 Brauer, A., 233, 295 Brawn, R. I., 31, 63 Breathnach, A. S., 315, 356 Brehme, K., 254, 293 Breider, H., 271, 287, 293 Bridges, C. B., 227, 229, 249, 250, 251, 254, 259, 261, 266, 295, 302, 343, 368 Briggle, L. W., 9, 37, 63 Briggs, J. H., 245, 296 Brooks, J. S., 27, 63 Brosseau, G. E., Jr., 252, 293 Brown, M. S., 61, 65, 66, 99, 107, 110 Brown, S. W., 60, 107 Brown, W. L., 41, 45, 63 Browne, E. B., 45, 64 Brunelli, G., 175, 187, 216 Brunet, P., 321, 325, 328, 330, 336 Brunet, P. C. J., 320, 321, 336 Buchert, J. G., 5, 6, 12, 42, 63 Buder, J. E., 159, 216 Bumpus, H. C., 124, 161 Bunting, E. S., 119, 161 Burns, G. P., 135, 161 Buzzell, R. L., 101, 108

C Calef, E., 357, 367 Callan, H. G., 261, 263, 293 Calvin, M., 322, 336 Cambier, R. K., 232, 233, 506 Cannon, W. B., 117, 147, 161 Cardcll, R. R., 315, 537 Carlson, E., 209, 222 Carr, D. H., 247, 295 Carson, H. L., 158, 160, 162, 177, 183, 216 Case, J. D., 320, 337 Caspari, E., 2, 4, 44, 63, 66, 266, 2BS Caspari, S. B., 343, 348, 351, 355, 367, 568 Caspersson, T., 261, 306 Castle, W. E., 228, 229, 230, 269, 293 Catcheside, D. G., 261, 293, 356, 367 Cattanach, B. M., 271, 272, 293 Cauderon, Y., 88, 108 Celada, F., 273, 274, 293 Centerwall, W. R., 239, 240, 306 Chakravartti, M. R., 233, 237, 297 Chandley, A. C., 198, 200, 202, 209, 216 Chang, T. T., 28, 38, 63 Chao, C-Y., 66, 108 Chapman, V., 58, 77, 82, 83, 85, 86, 88, 90, 92, 99, 112 Cheesman, E. A., 239, 307 Chheda, H. R., 102, 108 Chigot, P., 248, 308 Chinwuba, P. M., 23, 24, 65 Cholodkowsky, N., 160, 161, 216 Christakos, A. C., 247, SO7 Chu, E. H. Y., 272, 295, 506 Chung, C. S., 239, 302 Clapham, A. R., 133, 134, 161 Clark, A. M., 199, 203, 216 Clausen, J., 118, 122, 124, 129, 136, 139, 162 Clausen, R. E., 98, 108 Clayton, F. E., 170, 196, 216 Cleffmann, G., 331, 536 Clements, F. E., 133, 162 Clermont, Y., 159, 164, 167, 168, 169, 174, 185, 186, 216, 220 Cocchi, M., 212, 216 Cockayne, E. A., 231, 233, 294

361

AUTHOR INDEX

Cohen, M. M., 241, 294 Cole, P. A., 261, 294 Coleman, D. L., 321, 325, 327, 328, 329, 330, 336, 339 Conen, P. E., 244, 294 Cook, C. D. K., 135, 141, 142, 143, 152 Cook, S. A,, 131, 135, 152 Cooper, J. P., 130, 162 Cooper, K. W., 63, 108, 161, 163, 171, 181, 189,193, 194,216,217,249,250, 251, 253, 254, 255, 258, 259, 260, 261, 294 Cope, F. W., 323, 336, 438 Corbett, M. K., 51, 64 Correns, C., 276, 285, 294 Counce, S. J., 178, 179, 217 Court Brown, W. M., 247, 294 Creighton, M., 185, 217 Cretschmar, M., 163, 178, 185, d l 7 Crocker, W., 131, 166 Cross-White, L. H., 247, 306 Crow, J. F., 127, 152, 233, 294 Crowther, F., 122, 162 Cumming, B. G., 128, 162 Curth, H. O., 232, 294

D Dandy, J. E., 133, 134, 162 Danneel, R., 325, 327, 330, 356, SS6 Dark, S.O.S., 282, 294 Darlington, C. D., 58, 59, 62, 63, 79, 82, 97, 103, 108, 159, 186, 217, 231, 240, 255, 276, 287, 288, 694, 300 Davenport, C. B., 233, 294 Davidson, H., 281, 308 Davies, T. S., 247, 294, 300 Davis, H. P., 159, 160, 162, 165, 169, 175, 176, 187, 189, 217 Davy de Virville, M. A,, 132, 166 Dawson, C. D. R., 101, 108 De, D. N., 64, 108, 193, 218 De Almeida, J. C., 245, 696 de Beaumont, J., 249, 303 Deegner, P., 160, 217 de Grouchy, J., 247, 300 de la Chapelle, A., 244, 248, 290, 308 Delbruck, M., 64, 108 Delhanty, J. D. A., 247, 296

Demerec, M., 45, 63, 158, 200, 202, 217, 356, 367 Dent, T., 247, 295 Depdolla, P., 159, 160, 161, 173, 183, 187, 191, 217 De Robertis, E., 271, 305 Dc Winiwarter, H., 240, 296 de Zulueta, A., 269, 296 Dice, L. R., 334, 336 Dietz, R., 268, 308 di Pasquale, A., 258, 292 Ditlevsen, E., 236, 270, 271, 310 Dobzhansky, Th., 123,162,193,205,217, 249, 250,253, 256, 261, 296 Donald, C. M., 119, 146, 149, 162 Donoso, R. F., 196, 217 Douglas, A., 247, 307 Dowrick, G. J., 101, 108 Doyle, G. G., 97, 108 Dressler, O., 286, 295 Drochmans, P., 315, 3S6 Dronamraju, K. R., 233, 234, 235, 236 237, 238, 239, 296 Druzba, J., 270, 298 Dubinin, N. P., 260, 296 Duesberg, J., 164, 217 Duncan, A., 324, 336 Duncan, F. N., 171, 172, 217 Dunn, L. C., 332, 333, 336 Duvick, D. N., 2, 5, 7, 8, 11, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 34, 45, 48, 63, 64 Dyer, G. S., 233, 296

E Eberlein, W. R., 244, 299 Ebert, M., 212, 217 Eckhardt, R. C., 14, 15, 64 Ede, D. A., 178, 217 Edmondson, M., 163, 167, 171, 172, 179, 205, 210, 213, 214, 822 Edwards, J. H., 247, 296 Edwards, R. G., 186, 198, 199, 217 Edwardson, J. R., 2, 3, 4, 28, 29, 38, 41, 51, 64, 66 Eichwald, E. J., 272, 274, 296 Ellis, J. R., 247, 285, 296, 299 Endrizzi, J. E., 99, 108

362

AUTHOR INDEX

Engledow, F. L., 119, 152 Enns, H., 66, 68, 108 Enriques, P., 230, 296 Evans, B. H., 185, 217 Evans, H. M., 240, 296 Everall, J. D., 315, 336 Everett, H. L., 23, 26, 54 Ezrin, C., 244, 294

F FabergB, A. C., 63, 105, 106, 108 Fahmy, M. J., 198, 200, 203, 210, 211, 217 Fahmy, 0. G., 198, 200, 203, 210, 211, 217 Fantham, H. B., 23:3, 296 Farquharson, L. I., 39, 54 Fattorusso, E., 322, 324, 338 Fawcett, D. W., 187, 217 Feinberg, R. G., 262, 301 Fenasse, R., 247, 308 Ferguson-Smith, M. A., 240, 247, 249, 296, 307 Fincham, J. R. S., 357, 558 Fisher, F. J. F., 135, 136, 152 Fitzpatrick, T. B., 315, 320, 321, 325, 328, 330, 335, 320, 338 Fleming, A. A., 45, 54 Fling, M., 320, 336 Ford, C. E., 231, 240, 245, 296 Foster, M., 312, 314, 320, 321, 324, 325, 326, 327, 328, 329, 330, 331, 336 Fowden, L., 30, 54 Fox, A. S., 262, 263, 265, 272, 296 Franchi, L. L., 173, 174, 218 Frankel, 0. H., 123, 152 Frankel, R., 51, 54 Franzke, C. J., 67, 113 Friedman, E., 172, 197, 201, 202, 210, 212, 213, 214, 215 Friele, A., 161, 218 Friesen, H., 172, 198, 201, 202, 203, 204, 205, 207, 209, 210, 213, 215, 218 Fritz-Niggli, H., 200, 218 Frolova, S. L., 193, 218 Fujumura, W. H., 187, 194, 226 Fukasawa, H., 10, 29, 64 Fuseau-Braesch, S., 321, 336

G Gabe, D. R., 285, 296 Gabelman, W. H., 32, 54 Gajewski, W., 41, 54 Gajic, I)., 118, 128, 153 GalBn, F., 268, 284, 296 Galbraith, D. B., 329, 332, 336 Galinat, W. C., 40, 42, 54, 65 Gall, J. G., 64, 108, 263, 296 Gardner, L. I., 244, 308 Gates, R. R., 231, 232, 233, 234, 235, 237, 296, 297 Gay, E. H., 258, 260, 297 Gay, H., 161, 163, 171, 181, 189, 193, 194, 195, 198, 199, 200, 218, 219 Geerts, S. J., 201, 212, 218 Geigy, R., 161, 180, 218 Geitler, L., 159, 160, 218 GBrard, P., 159, 218 Gersh, E. S., 261, 297 Gershenson, S., 254, 256, 297, 303 Gerstel, D. U., 98, 100, 108, 113 Ghosh, R. R., 234, 305 Gilchrist, B. M., 268, 269, 297 Gilden, R. V., 93, 207 Giles, N. H., 357, 358 Ginsburg, B., 325, 328, 330, 336 Ginsburg, 8. G., 262, 501 Gleichauf, R., 181, 198, 218 Gloor, H., 161, 180, 218 Gliick, H., 135, 152 Gluecksohn-Waelsch, S., 313, 356 Goebel, K., 142, 144, 145, 152 Goldschmidt, R. B., 160, 218, 255, 258, 260, 266, 267, 271, 297, 307 Goldstein, M. N., 247, 298 Gooch, P. C . , 241, 292 Goodman, G. C., 64, 107 Goodrich, H. B., 270, 297 Goodsell, S. F., 37, 39, 54 Goodspeed, T. H., 98, 108 Gordon, M., 271, 297 Gowen, J. W., 66, 108,258,260,369,270, 283, 297 Cower, J. S., 271, 305 Grafius, J. E., 146, 149, 155 Greb, R. J., 356, 358 Gregor, J. W., 135, 137, 155 Grell, E. H., 97, 109

363

AUTHOR INDEX

Grell, R. F., 260, 297 Griffin, A. B., 256, 304 Gripenberg, U., 241, 297 Grogan, C. O., 23, 24, 63 Griinberg, K., 159, 160, 161, 162, 172, 190, 218 Grumbach, M. M., 293 Gugler, H. D., 180,194, 198,200, 202,219 Guyhot, E., 157, 162, 191, 193, 194, 218

H Habich, A., 249, 307 Haga, T., 60, 109 Haldane, J. B. S., 122, 163, 231, 235, 237, 268, 269, 274, 296, 297 Hamerton, J. L., 231, 240, 296 Hanna, B. L., 316, 336 Hanna, G. C., 285, 306 Hannah, A,, see Hannah-Alava, A. Hannah-Alava, A., 161, 167, 168, 171, 172, 181, 183, 185, 187,'189, 193, 194, 196, 198, 200, 201, 202, 203, 205, 207, 209, 210, 211, 213, 214, 218, 219, 255, 258, 297 Hanson, F. B., 197, 219 Hanson, W. D., 119, 163 Hague, A., 231, 240, 294 Harlan, J. R., 102, 107, 131, 141, 163 Harnden, D. G., 247, 294 Harper, J. L., 118, 128, 131, 132, 140, 146, 163 Harris, B. B., 159, 171, 172, 197, 211, 219 Harris, H., 237, 298 Hartmann, J. F., 315, 339 Harvey, P. H., 13, 14, 16, 63 Haskins, C. P., 270, 298 Hasson, J. E., 247, 298 Hathaway, D. S., 172, 179, 2lS Hauschka, T. S., 247, 272, 273, 298, 306 Hauschteck, E., 247, 291 Hayman, B. I., 77, 109 Hearne, E. M., 66, 109 Heilborn, O., 104, 109 Heilbronn, A., 284, 898 Heitz, E., 250, 255, 256, 298 Heller, R., 247, 291 Helwig, E. R., 209, 819 Henking, H., 228, 298 Heptner, M. A., 896

Herringham, N., 233, 298 Herskovitz, I. H., 254, 303 Hertwig, P., 198, 219 Hess, O., 194, 221, 251, 260, 263, 264, 265, 298, 302 Heys, F., 197, 219 Hiesey, W. M., 118, 122, 124, 129, 136, 139, 162 Hinson, K., 119, 163 Hinton, T., 64, 109, 256, 298 Hirata, K., 285, 298 Hirsch, H. M., 315, 336, 339 Hoagland, M., 52, 64 Hodge, B. A., ,292 Hodgson, G. L., 118, 163 Hoffmann, W., 285, 286, 298 Hofmeyer, J. D. J., 286, 287, 298 Holdridge, B. A., 272, 273, 298 Hollingshead, L., 72, 109 Holmgren, P., 130, 161 Horowitz, N. H., 320, 337 Hortling, H., 244, 294 Howard, A,, 186, 199, 212, 217, 223 Hoyer, B. H., 333, 337 Hsu, T. C . , 240, 299 Hu, F., 315, 337 Hu, W. L., 66, 108 Huang, R. C., 93, 107 Huckins, C., 173, 219 Huettner, A. F., 178, 180, 181, 191, 193, 194, 216 Hughes-Schrader, S., 269, 299 Huhnke, W., 286, 299 Hunter, H., 241, 244, 292 Huskins, C. L., 66, 82, 109 Hustinx, T. W. J., 247, 299 Huxley, J. S., 126, 163

I Imher, D., 92, 114, 126, 166 Ingram, D. J. E., 322, 323, 337 Isenberg, I., 322, S37 Ishida, H., 187, 194, 226 Ishihara, T., 247, 274, 275, 299, 300, 306 Ishmael, J., 247, 300 [to, S., 187, 194, 217, 219 Ives, P. T., 197, 200, 201, 203, 819 Iwasaki, 170, 176, 177, 178, 185, 190, 828 Iwashita, S., 315, 338

364

AUTHOR INDEX

J Jacobs, P. A., 245, 247, 294, 299 Jacobsen, P., 282, 299 Jain, H. K., 60, 109 Jain, K. B., 73, 109 Jain, S. K., 73, 10'7, 109 Janick, J., 286, 299 Janssens, F. A., 185, 219 Jenkins, M. T., 3, 37, 66 Jinks, J. L., 52, 64, 71, 109, 123, 163 Jorgensen, C. A., 101, 109 John, B., 59, 66,109,110 Johnson, B. L., 97, 109 Johnson, I. J., 5, 66 Johnston, G. S., 20, 23, 64 Jones, D. F., 3, 4, 5, 9, 14, 20, 23, 26, 30, 31, 49, 64, 66 Jones, K. W., 245, 296 Jonsson, U. B., 172, 181, 197, 200, 220 Jordan, C., 286, 299 Josephson, L. M., 3, 4, 20, 23, 26, 37, 66 Josephson, N, D., 270, 297

K Klllkn, B., 241, 299 Kajii, T., 241, 244, SO1 Kalinin, V. S., 262, 301 Kaplan, W. D., 180, 194, 198, 200, 202, 219

Karper, R. E., 119, 163 Kato, H., 170, 175, 177, 178, 185, 190, 222

Kaufmann, B. P., 161, 163,171, 181, 189, 193, 194, 195, 198, 199, 200, 202, 21'7, 218, 219, 250, 256, 299 Kawaguchi, E., 177, 190, 211, 219 Kayhart, M. E., 343, 368 Keck, D. D., 118, 122, 124, 129, 136, 139,

Key, J. A., 233, 299 Khan, M. A,, 118, 119, 120, 163 Khishin, A. E. F., 172, 180, 181,197, 198, 200, 204, 219 Khoo, U., 3, 5, 9, 14, 23, 28, 30, 31, 66 Kidd, K. K., 180, 194, 198, 200, 202, 219 Kiesselbach, T. A., 22, 66 Kihara, H., 110, 276, 282, 283, 299 Kimball, A. W., 214, 220 Kimball, R. F., 209, 212, 220 Kimber, G., 58, 72, 82, 83, 85, 88, 90, 98, 109,112

Kimura, M., 127, 163 Kincer, H. C., 20, 23, 26, 66 Kindred, B. M., 271, 272, 299 King, R. L., 269, 299 Kirpichnikov, W., 269, 199 Klebs, G., 117, 163 Klevit, H. D., 244, 299 Knaben, N., 170, 176, 178, 183, 210 Knapp, E., 288, 299 Kobayashi, T., 170, 177, 178, 220 Kodani, M., 231, 256, 300 Kocpf, G. F., 247,298, 306 Kogure, M., 203, 220 Koller, P. C., 231, 240, 300 Komai, T., 274, 300 Kondo, S., 159, 163, 167, 170, 190, 203, 211, 213, 214, 280, 226 Kostoff, D., 58, 110 Kozelnickey, G. M., 45, 64 Krivshenko, J., 252, 300 Kroning, F., 325, 33'7 Kuhn, E., 276, 284, 285, SO0 Kukita, A., 320, 321, 325, 328, 330, 336 Kumar, L. S. S., 286, 287, 300 Kvelland, I., 202, 210, 220

L

162

Kelly, E. M., 201, 203, 212, 224 Kemmer, M., 204, 214, 224 Kempanna, C., 58, 76, 77, 83, 85, 90, 95, 96, 104, 109, 112 Kerkis, J., 179, 180, 181, 193, 219 Kerkut, G. A., 323, 337 Kerner von Marilaun, A., 119, 132, 133, 136, 163 Keuneke, W., 213, 919

La Cour, L., 255, 294 Lafourcade, J., 248, 308 Lamb, A. B., 63,110 Lamm, R., 70, 110 Lammerts, W. E., 98, 99, 110 Lamy, M., 247, 300 Lamy, R., 254, 300 Langridge, J., 122, 163 Lantican, R. M., 26, 66

AUTHOR INDEX

Larter, E. N., 66, 68, 108 Laurence, K. M., 247, 300 LaVallette St. George, A. v., 159, 162, 220 Lawrence, C. W., 60, 110 Lawrence, W. J. C., 58, 103, 110 Leage, B. B., 193, 198, $10 Leblond, C. P., 159, 164, 167, 168, 174, 185, 186, 216, 220 Lefevre, G., Jr., 172, 181, 197, 200, 220 Lehtovaara, R., 248, 306 Lejeune, J., 248, 300, 308 Leng, E. R., 9, 66 Lerner, A. B., 320, 336, 337 Lerner, I. M., 123, 163 Levan, A., 100, 110,240, 241, 299, SOY Levenbrook, L., 258, 300, 308 Levins, R., 126, 163 Levit, S. G., 262, 300, 301 Lewin, R. A., 128, 163 Lewis, D., 276, 301 Lewis, E. B., 261, SO1 Lewis, H. S., 320, 337 Lewis, H. W., 320, 337 Lewis, K. R., 59,110 Lewis, M. R., 160, 187, 820 Lewontin, R. C., 117, 123, 125, 153 Li, C. H., 66, 68, 110 Li, D., 66, 108 Li, H. W., 66, 68, 110 Lilienfeld, F. A., 88, 101, 110 Lindsay, R. H., 286, 301 Lindsley, D. L., 254, 301 Little, C. C., 333, 337 Livers, R. W., 41, 65 Ljungdahl, H., 101, 110 Lloyd, L., 263, 293 Lave, A,, 282, 283, 301 Long, F. L., 133, 152 Loomis, N. H., 287, 301 Loud, A. V., 315, 33Y Loustalot, P., 315, 339 Luers, H., 172, 220 Luning, K. G., 198, 202, 220 Lustgraaf, E. C., 274, 296

M McCarthy, B. J., 333, 337 McCarthy, J. C., 200, 213, 220

365

McClintock, B., 58, 61, 63, 69, 110 McClung, C. E., 160, 161, 163, 175, 187, 220, 221, 228, 301 McDonald, H. A., 101, 111 McGregor, J. H., 185, 281 Mackay, E., 288, 301 Macklin, M. T., 231, 232, 294, 301 McKusick, V. A., 247, 248, 301 McLaren, A., 271, 272, 301 Maclean, N., 247, 294 McLeish, J., 58, 110 MacLeod, H., 320, 336 McNaughton, I. H., 131, 163 MacNeish, R. S., 40, 42, 66 McPhee, H. C., 285, 301 McSheehy, T. W., 214, 221 Makela, O., 248, 305 Mahoney, D. L., 286,299 Makino, S., 231, 241, 244, 271, 301 Makinodan, T., 274, 293 Malacarne, P., 247, 306 Maling, J. E., 322, 324, 336 Maltby, E. L., 312, 313, 337 Mandl, A. M., 173, 174, 216, 218 Mangelsdorf, P. C., 20, 40, 42, 66 Mantle, D. J., 247, 294 Manton, I., 63, 110 Margolis, E., 239, 301 Markert, C. L., 321, 337 Martin, E. V., 133, 162 Mason, H. S., 322, 323, 337 Massart, J., 135, 164 Mather, K., 77, 109, 116, 123, 125, 126, 148, 163, 164, 258, 301 Matthey, R., 231, 271, 302 Mayer, T. C., 312, 313, 337 Mayr, E., 148, 164 Mazoti, L. B., 45, 66 Mead, C. G., 262, 265, 296 Mellman, W. J., 244, 299 Mensel, M. Y., 65, 66, 110 Mercado, A. C., Jr., 26, 66 Mercer, E. H., 316, 336 Metz, C. W., 157, 193, 221, 256, 302 Meurman, O., 282, 286, 302 Meyer, G. F., 161,181,183,194,195,221, 251, 260, 263, 264, 265, 298, 302 Meyer, H. U., 163, 167, 171, 172, 179, 205, 210, 213, 214, 222 Michaelis, P., 51, 66

366

AUTHOR INDEX

Miller, A., 161, 193, 194, 200, 221 Miller, 0. J., 245, 247, 296, 302, 307 Miller, 0 . L., 66, 67, 110 Millington, W. F., 141, 142, 161 Minouchi, O., 240, 302 Mishima, Y., 315, 337 Mittwoch, U., 240, 245, 296, 302 Miya, K., 170, 177, 178, 221 Moewus, F., 285, 302 Moffet, A. A,, 58, 103, 108 Mohr, 0. L., 163, 164, 177, 185, 189, 221 Monesi, V., 164, 165, 167, 168, 169, 174, 183, 186, 198, 199, 203, 213, 221 Mooney, H. A., 130, 164 Moore, W. G., 197, 221 Morgan, L. V., 254, 302 Morgan, T. H., 259, 261, 302, 343, 368 Morishima, A,, 293 Morton, N. E., 231, 239, 302 Moses, M. J., 64, 110 Mossige, J. C . , 200, 221 Moyer, F. H., 314, 317. 337 Miintzing, A., 70, 72, 73, 77, 78, 100, 101, 110,111 Mukherjee, B. B., 247, 307 Mukherjee, D. R., 233, 237, 297 Muldal, S., 244, 247, 302 Muller, H. J., 63, 1 1 1 , 157, 163, 167, 171, 172, 179, 205, 209, 210, 211, 213, 214,221, 222, 239, 254, 255, 256, 288, 289, 300, 303 Muller, M., 247, 308 Munday, A., 123, 162 Munday, K. A., 323, 337 Munson, A. P., 26, 66 Munson, J. P., 159, 162, 222 Muramatsu, T., 88, 111 Muramoto, J., 241, 301 Murphy, M. M., 287, 301 Murray, M. J., 283, 284, 303 Murty, B. R., 66, 67, 113 Musset, R., 247, 300 Myers, W. M., 100, 102, 1 1 1

N Nachtsheim, H., 313, 333,337 Nafei, H., 200, 213, 220

Nakai, T., 315, 337, 338 Nakajima, M., 203, 220 Nakanishi, Y. H., 170, 176, 177, 178, 185, 190,222 Navashin, M. S., 240, 291 Naville, A., 157, 162, 191, 193, 194, 218, 249, 303 Naylor, B., 66, 109 Negrul, A. M., 287, 303 Nelson, L. A., 233, 294 Nelson, N. J., 357, 368 Nelson, 0. E., 159, 160, 162, 165, 167, 169, 172, 174, 175, 205, 222 Neuer, H., 286, 303 Neuhaus, M. E., 252, 253, 254, 256, 303 Nezelhoff, C., 247, 300 Nichols, S. E., Jr., 312, 338 Nicolaus, R. A,, 322, 324, 338 Nilsson-Ehle, H., 115, 164 Noble, S. W., 23, 26, 66 Norby, D. E., 275, 307 Nordenskidd, H., 100, 101, 111 Noujdin, N. I., 261, 303

0 Oakberg, E. F., 162, 168, 174, 186, 198, 199, 201, 203, 211, 212, 213, 922 Oberle, G. D., 287, 303 Ockey, C. H., 244, 247, 302 Oftedal, P., 197, 211, 212, 214, 222 Oguma, K., 240, 269, 296, 303 Ohta, T., 240, 302 Okamoto, M., 82, 83, 85, 111, 113 Oksala, T., 183, 185, 186, 222 Olive, L. S., 63, 111 Oliver, F. W., 119, 132, 133, 136, 163 Olivieri, A,, 172, 198, 200, 202, 203, 204, 205, 207, 209, 212, 213, 214, 222 Olivieri, G., 172, 198, 200, 202, 203, 204, 205, 207, 209, 212, 213, 214, 222 O'Mara, J. G., 77, 111 Omura, T., 163, 170, 185, 222 Ondraschek, H., 268, 304 Ono, T., 276, 277, 282, 283, 299, 304 Ortevant, It., 186, 222 Otte, H., 168, 175, 185, 22.9

367

AUTHOR INDEX

P

Reams, W. E., Jr., 312, 338 Reed, S. C., 232, 233, SO5 Rees, H., 59, 66, 70, 71, 74, 111,112, 122, Painter, T. S., 240, 255, 256, 303, 304 Pao, W. K., 66, 68, 110 154 Reeve, E. C. R., 123, 154 Parker, D. R., 198, 204, 228 Regaud, C., 159, 185, 223 Partridge, C. W. H., 357, 358 Rendel, J. M., 123, 154 Pateman, J. A , , 357, 358 Renkonen, K. O., 248, 305 Patterson, J. T., 204, 223 Renshaw, C. C., 26, 56 Patton, R. G., 244, 308 Resende, F., 255, 305 Pavan, C., 256, 304 Rhoades, M. M., 2, 9, 27, 32, 36, 37, 38, Paxman, G. J., 122, 154 55, 56, 64, 97, 112, 159, 172, 223 Payan, H., 247, 308 Rhyne, C. L., 98, 112 Payne, M. A., 201, 223 Ribbands, C. R., 58, 112 Pelc, S. R., 186, 199, 223 Ricci, N., 247, 305 Pelecanos, M., 197, 215 Rick, C. M., 285, 305 Penrose, L. S.,232, 245, 247, 296, 304 Ridler, M., 245, 296 Person, C., 58, 66, 73, 74, 111 Rieger, R., 58, 61, 112 Pfahler, P. L., 286, 299 Riley, R., 58, 72, 77, 78, 82, 83, 85, 86, Philip, U., 196, 22S, 254, 256, 304 88, 90, 92, 94, 96, 97, 99, 104, 109, Piatelli, M., 322, 324, 338 112 Piternick, L. K., 258, 297 Plunkett, E. R., 247, 293 Ritte, U., 232, 308 Polani, P. E., 245, 247, 296, 304 Rittenhouse, E. W., 318, 329, 338 Robertson, H. T., 72, 114 Polis, B. D., 323, 338 Pontecorvo, G., 60, 111, 171, 193, 223, Robertson, W. R. B., 160, 161, 163, 164, 168, 172, 183, 185, 187, 189, 220, 223 255, 304, 356, 358 Robinson, E., 240, 241, 305 Poostchi, I., 101, 111 Rodgers, J. S., 4, 56 Poulson, D. F., 178, 179, 223 Rojas, P., 271, 305 Powers, P. B., 183, 187, 223 Roosen-Runge, E. C., 159, 162, 163, 167, Prader, A., 247, 249, 291, 307 Prakken, R., 66, 100, 101, 111 168,169,173,174,183,185, 186, 223 Pritchard, R. H., 60, 111 Ross, J. G., 67, 113 Prokofyeva, A. A., see Prokofyeva-Bel- Russell, E. S., 313, 328, 331, 538, 339 govskaia, A. A. Russell, L. B., 201, 203, 212, 224, 271, 272, 305, 308, 325, 328, 330, 338 Prokofyeva-Belgovskaia, A. A., 250, 255, 256, 261, 271, 304, 305 Russell, W. A., 23, 26, 56 Prout, T., 123, 154 Russell, W. L., 201, 202, 203, 211, 212, 214, 223, 224, 625, 271, 305, 325, 328, Puro, J., 167, 171, 172, 18C, 200, 201, 202, 203, 204,209, 210,213,214,219,223 330, 338

Q Queal, M. L., 271, 292

R Rabinowitz, M., 178, 179, 923 Rao, P. D. P., 238, 291 Rappaport, H., 315, 337, 538 Rawles, M. E., 312, S38

S Sachs, L., 231, 305 Sado, T., 160, 161, 162, 163, 170, 177, 190, 197, 198, 200, 201, 203, 212, 224 Savhagen, R., 203, 204, 214, 224 Saez, F. A., 271, 305 Safir, S. R., 228, 251, 305 Salisbury, E. J., 116, 133, 154 Salmon, C., 248, 308

368

AUTHOR INDEX

Sandberg, A. A., 247, 298, 306 Sanders, M. E., 67, 113 Sansome, F. W., 285, 306 Santos, J. K., 276, 306 Sarkar, N., 282, 283, 301 Sarkar, P., 86, 113 Sarkar, S. S., 233, 234, 237, 239, 240, 306, 306 Sasaki, M. S., 241, 244, 301 Sastry, G. R. K., 99, 113 Saul, G. B., 345, 354, 368 Sax, K., 80, 113 Schacht, L. E., 200, 203, 209, 224 Schiirer, K., 247, 291 Schaffer, E. L., 185, 224 Schalet, A., 209, 222 Schaub, F. F., 315, 337 Schellenberg, A., 160, 177, 189, 224 Scheremetjewa, E. A., 193, 194, 226 Scheu, H., 287, 293 Schiemann, E., 101, 113 Schiemann, R., 101, 113 Schmalhausen, I. I., 125, 140, 164 Schmidt, J., 228, 269, 306 Schofield, R., 230, 233, 306 Schrader, F., 62, 113 Schulta, J., 211, 224, 251, 255, 256, 257, 258, 259, 261, 300, 302, 306, 308 Schwarta, D., 9, 66 Scott, G. E. B., 247, 296 Searle, A. G., 313, 333, 338 Sears, E. R., 60, 80, 82, 83, 111, 113 Sedgewick, W., 233, 306, 307 Seidel, S., 163, 171, 180, 181, 189, 193, 195, 198, 200, 284 Seiji, M., 315, 336, 338 Seiler, J., 267, 306 Selman, G. G., 172, 179, 219 Sever, R. J., 323, 338 Sficas, A. G., 98, 113 Shank, D. B., 123, 161 Shapiro, A., 245, 296 Shapiro, N. I., 205, 224 Sharma, A., 255, SO6 Sharma, A. K., 255, 306 Shaver, D. L., 9, 41, 42, 66 Shaver, S. L., 162, 199, 284 Sheldon, B. L., 123, 164 Shen, T. H., 251, 306 Shimao, K., 315, 338

Shimotamai, N., 101, 113 Shiwago, P. I., 240, 306 Shubik, P., 315, 338 Sidorov, B. N., 260, 296 Siebenmann, R. E., 247, 291 Siegel, B. V., 315, 339 Silmser, C. R., 272, 296 Silvers, W. K., 273, 292, 312, 313, 314, 317, 331, 936, 339 Simmonds, N. W., 120,164 Simmonin, R., 247, 308 Singleton, J. R., 63, 113 Sinnott, E. W., 142, 144, 164 Sirlin, J. L., 186, 198, 199, 217 Sisken, J. E., 180, 194, 198, 219 Slate, J. M., 270, 297 Slatis, H. M., 233, 237, 238, 306 Slautterbach, D., 187, 217 Slizynska, H., 254, 300 Smith, E. M., 233, 306 Smith, S. G., 61, 113 Sneep, J., 285, 306 Snyder, R. J., 14, 20, 23, 64 Sobele, F. H., 201, 202, 203, 210, 212, 224 S@rensen,T., 128, 147, 149, 164 Sofuni, T., 241, 301 Sonbati, E. M., 197, 203, 210, 211, 216 Sonnenblick, B. P., 162, 172, 178, 179, 180, 224 Soost, R. K., 66, 113 Speicher, B. R., 356, 368 Spencer, W. P., 264, 306 Spofford, J. B., 261, 292 Srinivasan, V. K., 286, 300 Staubli, W., 315, 339 Stahl, A., 247, 308 Stead, B., 16, 66 Stebbins, G. L., 80, 86, 97, 113, 117, 128, 132, 164 Steinitz-Sears, L. M., 60, 113 Stern, C., 181, 205, 224, 229, 232, 233, 235, 237, 239, 240, 252, 253, 266, 289, 30.4, 306, 306 Stevens, N. M., 190,286, 228, 229,307 Stevenson, A. C., 239, 286, 307 Stevenson, E. C., 299 Stinson, C . W., 247, 307 Stinson, H. T., Jr., 3, 5, 9, 14, 23, 26, 28, 30, 31, 37, 45, 66, 66

369

AUTHOR INDEX

Stolte, L. A. M., 241, 308 Stone, W. S., 196, 198, 215 Storey, W. B., 286, 287, 307 Strainer, M., 274, 296 Stringfield, G. H., 23, 56 Stromnaes, O., 200, 225 Strong, J. A,, 245, 299 Sturtevant, A. H., 251,253,260, 261,307, 343, 358 Suche, M., 204, 223 Suguira, T., 286, 307 Sutton, E., 261, 294 Sutton, W. S., 160, 175, 225, 240, 307 Swaminathan, M. S., 66, 67, 88, 99, 113,

Tjio, J. H., 240, SO7 Tobias, P. V., 160, 183, 185, 1S6, 225 Todd, N. B., 333, 339 Tokunaga, C., 268, 307 Tommasi, C., 231,232, 233,234, 237, 307 Toyama, K., 160, 177, 190, 225 Traut, H., 203, 125 Travaglini, E. C., 258, 300,308 Trinhaus, J. P., 270, 297 Tucker, J. M., 129, 164 Turesson, G., 135, 136, 137, 139, 164, 155 Turpin, R., 248, 308 Tutin, T. G., 133, 134, 161

U

114 Swan, G. A., 322, 339 Swanson, C. P., 64, 113 Swexy, O., 240, 296 Swift, H., 64, 113, 315, 338 Sxilard, L., 249, 307

T Takenaka, Y., 98, 113 Tanaka, M., 98, 113 Tanner, J. M., 249, SO7 Tates, A. D., 194, 201, 202, 624, 225 Taylor, H. L., 233, 294 Taylor, J. H., 64, 110 Taeima, Y., 170, 177, 178, 197, 198, 203, 211, 213, 225, 267, 268, 307 Thoday, J. M., 116, 123, 126, 136, 154 Thomas, W. I., 5, 66 Thompson, D. L., 13, 14, 16, 53 Thompson, D. W., 244, 294 Thompson, J. B., 70,71,74,112, 122,154 Thompson, M., 247, 302 Thompson, P. E., 203, 225 Thompson, W., 72, 114 Thomson, L., 325, 326, 328, 329, 336 Thuline, H. C., 275, 307 Thurston, J. M., 131, 145, 154 Tietx, W., 239, 307 Tihen, J. A., 159, 163, 164, 171, 180, 193, 194, 226 Tijdink, G. A. J., 241, 308 TimofBeff-Ressovsky, N. W., 211, 225 Tinderholt, V., 180, 194, 198, 200, 202, 319 Tinker, H., 333, 336

Uggeri, B., 212, 216 Uhl, C. H., 276, SO8 Ullerich, F. H., 268, 308 Underwood, G., 139, 156 Unrau, J., 86, 99, 112 Upadhya, M. D., 88,114 Uphof, J. C. T., 131, 141, 145, 155

V Vague, J., 247, 308 Vaharu, T., 244, 308 Vanderlyn, L., 255, 308 van Steenis, H., 203, 224 Van Wijck, J. A. M., 241, 308 Vaupel, J., 269, 308 Vella, F., 234, 235, 297, 308 Verson, E., 159, 225 Vetter, W., 247, SO7 Villareal, R. L., 26, 65 Vinson, C. G., 28, 66 Vishveshwaraiah, S., 278, 300 von Ebner, V., 159, 164, 225 von Hansemann, D., 240, 308 von Olphen, A. H. F., 247,299 von Sengbusch, R., 285, 903, 308 Voorhess, M. L., 244, 508

W Waddington, C. H., 116, 117, 123, 124, 125, 138, 139, 140, 147, 149, 165, 314, 339 Wahrman, J., 232, 308

370

AUTHOR INDEX

Waldeyer, W., 225 Wallace, B., 123, 162 Warburg, E. F., 133, 134, 161 Warburton, F. E., 139, I66 Warmke, H. E., 278, 280, 281, 285, 308 Watanabe, Y., 72, 73, 75, 114, 320, 337 Waterhouse, D. F., 178, 223 Watson, G. S., 44, 56 Watson, M. L., 186, 996 Watson, W. A. F., 211, 225 Wedvik, H., 200, 226 Weks, F., 233, 308 Weissman, I., 274, 296 Wellings, S. R., 315, 339 Welshons, W. J., 202, 214, 226, 271, 273, 274, 293, 308 Wennstrom, J., 248, 290, 308 Wenrich, D. H., 187, 189, 2$5 Went, F. W., 117,165 West, M., 130, 154 Westergaard, M., 162, 163, 226, 275, 276, 277,278, 279, 280, 281, 282, 284, 285, 287, 308, 309 White, L. L. R., 247, 302 White, M. J. D., 61, 62, 114, 159, 168, 169, 177, 183, 185, 187, 189, 226, 269, 309 Whitehead, F., 135, 155 Whiting, P. W., 342, 343, 344, 345, 348, 350, 351, 356, 357, 368 Whittinghill, M., 203, 226 Whittington, W. J., 66, 114 Wilkins, D. A., 138, 156 Williams, C. F., 287, 301 Williams, W., 120, 121, 123, 156 Wilsie, C. P., 101, 108

Wilson, E. B., 62, 114, 159, 160, 162, 165, 187, 226, 228, 229, 249, 250, 309 Wilson, G. B., 286, 292 Winge, g., 236, 269, 270, 271, 276, 277, 282, 309, 310 Witschi, E., 271, 310 Wolfe, H. G., 325, 328, 339 Woodward, D. O., 357, 368 Woskressensky, N. M., 193, 194, 996 Wright, S., 313, 314, 325, 327, 328, 339

Y Yaffe, S. A., 233, 310 Yamada, I., 285, 310 Yamada, K., 241, 244, 301 Yamamoto, T.-O., 271, 310 Yamamoto, Y., 283, 299, 310 Yasueumi, G., 187, 194, 226' Yokoyama, T., 268, 310 Yoon, S. B., 262, 263, 265, 296 Yoshida, V., 193, 218 Ytterborn, K. H., 171, 205, 296

Z Zahlan, A. B., 322, 324, 335 ZeEevic, L. M., 72, 114 Zelickson, A. S., 315, 336, 339 Zich, K., 161, 172, 226 Zimmer, K. G., 211, 226 Zohary, D., 60, 92, 107, 114, 126, 166 Zoschke, U., 286, 310 Zuber, M. S., 23, 24, 63 Zuckerman, S., 173, 218 Zujtin, A. I., 193, 626

SUBJECT INDEX A Acacia, plasticity in, 144 Aehillea, plasticity in, 118, 129 Acnida, sex determination in, 283-284 Acriflavine, cytoplasmic pollen sterility and, 31-32 Adenostoma, plasticity in, 144 Aegilops caudata, Triticum genetics and, 87 Aegilops comosa, Triticum genetics and, 87 Aegilops cylindrica, Triticum genetics and, 87 Aegilops longissima, Triticum genetics and, 85-90 Aegilops mulica, Triticuna genetics and, 86-88, 92 Aegilops ovata, Triticum genetics and, 87 Aegilops speltoides, Triticum genetics and, 82-83, 85-88, 92, 94 Aegilops squarrosa, Triticum genetics and, 82-83, 94 Aegilops triuncialis, Triticum genetics and, 87 Aegilops turcomanica, Triticum genetics and, 87 Aegilops umbellata, Triticum genetics and, 87 Age, frequency of hypertrichosis pinnae auris and, 238-240 Agouti pattern, biochemical studies of, 331-332 Agrostemma githago, plasticity in, 118-119, 128 Agrostis tenuis, plasticity in, 128 Albinism locus, physiological interpretation of, 327-328 Alisma plantago-aquatica, plasticity in, 144

Alleles, partial fertility restoration and, 15-19 Allopolyploids, pairing specificity in, 97-100 Aniphicarpum jloridanum, plasticity in, 145 Angiosperms, sex chromosomes of, 276-287 Animals, plasticity in, 124-126 Anthers, phenotypic appearance of, 6-9 Antigenic differences, Y-chromosome and, 262-263 Apical cell, definition of, 162 Apical complex, definition of, 162 Argentina, cytoplasmic pollen sterility from, 3 Arabidopsis, plasticity in, 122 Asparagus, sex determination in, 285 Atriplex latijolium, plasticity in, 136, 137 Atriplex littorale, plasticity in, 138 Atriplex patulum, plasticity in, 136, 137 Atriplex sarcophyllum, plasticity in, 138 Autopolyploids, bivalent formation in, 100-102 Autosomes, possible role in sex determination, 279-280 Avena fatua, plasticity in, 145 Avena ludoviciana, plasticity in, 145 Avena scabrivalvis, plasticity in, 141

B Barley, plasticity in, 118-119, 122 Betula pubescens, plasticity in, 131 Bivalent (s), secondary association of, 103-105 Bivalent formation, imposition of, 100-103 Bobbed character, Y-chromosome and, 252-255 371

372

SUBJECT INDEX

Bombyx mori, chronometry of spermatogenesis in, 199-200 sex determination in, 267-268 Bothriochloa intermedia, chromosome pairing in, 102-103 Breeding procedures, cytoplasmic pollen sterility and, 48-49 Bromus, plasticity in, 128 Bromus carinatus, plasticity in, 131, 141 Brown locus, physiological interpretation of, 328-329 Bryonia, sex determination in, 284-285 Bryophytes, sex determination in, 287-288

C Camelina sativa, plasticity in, 132 Campanula rotundifolia, plasticity in, 144 Cannabis, sex determination in, 285286 Capsella bursa-pastoris, plasticity in, 128, 147, 149 Carica papaya, sex determination in, 286-287 Cat, Y-chromosome of, 274-275 Centaurea jacea, plasticity in, 138 Cercidium jloridum, plasticity in, 129 Cereals, plasticity in, 119 Ceylon, frequency of hypertrichosis pinnae auris in, 238 Chenopodium album, plasticity in, 128, 132, 136 Chenopodium rubrum, plasticity in, 128 Chlorophyll, cytoplasmic inheritance and, 45 Chromosome pairing, allopolyploids and, 97-100 causal processes, hypotheses of, 61-65 complex control systems, 69-79 conclusions, 105-107 function in Triticum, 90-93 functions, preliminary consideration, 59-65 homologous, types of, 57-58 nonspecific, types of, 58 quantitative variation in, 65-80

secondary association of bivalents and, 103-105 specificity, extent of pairing and, 79-80 genetic control of, 81-105 timing of, 59-61 Chrysanthemum, chromosome pairing in, 101 Chrysanthemum segetum, plasticity in, 132 Coat color, comparative genetics of, 332-333 deer mouse, 334 Coccinea indica, sex determination in, 287 Color genes, evolutionary biology and, 332-334 hair bulb melanocytes of mouse and, 318-319 melanogenic activity and, 325-331 Color loci, roles, physiological interpretation of, 327-331 Color polymorphism, natural mouse populations in, 333-334 Corn, see also Maize, Zea mays cytoplasmic inheritance in, 45-46 hybrid, detasseling and, 47-48 Cotton, plasticity in, 122 Crop plants, plasticity in, 149 Cyst, definition of, 162 Cytology, melanin pigmentation and, 312-314 Cytoplasm, pollen sterility and 4-9 sterility-inducing, classification of, 9-12 creation of, 36-43 Texas, fertility restoration in, 12-20 morphological characters and, 20-26 USDA, fertility restoration in, 12 Cytoplasmic pollen sterility, Argentinian source of, 3 breeding procedures for use, 48-49 chemical studies of, 29-30 comparison of two cytoplasms, 4 4 concluding remarks, 50-52 cytological studies, electron microscope, 29

373

SUBJECT INDEX

light microscope, 27-29 discoveries and classification of, 9-12 economic usefulness of, 47-50 early discoveries of, 2-4 environment and, 13-14 evolutionary usefulness of, 43-44 hybrid production, patent of, 49-50 inbred 33-16, 3 origin and expression in maize, 27-47 Peruvian source of, 2-3 spontaneous origin of, 43 stability, empirical observations, 30-31 experimental treatments and, 31-32 restorer genes and, 31 segregation of particles and, 32-36 summary, 46-47 Texas source of, 3-4 U. S. Department of Agriculture source of, 3

D Dactylis glomerata, plasticity in, 136, 137 Deer mouse, coat color and adaptation in, 334 Definitive spermatogonia, development of, 183-196 types of, 163-164 Deoxyribonucleic acid, chromosome pairing and, 81, 93 Desynapsis, genetics of, 68-69 Detasseling, hybrid corn and, 47-48 Dicliptera assurgens, plasticity in, 131 Drosophila, indefinitive and definitive spermatogonia of, 190-196 plasticity in, 123-125, 136, 149 primordial germ cells of, 178-183 spermatogenesis in, 170-172 X-chromosome of, 250-251 Y-chromosome of, 249-266 Drosophila melanogaster, chronometry of spermatogenesis in, 200-201

E Environment, cytoplasmic pollen sterility and, 13-14

Eumelanin, biochemical pathway to, 320-321 chemical studies on, 322 structure, genes and, 322-324 Euphrasia, plasticity in, 138 Euploid autosome sets, sex determination and, 278-279 Evolution, color genes and, 332-334

F Fertility, Y-chromosome and, 251-252 Fertility restoration, complete, 14 genetics of, 12-20 modifier genes and, 14-15 partial, 15-19 requirements, classification of, 10-12 Fertility restorer lines, production of, 49 Fish, Y-chromosome of, 269-271 Fitness, plasticity and, 124-125 Flax, cytoplasmic pollen sterility in, 41 p-Fluorophenylalanine, chemical pollen sterility and, 31-32 Foreign species, cytoplasm, creation of sterilityinducing cytoplasm, 38-42 Fragaria elatior, chromosome pairing in, 101 Free radicals, human hair melanin and, 322-323

G Gene(s), biochemistry of melanin formation and, 319-332 color, house mouse, 317-318 eumelanin structure and, 322-324 infrared spectra of melanins and, 323-324 major, chromosome pairing and, 6569 melanin pigmentation and, 312-314 restoration of pollen fertility by, 4-5 restorer, geographical distribution of, 41-42 pleiotropic effects of, 26-27 stability, 31

374

SUBJECT INDEX

ultrastructural basis of melanin formation and, 314-319 Y-linked in plants, 276-277 Genetic criteria, chronometry of spermatogenesis, 201-21 1 Genetic induction, creation of sterilityinducing cytoplasm, 36-37 Genetics, comparative, coat color, 332-333 Genotypes, nonrestorer, 12 partial restorer, 13, 19 restorer, 12-13 other kinds, 19-20 Germ cells, definition of, 162 Germ line, maintenance, spermatogonial multiplications and, 159-196 Gossypium, chromosome pairing in, 65, 97-99 Gossypium barbadense, chromosome pairing in, 98 Gossypium hirsutum, chromosome pairing in, 98 Gossypium tomentosum, chromosome pairing in, 98 Grain yield, Texas cytoplasm and, 2326

H Heat, cytoplasmic pollen sterility and, 31 Hair, human, color, 315-317 Helianthus annuus, plasticity in, 118 Helminthosporium maydis, see Southern leaf blight Heterochromatin, polygenes and, 258 variegation and, 258-261 Y-chromosome and, 255-258 Heterosis, chiasmata and, 70-71 Heterozygosity, phenotypic plasticity and, 122-123 Heterozygous plants, “restored,’ ’ appearance of anthers of, 8-9 fertility of, 5-6

Hieracium umbellatum, plasticity in, 138 Histocompatibility antigen, Y-chromosome and, 272-274 Histone, chromosome pairing and, 64-65, 93 Homoeologs, pairing of, 82-83 Homeostasis, plasticity and, 117 Hordeum vulgare, desynapsis in, 68 Human, hair, differently colored, 315-317 hair melanin, free radical content of, 322-323 Y-chromosome, function of, 230-249 Humulus, sex determination in, 282 Hybrids, interspecific, chromosome pairing in, 80 production, patent of, 49-50 Hypertrichosis pinnae auris, frequency, age and, 238-240 in India and Ceylon, 238 later work on, 237-238 mode of inheritance, 236-237 phenotype of, 233-235 recent work, 235-236 Tommasi’s pedigree, 235

I Indefinitive spermatogonia, definition of, 163 development of, 183-196 India, frequency of hypertrichosis pinnae auris in, 238 Infection, creation of sterility-inducing cytoplasm, 38 Infrared spectra, melanins and, 323-324 Inheritance, cytoplasmic, 45-46 Insects, sex-determination, Y-chromosome in, 266-269 Instability, definition of, 116-117 Intermediate spermatogonia, definition of, 164

J Juncus effusus, plasticity in, 131

375

SUBJECT INDEX

Juncus heterophyllus, plasticity in, 119 Juncus obtusijlorus, plasticity in, 119

1 Leaves, number, Texas cytoplasm and, 22-23 Lepidoptera, indefinitive and definitive spermatogonia of, 190 primordial germ cells of, 177-178 spermatogenesis in, 170 Linseed, plasticity in, 119, 120 Linum usitatissimum, plasticity in, 118-120, 128 Lolium perenne, plasticity in, 130 Lotus corniculatus, chromosome pairing in, 101 Loxa JEavicolis, synapsis in, 64-65 Lymantria dispar, sex determination in, 26&267

studies without immediate genetic orientation, 314-315 Mercurialis, sex determination in, 285 Mimosa pudica, plasticity in, 143 Mormoniella, mutants, stock maintenance, 351-356 reproductive behavior of, 341-342 R locus of, 342-356 Mouse, chronometry of spermatogenesis in, 199 color genes and hair bulb melanocytes of, 318-319 color genes and retinal pigmentation of , 317-318 natural populations, color polymorphisms in, 333-334 Y-chromosome of, 271-274 Mutants, eye-color, R locus, 342-347 Mutations, identical, clusters of, 210-211

M

N Maize, see also Corn, Zea mays plasticity in, 123 Male-sterile lines, production of, 48 Mammals, indefinitive and definitive spermatogonia of, 185-186 primordial germ cells of, 173-174 spermatogenesis in, 168-169 Mayaca fluviatilis, plasticity in, 141 Melandrium, sex determination in, 277-282 Melanin, formation, genes and biochemistry of, 319-332 infrared spectra, genes and, 323-324 Melanin pigmentation, genes and cytological basis of, 312-314 Melanogenic activity, assay of, 325-327 color genes and, 325-331 Melanosome, definition of, 314 formation, genes and ultrastructural basis of, 314-319 genetically oriented studies, 315-319 hair bulb, color genes and, 318-319

Nicotiana, chromosome pairing in, 97-99 Nicotiana rustica, plasticity in, 122, 123 Nicotiana sylvestris, chromosome pairing in, 98 Nicotiana tabacum, chromosome pairing in, 98-100 Nicotiana tomentosijormis, chromosome pairing in, 98

0 Oenanthe aquatica, plasticity in, 141 Orthoptera, indefinitive and definitive spermatogonia of, 187-190 primordial germ cells of, 174-177 spermatogenesis in, 169-170 Oxalis acetosella, plasticity in, 143

P Panicum clandestinum, plasticity in, 145 Papaver, plasticity in, 128, 131

376

SUBJECT INDEX

Papaver rhoeas, plasticity in, 132 Papaver striatocarpum, chromosome pairing in, 101 Particles, sterility-inducing, segregation of, 32-36 Patent, hybrid production, 49-50 Peru, cytoplasmic pollen sterility in, 2-3 Petunia, cytoplasmic pollen sterility in, 51 Phaeomelanin, question of, 321-322 Phenotypic flexibility, definition of, 116 Phenotypic plasticity, conclusions, 145-149 conditions disfavoring, 138-140 conditions favoring, 125-138 crop plants and, 149 definition of, 115-116 directional selection and, 136-137 fitness and selection, 124125 fixed phenotypic variation and, 144145 general characteristics of, 145-146 genetic control of, 117-124 interrelationship of different characters, 146-147 mechanisms of, 140-144 open problems, 148-149 stabilizing selection and, 137-138 summary, 149-150 Phenotypic variation, fixed, plasticity and, 144-145 Phleum nodosum, autotetraploids, chromosome pairing in, 101-102 Phleum pratense, chromosome pairing in, 100-102 Photoperiod, plasticity and, 142, 143 Pink-eyed dilution locus, physiological interpretation of, 329-331 Plant (s), height, Texas cytoplasm and, 20-22 heterozygous, appearance of anthers, 8-9 partially fertile, appearance of anthers, 6-8 sex chromosomes of, 275-288 sterile, appearance of anthers, 6 Plantago lanceolata, plasticity in, 132 Plantago maritima, plasticity in, 135, 137

Plasticity, see Phenotypic plasticity Poa annua, plasticity in, 128 Poa pratensis, plasticity in, 128, 132 Poa trivialis, plasticity in, 132 Pollen, transmission of sterility-inducing cytoplasm through, 37-38 Pollen sterility, see Cytoplasmic pollen sterility Polygenes, heterochromatin and, 258 Polygonum amphibium, plasticity in, 135 Polypogon monspeliensis, plasticity in, 118, 128 Popcorn, cytoplasmic inheritance in, 45 Potamogeton, plasticity in, 133-135 Potamogeton lucens, plasticity in, 119 Potamogeton natans, plasticity in, 119 Potentilla, plasticity in, 118, 129-130 Predefinitive spermatogonia, definition of, 163 Drosophila, 178-183 lepidopteran, 177-178 mammalian, 173-174 orthopteran, 174-177 Primary spermatogonia, definition of, 163-164 Primordial spermatogonia, definition of, 162 Drosophila, 178-183 lepidopteran, 177-178 mammalian, 173-174 orthopteran, 174-177 Proserpinaca palustris, plasticity in, 135

Q Quercus douglasii, plasticity in, 129 Quercus ilex, plasticity in, 129 Quercus robur, plasticity in, 129 Quercus turbinella, plasticity in, 129

R Radiations, ionizing, cytoplasmic pollen sterility and, 32 Ranunculus aeris, plasticity in, 136, 137 Ranunculus aquatilis, plasticity in, 131, 134, 135, 142, 143 Ranunculus Batrachium, plasticity in, 133, 134

SUBJECT INDEX

Ranunculus jlabellaris, plasticity in, 141, 143 Ranunculus hederaceus, plasticity in, 119, 134 Ranunculus hirtus, plasticity in, 135136, 148-149 Ranunculus moseleyi, plasticity in, 131 Ranunculus peltatus, plasticity in, 119, 134, 140, 143 Ranunculus plebius, plasticity in, 136 Recombinants, clusters of, 203-210 Retinal pigmentation, house mouse, 317-3 18 R locus, Mormoniella, deleterious factors, 348-356 discussion of, 356-357 eye-color mutants, 342-347 unstable 0, 347-348 Rorippa amphibia, plasticity in, 141 Rumex, sex determination in, 282-283 Rye, plasticity in, 122

S Sagittaria sagittijolia, plasticity in, 141, 144, 145 Scleropoa rigida, plasticity in, 141 Scutigera forceps, synapsis in, 64-65 Secale cereale, breeding behavior of, 70-78 Triticum genetics and, 85-88, 90, 93 Secale montanum, Triticum genetics and, 87, 95, 96 Secondary spermatogonia, definition of, 164 Sedum maximum, plasticity in, 138 Selection, directional, plasticity and, 136-137 disruptive, plasticity and, 126-136 plasticity and, 124-125 stabilizing, plasticity and, 137-138 Sex chromosomes, angiosperm, 276-287 plant, 275-288 Szx determination, euploid autosome sets and, 278-279 fragmented Y-chromosomes and, 280-282 possible role of autosomes, 279-280

377

Y-chromosomes and, 245-247 in insects, 266-269 Sex differences, Y-chromosomes and, 262-263 Sex-linkage, partial, 231-232 Silene otites, sex determination in, 285 Sium latifolium, plasticity in, 141 Solanum nigrum, chromosome pairing in, 101 Solidago virgaurea, plasticity in, 130 Sonchus arvensis, plasticity in, 128 Sonchus oleraceus, plasticity in, 128 Sorghum, plasticity in, 119 Southern leaf blight, resistance, Texas cytoplasm and, 26 Soybeans, plasticity in, 119 Space, disruptive selection in, 133-136 Sparganium erectum, plasticity in, 119 Sparganium minimum, plasticity in, 119 Spermatid bundle, definition of, 162 Spermatogenesis, chronometry of stages of, 197-201 historical background, 157-159 relating temporal and spatial patterns, 196-211 genetic criteria for, 201-211 terminology of, 161-164 Spermatogonia, definition of, 162-164 radiosensitivity of, 211-215 renewal, methods of, 164-172 stages of, 172-196 Spermatogonial multiplication, germ line maintenance and, 159-196 Spinacea oleracea, sex determination in, 286 Sporoholus subinclusus, plasticity in, 132 Stability, definition of, 117 Stellaria media, plasticity in, 131 Sterility, excessive, chronometry of spermatogenesis and, 201-203 Stipa nevadensis, chromosome pairing in, 97 Streptomycin, cytoplasmic pollen sterility and, 31-32 Subterranean clover, plasticity in, 119 Succisa pratensis, plasticity in, 138

378

SUBJECT INDEX

T Teosinte, creation of sterility-inducing cytoplasm and, 39 Time, disruptive selection in, 126-133 Tomatoes, plasticity in, 120-123 Tommasi’s pedigree, hypertrichosis pinnae auris, 235 Trifolium fragiferum, plasticity in, 132 Trifolium repens, plasticity in, 132 Trifolium subterraneum, plasticity in, 132 Tripsacum dactyloides, creation of 1 sterility-inducing cytoplasm, 39-40 Triticale, chromosome pairing in, 77-78 Triticum, formal genetics of, 81-90 Triticum aestivum, breeding behavior of, 70, 72-78 chromosome pairing, evolution of, 94 function of, 90-93 relation between effects, 95-96 desynapsis in, 68 plasticity in, 123 secondary association of bivalents in, 104-105 Triticum monococcum, Triticum genetics and, 82-83, 94 Tyrosinase, structural gene for, 327-328

U Ulex, plasticity in, 144 Ultrastructure, 314

V Valeriana dioica, sex determination in, 285 Variegation, Y-linkage and, 277 Variegation, heterochromatin and, 258-261 Vicia fuba, plasticity in, 118-119 Viola odorata, plasticity in, 131, 141 Vitis, sex determination in, 287

W Wheat, cytoplasmic pollen sterility in, 41

X Xanthium, plasticity in, 145 X-chromosome, Drosophila, 250-251

Y Y-chromosome, bobbed character and, 252-255 bryophytes, 287-288 cat, 274-275 cytogenetics of 240-249 Drosophila, 249-266 fertility and, 251-252 fish, 269-271 fragmented, sex determination and, 280-282 general discussion, 288-290 genes in, 248-240 heterochromatin and, 255-258 historical considerations, 227-230 human, “inertness” of, 247-248 mouse, 271-274 partial sex-linkage and, 231-232 role in sex determination, 245-247 sex and antigenic differences, 262-263 sex determination in insects and, 266-269 variation in length, 241-245 XO-males and, 264 XYY-males and, 264-266 Y-lin kage, characteristics of, 230-240 complete, general, 232 hypertrichosis pinnae auris, 232-240 in plants, 276-277 traits concerned, 233

Z Zea mays, see also Corn, Maize asynapsis in, 67 chiasma frequency in, 72