Ma Received Accession Given By Place. rine Sept Biological . 7, 1950 Laboratory No. 64568 The Iviacmil an Co. -^ew Ycrk City THE ELEMENTS OF GENETICS by Dr. C. D. Darlington and Dr. K. Mather ESSAYS ON GENETICS by Dr. C. D. Darlington CHROMOSOMES AND PLANT BREEDING RECENT ADVANCES IN CYTOLOGY THE EVOLUTION OP GENETIC SYSTEMS by Dr. C. D. Darlington and L. F. La Cour THE HANDLING OF CHROMOSOMES by Dr. C. D. Darlington and E. K. Janaki Ammal CHROMOSOME ATLAS OF CULTIVATED PLANTS by Dr. K. Mather THE MEASUREMENT OF LINKAGE IN HEREDITY STATISTICAL ANALYSIS IN BIOLOGY BIOMETRICAL GENETICS THE ELEMENTS ^ OF GENETICS by C. D. DARLINGTON and K. MATHER NEW YORK THE MACMILLAN COMPANY 19^0 FIRST PUBLISHED IN I949 SECOND IMPRESSION I95O This book is copyright under the Bertie Convention. No portion of it may be reproduced by any process without written permission. Inquiries should he addressed to the publisher PRINTED IN GREAT BRITAIN in 12 point Bembo type BY UNWIN BROTHERS LTD WOKING AND LONDON Knowledge of all kinds is good. Conjecture as to things useful is good; but conjecture as to what it would be useless to know, such as whether men went on all four, is very idle. JOHNSON {BosweU's Life, 1791) For to pass the time this book shall be pleasant to read in; but for to give faith and believe that all is true that is contained herein, ye be at liberty. CAXTON (preface to MaUory, 1485) Except ye see signs and wonders, ye will not believe. JOHN IV. 48 1^ DEDICATED to the compatible and incompatible elements of Linnaeus and Darwin, of Pasteur and Weismann, of Mendel and Morgan, of Bateson and Pearson, which, being brought together in fertile combination by the passage of time and the intercourse of nations, have yielded the abundant harvest of Genetical Science PREFACE There are two ways of attempting to describe a part of nature in scientific terms. One is to deal with the area which has been exactly mapped by experiment, with the ensuing generaliza- tions and predictions, and to leave the rest empty. The other is to go further and use our knowledge of the mapped area to fill in the empty spaces according to the more likely assumptions. The first method is evasive, the second hazardous. We prefer the second and have adopted it. In the present account of Genetics we have tried to use what is known in order to find out what is unknown. As a consequence there stretches a complete range, in a gentle gradient or cline, from the old theories of Chapter i, which are called Laws of Nature, to the new theories of Chapter i6, which are called Dangerous Speculations, The reader will don his doubting glasses at the point he feels proper. He will do well, however, to recollect that this is the first attempt to represent the whole scope of genetics, the whole of what has always been needed. In the past open hypotheses have been replaced by concealed assumptions and such assumptions are far more dangerous than the statements we have laid before the reader in black and white. Events have lent a special urgency to the survey of the whole territory of genetics. They have at the same time made it a venture of special opportunity, one which offers unprecedented rewards. Genetics has now reached the point when its central position in the world of science is becoming generally understood. This isthmus is being found to join continents that have hitherto been unknown to one another. Across it now botanists and zoologists may venture to find common ground with bacteriologists and virologists. On its pathways the student of evolution may teach, and learn from, the investigator of cancer and the practical stock-breeder. Within its confines the physical chemist may verify some of his predictions and confound others. Breathing its air, the physiologist and the embryologist may come to agree that plants are organisms not too simple to be used in explaining the more elaborate mysteries of animal life. PREFACE It may be, of course, that we have over-siniphfied our story. It may be that, in seeing heredity as the outcome, as well as the material, of adaptive and evolutionary change, and in assuming in it a unity of principle which applies also to development and infection in all plants and animals (even in ourselves), we have travelled too far and too fast. We do not hope to satisfy the critic who prefers the small, the single, and the secluded, department. But we do hope that many, whether wise veterans or innocent enthusiasts, who read this book, will share some of the delight we have had in writing it. C. D. D. K. M. John Innes Horticultural Institution, Merton December 1947 ACKNOWLEDGEMENT We are indebted to the various authors and publishers of periodicals and books named in the References to each chapter for the use of their figures in the text. Note on Frontispiece It has been pointed out to us, by Professor R. A. Fisher, that the figures on this page of Mendel's notes suggest factor interaction. Thus the ratio of 343 : 92 : 166 in the middle of the page suggests the 9:3:4 ratio characteristic of an F^ segre- gating for two genes related in action by recessive cpistasy (see Fig. 38 on page 156). This is all the more likely as the class of 166 is recorded near the top of the page as "weiss" and denoted as W. If this inference of epistasy is correct, the results were perhaps from beans rather than from peas. Mendel mentions work with beans (including the species cross Phaseolus vulgaris X Ph. timltifiorus) in his paper, but gives httle detail of the results, which he evidently found difficult to interpret. He makes no mention of any epistatic colour relations in his peas and the notion of cpistasy was not introduced until 1907. 8 CONTENTS PAGE INTRODUCTION Till' Aim and Scope of Genetics 1 5 PART I INDIVIDUALS CHAPTER 1. The Chromosome Mechanism 23 2. The Mendclian Method 36 3. Continuous Variation 55 4. The Biometrical Analysis 78 5. Bases of Change 95 6. Consequences of Change 121 PART II CELLS 7. Genes, Molecules and Processes I45 8. The Cytoplasm 168 9. Development and Differentiation 189 10. Viruses, Proviruses and the Conflict of Systems 207 PART III POPULATIONS 11. Adjustment and Balance 227 12. Breeding Systems 239 1 3 . Selection and Variability 272 14. The Breakdown of Continuity 302 15. The Growth of Genes 327 16. Man and Mankind 346 CONCLUSION The Fiittire of Genetics 367 Appendix I. Glossary of Genctical Terms 375 Appendix 2. Symbols and Symbolism 428 Appendix 3. Genetical Books published in the English Language 433 Index 439 9 64568 TEXT FIGURES rCUKE PAGE 1. Nucleus and cytoplasm in Acetahulciria 17 2. Hybrid and merogon sea-urchins 18 3. The chromosome cycle in sexual reproduction 24 4. The chromosomes of Drosophila and Crocus 26 5. The salivary gland chromosomes of Droiop/ii'/a 27 6. Meiosis and crossing-over 32 7. Segregation in peas 39 8. Recombination in Chlamydomonas 44 9. Maps of the X. chromosome in Drosophila melanogaster 47 10. Sex-linked inheritance 48 11. Autosexing in poultry 50 12. Partial sex-linkage in fishes 51 13. The sex chromosomes of Dro50/)/!i7a and man 53 14. The normal frequency distribution of stature in man 57 15. Relation between genotype and phenotype in polygenic in- heritance 67 16. Polygenic segregation in Epilachtia 69 1 7. Segregation for corolla length in Nkotiana 71 18. The components of variation in height of Antirrhinum plants 87 19. The origin of triploids in Ascaris 97 20. The results of chromosome breakage and reunion loi 21. The Bar duplication in Drosophila io8 22. Chimaeras in So/aHKWi iii 23. A gynandromorph in Drosophila 112 24. The modes of origin of gynandromorphs 113 25. Pleiotropic action of a lethal gene in the rat 116 26. The Agouti series of allelomorphs in the mouse 118 27. Meiosis in polyploids 122 28. The pollen grain chromosomes of Crepis capillaris 124 29. Interchange, and meiosis in the interchange hybrid 128 30. Chromosome pairing in structural hybrids 129 31. Inversion distinguishing Drosophila melanogaster and sinnilaiis 131 32. Pachytene pairing and linkage map of Oenothera 133 33. Chromosome balance in Raphano-brassica 136 34. Polyploidy in Aescuhis 138 35. Segregation and fertihty in polyploids 140 36. Genes, chromosomes and nucleo-proteins 148 37. The A, B, O system of blood groups in man 153 38. The interactions of two genes 156 39. The synthesis of tryptophane in NeMro5porrt 162 40. The four relations of genes in action 165 41. Plastid inheritance in barley 172 42. The inheritance of Killer in Parameciutn 176 43. The inheritance of meUbiose fermentation in yeast 180 44. Dauermodification in Phaseohis 184 II TEXT FIGURES FIGURE PAGE 45. The inheritance of direction of coiling in snails 192 46. Mitosis in the red and wliitc blood precursor cells of man 194 47. The differentiation of nuclear behaviour in Scilla pollen grains 196 48. Cleavage and chromosome fragmentation in Asciiris 197 49. Co-operation bet\\-een pollen grains in Uvularia 199 50. Types of embryo-sac in terms of cell gradients 200 51. Haploid competition, including the Rcnner Effect 201 52. Nucleus and cytoplasm in differentiation 204 53. The three levels of genetic structure 221 54. Differing balances in the homo- and hetero-gametic sexes of hybrids 229 55. Sex balance in Drosophila 231 56. Chromosome behaviour and pollen fertility in Festtica-Lolium hybrids 233 57. Heterosis and inbreeding depression in maize 236 58. Incompatibility in sweet cherries 244 59. The mechanism of incompatibility 245 60. Hetcrothally governed by two loci in fungi 246 61. The efficiency of hetcrothally as an outbreeding mechanism 247 62. Distyly in Primula 250 63. Tristyly in Lythrtim 251 64. The breakdown of incompatibility in Petunia 257 65. Temperature and sex determination 264 66. Sexual and non-sexual forms in Artemia 265 67. The leaves o f triploid and hypo-triploid Taraxacum 266 68. Sexuality and apomixis in Taraxacum species 267 69. Types of twinning in plants and animals 270 70. The release of potential variability 280 71. The states of variability 281 72. Litter size and fertility in pigs 284 73. Selection in Drosophila 286 74. Seasonal variation of gene frequency in Adalia 295 75. The mechanism of correlated response to selection 299 76. Geographical variation in Microtus 304 77. Isolating mechanisms 307 78. Inversion phylogeny in Drosophila 313 79. The isolating effect of inversion 315 80. Three types of interchange evolution 317 81. Genetic changes in species formation 319 82. Chromosome phylogeny in woody plants 325 83. The Rhesus blood groups in man 333 84. Sex-ratio inversions in Drosophila azteca 337 85. The fatuoid series in oats 339 86. Heterocaryosis in fungi 343 87. Mating restrictions in plants, animals and man 357 88. A and b. The distributions of the O blood group gene and the TH sound in Europe 362-3 12 TABLES TABLE PAGE I. Mendel's seven characters in peas 37 3. The composition of F2's in peas 3 8 3. The nine genotypic classes in an F-, segregating for two factors 41 4. Weights of mother and daughter beans in Johannsen's experi- ments 63 5. Weights of coloured and white beans in an Fj 72 6. Effects of the three large chromosomes on hair production in Drosophila 74 7. The inheritance of height in Antirrhiimm majiis x glutiiiosiim 86 8. Balanced and unbalanced complements of chromosomes 98 9. The frequencies of chromosome mutants in tomatoes and newts 99 10. Primary and secondary chromosome changes 103 11. Four allelomorphs governing size of eye in flowers o{ Primula sinensis 115 12. Chromosomes in the pollen of triploid Crcp/5 123 13. The extra chromosomes in triploid Tulipa and Dattira 125 14. The fertility of male and female gametes from triploids 126 15. Chiasmata in Primula kewensis 137 16. Chromosome numbers in the pollen o£ Primula kewensis 139 17. The decay of dauermodification in Phaseohis 185 18. Inheritance of variegation in Scolopcndrium 186 19. The origin and transmission of viruses 219 20. Chromosome behaviour and pollen fertihty in Lolium-Festuca hybrids 232 21. Seed setting in the primrose 249 22. PoUen competition in Streptocarpus 254 23. The breakdown of incompatibility in Primula sinensis 255 24. Chromosome numbers in Poa pratensis 269 25. Chiasma formation and chromosome pairing in asynaptic maize 289 26. The geographical distribution of interchanges in Datura 316 27. Species in Paeonia 322 28. Frequencies of first cousin marriages in man 347 29. Percentages of concordance in human one-egg and two-egg twins 349 30. Tumours in one-egg and two-egg twins 350 31. Frequencies of affection in relatives of tuberculous individuals 351 13 JU, = ysi- V /; -.'o '•>fip^ t;uisr.M, tf ^' 2>HD I. A PAGE FROM THE NOTEBOOK OF GREGOR MENDEL RECORDING THE RESULTS OF HIS CROSSES (>rt' noic on p. S) Reproduced by kind permission of the Curator of the Museum Mendeliaiuim, Brumi ^^;^<^^, INTRODUCTION THE AIM AND SCOPE OF GENETICS Genotype mid Eiiviroiiiiiciit Nucleus and Cytoplasm Individuals, Cells and Populations When we sow an acorn we expect to raise an oak; when we breed dogs we expect to get puppies; and even when we culture a typhoid bacillus we expect to produce more bacteria of a kind that will cause typhoid. In a word, we expect that like will beget like. But the resemblance we look for is not absolute or unconditional. One oak is not exactly like another ; nor is one dog like another ; nor even, invariably, one typhoid bacillus. The causes of these unlikenesses are sometimes obviously external. Lack of iron, or lack of light, or :oo much light, may make the green leaves of a plant turn yellow. Disease, starvation, or training may alter the shape, habits, or abilities :>{ an animal. The causes of other unlikenesses, as of the likenesses, ire internal, although less obviously so. These inborn causes have o be discovered. By comparison and experiment they have to be eparated from the external causes. They have to be defmed as naterials or processes whose behaviour and effects we can predict nd control. This is the aim and scope of genetics. Our first task is thus to separate external from internal causes. .eaves may be yellow on account of the conditions under which the Jant is grown, that is on account of the environment. But we also now plants that have yellow leaves under any conditions. These lUSt be yeUow on account of their internal or inborn properties. V^hat makes these inborn properties? Popularly we say heredity. echnically, we must be stricter and say genotype (the type which reates). The genotype, according to Johannsen, is what gives the bserved development, the appearance, of a plant or animal in a articular environment. This appearance we call the phenotype (the 'pe which is seen). Differences between phenotypes (such as our sllow and green leaved plants) may be due to differences between motypes, to differences between environments, or even to dif- rences in both acting together. For example, there are strains of 15 ARY % INTRODUCTION oats, Ai'cna sativa, which bleach and die in strong sunlight, but, in a weaker light, seem normal, i.e. have a normal phenotype. The phenotype is the product of reaction of the genotype and the environment. Either can change it, and it is not to be traced to either separately. Thus, too, the study of differences in both heredity and environment, by permitting us to separate their effects, must be the foundation of our knowledge of genetics. The differences between individuals are not the only ones with which heredity is concerned. All organisms undergo development which consists of growth together with the origin of differences between the parts, differentiation as we call it. Heredity depends on a repetition of this development. Similar genotypes give or deter- mine similar sequences of development. Heredity at once determines that individuals are alike and that their parts are unlike. This is the paradox that baffled our forebears. We can now resolve it by examining the materials and processes concerned. When we cut the tail off a worm or the top off a dandelion, what is left grows again to replace the lost part: it regenerates. In spite of their differentiation, the parts of the animal or plant have some- thing inborn in them which is still the same. Similarly, fragments, cuttings and grafts, of particular animals and plants, Hydra, the pondweed Elodea, or a variety of apple or pineapple, can be propa- gated throughout the world with great and predictable uniformity. These vegetative individuals, or clones, demonstrate heredity in its simplest form. They have, as a rule, and so far as we can make out, the same genotype. And since the whole of each clone is derived from a fragment of one individual, we see that the differentiated parts of that individual must have had the same genotype. With sexual reproduction the matter is different. Here a new individual arises from the fusion of two germ cells, usually from the fertihzation of an egg by a sperm. No two individuals formed in this way are absolutely alike. In fact, if we fmd a pair of twin children (or eight armadillos) who are alike, we assume that they have been derived from single fertihzed eggs by mere fragmentation and growth. And we call them identical twins (or octuplets). What is it that remains constant in vegetative individuals, yet is liable to change in sexual reproduction? To know this, we must look at the cells of which plant and animal bodies are composed. i6 NUCLEUS AND CYTOPLASM Their multiplication underlies both kinds of reproduction. In all higher organisms these cells contain a dense spherical body, the nucleus, lying in the more fluid cell substance or cytoplasm. The nucleus has two primarily significant properties. In the first place it can be derived only from a pre-existing nucleus. It has the character ACETABULARIA CREN MEDN + CRENN->INT CREN/MEDN^MED MED/CRENN_>CREN I. — Diagram showing the forms produced by reciprocal grafting of the uni- MED Fig cellular algae Acetabularia mediterranea and crenulata. Where both nuclei are present the hat is regenerated in an intermediate form as if the plant were a sexual hybrid. Where the nucleus is of one species (indicated by N) its influence gradually pre- dominates over the cytoplasm of the stem in regeneration (based on Hammerling 1943). of heredity in itself. In the second place without a nucleus, or even sometimes with one if it is from a different species, cells die. Bu sometimes a nucleus of one species can be put into a cell of another. This can be done by transplantation in a green alga Acetabularia, whose single cell consists of three parts, hat, stem and base containing the single nucleus. When the stem of one species is grafted onto the base of another and the hat cut off, the new hat that grows is most like that of the species from which the base with the nucleus is taken. This, and similar experiments with various combinations of stem Eltments of Genetics 17 INTRODUCTION and nucleus, show that the nucleus must be deciding what kind of hat is grown. Tlie nucleus seems to bear the genotype (Fig. i). Sexual reproduction is just such a transplantation as we see in the alga. A sperm nucleus or generative nucleus of a pollen grain enters an egg cell hundreds or thousands of times larger, and fuses with the female nucleus. Thus the nucleus of the fertilized egg or zygote is of mixed origin; but its cytoplasm is almost exclusively from the mother. The new organism develops from this zygote and shows, S S?xEcJ [S]xEc^ E Fig. 2. — Larvae of sea urchins: S, Sphaerechinus granuhtus; E, Echinus microtuber- culatus; S $ x E (^ , the normal cross; [S] X E (^ , the haploid larva produced by the same cross when the nucleus of the egg has been removed. The larva is then scarcely anything other than a dwarf edition of the sperm parent which is the source of its nuclei (after Boveri i{ where they differ sufficiently, the characters of father and mother in equal measure. Going a step further, Boveri was able to fertilize a piece of egg, lacking a nucleus, of one species of sea urchin with the sperm of another. The resulting larva (termed a merogon) closely resembled a dwarf of its male parent from which it had derived its nuclei and showed no obvious influence of its female parent from which it had derived its cytoplasm (Fig. 2). We thus see the overriding action of the nucleus. The study of its structure and movements may therefore be expected to tell us why individuals are so constant within themselves in their inborn character and also why they differ from one another. 18 INDIVIDUALS, CELLS AND POPULATIONS Equipped with this knowledge of individuals we can return to consider their parts, to find out what happens within each individual. We can try to discover what the rest of the cell, the cytoplasm, has to do with the nucleus in determining heredity, and how it reacts with the nucleus to produce the differentiation of cells and tissues during development. And finally we can see the individual as part of something larger. We can recognise its heredity as part of a system of individuals, a population breeding together, descended from a long line of ancestors similarly related, and likely also to give rise to a long line of descendants. From the most minute and transient events we shall come to envisage the vast and enduring laws of change which we associate with the name of evolution. It is thus in three parts, in relation to individuals, to cells and to populations, that we shall have to unfold the working of genetics. REFERENCES AKERMAN, A. 1922. Untersuchungcn fiber cine in direkten SonneiJichte nicht lebensfahige Sippe von Avena sativa. Hereditas, 3 : i^rj-i']']. BOVERi, T. 1889. Ein geschlechtlich erzeugter Organismus ohne miitteriiche Eigenschaften. Sitz. Ber. Ges.Morph. Phys. Miincheti, 5. (Translation by T. H. Morgan, 1893. Am. Nat. 27: 222-232.) HALDANE, J. B. s. 1946. The interaction of nature and nurture. Atin. Eugenics, 13: 197-205. HAMMERLiNG, J. 1943 . Ein- und zweikemige Transplantc zwischen Acetahularia mediterranea und A. crenulata. Z.I.A.V., 8i: 1 14-180. JOHANNSEN, w. 1911. The genotype concept of heredity. Aw. Nat., 45: 129-159. 19 PART I INDIVIDUALS CHAPTER I THE CHROMOSOME MECHANISM Mitosis The Polytene Nucleus Meiosis Reduction and Recotiibination Mitosis The growth of the organism depends on the growth and multi- pHcation (or, as we call it paradoxically, division) of its cells and nuclei, which must evidently divide together if each cell is to have its one nucleus. The process by which they divide is known as mitosis, and it is during this process that we are able to study the structure of the nucleus. The first sign of mitosis is shown by a change in the nucleus. In the undividing cell the nucleus is said to be at rest. It is globular and shows no structure apart from one or two dense storage bodies, the nucleoli. When mitosis begins, dense and stainable threads appear in a more fluid medium. These threads are double. They shorten and thicken by forming a spiral, to give curved or bent double rods, the chromosomes, which are released into the cell when the membrane or boundary of the nucleus disappears. The release of the chromosomes marks the end o( prophase. At the same time, the beginning of metaphase is shown by the appearance of the spindle. The spindle is a liquid-crystal structure whose fibres lie parallel to its axis. The chromosomes, now at their shortest (25 n down to I /^ or less), come to lie in the wider middle of the spindle and form a flat plate. We can then see five of their important properties (Fig. 3). The first is that the number of chromosomes in the mitoses of one individual, and often indeed of one whole species, is constant, whether it be two thousand or only two. The second is that the chromosomes are also constant in shape and relative size. The third property is revealed by the second and is true of all cells in the higher plants and animals except the germ cells: there are two chromosomes of each kind or, if you like, two similar sets. The 23 THE CHROMOSOME MECHANISM Fig. 3. — The chromosome life cycle in sexually reproducing organisms showing the alternation of haploid and diploid phases of different duration according to the relative position of meiosis and fertilization. Of the three pairs of chromosomes the longest bears the nucleolar organizer. P, M, A, T: pro-, meta-, ana-, and telo-phase. 24 MITOSIS fourth is that each chromosome owes its characteristic shape to the possession of a constant point of attachment to the spindle, marked by a constriction where the chromosome bends. The main body of the chromosome may lie off the spindle and off the plate : only the point of attachment is fixed on the spindle. The fifth is that, except at these attachment constrictions, the chromosomes are double throughout. They consist of pairs of sister chromatids (Fig. 4). The importance of these structures becomes apparent at the next stage. All the chromosomes simultaneously begin to split at those very points of attachment where they have previously remained single. It can then be seen, after proper treatment, that the point of attachment consists of a minute particle, the centromere. It is the centromere which organizes the developing spindle. It is also the centromere which suddenly divides, or explodes, so that its two halves are driven apart along the fibres of the spindle, that is towards the poles. Each half drags behind it one of the two chromatids of its chromosome. Hence the whole body of the chromosomes lying on the plate is divided into two groups of daughter chromosomes, identical in number, shape and size. The separation of these daughter chromosomes is known as anaphase. Two daughter nuclei are reformed from the two groups. The chromatids now loosen their packed spirals, and their fme threads are lost once more in the optically homogeneous nucleus. One or more nucleoli reappear in each nucleus. A partition or wall develops across the equator of the spindle and separates the daughter cells. This is telophase. Mitosis is complete. This course of action shows us that it is through the chromosomes that the nucleus transmits its character to its daughters, that is from cell to cell. It shows us, too, how a complement of chromosomes inside a nucleus is divided into two daughter complements which are distributed to the two daughter nuclei. The significant fact for genetics is that these two complements are always exactly like one another. It was first noted so long ago as 1879 by Flemming, one of the discoverers of mitosis. This observation, however, leaves us with yet another question. How does each chromosome, single as it is when it disappears in telophase, come to be double when it reappears, consisting of two apparently identical halves, in the following prophase ? The obvious explanation, the one long ago suggested by 25 THE CHROMOSOME MECHANISM Boveri, was that the chromosomes remained in being in the resting nucleus. They remained as invisible threads, each of which split lengthwise, or reproduced itself, to give two identical chromatids which reappeared in the following mitosis. There are many ways of showing that the theory of the repro- duction of chromosomes, which at first seemed unnecessary and Fig. 4. — Diploid chromosomes at metaphase of mitosis in polar view to show the arrangement in a flat plate. Left, Drosophila melanogaster male with XY, two large, and one very small autosome pair. Right, Crocus hyemalis with five pairs. All chromosomes have centric constrictions and in addition one Crocus pair has a nucleolar constriction. X 3,000. fantastic, is both necessary and true. The simplest way is by com- paring the uncoiling threads of telophase with those which become visible at prophase. Then we see the uncoiling still going on. There are coils which straighten out and disappear only just before meta- phase, and these are obviously relic coils, the outward and visible signs of the inward and invisible coils of the preceding mitosis. As we proceed step by step new evidence of this fundamental principle of continuity will come to light. The Polytene Nucleus There is another kind of indication of what the chromosomes are doing, or can do, in the resting nucleus in certain large gland cells. Such nuclei, for example in the salivary glands of flies, con- tinue growing without ever dividing. Their chromosomes, however, remain stainable and divide again and again inside the nuclei. They reproduce while continuing their ordinary work. Instead of the double chromatids of prophase nucleus, there are then bundles of 26 THE POLYTENE NUCLEUS four, eight, sixteen, and higher doublings of threads. Each of these threads, when stained, has a characteristic beaded structure, and the identical beads of the multiplied sister threads form plates. Each polytene, or multiple chromosome, then looks like a banded ribbon CENTROMERES and HETEROCHROMATIN IV Fig. 5. — The paired and banded polytene chromosomes of a pressed saUvary gland nucleus in Drosophila mclanogastcr. AU the centromeres and heterochromatin are agglutinated in a single body at the top. The two arms of each of the long autosomes are marked. X ea. 600 (after Painter 1934). as seen from the side. Now these bands, of which there are hundreds in each chromosome, are different from one another, and constant in size, structure and number, just as are the chromosomes in a metaphase plate. They are the chromomeres (Fig. 5). The linear order of the chromomeres is recognizably constant from nucleus to nucleus and displays the individual permanence of the chromosomes. It shows us how they must maintain their struc- ture as a line of distinct particles within the resting nucleus. It also 27 THE CHROMOSOME MECHANISM shows US why the chromosomes must reproduce along a longitudinal axis. Only by linear order and longitudinal doubling and splitting can a chromosome, an aggregate of chromomeres, produce daughter chromosomes, daughter aggregates, identical with each other and with itself. The nucleus, therefore, evidently has the means of identical self-propagation. What do we now know of the nucleus ? We saw that it is, in some sense, paramount in the ceU; that is, in development and heredity. We now see that it is an aggregate of particles, dissimilar particles, and that this aggregate has the capacity of reproducing itself as an aggregate of the same particles in constant proportions and constant order. It undergoes great transformations in each cycle of nuclear division, but they have a chemical regularity. Underlying this regu- larity must be a constant molecular structure. It has a complex character but it has the capacity for propagating this character unaltered; in other words it has the capacity of heredity. The chromosome mechanism shown by mitosis makes it possible to understand the constancy of vegetative growth and propagation. It provides for that uniform genetic character of cells in a tissue and in an organism on which, as we shall see later, their co-operation in development is based. It shows why fragmentation of an organism, from buds, cuttings and vegetative spores, gives daughter individuals which are identical in heredity. It does not, however, explain the differences between individuals, or the variation amongst the progeny of the same individual or pair in sexual reproduction. To do this we must examine the history of the nucleus during the sexual processes. Meiosis We have seen that fertilization involves, or indeed consists in, the fusion of two nuclei. The nucleus of the zygote produced by fertilization thus contains the sum of the chromosomes of egg and sperm. In the nematode Ascaris megalocephala chromosomes from the two germ cells, one from each, combine to give two in the embryo. In the flowering plants, Crepis capillaris or Crocus graveolens, three chromosomes from the pollen nucleus combine with three corresponding ones from the egg nucleus in the embryo sac to give six in the embryo, three obvious pairs. In the formation of these 28 MEIOSIS germ cells by the parent zygotes there must therefore have been a process of reduction, a halving of the chromosome number to compensate for the addition in fertilization. Such a process, predicted by Weismann in 1887, is now knov^m or assumed to occur in the cell history w^herever there is sexual reproduction. It is called meiosis. In most algae, fungi and protozoa the diploid or double nucleus produced by fertilization undergoes meiosis at once to give plants or animals with haploid nuclei containing the half number of chromo- somes. Elsewhere meiosis is postponed so that a diploid stage is intercalated in the life history. In the mosses and liverworts the small diploid organism or sporophyte is, as it were, parasitic on the larger haploid. In the ferns the diploid has become more important than the haploid. In the flowering plants the haploid phases, the embryo- sac and poUen grain, are reduced to a parasitic state. Finally, the complete reversal, in which the haploid phase is eliminated as a separate organism and exists only as the germ cells themselves, eggs and sperm, is attained in all the higher animals. Through all these variations in relation to the life cycle the course and character of meiosis remain the same. It may be concerned with the formation of a fungus spore or a lily pollen grain, the maturation of a spider's egg or a human sperm; but it consists always of two divisions of the nucleus, with only one division of the chromosomes. From its very outset it differs from mitosis. The chromosomes are single when they emerge from the resting stage in the mother cell. Moreover, just as in polytene nuclei, where activity and reproduction are going on at once, the chromosomes appear in the form of strings of chromomeres. As soon as they appear, these strings begin to .pair. They come together side by side, chromomere by chromomere. The chromosomes make contact and begin to pair at their ends, or at their centromeres, or at both at the same time. From the contact point the pairing runs along them zip-fashion. Sometimes it runs the whole length. Sometimes it is interrupted so that only the part near the first point of contact is paired: pairing is then locaHzed. After pairing, the chromosomes coil round one another, and so they remain for an indefinite period. This is the thick-thread or pachytene stage. We then see that the chromosomes of each pair exactly correspond. Evidently one is from the sperm, the other from the egg, which 29 THE CHROMOSOME MECHANISM fused to give the zygote we are examining. Thus, instead of the diploid, 2M, number of chromosomes of mitosis, there is a reduced, haploid, n, number of chromosomes. And instead of the paired sister chromatids of mitosis produced by reproduction, there are paired homologous chromosomes brought together by attraction. Suddenly attraction gives way to repulsion. All the pairs fall apart. At the same time they are seen to be double. Each chromosome has divided into a pair of cJiromatids. The chromosomes seem to remain in contact at certain points and closer examination shows that their chromatids exchange partners at these points. Apparently, under the strain of coiling and at the moment of their origin by division, the chromatids have broken, pairs of partner chromatids breaking at corresponding points. They have uncoiled and rejoined in new com- binations. This mechanical transformation is called crossing-over, the observed change of partner is called a chiasma, and the stage is called diplotene. Some of the coiling of the chromosomes is undone in the forma- tion of chiasmata and some is lost afterwards by still more uncoiling. The chiasmata can then be seen and recorded in number and position. They are as a rule evenly distributed over those lengths of the chromosomes which were paired at pachytene. When pairing has been complete, their distribution is uniform throughout. When it has been locaHzed, the chiasmata are of course similarly localized, near the ends, or near the centromeres, or near both. The kind of distribution of chiasmata is characteristic of each species or even genus of plant or animal, e.g. complete and uniform in Lilium or Zea Mays and pro-terminal in Triton, Tradescantia or Oenothera. In Allium and Fritillaria there are some species with uniform, and others with pro-centric, distribution. As uncoiling proceeds, the chromosomes thicken and shorten even more than in mitosis. As this happens, adjoining loops between chiasmata come to lie at right angles. The distal chiasmata (nearest the ends) are pushed towards the ends of the chromosomes, which then appear merely to touch. The compact paired, or bivalent, chromosomes, as we may now call them, lie evenly in the nucleus. Small chromosomes, where the centromeres are very close to the nearest chiasma, are drawn into fme threads by the repulsion between their centromeres. This stage is diakinesis. It is followed by the dis- 30 MEIOSIS appearance of the nucleolus and the nuclear membrane, the appear- ance of the spindle, and the movement of the bivalents on to the metaphase plate, in other words, by metaphase. But this, the first metaphase of meiosis, is different from metaphase of an ordinary mitosis in two related and all-important respects. First, individual chromosomes are not now sufficient to themselves. Their centromeres do not orientate themselves separately or individu- ally on the spindle. They orientate themselves only in relation to their partner centromeres. The stretching between partner centromeres, already noticeable at diakinesis, is now exaggerated. Secondly, and consequently, anaphase does not begin by the division of the cen- tromeres. Just as the chromosomes remained undivided at the be- ginning of prophase, so, in turn, the centromeres remain undivided at the beginning of anaphase. It is not their division which sets the chromosomes moving to the poles. It is merely the lapse of attraction between the paired chromatids where they are hindering the move- ment apart of the paired chromosomes, i.e. on the far sides of the chiasmata from the centromeres. Such a lapse of attraction occurs at mitosis, but it is hardly noticeable because at mitosis there is nothing to puU the chromatids apart until the centromeres divide. Now the paired centromeres are already repelling one another in readiness for the lapse of attraction. When it occurs, the daughter bivalents, each double except for its centromere, run to opposite poles. The first division is complete. The situation as the daughter nuclei are formed is a remarkable one. Each nucleus has the haploid number of chromosomes, but the diploid number of chromatids. For the chromosomes are already divided. They are double as they are in the prophase, not in the telophase, of an ordinary mitosis. Nor are the two chromatids of each chromosome related to one another like those in prophase of mitosis. They are true sister chroma- tids between the centromere and the nearest chiasma. Beyond that chiasma, until the next one, they are chromatids from parmer chromosomes. Looking at it from another angle, we may say that the separation of the first division has been between partners next to the centromere: it has been reductional. Beyond the chiasma it has been between sister chromatids : it has been equational. An unexampled situation calls for an unexampled remedy. The 31 THE CHROMOSOME MECHANISM Prophase Tetrad of Spores or Gsrm Cells Fig. 6. — The structural and genctical history at meiosis of two pairs of chromosomes, one partner in each hatched, undergoing crossing-over and segregation. Note that all four products differ in the genetical origin of each of their chromosomes even where only one chiasma has been formed in a bivalent (from Darlington 1945). 3^ REDUCTION AND RECOMBINATION daughter nuclei at once divide again. But their chromosomes, already double, do not divide again. They lie on the second metaphase plate and look just like those of an ordinary mitosis, except that their chromatids, which are not true sister chromatids throughout their length, do not lie together throughout their length. Indeed they need not touch at all except at their centromeres. The second meiotic anaphase then enables us to see very clearly the essential character of an anaphase. Separation must depend on the division of the cen- tromeres, because elsewhere the chromatids are already separated. The result is four nuclei, each with the haploid number of chromo- somes, now normal single chromosomes, in fact four truly haploid nuclei (Fig. 6). Reduction and Recombination What does meiosis tell us ? The chromosomes which pair, indeed the individual chromomeres which pair, are generally of similar size and shape. They must therefore pair because they are like one another. And since the differences in the sizes and shapes of different pairs are constant from generation to generation, the pairing chromo- somes must be derived from opposite parents in which they have corresponding structures and functions. They must therefore (ulti- mately) be of common ancestry, or, as we say, homologous. Further- more the chromosomes which do not pair, whether from the same parent or from opposite parents, must be different in structure and function from one another. To some extent, the chromomeres which make up each chromosome may be supposed to be carried along by their neighbours in pairing. But, to some extent, they too must be differentiated particles with their own specific attractions for similar homologues. In other words, the chromosomes composing a haploid set must be different from one another and are likely to be made up of specific and different chromomeres in a constant linear arrangement. What are the results of meiosis ? The diploid mother-cell has given rise to four haploid spores or gametes. The number of the chromo- somes has been reduced to half. There was formerly a great deal of argument as to whether this reduction took place at the first division or at the second. We now see that it takes place at neither. It takes place by virtue of the fact that no division of the chromo- Elements of Genetics "IT. C THE CHROMOSOME MECHANISM somes intervenes between these two divisions of the nucleus. If either of the divisions fails to separate two daughter nuclei effectively, an unreduced or diploid germ cell is formed. And if both fail, the germ cell or spore is tetraploid. Now the four germ cells produced by a regular meiosis are alike in being haploid. But in all other respects their nuclei are different. The arrangement of the bivalents at first metaphase has been, as we can show, at random, so that maternal and paternal chromosomes will have been assorted at random. At the same time we have to realize that we can no longer speak of chromosomes as the units they were at mitosis. Their parts have also been reassorted and exchanged or recomhined by crossing-over. Those parts which separated rcduc- tionally at the first division, separate equationally at the second, and vice versa. Further, since every bivalent has been held together by at least one chiasma, and its chromatids have undergone at least one crossing-over, all its four chromatids distributed to the four nuclei are different combinations. And finally each bivalent in each mother cell differs from all others almost without limit, since each crossing-over can occur at hundreds of different positions. Hence each mother cell will have given rise to four spores or sperms dif- ferent from one another and different from those produced by any of the other mother cells. It was the consideration that the universal production of four cells at meiosis implied that the four must be universally different that led Janssens in 1909 to suppose that crossing- over was a universal concomitant of meiosis; that it was associated with the formation of chiasmata; and that it led to the recombination of hereditary differences. In short, by virtue of crossing-over, no two of the products of one meiosis will be alike. And since crossing-over can probably take place between any two chromomeres, it will be rare indeed for two identical haploid nuclei to be produced from different meioses— unless the pairing chromosomes are themselves identical. Still rarer will it be that two haploid gametes will combine in fertilization to restore the type of either of their parents. Here then we sec how variation, the occurrence of differences between individuals, can be maintained from generation to generation in sexual reproduction; and not only maintained, but rearranged and redistributed in such a way as to make every varying individual unique. 34 THE CHROMOSOME MECHANISM REFERENCES BOVERi, T. 1904. Ergebnisse iiber die Konstitution der Chroinatisclien Suhstanz des Zellkerns. Jena. DARLINGTON, c. D. 1937- Rcccnt Advances in Cytology. 2nd ed. London. DARLINGTON, c. D. 1945 . The chemical basis of heredity and development. Discovery, 6: 79-86. PAINTER, T. s. 1934. A new method for the study of chromosome aberrations and the plotting of chromosome maps in Drosophila molanogaster. Genetics, 19: 175-188. WEiSMANN, A. 1 893. The Genuplasm. London. 35 CHAPTER 2 THE MENDELIAN METHOD Mendel's Experiments The Segregation of Factors Linkage The Chromosome Basis Sex Determination and Sex-Linkage We have now seen how certain visible self-propagating structures are transmitted from cell to cell and from parent to offspring. We have seen the rule and order of these processes. How are they reflected in the gross structures, the phenotypes, of parents and offspring? The answer is given by the mendelian method. Mendel discovered the two requirements for success in planning a critical experiment on heredity — that is, on sexual heredity. First of all, crosses must be made between visibly and sharply different parents. Secondly, these parents must come from true-breeding lines, otherwise any differences visible in the progeny might be referable, not to the known differences between these parents, but to unknown differences between the ancestors of one of them. Such true-breeding lines he knew from experience could be obtained with certainty only in organisms with regular self-fertilization, i.e. where the male and female gametes which fuse are produced, generation after generation, by the same zygote, by the same or different flowers of one parent plant. Mendel's Experiments The garden pea, Pisum sativum, has varieties which meet these requirements and we cannot do better than describe the experiments Mendel made with them. In 1857 he took 34 varieties, and after two years' trial selected 22 of them for his experiments. These remained constant throughout the work. They gave him seven differences of character (or phenotype, as we may say) distinct enough for his purpose. These differences, or "characters" as they are conveniently called, showed themselves in the course of development in the order given by Table i. One character, that of purple pigmentation, expresses itself at 36 MENDEL S EXPERIMENTS different stages of development. It will be seen that the one structure of the seed contains parts of the parent generation (in the seed coat) and parts of the offspring (in the cotyledons). TABLE 1 MENDEL'S SEVEN CHARACTERS IN PEAS 1. Cotyledons yellow versus green in the ripe seed 2. Cotyledons round versus wrinkled in the ripe seed r Leaf-axils purple versus green in the seedling 3. < Petals purple versus white in the mature plant I Seed-coat purple versus white in the mature plant 4. Stem tall versus dwarf in the growing plant 5. Flowers axial versus terminal in the mature plant 6. Unripe pods green versus yellow in the mature plant 7. Ripe pods inflated non-sugary versus constricted sugary in the mature plant Mendel crossed these differing lines of peas and found that, in each cross, the whole of the seedlings of the first fiHal generation, or Fj, resembled one of the parents in respect of each of the character differences. The type which appeared in the crossed plants he termed dominant, and its latent alternative recessive. The dominant is the first of the two alternatives for each character in Table i, but it is impor- tant to note that the result is the same whichever way the cross is made : reciprocal crosses are alike. The Fi was uniform, as uniform as the parents had been. A second generation, Fg, was then raised from self-pollination of the Fj, and in the Fg famihes the recessive types reappeared together -With the dominants. But no transitional or intermediate types appeared amongst them. Furthermore, the dominants and recessives were always in the approximate proportion of three to one. The exact proportion, of course, varied from family to family according to the chances of sampUng. The examples of Table 2, showing the numbers of plants with alternative cotyledon characters, illustrate the principle. The next question to settle was the breeding properties of the different types, the dominant and the recessive, in the F2. F3 families were raised from individual F2 plants by self-pollination. All the recessives bred true, but only one third of the dominants did so. For example 166 bred true for yellow cotyledons and 353 gave yellow and green types again in the proportion of three to one. 37 THE MENDELIAN METHOD TABLE 2 COMPOSITION OF Fj's IN PEAS (MENDEL) Families from crossing No. of F| plants F2 Dominant Recessive Yellow-green Round-wrinkled . . 258 253 6,022 2,001 5,474 1,850 Thus the Fg consisted of three types: pure dominant (Hkc one parent), mixed dominant (Hke the Fj again), pure recessive (Hke the other parent), and they occurred in the proportions of I : 2 : i or, expressed as fractions of the whole, j : |^ : j. The Segregation of Factors How was Mendel, and how are we, to understand these simple rules and ratios ? In respect of each character the plants of the Fo are all either like one or other of the two parents, or like the Fj, which is of course itself merely the product of fusion of gametes from those two parents. We need therefore assume only that the Fj plants are producing gametes, pollen and eggs, of the types produced by both of the parents. Further, since plants of the two parental types are equally frequent in the Fg, gametes of the two parental types must be equally frequent in the pollen and eggs of the F^. Take as an example the case of round and wrinkled seeds. Half the pollen (and half the eggs) in F^ are of round type, R, and the other half of wrinkled type, r. Then R eggs will meet R pollen in ^ X ^ of the cases to give RR homozygotes (to use Bateson's term), and r eggs will similarly meet r pollen in ^ of the cases to give rr homozygotes. R eggs will meet r pollen in another ^ and r eggs R pollen in the fourth j; these two types, being both Rr hctero- zygotes, are indistinguishable and together constitute \ the cases (Fig- 7). Of course \ and i merely represents the chance of a gamete being of a given kind, or of a zygote arising in a given way. The vagaries of sampling will result in the exact ratios, the theoretical or expected ratios, being rarely realized. 38 ROUND WRINKLED 2n Meiosis n Fertilis*^ 2n Meiosis n Fertilis'^ 2n MEIOSIS GAMETES AND FERTILIS'* 2n Fig. 7. — Mendel's experiment of crossing round and wrinkled peas. The parent strains (P) were true breeding, giving only one kind of gamete (small circles) each. The hybrid between them (Fj) was round seeded, but gave two kinds of gamete in equal numbers, one like the gametes from the round parent and the other like the gametes from the wrinkled parent. When pollen of the two kinds fertilizes embryo sacs of the two kinds in random proportions, three round seeded peas are obtained on the average for every wrinkled seeded pea in Fg. The wrinkled peas and one-third of the round peas of F2 are true breeding like the original parents, but two-thirds of the round peas resemble F^ in constitution and again give the 3 : i segregation in F3. The chromosome cycle of reduction and fertilization is shown in relation to Mendel's observations on the right of the diagram, where 2» is the somatic and n the gametic number of chromosomes. THE MENDELIAN METHOD Now all these statements can be put much more simply if we say that the zygotes are double, and the gametes single, in respect of something which determines each of the characters and differences Mendel was studying. We must then say that the two alternative determinants or factors of each kind in a zygote may be alike or different; but even if different, these alternatives, or allelomorphs as they arc called, separate, or segregate, from one another unchanged to give pure gametes; that is each gamete has only one of them, and that one untainted and unblended. This is Mendel's so-caUed first law of inheritance. In applying this law we may note that it is quite indifferent for its transmission whether a factor is dominant or recessive, whether it comes from the male or the female parent, or whether it goes to male or female gamete. On this basis we can, as Mendel did, make certain predictions. When the Fj is crossed, back-crossed as we say, to one of its parents, offspring will be produced of two kinds in equal numbers. One kind wiU be like the parent in question and the other like the F^. With the dominant-carrying parent the two types will look alike, although they will breed differently {RR and Rr). With the recessive-carrying parent they will look different, as well as breed differently {rr and Rr). This expectation was realized. In a back-cross of an Fi to its round- seed parent, Mendel obtained 192 round seeds, and on testing 177 of these further 87 bred true and 90 proved to be of the Fj mixed type. In the back-cross to the wrinkled parent, he obtained 208 seeds ; 102 were wrinkled seeds breeding true, i.e. homozygous for the factor rr, 106 were round seeds, all of the mixed type, i.e. hetero- zygous for the factor Rr. So much for single differences. Where the homozygous parents differed in two characters, the factors determining the two differences were independent. They were independent equally in transmission and in action. Thus the F^ showed both dominant characters whether these came from the same or from different parents. And in both cases the ¥., showed a proportion which was the square of a 3 : i proportion, in other words 9:3:3:1. Thus Mendel crossed round yellow by wrinkled green peas {RRYY X rryy) and obtained a round yellow F^ (Rr Yy). This gave him an Fg of 556 plants in the following numbers: 40 LINKAGE Round Yellow Wrinkled Yellow Round Green Wrinkled Green 315 loi 108 32 All but 27 of these plants set seed by selfmg and their constitutions proved to be as shown in Table 3 : TABLE 3 THE NINE GENETICAL CLASSES IN AN F2 SEGREGATING FOR TWO FACTORS (MENDEL) R—r Y—y RR Rr rr Total YY 38 60 28 126 1 2 1 Yy 65 138 68 271 2 4 2 yy 35 67 30 132 1 2 1 Totals 138 265 126 529 Thus the segregation ratio, as we may call it, is i : 2 : i for R-r in each of the Y-y classes and vice versa. The factors are recombined at random, so that the whole table represents the square of a i : 2 : i ratio. Applying this square rule, if for one factor there are two kinds of gametes, for two factors there must be four kinds, produced in equal numbers {RY, Ry, rY, and ry). This conclusion Mendel also verified by back-crosses. He even extended it by testing the indepen- dence of three factors, where of course eight kinds of gametes are produced in equal numbers. This principle of independent recom- bination is known as Mendel's second law. Linkage Since Mendel's work was rediscovered in 1900, the application of these rules has been studied in nearly all groups of sexually repro- ducing organisms. They have been universally verified with one modification, itself also universal : different factors are not always independent of one another in their segregation. In the F2 of a cross between two strains of Sweet Pea, Lathyrus 41 THE MENDELIAN METHOD odoratus, Batcson and Punnett in 1902 observed segregation for flower colour (P-p) and pollen shape (L-/). Each factor gave a 3 : i ratio, three purples to one red, three long-pollen to one round- pollen. But the combinations of these characters were not in the 9:3 : 3 : I ratio that Mendel had found. They were as follows: Purple Long Purple Round Red Long Red Round 1,528 106 117 381 Evidently the four classes of gamete were not being produced in equal numbers. The double dominant and double recessive classes, PL and pi, must have been about seven times as numerous as their alternatives Pi and pL. Later, other cases were found in which the two classes of gamete with one dominant and one recessive (such as Ab and aB) were in excess. The reason for this behaviour was made clear by Morgan and his colleagues in their experiments with Drosophila melanogaster in 1910 and later years. In this small fruit fly, which raises its enormous progenies in a ten-day cycle, they studied the inheritance of some hundreds of factors, to which they gave Johannsen's name o£ genes. These, they found, fell into four groups. A gene from one group would segregate independently of any gene from the other three. But two genes from the same group showed linked inheritance. Certain of the combinations of these genes were more frequent in the gametes than their alternatives. And these favoured combinations (whether dominant with dominant or dominant with recessive) turned out to be the ones which had been present together in the parents. For example, let us take the case of females produced by crossing the double dominant wild or standard type B B Vg Vg, with the double recessive h h vg vg, having a black body and vestigial wings. These females were back-crossed to hh vg vg males and their progeny were as follows : Bb Vg vg ^ ^ ^S ^S ^^ ^S ^S ^ ^ ^'S ^S 586 106 III 465 Thus the old or parental types of gamete are about five times as common as the new or recombinant types. To be precise, in this experiment, 17 •! per cent of gametes show recombination. When, however, the doubly heterozygous females came from 42 LINKAGE the cross of black {bh Vg Vg) by vestigial {B B vg vg), the progeny from back-crossing to the double recessive were as follows : Bh Vg vg b^ ^S ^S ^ ^ ^S ^S h b vg vg 338 1,552 1,315 294 The preponderant types of gametes are again the parental ones and now 17*9 per cent show recombination (Morgan 1914). Thus the percentage of recombination is a property of the two genes recombining; it is a property fixed within narrow limits for each pair of genes (such as b and vg), and differing between different pairs of genes. All percentages occur up to 50, this limit being indistinguishable from Mendel's case of independence, which appears when the genes are in different linkage groups. What then happens when three genes in the same linkage group are recombined in the same experiment? We can obviously work out the recombination percentage or value for each pair of them. Taking a third gene, that for purple eye, pr* in addition to b and vg, Bridges found that it showed 6 '4. per cent of recombination with b and lo- 8 per cent with vg, while b and vg showed 16-3 per cent in this experiment. Thus two of the values add up to the third, or to a little more. It seems as if the genes were arrayed in a line, thus : b pr vg ^ 6-4 >,< 10-8 16-3 Now with such a linear arrangement, a recombination between b and pr must also give a recombination between b and vg. Similarly one between pr and vg must give one between b and vg, always provided that the recombinations between b and pr and between pr and vg do not take place simultaneously. Such double recom- binations were in fact found in 0-46 per cent of gametes, so giving a deficit 0-92 per cent (each double cancelling two singles) in the observed recombination of b and vg as compared with the sum of the two smaller recombination values. * In designating the gene as pr, the allelomorph which gives the normal or wild-type red eye is assumed. It is this wild-type allelomorph which we should designate by Pr on the convention we have been adopting. In the Drosophila system {see Appendix 2) no special symbol is assigned to the wild-type allelomorph, the gene being designated by the variant allelomorph. 43 ABC ABC abC abC aBC aBC ABC ABc ABC aBC Fig. 8. — Types of haploid spore produced by meicsis in an F^ hybrid between two species of the green alga Chlamydomonas. Top: the two spore types of the parents have segregated, i.e. Large (A), Thick-walled (B) and Loose-chloroplast (C) from Small (a). Thin-walled (b) and Adhercnt-chloroplast (c). A'liddle Row: two of the results where separation is reductional at the first division for all the three genes and in consequence only two different types are formed. This event ensues whenever there is no chiasma between any of the genes and the centromeres of the chromo- somes that are carrying them. N.B. on the left, recombination is of C-c with A-a and D-b; on the right, o( A~a with B-b and C-c. Bottom Row: two of the results where separation is cquational at the first division for at least one of tlie three genes and in consequence four different types are formed. This event ensues when a chiasma has been formed between the gene in question and the centromere of its chromosome. N.B. in each case the four types include the parental combinations, but they can all be recombinants (with kind permission, after Mocwus, 1940, and unpublished). THE CHROMOSOME BASIS Now this proportion, 0*46 per cent, is instructive. If double recombinations occurred in the proportion expected with random- ness there would be (6-4 X 10 -8)/ 100 or 0*69 per cent. The fact that only 0-46 per cent are recovered {i.e. found in the progeny) shows that recombination between b and pr reduces the chance of, or, as Muller put it, interferes with, that between pr and vg. It is an easy step from this kind of experiment to the business of testing and arranging in a line all the genes of one group at intervals proportional to their recombination values. In this way we get a map on which distance between any two points stands for frequency of recombination between the two genes they represent (Fig. 9). From this map the linkage properties of all the mapped genes can be, and nowadays regularly are, predicted in combinations which have not been tried. The Chromosome Basis The system of map prediction is useful but it is not enough. We want to know why it works. It must have a material basis, and this becomes plain when we return to the cell. The chromosomes of the fruit fly fall into four pairs. One of these is a very small pair (Figs. 4 and 5), just as one of the linkage groups contains very few genes. Two of the other chromosomes are roughly equal and somewhat longer than the remaining one. Again just like the linkage maps. Both the chromosomes and the linkage maps are linear. Both the chromosomes and the genes are double in the zygote, single in the gamete. The chromosomes, or rather their constituent chromatids, cross-over and separate in germ cell formation just as the genes segregate and recombine. And the recombination limit of 50 per cent follows simply from the fact that two of the four chromatids cross over at each chiasma. All in all we have to take it that we are observing the same chromosomes in two different ways, under the microscope and in the breeding experiment, cytologically and genetically. In other organisms than Drosophila the comparison can be carried even further. In maize {Zea mays), for example, not only can each of the ten linkage groups be shown to correspond with one of the ten pairs of chromosomes ; these chromosomes can be seen to pair and 45 THE MENDELIAN METHOD to cross-over with the frequency required by the breeding data. And we shall fmd later that they can be seen to undergo other changes whose results can be recognized by breeding. We can now turn back and look at the facts of heredity from the chromosome point of view. Our starting point must be the parental chromosome which can be passed on unchanged to the offspring. Recombination between linked genes is the result of a change in their chromosome, the change always produced by crossing-over. If there is no crossing-over the different chromosomes are recom- bined as wholes and the individual chromosome passes on from generation to generation as a unit of heredity. All its genes will then go in one block and appear like one gene, a super-gene, having many different effects. Strange to say this failure of crossing-over actually happens in the male of Drosophila and other flies. At meiosis in the sperm mother cell, the chromosomes pair but no chiasmata are formed and no crossing-over takes place. The chromosomes passed from father to offspring pass as unbroken units. Thus a chromosome can remain unbroken for any number of generations. It is not, however, likely to do so since the chance of being passed to a female, where crossing- over will occur, is a half in each generation, for half the flies are females. Of all the chromosomes in flies only i in 1,024 will have been free from crossing-over for ten generations or more. To this rule there is one exception. One chromosome is passed down only from father to son. This is known as the Y chromosome and its smaller partner, with which it pairs at meiosis, is known as the X. In the female there are two X's and these correspond to the middle-sized linkage group (Fig. 9). The male, XY, is thus hetero- zygous for this pair of whole chromosomes, the female homozygous. The male at meiosis produces four sperm from each mother cell; two have an X and two a Y. At the same time the female produces eggs all with one X. Thus the cross between male and female is a back-cross for the X-Y pair of chromosomes or, if you like, the X-Y super-gene, and half the offspring are of each sex. Further, we must notice, the daughter receives an X from each parent, the son an X from his mother and a Y from his father. It would be easy on this evidence to say that the difference between X and Y determines sex. It would 46 THE CHROMOSOME BASIS M P Fig. 9. — Maps showing the linear distribution of 13 genes and the centromere (cent) in the X-chromo- some of Drosophila melanogaster. C is the map ob- tained by arranging the genes according to the frequencies of crossing-over (as judged by genetic recombination) between them. M is the correspon- ding map where the genes are spaced according to the proportionate rates of occurrence of lethal mutations between them. P is the cytological map based on the positions of the genes in the polytene chromosomes of the salivary glands, and S that based on the positions of the genes in the mitotic chromosomes of the nerve ganglia cells. Only 7 of the genes have been localized on the mitotic map. The large block of heterochromatin near the centromere is shown black. It is hardly detectable in the genetic map, and is much smaller in the poly- tene than in the mitotic chromosomes. cent 47 THE MENDELIAN METHOD be more accurate, however, to say that it determines whether a fertihzed egg with a normal outfit of the other chromosomes, the autosomes, shall develop into male or female. How the autosomes affect the issue we shall see in a later chapter. Sex-Linkage Corresponding with this cytologically observed situation is the genetically observed mechanism of sex-linked inlieritance. When a wild type, red-eyed, female fly is crossed with a white-eyed male, Fig. 10. — Reciprocal crosses showing the transmission and expression of a gene pair in the X chromosome, having no allelomorph in the Y and with dominance, e.g. white-eye in Drosophila or haemophilia in man. In the black-yellow difference of the cat, the heterozygote is distinguishable as the tortoiseshell female. The solid circle below the X denotes the domuiant allelomorph, the empty circle the recessive. The thick outer circle denotes the dominant phenotype. Broken lines show the transmission of the Y. all the Fi are red : white is recessive. In the Fg (from crossing brothers and sisters in the F^) we get three red flies to every white. But all the white-eyed flies are males: the gene is sex-linked. When the cross is made the other way round, white female being crossed with red male, the sex linkage makes itself apparent in the Fj. As before, the females are red but the males are now white. In the F2 the males as before are half red and half white, but now so are the females too. What does this mean? The females in the F^ are the same no matter which way the cross is made. The females must therefore inherit the gene equally from both parents. The males in Fg are all 48 SEX-LINKAGE the same although their fathers are of the two kinds. They obtain the gene only from their mothers. Thus we see that the gene follows the path of the X chromosome. At the same time we see that the Y has no effect. Where the X chromosome carries the red-producing form or allelomorph of the gene, the male fly is red-eyed; where the X carries the white allelomorph the male fly is white-eyed. Figure lo makes this clear. In mammals, like flies, the male is the heterozygous sex, but in birds it is the female. Sex-linkage is of peculiar and practical interest in both. The gold-silver difference used in sexing crossed chicks depends on a gene in the X chromosome, and so shows criss-cross inlieritance like the red- white eye in the fly. Haemophilia and colour- blindness depend on recessive genes in the X of man and hence appear less often in the XX female. Their inheritance follows the same rules as that of white-eye in flies and their frequencies in the population can be deduced from these rules. Thus Pickford found that populations with 7 • 8 per cent of colour-blind men have 0 • 65 per cent of affected women. The tortoiseshell cat is heterozygous for the X-linked gene, one allelomorph of which gives black, the other yellow, when homozygous. In mammals a normal male cannot be heterozygous for an X-linked gene and tortoiseshell males occur only as rare and sterile abnormalities. In Drosophila the red-white eye difference showed that for the purpose of this sex-linked experiment the Y chromosome might as well be empty. No gene in the Y appeared to correspond with this gene, for no gene interfered with its action. This is a general property of the Y chromosome for, with the sole exception of the gene for "bobbed," the Y simply does not correspond. Nor does it show recognizable differences of its own. That is to say, different males do not carry Y chromosomes determining differences comparable with that between red and white eyes. What differences they can carry we shall see later. The fly is typical of a great many insects in the emptiness of the Y chromosome. In the vertebrates, however, the Y chromo- some shows more correspondence with the X. The corresponding genes are of two kinds. Some of them do not cross-over from one chromosome to the other. For example the Y chromosome in poultry seems to carry the normal allelomorph of the barring gene Elements of Genetics AQ D THE MENDELIAN METHOD in the X, but the two chromosomes never exchange allelomorphs in heterozygous females. The barring gene can be used, therefore, to disthiguish between the X and the Y, and so makes possible the "autosexing" breeds as shown in Fig. ii. O Cf Fig. II. — The gene for barred feathers (open circle) in poultry expresses itself only partially in females; so suggesting that the Y chromosome carries its non-barred allelomorph (filled circle). Where the basic pigmentation of the birds is brown, rather than black, this ctiect of the Y chromosome is clearly detectable by deeper colouration in newly hatched chicks. The combination of the gene for brown pigmentation and that for barring thus gives a true breeding strain of birds in which the sexes are distuiguishablc at hatching by the deeper intensity of down pigmentation, especially round the head, in females than in males. Such a breed is termed auto- scxine. Sometimes, however, as in certain fishes such as Lehistes and in man, the X and the Y chromosomes not only carry the same genes, but also exchange them by crossing-over. These genes, therefore, no longer show complete sex-linkage. Their association with the sex chromosomes is recognizable by the partial, rather than the com- plete, restriction of a particular allelomorph to a particular sex in SEX-LINKAGE any family. Instead of all the Y-linked genes of the father passing to the son, a proportion pass to his daughters. The proportion depends on the crossing-over distance between the gene in question and the segment with complete sex linkage (Fig. 12). 64,3 35.6 32.5 67.7 Fig. 12. — Segregation in a cross in the fish Xtphophorus helleri. Taken over the whole progeny the gene determining spotted v. spotless gives a good i : i ratio; but the two types are unequally distributed between the sexes. This is the type of segregation characteristic of partial sex-li:ikage, though the validity of this inter- pretation has not been fully established in the present case (from Kosswig, 1939). Finally, in Drosophila and in man, there are completely Y-linked genes without any allelomorph in the X. In man such genes have been recognized by their transmission only from father to son ; in Drosophila by the effects of absence of the whole or part of the Y chromosome. Flies are sometimes hatched without any Y. They are perfect-looking males but their sperm cannot swim, so that they are sterile. The Y chromosome is therefore doing something, although what it does cannot be shown by its relationship with mendelian differences in the X. There are thus three kinds of parts in the sex chromosomes: 51 THE MENDELIAN METHOD X-liiikcd, Y-linkcd and interchangeable. When we examine the sex chromosomes wc see that they have similar parts which pair and cross-over; these are the pairing segments. They also have parts w^hich are dissimilar and which do not pair; these are the differential segments belonging, one to X, and the other to Y, The differential segment of the X is usually larger than that of tlic Y. It usually, as in man, lies at one end, the pairing segment at the other. The distribution of the chiasmata in the pairing segment relative to the centromere and the differential segments then decides whether the first division is reductional for these segments or equational. In the male Drosophila, where crossing-over occurs in the sex chromosomes, although nowhere else, the pairing segment is in the middle of the Y chromosome (separating two differential segments) and at the end of the X. Crossing-over occurs doubly and reciprocally, the second exchange cancelling the first. It therefore keeps the two differential segments of the Y together and does not give any result detectable in ordinary breeding experiments (Fig. 13). The same principles of sex determination apply generally not only to animals but also to plants with separate sexes. In the dioecious Campions [Melandrinm dioica) as in Drosophila there are distinct sex chromosomes, the male having XY, and sex-linked genes are found in both X and Y. In different animals there are, however, many differences in detail. In grasshoppers (Orthoptera) and aphids (Hemiptera) there is no Y chromosome. Females have XX, males have one X only. The whole of the X is therefore differential. In some Hemiptera, and in spiders and Ostracoda the X is represented by several chromosomes which move and segregate as a unit in meiosis, the females having twice as many as the males. In Lepi- doptera, birds, and some fishes the female is the heterogametic or XY sex, the male having XX. With these modifications in its chromosome basis, sex-linked inlieritance is correspondingly modified. Altogether these observations on mendelian breeding and chromo- some behaviour show us the mechanism of heredity. They show us its common and universal principles. They also tell us something of the material structure and organization of heredity. They show it to be particulate and they show the units, or particles, or genes, into which it is separable to be smaller than the chromosomes. 52 SEX-LINKAGE IPROSOPHILA xx~9 Y ^^Sr^:^'^'''''' ^ /Reciprocal C/iiasma/a. CO. per ccnfT— O 5 9 14 28 55 Re5ucfionaI Se[)arof1on:^ EqualTonol X Y Tfc=^ ^ yI^ Fig. 13. — Crossing-over between the pairing segments of X and Y in heterogametic (male) sex oi Drosophila and man showing the structure of the bivalent at pachytene and first metaphase of meiosis. The pachytene pairing gives a metaphase orien- tation and consequent separation at the first anaphase which depends on the position of crossing-over and chiasma formation. In Drosophila the genes bb, bobbed, and B, Bar, etc., and in man the genes c.b., colour blindness, ha, haemophilia, iV, ichthyosis, etc., are marked. The crossing-over distances in man are from Haldane. Black, pairing segment with partial sex linkage through crossing-over between X and Y (as well as between X's in females). Hatched, differential segment with complete sex linkage in X and with crossmg-over between X's in females. White, differential segment with complete sex linkage in Y and no crossing-over whatever. 53 THE MENDELIAN METHOD Evidently the chromosomes which we see as materials are composed of units which we can recognize by their differences. We shall carry our analysis further and consider the properties of these units. But before we do so we must find out how far, and above all how rigorously, the whole of hereditary differences can be expressed in terms of such units. REFERENCES BATESON, w. 1909. Mendel's Principles of Heredity. Cambridge. DARLINGTON, c. D. 1934. Anomalous chromosome pairing in the male Drosophila melanogaster . Genetics, 19: 95-118. HALDANE, J. B. s. 1936. A scarch for incomplete sex-linkage in man. Ann. Engen., 7: 28-57. HEiTZ, E. 1935. Chromosomenstruktur und Gene. Z.I.A.V., 70: 402-447. ROLLER, p. c. 1937. The genetical and mechanical properties of sex chromosomes. Ill, Man. Proc. Roy. Soc. Edin. B., 57: 194-214. KOSSWiG, c. 1939. Die Geschlechtsbestimmung bei Zahnkarpfcn. Rev. Fac. Sci. Univ. Istambul, 4: 1-32. MATHER, K. 1938. Crossing-over. Biol. Revs., 13: 252-292. MENDEL, G. 1 865. Experiments in plant hybridisation. (Translation in Bateson, 1909.) MOEWUS, F. 1940. Die Analyse von 42 erblichen Eigenschaften des Chlamydomonas eugametos-Gruppe. Z.I.A.V., 78: 418-522. MORGAN, T. H. 1913. Heredity and Sex. New York. MORGAN, T. H. 1914. No crossing-over in the male o( Drosophila of genes in the second and third pairs of chromosomes. Biol. Bull., 26: 195-204. MULLER, H. J. 1916. The mechanism of crossing over. Amer. Nat. 50: 193 etsqq. PiCKFORD, R. w. 1947. Frequencies of sex-linked red-green colour vision defects. Nature, 160: 335. WINCE, 0., and ditlevsen, e. 1947. Colour inheritance and sex determination in Lehistes. Heredity, i: 65-83. 54 CHAPTER 3 CONTINUOUS VARIATION The Limitations oj Mendelism The Specification oj Continuous Variation Heritable and Non-heritable Diferences Cumulative Effects of Genes Polygenic Systems Linkage oj Polygenes Mendel's bequest to genetics was twofold. He gave us his principles of inheritance, and at the same time he gave us the experimental method by which he had been able to establish those principles and by which they could be tested and extended. This method, as we saw in the last chapter, has enabled us to show that Mendel's principles reflect the properties of inheritance through the nucleus. Wherever the hereditary determiners of variation are carried by the chromosomes we must therefore expect these principles to apply. We cannot, however, expect the experimental method to be equally widely applicable. This depends for its success, as Mendel himself clearly realized, on the differences under investigation (tall V. short, round v. wrinkled, etc.) being so large compared with any residual or extraneous variation in the character, that individuals could be assigned unambiguously to one or other of the alternative classes. Thus Mendel's short peas ranged in height from 9 inches to 18 inches, and his tall parents from 6 feet to 7 feet. The F^'s of tall and short even exceeded the tall parent with heights up to j\ feet. But in spite of this variation in both tails and shorts, there was never any doubt that a plant, whether in the parental, F^,' Fg, or any other generation, was either the one or the other. The variation was in fact what came to be called discontinuous: all below the discontinuity in the distribution of heights were shorts and all above it, tails. The residual variation, within the tails themselves and equally of course within the shorts, was evidently not of this kind. There was presumably a smooth, or at least an apparently smooth, gradation in height between the shortest and tallest tails and between the shortest and tallest shorts. The variation within these classes was 55 CONTINUOUS VARIATION contimtoHs. In any family where the gene of major effect was not segregating, the variation must have been wholly of this continuous kind. The variation that we normally see in stature in man, or indeed of size in any organism, is continuous in this way. Stature may vary between wide extremes, and every height between those extremes is represented. There is no discontinuity in the range of statures: the gradations are imperceptible. As a consequence we cannot classify people into tails and shorts in the way that Mendel was able to do with his peas. It is therefore impossible either to specify a family or population in respect of stature by a mendelian ratio of tall to short, or to investigate the inheritance of stature in man by the mendelian method. What method can we use to take its place ? The Specification of Continuous Variation Where variation in a character is continuous, the number of . classes into which individuals can be divided according to the mani- | festation of the character is limited only by the fmeness of the means we possess for measuring it. Each observation is unique or poten- tially so. We may, however, defme classes with arbitrarily chosen limits and describe any group of individuals by recording the num- bers which fall into each of these classes. Thus we might describe a human population by recording the numbers o£ people with heights between 4 feet 6 inches and 4 feet 7 inches, 4 feet 7 inches and 4 feet 8 inches, 4 feet 8 inches and 4 feet 9 inches, and so on. The members of each class will not, of course, all have exactly the same j height. The representation is therefore only approximate, and a .' closer approximation would be obtained by classifying into ranges of 1^ inch instead of i inch; for the frequency distribution of stature, as it is called, would then approach more closely to the true state . of continuity. The fmer the gradations we use, however, the fewer f individuals will fall into each class, and the more erratic will the distribution appear when limited numbers of individuals are avail- able for measurement. To overcome this difficulty we need a means of specifying the frequency distribution as a whole. We need a means of representing it by a few constants (known as parameters), know- ledge of whose values would enable us to determine the proportion 56 THE SPECIFICATION OF CONTINUOUS VARIATION of a large population which would fall within any specific range of heights; or to put it another way, which would enable us to calculate the chance of any individual taken at random falling within any specific range of heights. In Fig. 14 is shown the frequency distribution of height in 1,164 men, the grouping being into classes covering i-inch ranges. The 60 55 70 STATURE IN INCHES 75 Fig. 14. — The cross-hatched histogram shows the frequency distribution of stature as observed in 1,164 men. The data are grouped into classes of one inch, centred on the half-inches. The curve is the normal distribution fitted to these data, and from which they are assumed to depart only by sampling error. A normal distribution is specified by two parameters, jx which fixes the position of the centre point of the curve, and a which measures its spread by the distance from the centre to the points of maximum slope. These are estimated as the mean, .y , and the standard deviation, s, respectively. distribution is characteristic of continuous variation in natural popu- lations in that the middle heights are the most common and the frequencies fall off towards each extreme. In this particular case the distribution is also symmetrical; but symmetry depends on, among other things, the scale used in making the measurements, and adjust- ment of the scale may be necessary before a symmetrical distribution is obtained. This question of choice of scale is not encountered in mendelian genetics, for we do not need to specify how tall a tall pea 57 CONTINUOUS VARIATION is, or how short a short one is, provided we can classify into the tall and short categories. Scaling is, however, of obvious importance in the analysis of continuous variation, since the scale will, in part, determine the shape and properties of the distribution which are all we have to base the genetical conclusions upon. We shall return to it later. As we have seen, the type of grouped frequency distribution of Fig. 14 is to be regarded as an approximation to the underlying continuous distribution which we may seek to specify algebraically in terms of a few parameters. Where the distribution is symmetrical and the frequencies fall off on each side of the centre value, as in the figure, this ideal curve can be, and indeed usually is, taken as an example of the Gaussian or Normal Curve, whose general formula is I (x-M.)2 df-:^^e 2a^ dx aV2TT where df is the frequency of individuals falling within the infini- tesimal range, dx, of heights. Apart from f, the frequency, and x the heights, four quantities appear in this formula 77, e, a and yu,. The first two of these, 77 and e, are familiar mathematical constants, the ratio of the circumference of a circle to its diameter and the natural base of logarithms respectively. These will be the same for all normal curves. The other two, a and ju,, are the parameters which specify the particular normal curve in question, and they must be found for each distribution individually. They are the constants by means of which we can specify the variation of the character in which we are interested. The way in which ju, and a specify the curve can be seen from Fig. 14. /M is the value of the character at the centre of the dis- tribution and is estimated as the mean, or average, of the values of the character in all the individuals concerned. Thus where there are n individuals whose height has been measured, ^i is estimated S(x) by X = ' S standing for summation of the values of x from all individuals. yL, or its estimate x, fixes the position of the curve of distribution. The second parameter, a, is the distance of the point of maximum slope of the curve from the mean, /u,. There are, of course, two 58 THE SPECIFICATION OF CONTINUOUS VARIATION such points, one on each side, but as the curve is symmetrical they must lie at equal distances from /x. The estimate, s, of o- obtained from any set of data is called the standard deviation and is found as S(x-i)2 — I ^ ' the notation being as before. It is, however, more convenient for purposes o£ calculation to use the algebraically identical formula n — I where S^{x) stand for the square of the sum of all the x's. It will be observed that s is found as the square root of s^, which is generally called the variance, and is denoted by V. The variance is in fact of more use than the standard deviation in genetical work, because when variation is measured by the variance, the con- tributions made to it by independent causes of variation are additive. This obviously caimot be equally true when the variation is measured by the standard deviation. We shall, therefore, regard the normal curve as most conveniently specified by fx and a"; or rather by the estimates of these quantities, x and V, as obtained from a set of actual data. Not all frequency distributions of this general shape, with the central values the most common, are normal curves. Some are asymmetrical. And even some symmetrical curves do not conform to the normal type. Such curves require further parameters, esti- mated from the values of S (x— x)^ and S (x— x)*,for their full speci- fication; but even so, the mean and variance still retain their crucial importance for our understanding of these distributions. As we shall see, much can be learned of the genetical situation underlying con- tinuous variation by the analysis of means and variances, and this still remains true even where we know the frequency distribution to depart from strict normality. There is one further biometrical quantity which is of importance for the genetical study of continuous variation, since it helps to tell us directly the extent to which continuous differences are hereditary. Where we have simultaneous observations on pairs of relatives, say 59 CONTINUOUS VARIATION the heights of father and daughter, we can find the variance of fathers' heights and the variance of daughters' heights. Let us denote fathers' measurements by the suffix f and daughters' by the suffix d. I _, -V, ,„ I Then V, = -— ^S (x,- Xf)2 and V, - ^^— ^ S(xj - x,)2. n wiU be the s^me in both cases since for every father measured, a daughter was also measured. It is therefore clear that we can find S [ (x^— x^) (x^— x^)] in a way parallel to that used for S{x^—x^y- and S(xj — xj^, but multiplying the deviation of each father's height from its mean, (Xf— Xf), by the corresponding deviation of his daughter (x^— x^), instead of squaring the father's or the daughter's deviation, before summation. We can then calculate the covariance, W^^, of fathers' and daughters' height, as W,, = ^^S[(x,_x,)(x,-x,)] One important point should be noted about these covariances. Variances must always be positive since they are derived from sums of squares. Covariances, on the other hand, are derived from sums of cross-products of deviations and so may be either positive or negative. A positive value indicates that the deviations from the mean in one distribution, say fathers' heights, are preponderantly accompanied by deviations in the other, say daughters' heights, in the same direction, positive or negative. A negative covariance on the other hand indicates that deviations in the two distributions are preponderantly in opposite directions. Where a deviation in the one distribution is equally likely to be accompanied by deviation of like or opposite sign in the other, the covariance, apart from errors of random sampling, will be zero. The importance of the covariance in genetics will now be obvious. If variation in height is under genetical control we should expect tall fathers generally to have tall daughters and short fathers generally to have short daughters. In other words we should expect them to have a positive covariance. Lack of genetic control would produce^ a covariance of zero. It was by this means that Galton first showed' stature in man to be under genetic control. He found that the covariance of parent and offspring, and also that of pairs of siblings, was positive. 60 HERITABLE AND NON-HERITABLE DIFFERENCES The size of the covariance relative to some standard gives a measure of the strength of the association between the relatives. The standard taken is that afforded by the variances of the two separate distributions, in our case of fathers' and daughters' heights. We may compare the covariance with either of these variances separately, and we do this by calculating regression coefficients which have the W W form, — - (regression of daughters on fathers) or, less usefully, ~~ (regression of fathers on daughters). We may also compare the covariance with the two variances at once in a correlation coefficient W^ found as — =^=. Were the correlation coefficient to have its maximum value of i, it would signify a complete association, a full determination of a daughter's height by her father. Independence of father's and daughter's height would be shown by a coefficient of o, because, of course, W^y would itself then be o. Values between o and I show partial determination of a daughter's height by her father. The coefficient actually observed in man by Galton was just under 0-5, for reasons which we shall see later. Heritable and non-Heritable Differences In 1900, when Mendel's results first received wide 'attention, Galton and his successor Pearson had already established by means of correlation studies the heritable nature of continuous variation in a variety of characters in man, in dogs and even in sweet peas. The results were used as the foundation of the so-called Law of Ancestral Heredity. Their approach suffered, however, from one weakness which we can now see to have been fatal to any theory of hereditary transmission. While Galton had an idea that the hereditary materials might be particulate, neither he nor his fol- lowers ever grasped the distinction between the determinant and the effect, the genotype and the phenotype. Mendel supphed the missing principle of the determinant and demonstrated its vahdity in his peas. He was, however, dealing with discontinuous variation and it was held by the biometrical school that his principles would not apply to continuous variation. Indeed it was considered that the segregation of mendelian differences could 61 CONTINUOUS VARIATION not account for correlations between parent and offspring so high in value as those observed. It was also considered, in spite of Galton's earlier leanings towards a particulate theory, that discontinuity in segregation of the kind which the niendelian theory demanded was incompatible with continuous variation in the phenotype. This notion seems to have been shared by the early mendclian geneticists, for, just as Pearson and his school took the view that mendelian theory was limited in its application to such heritable variation as was not continuous, De Vries argued that the mendelian principles were of universal applicability because no continuous variation was ever heritable. This error was the only noticeable point of agreement between the two schools of thought. The basis was laid for the interpretation of continuous variation on mendelian principles by Johannsen in Denmark and Nilsson-Ehle in Sweden. Johannsen took as his experimental material the dwarf bean (Phaseolus vulgaris) which shares with peas the property of regular natural self-pollination. Thus any bean which is not descended from a deliberate cross-pollination will be expected, on the basis of simple mendelism, to be homozygous for all or nearly all its genes. Its progeny, in their turn, will be to the same extent like their parent and like one another. They, with their descendants, will constitute what Johannsen called a pure line. Johannsen started in 1900 with 19 beans, from which he derived 19 pure lines. In 1901 he had 524 beans whose individual weights he recorded in centigrams. These in turn yielded 5,494 beans in 1902, and again he determined their weights. The essential results are shown in Table 4. If we compare the weights of the 5,494 beans with those of their 524 mothers we obtain the upper of the two tables. It is clear that the average weight of daughter beans is related to that of the mother, though the differences amongst the daughters' averages are smaller than those amongst the mothers. Thus the mothers range from the 20-cg. to the 70-cg. class, but the daughters' averages range only from 43 '8 cgs. to 56-0 cgs. The mothers can be subdivided further according to the pure lines to which they belong. The lower of the two tables shows the average weights of the daughters from the mothers in the various weight classes of 9 out of the 19 lines. These 9 were the most informative, but the remaining 10 lines tell the same story so far as they go. 62 HERITABLE AND NON-HERITABLE DIFFERENCES No matter what the weights of the mother beans from any one Hne were, the averages of their daughters are the same within the Hmits of sampHng error. Even where the mother beans differed by as much TABLE 4 FREQUENCIES OF BEANS (PHASEOLUS VULGARIS) OF VARIOUS WEIGHTS, GROUPED INTO CLASSES CENTRED ON THE WEIGHTS SHOWN (JOHANNSEN 1909) Weight of Weight of daught er beans Average mother beans 10 20 30 40 50 60 70 80 90 20 1 15 90 63 11 43-8 30 — 15 95 322 310 91 2 — — 44-5 40 5 17 175 776 956 282 24 3 — 46-2 50 — 4 57 305 521 196 51 4 — 48-9 60 — 1 23 130 230 168 46 11 — 51-2 70 — — 5 53 175 180 64 15 2 560 Total . . 5 38 370 1,676 2,255 928 187 33 2 47-92 19 lines, 524 mother beans, 5,494 daughter beans. B. AVERAGE WEIGHTS OF DAUGHTER BEANS FOR THE VARIOUS CLASSES OF MOTHER BEAN IN NINE OF THE NINETEEN PURE LINES (JOHANNSEN 1909) Line . . I II V VII XII XIII XV XVIII XIX 1 20 _ _ _ 45-9 49-6 _ 46-9 41-0 _ S 30 — — — — — 47-5 — 40-7 35-8 "g 40 — 57-2 52-8 49-5 — 450 — 40-8 34-8 °^ 50 — 54-9 49-2 — 45-1 45-1 44-6 — — . •§) 60 63-1 56-5 — 48-2 44-0 45-8 450 — — i 70 64-9 55-5 50-2 — — — — — — Average of line 64-2 55-8 51-2 49-2 45-5 45-4 45-0 40-8 351 as 40 cgs. this is still true. The lines differ, however, from one to another, the heaviest averaging 64-2 cgs. over all daughter beans and the lightest only 35-1 cgs. Thus the variation in bean weight within a line was, as expected, non-heritable; the variation between lines being at least in part heritable. 63 CONTINUOUS VARIATION Four important conclusions follow from this experiment: In the first place a character can (and in fact nearly always does) show heritable and non-heritable variation simultaneously. Secondly, the two kinds of variation are indistinguishable by anything but a breeding test. Only experiment can tell us whether the bean is large because of its genotype or in spite of its genotype. Thirdly, the effects of the environment can either reinforce or counter-balance genetical differences. Thus 70-cg. mother beans from line II are larger than 20-cg. mother beans from line XVIII, partly because of the genetical difference, which their offspring reveal as averaging 15-cg., and partly because of non-heritable or external effects. At the same time 6 of the 9 lines of Table 4 B included mother beans in the 60-cg. class, even though the genetical values of these beans must have differed by nearly 20 cgs. in the extreme case. These genetical differences were counter-balanced by non-heritable effects. Finally, the genetical values of the lines show a graded series from 35 -I to 64*2 cgs., with no single great discontinuity. Indeed, the genetical differences that are here proved to exist between these lines are much smaller than the effects that environmental agencies are producing within each line. The genes that are acting must be genes of relatively slight effect. Cumulative Effects of Genes Johannsen's experiments with true-breeding lines enabled him to demonstrate all these effects and properties of non-heritable variability. They also, however, tell us that there must be many genotypes having different effects on the character; for, even when separated from the non-heritable, the genetical differences are not simply due to a single gene. Nilsson-Ehle showed by hybridization experiments how this could come about. He crossed varieties of wheat and of oats differing in various characters, which segregated clearly in Fg and later generations; but not always into the familiar mendelian ratios. For example, in wheat, an F2 from crossing red- and white-grained varieties might segregate in a ratio of 63 plants with red grain to i 64 CUMULATIVE EFFECTS OF GENES with white grain. The white-grained plants bred true. So did some of the red-grained ones — 37 out of the 63 of them, to be precise. But of the remaining red-grained plants, some gave 3 : i ratios in F3, some 15:1 ratios and some repeated the 63 : i ratio. Nilsson- Ehle reahzed that the parents must have differed by 3 genes (which we can now relate to their having six sets of chromosomes instead of the normal two), the red-producing allelomorph being dominant in each of them, and a single red-producing allelomorph being in itself sufficient, no matter to which gene it belonged, to give a red- grained plant. The genes were all like one another in their effects. This similarity went even deeper. The reds, though all having red grains, did not all display quite the same degree of redness. The palest reds generally gave 3 : i segregations in the next generation. They had, so to speak, only one dose of red. The next palest might breed true, but give 3 : i segregations in Fg after crossing with white ; or they might give a 1 5 : i segregation immediately in the next generation. They had two doses of red, either by being homozygous for one of the genes or by being heterozygous for two of them. It was dosage of red-producing allelomorphs that mattered, not the particular genes involved. Thus genes with effects large enough for them to be followed individually by the mendelian method, could be shown to have similar effects, and effects which were cumulative, that is to say supplementing one another, on the phenotype. It was realized by Nilsson-Ehle and independently by East that similar and supplementary action provided the basis for the graded genetical levels demanded by continuous variation. And, if the effects of the single gene differences were also small compared with the non-heritable variation, as Johannsen's experiments had show^n to be possible, no mendelian ratios would ever be obtained even though the genes segregated in the mendelian fashion. Mendelian inheritance was not limited to cases where it could be detected by the mendelian method. It might indeed be regarded as covering the whole of heritable variation. We can thus picture a spectrum of variation. At the one extreme are gene differences of so large an expression in the phenotype that they stand out both from their fellows and from the non-heritable agencies which also cause variation in the character. These genes will, I InihiilsofGcueiics 65 E CONTINUOUS VARIATION by their dirtcrences, cause discontinuities in the variation ot the phcnotype and they will therefore be traceable by the mendelian method. Included in this group are such genes as have a lethal eftect when homozygous. At the other extreme are genes whose differences cause effects so slight that they are obscured by the remaining variation, both heritable and non-heritable. By itself, the etfect of such a single gene might entirely escape detection, but a number rein- forcing one another in action might produce quite a large difference between the extreme genotypes. This difference will not, however, create a discontinuity in variation, for two reasons. Between the two extremes there will lie a graded series of genotypes; and the environment will smooth out the small differences between the members of this series. These genes cannot, therefore, be followed individually in mendelian experiment; they must be handled as groups by the biometrical method. Each character of the phenotype may show the effects ot gene differences covering the whole range of the spectrum. Any example of variation is likely to involve several, if not many, of the gene differences of small effect, even where the major part of the variation is due to one or more genes of large effect. Non-heritable variation is also ubiquitous in its occurrence. It may indeed be stated as a general rule that aU characters can and do show variation due to genes of small effect, variation due to genes of large effect, and variation due to non-heritable agencies. The susceptibility of a gene to detection by the mendelian method is thus dependent, not on its mode of inheritance, but on the size of its effect in relation to its fellows. We can in fact account for continuous variation on the basis of the simultaneous operation of a number of genes. These genes are inherited in the mendehan way, but their differences have effects which are small in relation to those of non-heritable agencies (or at least in relation to the total variation), similar to one another and supplementary to one another (Fig. 15). Such a set of genes constitutes a polygenic system, and its individual members may be conveniently termed polygenes. Genes such as those with which Nilsson-Ehle was concerned have two of the properties of polygenes. They are of similar and sup- plementary' action. But they cannot be described as polygenes because their effects are so large as to cause a sharp discontinuity in the 66 I CUMULATIVE EFFECTS OF GENES GENOTYPES DISTRIBUTION OF PHENOTYPES Fig. 15. — Diagram showing the relations between the genotypes and the frequency distribution of phenotypes where two genes of equal and additive effect are varying independently of one another and two allelomorphs of each gene are equally common. Dominance is assmned to be absent, so that the phenotypic expression is proportional to the number of capital letters in the genotype. Five phenotypic classes will be produced with two genes of equal effect, seven phenotypic classes with three genes, nine with four, and so on. As the number of classes increases, and as non-heritable influences exert their effect, the distribution of phenotypes approximates more closely to a continuous curve. 67 CONTINUOUS VARIATION variation, a discontinuity which permits the analysis of the system by the nicncichan method. Genes hke these are often termed poly- meric genes. All polygenes are therefore polymeric, but not all polymeric genes are polygenes. Polygenic Systems Two questions immediately arise about polygenic systems. First we see that such systems can explain continuous variation; but is continuous variation in fact to be ascribed solely to their operation ? Or to put it another way, we have been led to postulate systems of genes which cannot be analysed by the mendelian method. How then can we be sure that these genes are inherited in accordance with mendelian principles i Secondly, since we cannot follow the genes individually, we cannot describe the properties of these systems in terms of individual segregation ratios and linkage values. How then are we to understand and express and predict the behaviour of the systems ? To the first of these questions we must now turn. The second will be discussed in the next chapter. We have seen in the previous chapter that mendelian inheritance reflects the fact that the genes concerned are carried by the chromo- somes. The cytological study of meiosis shows that two properties must mark this type of transmission. All the genes must show segregation, and those which are borne in the same chromosome must show linkage with one another. If, therefore, we can establish that the determinants of heritable continuous variation show both segregation and linkage, we caimot avoid the conclusion that they are nuclear genes and that as such they will conform to mendelian principles. Where a stock or line of a species has long been inbred we must expect that its members will be homozygous for all or nearly all their genes and that differences between them will be non-heritable. Johannsen's experience confirmed this with beans. If, now, we cross two such lines with one another, the F^ will be genetically as uniform as its parents. Though it will be heterozygous for any genes in which the parental lines differed, all the members of this generation will be alike in their heterozygosity. Thus variation in the Fi will also be non-heritable. But in the Fg the individuals will not all be alike 68 POLYGENIC SYSTEMS genetically, for segregation will have occurred during the formation of gametes from the F^ zygotes. Heritable variation will have been added to the non-heritable, and we must expect therefore that the CORFU EGYPT Fig. i6. — Parents and Fj progeny of a cross between two races of the ladybird Epilachna chrysomclina showing polygenic segregation in respect of the black flecks and the dark colouring between them (after Bauer and Tiniofeeff-Ressovsky, 1943). frequency distribution of the Fg will cover a wider range, have a larger spread, than those of the individual parent lines and F^. In biometrical terms the F2 should have a larger variance than parents or Fj, because this variance will have a new, heritable component 69 CONTINUOUS VARIATION added to the non-heritable component which it shares with its predecessors. Now any gene segregating in Fg will, by Mendel's principles, be lioniozygoiis in half the F^ individuals and heterozygous in the other half. If, therefore, we raise Fg's by inbreeding the F., individuals, half will show segregation for each gene and half will not. Taking all genes into account we shall expect that the average variance of all the Fg's will lie between the variance of Fg on tlie one hand, and the variances of parents and F^ on the other. For the average heritable component of the F3 variance will be smaller than that of Fo, but not, of course, so small as those of parents and F^ where variation was solely, or nearly solely, non-heritable. But not all the Fg's will be alike in cither variances or means. The variances will reflect the different numbers of genes heterozygous in the different Fg mothers. Furthermore, for all genes in wliich the mother was homozygous, each F3 individual must carry the same allelomorphs. The mean expression of the character in each F3 will therefore be correlated with the expression in its Fg mother, just as Galton found the heights of fathers and daughters to be correlated in man. So, even though we cannot follow segregation by ratios of types in the mendelian fashion, we can still hope to detect its occurrence by the biometrical properties of frequency distributions. And we can make so many predictions about these properties, that if all of them are borne out in experiment we can entertain no doubt that mendelian segregation is occurring. A characteristic experiment of this kind is illustrated in Fig. 17. It shows the inheritance of corolla length in a cross between two lines o( Nicotiana longijiora as recorded by East (1915). The corolla lengths are expressed in millimetres and the data are grouped for convenience of presentation into classes each covering a range of 3 mm. and centred on 34, 37, 40, etc., mm. The frequencies arc shown as percentages of the individuals of the various families which fell into the different classes. The variances, measuring the spreads of the distributions, are much the same for the two parents and the Fj, whose mean is just about midway between those of the parents. The F2 has a much larger variance, while those of the four Fa's shown differ amongst themselves and lie between the values for Fg on tlie one hand and parents and F, on the other. The arrows 70 PERCENTAGE OF PLANTS -50 -25 -M) 1 40 55 70 85 100 COROLLA LENGTH IN MMS. Fig. 17. — Frequency distribution of corolla length in families of Nicotiaim longijlora, showing segregation for the polygenes controlling the differences between the twu parent strains (P) in this character. Variation in P and F^ is wholly non-heritable. To this is added in F, the heritable component depending on segregation. The spread of the frequency distribution is thus higher in Fo than in F^ and P. In F3 the average heritable component of variation is half that in Fj, but it varies from family to family according to the number of genes for which the Fg parent was heterozygous. The spread of the frequency distribution is thus variable in F^ though always lying between that of F2 on the one hand, and of F^ and P on the other. The mean corolla length of each F3 is related to the corolla length of its F2 parent, indicated by the source of the relevant arrow (based on East, 191 5). 71 CONTINUOUS VARIATION indicate the classes in which the Fg plants fell, from which the Fg's came. It is clear that the F3 mean is correlated with the value of the Fo individual from which it came. In this experiment all our predictions are verified, and we cannot doubt that the polygenes controlling corolla length show segregation in the way to be expected from Mendel's principles. We may observe, too, that in this experiment the F^'s from reciprocal crosses are alike. Male and female parents contribute equally to F^ as would be expected with nuclear inheritance. Many experiments of this kind have been performed and all have agreed, within the limitations imposed by their size and structure, with the behaviour expected. Linkage of Polygenes Not a few of the experiments which show polygenic segregation also reveal linkage of polygenes and major genes. The first case was that of Sax (1923) in dwarf beans. He made a cross between a strain with large coloured beans and another which had smaller white beans. In the F., there was a clear segregation into a ratio of 3 plants with coloured beans to i with white beans (Table 5). F3 families were grown and by their aid the coloured plants were classified into homozygotcs and heterozygotes. These appeared in the ratio of i : 2. Thus seed colour was clearly under the control of a single gene which has an effect large enough to be followed by the mendelian method. TABLE 5 AVERAGE WEIGHTS OF BEANS (in cg.) FROM COLOURED AND WHITE MEMBERS OF AN F, IN PHASEOLUS VULGARIS (SAX, 1923) Number of plants Colour constitution Average bean weight 45 80 41 { PP Coloured < ,. White pp 30-7 28-3 26-4 Seed size, on the other hand, proved to be a continuously varying character. Segregation could be detected by the greater spread of 72 LINKAGE OF POLYGENES the seed weight distribution in Fo, but no major discontinuities appeared. The heritable variation is thus polygenic. But when Sax determined the average weights of the seeds in his three classes, distinguished by the seed colour gene, viz. homozygous coloured (PP), heterozygous coloured {Pp) and white (pp), he found that they differed. The PP plants had the largest seeds and the pp plants the smallest, just as the coloured parental strain had larger seeds than the white one. Such an association between size and colour would be expected if some, at least, of the polygenes controlling seed size were linked with the major gene controlling colour. And since the major gene must be carried in the nucleus, so must the polygenes. On one point, however, the experiment is not decisive. That part of the difference in seed size which was associated with the colour difference could have been a secondary effect of the major gene which itself deter- mined the colour difference. In other experiments, however, this possibility can be ruled out. We may take, as an example, an experi- ment done by Mather and Harrison on the polygenic system con- trolling variation in the number of hairs on the undersides of the 4th and 5th abdominal segments of Drosophila melanogaster. With hair-number the class groupings to be used in the frequency distribution are of course decided for us, just as though we could not measure height except in whole inches. Drosophila melanogaster, it will be recalled, has three large pairs of chromosomes and one small pair. This last is, in fact, so small that it can be neglected for most purposes. Most of the genes belonging to any polygenic system will be found on the three larger ones. Now those three large chromosomes can be marked by certain gene differences which are capable of being followed by the mendelian method, and which are particularly convenient because hetero- zygotes for each of them can be recognized phenotypically. The genes used in the experiment were Bar (jB) affecting the shape of the eye. Plum (P/n) affecting the colour of the eye and Stubble [Sh) affecting the shape of certain bristles (though not, it should be remarked, altering the number of abdominal hairs so far as is known). B marks the X chromosomes, Pm the second chromosome (II) and Sh the third (III). These genes were combined with inverted pieces of chromosome, which as we shall see in Chapter 6 reduce, or in 73 CONTINUOUS VARIATION extreme cases suppress, recombination, so that the chromosomes tend to se2;rec;.ite as wholes at mciosis. Two wild-type strains o{ Drosophila were used in the experiment. These showed no differences from one another which could be followed by the mcndclian method, but they did differ from one another in their average numbers of abdominal hairs. The one, which we may denote as S, showed an average of 59*2 hairs on females and the other O, only 43 • 5 hairs per female. Tiiese strains were each crossed to a third carrying the marker genes, B, Pm and Sh. The Fl females, which showed the effects of jB, Pm and Sh in their phenotypes, were crossed back to males from their respective wild- type parental strains. Eight classes could be distinguished by the simultaneous segregation of 5, Pm and Sh in the backcross progenies, and the hairs were counted on a number of female flics in each of these classes. The averase hair numbers are shown in Table 6. TABLE 6 A. AVERAGE NUMBERS OF HAIRS ON FLIES OF EIGHT CLASSES DISTINGUISHED BY THE ANCESTRY OF THEIR CHROMOSOMES Average number of hairs Wild-type parent BPmSb B Pm BSb Class B PmSb Pm ^, + (wild- •^^ type) 51-9 52-4 48- 1 43-6 55-6 48-0 56-7 54-3 46-7 49-0 55-9 45-2 56-4 58-9 490 460 B. THE DIFFERENCES IN AVERAGE HAIR NUMBER ATTRIBUTABLE TO THE THREE LARGE CHROMO- SOMES Excess over tester in chromosome X u 111 Total S 0 2-66 0-73 3-35 0-95 1-64 - 3-08 7-65 - 1-40 Strain difference S — O 1-93 2-40 4-72 9 05 Now the B Pm Sh class has one representative of each of the three 74 LINKAGE OF POLYGENES large chromosomes from the B Pm Sb tester stock, and one repre- sentative of each from the wild-type strain under test. The B Pin class is in the same case for chromosomes X and II, but it has both its representatives of chromosome III from the wild-type strain. If, therefore, chromosome III from the wild strain differs from its mate, or homologue, from the tester stock in any genes affecting hair number, there must be a corresponding difference in the average numbers of hairs shown by the B Pm Sb and B Pm classes in the backcross. The only limitation is that completely dominant genes from the wild-type strain wiU obviously have no effects which can be detected in a backcross of this kind. Similar estimates of the difference in chromosome III can be found by the comparisons between the average hair numbers of the B and B Sb classes, the Pm and Pm Sb classes and the + (wild-type) and Sb classes. The average difference in hair number due to genes in chromosome III can be found therefore as i {B Pm— B Pm Sb+ B— B Sb+ Pm— PmSb+ { + )— Sb) where B Pm stands for the average hair number of the B Pm class and so on. The effects of the other two chromosomes can be found similarly as: — X chromosome J {Pm Sb— B Pm Sb-\- Pm — B Pm -j- Sb— BSb + {+)-B) II chromosome I {B Sb— B Pm Sb-^ B— B Pm+ Sb— Pm Sb + (+)-Pm). The excesses of the three chromosomes of the two strains, S and O, over the marked homologues from the tester, arrived at in this way, are shown in the lower part of the Table. The differences between the chromosomes of either wild-type stock and those of the tester might be influenced by a secondary action of the marker genes, B, Pm and Sb. Other observations on the effects of these genes would not lead us to expect such secondary action, and indeed we can rule it out decisively in another way. The S stock has an unmarked X chromosome which, so far as this experiment goes, gives rise to the production of 2-66 more hairs than the tester X chromosome marked by the gene B. The X chromo- 75 CONTINUOUS VARIATION some of the O stock shows a corresponding excess of only 0*73 over the same tester X. Then the X from S gives rise to 2-66 — 0-73 or 1-93 more hairs than does the X from O; and this difference cannot be due to secondary action of the B gene, or indeed to any other major gene, for O and S differed in no gene of effect sufficiently large to be followed by the mendelian method. In the same way chromosome II from S exceeded its homologue from O by 3*35 — 0'95 or 2-40, and chromosome III by 1-64+ 3 'oS or 4*72. Thus, we have observed a total excess of i -93 -f- 2-40-1- 4*72 or 9*05 hairs caused by the three large chromosomes of S as com- pared with those of O. This method of assaying the action of the various chromosomes by comparison with the tester depends on comparing hair number in flies homozygous for the chromosome under test with that of flies heterozygous for that same chromosome and for the marked chromosome. As we have seen, this prevents us observing the full effect of any gene whose allelomorph in the wild strain is not fully recessive to that in the tester stock. Indeed, fully dominant allelo- morphs must escape detection altogether by such a comparison. Now the Fi females in both crosses, S X tester and O X tester, differed in hair number from those in their parent wild strains. The genes from S and O were therefore not all, or not fully, dominant over their allelomorphs from the tester. These F^ females also, however, differed from one another. So the genes from S and O were furthermore not all, or not fully, recessive to their allelomorphs from the tester. We, therefore, expect our survey to reveal only part of the difference which would be seen between flies homozygous for chromosomes from the wild strains on the one hand and from the tester stock on the other. If we assume that dominance is absent or balanced in the two directions (an assumption agreeing well with our general experience of continuous variation), w^e should expect our tests to trace half the difference between each wild type strain and the tester. The comparison of the chromosomes from the two wild-type strains should then reveal half the effects of the genes by which O and S differ in their three large chromosomes. Now females within strain S have on the average 15*7 more hairs than those within O. We have found a grand difference of 9 hairs in our chromosome tests, and 76 LINKAGE OI- I'OLVGENHS since 9 exceeds half of 15-7, we have no reason to doubt that the heritable difference between S and O is wholly due to differences in nuclear genes What position have we now reached? First, we see that con- tinuous variation is capable of being caused by nuclear genes, which, to be sure, work in special systems. Furthermore, we see that the heritable part of this variation is wholly due to such genes. We see this both from the lack of differences between reciprocal crosses and from the balance-sheet of variation which the linkage experiments enable us to draw up. Finally, we have been able to infer the action of individual genes although these genes are not individually recog- nizable. We been able to do so simply because these individual genes are all transmitted in the same way, by the very mendelian inheritance whose principles they were at one time supposed to contradict. REFERENCES BAUR, H., and TiMorEErF-RESSOVSKY, N. w. 1942. Genctik und Evolutionsforschung bei Tieren. Die Euolutionen der Organismen (Ed. G. Heberer), 335-429. EAST, E. M. 1915. Studies on size inheritance in Nicotiana. Genetics, i: 164-176. GALTON, F. 1889. Natural Inheritance. London. JOHANNSEN, w. 1909. Ekmente dcr exakten Eihlichkcitslelirc. ]en^. MATHER, K. 1943. Polygenic inheritance and natural selection. Biol. Revs. i8 : 32-64. MATHER, K. 1949. BiomctHcal Genetics. London. MATHER, K., and HARRISON, B. J. 1949. The manifold effects of selection. Here^/Vy (in the press). NILSSON-EHLE, H. 1909. KreHzimgswitersucluiugen an Hafer und Weizen. Lund. PEARSON and LEE, quoted by fisher, r. a. 1947. Statistical Methods for Research Workers. loth ed., Edinburgh. sax, k. 1923. The association of size differences with seed-coat pattern and pigmentation in Phaseolus vulgaris. Genetics, 8: 552-560, 77 CHAPTER 4 THE BIOMETRICAL ANALYSIS AMit'we Scales Constitution of the Statistics The Test of Dominance and Linkable Randomly Breeding Populations and Correlations between Relatives Effective Factors or Units The Union of Genetics and Biometry The knowledge that the polygenes are borne on the chromo- somes provides us with a base from which to explore the genetical properties of continuous variation. We cannot follow these genes in their individual segregations by the mendelian method; we can nevertheless feel confident that they will segregate on mendelian principles. That is to say, the various types of family, raised by crossing and inbreeding, will contain the different homozygotes and heterozygotes in the proportions which mendelian experiments and a knowledge of chromosome numbers have led us to expect. Before we turn, however, to the interpretation of the biometrical quantities, means, variances and covariances, in terms of mendelian segregations, we must first consider one other problem. This is the problem of the scale to be used in measuring or representing the degree of expression of the character. This problem does not arise with the mendelian method where we compare the frequencies of distinct types, but it is basic to all biometrical analysis, where we compare the magnitudes of individual expressions. An appropriate scale will go far to ensure the success of an analysis: an inappropriate scale may lead to serious misjudgment. The scales that we use in measuring character expression are the ones which experience has shown to be most convenient. They do not bear any necessary relation to the ways in which genes act or supplement one another in action. They may be satisfactory for the purpose of genetic analysis or they may not. We need, in fact, some way of deciding whether a given scale, be it the one we customarily use for measuring length, weight or any other property, or be it some transformation of the customary scale, as for example into log measure, is satisfactory for our purpose. Our task is that of interpreting tiie means, variances and 78 ADDITIVE SCALES covariances in terms of which we can express the genetical properties of our faniihes. This is most easily done if the various agencies, genes and non-heritable factors, add together in their effects on the phenotype. That is to say, if the difference produced in the phenot^'pe by substituting one allelomorph of a gene for another, or one environment for another, is the same no matter what the effects of the other genes or environmental factors may be. This may, of course, be merely an ideal, for it may be impossible to find a scale on which all genes supplement one another's action in an additive way. Since, however, we cannot isolate the genes and discover their individual properties we can measure only their average behaviour, A scale will, therefore, be satisfactory on which the gene and non-heritable effects are simply additive on the average. Now, if we cross two true-breeding lines and then backcross the Fi to one of them, mendelian theory shows us that the backcross will contain equal numbers of individuals homozygous, like the parent to which the backcross is made, and heterozygous, like the Fi, in regard to each gene by which the parents differed — equal, that is, apart from sampling error. Then the average expression of the character in the backcross, will, in so far as any one of the genes is concerned, be mid-way between the parent and the Fj. If, and only if, the effects of the genes are additive on the scale used, will this relation also hold between the parental rriean, the Fj mean, and the backcross mean, for all the genes taken together. In other words the two backcrosses provide us with two tests of adequacy of the scale. Letting B^ be the mean measurement of the individuals in the backcross to the parent whose mean is Pj, the scale must be such that : — 2B1 = Pi + Fi and 2B2 = P2 + F^ In the same way one quarter of the individuals in an F2 are like each parent and half like the Fj in respect of each gene, so that if the effects of the genes are additive 4F2 ^ 2F1 + Pi + Pg = 2B1 -j- 2B2. Similar tests of the scale can be devised using F3, the mean measure- ment of Fg, and so on. The test of the additiveness of heritable and non-heritable con- tributions to the variation is somewhat different. The variation of 79 THE BIOMETRICAL ANALYSIS true-b reeding lines and of their F^'s is all non-heritable, but the lines and the F/s differ from one another in their genotypes. Then, if comparable environmental differences are to contribute to the variation (or rather to the quantities representing it) independently of the gcnetical differences, we must fmd a scale on which the variances of the frequency distributions of the lines and of their Fi's are the same, even though their means differ for genetical reasons. If the scale used in taking the measurements fails to satisfy these tests, the measurements may be transformed by taking logs, anti- logs, square-roots, squares, or in any other consistent way, and the tests repeated on the new scale. If, as may sometimes happen, no scale is found to be satisfactory on all counts, some suitable com- promise has to be adopted. Constitution of the Statistics Mendelian theory thus helps us to cope with this first problem of finding a scale suitable for genetical analysis. It is equally essential for our analysis of the statistics which are obtained from the measure- ments represented on that scale. Let us denote by 26.^ the difference in phcnotype between two individuals raised in the same environment and differing genetically only by being homozygous for different allelomorphs of the gene A-a. Then, if neither allelomorph is dominant, their heterozygote will have a phcnotype midway between the homozygotes. If there is dominance then let the heterozygote depart by h^ from this mid point. The phenotypcs corresponding to the three genotypes are then related in the following way : — aa Aa AA - d. K 4 The effect of gene B-h can be similarly measured in terms of d^ and h.^, and so on, the convention being adopted that the capital letter denotes the allelomorph making for increased manifestation of the character in all genes. Thus A is not of necessity dominant over a: h^^ may depart from the mid-point in either the positive or the negative direction. The same is true oi \\, and furthermore So CONSTITUTION OF THE STATISTICS the sign of hj, may not be the same as that of h^. The dominances may be reinforcing or opposing one another. Two true breeding Hnes raised in a comparable range of environ- ments will have mean phenotypes differing by: — 2[S(dJ-S(d.)] where S(d^) is the smn of the d increments added by all the genes represented by increasing allelomorphs (A, B, etc.) and S(d_) the sum of the increments added by all the genes represented by decreasing allelomorphs (a, b, etc.) in the parent with the larger manifestation of the character. The average of the two lines, the mid-parent as we may call it, will be the zero point from which the h increments can be measured. It is the natural origin of the scale. The Fi between the two lines will be uniformly heterozygous for all the genes in which its parents differed and so will differ from the mid-parent by h^+ h^^ • • • == S(h), taking the signs of the various h's into account. Thus S(h) can be zero even though each h is not zero, because of the opposing signs of the h's, or, in genetical terms, because of the opposing dominances. In the same way the parental difference may be zero, i.e. [S(d^) — S(d_)1 = O, no matter what values d^, d^^, etc., may have, because the increasing and decreasing allelomorphs of the different genes may be balanced in the parents. The simple comparison of an Fj with its parents is sufficient to establish the dominance relations of a gene in mendelian genetics. But, as we can now see, it is not sufficient to show even the average dominance relations of a polygenic system in biometrical genetics. For if we divide the departure of the Fj mean from the mid-parent by half the parental difference, a ratio which is commonly used to represent the degree of dominance of single genes, we obtain: — / , V „/ , X and this obviously bears no simple relation to the s(d^)-S(d_) ^ ^ •^ K , , . . ratios -t-> -y- and so on. It can vary between zero and an infmitely a b large value, according to the way in which the increasing and decreasing allelomorphs are distributed betw^een the parents, and the way in which the h's reinforce or oppose one another. One thing Elements uj Genetics g I F THL BIOiMI-TRICAL ANALYSIS it ain show us, however. If this fraction departs significantly from zero, then S(h) cannot be zero and at least some of the genes arc showing some dominance in the direction of the departure. In Fo the genotypes AA, Aa and aa occur with the relative frequencies |, 2, 4» so that the mean measurement as affected by this gene will be 4d3H- ^h_, — jd^ or Ih^^. Then, taking all genes into account, the mean of Fg will depart from the mid-parent by |S(h). The mean of F3 similarly departs from the mid-parent by 4S(h). We can therefore learn no more about the genes' domi- nance relations from the Fo and F3 means than we can from the Fj mean. The information to be gained merely from a study of the means is thus very limited. The different variances and covariances which we can calculate are, however, more helpful. The parental lines and the Fj will show only non-heritable variation. We can consider their variances as including only one component, E. The variance of F2 will, however, contain a heritable portion, to which each gene wdll contribute. The contribution of gene A-a to this variance can be ascertained by fmding the deviations from the mean of the measurements of AA, Aa and aa individuals, squaring them and adding them up. The F2 mean in respect of this gene is Ih,^, so that the deviation of an AA individual will be d^ — ^h^, the deviation of Aa, h.j — ih^, and that of aa, — d^ — Ih^. Now one quarter of the individuals will be AA, one half /It/ and one quarter aa, so that the contribution of gene A-a to the variance of F2 must be: — i-a- |hJ2+ i(h, - 4hJ^+ i(- d - il,J2 The remaining genes in which the parental lines differed will make similar contributions to the variance of Fg. Provided that the genes are unlinked, these contributions will be independent, and the total heritable variation will be the simple sum of the contributions made by the individual genes. Then if we write D = d^'^H- d^^-\- d^^ . . . and H = h^^ -|- h^^ -\- \\^ . . . the heritable variance of Fa becomes |D+ ^H. Since the variance will also contain a non- heritable component, the full formula must be ^D + ^H+ E. Two important properties of the variation are revealed by this formula. In the first place, the effect of dominance, as measured 82 CONSTITUTION OF THE STATISTICS by H, is separable from the effect of differences between the homo- zygous phases of the genes, as measured by D. In the second place, D is compounded of items like d/, and H of items like h^^. The value of D will therefore be the same no matter how the increasing and decreasing allelomorphs of the various genes were associated with one another in the parents. And the value of H will be unaffected by the signs, whether positive or negative, of the h increments, i.e. by the directions of dominance of the individual genes. H is, of course, expected to be O in the absence of dominance from all genes, and the ratio . / — provides us with a measure of the average dominance of the genes irrespective of whether dominance is in the same direction for all genes or not, and irrespective of the distribution of the genes between the parental lines. It provides us, in fact, with the information about dominance which could not be obtained by a simple comparison of the parental and F^ means. Were the variance of Fg the only statistic available it would obviously be impossible to estimate the separate values of D and H. Backcrosses and the F3 generation, however, help us here. They supply us with additional information about D and H, and they may be used in combination with Fg to effect the separation of D and H both from one another and from the non-heritable varia- tion E. Taken separately, the variances of the backcrosses cannot be expressed in terms of D, H and E ; but if we add together the variances of the two backcrosses, one to each parental line, they have the joint value of |DH- |H+ 2E. The environmental com- ponent is, of course, 2E because each backcross must show as much non-heritable variation as the single Fg family of the same size. It will be seen from Fig. 17 that the F3 generation is able to provide us with three statistics. These will be derived from (i) the variation between the different F3 families taken as wholes; (2) the variation within the different F3 famiHes; and (3) the relation between the expression of the character in the individual F3 families and their immediate parents in Fg. The statistics which express these relations are: — I. The variance of the means of F3 families, which has the value ID+J^H+E. 83 TlIK BIO. METRIC A I. ANALYSIS 2. The mean of the variances of different F3 families, which has thevakie|D+ JH+E. 3. The covariance of means of F3 famihcs with the measurements of the F2 parents, which has the value iDH- JH. In a properly designed experiment, the effects of the environment on average manifestation of the character in F3 will bear no corre- lated relation to the effect of the environment on the character in the Fo parent. The covariance, therefore, has no E component. The E component of the variance of F3 means will in general differ from that of the other statistics since the variation of an average value will be influenced to a different extent than that of a single value by the vagaries of the environment. We must thus distinguish between El, the non-heritable component of variation of single individuals, and Eg, the non-heritable component of variation of family means which will usually, though not always, be smaller than E^. The non-heritable component appropriate to individuals, E^, will appear in all the formulae except that for the variance of F3 means, which will contain E2. E^ and Eg can be estimated directly from the variation within the true-breeding parent lines and F^, E^ as the variance of single individuals and E., as the variance of the means of groups containing the same number of individuals as the F3 families. The Test of Dominance and Linkage An example will illustrate the use of these results. The two species o( Antirrliinmn, majus 3.nd gliitinostun, differ in, among other charac- ters, their heights. In a true-breeding strain of majus the average length of the leading shoot was 21-45 inches. No strain o£ glutinosum known to be true-breeding was available, but one which was empirically observed to be no more variable phenotypically than the strain of majus, had leading shoots whose average length was 9-12 inches. The F^ between the species gave a mean measurement of 17-08 inches. An F2 was raised which showed no segregation of genes with major effects on height, the variation being continuous and depending, presumably, on a polygenic system. The F^ was also backcrossed to both parents, and 19 F3 families were raised by 84 THE TEST OF DOMINANCE AND LINKAGE self-pollinating Fg plants. The values found for the various statistics are given in Table 7. The estimate of £3 obtained directly from the parents and Fj is lower than that of Ei, in the way generally expected to be the case. The seven observations provide us with seven equations for the estimation of the four quantities D, H, Ej and £3. The estimation is undertaken, therefore, by the method of least squares (Mather 1949) and we fmd: — D =25-708 H = - 10-778 El = 4*995 Eg = 0-146 These estimates can be substituted in the expectation formulae to give calculated values for the seven statistics as shown in column four of the Table. Two points are immediately striking about the results of this analysis; a negative value has been obtained for H which, like D, was defined as a sum of squares and therefore cannot be negative, except as a result of sampling variation; and the agreement of observation with expectation in the Table is rather poor, especially for the mean variance of F3 where the discrepancy is nearly 25 per cent of expectation. The analysis has not achieved full success. The reasons for this partial failure become apparent when we consider the consequences of linkage between the polygenes. When genes are unHnked their contributions to the various statistics are simply additive. This is not so when they are linked. If p is the frequency of recombination between the two genes A~a and B-b, the contribution to D as it appears in the variance of Fg is d^2+ ^b^zk ^d^ d^^(i — 2p), the third term being added when the genes are coupled and subtracted when they are repulsed. This reduces to d^^+ ^^~ when p has its free value of 0-5. In the same way, the contribution to H in the variance of Fo is h^- + h^'-^^" 2h^ \{i — 2p)-. Where more than two genes are involved we have as many terms of the kind 2d^ d^^ (i — 2p) in D and 2h^ h,^ (i — 2p)'^ in H, as there are pairs of genes. With three genes there are three such terms, with four genes six terms, and so on. All of these terms as they appear in D depend on (i — 2p) where p is the frequency 8i THE BIOMHTRICAL ANALYSIS of recombination between the two genes concerned, and all in H similarly depend on (i — ^p)^. It will thus be noted that the effects of linkage of polygenes are expressible independently of the linear order. Exactly the same linkage terms appear in D and H in variance of F2, variance of F3 means, and covariance of F2 and F3. But while the same number of linkage terms appears in the D and H of mean variance of Fg's, those in D now depend on (i — 2p)- instead of TABLE 7 THE INHERITANCE OF HEIGHT IN ANTIRRHINUM MAJUS X GLUTINOSUM Statistic Expectation Observed Calculated No linkage Linkage Variance of F2 -VD + -^H + El 15-838 15-155 15-663 Summed variances of back- crosses iD + iH + 2E, 16-750 17-455 16-750* Variance of F3 means iD + t'^H + E2 11-938 12-326 11-585 Mean variance of F3's iD + iH + E, 12-550 10075 12-550* Covariance of F2 parent and F3 family . . iD + iH 10-681 11-507 11-211 Variance in parents and Fj Single individuals . . E, 3-250 4-995 3-426 Means of groups . . E2 0-534 0-146 0-887 The assumption of linkage permits a perfect fit. on (i — 2p), and those in H on (i — 2p)- (i — 2p+ 2p-) instead of on (i — 2p)2. We can thus see how linkage may explain the discrepancies in the analysis of the Antirrhinum results. Similarly, although the linkage terms in D of the summed backcross variances are just like those of D in Fg variance, the linkage terms in H of the backcross depend on (i — 2p) as compared with (i — ^p)^- When p = o • 5 for free recombination, all these linkage terms vanish from all statistics and D and H arc constant over the whole range. The test of linkage is, therefore, the test of differences in the values of D and H over the various types of family. In the experiment with shoot length in Antirrhinum, D and H should be constant, even with linkage, over variance of F^, variance of F;> means and covariance 86 THE TEST OF DOMINANCE AND LINKAGE ot Fo and F3. The D and H of the summed variances of backcrosses and of the mean variance of Fg's will, however, differ both from one another and from those of variance of Fo with linkage. If, therefore, we find the best estimates of D and H, E^ and E2, omitting the ANTIRRHINUM VARIATION IN HEIGHT NON-HERITABLE -E -H FIXABLE HERITABLE -Q ^m UNFIXABLE HERITABLE Vi fl Vb,+ V82 "^Fz/n Fig. 18. — The components of variation in height of plants of Antirrhinum majus X ghitinosiirn. For each statistic the centre column of the histogram shows the value observed, the left column that expected on the assumption of no linkage, and the right column that expected assuming the possibility of linkage. The compositions of the expected values are shown in terms of D, H, and E. The best estimate of H is negative when linkage is assumed to be absent, so that the contribution of H is overlapped by the contributions of D and E in the left column. When the possibility of linkage is assumed, Vbi + Vb2 and Vps become perfect fits. Wps/ps has no E component. Vpo = variance of F2 Vbi + Vb2 = summed variances of backcrosses Vf3 — variance of F3 means Vfs = mean variance of Fs families Wf2/f3 = covariance of Fo measurement and F^, mean. summed backcross variances and the mean variance of Fg's, these should give a better fit than do the values found for them using all the data, should linkage be interfering with the result. If the fit is not improved there can be no evidence of linkage. 87 THE HIOMETRICAL ANALYSIS When we omit the summed variances of backcrosses and the mean variance of Fs's from the Antirrhinum results, we find: — D = 20-369 H= 8-209 El = 3 -426 Eg = 0-887, and again expected vakics can be calculated for the various statistics as shown in column five of the Table. The analysis is now much more satisfactory (Fig. 18). In the first place a positive value has been obtained for H, though it is too small to afford any reliable indication of dominance. Secondly, the discrepancies between observation and expectation have been very much reduced in Table 7. This is true, not merely for the summed variances of backcrosses and mean variance of Fg's, for which linkage allows us to assume a perfect fit, but also for the other statistics, notably E^ and the variance of F2. The results indicate that at least some of the polygenes are not rccombining freely. Furthermore, the mean variance of F3 is larger than would be expected from the F2, so that the linkage must be preponderantly in the repulsion phase, i.e. of the kind — • Large, therefore, as was the phenotypic difference in height between the species, it did not represent the fuU difference between the genotypes, for some of the genes must have been counter-balancing one another's effects in these repulsion linkages. Randomly Breeding Populations and Correlations between Relatives The types of family which were used in this experiment on shoot length are not, of course, exhaustive. In particular, we can raise families of the third generation by intercrossing pairs of individuals taken at random from Fg. These biparental families will, in fact, be the only type available in the third generation of experiments with most animals, where true Fg's are obviously impossible to obtain. Tlie same range of statistics is available from biparentals as from Fg's, viz. variance of biparental means, mean variance of biparentals, and covariance of F2 parents and their biparental RANDOMLY BREEDING POPULATIONS AND CORRELATIONS progenies. The values of these statistics also depend on D and H, thus: — Variance of biparental means jD + i^H + E^ Mean variance of biparentals ^D + i^eH + E^ Covariance of F2 and biparentals jD They may be used to supplement or to replace the statistics from F3 families in the estimation of D and H. When we distinguish between the biparental families obtained by the intercrossing of different pairs of individuals from Fg, we break the heritable variance down into two parts: the variance of the means, which measures variation between families, and the mean variance, which measures variation within families. The total herit- able variation of this generation is the sum of these two parts, viz. (iD+ r6H)+ (iD+ i%H) =|D+ JH. It is exactly the same as the heritable variance of F2 itself. And if we breed still another generation of biparental progenies, the pairs of parents being taken at random, without distinction of family, from the first biparental generation, we fmd that its heritable variance is once again ^D + jH. This variance is, in fact, characteristic of the random mating system used. The variances will be the same from generation to generation provided that the system of random mating is continued, and pro- vided also, of course, that the environmental variation, and with it the E component, is constant. The covariance of parent and offspring is also constantly jD under this system of mating, with no E component appearing in it. In the particular case under discussion we commenced with the Fg of a cross between two true-breeding lines, so that the frequencies of the two allelomorphs of each gene by which the parents differed, A-a, B-b, etc., must be equal. The variance and the parent-offspring covariance still retain their values of ^D+ 4H4- E and jD even, however, where the frequencies of yl and a, B and b, etc., are not equal in the group of individuals. The variation formulae are therefore characteristic of all randomly breeding groups. The effect of variation in the gene frequencies appears in a different way, viz. in the contributions which the genes make to D and H. With equal gene frequencies and no linkage we have D = S(d-) and H = S (h^) ; but when allelomorph A has the frequency u^, 89 THE BIOMETRICAL ANALYSIS and a the frequency v^ ( =: i — uj, etc., we find that the con- tribution of the gene ^ — d to D is 4 u,v^ [^a~r hgCv^— uj]^ and to H is 16 u^^^^-h^- so that the general fornmlac are D = S|4uv[d -f h(v - u)]2} and H = S(i6u2v2h2) These reduce to the original forms when u = v = o • 5 for all genes. The special virtue of equal gene frequencies for analysis now becomes clear. Unless u = v = 0*5 for all genes, the full effects of all the dominance relations is not represented by H; some of them appear inD. Using these general forms of D and H, we can represent the relations between parents and offspring of any randomly breeding group or population in terms of the variance, hT) + |-H + E, and the parent-offspring covariance, ^D. The parent-offspring correlation is given by r^^^ = ^ :. We can now see that Wp/„ = JD, while V^ = V^ = lD+ JH + E, so that: — iD 'p/o ID+IH+E D, H and E are quadratic quantities and so must always be positive. The maximum correlation which parents and offspring can show for simple genetic reasons within a randomly breeding population must, therefore, be r= 0*5 when H= E=: O. We can find the covariances between other pairs of relatives, of which the most useful is that between pairs of siblings. This, too, can be represented in terms of our three components of variation as: — Then the fraternal correlation will be: — ^s/s 4D + 1H+E This, too, will have a maximum of o • 5 when H =E = O. But when dominance is present, so that H > O, r^/^ must exceed rp^^ because of the additional term -^H in its numerator. 90 EFFECTIVE FACTORS OR UNITS Within human groups mating is not quite at random, but it is near enough to being so for the parental and fraternal correlations to show the relations we should expect. It has been found by Pearson and Lee, in respect of the human cubit measurement, that: — rp/o = <^*4i8o and 13/^ = 0-4619 The slight departure from random mating, and the likelihood that members of one family will develop in environments more alike than those of unrelated people, make detailed estimates of D, H and E somewhat untrustworthy. We can, however, draw some general conclusions from these correlations. They are both so near to their maximum value of o • 5 that the non-heritable component of varia- tion must be small relative to the heritable. And since the fraternal correlation exceeds the parental we have evidence of dominance of the genes controlling variation in human cubit measurement. Effective Factors or Units We can now return to crosses between true-breeding lines. Where these lines differ in such a way that the increasing allelomorphs {A, B, C, etc.) of all the genes by which they differ are assembled in one of them, and all the decreasing allelomorphs {a, b, c, etc.) in the other, the mean measurement of each will depart from the mid-parent by S(d). Now if we care to assume that all the genes have equally large effects, i.e. d^ = d^^ ^ d^ . . . = d, S(d) = kd where k is the number of genes. Now we can estimate D which, when the genes have equal effects and there is no linkage, will have the value kd^. Then the square of the departure of each line from the mid-parent, when divided by D, will give us an estimate of the number of genes, for it will be S^(d) _ (kd)-- _ D ~ kd2 ""^ The shortcomings of this estimate are obvious from the assump- tions which nmst be made to obtain it. In particular it will be an underestimate if the increasing and the decreasing allelomorphs are not concentrated in the opposite parents, or if the effects of the genes are not all equal. A second method of estimating k is based 91 THE BIOMETRICAL ANALYSIS on the variances of F3 families. It overcomes the first difficulty I because it demands no assumption about the distribution of allelo- morphs between the parental lines; but it magnifies the second dirticulty since differences between the effects of the individual genes reduce this estimate of k to an even greater extent than the first one. Both estimates are also reduced by linkage. When all these reservations have been made, the estimates ot k are still instructive. Our experience of the fine gradations which continuously varying characters show, would lead us to expect that many genes w^ould be concerned in the polygenic system governing any particular variation. We find, however, that the estimate of k is never large and is commonly as low as 5 or even less. The value given by the Autirrhi}inin data^ using the first method of estimation, is, for example, k = i -9. The reason for this is that k is not truly an estimate of the number of genes, but of a different unit which we may call the effective factor. To see what effective factors are, let us consider a hypothetical case when the parental lines differ by a large number of genes, scattered along the length of every chromosome. Genes which do not recombine must always appear as one in segregation. Now the number of chiasmata, upon the formation of which depends recom- bination of genes within a chromosome, is usually no more than one or two per bivalent. Not all the genes, then, will be separated from their neighbours in the chromosome by chiasmata. In fact, one chiasma will break the chromosome into two pieces, the genes within each of which will segregate together. Two chiasmata will give three such segments, and so on. These segments are the physical basis of the effective factors whose number is estimated by k. At most this number cannot exceed the haploid number of chromo- somes plus the mean number of chiasmata in the nucleus. It must generally be less, for some segments will be unmarked by segregating genes. Furthermore any variation, such as we know normally to occur, in the positions in which chiasmata form, will lead to the effective factors varying in their genie content and effect. This, too, as we have already seen, will reduce the estimate of k. The effective factors which emerge from the segregation seen in an F2 may be broken down further in an F3 owing to the formation of chiasmata in new positions at meiosis in the Fo individuals. Thus 92 THE UNION OF GENETICS AND BIOMETRY the effective factors are not necessarily fixed in the course of an experiment and may increase in number. Their history will depend on the circumstances of localization of crossing-over which we have already noticed, and also, as we shall see later, on various restrictions of crossing-over which arise in hybrids. When the individual genes have large effects unlike one another, we can follow them and count them as individuals by the mcndclian method. A single recombination between them, however many tests must be made before it is found, is sufficient to show that two distinct genes are at work. Such a unique recombination can be detected without ambiguity by the mendelian technique when the genes have different effects on the phenotype. But the members of a polygenic system cannot be followed individually in inheritance, nor their recombinations identified as individual events. In counting the units of polygenic inheritance we are therefore forced to use a coarser criterion, that of the occurrence of a particular recom- bination frequency, usually 50 per cent. The effective factors whose number we estimate are thus, in the general case, not ultimate polygenes. Indeed we can have no certainty that any unit of polygenic inheritance, which we may find and whose properties we may determine, is an ultimate unit. We are debarred from studying polygenes as individuals by the inherent limitations of genetical method. Rather we must follow them as they are organized into effective factors. These units are not final in the way that individual major genes are. They can be broken down into smaller parts by recombination and they can presumably be synthesized by bringing together their parts through recombination. Their properties must depend on the way the genes are put together to make the factor as much as on the genes them- selves. The Union of Genetics and Biometry Biometrical genetics is built upon the foundation of mendelism. It could exist in no other way, and the relative failure of Galton and the early biometricians to achieve an understanding of inlieritance was due to the lack of the foundation which Mendel, by an entirely different technique, was able to supply. There is thus no conflict between biometrics and mendelism. On the contrary, biometrical 93 THE BIOMETRICAL ANALYSIS genetics extends mendelism to all, or nearly all, variation. In making this extension we learn to measure variation by new quantities and to understand it in terms of new units: quantities and units as appropriate to the study of continuous variation as the segregation ratios, recombination frequencies and individual genes are to the study of discontinuous variation. The mcndelian foundation upon which the biometrical method rests has been laid by the study of major differences. The geneticist has chosen the variants which he needed for this purpose from amongst the wealth of variation which living species offer him. He has not been concerned with the phenotype except in so far as its changes and differences marked for him changes and differences of the genotype whose understanding was his primary aim. No such choice is open to the breeder of crops and stock. His aim must always be to adjust and improve the performance of his plants and animals in respect of some character, yield, quality, disease resistance or whatever it may be, which is chosen for him. He must be pre- pared to make good use of whatever heritable variation, continuous or discontinuous, his individuals may show. It is for this purpose that he, no less than the student of ultimate principles, needs the integration of the mendelian and biometrical techniques which we can now attain. REFERENCES nsHER, R. A. 1918. The correlation between relatives on the supposition of mcn- delian inheritance. Trans. Roy. Soc. Eiiin., 52: 399-433. nsHER, R. A., IMMER, F. R., and TEDIN, o. 1932. Thc genetical interpretation of statistics of the tliird degree in the study of quantitative inheritance. Genetics, 17: 107-124. MATHER, K. 1949. BiowetricaJ Genetics. London. PANSE, V. G. 1940. The application of genetics to plant breeding. II. The inheritance of quantitative characters and plant breeding. _/. Genet., 40: 2S3-302. 94 CHAPTER 5 BASES OF CHANGE Polyploidy and Polysoiiiy Structural Cliaugc Misd'wision oftiie Cciilroiiwrc Deficieticy and Biilancc Intergeuic and Ititragcnic Clmiigc: Presence and Absence Somatic Mutation The Gene as a Unit oj Clianqe So FAR WE HAVE CONSIDERED experiments in which genes have appeared as fixed in the chromosome, and chromosomes, apart from crossing-over, as fixed in the nucleus. In any large experiment, however, we find individuals which do not correspond to the predictions that we are justified in making on the basis of the simple rules of segregation and recombination. We fmd new and unex- pected phenotypes. The^e variants, sports or mutants have changed in their heredity. What kinds of changes occur and how do they come about ? To answer these questions we call on a variety of methods and observations. Polyploidy and Polysomy If we cut down a plant of the tomato, Lycopersicum escidentum, a proportion of the shoots which subsequently grow from the cut surface are of somewhat stouter growth. They have larger flowers, but the ripe fruits are smaller with fewer seeds. These shoots, which can be propagated as cuttings, and from their seeds, have 48 chromo- somes instead of the usual 24. They are tetraploid with four sets of chromosomes (2n = 4x) instead of diploid (2n = 2x). Doubling of the whole nucleus arises from a failure of mitosis to complete itself: the chromosomes have divided without the daughter nuclei separating and without the cell dividing. This fiilure is very common in plants and can be readily induced by treatment of the seed, the growing point of the shoot, the young embryo, or the germ mother-cells with colchicine, with high or low temperatures, and in other ways. Tetraploids have larger cells and are generally larger and more robust than their diploid forbears. Their fertility is usually reduced. Otherwise their resemblance to 95 BASES OF CHANGE the diploids is close and they are consequently common in horti- culture. In a great many plants and a few animals triploid (sx) types occur. They arise by failure of meiosis in one of their parents. Gametes containing the diploid instead of the haploid number of chromo- somes are produced, and by uniting with haploid mates give triploid progeny (Fig. 19). A cross between diploid and tetraploid will, of course, give the same result. Triploids are again more robust than their parents which they otherwise resemble closely except in being, as a rule, highly infertile. Triploid apples and pears, however, set just enough of the ten seeds in each fruit to provide a satisfactory crop without overburdening the tree and this places them amongst the most valuable varieties. The immediate cause of the infertility of triploids is seen when we examine such of their progeny as survive. For the triploid rarely, if ever, produces triploid offspring. Its gametes contain all the possible combinations of the extra or odd chromosomes (Fig. 28); but most of these die either in the pollen or, if carried by the eggs, in the embryo-sac or young embryo, and the survivors have, as a rule, only one or two beyond the diploid number. These survivors are always of reduced vigour and abnormal form, but their abnormalities are different from those of triploids or tetraploids. They are no longer general in character, but rather specific. They affect different parts of the plant or animal, which consequently seems unbalanced. The type of its unbalance, we find, goes with the particular extra chromo- somes which it gets from the extra chromosome set of the parent. In Datura stramonium and the tomato (2x = 24) there are 12 types of trisomic (with 24 -|- i chromosomes) corresponding to each of the 12 chromosomes in the haploid set {cf. Fig. 67). The trisomies stand between the diploid and the triploid in fertility. Similar off-types also come directly from the diploid in many plants and animals. In Drosophila melanogaster, triploid flies occasionally appear amongst the diploid males and females. And also some with the small fourth chromosome represented once or three times instead of twice. Such monosomic and trisomic flies are again distinct from the disomic type. They result, presumably, from the failure of the fourth chromosome to pair at meiosis, in which case the two partners behave independently and may pass to the same 96 POLYPLOIDY AND POLYSOMY ME TE Fusion (^ Fig. 19. — Meiosis in the egg of the horse thread worm Ascaris megalocephala, a race with two diploid chromosomes. Left: the normal series ending in fusion of single egg and sperm chromosomes. Right: the result of a misorientated first division spindle is the failure of expulsion of the first polar body which fuses with its sister nucleus. The second division leaves a diploid egg which is fertilized to give a triploid embryo. Fusion with a similarly exceptional diploid sperm would give a tetraploid of the race which is indeed found in nature (after Boveri, 1904). EUmenli of (j::i!<:ik!i 97 BASILS OI- CHANGE gamete. The abnormality is not seen in respect of the larger chromo- somes, whose unbalance presumably leads to too great a disturbance for the fly to live. Except, that is, for the Y. An extra Y does no harm and lack of the Y, as we saw, does not kill the fly, but produces a male with immobile sperm. TABLE 8 THE TYPES OF BALANCED AND UNBALANCED CHROMO- SOME COMPLEMENTS WHERE ABCD STAND FOR A SET OF FOUR Haploid X ABCD Diploid ABCD 2x ABCD Triploid ABCD 3x ABCD ABCD Autotetraploid 4x ABCD ABCD ABCD ABCD Allotetraploid 4x ABCD ABCD A'B'C'D' A'B'C'D' Deficient Gamete X - 1 ABC - Monosomic Diploid 2x - I ABCD ABC- Trisoniic Diploid 2x + 1 ABCD ABCD D Tetrasomic Diploid 2x + 2 ABCD ABCD D D Doubly Trisomic Diploid 2x + 1 + 1 ABCD ABCD A--D Unbalanced or Secondary Polyploid ABCD ABCD A'B'C'D' A'B'C'D' A''B' A"B" No such change of balance occurs in another whole-nucleus variation, that due to the development ot an egg without fertiliza- tion. Such parthenogenesis gives a haploid individual which, with inbreeding and therefore nearly homozygous stocks, often develops to maturity, though of reduced size. We have already seen one produced artificially in a sea-urchin (Fig. 2). At meiosis in haploids the chromosomes, lacking any regular partners, are scattered like the 98 POLYPLOIDY AND POLYSOMY extra set in a triploid to give spores or gametes with, as a rule, ever^' number up to the haploid. In consequence haploids are highly sterile and their rare progeny are diploids. Entirely homoz)^gous diploids have been produced in this way in the tomato and in Datura. In plants, and perhaps even more in animals, changes in chromo- some numbers are a continual source of new variation which may be recognized by change in size, form or fertihty of the variants. Their frequency will depend largely on the conditions of temperature at the time of formation of the parental germ cells and of their fertilization, so that the results recorded in Table 9 might no doubt be multiphed or divided by ten in special circumstances. In plants the polyploid, at least the tetraploid, mutants may estab- lish new races. TABLE 9 WHOLE CHROMOSOME MUTANTS IN POPULATIONS OF PLANTS AND ANIMALS: LYCOPERSICUM FROM RICK (1945) AND TRITURUS FROM FANKHAUSER (1941) Type of mutant 55,000 tomatoes: 66 unfruitful plants examined 1,074 newts: all examined Haploid : X Trisomic: 2x + 1 2x + 1 + 1 . . Triploid : 3x . . Tetraploid : 4x . . Pentaploid: 5x . . Chimaeras: x/2x 2x/3x + . . Abnormal 2x Deformed Sterile . . Died . . 2 2 45 3 3 8 3 1 1 10 1 3 1 1 not recorded not recorded not recorded Total 66 18 Amongst animals on the other hand, polyploidy is of little account. Where the sexes are separate, obviously the first polyploid has no mate of its own kind. In experiment healthy and vigorous polyploids can be readily produced in Drosophila, species of Triton and elsewhere. They cannot maintain themselves by reproduction. In nature, there- fore, although they may occur in all species, they establish them- selves only where sexual reproduction has been abandoned (as in 99 BASES or CHANCE the shrimp Artemia salina) or where sexual differentiation is replaced by hermaphroditism (as in nematodes and molluscs). Structural Change Apart trom clianges in the number of whole chromosomes, changes also occur within the chromosomes, structural changes. Wc have already an indication of such changes in the normal crossing- over at meiosis; but of course crossing-over is reciprocal and can do no more than rccombine differences that are already there. The results of new changes that have taken place in the resting nucleus we might see at the ensuing mitosis, especially at metaphase. But examination of hundreds of individuals and thousands of mitoses will fail to discover any spontaneous changes within chromosomes. Only in abnormal individuals or under abnormal conditions, as from sudden changes of temperature, do we find the chromosomes breaking up, and then the break-up is usually frequent and extensive. Its common characteristic is the breakage of the chromosomes or chromatids during the resting stage, with results that are seen at the following metaphase. Such spontaneous breakage has been described, for example, in the pollen grains of Tulipa fragrans. Breakage can also be induced by X-rays or other ionizing radiations, or by chemical poisons such as mustard gas. The same description, however, can apply to all three, since they follow the same rules. The simplest change, and the one which precedes all others, is breakage of the chromosome fibre into two or more parts called fragments. The breakage occurs in the resting nucleus. Now the chromosome reproduces, its single thread becoming doubled, in the resting nucleus. The breakage therefore affects either the whole chromosome before its reproduction (B"), or only one of its two daughter chromatids after its reproduction (B'). B" always gives two kinds of fragments, one with the centromere and one or more without (Fig. 20). Acentric fragments cannot move spontaneously on the spindle: they lag and are usually left out of the daughter nuclei, like unpaired chromosomes at meiosis. They degenerate in the cytoplasm. Evi- dently they cannot develop a new centromere. It seems, indeed, that we have to look upon the centromere as a specific and permanent 100 STRUCTURAL CHANGE self-propagating organ like any other gene or group of genes, concerned with the organization of proteins on the spindle just as others are in the nucleus. Centric fragments on the other hand, move normally on the spindle. They are distributed to the daughter nuclei and appear in hi Split r <■-- 2.b" — > II (T^ f^ Split SR Post Split K <--- -> Fig. 20. — The chief successions of change that may follow two spontaneous or induced breaks (B") in two chromosomes with restitution, chromosome reunion (R"), sister reunion (SR) and chromatid reunion (R') — in this order — at later stages. The pre-split stage is represented with the chromosomes divided for ease of comparison. Each step may be fmal and hence observable at the ensuing metaphase. SR is independent on the centric and acentric sides of a break (from Darlington and KoUer, 1947). inheritance as chromosomes lacking particular blocks of genes. If too many genes are missing from a fragment the unbalance from this deficiency may be fatal even where its homologue is intact. Indeed X-ray experiments show that in diploid organisms most cells whose chromosomes are deficient, owing to breakage, never divide again after the mitosis where the fragment is lost. A single break can lead to nothing but loss; two breaks begin to show us more fruitful possibilities. The broken ends can rejoin lOI BASES OI- CHANGE with one another to give two new combinations. Mechanically this reunion is just like that of crossing-over: physiologically, on the other hand, it is something quite new. Since the breaks can occur in any parts of any chromosomes, reunion can give a prodigious variety of new combinations. It can occur between ends within one arm of one chromosome, between the two arms of one chromosome, or between the arms of two chromosomes, homologous or non- homologous. Some of these new combinations arc unv/orkablc : the new chromosomes have no centromere; or they have two centromeres which act independently and so cause yet another breakage by passing to opposite poles at anaphase. But other combinations have only single centromeres and these are workable and important (Fig. 20). The workable combinations arc of two kinds: an inversion happens where the two breaks are in the same chromosome, an interchange where they are in different chromosomes. Where they are in oppo- site arms of the same chromosome we may look upon the change cither as an interchange or an inversion. These types of reunion are found wherever there is breakage. And, when the breakage is of chromatids, the association of the changed and unchanged chroma- tids remains at the following metaphase of mitosis to bear witness to the course that events have followed. For example an interchange of chromatids gives the appearance of a chiasma, and an inversion of a chromatid gives a loop in addition to a chiasma (Fig. 30). Two chromosomes undergoing interchange may be represented as of two segments each, AB and CD, which acquire the new order, AD and BC or AC and BD. For inversion we need four such segments to represent one chromosome, abed which changes into acbd. The homozygous types derived from these changes show new linkage relations. Experimental interchange in X-rayed Drosophila moves blocks of genes from one linkage group to another. Inversion leaves them in the same groups but their recombination frequencies correspond to a new order on the map. Two breaks suffice to produce inversion or interchange. Three will give more complicated changes. A piece may be taken out of a chromosome and inserted or translocated wherever else the same or another chromosome may be broken. Thus two chromosomes iihc and def c^iu become ac and dbcj. In a succeeding generation this 102 STRUCTURAL CHANGE cliange can give homozygotes for the two new cliromosome types. These are still balanced. Indeed all these primary structural changes, apart from simple loss, which eliminates itself, merely rearrange the materials in the chromosomes. New chromosomes are created but there is no change in their aggregate content. But by recombination with normal homologues at meiosis secondary changes arise. Our translocation will give heterozygotes and homozygotes for ac and ^t^ which will be deficient in the segment h and meet the fate of deficiencies. And it will give heterozygotes and homozygotes for abc and dbef which will have a duplication o£h. Such individuals will usually be viable but they will be different from the original type. Again, by translocation within itself abcde can become adbce. On crossing-over with the normal homologue in the heteroz)'gote, such a changed chromosome will likewise give deficient (ade) and dupli- cated (abcdbce) progeny from crossing-over in d, and similarly abce and adbcde from crossing-over in be. TABLE 10 THE CLASSIFICATION OF THE TWO ORDERS OF CHROMOSOME CHANGE IN THEIR PRIMARY AND, FOLLOWING RECOMBINATION, SECONDARY CON- DITIONS Type of Change Primary: Not changing balance Secondary: Changing balance Numerical heteroz>'gous homozygous Nuclear Triploid 3x Tetraploid 4x Chromosomal Trisomic, etc., 2x + 1, 4x ± 1 Tetrasomic, etc., 2x + 2, 4x ± 2 Structural (heterozygous or homozygous) Sub-chromosomal Jnversion Deficiency Interchange (reversion to type) Translocation f Duplication \ Deficiency Numerical + Structural Misdivision of the centro- mere and Formation of two telocentric chromo- somes Origin of an iso-chromosome from a telocentric 103 BASES OF CHANCE Misdivision of the Centromere There is one change in the hereditary mechanism, and one only, which affects both the number and the structure of the chromosomes at a single stroke. That change is the misdivision of the centromere. The unpaired chromosome at the first anaphase of mciosis is in an equivocal position. Its centromere is unable to co-orientate w^ith, or segregate from, a partner. Nor can it divide at the same time as the paired chromosomes segregate. It is therefore frequently lost. Sometimes, however, its difficulty is resolved by division within itself. Instead of separating lengthwise from the product of its own reproduction (which is not yet available) it separates crosswise into two components. It thus proves to be a compound or repetitive gene. Sometimes it explodes to give useless fragments, but often it divides into what must be two nearly equal halves, for each part successfully carries its chromosome arm to the pole and undergoes a second division of its new chromosome. The new chromosome is telocentric and in some circumstances is stable. Both in Orthoptera and in the plant Campanula persicifolia races have been found where a particular chromosome is replaced by its two telocentric arms so as to add one more to the haploid number. But this is not always so. At the next mitosis (in the pollen grain ofFritillaria for example) reunion of the two daughter chroma- tids is found to have taken place within the centromere. The new chromosome, which passes to one pole without division, is then twice the size of the telocentric. It has a double-sized centromere and two identical arms. It is known as an iso-cliromosomc. A pair of iso- chomosomes are in effect tetrasomic. They have been found as part of the regular complement in Nicatnlra and as supernumeraries varying in number in different plants in Sorghttm, Sccale, and Datura. Thus the primary changes of inversion, interchange, translocation and misdivision can never change the balance. But by recombination secondary changes occur, producing a new content for the nucleus as a whole. Deficiency and Balance Deficiencies in parts o{ single chromosomes, if they arc small enough, are not always fatal to diploid nuclei. But when such a 104 DEFICIENCY AND BALANCE deficient chromosome passes through meiosis and reaches the haploid generation it may still prove fatal. The danger is greater when this generation has to undergo several cell divisions: thus, as we shall see in detail in the next chapter, the pollen is more sensitive than the embryo-sacs, and both are more sensitive than the sperm in animals. The haploid egg nucleus in animals, since it is immediately fertiHzed, is never expo'sed to any trial of sensitivity. Sperm of Drosophila lacking whole chromosomes, even the large autosomes, can fertilize eggs. But pollen grains of lilies so lacking will never even pass through their vegetative mitosis. And, even where deficient gametes survive, a diploid homozygous for their deficiency cannot do so. Evidently, as we might expect, nearly all the genes are indispensable for the life of the cell in one dose and are most efficient in tw^o doses. The loss of a small part of a chromosome, and even of a whole chromosome, must be of some importance in the origin of natural variations as the sex chromosome differences show. The Y chromo- some of mammals is little more than a deficient X. The male is therefore a deficiency heterozygote. The situation is even clearer in those insects and spiders with no Y chromosome at all. With them the male is heterozygous for the deficiency of the whole X. It is monosomic or XO. Heterozygous deficiencies and duplications are, obviously, for segments what monosomies and trisomies are for w4iole chromosomes. Corresponding to the homozygous deficiencies and duplications are nullisomics (zx — 2) and tetrasomics (2X+ 2). Nullisomics cannot survive in diploids, but they appear in analogous forms in polyploids {e.g. 6x — 2). Tetrasomics in Datura can survive, but are even poorer and more abnormal than the corresponding trisomies. The homozygous duplication, in Drosophila and maize, bears a similar relation to the heterozygote. Two laws of gene balance emerge from these considerations. First, the larger the proportion of the chromosome outfit, up to a half, which departs from the numerical proportions of the rest, the greater the disturbance of development. A trisomic for two chromosomes, or double trisomic, is more abnormal than a single trisomic — except in Crepis capillaris (x = 3) where 2x+ i + i = 3x— I. Secondly, the larger the departure in proportion of the unbalanced element the greater the disturbance. A monosomic or 105 BASES OF CHANGE tctra5omic diploid is more deranged than a trisomic, or, of course, than a monosomic or tetrasoniic polyploid in a group of organisms working with the same chromosome series. Intergcuic and Intragenic Change: Presence and Absence There is one other genetic effect of structural change. This is the physiological effect of change of position itself. Changes of order might be expected to produce no effect on the phcnotypc. Indeed in maize, where some two hundred have been produced by breeding with X-rayed pollen, it is doubtful whether they ever do so. In Drosophila and Oenothera, however, an effect often has to be inferred as the result of the mere change in order itself, a position effect. The position effect is now known to be responsible for many changes which were described as gene-differences in breeding experi- ments with Drosophila before the chromosomes could be directly examined in polytenc nuclei (that is, before 1933). Allelomorphs of the gene scute affect the number of bristles on the thorax and scutellum, and several of them are due to inversions of various lengths, following breakage by X-rays. The Pale gene likewise goes with a spontaneous translocation from the second to the third chromosome and is probably just due to the change of position. The scute mutations are simple recessives having no special effect on the hcterozygote ; the Pale gene is lethal when homozygous and varies in its effect on the heterozygote. Another side of the picture is shown by the fact that a large proportion of inversions, which have arisen spontaneously as well as by X-raying in laboratory cultures of Drosophila, are lethal in the homozygous state, while some of them are invariable concomi- tants of visible mutations like Curly and Plum. It thus seems that the action of one gene may be modified, directly or indirectly, by its neighbours. And, when it changes its neighbours, it does not necessarily work as it did before. Either the shapes of the chromo- some constituents themselves, or at least of their products inside the nucleus depend on their linear arrangement. To this extent whole segments, or even whole chromosomes, must be considered as units of action in physiology just as they are units of structure, single giant moleailes, in chemistry. 106 INTERGENIC AND INTRAGENIC CHANGE Changes of balance likewise mimic gene differences in Dwsophila. Notched wings and Minute bristles are produced by deficiencies. They can usually be recognized in two ways : by the absence of from one to ten bands from the polytene chromosomes, and by the absence of the allelomorphs of particular genes as shown by the failure to "cover" a recessive in breeding experiments. Some 70 varieties of Notch and 80 of Minute, both spontaneous and induced, have been described; all the Notches are at the end of the X chromo- some while the Minutes may lie in any of the different chromosomes. Minutes seem to be lethal when homozygous; Notches are lethal to the male (which has no allelomorph to them in the Y) and they camiot, therefore, be obtained homozygous in the female. All these deficiencies are dominant (for which reason they are given capital letters in the Drosophila usage*) and are known by the phenotype they give in the heterozygotc. Thus a deficiency is recognizable in breeding by its drastic effect and its segregation as an allelomorph of a group of genes. In these two respects duplication is similar, but of course its effect is less drastic and its relation to other genes is the reverse. Deficiency "uncovers" a recessive allelomorph. Duplication "covers" even a homozygous recessive. Duplications also resemble deficiencies in usually affecting the bristles and the wings. The most interesting of them, however, is the narrow-eye or Bar factor in Drosophila (Fig. 21). For 25 years Bar was regarded as a simple gene-difference in the X chromosome. It was odd merely in its property of unequal crossing-over. Homozygous Bar females sometimes gave progeny in which the Bar gene was replaced either by the normal allelomorph, or by a new one of greater strength (giving still smaller eyes) called Double-Bar. Finally the polytene chromosomes showed Bar to have two like pieces side by side instead of one, as it were ABBC instead of ABC. Then unequal crossing-over in the homozygote ABBC/ ABBC reconstructs the chromosomes, so as to give the types ABC and ABBBC detectable genetically and cytologically in the next generation. A remarkable incidental observation in these experiments was that the heterozygous combination, ABCjABBBC, although the same * Sec Appendix 2, on Symbols and Symbolism. 107 BAStS 01= CHANGE ALLELO- MORPH Wild -Type (+) Bar (B) Double- Bar (BB) SALIVARY CHROMOSOME REDUPLI. CATION ORIGIN UNEQUAL CROSSING OVER Average Number of Facets Per Eye in Females Males PHENO- TYPES BB/+45^56-f-/B 25 36\ 68 BB/BB BB/B V B/B + 738 B 91 BB 29 THESE TWO TYPES HAVE THE SAME GENIC CONTENT. THE PHENOTYPIC DIFFERENCE IS DUE TO DIFFERENCE IN ARRANGEMENT The Position Effect Fig. 21. — The Bar gene, which reduces the size of the eye in Drosophila mclanogastcr , is itself a duphcation for a segment of the X clironiosonic containing at least tour distuict bands in the salivary chromosome. Unequal crossing-over in females homozygous for the Bar duplication leads to triplication of the segment, which thus gives the enlianccd eftcxt described as l^ouble Bar. Females heterozygous for the wild type chromosome and Double Bar have the same total gene content as females homozygous for Bar, though the genes are distributed differently between the chromosomes. The eyes of these two classes of fly have different average sizes, showing that the arrangement of the genes in the chromosomes has its effect on their action. This is the Position Effect. 108 INTERCLNIC AND INTRAGENIC CHANGE in total B content, has more Bar effect than docs ABBC/ABBC. Its eyes have fewer facets. Evidently the B's are disproportionately effective when they are concentrated on one chromosome. The effect of their unbalance is not additive but multiplicative. This is another and special example of the position effect, and indeed it was the first that could be proved, since it could be done and undone so readily by crossing-over. Thus simple additions, subtractions, and dislocations of genes produce unit hereditary effects. At one time Bateson considered that plus and minus differences, due to mere presence and absence of determinants, were the essence of all hereditary change, the presence being dominant to the absence. This led to a difficulty because mere loss (or even perhaps gain) could not ultimately produce anything new. Unpacking requires a previous packing. Furthermore, crucial evidence against Bateson's view is that the two allelomorphs of many genes (like white eye and red eye in Drosopliila) can each mutate to the other. Change has often been observed in this way in both directions ; and where it has been seen only in one, that is doubtless sonietimes due to the frequency being low, and lower one way than the other. Yet the absence could hardly be expected to mutate to the presence. Unless, like the normal allelomorph of Bar, it is only half an absence. Finally, as we have seen, the absence, the deficiency, can be at least partly dominant. It is not, therefore, surprising that when most mendelian hetero- zygotes are examined, for example, at pachytene in maize or at polytene in Drosopliila, we find no visible differences between their pairing chromosomes. Both dominant and recessive thus prove to be present. There is evidently a residual class of differences which cannot be shown to be due to structural change. Ultimately, of course, we must assume that all changes are structural in the sense of being changes in number or arrangement of some constituents. Since a linear order has been established for the genes, changes between genes must always be longitudinal. We can still suppose, however, that genes have additional dimensions, and that lateral change occurs within them. To put the argument in another way, the occurrence of a second kind of change other than one of mere arrangement is inevitable. Arrangement of units has no meaning unless there are differences 109 BASES OF CHANGE between the units to rearrange, differences such as we actually see bet\vecn the parts of the cliromosomes. These differences between units cannot, therefore, have arisen by the changes of arrangement themselves. Now crossing-over is a change in arrangement between genes. Changes within genes should, therefore, have no direct effects on their own crossing-over. Many genes, in fact, have an indirect or physio- logical effect on the process of chromosome pairing and on the observed recombination. For example, a so-called asynaptic gene often (in maize, pea, or Drosophila) produces a general reduction in the crossing-over of all the chromosome pairs when homozygous by causing incomplete pairing with results that we shall see later. But of direct effects produced in the heterozygote these residual intragenic mutations have none. They are apparently changes in indivisible points or loci on the linkage map, and are accordingly known as point mutations. Somatic Mutation Instances are known, in Antirrhinum and elsewhere, of two allelomorphic genes or two homologous chromosomes mutating in the cell simultaneously to the same new allelomorphs. Such events suggest a chemical determinacy in the mutation even if they arise in two steps. These instances are, however, very rare and it is a remarkable fact, or even principle, that allelomorphs are (with these few exceptions) independent in their mutations. The original form of any change or mutation in the body o{ a zygote must therefore be a heterozygote. And the original form of any hetero- zygote must be a mutation. Only later can the heterozygote appear as we characteristically fmd it, that is as a cross between two homozygotes, as a hybrid in the restricted pre-mendelian sense. It is also worth noting that most mutations occur somatically, that is in the nuclei of a growing body. Hence the new type of cell is not merely heterozygous. It is also incomplete so far as the body is concerned : it is a patch. In plants where the germinal tissue is not separated early in development a change may or may not affect the no SOMATIC MUTATION germ layer. In any case only one layer is changed and hence gives rise to a chimaera with, mixed tissues: a layer of mutant lies inside or outside a layer of the unchanged type. Chimaeras can be produced artificially as graft-hybrids or by any treatment which alters the chromosomes. Most old vegetatively i Fig. 22. — Leaves and fruits of 2x Solatium sisymbrifolium {.<), 6x S. nigrum (/;) and the graft-hybrid or chimaera with one layer of ^ over n which sorts out to give pure nigrum in parts of its leaves (after Jorgensen and Crane, 1927). propagated plants such as pelargoniums and potatoes have become chimaeras owing to somatic mutation at some time in their history. When they are propagated from root-cuttings (or from disbudded tubers) shoots grow out of the concealed irmer layers which reveal the chimerical structure and the ancient mutations of these plants. The elucidation of these properties by Baur, Bateson, Winkler and Crane has enabled us to understand that natural changes in the arrangement of tissues lead to the repeated appearance of "sports" III BASES OF CHANGH in chimaeras which themselves have arisen from a single original mutation {cf. Fig. 22). In animals the effects of somatic mutation are slightly different. Development and life being limited, the mixture is not a permanent one. Development being less simple the mixture is also less regular. As a result, the changed cells give flakes and sectors instead of layers Fig. 23. — Gynandromorph of Drosophila melanogastcr. The left side is XX and female. One X carrying the dominant gene Notch, whose effect appears in the left wing, has been lost on the right side which is therefore male and shows the effects of the recessive genes of the other X, viz. ruby (eye colour), scute (bristle reduction), broad (wing), and forked (bristle gnarling). The whole fly is slightly warped owing to the male side being shorter than the female (from Morgan, Bridges and Sturtevant, 1925). and the product is known as a mosaic instead of a chimaera. There is, however, one regular type of mixture of particular interest and this is the gynandromorph. Such monsters, male in one part and female in the rest, arise from a genetic difference between a pair of nuclei produced at an early mitosis in the embryo. They give various mixtures according to the stages and relative positions of the mitoses, but perhaps the commonest is the half-sidcr of the type shown in Fig. 23. They come about in a variety of ways as can be proved by genetic as well as by cytological evidence (Fig. 24). 1J2 ORIGINS OF GYNANDROMORPHS Fig. 24.--Diagram showing the modes of origin of gynandromorphs in different insects directly observed in the egg or inferred from the genetic character of the two sides of the body which are of opposite sex. In Bombyx (after Goldschmidt and Katsuki, 193 1) two sperm may fertilize the two dissimilar products of the second meiotic division in the XY female. In Habrobracon (after Whiting, 1943) a sperm may fertilize one of the products of the second meiotic division to give a diploid temale side, a haploid and parthcnogenetic male side being derived from the other product. More rarely a second sperm may cleave side by side with the fertilized egg. In Drosophila one of the two X's in a fertilized female egg may be lost at the first cleavage division to give an XO side which is morphologically male (after Morgan, Bridges and Sturtevant, 1925). More rarely two sperms, one X- and the other Y-bearing, may fertilize the two first products of cleavage to give a perfect gynandromorph. Thus all the different irregularities occur which will give workable results in each organism. LkmcnU of Gaieties 113 II BASES or CHANGIi The Gene as a Unit oj Cliaiigc How can wc find out something about what is inside that point, the gene ? The evidence of the visible or breakable structure of the chromosome, or of the linkage map inferred from the breeding experiments, clearly no longer helps us. We have gone beyond the limits of resolution by such means. Only by the study ot action and of changes in action, that is of mutation, can we get any further light on the matter. At most loci in the mapped chromosomes of maize or man, Drosophila or Oenothera, we have discovered only two allelomorphs of recognizably different effect. In addition to such simple allelo- morphs, however, particular loci in scores of species show whole series of alternatives, multiple allelomorphs as they are called. In many such series the effect of the mutation of one allelomorph into another has been seen in experiment. Bar eye is an extreme example, where we have crossed the boundary into structural change. Most of the differences, however, are intra-gcnic. Multiple allelomorphs are of particular importance when they control the breeding systems of plants. One set or series then have to be closely related in their actions, in order, as we shall see later, to control the growth of the pollen down the style. This physiolog- ical relationship is also seen in other series such as those at the white- eye locus in Drosophila. Here, a number of allelomorphs produce a range of intensity of pigmentation between none at all and the full red of the dominant wild type. A dozen have been recorded and there may well be more grades, and therefore more allelomorphs, than have yet been separated by eye. Evidently all the allelomorphs arc doing the same thing but to different degrees. One unit of action therefore suffices to account for the range of behaviour and mutation of this gene, although the range of its mutants shows the complexity of its parts. Not all multiple allelomorphs, however, are so simple. A series of four in Priinida sinensis includes three which affect only the size of the greenish "eye" round the mouth of the corolla tube; the fourth, which is recessive to all the rest, has the same effect on the eye as the next most recessive but, in addition, shortens the style. The series may be arranged, therefore, as in Table ii. 114 THE CENt AS A UNIT OF CHANGE TABLE 11 FOUR ALLELOMORPHS GOVERNING SIZE OF EYE IN FLOWERS OF PRIMULA SINENSIS Allelomorph Symbol Effect Alexandra Original type . . New type (1938) Primrose Queen A' A a" a No eye and normal style Normal eye and normal style Large eye and normal style Large eye and short style Two interpretations are clearly possible. Either (i) a has two effects independent from the beginning and one of them distinct from that shown by any of the others, even the otherwise indistinguishable a". This means two units of action, separable in their changes, but inseparable in recombination, so far as our experience goes. For if short-style could occur with normal eye we have not yet recog- nized it. Or (ii) a" does the same thing as a but does it more effectively, thereby passing a threshold which introduces a new or secondary ultimate effect, the visible change in style length. This means one unit of action as well as of recombination, but supposes a relation in development between eye and style of which we other- wise know nothing. The distinction betw^een these alternatives is thus as follows: Complete Linkage Two genes having distinct initial effects but inseparable by recombination. Pleiotropic Action One gene having a single initial effect with manifold expression. The choice between these alternatives is important wherever manifold expression is concerned, whether multiple allelomorphs are present or not. In every case it must depend on our knowledge of the whole series of steps from the gene to its expression. This knowledge is perhaps available in certain simple cases. In other more complicated cases we must turn to new ways of attacking the prob- lem. If we can show that the series of steps that we do know can be traced back to one unit action, a single gene of manifold effect must be at work. The tracing of gene action has been done in two ways. Embryo- 115 BASI'.S ()!• CIlANCr. logically, Griineberg has shown, for example, the common and simple origin of a number o{ defects occurring together in the rat. Distortion of the blood system and lungs, blockage of the nostrils, inability to suckle and deformity of the bones of the forelegs, all Slight changes in Larynx and Nose GENE Anomaly of Cartilage Thickened Ribs Spur on Deltoid Ridge of . * . Humerus _ Fixation of Thorax in Inspiration Fixation of Thoracic Vertebrae. Displacement of Thoracic Viscera Dilation of Lung Cavities and Passages / \ Increased Resistance Arrest of Pevelopment Slow Suffocation in Pulmonary Circulation J Blocked Nostrils / 7 Blunt Snout I / Compensatory Overgrowth I Inability to Suckle I of Right Ventricle Faulty cutting of I Coma ^ \, Incisor Teeth | Capillary Bleeding Heart 1 Starvation Fig. 25. — The piciotropic effect of the grey-lethal gene in the rat (after Griineberg, 1938). these arise during development from a single abnormality in the formation of the cartilage. The gene is single and pleiotropic (Fig. 25). Genetically, we can get the evidence from the control of dominance and the effects of other genes in modifying the action of the one we are chiefly concerned with. The agouti series in the mouse is a case in point. n6 THE GENE AS A UNIT OF CHANGE The common type of wild mouse has entirely agouti fur; each hair is black with a yellow band near the tip. Some wild mice are agouti with light bellies, and in fancy mice there are corresponding types, a plain black and black with light belly (black and tan). Finally, there is an all-yellow type in the fancy. All these types behave as though governed by a multiple allelomorph series. Yellow stands by itself: the homozygote is never born alive, so that all yellow mice are heterozygous. It is like the Notch effect in DrosopJiila and, like it too, may well be due to a deficiency. The four other types evidently consist of combinations of light or dark belly with agouti or non-agouti (black). We might, therefore, again say that they were due to the combination of two gene differences completely linked in all known experiments. The evidence for the two-gene view is better in the mouse than in Primula, for all four combinations are found. Furthermore, how- ever they are combined, light belly is always dominant to dark belly, and agouti hair to non-agouti hair. Thus the mouse hetero- zygous for agouti and black-and-tan looks just the same as a light-bellied agouti. Finally, there is another gene which has the effect of reducing the dominance of agouti over non-agouti and this gene has no effect on the dominance of light over dark belly. Hence the dominance relations accord with the two-gene view. A similar two-gene explanation may be given for the dumpy multiple allelomorph series affecting the development of wings and hairs in DrosopJiila. But here the two possibly independent com- ponents are afiected in the same way by the same dominance modifiers. How then are we to resolve the question of one gene or two ? The answer is that it often cannot be resolved. What behave as two units of recombination under one set of conditions may, it seems, behave as one under others. Suppression or even reduction of the frequency of crossing-over, either general or local, may have this effect. Such a structural change as the inversion of a couple of genes, will, as we shall see, prevent crossing-over, or at least effective recombination, between them except when the two genes are homozygous and the recombination is, therefore, ineffective with respect to these two genes. The absence of crossing-over in the agouti group might be due merely to an inversion which constantly 117 BASES or CHANGli distinguishes all agouti from all non-agouti or all dark-bellies from all light-bellies. Fisher has suggested that an inversion is associated with the agouti difference (Fig. 26) and is putting this view to an experimental test. 1 s ^ 111 UJ \- (rt > l/> ut Z 10 UJ 0 Z 0 LU 5 n ►- 1 1 lU a 3 in iij z 0 AGOUTI LIGHT BELLY H j-H ^ -O Wild type, U.S.A. AGOUTI dark belly -HH -—& -OVVild type. G.B. non-agouti LIGHT BELLY { O ^^o^ £ tan non-agouti dark belly ~ 1 L -L .1 U L. U I I .1 -L _ I " ■ I 1 ' !"■"" — ■-- I' -OAII black i INVERSION /- LIGHT BELLY non-aqouti I-H h non-aqouti H Z Y dark belly -X Black I tan -X All black DEF/CIENCY Yellow Fig. 26. — The possible rcLition of structural changes to gene recombination in the Agouti scries of the mouse, where four allelomorphs represent all recombinations of two ditferences between which recombuiation has not, however, been observed. The differences could be interpreted as two gene ditferences so closely linked as never to recombine (two-gene system). Or one of them could be regarded as associated with an inversion inhibiting recombination (one super-gene system). Further, it might be that what behave as two units of action under one set ot external conditions, or in one set of internal relations, behave as one unit of action under others. Indeed the position-effect has already shown us this in another way. Finally, it seems that recombination may be mistaken for mutation if it is rare enough. This we have seen already at the Bar locus and wc shall see it again in Oenothera. iS THE GENr. AS A UNIT OT CHANGE All these residual or minimal units of genetic structure are therefore conditional and there is no reason why they should be proved to correspond in regard to action and change in different experiments. Unless, that is, the unit of action is so built up that any change of its structure whatever has the same effect; then, of course, all its mutations would be alike and no recombination would be detectable within it. The genetical unit into which we divide the chromosome, there- fore, depends on our experimental technique or our theoretical purpose. The cytological unit must also depend on the wave-length of light used in the resolution of chromomeres. In ultra-violet photo- graphs of polytene bands in Drosophila we must be near to an ultimate analysis, for Muller and Prokofieva have found them to agree with the limits of X-ray breakage. This correspondence, however, is not so important as the general principle that the chromosome consists of a linear arrangement of particles which are frequently, or even usually, different from their neighbours in structure, in action, and in capacity for change. But though different, they are not wholly independent, for they have to move and to work together. The question of how they move and work together is on the border-line of chemistry and to answer it we shall have to get down to the chemical level of approach. Meanwhile we can admit that the visible chromomeres of the cytologist, and the genes as units of crossing-over and mutation of the geneticist, agree to a first approximation. They are the ultimate units of structure, change, and action in the chromosome and in heredity. REFERENCES BATTAGLiA, E. 1947- La "Seniigamia," singolare comportamcnto del nucleo spermatico nelle uova diploidi delle specie apomittiche del genere Rudheckia (Asteraceae) e consequente embriogenesi di tipo chimerico. Rendiconti Acad. Naz. Lined, Ser. 8, 2: 63-67. BRIDGES, c. B. 1936. The bar "gene" a duplication. Science, 83: 210-211. DARLINGTON, c. D. 1940. The origin of iso-chromosomes. J. Genet., 39: 351-361. DARLINGTON, c. D., and ROLLER, P. C. 1947. The chemical breakage of chromosomes. Heredity, i: 187-222. DARLINGTON, c. D., and LA COUR, L. F. 1945. Chromosome breakage and the nucleic acid cycle./. Genet., 46: 180-267. TT9 BASES or CHANCE FANKHAUSi-R, G. 1941. The frequency of polyploidy, etc., in the newt. Proc. Nat. Acad. Sci. Wash., 27: 507-512. GOLDSCHMIDT, R., and KATSUKi, K. 1931. Vicrtc Mitccilung iibcr erblichcn GvTian- dromorphisnius und somatischc Mosaikbildung bci Boinhyx mori L. Biol. Zent., 51: 58-74. GRUNEBERG, H. 193 8. -An analysis of the "plciotropic" effects of a new lethal mutation in the rat {Mhs twrvcqicus). Proc. Roy. Soc. B., 125: 123-143. J0RGENSEN, c. A., and CRANE, M. B. 1927. Formation and morphology of Solanum chimacras. J. Genet., 18: 247-273. MORGAN, T. H., BRIDGES, c. B., and STURTEVANT, A. H. 1925. The genetics of Drosophiia. Bihliogr. Genet., 2: 1-262. MULLER, H. J., and PROKOFYEVA, A. A. 1935- Thc individual gene in relation to the chromomcre and the chromosome. Proc. Nat. Acad. Sci. Wash., 21: 16-26. RICK, c. M. 1945. A survey of cytogenetic causes of unfruitfulncss in the tomato. Genetics, 30: 347-362. STURTEVANT, A. H. 1928. A further study of the so-called mutation at the bar locus o£ Drosophiia. Genetics, 13: 401-409. WHITING, P. w. 1943. Androgenesis in the parasitic wasp Hahrohracon. J. Hered., 34: 355-366. 120 CHAPTER 6 CONSEQUENCES OF CHANGE Meiosis and Fertility in Polyploids Structural Hyhridity Units of Recombination Allopolyploidy The Integration of Differences The different polyploid and polysomic types, and such of the new structural types as are viable, behave normally at mitosis. Their vegetative life follows an even course. At meiosis, in their germ cell formation, however, we fmd new types of behaviour which lead to abnormalities of breeding and the loss of fertility. Meiosis and Fertility in Polyploids. Triploids, or trisomies which are merely triploid for one chromo- some, show the principles governing all abnormalities of meiosis, and indeed governing meiosis itself There are three chromosomes of each type capable of pairing with one another throughout their length at prophase. In the polytene nuclei of a triploid Drosophila this expectation is fulfilled. But in all triploids, at the prophase of meiosis, association is limited to two. One is left out, but this is usually a different one at different places; the chromosomes make contact in pairs at random either near the ends or near the centro- meres; each association runs along the chromosomes until it meets another and they consequently exchange partners according to the chances of their original contacts. Crossing-over takes place and chiasmata are consequently formed which hold together the paired chromosomes at the points of crossing-over. If all three chromosomes have been paired, and if one has formed chiasmata with both the other two, an association of three, a trivalent, is held together until metaphase. If one fails to cross-over with either of the other two it is left out completely, a univalent, while the other two form a bivalent (Fig, 27). At first metaphase in triploids the bivalents arrange themselves on the plate, and the trivalents also with their centromeres either 121 CONSEQUENCES OF CHANGE 122 MEIOSIS AND lEUTILITY IN POLYPLOIDS in one line or in a convergent V. With convergence tv^o cliromo- somes go to one pole and one to the other; with linearity the middle chromosome may go to either pole or it may be left behind and act as though it had been unpaired. Univalents either divide late at the first division and their daughters lag and arc lost in the cytoplasm at the second; or they lag and are lost at the first; or again they are included without division in one of the daughter nuclei at the end of the first division as though they were daughter bivalents; or even, as we saw, they may misdivide. TABLE 12 A. CHROMOSOMES OF POLLEN GRAINS IN SAMPLES TAKEN UNDER FAVOURABLE (F — 502 GRAINS), AND LATE SEASON OR UNFAVOURABLE (U — 116 GRAINS) CONDITIONS, FROM TRIPLOID CREPIS CAPILLARIS Ox = 9). (FROM DATA OF CHUKSANOVA, 1939) Percentage of pollen grains Number of chromosomes Percentage 3 4 5 6 Mean grains Expected 12-5 37-5 37-5 12-5 4-50 25-0 F 17-2 38-6 37-8 6-4 4-33 11-1% 23-6 U 32-6 23-4 28-4 15-6 4-11 15-5% 48-2 B. PROPORTION OF THE EXTRA FREQUENCY PER GRAIN (0-5) ACTUALLY FOUND FOR THE THREE EXTRA CHROMOSOMES, A, C AND D Chromosome + D + A + c Mean Loss F U 100 0-89 0-88 1-02 0-79 0-62 0-89 0-84 16% Weighted Mean 0-98 0-90 0-76 0-88 — Loss 2% 10% 24% 12% — Note: "Loss" is the deficiency of the mean with respect to the binomial ex- pectation. As in Hyacinthus, it is highest for the shortest chromosome because this forms fewest chiasmata and therefore fewest trivalects. As a rule the different trivalents orientate themselves at random with respect to one another on the fi.rst m.etaphase spindle. In con- T-3 CONSEQUENCES OF CHANGE sequence the distribution of the odd set is a binomial one with halt the triploid number expected to be the most frequent type. The mean and the mode, however, are usually below 3X/2 on account of the loss of a varying number of chromosomes, usually 10 or 20 per cent. The loss is, of course, highest in species with the x=3 -y<^ ADC x+A ^J^ +c vf -D -C 2x = 6 AADDCC Fig. 28. — Mitosis in types of balanced and unbalanced pollen grain produced by a 3.V Crcpis capillaris in the proportions shown in Tabic 12 (after Chuksanova, 1939). smallest chromosomes, and for the smallest chromosomes in a set, since these have the fewest changes of partner, the fewest chiasmata, and therefore the most failure of pairing. Loss is also higher in the larger embryo-sac mother-cell than in the smaller pollen mother- cell. The course of events at mciosis in triploids is reflected in the frequencies of the pollen grains having different chromosome num- bers (Fig. 28 and Table 12). At mitosis in the pollen grains of Crepis capillaris each of the three chromosomes can be recognized. 124 MIIOSIS AND riiRTILITY IN POLYPLOIDS In the triploid the numbers of grains with an extra one of each kind can be recorded. The average number of chromosomes should be 4*5, but is always less owing to loss of unpaired chromosomes at meiosis (Table i2a). Samples also differ owing to the fact that the balanced grains develop more quickly than the unbalanced ones and some chromosomes even differ from others in their effect on balance and development (Table I2b). TABLE 13 PERCENTAGE DISTRIBUTION OF EXTRA CHROMOSOMES AS SEEN IN THE POLLEN GRAINS (P.G.) AND IN THE PROGENY (3>: X 2x AND 2;c x 3a:) OF TULIPA, WITH LARGE, AND OF DATURA, WITH SMALL CHROMO- SOMES. THE NUMBER OF OBSERVATIONS IS GIVEN IN PARENTHESIS IN THE FIRST COLUMN. (UPCOTT AND PHILP, 1939) No. of chromosomes 0 1 0 3 4 5 6 7 8 9 10 11 12 Mean Tulipa P.G. (100) 3jc X 2x (50) 2x X 3x (25) 20 40 3 14 32 12 9 10 22 14 19 16 20 6 14 10 6 4 9 4 6 4 2 4 — 6-17 5-04 1-90 Datura P.G. (500) 3x X 2x (285) 2x X 3x (7) 3 4 20 48 86 14 7 28 11 15 4 — 16 11 11 9 5 4 3 1 5-35 I- 15 0-14 Note: The basic number for both is 12, so that the mean number of extra chromosomes without loss would be 6. What happens to the pollen and eggs with different numbers is shown by the frequencies of plants with correspondingly different numbers when these pollen grains and eggs are united with haploid gametes, that is to say, in reciprocal crosses with diploids (Tabic 13). We then fmd that there is a reduction of the types with the middle numbers (which are the most unbalanced) and with the higher numbers (which are most unlike the gametes of the diploid parent). The elimination of unfavoured types is much stronger when it is the triploid parent which is providing the pollen. Evidently the pollen is subjected to a stricter test than the eggs. The reason for this might be that the numbers of pollen grains on the stigma are always greater than the number of ovules to be fertiUzed. Or it might be 125 CONSLQUENCES OT CIIANCr. that a proporiioii of unbalanced types which can survive as eggs if fertilized are unable to grow dowm the style at all as pollen grains. This is evidently the case, for the fertility of triploids is actually lower as pollen than as egg parents (Table 14). TABLE 14 THE FERTILITY OF RECIPROCAL CROSSES OF DIPLOIDS AND TRIPLOIDS, SHOWING THAT IT IS LOWER WHEN THE TRIPLOID IS THE POLLEN PARENT, AND ALSO IN PLANTS WITH HIGHER BASIC NUMBERS OF CHROMOSOMES. THE DATA ARE EXPRESSED AS PER- CENTAGES OF THE INFERRED NORMAL SEED SET. (UPCOTT AND PHILP, 1939) Species X 3.1c X 2.x Ripe Germin- Sur- seeds ated vived 2.x X 3x Ripe Germin- Sur- seeds ated vi\ed Campanula persicifoUa Zea mays Tiilipa gesneiiana Datura stramonium . . Primula sinensis Pyrus malus 8 10 12 12 12 17 23-3 2-5 — 140 5-3 — 19-6 7-8 2-7 4.4 4. J 2-9 006 003 0-00 215 1-40 1-25 9-8 J-8 — 6-8 4-4 — 5-5 2-8 1-0 _ — 1-2 019 000 000 0-29 0-19 013 — indicates that no d;ita are available. There is another interesting sequel to the random segregation of extra chromosomes and the selection of balanced types in triploids. As the basic number increases, the proportion of haploid gametes remains always about J\ Therefore fertility declines with increasing basic number. It is high in Drosophila or Crcpis, low in Pyrus (Table 14). ^ u • • 1 Tetraploids behave as we should expect on these principles (Fig. 27). The chromosomes pair, that is each member of the set forms two pairs; but they pair differently at different points and therefore have to change partners as they do in triploids. Their association at pachytene then resembles diplotene in a diploid. Where they arc paired they may form chiasmata. Hence associations of four appear at metaphase wherever changes of partner and chiasmata permit. Failing such quadrivalents we find pairs of bivalents or trivalcnts and univalents. The quadrivalents arrange themselves on the spindle at metaphase, like the trivalents, either in a line or zig-zag, that is T26 MEIOSIS AND I-LRTILITY IN POLYPLOIDS convcrgently. Or, like two bivalents, they can lie parallel in a rectangle. Again, middle members of these configurations can lag and be lost so that separation may be two-and-two or three-and-one or two-and-onc with one lost. The products of meiosis are thus sometimes equal in their numbers of chromosomes, but perhaps equally often they show an error of one or two above or below. This error is what reduces the fertility of tetraploids, but the reduction is o£ the order of one quarter or less, not as with triploids ot the order of one half, for each chromosome of the basic set. We learn several new principles from these observations. Li the first place, we see that the triploid is a numerical hybrid. It has two nuclear units from one side and only one from the other. Like other hybrids or heterozygotes it is incapable of breeding true and even its loss of fertility, as we shall find later, is characteristic of hybrids which arise from crossing species. The genetic system of the triploid is adjusted until it comes to the production of germ cells. It then breaks down. It does so because at meiosis new individuals, new self-propagating systems, with new and untried combinations of chromosomes are brought into being. Unbalance, which appears in mature plants and animals as the cause of abnormal forms, appears in immature individuals as the cause of death. The fertility of the parent is the inverse measure of the frequency of death of the offspring. The segregation of differences at meiosis is thus a prime cause of sterilitv. Secondly, we see that development follows a standard course in organisms with the standard set of chromosomes equally multiplied. But where the multiplication is unbalanced, development is unbalanced and disturbed. Evidently all the cliromosomes are different in their effects on growth, just as they and their parts are different in their specific attractions for one another at meiosis. This disturbance accounts for the shghtly reduced fertility of trisomies and of tetraploids, whose segregation is slightly irre^^ular, and for the greatly reduced fertihty of triploids, whose segregation is grossly irregular. An exception proves this comiexion between balance and fertility. In triploid hyacinths, HyaciiitJms orientalis, fertihty is not greatly reduced, and plants with all intermediate numbers between diploid and tetraploid appear in their progeny. The development 127 CONSLQUENCl:S OF CHANGl: of these plants is not disturbed too much to prevent them providing some of our best garden varieties. Thus in this species the chromo- somes are less sharply differentiated than usual. Or, to put it the other way round, each chromosome is balanced in much the same way as the chromosome set in the aggregate is balanced. The fertility of the triploid and the normality of the trisomic follow from this unusual condition. Structural Hyhridity The product of structural change in the chromosomes, like the product of numerical change, reveals its peculiar properties at meiosis. We then see that it is a structural hybrid, an individual UnchanjeS Chromosomes Clirs. af mitosis after Pairing of two inferclian^eS Intercnan^chif DKaUage Vtwo unchanged Chrs. VRcunlon of Chromatids aff^e ?hano-' brossiea <- ■< In crccisi "A >■ -> Ferftlify Fig. 35. — Pairing at mciosis in diploids of different degrees of hybridity and scgregational sterility compared with that in the tetraploids derived from them: to illustrate the law of the negative correlation of fertility in diploid and tetraploid. them for new conditions, fit them for colonization. Occasionally they segregate differences by accidental pairing of chromosomes from the ultimate diploid parents and this also gives them adaptability. But yet they remain highly fertile. It is not surprising, therefore, that a large proportion of the species of flowering plants owe their origin to alloploidy, and more especially a large proportion of the new types of plants that have covered the cultivated part of the earth in the last 10,000 years {cf., Fig. 82). 140 THE INTEGRATION OF DIFFERENCES The Integration of Differences We have seen that the hereditary materials, the chromosome com- plement contained within the nucleus, can undergo changes of three types, in number, in structure, and in a residual class of point mutations. The distinction between the structural, or inter-genic, and the intra-genic changes follows inevitably in theory from the fact that the chromosome is a linear arrangement of dissimilar particles. But it cannot always be made in practice owing to the fact that the two types can be combined in one integrated difference. We have seen that the combination of the dissimilar chromosomes and parts of chromosomes in a working set constitutes balance. The primary changes that occur in their numbers and in the arrangement of their parts do not upset this balance in vegetative life. But when meiosis takes place these primary changes are broken up and recom- bined so as to produce secondary changes with new kinds of balance. Many of these new combinations fail to work. The gametes and zygotes carrying them die and fertility is reduced. We have also seen how structural hybridity will reduce chromo- some pairing, crossing-over and recombination, or even abolish them altogether, and in so doing remedy the infertility of a polyploid on the one hand and of a gene hybrid on the other. Hence all three types of change, genie, structural and numerical are concerned with one another in permitting the principle of variation to operate in a system of heredity. REFERENCES CHUKSANOVA, N. 1939. Karyotypes of pollen grains in triploid Crcpis capilUnis. Coup. Ranis. [Dokhidy) Acad. Sci. U.S.S.R., 25: 232-235. DARLINGTON, c. D. 1936. The limitation of crossing-over in Oenothera. J. Genet., 32: 343-352. DARLINGTON, c. D. 1940. The prime variables of meiosis. Biol. Rev., 15: 307-322. DARLINGTON, c. D. and GALRDNER, A. E. 193?. The Variation system in Campanula. J. Genet., 35: 97-128. DARLINGTON, c. D., and MATHER, K. 1944. Cliromosome balance and interaction in Hyacinthus. J. Genet., 46: 52-61. KARPECHEKKO, c. D. 1927. Polyploid hybrids of Raphaniis scitivus L. X Brasska oleracea L. B. Ap. Botany, 17, 305-408. 141 CONSEQUENCES OF CHANGE PATAU, K. 1935. Chromosonicnmorphologie bci Drosophila mclanogastcr und Drosophila siinulans und ihre Bedeutung. Naturwiss., 23: 537-543- UPCOTT, M. 1936. The parents and progeny o( Acsculus camca.J. Genet., 33 : 135-149. UPCOTT, M. 1939. The nature of tetraploidy in Primula kcwensis. J. Genet., 39: 79-100. UPCOTT, M., and philp, j. 1939. The genetic structure of Tulipa. IV Balance, selection and fertility. _/. Genet., 38: 91-123. 142 PART II CELLS CHAPTER 7 GENES, MOLECULES AND PROCESSES Chromosome Structure Chromomeres and Proteins HctcrocJiromatin and Polygenes What Major Genes do The Interaction of Genes The Expression oj Genes Synthetic Seq^uences: Supply and Demand We have learnt something of the resemblances and differences that occur between parents and their offspring, and of the materials and processes which are ultimately responsible for these relationships. But we also have to discover how these materials, lying in the cells of animals and plants, are so organized that they determine the effects we observe, and by what steps they do so. How does the cell nucleus with its outfit of genes and chromosomes act on the cell with the constancy that we recognize in heredity; repeating in each generation the elaborate processes of development which seem to contradict all possibility of a constant basis ? What part does the cytoplasm play in this paradox ; To these questions we must now address ourselves. Chromosome Structure The nucleus, as we have seen, is a body constituted of chromo- somes. The chromosome is a body constituted of a protein fibre to which active groups or genes are attached. Pepsin disintegrates this fibre and it must therefore be based on a polypeptide chain. This chain may be single or multiple but it must be highly folded in the ordinary resting nucleus since, when the nucleus grows (as in the salivary glands), the chain itself stretches to many times its early prophase length. During mitosis, nucleic acid is attached to the genes which, owing to the folding of the thread between them, form an almost con- tinuous chain. The nucleic acid consists of an unlimited polymeriza- tion of desoxyribose nucleotides to form a colunm. The nucleotides are spaced at the same distance of 3 • 3 Angstrom units apart as are the repeats in the stretched polypeptide chain. Desoxyribose nucleic Elements of Genetics 145 K GENES, MOLECULES AND PROCESSES acid is tound only attaciicd to chroinosonics and in bacteria and certain animal and bacterial viruses. In all of these it must indicate the presence of genetically analogous structures. At low temperatures in plants, or with low feeding in animals, the chromosomes can be starved of nucleic acid. Two consequences follow. First the chromosomes, especially their end genes, fail to reproduce, or at least to separate the products of their reproduction, in mitosis. The daughter chromosomes consequently sometimes stick together at anaphase. Secondly, certain short segments of the chromosomes are undercharged and therefore fail to stain. In extreme cases the coiling or spiralization of the chromosome thread fails. It remains uncoiled at metaphase. Such a failure of coiling is usual during rapid mitoses in certain protozoa but would be highly inconvenient in the specialized cells of higher organisms if it affected the whole chromosomes. Thus the attachment of nucleic acid regulates the reproduction of the chromosomes, possibly, as Astbury suggests, by acting as a template. It also locks them up in spiral bundles; and these are not merely neat bundles for movement and distribution. They are strong bundles protected against the dangers of extra-nuclear life, even, as experiment shows, against being broken by X-ray ionization or chemical poisons. The uncoiling of the chromosomes at telophase depends on their throwing off this coat of nucleic acid. How do they do this? Evidently the nucleic acid is depolymerized and broken down. At the same time the products of action of the genes, now uncovered, fill the nucleus; and the simple proteins produced, together with the ribosc nucleic acid derived by reconstruction from the desoxyribose, are assembled by a particular gene or organizer to reconstitute a nucleolus. We now fmd, however, that in some cells of some organisms certain constant parts of the chromosomes do not throw off their coat. The nucleic acid becomes sticky, owing perhaps to depolymerization, but remains attached throughout the resting stage. These parts are known as heterochromatin as opposed to the normally behaving euchromatin. It is these parts or blocks which in some organisms, under the conditions we saw, are liable to be starved of nucleic acid at mitosis. 146 CHROMOMERES AND PROTEINS Chromotneres and Proteins There are, as we saw in the first chapter, two types of nucleus which reveal their chromosome structure while they are still active in producing proteins. They are the polytene and the pachytene nuclei. In both of these, chromomeres appear. The explanation of these chromomeres, advanced by Caspersson, is that the nucleic acid is attached to the active genes whose products stretch the normally folded thread between them. Their gradual emergence from a homogeneous thread at the beginning of the prophase of meiosis confirms this view (Fig, 36). Thus chromomeres are genes making themselves visible as working units. This is not so in the diffuse resting nucleus where nucleic acid is not attached and where proteins are pushed straight outwards to give the appearance sometimes described as a lamp- brush chromosome. In the polytene nuclei these products of gene (or chromomere) activity can be observed. Heterochromatin, which is active in these nuclei, produces simpler proteins of a histone type, while euchromatin produces larger, more complex and therefore, we must suppose, more specific proteins of a globulin type. The chromomeres of heterochromatin are accordingly smaller and less exactly paired than those of euchromatin. There are chromosomes which contain little but heterochromatin, for example the Y chromosome in Drosophila, and supernumerary chromosomes in maize and other plants. These appear to have little specific effect on the organism. When extra ones are added, however, other things being equal, the size of the nucleolus and the quantity of nucleic acid in the cytoplasm are said to be increased. Such an increase within the organism can be shown to go with increased protein production. For example, ribose nucleic acid, which is absent from actively fermenting yeast in the absence of a source of nitrogen, immediately appears when nitrogen is made available and proteins begin to be produced. Heterochromatin, ribose nucleotides, and protein production are thus closely bound up together. Ribose nucleotides are abundant, not only in embryonic and cancerous tissue, but also in the egg cells of plants and animals where protein production is not immediately taking place. But here, as Brachet has shown, it is 147 GENES, MOLECULES AND PROCESSES o o Z (S)(fe(fe(t) O O o o o o o 6 o in O O o o^ o o 0 S 2 S - o . i-i T= (J ^ 1) O rt -2 bD 3 = O J3 o o I-I j3 O - J2 'A • -C *-• .S3 o i^ c 5i -a o o O ■— _Q £ 'o o « o C «J rt M "■ T! -S o o u- -s -5 >< O O (j_, w ^ -^ o ..^ S a o c SJ 4j I" W ^-^ I -C r3 u (J -O tl rt rt o Q_ bO Oh CU C p ^ P o O Jo l- " 2-c o^ 5 "-^ - ° s o ' c/0 Fi {J JJ Ui Q- r- ^ O o s H S S « 2 I 2 2-5t; ^" -6 c i! Ji -2 -T3 tu -c U 148 CHROMOMERES AND PROTEINS necessary for conversion into the desoxyribose form required for the rapid multipHcation of nuclei which follows fertilization. Ribose nucleotides are capable of some polymerization. Their main work, however, seems to be less mechanical and more physiological than that of their desoxyribose relatives. They are concerned with protein reproduction. They are regularly found in the smaller viruses, in the plastids of plants and in other small protein bodies of the cytoplasm. The viruses and plastids are characteristically self-propagating bodies like the genes of the nucleus. Ribose nucleic acid, therefore, may well do for these proteins in a small way what the desoxyribose does for the more highly organized chromosome, namely, act as the agent of their reproduction. What picture do we then get of the chemical relations of the nucleus and the cell? The nucleus is a differentiated and highly organized, mechanically stable and totally balanced system of protein-producing units. The cytoplasm has no mechanical stability to give it a permanent and total balance. Indeed, as we shall see, differentiation between cells depends on changes in its balance. It is not surprising, therefore, that the permanence of control in heredity and development so largely comes from the nucleus. At the same time the existence of self-propagating bodies in the cytoplasm would indicate that the control of the nucleus was not total, im- mediate and absolute. The cytoplasm must always feed the nucleus with the materials from which it builds up its specialized structures and with which it in turn feeds the cytoplasm. Each must feed the right materials to the other. When, as we saw, the nucleus of one species is put in the cytoplasm of another, breakdown often results from the failure of this reciprocal process. In development, therefore, we camiot argue for the greater importance of one or the other. It is only the long-term relations concerned with the per- manence of heredity that enable us to take their interactions to pieces and show where precedence and authority lie. This picture opens our eyes to a number of new possibilities. First, although there are many animals and plants in which the differentiation of the heterochromatin is not visible, it seems likely that the two kinds of genes are always present. The one kind gives the simpler products and should have less specific, and individually 149 GENES, MOLECULES AND PROCESSES less pronounced, effects. But these effects should include, as they do, an influence on the other kind which gives the more complex products and should have the more specific and elaborate effects. Secondly, we must expect the actions of genes to depend on each other and on the varying character of the cytoplasm as reflecting their earlier action. Differentiation will depend to some extent on the limitations of diffusion within the nucleus and the cell. Finally, we must expect that the cytoplasm as well as the nucleus will contain self-propagating particles, although they are likely to be subordinate to the nucleus in general since they have not the same powers of orderly reproduction and transmission that are given by the giant chromosome molecule. Heterochromatin and Polygenes The difference between genes in heterochromatin and in euchromatin is in fact made clear in several ways. As we saw, the Y chromosome in Drosophila and the supernumeraries in plants, both heterochromatic, have few or no drastic effects. The super- numeraries are not even indispensable. And none show mutations of the sharp and specific kind that are used in mendelian experiments. These principles apply whether the heterochromatin composes the whole chromosome, or, as in the X o£ Drosophila, a part of it. On these grounds, wherever it has been found, heterochromatin has been described as inert. Now, however, we have two means of demonstrating its peculiar activity, hi maize many cultivated varieties regularly have supernumeraries which vary in number from plant to plant. Yet these chromosomes often have defective centromeres and so tend to be lost at meiosis or even at mitosis. Their maintenance must therefore depend on a selection which favours increase in number of chromosomes, and this can come about only if these chromosomes actively affect the character, the phenotype, of the plant. The same principle applies to super- numerar)' fragments of the X chromosome found up to the number of 14 in the bed bug, Cimex lectularitds, as well as in many other Heteroptera. The second means of approach is through the polygenic systems which we have seen to govern continuous variation. The members 150 HETEROCHROMATIN AND POLYGENES of such a system have small, similar and supplementary effects, because, we may presume, they have less differentiated products and less elaborate action. The simpler action of these genes at once recalls the simpler products of heterochromatin as seen in the cell. The relationship is brought closer when we fmd that heterochromatic chromosomes, hitherto regarded as inert, affect quantitative variation. In a diploid millet, supernumeraries, which are lost except in the stem, lead to extra mitoses in the pollen. The larger the number of supernumeraries, the larger the proportion of pollen which is killed in this way. Nor is this an isolated example, for supernumeraries are now known to affect the phenotype of a number of grasses and cereals in a quantitative way. Quantitative effects are also produced by the Y chromosome in Drosophila melanogaster. Y chromosomes from different strains vary in their effects on the numbers of bristles. But the greater part of this variation can be destroyed by association with a common X chromosome partner. It thus seems that genes in the Y, affecting bristle number, can be exchanged with corresponding genes in the X. And, since only the heterochromatic portion of the X lies in the pairing segment and is homologous with part of the Y, we can see that X-borne, as well as Y-bome, hetero- chromatin is active in this way. The crossing-over that would account for such exchange has, as we saw, been cytologically inferred from the occurrence of reciprocal chiasmata between their pairing segments. The heterochromatin is thus active; it is variable in its activity; and this variability is divisible by crossing-over. In other words it is composed of genes similar in linear organization to the major genes, but differing in kind or order of physiological effect. Indeed the smallness of their individual effects implies the largeness of the number of these polygenes. We can now recognize two types of gene and two types of chromatin. The major genes occur only in euchromatin and hetero- chromatin contains only polygenes. Euchromatin may well also contain polygenes, perhaps in the euchromatin proper or perhaps lying in segments not distinguishable as heterochromatin owing to their small size or for some other reason. This is all the more probable since, as we saw, many plants and animals have no 151 GENES, MOLECULES AND PROCESSES recognizable lieterochromatin. In the onion Allium cepa, for instance, some varieties have visible hcterochromatin and others not. And in species with blocks of hcterochromatin, these blocks are always more variable in size and number among individuals than are the parts of the euchromatin. What Major Genes Do The mode of action of polygenes can, as yet, only be inferred from the activity of hcterochromatin in producing small proteins and ribose nucleic acid. The modes of action of the major genes, on the other hand, are recognizable by other means. The most important of these is the dosage method of MuUer, By X-raying sperm, Muller produced flies with broken chromosomes and, combining these with normal chromosomes, he was able to obtain particular genes or their mutant allelomorphs present in one, or three, or four doses instead of the proper two. He also obtained combinations of wild type and mutant in different doses. What he then found was that most mutant genes, whether themselves spontaneous or induced by X-rays, had the same action as their normal allelomorphs, only to a lesser degree. Similarity of action of the normal and mutant gene could be shown in the following way. The mutant gene scute-i normally removes, or appears to remove, certain of the wild-type bristles. But, when an extra scute-i gene is added, the number of bristles is almost normal. With two extra scute-i genes the fly actually has more bristles than the normal. Thus, far from removing bristles, scute-i helps to produce them. It does the same job as the wild type, but less than half as effectively. Such a gene Muller calls a hypomorph or, in the extreme case, when it does nothing, an amorph. All deficiencies will, of course, appear as amorphs. Other types of action are also shown by this method. The hypermorph is more efficient than the wild-type gene. The antimorph opposes the action of the wild-type gene. The neomorph does some- thing new. Clearly these terms are comparative. The wild-type gene is hypomorphic to its hypermorphic mutant and amorphic to its neomorphic mutant. Antimorphs and neomorphs frequently, if not always, involve structural changes, though these may be only small duplications as in the neomorph Bar. It may well be, therefore, 152 WHAT MAJOR GENES DO that they depend on a position effect, that is on the integrated action of more than one genie constituent. Of these types, all but hypomorphs are rare. What does this SPERM A 25°/c O 70% ^\\\\\\- B 0 xWXVvXWSb Fig. 37. — The A, B, O blood group system governing blood transfusion in man. The approximate frequencies of the three allelomorphs in the gametes of the English population are shown along the margins of the diagram. Six genetically different zygotes will then occur with frequencies proportional to the corresponding areas in the diagram. A and B are, however, each dominant to O, though not to one another. Thus genotypes AA and AO are indistinguishable in the phenotypes they give, as are BB and BO. There are then four groups distinguishable in trans- fusion behaviour, viz. AB, B, A and O, as shown by the cross-hatching. The directions in which blood can be successfully transfused are shown by the arrows. Group O is the universal donor and AB the universal recipient. mean? It means that mutation, in the major genes, is generally a loss of efficiency, a breakdown in the working of a complex mechanism, a pathological change in a healthy system. Thus, by a 153 GENES, MOLECULES AND PROCESSES new method wc have additional evidence that the elements of the cuchroniatin are producing complex proteins and therefore have a complex and delicately adjusted organization. In such a system undirected change is likely to break down the old rather than to build up the new. Another way of getting at this problem is by examining what appears to be the closest to the gene of all the products of its activity. Antigen production is the most specific and unconditional activity of major genes. The chain of reactions from the gene in the nucleus to the antigen in the cell is therefore the shortest we can infer. All organisms contain antigens since any protein can behave as an antigen. But in the blood groups which govern the possibilities of transfusion in man and other animals, different antigens are directly associated with corresponding genes. If the gene is present so is the antigen. There are many sets of blood-group alleloniorphs such as M-N, and the Rliesus series. In a heterozygote of any of them the antigens of both allelomorphs are present in the cell — notably, of course, in the red blood cells where they are recognizable if the appropriate antibody is induced. In the important A-B-O series in man and the apes, it happens, for a reason that is not yet understood, that the antibodies to A and B occur spontaneously, whereas O cannot even induce one easily. Thus O is recessive to A and B and serologically appears merely as their absence (Fig. 37). On the other hand, in certain species hybrids of doves new antigens appear to arise which are found in neither parent; thus the co-operation of more than one gene in their production is implied. Otherwise, so close is the coimexion, that the distinction between gene and antigen is formally maintained only because of the necessity for disting- uishing between determinant and product which genetics has taught us to respect. Now the blood antigens, and likewise the antibodies they call forth, are complex proteins; we therefore have the best evidence that such proteins can be the immediate products of gene activity. The Interaction of Genes The relation between the gene and the antigen it produces seems generally to be a simple one; as simple indeed as that between the 154 THE INTERACTION OF GENES gene and the daughter it produces in its own reproduction. Gene reproduction makes use of the special mechanism, we might almost say of the midwife molecule, of nucleic acid. But nucleic acid appears not to enter into the production of other molecules by the genes. We should not, therefore, expect the same simple correspondence between the gene and its effect in action, as between the gene and its daughter in reproduction. Furthermore it is hardly likely that genes will fail to interfere or react with one another, either in the consumption of materials or in the release of products. On the contrary, we should expect genes to interact with one another, both within the nucleus and outside it in the cytoplasm. One type of interaction between genes in their work we have seen in the position effect. This effect not only shows interaction; it shows that the interaction can occur within the nucleus. Another interaction that is probably within the nucleus is that whereby one gene modifies the rate of mutation of another. In Drosophila this happens at times as a result of a position effect. A gene of normal stability is moved close to the heterochromatin and becomes highly mutable, so much so that it regularly produces a mixture or mosaic of mutant and non-mutant tissues, such as that shown by Plum-eye. The control of mutability of one gene by others is common, and, though not generally acting by position effect, the modification of mutation rate may have something to do with the heterochromatin. For example, in maize the Dotted gene, which is recognized by its effect on the mutability of an anthocyanin gene, is shown by linkage tests to lie in or near the heterochromatic end "knob" of chromosome lo. Mutability control being so widespread we should expect that an upset in the genetic organization or balance would be reflected in changed mutation rates. It is, in fact, often claimed that F^ species crosses show a greater frequency of mutation — visible somatically — than either parent. The first types of genie interaction were recognized by Bateson in the course of the series of studies between 1900 and 1 910 in which he estabhshed the general validity of mendelism. These interactions proved to be ones which cannot be traced back to the nucleus, and indeed probably arise very far away from it. They are the types which reveal themselves in the segregations of whole individuals in 155 BB Bb bb GENES, MOLECULES AND PROCESSES AA Aa aa Fj RATIOS AS MODIFIED BY GENE INTERACTION 1 2 2 4 J 2 1 2 1 @ AB Ab aB ab Additive AB Ab aB ab Rec.Suppressor AB aB Ab ab <4) Rec.Epistatic AB Ab ^ ab DOM EPISTATrC (D Duplicate Fig. 38. — Where two genes are segregating in Fj, nine genotypes are expected with the frequencies shown at the top left of the diagram. With dominance of each gene they give four phenotypcs in the characteristic 9:7:3:1 ratio of the centre of the diagram. This ratio may be modified by genie interaction in the six ways shown. Three of these, complementary action, duplicate action and recessive suppressor Continued at foot of page i$7 156 THE INTERACTION OF GENES F2 families. Where two genes which segregate independently do not interact, they give Mendel's familiar 9:3:3:1 ratio. When they interact this ratio is simplified; classes are combined in most of the ways possible with complete dominance (Fig. 38). Interaction in the simplest case consists of a mere anticipation: the effect of one gene may be cut out by that of another which is said to be epistatic to it. Epistasy comes in at all stages of development, early and obvious or late and inferential. A gene removing life, a lethal gene, must obviously be epistatic to any other genes affecting the life it cuts short. A gene removing an organ is likewise obviously epistatic to one modifying that organ. A gene suppressing, or failing to produce, a substance, is obviously epistatic to one modifying that substance, or requiring it for its own activity. But the conclusion whether the substance is modified on the one hand, or is required on the other, is usually to be deduced only from the epistasy itself. Epistasy expresses itself in two types of F2 ratio according to whether the epistatic allelomorph is dominant or recessive. Albinism in rodents is due to a gene which is recessive, and also of course epistatic to the various colour and pattern genes. The 9:3:4 ratio is therefore characteristic of Fg's segregating for albinism. White leghorn fowls, on the other hand, owe their absence of colour to a gene, which is, again, epistatic to other colour and pattern genes just like the albino gene in rodents. But it is dominant. Thus they will give a 12 : 3 : i ratio in Fg. Fig. j8. — Continued from page 156 action, give two phenotypic classes understandable on the assumption that the appear- ance of the character in question requires a particular allelomorph of each gene to be present. Failure in either or both genes results in absence of the character. These three cases differ only in the dominance relations of the effective allelomorphs, both being dominant in complementary action, both recessive in duplicate action, and one of each kind in recessive suppression. Tw^o other cases, dominant and recessive epistatic action, give three classes, one gene, said to be hypostatic, causing a difference in phenotype only in the presence of a particular allelomorph of the other gene, said to be epistatic. The cases differ only in the dominant or recessiveness of the effective allelomorph of the epistatic gene. In the sixth case, of additive action, the two genes have individually indistin- guishable, but cumulative, actions. The classes which are confounded are separated only by dotted lines in the repre- sentation of their various interactions, and the relative frequencies of the pooled classes as they would appear in F.j segregations are shown in the small circles. 157 GENES, MOLECULES AND PROCESSES In cpistasy the expression of one gene difference conditions that of another. The relationship is not reciprocal. With reciprocal relations other Fg ratios are found. They are of three kinds according to whether it is the two dominants, the two recessives, or one of each which are needed for the combined effect. In any case only two classes are recovered instead ot the three with epistasy. When two dominants are needed we have complementary genes. The classical case, indeed the first case, of any interaction to be described and understood, is the production of coloured sweet peas from a cross between two whites, the coloured-white ratio in Fg being 9 : 7. When two recessives are needed we have duplicate genes, in a number of characters such as ligule — liguleless in wheat and oats. The Fi may resemble both parents (when it is Ab X aB) but the F2 gives a 15 : I segregation. The duplicate genes are so-called because the allelomorphs appeared to be doing the same thing, whereas complementary genes, being themselves dominant, were supposed to be doing different things. In cereals the duplicate (or triplicate) genes are, no doubt, doing the same thing since these plants are polyploid. But, in diploid plants and animals, the 15:1 segregation may just as well be taken to indicate a complementary action of recessives. This interpretation is all the more plausible in view of the occurrence of yet a third type, the complementary action of dominant and recessive giving the 13 : 3 ratio which is shown by segregation for ivory and yellow flower colour in suitable Antirrhinum crosses. The recessive allelomorph of one gene is here called a suppressor of the recessive allelomorph of the other. The term suppressor is again, of course, merely an accidental tradition in this connexion. It might just as well be applied to the complementary and duplicate relation- ships, where it is the dominant allelomorph of one gene which, in effect, suppresses the recessive of the other. Since dominance is itself modifiable, the same kinds of interaction may underlie all three. The difference may depend merely on which combinations of dominants and recessives yield similar groups of phenotypes. Distinct from the five types of interaction discussed so far is that where the Ab and aB combinations are not distinguishable. We then have a 9 : 6 : i ratio and A and B are said to be additive. Simple additive interactions arc rare, amongst major genes at least, 158 THE EXPRESSION OF GENES but appear to account for the fact that two types of sandy pig have been seen in Fg's from crosses between red and white. The genes governing red and white chaff in wheat show additive action but the ratios arc coniphcated by incomplete dominance which leads, with two genes, to five distinguishable classes instead of three. All these phenotypic groupings which give simplified ratios indicate gene interaction. The groups can, of course, be broken up into their genetic classes, in the same way as Mendel broke up his F2 classes, by appropriate breeding tests. They can also sometimes be broken up by closer microscopic examination or chemical tests. For example two races of Rudbeckia with yellow bud cones give purple bud cones on crossing and 9 purple to 7 yellow in Fg. But of the 7, 4 turn red and 3 turn black on treatment with caustic potash solution. At this new level of analysis, what had appeared to be complementary interaction is seen as recessive epistasy (see also Fig. 39). Clearly, therefore, classification of phenotypes in an F2, even aided by chemical analysis, can only give us a provisional account of the order and relationship of the individual processes at work. Further into this question we shall see later. The Expression of Genes So far we have considered the interactions of separably demon- strable genes, of major genes. The expression of all major genes, however, is affected by modifiers, presumably polygenes, which are otherwise not readily detectable. For example the gene "eyeless" reduces the eye in Drosophila melanogaster to a greater or less degree, characteristic of each laboratory stock. When flies from a particular eyeless stock are crossed with wild-type flies, the eyeless part of Fg shows a sharp increase in the range of reduction. Eyeless stocks can be selected from such a family with eyes differing in size from one another and from their eyeless ancestors. Thus, the wild-type fly must always carry genes capable of modifying, even of enhancing, the expression of "eyeless." The many grades of eyelessness in Fg show that many genes are concerned in this way in controlling what Timofeeff-Ressovsky calls the expressivity of the eyeless gene. These comparisons, of course, apply to experiments under constant conditions of nutrition, temperature and so forth. Variations in the 159 GENES, MOLECULES AND PROCESSES environment have often been shown to affect the expressivity of genes. In fact genotype and environment work in parallel. Sometimes variation in the expression of a gene is such that it is not shown by all the individuals, even the homozygotes, carrying it; it does not, as it were, penetrate the whole population of individuals carrying it. The gene "antennalcss," for example, studied in Drosophila by Gordon and Sang, removes either both or only one of the fly's antemiae according to its degree of expressivity. A proportion of the antennaless flies even show no effect of the gene at all. Its penetrance is incomplete. But the degree of penetrance (and expressivity) varies with both genotype and environment, and both effects are shown in relation to sex. In the early emerging flies, penetrance is lower in males than in females; the reverse is true in the later part of the hatch. In both sexes the penetrance is at a minimum in a culture at about the fourth day of emergence and at a maximum at eight days. These variations in expression are due to changes in the food of the larvae which arise from staling of the culture medium. This, in turn, is partly explained by the discovery that increase in the supply of vitamin Bg reduces penetrance. Indeed this vitamin enables all the flies to get over some of the difficulties, and some of the flies to get over all of the difficulties, presented by the shortcomings of the antennaless gene. These experiments have had to do with genes in the homozygous conditions. When we examine heterozygotes we are concerned with dominance and we can regard dominance as a measure of the expressivity and penetrance of the heterozygous gene pair. We might, therefore, expect that dominance would also be subject to modification by genotype and environment. The effect of the genotype has indeed often been established — for example, in cotton, butterflies, and poultry. In the mouse, as we saw, agouti is normally dominant to non-agouti. The gene umbrous, however, shifts the expression of the agouti heterozygote towards non-agouti. The Fg ratio of 3 agouti to i non-agouti becomes 1:2:1. Umbrous thus modifies the dominance of agouti and, indeed, this is the only known effect of the gene. The effect on dominance of the environment is, on the other hand, less widely known. The recessive gene cubitus interruptus in 160 SYNTHETIC SEQUENCES Drosophila melanogaster breaks a wing vein of the flies. At 19° C. it affects all homozygotes but it is expressed in only half of them at 25° C. In the heterozygote, according to Stern, it does not express itself at all at 25° C. but at 13° C. it is seen in about 10 per cent of the flies. Another gene, Scutenick, which is lethal in the homozygote, produces multiple effects in the heterozygote at 25° C. As the temperature rises its effect on the eye decreases, while its effect on the scutellum increases. Both of these modifiable heterozygotes belong to genes in the small fourth chromosome near a block of heterochromatin, whose behaviour, as we saw, is sensitive to changes of temperature. They are, therefore, special cases. The reason for the general lack of outside effects on dominance is not far to seek. The normal or wild- type gene exists as part of a genotype long adapted to give a constant expression in varying environments. Now this normal and buffered gene is generally the dominant. Its heterozygote would, therefore, be expected to give a more stable result than would be given by its new untried allelomorph when homozygous. In fact, as Fisher has pointed out, the genotype, whose effects we have seen, must have been selected to give stable dominance. Are there any general physiological principles that we may infer from these actions and interactions of genes ? One most general principle is at once clear. Every gene depends, for its action, on that of others; of many others, possibly of all the others. The phenotype as a whole depends on the co-ordination of all genes, and this co-ordination, in the fit organism, is what we call balance. Hence we can see why polyploidy has a balanced effect on the genotype and why polysomy, duplication and single gene changes have unbalanced effects. Now, in the terms of materials and processes, co-ordination means that each gene is fed by others, and, in turn, feeds others. How, we must ask, is their feeding adjusted? The answer to this question is given by the precise chemistry of gene effects. Synthetic Sequences: Supply and Demand We can frequently see what the substitution of a particular gene by its mutant does to the chemical processes of the organism. A recessive dwarf in maize owes its dwarfhess to a characteristic Elenttnts of Genetics l6l L GENES, MOLECULES AND PROCESSES excessive ability to destroy auxin by oxidation. On the other hand the recessive alcaptonuric in man owes his pecuUarity to a character- istic inabiUty to destroy honiogentisic acid by oxidation. Again the Dahiiatiandog is distinguished from other breeds by a single recessive gene, w^hich reduces the transformation of uric acid into allantoin, and so increases the concentration of uric acid in the urine about ten times. SERINE HOCHjCHNHzCOOH' CHzCHNHjCOOH J i ANTHRANILIC i INDOLE TRYPTOPHANE ACID ICENEll |gen"e2| Fig. 39. — The bio-synthesis of tryptophane proceeds in three stages in Neurospora crassa which can be blocked by genes at two points at least. Mutation of Gene i blocks the first stages, that of production of the first precursor, anthranilic acid. Mutation of Gene 2 blocks the second stage, conversion of anthranilic acid into indole. Thus Gene i is considered to govern the production of an enzyme catalyzing the production of anthranilic acid, and Gene 2 the production of a second enzyme catalyzing the conversion into indole. Where only indole or tryptophane was recognizable, the genes would appear complementary in action, but where anthranilic acid was also detectable Gene i would appear in its true light, as epistatic to Gene 2 (see Fig. 38) (based on Beadle, 1945). So much for single genes and simple processes. The combination of genes and processes can best be seen in simple organisms such as bacteria and fungi. By X-raying spores o£ Neurospora crassa Beadle, Tatum, Horowitz and others have produced single-gene recessive mutants, which differ from the wild type in their inability to grow, or at least to grow well, on media lacking certain substances, amino- acids, vitamins, pyrimidine bases and so on. Now the antennaless fly seems unable to make efficient use of vitamin Bg, and therefore requires it in greater quantity to produce anteimae. In the same way these mutant moulds are unable to make use of precursors of these various substances, and so require a supply of them ready- made in their food. Different genes may, by mutation, lead to different kinds of 162 SYNTHETIC SEQUENCES failure in producing the same substance. As a consequence two mutant genes in Neurospora with the same apparent effect may be carried by two nuclei combined by vegetative fusion in one hyphal cell, a Jieterocaryon; they can then compensate for one another, each making good the other's deficiency, so that the hypha seems normal. Many of these mutants appear complementary in their effects. Some, no doubt, act in parallel, but this is not true of all. Thus for two different mutant strains tryptophane is a necessary raw material; but it is apparently necessary in different ways, for one will grow on medium with indole, but not on medium with anthranilic acid, whereas the other will grow on either. Since both these are likely precursors of tryptophane, the final step in its synthesis is, we must suppose, unaffected by either mutant. Furthermore, the mutant gene in the strain which can utilize either substance must affect an earlier stage than the one which can utilize only the indole. In other words the chain of synthesis must follow the course shown in Fig. 39. And the one mutant must break the chain before anthranilic acid, while the other breaks it only before indole. This inference of order is confirmed in a highly significant way. The mutant which grows only with indole accumulates anthranilic acid. New side chains of development may thus arise from blockage. For example, one mutant, failing to produce adenine, develops a new pigment, a polymerized purine. Here, then, we have our genes feeding one another, acting in succession, acting as we should say epistatically. They act not merely in fixed order in regard to the general sequence of development like the genes in Mendel's peas (Table i): they act in a fixed order in regard to particular other genes, wherever they take effect in development. The effects of a mutation may be conditional. For example, as we saw, one mutation blocks the synthesis of adenine. But it does so only at temperatures over 32° C. In such a case the mutant allelomorph may be merely hypomorphic or less efficient than the normal, or it may affect only an accessory process. More complex series of relations are shown in the synthesis of pigments in higher organisms. The chemical structures of these pigments and of their probable precursors are well understood in the flowering plants, and are uniform throughout the group. The sap soluble pigments are of two main kinds, anthocyanins, which 163 GENES, MOLECULES AND PROCESSES arc the red and blue pigments, and anthoxanthins, which are the ivory and yellow flavoncs and flavonols. These two classes have the same basic structure and both are assumed on chemical grounds to be built up by parallel processes from sugars. What does genetics tell us of these processes ? In Pliarhitis nil, two gene mutants, c and ca, interfere with production of both anthocyanin and anthoxanthin. They must, therefore, act before the synthetic chains branch. Other mutants, a and r, have a direct effect only on the anthocyanins. They act after the chains branch, as do most of the known pigment genes. Now if a gene acting on one branch sequence is reduced in activity we must suppose that an extra supply of materials will be available for the other sequence. Blockage should lead to diversion. This possibility has been tested in the autotetraploid Dalilia variabilis where every gene can be tested in five doses [a^, Aa^, A^az, A^a, A^). The gene B, according to Lawrence and Price, produces anthocyanin, I produces the anthoxanthin apigenin. Bh^i^ has, of course, antho- cyanin pigmentation as has Bh^Ii^. But, still keeping Bh^, with I^i or I^ the anthocyanin is much reduced. Thus B and / are evidently competing for a precursor. Further, I shows the effect, even more strongly, with another anthocyanin gene A; Aa^I^ produces almost no anthocyanin at all. There is also evidence that each of these genes A, B, I and a fourth, Y, appears to contribute to a pool of common precursor. In fact, of course, they must increase the supply by increasing the demand: they act backwards, as well as forwards, in development. When we say that the gene works backwards this is, of course, a figure of speech. It means that what is happening in the cytoplasm reacts on the nucleus. The utilization in the cytoplasm of materials produced by genes in the nucleus stimulates the activity of these genes. The reaction of nucleus and cytoplasm must be reciprocal, so far as the feeding of the nucleus is concerned. There seems little doubt that it must also be reciprocal so far as the feeding of the cytoplasm is concerned. Yet another point is shown by the anthocyanin genes A and J5 in Dahlia. In small doses they produce only cyanin, which is regarded as the primitive or precursor type of anthocyanin since it is commonest in the leaves, while its derivatives are commonest in the flowers, 164 SYNTHETIC SEQUENCES of flowering plants. The higher the dose of A or B, the more the crimson cyanin is replaced by its reduced form the scarlet pelargonin. The quantity, or more strictly the proportion, of the determinant, lengthens the chain of production and changes the quality of the product. Thus we see how balance controls particular processes. ACTION Direcl' Successive Cooperafive CompetltTve GENES ® ® K) (ft VCENES (D END PRODUCTS A. O X Y ANALYSISj examples"* __Si(T|^Ie EpisfafTc Complementary QuantlVaftvc {Anfi£ens Develojjmoifal Piemen T AnfTiocyanin lncom{3afTlMli^ Sequences \. Ho pigment v.Anf1io;(anff)in Fig. 40. — The four modes of gene action. In direct action, the end product must be very close to the gene itself. As the chain^of processes lengthens the action of differ- ent genes combine in the synthesis of one end product. The product of one gene may be the raw material for a second, in successive action. Or the immediate products of two genes may interact directly to give a common end product, in complementary action, or two genes may draw on a limited quantity of a common raw material, itself perhaps the product of a third gene, in competitive action. The appearances of the four types of action in genetical analysis are shown together with examples of characters in the production of which they have been recognized. More complex cases of gene interaction can be resolved into combinations of these four basic types. Looking back over these types of action and interaction we see that they fall into four main systems (Fig. 40). These we may call direct, successive, co-operative, and competitive. The first three correspond roughly with simple action, and with epistatic and complementary interaction. The fourth is merely a modification of complementary interaction. In complex processes all these types may well be combined. Indeed they seem to be combined in the production of eye-pigment in Drosophila where, in addition, 165 GENES, MOLECULES AND PROCESSES Ephrussi and Beadle were able to show by transplantation experi- ments that some of the necessary substances are produced on the spot, and others are brought in from elsewhere. Finally we must notice again that the majority of gene changes do not entirely stop or block a process; they merely slow it down. They are hypomorphs rather than amorphs. Indeed they often change a rate of activity in a way that can be exactly measured. The change S-s in the shrimp Gamrnarus chevreuxi reduces to half the rate of deposition of melanin in the eyes of the young larva. The rate falls off with growth, but more rapidly in the quickly depositing type so that in the end the product is not very different. A difference in rate may appear as a difference in time of expres- sion. For example in some local races of the moth Lyniantria dispar Goldschmidt found that the skin of the caterpillar has pigment throughout development; in others deposition begins half-way towards pupation, and even in these proceeds at different rates in different races. With the more highly integrated processes, for example, of sexual differentiation, all differences of races and hybrids become referable, as Goldschmidt has shown, to differences in rates of co-operating and competing processes common to all races and both sexes. In these terms we begin to see the material and mechanical basis of differentiation. The actions of genes depend on the simultaneous actions of one another. But even more, and somewhat differently, they depend on what other genes have done earlier and have done elsewhere. Even if the immediate products of a gene's action were always constant in amount — which is unlikely — its derivative effects would depend on when and where it was acting, on conditions in the nucleus, and, of course, in the cytoplasm too. These considerations bring us back to the cell as the sphere of action of the nucleus. If the cell feeds the nucleus and the nucleus feeds the cell, the separation of cells gives each of them its specific type of feeding relationship, its own pattern of reactions and development. Between the fertilized egg, and its mature develop- ment, the whole course is determined by the supply of raw materials, the rates of gene action and the rates of diffusion of its products. If the supply of raw materials never changed, or if the rates of gene action never varied, or if diffusion were instantaneous, differentiation i66 SYNTHETIC SEQUENCES could never occur. Before we come to grips with the problem of differentiation, however, we must look at the whole question from the other side. We must find out what part the cytoplasm plays in heredity. REFERENCES BEADLE, G. w. 1945- Biochemical genetics. Chemical Reus., 37: 15-96. BEADLE, G. w., AND cooNRADT, V. L. 1944. Heterocaryosis in Neurospora crassa. Genetics, 29: 291-308. BEALE, G. H. 1941. Gene relations and synthetic processes. J. Genet., 42: 197-214. BKACHET, J. 1944. Einbryologie Chimique. Liege and Paris. CASPERSSON, T. 1947. The relations between nucleic acid and protein synthesis. Symp. Soc. Exp. Biol., i: 127-151. DARLINGTON, c. D. 1947- Nucleic acid and the chromosomes. Symp. Soc. Exp. Biol., 1 : 252-269. DARLINGTON, c. D., AND upcoTT, M. B. 1941. The activity of inert chromosomes in Zea mays.]. Genet., 41 : 275-296. nsHER, R. A. 193 1. The evolution of dominance. Biol. Reus., 6: 345-368. FORD, E. B., AND HUXLEY, J. s. 1929. Genetic rate-factors in Gammarus. Arch. Ent. Mech., 117: 67. GOLDSCHMIDT, R. 193 8. Physiological Genetics. New York. GORDON, c., AND SANG, J. H. 1941. The relation between nutrition and exhibition of the gene Antermaless {DrosopJiila). Proc. Roy. Soc. B., 130: 1 51-184. GULiCK, A. 1944. The chemical formulation of gene structure and gene action. Advs. Enzymology, 4: 1-39. HEITZ, E. 1935. Chromosomenstruktur und Gene. Z.I.A.V., 70: 402-447. HOROWITZ, N. H. et. al. 1945. Genie control of biochemical reactions in Neurospora. Am. Nat., 79: 304-317. KUHN, A. 1941. Qber eine Gene-Wirkkette der Pigmentbildung bei Insekten. Nachr. Akad. Wiss. Gottingen, Math.-Phys. Kl., 6: 231-261. LAWRENCE, w. J. c, AND PRICE, J. R. 1940. The genetics and chemistry of flower colour variation. Biol. Reus., 15: 35-58. MATHER, K. 1944. The genetical activity of heterochromatin. Proc. Roy. Soc. B., 132: 308-332. MATHER, K., AND NORTH, s. B. 1940. Umbrous: a case of dominance modification in mice. _/. Genet., 40: 229-241. MULLER, H. J. 1932. Further studies on the nature and causes of gene mutations. Proc. 6th. Int. Cong. Genetics, i: 213-255. OSTERGREN, G. 1947. Heterochromatic B-chromosomes in Anthoxanthum. Hereditas, 33: 261-296. STERN, c. 1943. Genie action as studied by means of the effects of different doses and combinations of alleles. Genetics, 28: 441-475. TIMOFEEFF-RESSOVSKY, N. w. 1 93 1. Gerichtetes Variieren in der phanotypischen Manifestierung einiger Genovariationen von Drosophila funebris. Naturwiss., 19:493-497. 167 CHAPTER 8 THE CYTOPLASM Plastogeties Plasmagenes Cytoplasmic Equilibrium Cytoplasm, Nucleus and Etwironment Dauermodification Suppressiveness and Amhilinearity The cytoplasm must be the agent of the nucleus in development for it is only from the cytoplasm that the nucleus can obtain its raw materials, and it is only to the cytoplasm that it can pass on the products of gene action. But the cytoplasm might also act as an agent in its own right, by virtue of its own capacities of self- propagation. Clearly the way of testing this possibility is by bringing together cytoplasm and nucleus in new associations. There are two ways of effecting this re-association. The first is by grafting, whose consequences we have seen in Acetahularia. The second is by breeding. Sometimes the nucleus of one species can be associated with the cytoplasm of another. Thus an occasional seed borne on the lentil plant. Lens esculenta, in cultivation grows into a common vetch, Vicia sativa. Vetch pollen has entered the lentil embryo-sac. The male generative nucleus has supplanted the egg nucleus, doubled its chromosomes, and developed as a diploid vetch in a lentil seed coat. These intruders are entirely normal and show no effect of the cytoplasm in heredity. There are also instances of such exceptional behaviour in Fragaria and Nicotiana. These rare examples do not, however, offer a sure basis for generalization, as we can see if we turn to animal merogons. Sometimes development is successful and, in the long run, patro- clinal, as we saw earlier in sea-urchin crosses where the egg-nucleus has been artificially removed before fertilization (Fig. 2). In similar merogons between the newts Triton palmatns and T. cristatus the outcome is less successful. These develop under the control of the cytoplasm, that is maternally, for some time. But in the end they become malformed and development ceases. Evidently at the beginning, the cytoplasm had enough of the products of its past 168 PLASTOGENES nucleus to carry it on. Development was harmonious because these products were harmonious. Later, when the new and foreign nucleus begins to pass its products into the cytoplasm, the mixture is no longer in harmony and development breaks down. In hybrids, too, such cases may be much more common than those of successful development as in Lens, for they will normally escape detection through their failure to survive. The rare exceptions which are successful enough to be found may give a false picture. Plastogenes The disharmony that we see between cytoplasm and nucleus in Triton shows that materials of the cytoplasm of the egg must have some permanency, but this need be nothing more than the per- sistence of already existing materials. The experiment is terminated by the death of the hybrid too soon to provide us with the evidence we are seeking of self-propagation in the cytoplasm. Rather than consider the cytoplasm as a whole, we must examine its parts, and the most obvious of these are the plastids which are responsible for the chlorophyll and carotinoid pigments of plants. The plastids are visibly self-propagating bodies in the lower plants and the same property is to be inferred in the higher plants, where they multiply as pro-plastids in a small and colourless stage. The plastids, moreover, contain ribose nucleic acid. Breeding experiments confirm the genetic character first attributed to them by Baur in 1909. Primula sinensis, in common with hundreds or thousands of other species of green plants, has a chlorophyll-less or albino variant which is mendelian in inheritance. The homozygote is so pale that it dies; the heterozygote is yellower than the normal, but survives. Thus albinism is here due to something having gone wrong with a nuclear gene which supplies materials necessary for the production or stability of clilorophyll. In the same Primula, however, there is another yellow-leaved variant which passes on its character in a different way. All the offspring from such a yellow mother are themselves yellow, no matter what the character of the pollen parent. A yellow father, on the other hand, does not transmit this character to his offspring. Thus the colour of the offspring depends purely on that of the mother plant and the results of a cross depend 169 THE CYTOPLASM on which way the cross is made, thus (putting the female parent first): — yellow X green > yellow green X yellow >- green Evidently this inheritance is non-mcndelian and therefore non- nuclear. The effective agent must be in the cytoplasm, though whether it is attached to the plastids or is free from them and, like the gene in the other case, acting on them, we cannot yet say. Crosses between species of Oenothera answer this question. Two green species when crossed together sometimes give yellow off- spring— but only one way. There are always differences between the reciprocal crosses. In Reimer's experiments, for example: — Oe. hookeri X muricata {curvans) > yellow Oe. muricata [curvans) X hookeri > green Thus the plastids o£ hookeri, which are green in the parent species, are yellow with the nucleus of the hybrid. But the plastids of muricata remain green in the hybrid. Now the nuclei in the reciprocal hybrids must be the same. It must, therefore, be the plastids or the cytoplasms that differ. One of them gets on with its parental nucleus but fails to get on with that of the hybrid. Thus both the nucleus and certain agents outside it play a part in the reaction. This is not all. The yellow F^ seedlings die but the green seedlings from the muricata mother occasionally develop flakes of yellow in their shoots. These are evidently derived from odd hookeri plastids which have come in with the pollen and sorted themselves out during development to make the full plastid complements of certain cells. Now, these yellow flakes can form the sub-epidermis of a shoot; they then produce the germ cells and can be used for breeding. When sclfed, such a shoot gives two kinds of offspring; there is a simple segregation of the hookeri-muricata [curvans) nuclear difference, to give two types: one homozygous hookeri and the other heterozygous like the F^ hybrids; the homozygous muricata [curvans), dies in embryo. The two kinds are alike in having hookeri plastids and differ only in the nucleus. The hcterozygote is again yellow; but the homozygote is green. Thus the hookeri plastids 170 PLASTOGENES which turned yellow with the hybrid nucleus turn green again when restored to their own proper nucleus. The sorting out of the colour determinant from mixed cells is a characteristic defect of cytoplasmic transmission. Now if the extra- nuclear determinant were of a molecular size it would scarcely be expected to sort out so quickly. It seems likely therefore, although mixed plastids have not so far been seen in the cells of variegated plants that it is the plastid itself which carries the determinant of its own character. This story shows that the plastids are permanent in their character. It also shows that this character is adjusted in the green plant to the character of the nucleus with which it has to do its work. The interaction of the two is that of two complementary genes, one mendelian, the other not mendehan. To what does a plastid owe its permanent or self-governing genetic character? So far we have attributed all permanence to genes, tacitly assuming that they were all nuclear genes; an assumption, incidentally, which Johannsen refused to make when he proposed the term. Now, it seems, we must recognize the existence of determinants having the properties of genes but existing outside the nucleus, and even capable of surviving their disagreement with the nucleus. They are speciaHzed by their attachment to the plastids and Imai has called them plastogenes. Since, as we saw, the plastids of different species differ in genetic character, their plastogenes, like other genes, must be liable to mutation. Like ordinary gene mutation it is in most plants a rare event. In most Oenothera species a mutation is seen once in about 2,000 plants. But occasionally, as with gene mutation, it may become frequent, and it is then seen to be under a genetic control which is partly external to the plastid itself The inheritance of certain kinds of variegation shows how this control works. In barley, Hordeum vulgare, Imai found a variety in which the plastids are characteristically unstable. They often change during growth from green to white. Sorting out then gives green and white cells, and later green and white variegated tissues. Most of the eggs contain green plastids and most of the selfed seedlings, therefore, give rise once more to variegated plants. But some eggs contain only white plastids and these eggs give rise to albino seedhngs which 171 THE CYTOPLASM die. A few (too few to show in the diagram, Fig. 41) even contain plastids of both kinds and produce plants which are variegated already as seedlings. Evidently a white plastid never changes back to green. The same proportions of white and variegated seedlings occur in GG CC p F Fig. 41. — The inheritance of variegation in barley, Hordeum vulgare, according to Imai (1928). Black circles arc green plants, white circles white plants, and hatched circles variegated. Size of circle indicates frequency of the type amongst seedlings. Superimposed on mendelian segregation is the purely maternal inheritance of irrevocably white plastids in a small proportion of the progeny of mutating (variegated) plants whether selfed or crossed (after Darlington, 1944). the cross with pollen of a normal green barley as in the self of the variegated plant. The remaining plants from this cross, however, stay purely green instead of developing variegation at a later stage. Plastid mutation has been suppressed. The suppressor must be a dominant nuclear gene, because in the Fg a quarter of the plants are variegated like their grandparent. Mutation begins again with the 172 PLASMAGENES correct nucleus. The reciprocal cross, green by variegated, gives exactly the same result except that there are, of course, no white eggs from the green mother and no white seedlings in the F^. In this cross the plastids which begin to mutate in the Fg have no ancestry of mutability. The mutability of the plastids is the same, no matter what their ancestry may have been. It is determined by the nucleus, although their character before and after mutation is determined by themselves within the range of nuclear variation studied in this experiment. This kind of situation (which is known also in maize and rice) puts control and independence in a new light. They are not so simple as they might seem to be. They exist in three spheres of action. The first is reproduction, and here each organ of the cell is controlled by the whole. The second is activity or production, and here again control is by the whole, but in this control the cytoplasm is the immediate, and the nuclear genes together with extra-nuclear genes the ultimate authority. The third is mutation, which is similar in principle to the second, although the detailed processes are entirely different and the predominance of the nucleus is even more obvious. We may indeed speak of certain genes inside the nucleus as being mutafacient with respect to particular other genes, either inside or outside the nucleus. Plasmagenes Not all extra-nuclear determinants are conveniently attached to markers so easy to pick out as plastids, or so clear in what they do. Their existence, however, is vouched for by the non-mendelian inheritance, of which reciprocal differences are the simplest evidence, appearing in a great number of crosses in flowering plants, ferns, mosses, and protozoa. The moss Fiinaria provides an example of a reciprocal difference in crossing. Crosses made by Wettstein between F. mediterranea and F. hygrometrica differ in the shape of the diploid structure, the sporo- carp, according to the direction of the cross. A diploid gametophyte can be produced from the sporocarp by regeneration following injury. Meiosis and segregation are thus avoided. Like the sporocarps whose unchanged nuclei they bear, these gametophytes differ in leaf-shape. Evidently the cytoplasms provided by the two species 173 THE CYTOPLASM differ, and indeed they continue to show this difference after thirteen years of vegetative propagation. The effect of the cytoplasm is maintained even through sexual reproduction. Ordinary haploid gametophytcs obtained as segregates from the hybrid sporocarp can be back-crossed as females to the male parent. After several generations of such back-crossing, the nucleus, of course, becomes effectively identical with that of the male parent. Yet, after eight generations, Wettstein found that the plants were still different from the male parent. The cytoplasmic determinant was still unchanged, unimpaired by the continuous government of an alien nucleus. In other words it has the properties of a gene, a plasmagene. The plasmagene, or plasmagenes, in Funaria are unconditional in their effect, so far as the experiment goes, but elsewhere the nucleus also plays a part. The Fj cross between tall and procumbent flax, Linum usitatissimum, is normal both ways. But, when the procumbent variety is the female or cytoplasmic parent, one quarter of the Fg is male-sterile ; the anthers abort. Here a nuclear gene of the tall flax is reacting with a plasmagene of the procumbent. Or perhaps it is failing to react because a plasmagene of the tall flax is missing in the procumbent. No other combination gives this defect. And it is constant over many generations back-crossed with pollen from the tall. In Linum, just as with the plastids in Oenothera, the nucleus and the extra-nuclear component in heredity fit one another in their ancestral or habitual combinations, but fail to fit in some of the combinations or re-combinations that can be produced by crossing. The commonest symptom of this failure in the flowering plants appears at the last and most delicate stage of development, the formation of the pollen. Where, in Petunia and Nicotiana, self- compatible and self-incompatible species (whose properties, as we shall see, depend on pollen adjustment) are crossed, it is the nucleus of the self-incompatible parent whose activity fails in the cytoplasm of the self-compatible one. Thus it seems that the incompatibility mechanism depends on the adjusted properties of the two com- ponents, nucleus and cytoplasm. Changes in the cytoplasm can be related to constructive adaptation even though they may not be generally necessary for it. 174 CYTOPLASMIC EQUILIBRIUM The first evidence of the particulate transmission of plasmagenes we find ill Epilohimn. Again, malc-steriHty appears when the nucleus of one species, E. hirsutum, lies in the cytoplasm of another, E. luteum, as a result of continued back-crossing of the hybrid E. luteum X hirsutum with pollen of hirsutum. But this pollen must occasionally carry over the operative particles of paternal cytoplasm in fertiliza- tion, for in the fourteenth generation of back-crossing about one plant in 400 has some shoots with fertile anthers. Self-pollination of these plants gives fewer male-sterile progeny when the male-fertile flowers are used, instead of the male-sterile flowers. The plasmagenes, therefore, sort themselves out in the growth of the plant, although they do so less rapidly than the plastids: they are probably more numerous. Cytoplasmic Equilibrium In the infusorian Paramecium aurelia, plasmagenes have revealed still more specific properties. Of the seven varieties which Somieborn has described, four show nothing but mendelian inheritance of their differences. In the other three, however, there is always a cyto- plasmic element in inheritance. This has been most fuUy investigated in the case of two antagonistic types: one of these, the Killer, poisons the other, the Sensitive, type when they are put together in the same water. They can, however, be crossed and it is some- times found that the difference is wholly cytoplasmic in inheritance. In other cases a nuclear gene also comes into play. Where this happens each of the two Fj individuals from con- jugation, in spite of their exchange of nuclei, resembles in character the cell from which it came, so revealing the cytoplasmic element in transmission. The offspring of the Sensitive individual continue to be entirely sensitive in the Fg ; but in the Killer line one quarter of the Fg goes back to the Sensitive character of the male grandparent (Fig. 42). Clearly the Killer character can be lost by virtue of a change in the nucleus as well as in the cytoplasm. Or to put it another way we have, as in flax, a character, Killer, which depends on the simultaneous presence of a nuclear gene K and a plasmagene, termed kappa. The relation between K and kappa is not confmed to their action, for it affects the reproduction of kappa. Where Kis introduced into 175 THE CYTOPLASM a cytoplasm devoid of kappa, there is no development of Killer; but K, in the way characteristic of nuclear genes, continues to maintain itsclt indefinitely, so that, should conjugation or any other process restore kappa to the cytoplasm, Killer behaviour would once more begin to appear. /^CMCD AXIOM Female or Cytoplasm Lines VjtrNtryA 1 IVJIN SENSITIVE KILLER P .._..„._... 1 El F2 / 1 • • 2 • • it @ © © Fig. 42. — The inheritance of the Killer property as shown in certain crosses between strains o{ Panmiccium aurclia by Sonneboni (1947). Witliin the circles is the square Killer gene dominant to its round allelomorph ; outside them is the square cytoplasm with kappa which depends on both the action of the gene in the nucleus and the presence of its prototype plasmagene in the cytoplasm to maintain it (after Darlington, 1944). The reproduction of K is independent of kappa, and indeed so far as we know of anything else in which the cytoplasms may differ. The reproduction of kappa is not, however, independent of K, for if iCis replaced by k in the nucleus of an individual whose cytoplasm carries kappa, not only is there a loss of the Killer behaviour, but kappa also disappears after four or five fissions. Evidently kappa cannot reproduce in the absence of K, and so disappears as soon 176 CYTOPLASMIC EQUILIBRIUM as the existing amount has been dissipated. Thus K, or rather some product of its activity, is necessary for the reproduction of kappa, but it cannot initiate the formation of kappa. The relevant product of K is one, but only one, of the precursors of kappa, and these precursors can be welded together to give kappa only by kappa itself. We have therefore to assume at least four substances in the cytoplasm concerned v^rith the Killer behaviour, viz: (i) the dif- fusible Killer substance; (ii) the reproductive particle, kappa; (iii) the product of the nuclear gene K; and (iv) at least one other precursor substance. These substances must have the following minimal relation : — Precursor ^^ ^ reaction governed by kappa kappa > Killer / 1 K product' Nuclear genes are co-ordinated in reproduction both with one another and with the cell. Each gene appears, therefore, with the same dose in each nucleus, and, subject to the number of nuclei being constant, in each cell. For a plasmagene like kappa, whose reproduction is under chemical rather than mechanical control, this is no longer true. The amount of kappa is not constant from cell to cell, or even within a single cell. It depends on the rate of reproduction of kappa relative to cell fission. In variety 2 of Paramecium aurelia, by raising the food supply, Freer was able to increase the rate of cell fission to 3 -4 times a day, whereas kappa could double itself only i -9 times a day. In these circumstances the concentration of kappa diminishes until it is finally lost. When the average concentration has become small, the pro- portion of cells devoid of kappa is found to be that expected from the random sorting out of particles. In this way Freer was able to demonstrate the particulate nature of kappa and to show that the concentration necessary for the Killer behaviour to be shown was 200-300 particles per cell. It was also evident that, so long as one particle remained in the cell, the full concentration, and with it Killer behaviour, would be restored when conditions supervened Elements of Genetics 177 M THE CYTOPLASM less favourable to fission, so that kappa reproduced more quickly than the cell. The concentration of kappa increases most rapidly when the cell is not undergoing any fission at all. This diminution of kappa by rapid fission proved impossible in variety 4, where the reproduction of cells and of kappa remained in step even when each doubled itself six times a day. The relation between cell multiplication and the concentration of kappa can be observed in another way. When the cells are heated up to 38 '5° C. kappa is destroyed before the cell is killed. Thus, by adjusting the time of exposure, the concentration of kappa can be reduced to a very few particles and the Killer behaviour lost. If the cell is restored to conditions favouring rapid fission, kappa will stay at this low level. But if the rate of fission is reduced by reduction of food, old age, or sexual processes kappa increases in concen- tration until it is back to the 200-300 particles per cell required for Killer behaviour. When all the kappa particles are destroyed by heat treatment the ability even to develop Killer behaviour is irretrievably lost, just as it is when kappa is totally lost by sorting out in variety 2. That the loss is due merely to the absence of kappa can be shown in another way. Normally conjugating cells separate in less than 3^ minutes and no cytoplasm is exchanged. If, however, the cells remain connected for 4 or more minutes (and this connection can be encouraged by heat treatment), cytoplasm may be exchanged. When this occurs between a Killer and a Sensitive, the Sensitive can develop into a Killer if its nucleus contains K. Its cytoplasm must have received kappa by exchange from the Killer, and once infected (as we may say) with kappa. Killer behaviour can ensue. The time taken for it to develop depends on the relative reproduction rates of kappa and cell, in the way we have already seen. It also depends on the length of time the conjugants have remained connected. When this is for only 6 or 9 minutes. Killer behaviour develops more slowly than when the connexion is maintained for a longer period. Evidently, as one might expect, fewer particles of kappa pass into the formerly Sensitive cell in the shorter space of time. The extra-nuclear component in the heredity of the Killer- Sensitive difference can, as we have seen, be understood in terms of a single type of plasmagene, kappa, characteristically associated 178 CYTOPLASMIC EQUILIBRIUM with Killer behaviour. There is no need to postulate a second type of plasmagene associated with Sensitive. This state is merely the absence of Killer and is associated consequently with absence, or at least over-low presence, of kappa. We must, however, recognize the more general possibility of two different plasmagenes, or alter- native phases of the same plasmagene, being associated with the alternative characters. We should then be forced to interpret changes in plasmagene constitution as being, at least in part, due to the suppression of one plasmagene or phase by the successful competition of the other. Such suppressiveness might at first sight resemble the dominance of nuclear genes ; but it must be different in that it will preclude, at least if complete, all sorting out of the particles or determinants that are suppressed. The suppressor must displace the suppressed. Whether in competition with some alternative type of plasmagene in this way, or whether reproducing without any direct and imme- diate competition, the level or concentration of a plasmagene must depend on its rate of reproduction relative to those other constituents which determine the division of the cell itself. We have seen how the increase and decrease of kappa are governed in this way. In yeast we can go even further and see how the supply of a particular substance from outside can govern the concentration of a plasmagene. Both Saccharomyces carlshergensis and S. cerevisiae, when placed in a medium containing melibiose, are unable to ferment this sugar. But carlshergensis soon becomes habituated or "adapted" to doing so, while cerevisiae is quite incapable of adaptation. When the two species are crossed, the diploid hybrid is found to be adaptable, and segregation into adaptable and unadaptable haploids occurs in its asci. It would appear that one, or possibly more, genes govern the difference in adaptability between the species. The gene itself cannot, however, be responsible for the immediate production of all the enzyme in an adapted individual. Rather, as Spiegelmann points out, we should regard the allelomorph, which confers adaptability, as constantly seeding the cytoplasm with small quantities of the enzyme, or of a precursor which, in conjunction with some substance available from the cytoplasm, can give rise to the enzyme. In the absence of melibiose, the enzyme or precursor 179 THE CYTOPLASM breaks down as soon as it is formed so that the concentration at equihbrium is so low as to be indetectable by the fermentation it causes. When mehbiose is added, the concentration of enzyme rapidly SACCHAROMYCES CARLSBERGENSIS CEREVISI/E P n t'% z z 3 3 NO MELIBIOSE IN SUBSTRATE MELIBIOSE RESTORED Fig. 43. — The inheritance of the capacity to ferment mehbiose in yeast crosses according to Lindegrcn and Spiegehiian (1944). The plasmagene responsible depends on the action of a nuclear gene for its origin, while for its survival in the absence of the nuclear gene it depends on the presence of mehbiose in the substrate. grows; so rapidly that it cannot be due to the mere accumulation of the output from the nucleus by removal of the counter-balancing destructive process. On the contrary the enzyme, or precursor, in the cytoplasm must take on the self-propagating properties of a plasmagene when mehbiose is added. The mehbiose itself conditions the plasmagenic properties of the enzyme which ferments it. 180 CYTOPLASM, NUCLEUS AND ENVIRONMENT We should expect, on this view, that the enzyme could persist and multiply in the cell eve aM^ay from the gene which was its ultimate origin, always provided that the supply of melibiose was maintained. That this is indeed the case is suggested by an experiment carried out by Spiegelmann, Lindegren and Lindegren (Fig. 43). They crossed the two species of yeast and allowed the diploid hybrid to form asci in the presence of melibiose. Instead of the ascospores segregating into ferm ntors and non-fermentors, all were fermen- tors so long as melibiose was supplied. When the melibiose was removed, however, so that the ability to ferment was lost, only half of the lines were able to readapt to its fermentation. The habit can persist with the plasmagene in the cytoplasm, in the correct chemical conditions; but once the plasmagene is lost it can be re- initiated only with the help of a nuclear gene. Thus, unless the experimental evidence is at fault, we must conclude that in yeasts a plasmagene which is produced or created by a nuclear gene can reproduce itself indefinitely without that nuclear gene. Cytoplasm, Nucleus and Environment The experiments with Paramecium and yeast together tell us a great deal about the properties of plasmagenes. Those with Para- mecium are most comprehensive in showing us how anything which affects the rate of cell fission correspondingly affects the plasmagene concentration. In the yeast experiments a precise chemical footing has been achieved, and two new relationships revealed. One is that between the nuclear and cytoplasmic determinants. We have always supposed that the nucleus can pass out materials into the cytoplasm. We now fmd that those materials can sometimes cut adrift. They can propagate themselves independently, with this proviso: they seem to require more specific precursors or food materials than does the nucleus as a whole, which is assisted in this respect by the co-ordinated supply and demand of its constituent genes. Plasmagenes are therefore liable to extinction by changes either in the nuclear genes or (in micro-organisms) in the environment. The substance produced by a nuclear gene and necessary for the development of a particular plasmagene may be described as a specific precursor. It might be supposed that, in requiring such a 181 THE CYTOPLASM precursor, plasmagenes differ from nuclear genes which multiply with even pace in all living conditions. This, however, would be a misconception. The nuclear gene also requires special precursors to live, but the only evidence we have of their absence is the breakdown of nuclear activity and reproduction, as a whole. This breakdown is always found in deficient, and often found in unbalanced, nuclei. The nuclear genes are specific in their food requirements. They are even specific in the balance of these requirements. They are non-specific only in the mechanical control of their reproduction, which compels them all to live and multiply in step with one another or all to die in step with one another. The second relationship which we can now see is that between the plasmagene and the outside world. The plasmagene can adapt itself directly to change outside the unprotected simple cell. But it can do so only by suicide. The effect is Lamarckian, but it is an effect of disuse only and not of use. In these circumstances we are bound to ask ourselves what the unicellular situation would look like in a multicellular organism undergoing differentiation. In flowering plants there are abnormal- ities which develop during differentiation and affect different parts of an individual, or of a divided clone, in different degrees. These abnormalities must depend in an unspecified way on environmental differences. Plasmagenes, being dependent for their multiplication in the cell on its chemical equilibrium, should be subject to change in external conditions. For single cells in the multicellular organism these external conditions include development. A precise example is afforded by the heredity and development of rogues. These rogues are off-types which turn up unexpectedly as sports or mutants in inbred strains or clones of many cultivated plants, such as peas and tomatoes where they come from seed, and potatoes and tulips where they come from tubers and bulbs. In peas their mode of inheritance was made clear by the work of Bateson. The rogue pea appears in most garden varieties, and has more pointed leaves and more curved pods than the type of the variety. It breeds true when selfed. It also breeds true, as a rule, when crossed either way with the type. There is no segregation in Fg, and we must suppose that a plasmagene is at work. It must clearly be sup- 182 DAUERMODIFI CATION pressive of its normal alternative And in addition it must be carried by pollen as well as by eggs : it is amhiliiiear. In one variety of rogue pea a difference shows itself between reciprocal F^'s in the early stages of growth. In both crosses some of the plants begin, not as fuU rogues, but as intermediates. When the rogue character has been carried by the pollen there are more intermediates; the rogue effect is diluted and the dilution, as we might expect, is greater in the male contribution. Now, the inter- mediates turn into full rogues as they grow up, but the intermediate parts bear flowers which can be bred from. The progeny of these flowers includes both rogues and types and the proportion of rogues varies : it is correlated with the degree of expression of the rogue character at the node where the flower was borne. Thus the genetic properties of the plant must change and develop with its external character. For the first time we see heredity, unstable heredity, going hand in hand with differentiation, abnormal differentiation. Dauermodification Since the plasmagene system depends on a chemical equilibrium and is sometimes unstable in development it should be possible to change it by special chemical or physical treatments during develop- ment. Such treatments, inaugurated by Jollos, have in fact been successful in producing what he called Dauermodifications, changes in cytoplasmic heredity: with protista such as Paramecium, arsenious acid and heat shocks were used, with Phaseoliis chloral hydrate, and with Drosophila merely high temperatures. These changes are inherited in the female line but, even with selection of the individuals most strongly affected in each generation, the changed type has always hitherto been found to disappear, after a few sexual generations {see Table 17). The mutant plasmagene has not the hereditary staying power of the natural ones we have considered. Does this imply that it falls into another and inferior category? Probably not. These new effects are usually deleterious, at least when the determining conditions have been removed. This means that the conditions of chemical equilibrium governing the new plasmagene will favour its disappearance. And since it can scarcely be indispensable to the cell, those cells which have lost it 183 THE CYTOPLASM by sorting out will inevitably enjoy a selective advantage during development. Alternatively we may say that the mutation is due, as with kappa, to a reduced concentration in a plasmagene that is Tl Fig. 44. — The origin and persistence of a defective capacity for development in Phascolus vulgaris after treatment with chloral hydrate. P, parent; PT, treated parent; T 1-6, derived generations (after Hotfmami, 1927). normally present, its regular concentration being recovered, as Somieborn has suggested, when sexual reproduction affords a respite to growth. Severe external shocks may have an effect, similar to the dauer- modification of plasmagenes, on self-propagating substances released into the cytoplasm by nuclear genes. Goldschmidt has found in Drosophila that it is possible in this way to imitate the effects of gene change by treatment of the pupae, producing what he has described 184 SUPPRESSIVENESS AND AMBILINEARITY as phenocopies. These, of course, differ from dauermodifications in not being heritable. TABLE 17 DECAY OF THE DAUERMODIFICATION OF PHASEOLUS VULGARIS AFTER TREATMENT WITH 0-75 PER CENT CHLORAL HYDRATE: 200 PLANTS IN EACH GENERATION, SELECTED FROM THE MOST AB- NORMAL INDIVIDUALS (HOFFMANN, 1927) Generation after Percentage of abnormal treatment progeny 1 73 2 67 3 47 4 52 5 8 6 4 7 0 Suppress'weness and Amhilinearity The working of the plasmagene system now begins to take shape. Intermediates show dilution and, in so doing, show that the system is divisible. There must be a number, a varying number, of like plasmagenes in each cell of the multicellular, as well as of the unicellular, organism. This number changes with development and its change appears as suppressiveness. The action of suppressiveness is revealed also in the origin of rogues. In one variety, where it happens that the mutation occurs later or more slowly than in others, the young rogue plants begin as intermediates. Evidently the rogue plasmagenes have not established themselves and reached equilibrium in the seedling. In crosses, provided that a necessary minimum number are carried over by the pollen, and in new rogues, provided that mutation is early enough, suppressiveness will ensure that the plant will ultimately be a rogue just as Killer admixture swamps non-Killer in Paramecium. The rate at which a plant becomes a rogue is a measure of suppressiveness. Thus suppressiveness implies amhilinearity even where, as with plasmagenes, inheritance carmot be equilinear. And if there were varieties having a nuclear genotype in whose company rogue plasmagenes were not suppressive, the change to rogue would never come about in them and the rogue mutation would never appear. i85 THE CYTOPLASM The relationship between ambilinearity and suppressiveness is further clarified by the inheritance of one of the many types of variegation of the fern Scolopendriiun vulgare studied by Anderson- Kotto. In this type of variegation the diploid sporophytes are pale green with dark green sectors, which are produced by genetic change early in development. There is some determinant which bleaches or starves the clilorophyll in the plastids and this determinant is unstable; or perhaps, by sorting out, it fails to get into every cell of the rapidly growing young fern plant. Whole sporangia carrying this determinant give purely pale haploid gametophytes. Other whole sporangia give purely green gametophytes. Since there is no segregation within sporangia where meiosis occurs, the deter- minant cannot be nuclear. Now the green gametophytes breed true, and when self-fertilized the pale gametophytes give variegated sporophytes once more. It is thus only the pale determinant which is unstable or is sorted out, and that is only in the young sporophyte. When the pale and the green gametophytes are crossed, however, the sporophytes are variegated and the result is the same both ways ; thus the scheme is as in Table i8. TABLE 18 INHERITANCE OF VARIEGATION IN SCOLOPEN DRI UM VULGARE 2x variegated X green X pale reciprocally I I 1 2x green variegated variegated The unstable determinant, being carried with equal effect by eggs and spermatozoids, cannot be in the plastids. Nor, in the absence of segregation after meiosis, can it be in the nucleus. It must be a plasmagene, and one which is again both ambilinear and, in the fertilized egg, suppressive. As a plasmagene the bleaching agent of Scolopendrium tells us two new things, A plasmagene, like a nuclear gene, can control the plastids. It can also be unstable, but its instability is no doubt due i86 SUPPRESSIVENESS AND AMBILINE ARITY merely to the negative aspect of suppressiveness already seen in Paramecium. In one stage of development it may fall as far behind the average rate of multiplication of their cells as, in another stage, it is in advance of it. And once it is sorted out and omitted from a cell, that cell and its posterity will be free of it. Summing up: teclinically we recognize cytoplasmic heredity by its non-mendelian habits. Alternative conditions are found, but they are not alternatively inlierited in the sense that their determinants are subject to segregation at meiosis. Further, their transmission is not equally by egg and sperm, but as a rule bears some relation to the volume of cytoplasm contributed. They are either matrilinear or ambilinear. A determinant that succeeds in being carried effec- tively by way of the pollen does so by virtue of being suppressive of its alternative." Plastogenes obey the rules of plastid distribution: they are carried by the pollen in some plants and not in others. Plasmagenes, on the other hand, vary in effect in the same individual, that is to say, in their concentration in the cell during development. And when in low concentration, plasmagenes (like the plastids) are liable to uneven sorting out and consequent somatic segregation. At higher concentrations they are subject to the chemical equilibrium of the cell, an equihbrium imposed by the total reactions of genotype and environment. It follows from these conditions that plasmagenes must be much easier to distinguish in one-celled organisms, whose environment can be changed experimentally than in the higher organisms, where the tissue is the invariable substrate o£ the cell. What we call plasmagenes in micro-organisms must have chemical counterparts in the cells of higher organisms. The reproduction of these counter- parts must be under the control of the whole organism during development, and they themselves must be concerned in determining the processes distinguishing one kind of cell from another. The parts played by the cytoplasm and the nucleus must be different in development just as they are in heredity. What those parts are we must now examine. REFERENCES ANDERSSON-KOTTO, I. 1930. Variegation in three species of fems. Z.I.A.V., 56: I 17-201. 187 THE CYTOPLASM BATESON, w. 1926. Segregation, y. Genet., 16: 201-235. BATESON, w., and GAIRDNER, A. E. 1921. Male sterility in flax, subject to two types of segregation. _/. Genet., ii: 269-275. BAUR, E. 1909. Das Wesen und die Erblichkeitsverhaltnisse dcr "Varictates albomarginatae hort" von Pclor^onium zonale. Z.I.A.V., i: 330-351. BLEiER, H. 1928. Karyologische Untersuchungen an Linscn-Wicken Bastarden. Genetica, 11: 111-118. DARLINGTON, c. D. 1944. Hcrcdity, development and infection. Nature, 154: 164-168. HADORN, E. 1937. Die Entwicklungsphysiologische Auswirkung einer dishar- monischen Kemkonibination beim Bastardmerogon Triton palniatus $ X Triton aistatus <^. Arch.f. Entw. mech., 136. HOFFMANN, F. w. 1927. Some attempts to modify the germ plasm of Phaseolus vulgaris. Genetics, 12: 284-294. IMAI, Y. 1937. The behaviour of the plastid as a hereditary unit: the theory of the plastogene. Cytologia, Fujii Jub. I'oL, 934-947. JOLLOS, V. 1938. Dauermodijikation. Handb. Vererb., Berlin. MiCHAELis, p. 1937. Untersuchungen zum Problem der Plasmavererbung. Protoplasma, 27: 284-289. FREER, J. R. 1946. Some properties of a genetic cytoplasmic factor in Paramecium. Proc. Nat. Acad. Sci., Wash., 32: 247-253. RENNER, o. 1934. Die pflanzhchen Plastiden als selbstandige Elemente der genetischen Konstitution. Ber. Math.-Phys. Kl. Sachs. Akad., 86: 241-266. SONNEBORN, T. M. 1 947. Recent advances in the genetics of Paramecium and Euplotes. Advs. Genetics, i: 263-358. SPIEGELMAN, s. 1946. Nuclear and cytoplasmic factors controlling enzymatic constitution. C.S.H. Symp. Quant. Biol., ii: 256-277. WETTSTEiN, F. v. 193 7. Die genetische und entwicklungsphysiologische Bedeutung des Cytoplasmas. Z.I.A V., 73 : 349-366. 188 CHAPTER 9 DEVELOPMENT AND DIFFERENTIATION How the Nucleus Acts: Lag How the Cytoplasm Acts: Gradients Co-operation and Competition The Sequence of Events The development of a many-celled organism from an egg or spore does not consist merely of a multiplication of cells ; it consists also of the development of differences in structures and properties between the cells making different tissues. The mature organism is differentiated. What is this differentiation due to ? Two facts assure us that it is not due to changes in the nucleus. The first is the capacity of different parts of plants and animals to propagate or regenerate the whole character of the organism unchanged. The second is the corresponding capacity of the nucleus and its constituent chromosomes to propagate themselves unchanged by mitosis. These two properties are opposite aspects of the principle of the genetic uniformity of the parts of an individual. It is a principle to which, as we have seen or shall see, there are many exceptions, due mostly to chromosome, gene, and plasmagene mutations; but they are rare exceptions which do not determine differentiation, and are not usually even connected with it. They merely prove the rule. We are left, therefore, with the cytoplasm as the changing factor in differentiation. The power of the cytoplasm in guiding the course of events is very well shown by the alternation of gametophyte and sporophyte in the mosses, ferns and higher plants. Normally the one is haploid and the other diploid. But this momentous difference does not decide the line which development will take in the haploid spore or the diploid egg. For when the spore happens to be diploid (through the failure of meiosis) or the egg happens to be haploid (through the failure of fertilization) the alternation of generations follows its regular sequence undisturbed. Indeed, by injury, in certain mosses and ferns the gametophytic tissue can be made to develop directly or "regenerate" on the sporophyte (as we saw in the last chapter) without the intervention of a spore. It is the ordinary process of development through the alternation 189 DEVELOPMENT AND DIFFERENTIATION of generations which seems to present the organism with more difficiik problems than its suppression. In each pollen grain or embryo-sac or fertilized egg a nucleus of a new type produced by mciosis or fertilization is suddenly implanted in a cytoplasm accus- tomed to, associated with, or generated by, a previous and different nucleus. This situation requires, as we have already seen, that the cytoplasm has to take the lead in deciding the course of development, sporophytic or gametophytic. It also has the consequence that these cells are the most sensitive of aU to upset in the relationship of nucleus and cytoplasm. In the case of embryo-sac and egg the large bulk of the cytoplasm, or the continued nursing by the mother tissue, relieves the crisis. But the pollen grain has to fend for itself. It is the pollen grain, therefore, which is the first to suffer for malad- justment, Male-sterility is the prevailing failure of plant hybrids and the prevailing outcome of cytoplasmic mutation. Of this principle we have seen some instances and we shall see more. How the Nucleus Acts: Lag The determination of gametophytic or sporophytic development is immediately cytoplasmic, but the observations of Andersson- Kotto on Scolopendrium vulgare show that it is ultimately genie. A single dose of the gene "peculiar" turns some of the spores into sperm. A double dose has the still more drastic effect of short- circuiting the reproductive cycle by causing the sporophyte to develop gametophytic sprouts. Similar transformations are, of course, a commonplace of genetics. Any number of genes are known which convert stamens into petals or carpels, petals into stamens or sepals and so on. In Drosophila one gene will restore the four wings of its ancestor, while another will turn the feelers, and another again the mouth parts, into legs. Thus, in the very process of proving that the cytoplasm is immediately responsible for differentiation, we are compelled to show that nuclear genes exercise the ultimate control. The cytoplasm is their agent. The way in which the nucleus acts through the cytoplasm has been well displayed by Hiimmerling's experiment with the single- celled Acctahularia to which we have already had cause to refer. An individual deprived of its hat will grow a new one. It wiU still do 190 HOW THE NUCLEUS ACTS SO if deprived of its nucleus in the rhizoid, provided that no stem w^as taken away with the hat. In other words there is a hat-forming substance near the hat. If the hat is cut away with a large part of the stem, it can still be formed again provided that the nucleus remains present. But the regeneration then takes time — time for the nucleus to make some more hat substance. This argument is clinched by species transplants. If the hat and stem of one species is grafted onto the rhizoid and nucleus of another the existing hat is unaffected. But, as we have already had occasion to observe, any new hat is modified in its development, taking more and more the form laid down by the nucleus as the length of its association with that nucleus increases. Thus the nucleus is producing the materials, the proteins, which eventually decide the character of the cell; but time is needed for these proteins to accumulate and to become effective by replacing those produced by the previous nucleus. The lag before a gene becomes effective is important in the ordinary life of an organism. There are some genes which become visibly effective within a single cell generation. In the pollen grains, following segregation of waxy from non-waxy at meiosis in a heterozygous maize, the two types are distinguished before the pollen is ripe by the presence or absence of starch. In Paramecium, on the other hand, 36 or more generations may elapse before a gene takes effect. Correspondingly, in the multicellular echinoderms, hybrids have been found to cleave at the maternal rate until the gastrula stage is reached, when the paternal genes make themselves felt. Their effects may then appear as a breakdown of development when the cross has been too wide, that is, when the hybrid genes act too dissimilarly from the maternal ones which have laid down the character of the cytoplasm. Most of the characteristic genes of mendelian experiments, as we have seen, act later in development; not indeed until the individual is approaching maturity. Those which act more quickly act too strongly : they are lethal. A few act more slowly and do not manifest themselves until the next generation. The best known example is in the determination of the direction of coiling of the snail's shell which follows the mother's genotype whatever the character of the father (Fig. 45). 191 COILING RIGHT LEFT Rr Rr F, lRR+2Rr+lrr 1 £ s£i 3 Families all right 1 Family ALL left Fig. 45. — The direction of coiling of the shell in Liimica pcrc^ra is determined by a single gene difference, the allelomorph giving right-hand coiling being dominant to that giving left. The direction of coiling reflects, however, not the snail's ov^n genotype but that of its mother. Thus all the offspring of a female have the same direction of coiling, even though she be a heterozygote. The segregation will appear in the phenotype as differences between the families of grandchildren. In this way the reciprocal F^'s differ in the cross between true breeding strains of right and left coiled snails. The F2's from these reciprocals, however, are alike and are also uniform. The segregation among the genotypes of the F.j's shows itself only in the F3 generation which consists of 3 families of uniformly right coiled snails to i of uniformly left coiled snails, no matter which way the original cross was made. 192 HOW THE NUCLEUS ACTS The same principle applies to the sex determination of the midge Sciara. Some females produce only male offspring, and the rest only female. The females are again of the two types: those like the mother and those like the father's mother. Sometimes we can see that the delay in the time at which a gene becomes apparent depends on the amount of store, or the degree of multiplication, of the substance it produces in the cytoplasm. The disappearance of the old gene's products and the accumulation of the new gene's products may be proportionately slow. Thus, with the moth Ephestia, Kiihn (1937) found that, in the progeny of pig- mentless aa mothers (crossed with Aa), the pigmentless a^eyes were at once distinguishable from the pigmented Aa eyes. But in the young progeny of pigmented Aa mothers the distinction was smothered. The pigment determinants in the cytoplasm of the egg provided full coloration until a late larval instar, so that only then did the segregation appear. These determinants, by the way, were diffusible, for transplanted Aa testes could darken the eyes of an aa host and of its young aa offspring. The extreme slowness in action, or in accumulation for action, is attained by the gene ' grandchildless" in Drosophila suhohscura. Females homozygous for this gene are fertile; but no matter to what male they are mated, their offspring are sterile. They can have children but no grandchildren. This gene's normal allelomorph must be producing something necessary for fertility, but producing it so slowly that its absence is not felt until a generation has elapsed. Many genes will obviously depend for their manifestation not merely on a threshold value of their product, but also on the oppor- tunity for its giving a different result from that of its allelomorph. The direction of coihng in the snail is determined at the second cleavage division of the fertihzed egg. If this opportunity is missed, it will not recur for a whole lifetime. All we can know of the gene's lag, therefore, is that it is somewhere between two cell genera- tions and a whole life-cycle in length. Since a gene's opportunity IS itself determined by the totahty of genes, it is easy to see how each gene depends for its expression on its fellows. LUimnts nfOcfuUics 193 N DLVLLUPMLNT AND DIl FERENTI ATION How the Cytoplasjn Acts: Gradients The genes thus express tliemselves by what they give to the cytoplasm. But they vary in how quickly they give it; and it varies in how soon it finds something to react with. Genes express them- selves consequently at different times in the course of development. Wc next have to see what the cytoplasm gives to the nucleus and Fig. 46. — Blood precursor cells from the bone marrow of a man with pernicious anaemia, the normal contrast in nucleic acid charge between reds and whites being exaggerated. Left, over-charged red with thick chromosomes and over-developed and multipolar spindle which will give several non-viable hypo-diploid cells. Right, under-charged white with long thin chromosomes and undcr-dcvclopcd open spindle which will give a single tetraploid cell by failure of anaphase. X 2,500 (after La Cour, 1944). how this varies with development. The control of the nucleus by the cytoplasm is most readily seen in the synchronization of mitosis (and meiosis) in groups of cells. Such a synchronization is found in all tissues where cell walls are poorly developed or absent. In the cleaving animal egg, and within follicles of the testis or anther, in the endosperm, in the twin diploidized cells of fungi, and even in the feebly-walled pollen grains in the orchids, all the mitoses move in step. They do so, apparently, because the supplies of materials necessary' for mitosis, especially proteins and nucleic acid precursors, are uniformly diffused in the cytoplasm of the whole group of cells. 194 J HOW THE CYTOPLASM ACTS How this works has been shown by La Cour in the differentiation of the bone-marrow of mammals. Here two opposite types of cell- lineage are separated; one, with high nucleic acid content in the cytoplasm, has rapid divisions of its nuclei, a strongly developed spindle and chromosomes heavily charged and strongly spiralized. This type gives rise to the red blood corpuscles. The other, with low nucleic acid content, has slow divisions, a poor open spindle scarcely capable of executing the anaphase movement, and chromo- somes feebly charged and weakly spiralized. This gives rise to the white blood corpuscles in their far smaller numbers. Thus two lines of development are set going in the same ancestral nuclei by two conditions of the cytoplasm, arising no doubt in different positions in the tissue (Fig. 46). The differentiation of the embryo-sacs and pollen grains of flowering plants exactly parallels that of the blood precursors. In the pollen of Angiosperms one of the daughter nuclei is pressed by the first mitotic spindle against the wall, while the other is left in the middle of the cell. The peripheral, or generative, nucleus forms a small cell and is rich in nucleic acid: it divides again to give the two condensed sperm nuclei, often before the germination of the grain. The central, or vegetative, one forms a large cell and is poor in nucleic acid : it becomes large and diffuse and loses first its staining power, and then its coherence. It vanishes without further mitosis. (The nucleus of the ripe red blood corpuscle, similarly drained of nucleic acid, similarly dissolves.) This differentiation must depend on the fact that the distribution of materials in the pollen grain before mitosis is not uniform. There is a gradient and the position of the mitotic spindle is adjusted to lie along this gradient. If the axis of the spindle lies crosswise to the normal (following heat shock) cells and nuclei with similar properties are produced: differentiation fails (Fig- 47). In a species of Sorghum extra heterochromatic chromosomes have been found to cause the vegetative, as well as the generative, nuclei to divide again. Extra nucleic acid, which the additional chromosomes make available in the cytoplasm, stimulates nuclear division in a way which recalls the plan of development of the gametophyte in the lower plants. In extreme cases the nuclear division may give four or five generative nuclei and thereby kill 195 DEVELOPMENT AND UI FFERENTI ATION the pollen grain, much as the excessive mitosis of a tumour may kill an animal. Pollen grains provide us, both internally and, as we saw earlier, externally, with crucial tests of developmental principles simply on account of their unique property of being independent organisms limited as a rule to two or three cells. The different sequences of their development arise from the reactions of different types of Fig. 47. — The four pollen grains of a tetrad in Scilla sibirica still exceptionally adhering attcr the first mitosis. As in Ascaris (Fig. 48), a radial spindle has given normal differentiation in three cells; an exceptional tangential spindle has given no dif- ferentiation in the fourth. The concentration of gcnerative-nucleus-forming substances, represented by stippling, is evidently central in the pollen mother cell (La Cour, original). nuclei, symmetrically or asymmetrically placed with respect to the spatially differentiated cytoplasm of a single cell. The immense range of types of embryo-sacs in flowering plants depends on the same principles operating in more elaborate settings, showing a range of behaviour depending, no doubt, on varying gradients in the distribution of nucleic acid in the mother cell (Fig. 50). Similar to the differentiation in space in the blood and pollen is that in time, whereby the series of mitoses in the development of a plant or animal is interrupted by meiosis, as a result, perhaps, of flooding of the nucleus with nucleic acid and the charging of the 196 HOW THE CYTOPLASM ACTS Fig. 48. — Cleavage, blastula and gastrula stages in the development of the egg of the thread worm Ascaris megaloccphala (diploid race with one gametic chromosome). A, the maternal and paternal chromosomes meet on the first cleavage spindle. B, formation of two cells, ditferentiated in cytoplasmic determinants, the dorsal with the potentiality for more rapid division, leading to fragmentation of its chromosomes at anaphase and loss of the distal heterochromatin, the ventral with the potentiality for regular mitosis without breakage. Cj and C._, views of the second divisions: the differentiation continues with progeny of the cell which divides in the same axis as that of the first mitosis. D, the T-shaped embryo rounded off to form the blastula. E, F, third cleavage division. G, the gastrula stage: 32 cells of which one only has the complete set of chromosomes and will give rise to the germ cells. The cytoplasmic germ-line determinants, wliich are directly visible in copepods and are here marked only by the presence of fewer vacuoles are shown here by stippling (cf. Bounure, 1939). Note. — The difference in mitotic rates suggests a gradient in nucleic acid content which should be exaggerated by the absorption of lost heterochromatin in the cytoplasm (after Boveri's figures 1910 et al., but conflicting with the diagrams in Wihon, 1900; Bovcri, 1904; Morgan, 1913; and Belar, 1928). 197 Ui;VELOPMENT AND Dli^I HREN TI ATIO N chromosomes before they have reproduced themselves. Diftbrentia- tioii in space as well as in time is responsible for the fragmentation of the chromosomes in particular embryonic cells in Ascaris. In those which arise from one region of the egg, and are predestined not to form the germ cells, the chromosomes break up at the anaphase ot mitosis and leave some unessential pieces of heterochromatin (lacking centromeres) on the equator (Fig. 48). hi all these instances the cytoplasm of particular cells is telling the chromosomes what to do. But paradoxically enough, it can only be passing on what it has had from the nucleus. For in polymitotic maize, a recessive gene compels all the pollen grain nuclei produced by a plant homozygous for it to divide again as soon as they are formed, and to go on dividing until the whole nuclear organization is destroyed. In this case it is clear that the gene has acted on the cytoplasm (already in the mother plant, for the effect is not seen in the individual pollen grains of the heterozygote) and the cytoplasm has acted in turn on the nucleus, a different nucleus. Finally, it should not be lost sight of that the cytoplasm is in immediate control of all the everyday details of mitosis. Each centromere and nucleolar organizer is a gene, simple or compound, and when all ot them keep time in nuclear cycle they are illustrating the unity of reaction of any species of gene to the changes in the common substrate, the cytoplasm. Other genes whose activities are less minutely observable evidently behave in the same way to give the disciplined uniformity of nuclear propagation from which the authority of nuclear action derives. Co-operation and Competition The interaction of the cytoplasm and the genes in the nucleus, each modifying the behaviour and activities of the other, is now becoming clear. It expresses itself also in the relations of cells of different genetic character. When, as Barber found in Uvularia, two pollen grains are formed at meiosis, one having extra chromosomes, and the other lacking these same chromosomes, the first survives poorly and the second survives not at all. If, however, the cell-wall that should have separated them is inhibited by a heat-shock, so that the two complementary nuclei are lying in tlie same C)l:oplasm, 198 CO-OPERATION AND COMPETITION COOPERATION OF NUCLEf 8 + f/5 Fig. 49. — Pollen grains adhering in dyads and tetrads in Uvuhria grandijlora (x = 7) after a heat shock which has deranged the second division spindle and consequently anaphase separation and wall-formation. The pairs of nuclei in the incompletely divided cells are unequal but complementary and therefore come into mitosis at the same time. In the two bottom cells an acentric fragment is present and the sum of the pair of nuclei is unbalanced, with less (in the prophase on the left) or more (in the metaphase on the right) than a diploid complement. One of the resting cells shows double nuclei, but all have perfect wall formation and are not synchronized (after Barber, 1941). 199 DEVELOPMENT AND DIH E RLNTI ATION then both arc entirely successful. More than that, the two arc per- fectly synchronized in mitosis. Thus each nucleus must be providing something necessary or useful to the other, and providing it by way of the cytoplasm (Fig. 49). none Plumhago yremivm n- hre-fushtK 4. -/ e xc ess '(Sync/jo/azi/ ^"|hy|bcrc/io/- hyfyochaJazy Fig. 50. — Variations in the development of the embryo sac in Angiosperms dis- regarding the number of spores which take part in it. Egg cell nucleus solid, other nuclei with one dot for each haploid set. 8 means 3 mitoses before egg cell formation, 16 means 4. -f and — refer to excess or deficiency in the number of mitoses at the chalazal end of the embryo sac and thus imply a gradient. E, the endosperm which varies in ploidy according to the number of nuclei fusing in the centre. Zca, Cooper, 1938. Ocnonc, Went-Maheshwari, 1936. Plumbago, Haupt, 193 1, rritillnria, Westfall, 1940. Pyrcthrum, Martinoli, 1939 ( Haploid Competition -p fron\ FIura//fy <^/ ■'- 9 is: Lmoiyo-sQcs (n-2n) Ai.1. TrfSi AfoA/ot/'OA/c £.S 25% ro/yji>e/b/ce g'4 Symjiefa/cp Ca.3 ^ C YMNOSPEKMS mosf ortieri j occasior>Qll>l f>ro-microf)y)ar: Onacrace/^ Rosa pro-cholaZQl: 'Jug Jans Galiu/n /\speruIcL TaKus TCTKASPORIC £.S. D: Oe/ione 7". rCa^ea ' ( Plun^baqo \ Leonfod on Act. Types Ahosjbor/es Cnerum 5yA/5/»o/t/c j/J-o/,/«/n f-/ierac/un\ Art€n\isici OcAr\.a. Citrus So r bus Fig. 51. — Competition of cells depending on the interaction of the genetically different products of segregation with physiologically determined gradients at different stages in development of the embryo sac in the flowering plants. Rubiaceae, Fagerlind, 1937. Juglans, Nast, 1935. Oenothera, Rudloff and Schmidt, 1932 {cf. Schnarf, 1936; Darlington and La Cour, 1941). other, the one at the micropylar end is favoured by its position in the gradient, that is by its cytoplasmic content. If a B is at the more favourable end it always grows. If it is at the less favourable end there is conflict between the relative advantages of the cyto- plasmic gradient and the nuclear genotype. After a short contest the nucleus wins and A is strangled by B. Or vice versa. The proportion of victories one way or the other is characteristic of each species (Fig- 51). This Renner Effect shows that nuclei and cells of different gcno- 20 T DF.VELOPMENT AND DIFFERENTIATION type co-operate only where they arc equally un£t, as in Uvularia, or equally fit, as in Ascaris, where presumably the complete and the diminished types of nucleus are, each of them, fitter for their own types of tissue. But where the nuclei arc unequally fit, competition at once eliminates the weaker and establishes genetic uniformity. The Rcnner Effect shows also that one nucleus is not inherently fitter or stronger than another; it is merely better fitted to the cytoplasm in which it lies — A to that of the pollen, B to that of the eggs. But both cytoplasms have been produced under the control of the same AB nucleus. They differ merely in their developmental history, in their cellular antecedents, in their chemical character as parts of different tissues. Thus tissues, owing to their different relative positions in the organisms, come to differ in the character of their cells, as shown by the reactions of nuclei and cytoplasm. This is just the same principle that we have already seen at work in Griineberg's lethal in the rat, whose manifold effects on different organs can be traced to a specific initial action on cartilage. The working of the nucleus cartying this mutant gene breaks down when it is faced with a specific job, that is when it lies in a specific cytoplasm. Transplantation experiments show us another aspect of the genetic specificity of tissues. Hadorn transplanted into normal larvae organs of the slowly developing "lethal-giant" Drosophila, at a time when its imaginal discs were degenerating. He found that transplanted testes degenerated, but the ovaries continued to develop and even formed eggs. The cytoplasm thus determines whether and how the gene will express itself. Further, Hadorn was able to select two lines throwing the homozygous recessive lethal-giants. These giants died, in the one early, and in the other late, in larval development. The lethal-giants thrown by reciprocal crosses between these two lines took after the mother line in the time of death. In other words the eggs differed in cytoplasmic constituents which reacted differently with the lethal gene and so determined the different times of death. These constituents must themselves have been slowly produced gene products, since the reciprocal difference was not continued in the grandchildren. 202 THE SEQUENCE OF EVENTS The Sequence of Events These experiments show something more than nuclcar-cyto- plasmic reaction. They show the cytoplasm as the field of reaction of products of different genes accumulated at different rates and over different periods. We may therefore ask, at this point, how it is that nuclear products accumulate in the cytoplasm over long periods. We have already seen that certain particles or determinants, the plasmagenes, can propagate themselves in the cytoplasm with a permanency and autonomy scarcely less than that of the nuclear genes. Wright has argued that growth could not proceed auto- catalytically, nor could differentiated tissues retain their uniform character, without some similar propagation of other cytoplasmic particles. Such particles would not necessarily continue permanently and independently. Indeed we have evidence of gradations between a high rate of propagation which leads to permanence and a low rate which leads to transience. These gradations are themselves under genetic control. Thus, as we have already seen. Freer found that in one variety of Paramecium the rate could be forced up to the point at which the kappa particles were unable to keep pace and were gradually lost. In another, the kappa particles reproduced so rapidly that they were never lost, even though the cells of this variety divided nearly twice as rapidly as those of the first one. These differences in rates of propagation of cytoplasmic particles — some autonomous and some presumably nuclear products — show how the cell may, at one time, maintain itself and, at another, vary in constitution in the course of development, in the way that we observe it to do. The propagation of any one part of the cytoplasm is thus conditioned by the whole; and, since the whole is modified by the activity of the nucleus, the nucleus is in effect modifying its own activity (Fig. 52). It does so after a delay which must depend on such physico-chemical conditions as the permeability of cell and nuclear membranes, the rates of diffusion of particles of different sizes, and the rates of propagation of nucleo-proteins of different shapes. These are the fundamental conditions of differentiation, and their essential consequence is the lag berv\'een determination and 203 DEVELOPMENT AND DIFI ERENTI ATION eficct, or between one reaction and the next in the chain of processes. We can now see how the reactions of nucleus and cytoplasm give a progression of events in the cell. But the progression must be an UJ OL >- o z UJ Fig. 52. — The genes of the nucleus draw upon the cytoplasm for the raw materials of their own reproduction and action. The products of this action pass back into the cytoplasm, where these products may have only a short life or may further multiply themselves by self-reproduction, in association with nucleic acid, according to whether the composition of the cytoplasm be fivourablc. hi so doing they change the composition of the cytoplasm and thereby affect the fate of the further gene products which pass into the cytoplasm, hi this way a constant nucleus can be associated with a changing cytoplasm, and hence with a differentiating phenotype, for the cytoplasm is the agent of the nucleus in action. hi the diagram the gene products are represented by lines in the cytoplasm, and their varying fates by varyuig lengths of these lines. The products interact with one another and with the plasmagencs (dotted line). The phenotype reflects the totality of the properties of the cytoplasm as shown by the wide arrows. The cytoplasm is also the chamiel by which external materials and conditions reach the nucleus or affect its action (after Mather, 1948). orderly one which passes through a regular lite cycle, in the general case, with sexual reproduction. This orderliness is achieved by the adjustment of the hereditary materials, especially of the genes in the nucleus, and it is lost when the genie adjustment is destroyed. One example will illustrate this very general principle. In the morning and evening campions, sub-species ofAIelandrium dioicum, the sexes are separated with XX females and XY males. In the F.^ 204 THE SEQUENCE OP EVENTS o{ a cross between them, however, some XY plants appear which bear female organs. From these Winge has been able to establish new hermaphrodite lines. Thus the regular progression of develop- ment must be determined by genes, for the ancestral order had been broken by the mixing of genes from the two stocks. The males were no longer clear males: they had lost some of their distinctive features. In each sub-species the genes had been balanced to give the same result, but they had been balanced in different ways. How this can happen is part of a much larger problem into which we shall have to inquire later. Summing up : we may examine development and differentiation in plants or in animals or in micro-organisms, in individual cells or in vast aggregates; we may approach the problem by way of hybridization or of transplantation. But whichever way we go about it, we fmd that the centre of the control of development is the nucleus whose sphere of action is the cell. The nucleus, by way of the cell, controls both heredity and development. In development the nucleus provides the constant basis for the regular changes in the cytoplasm, changes which could not be regular unless they had that constant basis. The nucleus and the cytoplasm have long seemed to be the opposite poles of biological study; but their new unity need no longer surprise us. Heredity now appears as the repetition of the series of changes which constitutes development, and the study of each is bound to require, more and more, the understanding of the other. REFERENCES ANDERSSON-KOTTO, I., and GAIRDNER, A. E. 1936. The inlieritance of apospory in Scolopendrium vulgare. J. Genet., 32: 189-228. BELAR, K. 1928. Die Cytologischen Grimdlagen der Vererbung. Berlin. BOUNURE, L. 1939. VOrigine des Cellules Reproductrkes. Paris. BOVERi, T. 1910. Die Potenzen der /l^cdrii-Blastomeren bei abgeanderten For- schung. Fest. R. Hertwig (Jena), 3: 133-214. BRACKET, J. 1947. Nucleic acid in the cell and the embryo. Syiiip. Soc. Exp. Biol, 1 : 207-224. BARBER, H. N. 1941. Chromosome behaviour in Uvularia. J. Genet., 42: 223-257. BARBER, H. N. 1942. The pollen-grain division in the Orchidaceae. J. Genet., 43: 97-103. DARLINGTON, c. D., and LA COUR, L. F. 1941. The genetics of embryo-sac develop- ment. Ann. Bot., N.S., 5 : 547-562. 205 DEVELOPMENT AND DIFFERENTIATION DARLiMGTON, c. D., and THOMAS, P. T. 1941. Morbid mitosis and the activity of inert chromosomes in Sorghum. Proc. Roy. Soc. B., 130: 127-150. GRUNEBERG, H. 1943- Congenital hydrocephalus in the mouse: a case of spurious pleiotropism. J. Genet., 45: 1-21. HADORN, E. 1937. Transplantation of gonads from lethal to normal larvae in Drosophila melanogaster. Proc. Soc. Exp. Biol, and Med., 36: 63Z ^34. HADORN, E. 1940. Pradetermination des Letalitatsgrades eincr Drosophila-Rassc durch den miitterlichcn Genotypus. Rev. Suisse Zool., 47: I3-I7'5- HADORN, E. 1945. Zur Pleiotropie dcr Genwirkung. Archiu. d. Julius Klaus Stiff., 20: 82-95. HALDANE, J. B. s. 1932. The time of action of genes, and its bearing on some evolutionary problems. Am. Nat., 66: 5-24. KUHN, A. 1937. Entwicklungsphysiologisch-genetische Ergebnisse an Ephestia kuhniella. Z.I.A.V., 73: 419-455. LA COUR, L. F. 1944. Mitosis and cell differentiation in the blood. Proc. Roy. Soc. Edin. B.,62: 73-85. MATHER, K. 1948. Nucleus and cytoplasm in differentiation. Symp. Soc. Exp. Biol., 2: 196-216. METZ, c. w. 1938. Chromosome behaviour, inheritance and sex determination in Sciara. Am. Nat., 72: 4SS-S20. RENNER, o. 1940. Kurze Mitteilungen iiber Oenothera. IV Uber die Beziehung zwischen Heterogamie und Embryosackentwicklung und iiber diplarrhene Verbindung. Flora, N.F., 34: 145-158. SAX, K. 1935. The effect of temperature on nuclear differentiation in microspore development. 7. Am. Arb., 16: 301-310. SCHNARF, K. 1 93 6. Contemporary understanding of embryo-sac development among angiosperms. Bot. Rev., 2: 565-585. SPURWAY, H. 1947. An extreme example of delay in gene action in Drosophila suh-ohscura. Drosophila Information Service, 20: 91. WILSON, E. B. 1925. The Cell in Development and Heredity. 3rd ed. New York. wiNGE,0. 193 1. X- and Y-linked inheritance in Mc/iJfiJriMm. Hereditas, 15: 127-165. WRIGHT, s. 1945. Genes as physiological agents. Am. Nat., 79: 289-303. 206 CHAPTER 10 VIRUSES, PROVIRUSES AND THE CONFLICT OF SYSTEMS The Propagation of Viruses and their Getietics Animal Tumours Neutral Hereditary Infection The Origin of Viruses Nuclear and Cytoplasmic Systems Outside the nucleus there are, as genetic evidence has shown us, reproductive bodies in the cell. The plastogenes we can distinguish as bodies, which must He in the proplastids or the mitochondria. These bodies can be stained differentially and they are proteins with nucleic acid attached, sometimes combined also with phospholipids. The nucleic acid is the ribose form, not the desoxyribose of the chromosomes. Other bodies, "microsomes" 50-200 m^u, in diameter, Claude has separated by centrifuging lung, pancreas and plant cells. The ribose nucleic acid in the cell, whose quantity is correlated with the rate of protein production, is largely localized on these bodies. They, therefore, probably include both the plasmagenes and the other self-reproductive proteins important in development and differentiation. The Propagation of Viruses Bodies or particles also occur in cells which manifest heredity in themselves, but do not determine it in the organism which includes them; for, characteristically, they get where they are, not through the egg or the spore, but by infection or invasion or even the transplantation of tissues. The most obvious of these bodies are the recognized viruses, parasites of animals, plants and bacteria. The larger viruses are complex in organization. Vaccinia, for example, contains tat, carbohydrate and even copper, as well as nucleoprotein. It also contains desoxyribose nucleic acid. Its particles are about 150 lUfx in diameter. The simpler forms are also proteins, but they usually have ribose nucleic acid attached to them. They exist in the cell as particles, either globular and 20-30 m/x in diameter, or rod- shaped, 15 mfjL thick and 125-1,000 m/x long. They may be separated 207 VIRUSES, PROVIUUSLS AND THE CONFLICT OF SYSTEMS troin the cell and even precipitated as crystals or liquid crystals. But they cannot propagate outside the cells of their host. Now, particles transmitted in heredity as nuclear genes or plas- inagenes are necessarily more or less suited to the organism of which they form a part. Any organism or race carrying such inescapable particles is likely to be eliminated by natural selection if they are not so suited. Viruses are under no such compulsion. They infect their hosts through air or water, or through the body of a specialized insect carrier or vector. Their effects on a host are, therefore, within a wide range inditFerent to the viruses themselves, and we find that these effects indeed cover a very wide range. At one extreme, perhaps, is the bacteriophage, which destroys its bacterial victim in a few hours. At the other are the viruses which cause variegation in Ahutilon or "breaking" in tulips. They have existed in equilibrium in the cells of the host for hundreds of years. If, as sometimes happens, they can be carried by the egg, they either eliminate all susceptible stocks, or they will become, and may have become, universal in a species and indistinguishable from its plasmagenes. The general effect of viruses consists in a distortion, great or small, of the metabolism of the host cell. The virus may act as a phos- phatase or other enzyme, which pushes the cell processes of the host in a new direction. In doing this it multiplies itself and thereby greatly increases, for example, the protein content of an infected plant. Like the tryptophanelcss gene in Neurospora, as Rischkov found, it piles up its by-products beside it as a body of inert protein. Healthy processes, as we saw, are such as lead to the least accumu- lation of useless or inactive product. The virus starves or deforms the normal or healthy synthetic processes and replaces them by new ones. A bacterial infection may consequently benefit from the co-operation of a virus (as in Hog Flu). In green plants, we fmd that the plastids and pigments suffer most obviously. With its specialized activity the virus naturally favours special tissues. Just as cholera is almost harmless when injected under the skin, so curly-top of tomatoes and sugar beet requires the phloem for its propagation. In animals which survive the crisis ot a virus attack, diffusible anti-bodies are produced which restrain its multi- plication and lead to recovery. Li plants anti-bodies, perhaps owing to their lack of diffusion, are knowm only in one instance. But 208 THE GENETICS OF VIRUSES commonly the activity of the virus is starved by its own success, and equihbrium can often be restored to produce the kind of situation we have in the broken tuhps. Plants can also be cured of viruses by growing away from them under conditions favouring the host at the expense of the parasite, just as the tissue of Scolopendrium or the stock of Paramecium gets rid of the plasmagene it has carried. In the extreme case, water at 45° C. will kill a Vinca virus in a few hours without killing the plant, just as kappa can be killed in Paramecium. The relations of viruses with one another, relations which are best seen in plants, are not without genetic interest. Many pairs of viruses have no effect on one another in the same host. They are neutral or independent. A second type of reaction is co-operation. For example, K. M. Smith has shown that Rosette Disease of tobacco is due to two viruses. These can be separated by inoculation, and also by the insect vector, which carries one of them only in the presence of the other; but they act together like two complementary genes in producing their effect, and in the self-multiplication on which this effect depends. A third type of reaction is antagonism. This is commonly shown more strongly between related or mutant strains of the same virus. Thus an infection with a less virulent strain may enable a plant, which does not develop anti-bodies, to repel an infection by a more virulent strain. Such a situation is obviously due to the two strains needing the same food— or, in terms of gene physiology, to their having the same precursors. And finally, as Bawden and Kassanis found. Severe Etch actively replaces Potato virus Y and Hyoscyamus virus 3 in plants in which these viruses are already estabhshed. The one virus, as it were, digests the other. Here we see an analogy with suppressiveness as between plasmagenes, and also with the synthetic sequences in Neurospora. The Genetics of Viruses Two aspects of the behaviour of viruses are of genetic importance. The first is that of the virus itself Viruses that have been most studied have been found, like all other pests and parasites, to exist in strains of varying virulence just as their hosts are of var\'ing liUmeitl.s o/Geiictiti 200 O VIRUSES, PUOVIRUSES AND THIi CONFLICT OF SYSTEMS susceptibility. Many of these, like the 50 tested strains of Tobacco Mosiac, have arisen under experiment and, we may say, by mutation analogous, as Muller first pointed out, to that of nuclear genes. The question arises as to whether this mutation is in some sense free and uncontrolled, or whether some control is exercised by the host over its frequency or direction such as we saw in the property of mutafacience. The answer seems to be that all degrees of control occur. Bacteria, infected by viruses, mutate to become resistant to them. The viruses can then mutate, becoming adsorbable on the bacteria, so as to be once more successfully virulent. In this situation, described by Luria, we seem to have free mutation on both sides followed by the survival of the fittest mutants. On the other hand there are many types of genetic change in which viruses, like bacteria, react so characteristically to a particular type of change of host or diet that control seems to be indicated. Of this kind is perhaps the weakening or attenuation of rabies, secured by Pasteur when he inoculated a series o£ rabbits with the virus, passing it from one to another. This weakening varies in rate with the different strains of rabies. There is also the fortification of other viruses by rapid passage through higlily susceptible hosts, as described by Holmes and Pirie. Similar changes have lately been induced in plant viruses. Certain recent studies of mutation in viruses and bacteria (which in this respect seem to be analogous) give us an opportunity of discovering the means of mutation itself. Two experiments have a parallel significance. Different strains o£ Pneumococcus differ in the specificity of their antigens, which in this case are polysaccharides carried in the capsule. Avery and others found that if the capsule is lost, the specificity of a strain A can be transferred to a strain B, by feeding it with dead cells of A, or, even more precisely, by feeding it with desoxyribose nucleic acid from the capsules of A. We have already seen that this nucleic acid is necessary for the reproduction of nuclear genes. We now see that it is necessary for the specificity of this reproduction. It provides the pattern. Similar transformations can be produced amongst the viruses causing different kinds of tumour in rabbits. Dead myxoma virus, in a large excess, can be used to convert living fibroma virus into one producing myxoma of the same strain-type as the dead virus. 210 ANIMAL TUMOURS In both bacterium and virus the living is copying the dead pattern, a pattern provided presumably, as in the chromosomes, by a nucleic acid template. But in the bacterium a gap is being filled in the structure, whereas in the virus a part is being taken down and replaced under the pressure of an excess of myxoma. Also the agent in the virus is presumably ribose and not desoxyribose nucleic acid. Thus at this molecular level we can again produce a pseudo- Lamarckian effect. We can control heredity from the outside and control it this time constructively. The second aspect of virus genetics concerns the variation of the host. Variegated Ahutilon striatum and the green A. indicum were both grafted by Baur onto different parts of a plant of a third species, A. arhoreum. The virus passed from striatum through the arboreum, without seeming to affect it, and entered the indicum, turning it variegated. Thus the virus multiplied in arboreum enough to travel through it, but not enough to injure it. Indeed it behaved as though it were an ordinary cell protein of arboreum. But if A. arboreum is replaced by a species o( Lavatera the virus cannot get across. Thus different species or varieties of host react in widely different ways with a particular virus, and in the intermediate case they may be "carriers" which show no effect. The difference between carrier and susceptible has been shown to segregate as a simple mendelian difference in the potato. The difference between carrier and non- carrier in the insect vector of Maize-Streak virus is likewise geneti- cally determined. And in the Potato Yellow Dwarf virus there are strains fitted to two different insect vectors. There is thus a triple genetic adaptation of host, virus and vector, a relationship we shall examine later. Animal Tumours The connexions between viruses and other cytoplasmic deter- minants are revealed by the different modes of origin or causation of animal tumours. These tumours, we must recall, are all abnor- malities of development inasmuch as they lead to some degree of return of differentiated or mature cells to an undifferentiated or embryonic condition with rapid mitoses, large nucleoli, and high nucleic acid and protein production. They are also abnormalities of heredity: they arise from genetic changes of particular cells, 211 VIUUSLS, PROVIRUSllS AND THE CONFLICT OF SYSTEMS inasmuch as the specific properties of a tumour, fibrous, epithelial, and so on, can be transmitted from one organism to another through a constant lineage of cells by transplantation. In its physiological character of excessive growth and unlimited mitosis, the animal tumour is analogous to certain conditions in plants. But properties of increased and even excessive grow^th can arise in plants by genetic changes of various kinds. In the liigher plants development can be extended by extra mitosis in the pollen grains, through the action of particular "polymitotic" genes or of extra heterochromatic chromosomes. In Sorghum, as we have seen, the nuclei formed by polymitosis kill the pollen grains by mere exhaustion of materials. Here, then, a nuclear change leads to a cancerous condition, doubtless by favouring excessive ribose nucleic acid formation. In the yeast Torula utilis, on the other hand, treat- ment by camphor produces a permanent genetic change, not affecting the chromosomes, which, according to Thomas, increases the growth rate and doubles the size of the cell. The yeast, then, must undergo an induced plasmagene mutation. In a single-celled yeast or pollen grain, high mitotic rate and high growth rate correspond with tumour formation in many-celled animals, where changes from a normal to the potentially unlimited type of growth can arise, and be detected, only somatically. In this unlimited growth there is a continuous transition from the encapsulated tumour or the wart, which is limited or merely cut off from further growth by its owai effects, at one extreme, to the other extreme of the malignant tumour whose cells are some- times capable of metastasis or migration to new sites. Correspondingly there is a transition in the type of cell division between the rapid mitosis like that of embryonic tissue and the hurricane mitosis like that found in the nucleic-acid-flooded nurse cells of plants and animals, where mitosis is rarely completed in order, and a jumble of haploid and polyploid cells is formed, strewn with fragments and micronuclci. These graded differences probably do not depend on any genetic distinction in principle. There is another type of difference, however, which matters a great deal genetically: that is the mode of transmission. There are three orders or levels of transmission of tumours. 212 A ANIMAL TUMOURS The first is that where the tumour-promoting property can be transplanted only in or by whole cells. This condition arises in mammals both spontaneously and artificially. It can be induced by radium, ultra-violet and X-radiation, and also by the action of specific chemical agents. These carcinogens, apart from the sterols, produce their effects on particular tissues. Thus p-dimethyl- aminoazobenzene (or butter-yellow for short) acts only on the livers of rats or mice, and dibenzanthracene only on the skin of mice. Moreover, some carcinogens act with some strains of mice and not with others. Evidently, therefore, the change is a specific reaction between the carcinogen and some protein or protein-system. And in respect of this reaction cells differ as between tissues and as between races, just as they do with regard to spontaneous tumours. The carcinogen is a mutafacient agent, a highly specific one. The new particles produced in this way must be self-propagating, since the growth they induce is characteristic and unlimited and it persists when the carcinogen is removed. As particles, however, they have not the capacity of invasion. They spread by cell division and they are inherited by cell-lineage: they are transmitted only by transplantation, that is by grafts. Their spreading through the body is due simply to the migration of whole cells. In all these respects the spontaneous tumours of man and other mammals agree with the induced tumours. All of them have obviously arisen in the stocks in which they were found. The second order of transmission is found chiefly in fowls. Several types of spontaneous tumours in fowls can be passed on by injection of filtrable particles of known properties. Thus the Rous Sarcoma particles are known to be about 70 m/x in diameter, and to contain a specific antigen in addition to a normal fowl antigen. These particles have the properties of invasion of cells, although not of infection. They attack only damaged cells and they can be trans- mitted only artificially. They must, therefore, again have arisen where they were first found. The third order of transmission is found in a type of breast cancer of the mouse, which is conditioned in its occurrence, as we might expect, by hormonal activity and by race, that is by nuclear geno- type. Transmission is by the milk of the mother or foster-mother. It follows the milk line, not, like a plasmagene, the egg-line. Again 21; VIRUSES, PROVIRUStS AND THE CONFLICT OF SYSTEMS tliis determinant must have arisen by the creation or mutation of a reproductive particle in the cell. But here we have just (only just) crossed the boundary from heredity to infection: we have a true virus. Beyond the milk virus there arc ordinary infectious principles such as those inducing benign epidermal tumours or warts. The only difference between them is that it is difficult to imagine the milk virus arising otherwise than from its host's proteins whereas the wart, owing to its infection, might have done so. In this last group the Shope papilloma of the cottontail rabbit, a known nuclcoprotcin, deserves special credit. It serves to bind the whole series together. For on inoculation into the domestic rabbit it loses its capacity for infection and acquires malignancy: it is transmuted from a virus to a plasmagene. In this scries we are dealing almost entirely with modified cell proteins of the animals which develop the tumours. We begin with something not inherited through the egg (which it would presum- ably kill) yet as close as possible to a plasmagene. And we end with something infectious without limit and a true virus. In the middle stage we have something half-way between, something with all the properties of a virus save that of natural infection. We have a prouirus. The genetic conditions of cancer development can now be provisionally defined. Excessive growth, either new growth or continuance of growth, may be directly determined by the genotype (nuclcus-cum-cytoplasm) of an individual at fertilization, and arise without any somatic mutation. This is true particularly of plants. But the typical development of tumour growth in animals is due, and could only be due, to somatic mutation. This mutation is itself determined by the customary interaction of genotype and environ- ment. Spontaneous tumours are so called because the genotype is the predominant variable in determining the mutation; induced tumours because the environment is predominant. A first class oi these tumours are capable of transplantation only as whole cells. A second class can be transmitted by cell-free filtrates capable of invasion, that is as proviruses. The possibility that nuclear mutation plays a part cannot be excluded in the first class. But all may have arisen, and the second class have certainly arisen, by change in selt- 214 NEUTRAL HEREDITARY INFECTION propagating proteins in the cytoplasm. Since the proteins in question occur only in differentiated cells and are not transmitted in heredity, we may regard them as analogous to a substrate-conditioned plas- magene like the melibiozymasc of yeast. And in their relation to the cell, their changes are analogous to the mutations of plasmagenes. This is the basis of origin of cancer expressed in genetic terms. An interesting paradox now arises with regard to the treatment of cancer. The cancerous cells owe their condition to a change in their self-propagating cytoplasmic proteins. To break them of their vicious habit we can do nothing directly. They are too numerous and are, in any case, chemically self-adjusting. Reversion is unkno\vn. Our sole remedy is to render their nuclei incompetent of supporting cell life. This we do by breaking the chromosomes in the resting nucleus (whose rapid development makes them singularly susceptible) so that when it comes to divide its wrecked products immediately die. X-rays or radium are the means of breakage. (Koller, 1947.) The genetic analysis of the origin and transmission of cancer puts us in a position for the first time to see and understand the triangle plasmagene-cell-protein-virus, or (if you will) heredity-develop- ment-infection in its true perspective. Tumour formation indubitably arises by mutation in the first instance, but its properties are in- herently untestable by the techniques of breeding. The distinction between the diffusible and the non-diffusible agent of tumour formation, on the other hand, throws into relief the distinction between the cell-protein which provides the basis of differentiation, and whose diffusion must be prohibited or at least controlled, and that which provides the basis of infection whose diffusion is inherently out of control. Thus cancer research has some fundamental importance: it lies at the meeting of the ways. Neutral Hereditary Infection The conflict between the requirements of heredity, development and infection becomes less as the particle or determinant becomes less decisive in its effect. L'Heritier and Teissier discovered in the cytoplasm of Drosophila the most neutral and indecisive of all par- ticles, and it has proved to have the ambiguity of transmission that might be expected. 215 VIRUSl.S. I'RO VI RUSES AND THE CONTLICT OF SYSTEMS Certain stocks of flies were found to die in the presence of moderate doses of carbon dioxide. This unique susccptibihty was a mere biological freak which, in nature, neither helped nor hindered its possessors. The property was inherited largely in the female line. But it could be transmitted to a proportion of the offspring by the sperm of susceptible males fertilizing normal eggs. It was ambilinear although not cquilincar. The susceptible offspring, however, could be cured by subjecting them to a temperature of 33"^ C. for 24 hours either immediately on laying or during pupation, i.e. during the periods of most rapid cell-division. Thus the COg-sensitive plasmagene failed to keep pace with the rest of the body proteins at this temperature, while at a normal temperature the reverse was the case: it was suppressive. So far we see a close analogy with peas and Paramecium. But there is something more. Supernumerary sperm will carry the plasmagene over to the normal egg. And finally, transplanted ovaries, or even injected lymph, of susceptible flics will infect normal ones and the susceptibility will show in a part of their progeny. In other words the determinant is a provirus as well as a plasmagene. It must have arisen by mutation in the ancestors of the susceptible stock and its properties of infection and diffusion indicate that it is of no more consequence in differentiation than it is in heredity. Neutrality again enables us to find an overlapping of the proper- ties of heredity and infection in the case of the piebald guinea pig, a fancier's mutant. The black pigment is produced both in the hairs and in the skin of the coloured areas. Pieces of coloured skin grafted in white areas lead to pigmentation of the adjoining unpigmented cells and these will, in tum, infect more white cells after a second transplantation. Thus, as Billingham and Medawar have pointed out, there are cytoplasmic determinants, expressing hereditary characters and capable of indefinite self-propagation, which never- theless are capable of difflision, invasion or infection. It seems likely that in animals, at least, invasions of this kind, but of limited scope, are concerned in processes of normal differentiation. The Origin of Viruses In both animals and plants there are a number of diseases which have not arisen by infection although they can certainly be propa- 216 THE ORIGIN or VIRUSES gated afterwards by infection or by inoculation. Such diseases as virus III in rabbits and perhaps Herpes zoster in man, referred to by Hobiies and Pirie, may appear spontaneously. Sometimes a nutri- tional defect or a bacterial infection seems to favour their appearance. In these cases the virus probably results from a distortion of protein metabolism in the individual in which it first appears. The new proteins may arise from, or instead of, normal self-propagating cell proteins which need not be transmissible as such in heredity and need not, therefore, be called plasmagenes. But their replacement is most easily described as mutation since the result produced is indistinguishable from plasmagene mutation in a plant. One step further on from these intrinsic viruses are those which are normal proteins in the individual of their origin, but become unfriendly in an alien cell. Such is, no doubt, the virus producing ascending myelitis in man after a bite by a healthy monkey, a monkey which can scarcely have been bitten by a man suffering from this disease. Here we have the origin of a virus by transplantation as opposed to mutation. Its fullest verification is in plants. Plant viruses have, like tumour viruses, several orders of trans- mission. A few may be conveyed by contact of stems or roots : the important natural ones are carried by parasitic insects. There are others, however, like those causing the yellowing o{Ahutilon, privet, and laburnum, which can be passed on only by grafting. As diseases, therefore, they are again artificial, and their origin must be due, either to a change in a cell protein, or more probably to the grafting itself, that is either to mutation or to invasion. They are not naturally infecting viruses, but proviruses. Their origin has been shown, in various groups, by grafting different varieties and species. Indeed, though the possibility of the infection of scion by stock as opposed to the incompatibility of the two has long been overlooked, it was first pointed out by Patrick Blair in 1720. The types of abnormality resulting from the graft-creation of viruses are manifold. Some instances of graft incompatibility are, it seems, due to the scion which produces substances poisonous to the stock, the effect being restricted even to particular localities as in Quick Decline or Tristeza of sweet oranges on sour stock, or Graft BHght ot Lilac on privet stock. 217 VIRUSES, 1'1U)VIRUSI;S AND Till- CONTIICT OF SYSTEMS Other instances are due to the release and invasion of self-propa- gating particles. All plants o( Lathynis tingitanns, in the experiments of Johnson, produced infective symptoms when their sap was injected into the bean Phascolus vulgaris. All plants of the potato clone King Edward, in experiments of Salaman and LePellcy, produced infective symptoms when grafted onto other clones. Similarly the variety of apple, Lord Lambourne, when grafted on different stocks, according to Crane, develops different characteristic malformations, rubbery wood and stunted chat fruit, which spread through the whole tree and, like the Ahutilon virus, will pass through wood of another variety. The mode and degree of infection varies in these various plants. The principle, however, is the same in all. Just as reciprocal crosses enable us to test the reaction of a plasmagene in the cytoplasm of one species with the nucleus of another, so grafting enables us to test the reaction of something reproductive in the cytoplasm of one species with the nucleus of another, indeed of many others and over a range of many genera. A protein produced in the cells of one organism can propagate itself injuriously in the cells of others. Invasion, a characteristic of true viruses, is a necessary means of demonstration of these agents. In the animal tumours, on the other hand, the principle is slightly different. A protein produced in one organism mutates so as to propagate itself injuriously in the same organism. Here invasion is no longer necessary for demonstration and does not always occur. We are bound to make the distinction between provirus and plasmagene on the basis of capacity for invasion following grafting. Yet this criterion is, of course, a purely practical one arising from diverse conditions. In some instances the difference between invasion and non-invasion may be simply one of the size of particles. In other instances the difference may be simply one between presence and absence, as it sometimes is with nuclear genes. The case of Yellows in ever-bearing strawberries demonstrates the great theoretical and practical importance — and difficulty — o£ the question of origin and transmission in making the distinction between plasmagenes and viruses. Some 30 clonal varieties of these strawberries are subject to this disease and many have been destroyed by it. The variety Progressive, made in 1908, first showed symp- 218 THE ORIGIN OF VIRUSES toiiis of Yellows in 1925. Other varieties have become affected as soon as three years, or as late as fifty years, after introduction. In symptoms of variegation it resembles those supposed virus diseases which lead to chlorophyll breakdown in other forms ot Fragaria and Riibus. But it is not transmissible by grafts; and it is transmissible by seeds. In inlieritance Yellows resembles the rogues in peas. Thus 82 selfed seedlings from 5 healthy plants were aU healthy at 27 months. TABLE 19 THE THREE ORDERS OF TRANSMISSION OF PROTEINS AND THE TWO MODES OF ORIGIN OF VIRUSES Transmission Plant Animal No true transmission, i.e. retained in transplanted cells without invasion (Plasmagenes) Rogue peas, etc. Strawberry Yellows Most Mammalian tumours, natural and chemically induced. (Cells invade but determinants do not) ARISING BY MUTATION By artificial filtration or diffusion from cells after grafting or trans- plantation (Proviruses) King Edward Lord Lam- bourne Rous sarcoma (fowl) CO2 — sensitive (Drosophila) Killer (Paramecium) By natural infection (Viruses) Milk-carried cancer (mouse) Warts Shope papilloma (rabbit) Insect-carr led plant and animal viruses ARISING BY TRANSPLANTATION But of 412 seedlings of affected plants, 21 per cent showed symptoms at 4 months and 82 per cent at 27 months (Morris and Afanasiev). It seems that yellowed plants undergo an irreversible cytoplasmic change. Their chance of doing so must depend on the nuclear genotype, for some seedlings are more quickly aiiected than others. The character of their progeny also depends on whether the cyto- plasm of their germ cells is healthy or diseased. Finally, external conditions, although none have been proved to influence the result, must account for the fact that some members of a clone succumb earlier than others. At a first glance we should say that the Yellows 219 VIkUSLS, PUOVIRUSES AND THE CONILICT OF SYSTEMS was a nucleus-controlled plasmagene mutation because it is not transmitted by grafting. Now, if it were an innovation or a presence, it might invade and be classifiable with the Abutilon principle as a provirus. But what if it is due to an absence, a deficiency in a necessary self-propagating protein which propagates itself too slowly ? A deficiency could not well invade. Our classification for the time being must therefore be one of convenience. And our difficulty is not so much to show the relationship of plasmagene and virus as to make a distinction between them of any validity beyond the requirements of their adaptation to heredity and infection (Table 19). Taken together the two scries of observations and experiments, animal and plant, show that all the general properties of the natural and noxious virus have arisen under experimental conditions. The special property of insect-carriage alone has not been demonstrated. Nor will it be readily demonstrated until the number of experimen- ters is comparable with the number of insects. We may say, therefore, that the properties of the reproductive particle, which are combined naturally in the plasmagene with heredity, and in the virus with infection, may, in these artificial conditions, be seen stripped of such accessories. Conditions produced by the CO2 test in Drosophila, by carcinogens in vertebrates, and by grafting and breeding in plants, demonstrate the two modes of origin of the natural virus: by mutation and by transplantation. Nuclear and Cytoplasmic Systems The close relationship between plasmagene and virus enables us to put the virus in its proper place in the scheme of things. But it does much more. It enables us to put the different agents of heredity, development and infection in their proper relationship. They all depend on self-propagating particles. These particles have different kinds of control over one another according to the systems in which they are organized and the methods by which they are propagated. These seem to be essentially of two kinds, the nuclear and the cytoplasmic, distinguished from one another in their intrinsic properties and separated from one another by a protective boundary. 220 NUCLEAR AND CYTOPLASMIC SYSTEMS The nuclear system depends on the protein fibres of its chromo- somes for its mechanical permanence and on desoxyribose nucleic acid for its propagation. It is protected from outside influences by the nuclear membrane in the resting stage, and, during mitosis, it is locked up by its spiralization and by its nucleic acid charge. It consists of linear arrangements of genes, which are of two kinds, LEVELS OF GENETIC STRUCTURE Desoxy- /e. A/. A. 1 /^ ^ 0 0 rO Inlegraffe6 GENES f'-OtsOrCf:^\ -O ti Simple GENES Vooooo-/ ; \ k \_^ RfBOSE Nucleic Acid -i 5 PLASTOCENESl < ) ^ / k — > J CI •«% 0 0 PLASM Diff "5 b) ACENES 1 HercSify VIRUS ES ! DiffSbylnfecnon \ ti ... — * .s*.^ ► CELL- PROTEINS Di'ffcrenfTafcS '\x\ Develojjmcnf 5 IG. 53. — The relationships'of position, propagation, and interaction in the cell, of the two types of nucleo-protein responsible for heredity, development and infection. The downward arrows (except within the nucleus) are physiological, the upward evolutionary (after Darlington, 1944). or at least lie between two extremes represented by heterochromatin and euchromatin in the cell, and by polygenes and major genes in heredity. The former are the small determinants with the simple products; the latter are the large determinants with the complex products. Between the two there are doubtless intermediates, tran- sitional forms which are also transitory and, therefore, elusive. It is natural to suppose that the more complex is built up from the more simple, by integration of different simples (Fig. 53). Later we shall 221 VIRUSES, PROVIRUSFS AND THK CONFLICT OF SYSTEMS come across evidence of this integration on a larger scale, at a higher level, when we come to the growth of genes in evolution. The cytoplasmic system depends for its propagation largely (and originally) on ribose nucleic acid, which, not having the indefinite power of polymerization of its nuclear congener, cannot organize a differentiated fibrous structure such as a chromosome. The cytoplasmic units arc, therefore, liniited in organization and size, corresponding in range to genes rather than to chromosomes. They depend, moreover, on chemical equilibrium, co-operation and com- petition, for the adjustment of their proportions and distribution. The larger and scarcer ones, or the ones associated with larger and scarcer corpuscles like plastids, can therefore be accidentally sorted out. Again, the larger ones may be derived from smaller ones; and both, in the course of development, may be derived as cell-products from the nucleus; and both may in the end be lost. In these changes the cytoplasmic system is subject to the nucleus. It is also, to a greater extent than the nucleus, exposed to the environment and, of course, to the effects of differentiation. Lastly, for their permanent trans- mission, cytoplasmic elements have another channel open to them than heredity: they can spread by infection. By this piracy they separate themselves, in adaptation and evolution, from the organism of which they form a part. And the same separation occurs when by mutation they come to nmltiply in such a way as to injure or kill their own mother-body. Comparing the two systems, we sec why the cytoplasmic system has decayed as a meclianism of heredity step by step as development and differentiation grew stronger. Already in the higher plants, in the rogue, there is a conflict between the requirements of heredity and differentiation. And in the higher animals heredity has been reduced to complete, or almost complete, nuclear control. In this process the efficiency of the fibrous organization of chromosomes in the nucleus as a basis of heredity and of recombination has given it a long-term advantage over the cytoplasm. Only in the bacteria do we perhaps see an organization of life balanced between the cytoplasmic and the nuclear levels. Within the individual there are thus systems of different kinds at work in determining heredity, development and infection. In their foundations, as self-propagating proteins in the cytoplasm, they are 222 NUCLEAR AND CYTOPLASMIC SYSTEMS indistinguishable. But in their work at difterent levels of integration, each has its own rules of behaviour, and each is related to the others by yet other rules of conflict and co-operation. With all these the genetics of individuals is deeply concerned, REFERENCES AVERY, o. T. et. al. 1944. Studies on the chemical nature of the substance inducing transformation in Pneumococcus types. J. Exp. Med., 79: 137-150. BAiTR, E. 1906. Weitere Mitteilungen iibei die infectiose Chlorose der Malvaceen und iiber einige analoge Erscheinungen bei Ligustnim und Laburnum. Ber. Deut. Bot. Gesel, 24: 416-428. BAWDEN, F. c. 1943- Plant Viruses and Virus Diseases. 2nd ed. Waltham, Mass. BILLINGHAM, R. E., and MEDAWAH, P. B. 1948. Pigment spread and cell heredity in the guinea pig's skin. Heredity, 2: 1-20. BURNET, F. M. 1946. Virus as Organism. Cambridge, Mass. CLAUDE, A. 1943. The constitution of protoplasm. Science, 97: 451-456. CRANE, M. B. 1945. Origin of virus. Nature, 155: 115. DARLINGTON, c. D. 1944. Heredit)', development and infection. Nature, 154: 164- 168. DARLINGTON, c. D. 1949. The plasmageuc theory ot the origin of cancer. Brit. J. Cancer (in the press). DUBUY, H. G., and woods, m. w. 1943. Evidence for the evolution of phytopatho- genic viruses from mitochondria and their derivatives. II Chemical evidence. Phytopathology, 3 3 : 766-777. HADDOW, A. 1944. Transformation of cells and viruses. Nature, 154: 194-199. HADDOW, A., KON, G. A. R. ct. al. 1947- Chemical carcinogens. Br. Med. Bull., 4: 309-426. HESTON, w. E., DERiNGER, M. K., and ANDERVOUT, H. B. 1945. Gene-milk agent relationship in mammary tumour development. J. Nat. Cancer Inst., 5: 289-307. HOLMES, B., and pmiE, A. 1937. Biochemistry and the pathogenic viruses. Perspectives in Biochemistry. Cambridge. JOHNSON, J. 1942. Studies on. the viroplasm hypothesis. J. Agr. Res., 64: 443-454. KIDD, J. G. 1946. Distinctive constituents of tumors and their relation to autonomy, anaplasia and causation. C.S.H. Symp. Quant. Biol., 11: 94-110. ROLLER, p. c. 1947. Abnormal mitosis in tumours. Brit. J. Cancer, i : 38-47. l'heritler, p., 1948. COo sensitivity in Drosophila. Heredity, 2: 300. LLTRiA, s. e. 1945. Mutations of bacterial viruses affectmg their host range. Genetics, 30: 84-99. MORRIS, H. E., and AFANASiEV, M. M. 1944. Yellows, a non-infectious disease of the progressive everbearing strawberry in Montana. Montana State Coll. Bulletin, 424. RISCHKOV, v. L. 1943. The nature of ultra-viruses and their biological activity. Phytopath., 33: 950-955- 223 VIRUSES, PROVIRUSES AND THE CONFLICT OP SYSTEMS ROUS V 1936. Virus tumors and the tumor problem. Am. J. Cancer, 28: 233-272. SMITH. K. M. 1945. Transmission by insects of a plant virus complex. Nature, IS5: 174- - ] • N- THOMAS, P. T. 1945. Experimental mutation ot tumour condition^. Mature, 156:738-740. 224 PART III POPULATIONS Elements of Genetics CHAPTER I I ADJUSTMENT AND BALANCE Levels of Adjustment — Haldane's Rule — Genotypic and Segregational Sterility — Inbreeding Depression and the Hyhridity Optimum Levels of Adjustment Heredity, as we have now come to see it, depends on an organiza- tion of chromosomes, genes and cytoplasmic determinants which are adjusted to one another in the development and reproduction of each individual. We have recognized this adjustment at three levels; the organismal adjustment of cells with one another; the cellular adjustment of the parts of single cells, in particular of nucleus and cytoplasm; and the nuclear adjustment, or balance as we have called it, of the genes within the nucleus. There is, of course, a further adjustment of the whole system with the environment. Each level of adjustment has its own rules. The cells which work together as parts of the same body or soma normally have identical nuclear genotypes. This relation may, as we saw, be altered by gene or chromosome mutation in the body, with consequences ranging from a mere mosaicism of colour to a mixing of tissues normally confmed to distinct individuals, as in gynandromorphs. The mixture of unlike cells may also lead to intersexuality and sterility. The disparity of genotypes that can be associated in one soma has been investigated by grafting experiments. In mammals and birds, such grafts are successful only when the genotypes are very closely related. Otherwise antigenic differences come into play and the transplant is sloughed off. In other animals the tissues seem to be more nearly autonomous in their behaviour, for transplantation between genetically dissimilar individuals is more widely successful and in the insects, as we have seen, it has even led us to a clearer understanding of how genes act. In plants grafting can be accomplished between still more widely separated forms, even between genera, to give more or less stable graft-hybrids. We can put the aerial parts of one species onto the 227 ai)justmi;nt and haiance roots of another. Or we can put a skin of one round a core of the other, as in Crataegoniespilus, the graft hybrid of the hawthorn and the medlar. In this way, too, as we saw, the hcxaploid and diploid species of Solamim may be combined to give results whose appear- ance depends on which is inside and which is outside, and on how many layers of each there are. These chimaeras are not always fertile, nor are they always stable. An irregularity at the meristem in Cytisus adami frequently exposes the yellow flowers of its Laburnum core; and less often the purple flowers of the Cytisus pmptireus skin succeed in displacing the mongrel colour. The chimaeras produced by somatic mutation may also be of these various kinds, and they show the same characteristic instabilities. Indeed many ornamental plants surprise the gardener when they reveal their chimerical condition, and its origin by mutation of gene or plastogene, in this way. The normal adjustment of cell parts, of nucleus with cytoplasm, is not constant like that of cell with cell. On the contrary, it is regularly changing as the cytoplasm changes; and, as we have seen, it is on these changes that the processes of development and differen- tiation depend. Nevertheless, the changes must follow a fixed path if the outcome of differentiation is to be an organism that will work, and this is secured by the subordination of the changing cytoplasm to the constant nuclear genotype. If we put a nucleus into a cytoplasm which is not of its accustomed type, one of the two has to give way or the disharmony will lead to a failure of the normal sequence of changes and so to early death, as we saw in the various merogons with the cytoplasm of one species and the nucleus of another. When one gives way it always has to be the cytoplasm: the nucleus forces the cytoplasm into its own pattern, as in the Acetabtdaria grafts. Disharmony of nucleus and cytoplasm can be produced by hybridization as well as by grafting. When made one way, a species cross in Epilobium or Streptocarpus may be successful in giving a hybrid capable of adequate development. Yet when made the other way the hybrid may be abnormal in greater or less degree. The hybrid nucleus can work with cytoplasm, or more precisely with the plasmagenes, from one parent, but not with those from the other. Such disharmonv is not, however, the usual cause of abnor- 22S IIALDANL S RULli iiiality, or failure in some vital function, in wide hybrids. Much more commonly these are to be traced to disharmony at the third and most fundamental level, that of the genes within the nucleus. The father's chromosomes and the mother's cannot work together properly in the hybrid. HaUaiies Rule Disharmony, or unbalance of the genes, arising from hybridiza- tion affects every nucleus of the soma, from that of the fertilized egg onwards. It may therefore express itself in any stage of develop- Homogametic Sex X Heterogametic Sex AAXX X aaxy AaXx AaXy F, of HOMOGAMETIC sex with balance intermediate between that of the two parents aaxx X AAXY F, of HETEROGAMETIC sex with new balances different in the reciprocal crosses FEMALE . . XX and MALE . MALE ... XX and FEMALE . XY in Mammals and Flies XY in Moths and Birds Fig. 54. — Diagram to show the relation of balance and fertility in reciprocal crosses between species in animals. Chromosomes of opposite species shown in large and small type. nicnt, by untimely death or by loss of vigour; or in reproduction, by sterility of greater or less degree. The genie balance should be least disturbed where the contributions of father and mother are each complete and balanced, as in the case of a first cross between two races or species. And the greater the departure from this balanced 129 ADJUSTMliNT AND BALANCE completeness of the parental contributions, the greater should be the loss of vigour and fertility in the hybrid. One consequence of this rule is to be seen in crosses between sexually differentiated species, hi such cases the sexes very commonly differ in their vigour and fertility; and, as Haldane pointed out, it is the XY or heterogametic sex which is less vigorous and less fertile and even less frequent. The difference may be so extreme that only one sex survives to maturity, as in the moth crosses Chacrocampa clpenor X Mctopsiius porcellus and Deildphila euphorhiae X -D. galli. Here Federley has found that although the males survive the hetero- gametic females die as pupae. Or the difference may not appear until germ cell formation, as in Drosophila pscudoohscura X D. persimilis, where the females arc fertile but the males, otherwise normal, are made sterile by a wholly irregular process of meiosis. Why should disharmony affect the heterogametic sex more strongly l In the homogametic sex both autosomes and sex chromo- somes are derived equally from the two parents. In the heterogametic sex they are derived unequally; indeed the sex chromosome contribution may come entirely from one parent. The sex-autosome balance can be neither that of one parent, nor intermediate between those of the two. It is much more likely, therefore, to be out of gear in the heterogametic than in the homogametic sex (Fig. 54). The difference between the two is thus an expression of the same kind of balance between sex chromosomes and autosomes as that which is responsible for a DwsopJiila with two X chromosomes growing into a female if diploid, but into an intersex if triploid, for the autosomes (Fig. 55). Now comparison shows that the composition of the sex chromo- somes varies between species. One arm in the X chromosome of both D. pscudoohscura and D. persimilis, for example, corresponds to an arm of an autosome in D. mclanogaster: there has been a translocation in the history of the species. Smaller structural changes and genie changes will be even more characteristic of the relations between the sex chromosomes of different species. The balance of sex chromosomes and autosomes must therefore often differ between species, and we might expect to fmd that reciprocal species crosses give similar results in regard to the liomogamctic sex, but differ in their heterogametic offspring (Fig. 54). This is in fact true. In 230 GENOTYPIC AND SEGREG ATION A L STERILITY Federley's moths, the crosses reciprocal to those mentioned gave offspring of both sexes that survived to maturity. The males from the Drosophila cross were sterile, no matter which way it was made. But the testes, which were of normal size from psciidoohscum X persimilis, were abnormally small in tlie reciprocal progeny. X XX XXX A ? — — AA S ? ? AAA 6 f ? Fig. 55. — Diagram showing the relation of the X/Autosome balance to the sexual character in Drosophila. cJ = supermale; 9 = superfemale ; rf = intersex (all sterile). Note. — (i) Y is without effect on sex-grading. (2) Haploid is known as ovarian tissue. (3) The homogametic balance can be produced in three ways, the hcterogametic in only one. Genotypic and Segregational Sterility Sterility is the only outcome of hybridity in these male flies. Nevertheless it is to be traced to the same kind of disharmony that causes early death of the female moths. It is a direct result of unbalance in the zygote's genotype, and we may therefore refer to it as genotypic sterility. The sterility of the diploid RapJiano-hrassica and of the diploid Primula kewensis might appear at first sight to be due to the same cause, but one significant observation shows that this cannot be so. Both of these plant hybrids recover fertility when the number of their chromosomes is doubled; whereas tetraploidy leads to no such recovery in the male flies. Now tetraploidy does not cause any essential alteration of genie balance: it caimot therefore remedy genotypic sterility. It does, however, suppress segregation, and so it would appear that, in contrast to the 23 T A DJ U S T M r. N T AND I! A I A N CE sexually ditfcrentiated animals, the plants show segregatioiwl sterility. hi Raphano-hrassica, pairing of the chromosomes is almost absent from meiosis, so that gametes may receive any number, as well as TABLE 20 AVERAGE FREQUENCIES OF CHIASMATA IN THE BI- VALENT CHROMOSOMES IN RELATION TO THE FAILURE OF METAPHASE PAIRING AND THE POLLEN FERTILITY OF DIPLOID (;c = 7) GRASS SPECIES AND THEIR DIPLOID HYBRIDS (PETO, 1933) Plants Cross I (L.p. $) Cross II {F.p. ?) Xta.p.biv. Good pollen % Xta.p.biv. Good pollen "/, Loliiim perenne 1-81 92 1-59 25* Festuca pratensis 1-88 81 1-59 87 Fj (all male-sterile) . . 1-71 13 l-62t 1-62(2%) 1-61(2%) 1-57 0 0 0 0 Backcross to L.p. q . . 1-80 85 1-76 74 1-66 92 1-50 61 1-66 90 1-66 77 1-57 84 0-80(35%) 40 0-62(48%) 0 Backcross to F.p. q . . 1-43 0-88(33%) 37 23 Note: Xta - chiasmata. The percentage figures are univalents where these occur. * The LoUum parent in this cross was itself a varietal cross, hence the low pollen fertility. I Used for backcrossing. any combination, of chromosomes. In Primula kewensis pairing is sufficiently regular for the great majority of the gametes to receive the customary 9 chromosomes, one from each pair, but these 9 may represent any combination of the maternal and paternal sets. Evidently the property of harmonious working, which is shown by the complete paternal and maternal sets, is not shared by their individual parts. Owing to structural changes in their ancestry these 232 GENOTYPIC AND SEGREGATION AL STERILITY parts are differently distributed in the two species. So segregation leads to unbalance, either numerical or genie, in the gametes of these hybrids, just as it does in those of triploids and interchange hybrids. The unbalanced gametes cannot survive, and sterility is the result — until segregation is suppressed by tctraploidy. 1.0 o.9\ o.s 0-7- 0.6\ 0.5- 04- 0.3- 0.2- 0.1 / X xv / & ti/ / Y .... / f FL-L FL-F,-' 4'/ & (67) LF-L.- C(S2) LF FL -BSIft- o.S CHIASMATA per BIVALENT Fig. 56. — Relationship of chiasma frequency, chromosome pairing and pollen fertility in LoUum-Festuca hybrids. The graph shows that: (i) The upper limit is set by chiasmata to pairing and by pairing to fertility, (ii) The F^ is male-sterile probably owing to a defective reaction of the hybrid nucleus with both parental cytoplasms, in respect Festiica is regularly worse than LoUum. (iii) The backcrosses show segregation controlling chiasma frequency and hence fertility. Each point represents an individual: circles for plants used as male and female parents; F, Festuca pratcnsis; L, LoJiuin pcrcnne; squares for F^; triangles for backcrosses; numbers in brackets for percentage of potential bivalents formed where first metaphase pairing is incomplete (based on Peto, 1933). The distinction between segregational and genotypic sterility is thus seen to lie in the time of taking effect. Genotypic sterility is due to relative unbalance of the contributions of the two parents as wholes, which gives absolute unbalance of the diploid Fj hybrid. This is especially noticeable when sex chromosomes are concerned, for in them the Fj has something of the nature of a backcross. 23: ADJUSTMENT AND BALANCE Scgregational sterility, on the other hand, results where the con- tributions of the two parents are relatively balanced as wholes, but balanced in different ways, so that segregation and recombination of their parts give unbalance. This unbalance expresses itself gcno- typically in plants in the haploid generation, in the gametes that will give the Fo or backcross; but in animals where, as we have seen, unbalanced gametes regularly survive and function, scgre- gational sterility appears only as the death of the zygotes of the Fo or backcross. When the unbalance due to segregation is not so drastic as to lead to death, it may still cause a developmental upset in the individuals obtained in a backcross or Fg. This upset may, of course, express itself at germ cell formation so that it leads to a reduction in fertility. In this way segregation in the F^ may lead to genotypic sterility in the next generation (Fig. 77). These relationships are well shown by the comparison of the Fj and the backcross generations in the cross between the two grasses Lolitim pcrennc and Fcstuca pratcnsis examined by Peto (Table 20 and Fig. 56). Reciprocal crosses were made. The parents were different indi- viduals with slightly different chiasma frequencies but they were all highly fertile except one, which was itself a varietal hybrid and doubtless owed its bad pollen to segregation. Both of the reciprocal Fj's had chiasma frequencies similar to the parents, and showed no evidence of structural hybridity, apart from 2 per cent of potential bivalents unpaired in two plants. There was, however, a great reduction in pollen fertility, again presumably due to segregation. The results of this segregation appeared clearly, though in a dif- ferent way, in the next generation, which was the first backcross to Lolimn pcretine. Here 5 plants had normal chiasma frequencies, no failure of pairing and high fertility; but 2 plants had chiasma frequencies greatly reduced, in fact below the level necessary for regular pairing. Their fertility was therefore greatly reduced. But this reduction in fertility, in contrast to that of Fi, was presumably due in a great part to the failure of pairing. Now the dissimilarity of the pairing chromosomes must have been at its greatest in Fj . The decrease of chiasmata and pairing cannot therefore itself have been due to an increase in dissimilarity. Rather the failure of pairing, and with it the sterility, must have arisen from a genie upset 234 INBREEDING DEPRESSION AND THE IIYBRIDITY OPTIMUM following segregation in the gametes of the F^ hybrids. In other words the sterility of the backcross is largely genotypic, but it is a consequence of the segregation which caused an even greater sterility in Fi. It is also, we may notice, superimposed on a small amount of segregational sterility in the backcross plants themselves. A single mendelian difference, which might be supergenic and distributed over most of one chromosome, would account for the sesreeation seen in this backcross of the Fi from the Lolitim mother. In the second cross, where Festuca was used as the mother of the Fj, segregation also appears, but it is not so clear as in the first. Probably a larger number of differences are being recombined. For this reason, however, the correlation between chiasma and univalent frequency and pollen fertility are all the more clearly manifested (Fig. 56). There is also a small overall difference between the two reciprocal crosses and their progenies. The F^ from the Lolium mother has a little good pollen, while that from the Festuca mother has none. In the same way the progeny whose chromosomes show full pairing are more pollen-fertile in the backcross to Lolium of the F^ with Lolium cytoplasm than in the backcross of the F^ with Festuca cytoplasm. This difference may well be due to failure of the other type of adjustment, that between nucleus and cytoplasm. If so, however, it merely serves to emphasize the point that disharmonies at the cellular level generally cause less disturbance than do disharmonies within the nucleus, genie disharmonies such as are revealed by segregation in both these backcrosses. Inbreeding Depression and the Hyhridity Optimum Hybrid sterility may thus be due, either immediately to geno- typic unbalance of the hybrid, or less immediately to the inability of such wide hybrids to avoid the segregation of unbalanced gametes. Such segregation must, of course, be peculiar to hybrids. But geno- typic sterility, and equally loss of vigour, is not the property of hybrid nuclei alone. If we inbreed maize by self-fertilization, the result is invariably a loss of vigour, or of fertility, or of both, in succeeding generations of the inbred lines. This inbreeding depression, as it is called, is rapid at first but soon begins to slow down (Fig. 57). After five or six generations no great increase o^ the depression is 235 ADJUSTMENT AND BALANCE to be observed, no matter how long the inbreeding may be continued: something like the maximum depression has been reached. At the same time that vigour and fertility are decreasing, so is the variation between the plants of any line in each generation. This decrease also virtually ceases after the first five or six generations. Evidently the increasing depression is due to increasing homo- Fa F3 F4 Fig. 57. — Characteristic plants to show the relative heights of two inbred lines (P) of maize, of the Fj^ obtained by crossing them, and of the subsequent generations up to Fj, obtained by continued sclfing. The maximum hybridity, following crossing, is associated with maximum height, and the reduction in hybridity by selfmg is paralleled by a reduction in height. In F7 and Fg, when homozygosis in almost complete, height is reduced once more to the minimum. The increase with crossing is termed heterosis, and the decrease is termed inbreeding depression (based on Jones, in Sinnot and Dunn, 1939). zygosity and to decreasing heterozygosity. The heritable variation will then decrease until full, or nearly full, homozygosity is reached and only non-heritable variation remains. Other things being equal, an average of only i part in 32 of the heterozygosity will remain after five generations of selfing, and only i in 64 after six generations. As we should expect, the vigour of the original parents is restored, even exceeded, by crossing inbred lines derived from different varieties, or, if from tlie same variety, separated early in the inbreeding programme (Fig. 57). Though each line is homozygous, or nearly so, different lines are homozygous for different allelo- morphs of many genes, with consequent heterozygosity in the F/s 236 INBREEDINC: DEPRLSSION AND TUC IIYBRIDITY Ol'TIMUM between them. By the same token, crossing plants within the same inbred line has no effect in restoring vigour. The vigour is hybrid vigour, or Jieterosis as it is sometimes called. In maize, therefore, it is possible to show that full vigour and fertility are the result of a hybrid condition : loss of hybridity leads to loss of vigour and fertility. The same principle applies in rye and sugar beet, in pigs and flies, indeed, as we shall sec later, in nearly all cross-fertilizing species. In this way we are led to see that both undue hybridity, that is unaccustomed hybridity, and unaccustomed lack of it, may lead to genotypic unbalance. Hybridity is dangerous only where its kind or its degree is unaccustomed. Each species has, it seems, an optimum degree of hybridity to which it is accustomed. Any departure from this optimum, no matter in which direction, is followed by unbalance. Now, with rare exceptions, species can maintain hybridity only by the crossing of different individuals in every generation. Equally, of course, excessively wide crossing will lead to undue hybridity. Thus the adjustment of the genes within the nucleus, the ultimate one of the three adjustments we have been led to recognize, itself demands a fourth, and one not between parts of the cell or even of cell with cell, but of individual with individual. There must be a relationship between the hereditary materials, and their behaviour, throughout the whole group or species; and it is on this relationship that the genetic system depends for its character. Hybridity optimum, segregation, and recombination must all be related throughout the group. Furthermore, they must be related not merely amongst themselves, but also to the mating habits of the individuals which compose the group, and to the barriers which make that group by separating or isolating it from others. If we are to understand the nature of heredity and variation, all these relations require our consideration. We shall commence with the breeding system, the habit of mating within the group, in its simplest and most general characteristics. REFERENCES BRIDGES, c. B. 1922. The Origin of variation in sexual and sex-limited characters. Am. Nat., $6: 51-63. DOBZHANSKY, T. T941. Genetics and the Origin of Specief. 2nd cd. New York. 237 ADJUSTMENT AND BALANCE EAST, E. M., and JONLS, D. F. IQIQ. Iiibreeditig and Outbreeding. Philadelphia. FEDERLEY, H. 1929. Uber sublctalc und disharmonische Chromosomenkombina- tioncn. Hacditas, 12: 271-293. HALDANE, J. B. s. 1922. Scx ratio and unisexual sterility in hybrid animals. J. Genet., 12: 101-109. PETO, P. H. 1933. The cytology of certain intergencric hybrids between Festuca and LoJiuin.J. Genet., 28: 11 3-1 56. SINNOT, E. w., and DUNN, L. c. 1939. Principles of Genetics. 3rd ed. New York. 238 CHAPTER 12 BREEDING SYSTEMS Breeding Systems Iiibreediiio and Outbreeduio Devices Incompatibility Hctcrostyly Mating Discrimination The Breakdown of Control Stratification The Inbreeding Hybrid Apomixis The gametes which pair and fuse in sexual union can differ from one another in two ways; in form and in origin. In regard to form, there is a complete range, from the eqiiaUty of some algae, fungi and protista, to the vast inequality between male and female germ cells reached in birds and reptiles. This extreme of differentiation represents a division of labour, for it enables the organism to combine the mobility of the sperm, which makes fertilization possible, with the food storage of the egg, which gives the embryo a start in life. Like other differentiation, this difference between the male and female gametes is not to be traced to any corresponding difference in tlie genes which the cells carry. Gametic differentiation can be as complete when the gametes are genetically alike as when they are genetically unlike; when they are produced by the same individual as when they are produced by different ones. Of greater genetical significance is the origin of the gametes. Whether they are equal or unequal, the two which fuse may come from the same, or from different, diploid individuals. And if, as in most animals, from different individuals, these may have different degrees of relationship with one another. Where, as in some plants like Johannsen's beans, they regularly come from the same indi- vidual, generation after generation, the product oi fusion wiU be homozygous, or nearly so. Where, on the other hand, they come from different individuals the product will be in some measure heterozygous or hybrid. Now, in particular populations or mating groups of plants and animals there will be an average degree of hybridity. This will foUow from the average relationships of the parents. And this in turn will depend on a variety of conditions of which the most obvious and most important is the relative frequency of self- and 239 BREEDING SYSTEMS cross-fertilization. This habit is the breeding system of the population, or group, or species. We now want to know what breeding systems exist, how thcv arc controlled, and what arc their effects. Breeding Systems Breeding systems work at different levels with different methods of reproduction. In some fungi, such as the phycomycete Allomyces javatiicus, two cells of a single haploid hypha fuse in sexual repro- duction. This is called homothally and we can see that its result is haploid self-fertilizatioth All the haploid gametes produced by a haploid are identical and, mutations apart, this type of mating must therefore give homozygosis, complete and immediate. In other fungi, such as Mucor, there is a special device, hetcrothally, which prevents self-fcrtiHzation, and ensures that every fusion shall be the result of a cross between different haploid individuals. We shall examine hetcrothally in more detail later. In the higher plants and animals, all sexual reproduction demands the crossing of different haploid individuals, since each haploid indi- vidual produces, or even consists of, only one germ cell. But these may or may not come from the same diploid individual. In peas and barley they usually do: diploid selj-fertilizatioti is the rule, even if the parent is heterozygous. This process, like haploid self-fertiliza- tion, leads to homozygosis ; but it does not do so immediately. On mating at random, the haploid gametes produced after meiosis by a heterozygous diploid give zygotes half of which are heterozygous, and half homozygous, in respect of each gene pair which is hetero- zygous in the parent diploid. They give Fo proportions of a half AA and aa taken together, and a half Aa. Diploid self-fertihzation, therefore, does not give immediate homozygosis. Instead it moves (on the average) half-way towards it in each generation. In the flowering plants devices exist which encourage self- fertilization. Far more exist, in both plants and animals, which discourage or prohibit it. Of these the most striking, especially in animals, is sexual differentiation, or dioecy, of the diploid organism. Even in these conditions, however, with regular brother-sister mating, heterozygotes will give place to homozygotes just as they do when self-fertilized, though again not so rapidly. For example, 240 I INBREEDING AND OUTBREEDING DEVICES in the grass-mite, Pediculopsis, copulation of brothers and sisters occurs in the uterus of the mother with the same regularity as it can do in experimental breeding. Further, in man, where brother- sister mating is normally prohibited by social conventions, the same conventions in some societies ensure a regular first-cousin marriage. This, too, when starting with heterozygotes, will bring about homozygosis, though it will do so only very slowly. In short, heterozygosity will at once follow any lapse from haploid or diploid self-fertilization, brother-sister mating or cousin marriage. Homozygosity will be gradually recovered when the system of inbreeding is restored. But it will be recovered at different rates, each characteristic of its own system. Hence, where a propor- tion of sexual unions habitually depart from the strict inbreeding habit (as they do even in peas and barley) homozygosity and heterozygosity will always exist side by side : they will be in equili- brium. This liyhridity eqiiilihriiiin will depend on (i) the rate of recovery of homozygosity, which, as we have seen, depends in turn on the type of inbreeding, (ii) the frequency of lapses from inbreeding, and (iii) the amount of heterozygosity produced by lapses, which depends in turn on (iv) the amount of genetical variation in the breeding group, which of course is influenced by mutation. These varying factors jointly constitute what has been called the closeness of inbreeding of the group. Inbreeding and Outbreeding Devices The hybridity equilibrium can be controlled in a great variety of ways, so great a variety that their common effect is usually over- looked. This is particularly true of the flowering plants. To begin with, the primary function of the pollen mechanism is that it allows crossing; other secondary functions we shall see later. Superimposed on this mechanism we fmd devices favouring both selfmg and crossing;. Mechanisms favouring close inbreeding generally depend on the development and structure of the flowers. The failure of flowers to open (deistogamy), best known in Viola, is widespread, and obviously favours self-fertilization. In other cases (like our peas and barley) the flower opens, but only after the pollen has been shed LUmciitsofGendiiS 24.1 O niUir.UlN(. SYSThMS on the stigma. This elcvicc is therefore ahnost as eftectivc as cleisto- gamy in securing inbreeding. Again, in the tomato, poUination follows the opening of the flower, but the stigma is so enclosed in the cone of stamens that it always receives pollen from the same flower. That is, except in stocks where the enclosure is incomplete (or in countries, like its native Peru, where certain insects specialize in cross-pollinating this flower). Some cross-pollination then occurs. British glasshouse commercial varieties are mostly ot a rigorously inbreeding type; American field-grown varieties of the occasionally crossing type. Slight genetic variations in the growth of the style (m tomatoes) or time of pollen-shedding (in the cereals) alter the regularity of self-fertiHzation and thus must control the amount of inbreeding. A more extreme effect has been described by Rick in the tomato. He discovered a recessive gene which partiaDy removes the hairs from the plant and, since the cone of anthers is held together by hairs, thereby deprives the flower of its means of regular self- fertilization, and so of some of its fcrtiHty. The absolute rate of outcrossing is unaffected so that, of the reduced amount of seed produced, a greater proportion is crossed; in fact nearly 50 per cent instead of the usual i or 2 per cent. Regular cross-breeding, or outbreeding as it is perhaps better called, is more difficult to secure than inbreeding, and is in fact secured by more elaborate devices which act at every stage of the reproductive cycle. The most obvious of these is the formation of unisexual flowers, with the sexes borne on different plants, known as dioecy, or on the same plant, known as monoecy. Dioecy ensures outbreeding. Monoecy only favours it; but its effect may be rein- forced by a timing difference in the production of male and female flowers, as in maize. This timing difference is also common in hermaphrodite flowers. The pollen is shed before [protandry] or after [protogyny) the stigma of the same flower is receptive. Self-fertiliza- tion is thus prevented in the place where, with insect pollination, it is most likely to occur— in the same flower. This timing difference does not, of course, prevent pollination between different flowers on the same plant. In genetic effect this is still, as Darwin showed, self-fertilization, am! the outbreeding mechanism is therefore not fully efficient. 242 INCOMPATIBILITY Incompatibility Secondary systems favouring either self- or cross-pollination in bisexual flowers depend on control of a habit which is uniform for all individuals, and therefore adapted to the use o( the species very much as are the shape and colour of petals. We now have to deal with another system which depends, like dioecy, on genetic dif- ferences, not genetic uniformity, for its maintenance, but which operates in bisexual flowers. This system operates through incompatihiUty of pollen and style. Separation of pollen from the styles of the same flowers — separation either in space or time — can only hinder, not preclude, self-fertilization. Incompatibility acts later and can be absolute in its effect, as we can see if we consider an example. The Sweet Cherry, Pmnus avium, is a diploid species. It consists wholly of individuals, wild or cultivated, which are incapable of setting fruit or seed when self-pollinated (with which we include, of course, pollination with trees of the same vegetatively propagated variety). When cross-poUinated one of two things may happen. The seed set is either complete or is as much a failure as with selfmg. For example, Bedford Prolific is completely successful with Napoleon or Waterloo, but it fails with Early Rivers. With reciprocal crosses the success or the failure is always the same. Moreover there are groups of varieties (one of them includes 13 names) which are mutually unsuccessful or incompatible, while being compatible with all other groups of varieties of which some 16 are known (Fig. 58). Thus to ensure a crop it is necessary to mix together compatible varieties from different groups when planting the orchard. How does this incompatibility work; It is clearly due to like things failing to agree in pollen and style. When we look into the matter we fmd that witl; incompatible pollination the pollen grows too slowly down the style ever to complete its journey to the egg. With compatible pollinations, we fmd one of two situations. Some- times, as in Bedford Prolific by Napoleon or vice versa, all the pollen grows quickly and well; and sometimes, as in Bedford Prohfic by Waterloo and vice versa, half of it grows well, and the other half behaves as though it is incompatible. The good half, however, 243 in?rrDiNc systi-ms is enough to fertilize every egg and so give a full crop of fruit. This second type, half-and-half, is always found in the relationship o( parent and offspring. ^1^2 '<% Vs 'z%',% c/ 2 {EARLY BLACK BEDFORD PROUf/C BLACK TARTARIAN EARLY RIVERS fSCHRECKEN FRO GM ORE B/C. WATERLOO j NAPOLEON \ EMPEROR FRANC/S [ KENTISH B/C. ( ELTON \g OYER NOR WOOD X I Ha Ui Q) OQ li] O) 5 1 § I I — — — — 4- 4- 4- 4--i- 4- 4- — — 4- 4- + 4-4- -f 4-4- — — 4- 4- 4- 4- 4- 4- — — 4- 4- 4- 4-4- 4- 4- H- + 4- -h — — — 4-4-4-4-4- 4- 4- 4- -h — -h 4- -h -+- + -4-4-4-4- — 4- 4- 4- 4-4-4-4- 4- 4- 4- 4-4-4- 4-4-4-4-4- 4-4- 4- 4-4-4-4- 4- 4- 4- 4- 4- 4- 4- 4- — 4- 4- 4-4- -f — — 4- 4- 4- -f 4- 4- 4- 4-4- -f Fig. 58. — The compatibility relations of 12 varieties of sweet cherry, falling into 5 incompatibility groups. + indicates successful pollination, — unsuccessful pol- lination, and a blank that the pollination has not been tried. The compositions of the varieties relative to the S gene are shown above (after Crane and Lawrence, 1947). These results give us the clue to the genetic problem. The half- and-half types of pollen are obviously produced by single gene heterozygotes. Since the parent's style rejects all its own pollen and only half that of its offspring, the rejected pollen must be that which 244 INCOMPATIBILITY carries a gene, an incompatibility gene, also present in the style. Completely incompatible pairs of plants will then always be such as have both allelomorphs the same. Compatible ones will be such as differ in respect of one or both (Fig. 59). /Vt\ SEED Fig. 59. — Diagram showing the physiological relationship between pollen and style and the genetic relationship between parent and offspring with homomorphic incompatibility. Sj S.j styles receive, (i) self or similar pollen on the left and their ovules remain unfertilized, (ii) in the centre mixed pollen part of which, if in sufficient quantity, can fertilize all ovules, and (iii) on the right wholly compatible pollen all of which can fertilize the ovules. Note. — No progeny can be incompatible with their mother, but in the middle case a half will be incompatible with their father. This explanation suggested by Prell in 1921 was confirmed, a few years later, by East and Mangelsdorf with Nicotiana alata and by Filzer with Veronica syriaca. On its basis the inheritance of incom- patibility has been successfully predicted in a large number of diploid plants, hideed the mechanism probably occurs in some stage of development or decay in about half the species of flowering plants. MS UKl.LDlNCi SVSTi;MS The controlling incompatibility gene, it will be noticed, takes us a new step in genetical inference. We recognize its allelomorphs as distinct, not by a difference in behaviour of the various genetical kinds of pollen in styles carrying the same allelomorphs, for these are alike in that none will grow; but by the difference between such behaviour, on the one hand, and the growth of pollen carrying any allelomorph in a style carrying different allelomorphs, on the other. It is just the same as the way in which we sec the effects of inversion AB Ab aB ab AB Ab + — — aB — ab Fig. 6o. — Hctcrothally determined by two loci in fungi. Successful fusion, indicated by +, occurs only where the two hyphae ditl:er at both loci. All other combinations, whether alike at both or only one of the loci, fail to achieve fusion, indicated by — . or interchange at meiosis; not in homozygotes, which show normal behaviour, but in the structural heterozygotes. With suitable breeding experiments the linkage of this gene, S as it is called, with other genes can be established and its mutations recorded. Of course in a fully operative system the number of allelomorphs of the S gene can never be less than three and is always in fact much more numerous, hi the small species Oenothera organciisis, with a total wild population of perhaps no more than 500 plants, 35 allelomorphs have been identified. In the large species of Red Clover, Tiifoliuiii pratensc, one group o^ 24 plants had 41 dift'crcnt allelomorphs and another group ol 20 j->lants luid 37 246 INCOMPATIBILITY allelomorphs. And, although closely related, the 60 cultivated varieties of Sweet Cherry examined must have over 20 allelomorphs. The S gene tells us many things of importance. Its large number of allelomorphs is without parallel. The specificity of their action 04 0-6 0-8 AVAILABILITY OF NON-SISTERS Fig. 61. — Hetcrothally is an outbreeding mechanism in fungi. The proportion of sister haploids (i.e. from the same diploid parent) available for mating is plotted against the proportion of non-sister haploids available for mating. Thus with two loci each of three allelomorphs, ] of the sisters will be available but ^ of non-sisters. A line projected from the origin through this point shows that the outbreeduig bias, found as the ratio of availability of non-sisters to availability of sisters, is ^ -^ l or 1-78. The maximum outbreeding bias with one locus is 2, with two loci is 4, and with three loci is 8. Nothing short of intervention of diploid tissue, as in incompatibility in flower plants, can give an outbreeding bias of oo. in the style is nearly always complete, since S^ pollen fails on a style carrying S^ no matter what the other allelomorph may be. In some cases this specificity extends to the strength of action, for some allelomorphs are stronger than others whatever others are present, though in other cases the allelomorphs can strengthen one another's 247 BRrF.DING SYSTEMS action. Moreover the properties of the pollen itself are determined by the single allelomorph carried in its own nucleus after segregation : tliere is no delayed effect from the other allelomorph present in its diploid parent (and in its sister pollen). This rapid and specific action puts one in mind of the relation between gene and antigen in the determination ot blood groups. The analogy is still more evident from the effect of a rise of temperature which, so Lewis found, speeds up the growth of compatible pollen, yet slows down the growth of the incompatible. Incompatibility is thus due to a positive blocking reaction. In the haploid fungi, heterothaUy seems to work in the same way as incompatibility in the flowering plants. In Coprinus rostntpianus, for example, multiple allelomorphs exist within the species which prevent the fusion of likes. In Coprinus lagopus and elsewhere there are two series of allelomorphs, similarity in cither of which is sufficient to prevent fusion (Fig. 60). This double scries in the haploid plants, as opposed to the universal single series of allelomorphs in the diploid plants, is significant. In the diploid plants the action of the S gene in the style prevents any fusion of gametes from the same diploid mother. In the haploid plants such fusions can never be wholly prevented (Fig. 61). Indeed, with a single series of alle- lomorphs, sister gametes can fuse in half the cases. A double series is needed to reduce this chance to a quarter. A third series, of which no case is known, would reduce it only to an eighth. Thus the different systems of incompatibility genes are clearly related, we may say adapted, to their effect in reducing inbreeding, or enforcing outbreeding, in organisms with different systems of reproduction. Hctcrostyly Not all systems of incompatibility depend on the genetically autonomous behaviour of the haploid pollen grains. There is another type in which the properties of the pollen are controlled by the diploid plant bearing it. The control is exercised by way of the differentiation of the flower. The morphological aspects of this system have long been recognized under the name of hctcrostyly proposed by Hildebrand in 1864, Its physiology was first described in detail by Darwin in his "Forms of Flowers" in 1877. 248 HETEROSTYLY Heterostyly takes various forms. The simplest and best known is that found in most of the diploid species of Primula. Here there are two types of plant distinguished by the shape of their flowers: the thrum with a short style has the neck of the corolla filled with a thrum of anthers; the pin with a long style has the anthers halfway down the corolla (Fig. 62). In pin and thrum types opposite organs are in corresponding positions, so that a pollinating insect naturally transfers the pollen of one to the style of the other. This simple mechanical means of encouraging cross-pollination is not, however, all. The pin flower has a rougher stigma and smaller pollen grains than the thrum. Thus there is evidently some physiolo- gical difference beneath the morphological one. This difference was revealed by Darwin's experiment of comparing the amount of seed set when pin and thrum plants are crossed and when each is bred with its own kind. The one, the "legitimate" mating gives high fertility; the other, the "illegitimate" mating gives lower fertility (Table 21). TABLE 21 SEED SETTING IN THE PRIMROSE (DARWIN, 1877) Mating Flowers pollinated Capsules set Average seeds per flower poi.inated LEGmMATE Thrum x Pin . . Pin X Thrum . . Illegitimate Thrum x Thrum . . Pin X Pin . . 8 12 18 21 7 11 7 14 56-9 61-3 7-3 34-8 We now know that this effect is due to a difference of growth rate of pollen tubes, as with ordinary incompatibility. The illegiti- mate is slower and, if the two are mixed together, the legitimate gets there first. But there is one striking difference from ordinary incompatibility: all the pollen of one plant behaves in the same way. Yet the pollen (and eggs) of one of the types must be of two kinds in the heredity it carries, because crossing pins and thrums gives equal numbers of pins and thrums. Intercrossing pins gives only pins. Intercrossing thrums, as found in nature, gives 3 thrums to i pin. Thus thrum, in nature, is always heterozygous, and the thrum 249 BREEDING SYSTEMS allelomorph (S) is dominant over the pin allelomorph (s). But 5, as much as S, pollen grains of Ss plants behave in a thrum way, while 5 pollen grains of 55 plants behave in a pin way. Thus, either o£ two different mechanisms of gene action with which we are familiar elsewhere can determine incompatibility. One acts directly through the haploid pollen grain; the other is imposed upon the pollen through the cytoplasm of its diploid parent. LEGITIMATE UNION Complete Fertility ^ o^-- ^ ILLEGITIMATE UNION Incomplete Fertility kt I -•> LEGITIMATE UNION Complete Fertility ILLEGITIMATE UNION Incomplete Fertility PIN Small Pollen THRUM Large Pollen Fig. 62. — Distyly in Priinula, showing the incompatibihty associated with it. The illegitimate unions, of pin X pin and thrum x thrum, are incompatible whether the pollination is made within a flower, as shown in the diagram, or between flowers, whether from the same or dilferent plants (after Darwin, 1877). This delayed action is made still clearer by a further elaboration in the Purple Loosestrife, Lythrum salkaria, also studied by Darwin. Here there arc three types of plants, each with its own form of flower. There are three levels for the sexual organs within each flower, two of which are occupied by anthers and one by the stigma, so that the three types have long, mid, and short styles. Legitimate, that is fertile, unions are always those between anthers and stigmata of 2S0 HETliROSTYLY difterciit plants borne on the same level (Fig. 63). All others are sterile or nearly so. Thus a short stigma accepts pollen from short stamens of both long and mid-styled flowers. It will not accept pollen from the other stamens (mid or long) of these same flowers, any more than from its own or any other mid and long stamens. LONG MID SHORT Fig. 6^. — Tristyly in LYthruin salicaria. Oiily the pollinations indicated by arrows are compatible. AH others fail or virtually fail. Thus the two tiers of anthers in one flower give pollen having dift'erent properties in incompatibility, while anthers from tiers at the same level in different flowers give pollen having the same properties in incompatibility (after Darwin, 1877). Before we return to our general genetic problem we must note three highly instructive physiological properties of this system. First, each parental genotype has two paths along which it can drive its own pollen. Secondly, pollen from parents of diflerent genotypes may be driven along the same path. And thirdly, the genotype of the pollen itself never comes into the question. Tristyly in Lytlimm (or in O.xalis or Ndrcissus) could scarcely be 251 BREEDING SYSTEMS worked on the basis of a scries of multiple allelomorphs, and in fact it depends on two unlinked genes. One decides the difference between the short-style and the not-short-style. The other has no action on short-style, but decides whether a not-short-style shall be mid- or long-style. It is a case of epistasy. The same genetic and physiological organization as with hetero- styly is revealed by CapscUa grandifora. The pollen depends for its behaviour on its diploid parent, and two gene differences work the device as in Lytlirnm. But morphologically the flowers of the different types are all alike. The heterostyly is cryptic. Thus the physiological difference, which in the other instances goes with a, difference of position, can be achieved without the help of such a difference. Mating Discrimination The diversity of outbreeding mechanism in plants, dioecyJ monoecy, protandry, incompatibility in its various forms, is not] matched in animals. With them dioecy, sexual differentiation of the] diploid organism, is the basic device almost without exception. Even the hermaphrodite oyster can separate its sexes in time by the cyclical! succession of male and female phases, and thereby become effectively dioecious. Dioecy excludes the extreme inbreeding mechanism of self-fertilization so common in plants. But, as we saw, dioecy can! be determined by many sex-chromosome and other mechanisms,] and on it can be superimposed a number of devices working both ways, favouring either inbreeding or outbreeding. We have already noticed intra-uterine copulation and first cousin marriage as favour- ing inbreeding. We can now consider the outbreeding mechanisms, j Animals move about, but their movement is limited. Dioecy forbids self-fertilization, but it does not forbid brother-sister mating such as is most likely where eggs are laid in clutches. In some insects, e.g. Sciara, the dimg-fly, this incest is avoided by the unisexual brood. By delayed action, as with heterostyly, the mother directly determines the sex of her offspring, which may be all male or all female. The brood of one sex is thus excluded from incest by the generic properties of the mother. The habits of individuals or the laws of society may have the , same effect on the breeding system as delayed gene action. Where- ^5^ MATING DISCRIMINATION flies are offered a choice of mate they may exercise that choice in the avoidance of extreme inbreeding and extreme outbreedins. Courtship behaviour in fishes, birds and mammals elaborately testifies to the same exercise of discrimination. In man the prejudice of individuals against incest and bestiality has become hardened and generalized into social tabu and legal prohibition. Looking back on all these different systems we see that in a hundred different ways the same end is achieved, the end being the control of the mating system. The means adopted reflect the circumstances and capacities of the species. Plants make use especially of the diploid style as a sieve for sorting the pollen delivered to it by a pollinating agency, over which it can exercise no direct control. Animals have powers of perception which they use in accepting or rejecting mates. And lastly man uses his unique power of social coercion for the same acknowledged purpose. The control of the mating system is evidently such as will give a controlled degree of hybridity generally intermediate between homozygosity and that extreme heterozygosity which we see to be disastrous in crosses between species. In other words it is capable of giving the kind of hybridity optimum we were led to expect. Two consequences of this state of things may now be seen. First, bars to crossing between species are, in effect, inbreeding devices and should therefore show a dependence on the mechanisms which control mating within the species. This is clearly seen in discriminative mating which is as strong in flies as it is in mammals. When in Drosophila a male of melanogaster is presented with females o£ simulans and of his own species (which are alike to us) he never makes a mistake. Similarly, an experiment with Petunia shows how the plant style makes its choice. Pollen of the Fj between the species axillaris and violacea, put on the style of either parental species, is sorted out so that the grains carrying the chromosomes prepon- derantly of the same parent get through to the egg more often. This effect is even clearer when mixed pollen of two distinct types is used in Streptocarpus. Self-pollen then has an advantage over other species' pollen (an advantage proportional to the remoteness of the other species): but cousin-poUen has an advantage over both. 253 BREEDING SYSTEMS TABLE 22 RESULTS OF POLLINATING THE TYPE VARIETY (FROM KNYSNA) OF STREPTOCA RP US REXII WITH EQUAL MIXTURES OF ITS OWN POLLEN AND THAT OF OTHER VARIETIES AND SPECIES IN ORDER OF INCREASING DISTANCE OF RELATIONSHIP (LAW- RENCE, UNPUBLISHED) Admixed pollen Percentage of total seedlings which are selfed as opposed to crossed rexii Var. from Stutterheim . . „ „ 711 East London . . cyaneus gardeni ■ ■ dunnii ■ ■ 2 13 37 45 79 The Breakdown of Control The second consequence of the relationship of the mating system to a hybridity optimum is that, in so far as we expect the hybridity optimum to change, so we must expect the mating system to change. Mating systems must be built up and broken down with clianging conditions. Of these the conditions of cultivation in plants are the most easily verifiable. The wild tomato growing in Peru is, as we saw, regularly cross-pollinated. In England there is no pollinating insect — a common result of acclimatization. In consequence the cultivation of tomatoes, especially in glasshouses, has been contingent on the acquirement of a property of automatic self-pollination : a property which has therefore been developed by the preference of the grower for plants setting most fruit, and of nature for plants setting most seed. Hence the property we saw of modern glasshouse tomatoes, which have flowers whose structures ensure self-pollination, at least with a little shaking. Outbreeding has been replaced by inbreeding. Primula sinensis has been cultivated in glasshouses since 1824. Being hcterostyled, it presumably had, at first, a strong outbreeding mechanism, more rigorous than the tomato. Now the seed which the glasshouse grower saves is always from pin plants because, in dropping off, the corolla self-pollinates the pin flowers, and even 254 THE BUiiAKDOWN OF CONTROL a small seed-set obtained in this way is easier than a large seed-set obtained from legitimate pollinations by hand. This process of un- conscious selection for self-compatibility has had its results recorded at intervals over nearly half the 120 years it has been at work. They show a steady increase in the success of self-pollination of the pin plants, a steady decay in the effectiveness of incompatibility (Table 23). TABLE 23 THE BREAKDOWN OF INCOMPATIBILITY IN PRIMULA SINENSIS (MATHER AND DE WINTON, 1941) Mating Hildebrand (1864) Darwin (1877) Merton (1910-1940) Flowers fertilized Average seeds per flower Flowers fertilized Average seeds per flower Average Flowers seeds fertilized per flower Legitimate ia) Thrum x Pin . . 14 41 24 33 929 30 (b) Pin X Thrum . . 14 44 8 64 889 18 Illegitimate ic) Thrum x Thrum 53 16 20 23 2,444 16 id) Pin X Pin 37 11 7 14 24,770 14 0- 31 0 39 0 63 * Measure of fertility of illegitimate pollinations relative to legitimate ones, used in order to correct for the decline in fertility introduced into the later results by the accumulation of recessives genes in material used for genetical experiments. In spite of this partial breakdown of the incompatibility mechan- ism, its action is still controlled by the pin-thrum gene, S-s. This gene can therefore control incompatibilities of different strengths, whose differences are governed by other genes. In other words the S-s gene acts as a switch directing development into one of two alternative paths, of which the precise course depends on other genes, or rather polygenic systems, whose variation is not normally detectable. Ordinary incompatibility is controlled in the same way: it has the same genetic structure as heterostyly. For example. Petunia violacea has the usual multiple allelomorph system of pollen-style 255 BRLEDING SYSTEMS relationships : self-pollination rarely succeeds. P. axillaris, on the other hand, shows no trace of incompatibility : selfing and crossing succeed equally well. In the F^ different 5 allelomorphs from violacea vary in eftectiveness and plants differ in the degree of self-compatibility. By crossing together F^ plants with different S allelomorphs, or by backcrossing them to their violacea parent, we can get plants with the same 5 constitution as violacea but with other genes, half in the F2 and a quarter, or nearly a quarter, in the backcross, from axillaris. Although alike in regard to S these three types of plants show incompatibility relations differing in two ways (Fig. 64). First, the amount of seed set on self-fertihzation increases with the proportion of axillaris genes. These genes must therefore be under- mining the operation which the 5 genes control. Secondly, the amount of seed set on pollination of backcross or Fo plants by violacea is greater than any produced by self-pol- lination, although the same 5 genes are at work. Thus the axillaris genes not merely weaken the operation of the S genes; they shift their operation so as to put it out of step in plants with different proportions o£ axillaris genes. Or, the other way round, we may say the same 5 genes can not merely control systems of different strengths, but systems wliich are less efficient with one another than each is within itself. Now, we may ask, what happens when we cross two self-incom- patible species, each with the S genes? This has been done for Nicotiana alata and N. forgetiana in the production of the garden form N. sanderae. Pseudo-compatibility, that is successful fertiliza- tion with pollen having an S allelomorph the same as in the style and therefore not legitimately capable of growth, is unknown in forgetiana. It occurs only rarely in alata. In their derivative it is common, especially with certain of the weaker allelomorphs. Thus the recombination between the general gene systems of two species has robbed each of its efficiency as a basis for the action of the 5 series. The best known example of all switch genes, or gene complexes, is of course that determining sex. We have already seen how on crossing the two sub-species of Melandriwn dioicinn Winge was able to obtain in Fg plants male in regard to the sex chromosomes but bearing female as well as male organs. As with Petunia and Nicotiana 2^6 THE BREAKDOWN OF CONTROL INCOMPATIBILITY IN PETUNIA AXILLARIS X VIOLACFA PLANTS FROM VIOLACEA BACKCROSS fAxVJxV Fa (AxV)x(AxVJ ALL S1S2 POLLINATIONS SELFED SET ^-FAILED SELFED X VIOLACEA . k. SELFED X VIOLACEA ALL S1S2X S,?a Fig. 64. — Breakdown of the genetic determination of incompatibility in Petunia. P. violacca plants of the constitution SjSo set seed from only about i per cent of flowers which have been self-fertilized (indicated by size of black square relative to the white one containing it). S1S2 plants recovered in the F2 of the cross with the self- compatible P. axillaris set seed from about 25 per cent of flow^ers after self-pollination and S^So plants recovered in the backcross to P. violacea are intermediate in behaviour. Thus the addition of genes from P. axillaris causes progressive breakdown of the incompatibility mechanism of P. violacca. It also causes some change in the operation of the mechanism which is left, as pollination of the backcross or F.^ plants by P. violacea gives a greater set of seed than does selfmg, with which the cross is identical in respect of Sj and S.^ (based on Mather, 1943). Elements of Genetics 257 BREEDING SYSTEMS certain recombinations amongst the general genes had broken down the action of the switch gene. They had in fact prevented the XY combination from inhibiting tlic development of the ovary. They had thus turned males back into hermaphrodites, the condition from which maleness and fenialeness must have recently arisen in both the parent plants. In animals with long-established dioecy, the same breakdown occurs in hybrids, but it produces sterility. Race crosses in the moth Lymantria dispar, for example, give intersexes. In these, female development is superseded by male, or vice versa, in the course of growth, the time of the change determining the grade of the intersexuality, as we saw in Chapter 7. In Drosopliila we can go two steps further. Intersexes can be produced by altering the numbers of chromosomes present. As we have already seen, an extra set of autosomes with the ordinary two X's of the female turns it into an intersex. The presence or absence of Y makes no difference. Evidently, therefore, the switch gene effect of X-Y segregation depends on the differences in pro- portion of autosomes and X chromosomes in XX and XY flies (Fig. 55). Again, as in Petunia and Nicotiana, genes whose differences are not segregating are making the switch mechanism work. The second step in Drosopliila is in shov/ing that the switch gene itself is compound. When the X chromosome is broken by X-rays, and its fragments are lost, its effect is diminished: females are turned into intersexes. The X chromosome, or at least its differential segment, is thus a super-gene in respect of sex deter- mination, although an aggregate of genes in other respects. In genetic terms, breeding systems are now seen to be of two types. First, all those giving inbreeding and some giving outbreeding, like protandry and cyclical hermaphroditism, are controlled by general gene systems which do not show any special variation within the species. Their operation does not depend on diversity, that is on segregation. Secondly, the chief cross-breeding systems, sex and incompatibility, on the other hand, require segregation and therefore depend on the action of switch genes. But the existence of the mechanism which these genes switch, depends on a general gene system which does not need to show segregation. On this basis new systems can come into existence by adjustment, 258 STRATIFICATION through selection, of the general gene system; the switch gene, where it exists, is thereby given a progressively stronger effect. The evidence that this is at least sometimes the case arises from the method of breakdown seen already in Primula sinensis. Sudden breakdown can, of course, occur in the switch gene itself Various species o£ Primula, such as the common primrose, sometimes have the pin-thrum type replaced, in nature, by a homostyle type with anthers and stigma at the same level. This change was found by Ernst in one case to be due to a change of the switch gene to a third allelomorph. The new type must inbreed with a regularity which is equal to the outbreeding of its predecessor, because, of course, the genetic background remains the same. Similarly, in ordinary incompatibility, the usual 5 allelomorphs can be replaced by a so-called fertility allelomorph, S, which no longer inhibits self-pollination or even any kind of cross-pollination. Most fertihty allelomorphs have been found in self-compatible species related to other species which are uniformly self-incompatible. But mutations to Sj- have appeared in the otherwise regularly incompatible red clover. Moreover, in Antirrhinum majus, but nowhere else so far as is known, a fertility gene exists, overriding the S system, but not allelomorphic to it. These various cases of breakdown show that a system which is built up gradually of many elements including an operator, the switch gene, can be knocked down by removing any one of them, especially, of course, by removing the operator. Stratification All these facts and arguments relate to changes in the breeding system at one level, or at one time of operation. But, as we know, control occurs at many levels. How do they interact ? One system can be superimposed on another, while leaving all the evidence of the other plainly revealed to us. We then have a stratified system. For example consider wheat, any species of Triticum. The anthers and stigmata are thrust out into the wind to allow of cross-pollination, or so one would suppose ; but in fact the anthers burst and pollinate the stigma inside the flower before it opens. Inbreeding is superimposed on outbreeding: a low hybridity optimum can replace a high one. This sequence of systems 259 BREEDING SYSTEMS is coiifirnied when we look at a closely related cereal. Rye pushes out its anthers and stigma in just the same way; but instead of vitiating the crossing mechanisms by premature bursting, it rein- forces it by incompatibility. Wheat has developed one way and rye the other, after their ancestors diverged. Of peas the same story can be told. To be sure, the cross-pollination was to have been by insects, but its vitiation is still by premature bursting of the anthers. And when we return to the mite Pediculopsis, premature mating is again the means by which outbreeding is suppressed while the sex system adapted to outbreeding is retained. Outbreeding, so far as we know, never supervenes on inbreeding. Always it is the reverse. Complete inbreeding is evidently a dead end. The reasons for this we shall touch on later. But there are a variety of ways in which the effect of cross-breeding, the high degree of hybridity, may be maintained while the ancient system of crossing is allowed to lapse. One of these we have already seen in the allopolyploid. A second method is that of the true- breeding hybrid or complex heterozy^^ote. The Inbreeding Hybrid The true-breeding hybrid is known by the exact breeding analysis of Renner, as well as by chromosome study, in a hundred or so species of the American Evening Primrose, Oenothera. These species are characterized by having 6, 8, lo, 12, or all 14 of their chromo- somes linked in a ring at meiosis. They are interchange hybrids. They are hybrid not for one only, but for two, three, four, five or, in the extreme case of the complete ring, six, interchanges. Take a ring of 6 resulting from two interchanges. We can represent its chromosomes as follows: — a AB CD EF j8 BC DE FA The ring, in fact, usually arranges itself on the spindle in this alternating way, so that two kinds of workable germ cell are formed, both in eggs and pollen. They bear the two complexes a, or AB 4- CD -h EF, and /3, or BC + DE + FA. When a complex 260 THE INBREEDING HYBRID heterozygous Oenothera is selfed, however, it does not produce a mendchan combination of i : 2 : i. The two classes of homozygotes fail to appear. The hybrid breeds true. Indeed it is only when a heterozygous species is crossed with another, heterozygous or homozygous, that its heterozygosity is shown by the mixture in the progeny, by the production of twin hybrids. Thus if the second species is homozygous for a set y with the pairs: AB CE DF AB CE DF then the crossed seedlings will be of two kinds : — AB CE FD \y AB CE DF \ / \ / and \ \ /\/\/ AB EF DC U BC ED FA These two kinds will be recognizable by the differences in the size of the ring and in their external appearance. These observations present us with two problems. Why do sets or complexes, with different arrangements of chromosomes pro- duced by interchange, differ in their phenotypic effects ? And why do their homozygous combinations so often fail to appear, or at least to live? Renner showed that sometimes, as we have already seen, one complex fails in the pollen and the other in the egg. He also showed that, in many other cases, homozygotes were often formed, but quickly died. Evidently each set is defective in some way or other, and its defect is covered by its partner. If we can find out what this defect is we shall have explained why the different sets have different effects. A moment's consideration will show that our symbols AB, BC and so on make an unjustifiable assumption. Chromosomes, as we saw, can and do break and rejoin at various points, indeed between any two genes. They are not each composed of two unbreakable arms exchangeable only at the centromere. There are segments therefore (as we saw earlier in Fig. 32) which are omitted from our diagram between A and B, between B and C, and so on. The sum of these segments in one complex would be equal originally to the sum of those in its partner complex, provided that no super- fluous breaks or mutations had ever occurred. But it is of the nature of chromosomes that superfluous breaks and mutations do occur. When they occur in the complex heterozygote their consequences 261 BREEDING SYSTEMS are something quite dijOferent from what arises with ordinary sexual reproduction. The whole set acts as a single linkage group, which is broken into two parts if crossing-over occurs at any point between its middle segments. A new type of gamete is then produced which is only half-hctcrozygous. Thus crossing-over in the middle of dif- ferential segments must have been suppressed in all the ancestors of a complex hcterozygotc during its building up. The differential segments of opposite complexes in the heterozygous Oenothera species have been separated from one another, isolated, during this evolutionary process, just as much as the chromosomes of different species. Since the Oenothera species have incomplete chromosome pairing beginning near the ends, crossing-over is localized near the ends. The situation is therefore prepared for the development of the complex heterozygote before any interchange takes place. The complexes of Oenothera are differently adapted. Some fail as pollen. Others fail as embryosacs, and are displaced by their more female-vigorous sister segregates. So different, indeed, are the com- plexes that a triploid endosperm with a 2 : i balance would probably have been an embarrassment in building up the system; and we fmd in fact, that the Oenothera family is remarkable for having a diploid endosperm. The complex heterozygous species of Oenothera are, in the aggregate differences between their complexes, at least the equivalent of hybrids between pairs of species. But the differences are mechani- cally concentrated in the differential segments and physiologically adjusted to balance one another. They cannot, therefore, have arisen by hybridization in the ordinary sense. Crossing different stocks enables us to build up by steps a stock of Campatmla persicifoUa with a ring of twelve chromosomes. But the two sets of six into which this ring segregates do not constitute two mutually adapted complexes. The complexes of Oenothera have evidently been built up gradually by internal change. Thousands of generations of almost uninterrupted self-fertilization in species which, as we shall see, were previously cross-fertiUzed, accompanied by the continual elimination of homo- zygotes, have resulted in the production of hybrid species. Hybridity in Oenothera is a matter of crossing different gametes, not different zygotes; a distinaion pointed out by Mendel but still not under- 262 APOMIXIS Stood by all mcndelians; a distinction to which wc shall have to return. It was the mutations, of course, which first attracted De Vries' attention to Oenothera lamarckiana in 1886 and led him to propound his Mutation Theory of Evolution. What part do the famous mutations in fact play in this genetic system? They represent the breakdown in the mechanism of the true-breeding hybrid. The ring of chromosomes sometimes fails to arrange itself alternatingly on the spindle, and germ cells are then produced with 8 and 6 instead of only 7 chromosomes. Hence the series of trisomies with 15 chromosomes of which 68 types are to be expected in Oenothera lamarckiana, whose chromosomes normally form a ring of 12 and one pair. Again, segregation may fail altogether and unreduced germ cells will then give triploids and tetraploids. And finally the ring may break down by crossing-over, as we saw, to give new half- heterozygous forms. The mutants o£ Oenothera are therefore nothing more than symptoms of its peculiar hybridity and as such are of little significance in evolution. Later we shall see the ways in which the Oenothera system is significant. Apomixis The third way in which a high degree of hybridity may be main- tained while the ancient system of crossing is allowed to lapse, is by apomixis, the suppression of sexual reproduction. Apomixis comes about in two ways. Some organisms achieve apomixis, others have it thrust upon them by the circumstances of their birth. The first are of less importance to us. Many organisms, in producing their female spores or eggs under certain external conditions, are compelled to forgo meiosis. A single mitotic division replaces meiosis and gives a diploid egg which can develop without fertilization by a male germ cell. So is it with many blackberries and other plants. So it is also with the summer-brood females of aphides, which thereby continue to multiply vegetatively like a plant propagating itself by suckers.When the colder weather comes, a first symptom of meiosis begins to appear in some females. One of the two sex chromosomes is lost at the single maturation division of the egg, so that diploid XO eggs 263 BRliLDING SYSTEMS develop. They are sexual males. At the same time females arc produced whose eggs undergo regular mciosis and are therefore sexual. Both types undergo perfect reduction, but only the X-bearing sperm of the males function. The haploid eggs after fertilization, therefore, give only females, which re-establish the parthenogenetic summer series (cf Fig. 65). Fig. 65. — The effect of temperature on sex determination in the egg of the psychid moth Talaeporia tubidosa where the female with one X is heterogamctic according to Sciler, 1920. At the higher temperature or when over-ripe the unpaired X chromosome passes more often to the egg than to the polar body at the first meiotic division and therefore gives a preponderance of males. An adaptable sex-ratio is important in these moths where the female is immobile. This whole process of cyclical parthenogenesis is a means of economizing on sexual reproduction during the summer period of rapid expansion of numbers. If, of course, such a species moves south into a region of perpetual summer, as seems to happen in the United States, then the species will not enjoy its sexual season. It had achieved facultative apomixis but it will have had obligatory apomixis thrust upon it. Quite a different story is it with those plants and animals which find themselves suddenly deprived of means of regular sexual reproduction by the circumstances of their birth. A triploid of the 264 APOMIXIS crustacean Trichoniscus, or of the dandelion Taraxacum, arises sud- denly owing to the fertilization of an egg with the unreduced 1 6 chromosomes by a normal sperm with 8 (or vice versa). Such a triploid, although only a number-hybrid, suffers all the misfortunes of the authentic fruit of cross-fertilization. The lagging of unpaired ARTEMIA 5ALINA Sexual ->- Parthenogenetic (l)subsexual 2x nnnnnn □□□ nnnnDD □□□ Mediterraneans N.America 3x S.France £- Egypt Portorose 4x D Odessa (2)non- sexual Mediterranean 8x 3 Fig. 66. — Evolution of sexual, sub-sexual and non-sexual forms in the brine shrimp Artemia salina. The sub-sexual forms have meiosis followed by fusion of its products. The non-sexual ones suppress both meiosis and fusion: the unreduced egg develops. Each square represents a tested location (after Barigozzi, 1946). chromosomes at meiosis in its mother cells prevents the separation of the two daughter nuclei. A single restitution nucleus is reformed with the whole triploid complement. The second division then follows its normal course and two triploid nuclei are formed. In other words the reduction of chromosome number is evaded and a female germ cell formed in this way, and developing as it often can, especially if unreduced, without fertihzation, reproduces its 265 BREEDING SYSTEMS female parent and continues to do so generation after generation. Such is the undoubted origin of triploid apomictic "species" of plants and animals. There is, however, an omission in this account which would lead to an error if it were to go uncorrected. The pairing chromosomes in a triploid pair because they have crossed over and formed chiasmata. The sister chromatids at the second division, in a triploid as in a diploid, are therefore in part derived from partner chromo- 3x 3x-1 Fig. 67. — Leaves of the normal triploid Taraxacum polyodon and its eight types of disomic mutants occurring as apomictic seedlings in nature. X J (after Gudjonsson, 1946). somes, i.e. they are dissimilar; their separation thus leads to the segregation of differences. The triploid egg cells of the original triploid apomict are not therefore genetically identical with it, or with one another. As compared with sexual species variation is much reduced, but it still occurs. The new apomictic species is thus often subsexual (Fig. 66). The adaptation of apomicts for fertility in Taraxacum has evidently taken the form of making the suppression of meiosis more complete and therefore more regular. Efficient meiosis so desirable in a diploid has become a mere nuisance in a triploid. Triploid apomicts, in fact, show almost complete suppression of cliromosome pairing and of the consequent reduction of number. 266 APOMIXIS The evidence of variation is worth returning to. It has been revealed most comprehensively in some apomictic stocks of common descent — for we must not give them the name of race or species intended for biparental groups — in Taraxacum. The range is not unlike that in Oenothera. Some of the variation is attributable to the oc^8 Species in Tap.axacum EXUAL A p o M I C T I -subsejiiUQl- I \ \ (5x;-(A^BK:))erc. 3ac-A Fig. 68. — The origin of apomictic from sexual species in Taraxacum and their subsequent Umited and irreversible evolution under conditions of sub-sexual repro- duction, i.e. by recombination, loss and polyploidy at the suppressed meiosis. The unbalanced forms are homologous in different triploid series and are increasingly dwarfed with increasing unbalance (based on Sorensen and Gudjonsson, 1946). mere effects of the crossing-over without reduction seen at the suppressed meiosis : the variants have the normal triploid complement of 24. Others are due to grosser aberrations. Chromosomes are lost to give types with 23, i.e. one short. And these are recog- nizably of the 8 expected kinds, thus giving 8 parallel variants in each group (Fig. 67). Or again both meiotic divisions may fail so that the chromosome number is doubled and plants with 48 267 BRLLDINC; SVSTLMS (or 40) chroinosonics arise. Again there are parallel variants in the different stocks. Thus each stock of apomicts is a complex and self-sustaining system of variation (and adaptation) outside the ordinary sexual system, but nevertheless owing its character to an imperfect suppression of meiosis, whose imperfection inherently provides the means of perfecting itself (Fig. 68). The breakdown of sexual reproduction comes about in many other ways. One further example will illustrate certain useful principles. In the flowering plants, as a rule, every embryonic seed or ovule contains only a single embryo-sac mother-cell. This cell gives by meiosis four haploid cells or spores. Only one of these spores can, as a rule, develop into an embryo-sac and, as we have seen, the spores may compete for this opportunity, thereby giving the Rermer effect. The embryo-sac in its turn contains a number of cells, only one of which can, as a rule, develop into an egg capable of fertilization. The choice of the egg cell therefore depends on a series of choices during the development of the ovule (Figs. 50 and 51). Many and entertaining volumes have been written in a language of their own to describe variations and exceptions in this pre- destined course of differentiation, even in the circumstances of normal sexual reproduction. We have already had cause to relate the choice of spore in embryo-sac formation to protein and nucleic acid gradients, and the variations and exceptions no doubt also depend on the variable concentrations and distributions of proteins and nucleic acid available in the ovule. But from our present point of view what is important is that the issue is not always exclusively decided and several cells are not uncommonly available for fertili- zation within one or more embryo-sacs; and at the same time some of these, and others outside the embryo-sacs, whether derived from meiosis or not, arc often available for development without fertilization. Between these potential eggs and potential embryos there must be competition for development; competition whose outcome will depend on the two interacting factors of genotype and of position within the ovule. The Renner effect is thus but a special case of a more general phenomenon. The consequences of tliis free enterprise in development are found in many apomictic plants. Perhaps they appear best in Poa pratensis. 268 APOMIXIS Here a plant with 42 chromosomes can give progeny with 63 and 84 as well as 42 ; and a plant with 84 can give progeny with 42 and 63 and nearly 126 as well as with its own number. One plant with 72 even gave a vigorous seedling with 18. It is not surprising, there- fore, that a world collection of 56 plants of Poa pratensis revealed such a distribution as that shown in Table 24. It follows a normal curve, produced evidently by continual variation from a mode between 60 and 70. TABLE 24 THE WORLD FREQUENCY DISTRIBUTION OF CHROMO- SOME NUMBERS IN POA PRATENSIS (HARTUNG, 1946) Chromosome number 40 50 60 70 80 90 Frequency 13 25 12 This is an extreme instance of versatile reproduction, but great numbers of normally sexual species are known in which exceptional seeds reveal the same principle, since they contain twin embryos. One of these embryos is usually a normal sexual diploid, while the second is usually a haploid or a triploid which has arisen illegiti- mately in competition with its lawful twin (Fig. 69). These examples show that many plants, perhaps indeed individuals in nearly all species of flowering plants, have the capacity for evading meiosis or fertilization or both, and thereby giving rise, in the right circum- stances of sexual sterility, to the obligatory apomict which cannot do otherwise. In the normal sexual system, the hybridity optimum is related to the mode of breeding. The inbreeder has low hybridity, the outbreeder high hybridity, and the breeding system is genetically adjusted to secure these ends. This relation between hybridity and breeding system can be broken only by such extreme devices as complex hybridity or the abandonment of sexual reproduction. Complex hybridity and apomixis must have been derived from the normal sexual system, within which we can also see that the inbreeding system may replace outbreeding. What is it that determines these changes ? What advantages do inbreeding and 269 BREEDING SYSTEMS outbreeding confer on their possessors? Indeed, what is the advantage of the normal sexual system ? These questions we shall examine in the next two chapters. KINDS OF "TWINS 2x-2x. 2oz-, 2.x-S,x 2.X-OX. 2nc-3oc 2oc-cc Zoc-2.x C I or\al Fig. 69. — Twins produced in flowering plants classified according to their genetic relationship. One body in the nucleus stands for one haploid set. Arrows represent sperm which are assumed to be haploid (but can be diploid). Apomictic nuclei or embryos (which will develop without fertilization) are stippled. A, the two types formed in animals except that the type giving identical twins is not clonal but the result of fertilization. The lower group are fraternal or semi-fraternal, i.e. identical on the female side only (based on Linum, Kappert, 1933; Poa, Secalc, etc., Miintzing, 1933; Gossypium, TrifoUum, etc., cf. Skovsted, 1939). REFERENCES BARMGOZZI, c. 1 946. Uber die geographische Verbreitung der Mutanten von Artemia salina Leach. Arcbiv. d. Julius Klaus-Stift., 21: 479-482. BRiEGER, F. 1930. Selhsterihtdt und Kreuzungsterilitat in Pflanzenreich und Tierreich. Berlin. COOPER, K. w. 1937. Reproductive behaviour and haploid parthenogenesis in the grass mite, Pediculopsis ^raminum. Proc. Nat. Acad. Sci. Wash., 23 : 41-44. CRANE, M. B., and LAWRENCE, w. J. c. 1947. The Genetics of Garden Plants. 3rd ed. London. CRANE, M. B., and THOMAS, P. T. 1940. Reproductive versatility in Rubus.J. Genet., 40: 109-128. DARLINGTON, c. D. 193 1. The cytological theory of inheritance in Oettothcra. J. Genet., 24: 405-474- 270 BREEDING SYSTEMS DARWIN, c. 1877. The Different Forms of Flowers on Plants of the Same Species. London. DAVIDSON, J. 1927. The biological and ecological aspect of migration in aphides. Set. Prog., 85. EAST, E. M. 1929. Self-Sterility. Bihliogr. Genet., 5: 331-370. EMERSON, s. 1939. A preUminary survey of the Oenothera organcnsis population. Genetics, 24: 524-537. FISHER, R. A., and MATHER, K. 1943 . The inlieritance of style length in Lythrum salicaria. Ann. Eugen., London, 12: 1-23. GOLDSCHMiDT, R. 1934. Lymanttia. Bibliogr. Genet., ii: 1-186. HARTUNG, M. E. 1946. Chromosome numbers in Poa, Agropyron and Elymus. Am. J. Bot., 33: 516-531. LEWIS, D. 1944. Incompatibility in plants: its genetical and physiological synthesis. Nature, 153: 575-578. MATHER, K. 1943 . Specific differences in Petunia. I. Incompatibihty. /. Genet., 45:215-235. MATHER, K., 1944. Genetical control of incompatibihty in Angiosperms and Fungi. Nature, 153: 392-394- MATHER, K., and DE wiNTON, D. 1941. Adaptation and counter-adaptation of the breeding system in Primula. Ann. Bot., N.S., 5: 297-311. MULLER, H. J. 191 8. Genetic variabihty, twin hybrids and constant hybrids in a case of balanced lethal factors. Genetics, 3 : 422-499. RENNER, o. 1946. Artbildung in der Gattung Oenothera. Naturwiss., 33: 211-218. RICK, c. M. 1947. Partial suppression of hair development indirectly affecting fruitfulness and the proportion of cross-pollination in a tomato mutant. Am. Nat., 81: 185-202. SEiLER, J. 1920. Geschlechtschromosomen-Untersuchungen an Psychiden I. Arch. Zf, 15: 249-268. SKOVSTED, A. 1939. Cytological studies in twin plants. Comp. Rend. Lab. Carlsberg, 22: 427-446. S0RENSEN, T., and GUDJONSSON, G. 1946. Spontaneous chromosome aberrants in apomictic Taraxaca. Kong. Dansk Vid. Selsk. Biol. Skr., 4 (2). wmTEHOUSE, H. L. K. 1948. Sex and heterothaUism in the fungi. Biol. Reus, (in the press). WILLIAMS, R. D. 1939. Incompatibihty alleles in Trifolium pratense L; their frequency and linkage relationships. Proc. jth. Int. Cong. Genetics, 316. 271 CHAPTER 13 SELECTION AND VARIABILITY Selection Darwinism and Genetics The States of Variability Fitness and Flexibility The Effect of Linkable The Control of Recombination Balance in Homozygotes and Heterozyqotes Selection and the Reservoir of Variability Change of Genetic Systems : Inertia Correlated Response: Capital and Subordinate Characters Almost from the beginning of genetics we are bound to examine and discuss variation. New forms of plants and animals are the materials for the study of heredity. But what happens to these forms in nature ? It is obvious that a lethal mutation extinguishes the individual in which it expresses itself. Those which cannot live must die. It is less obvious, but no less certain, that many mutations which are not lethal make their possessor's survival doubtful, or at least reduce his chance of leaving progeny. Drosophila subobsaira, for example, uses its power of sight in courtship, and Rendel finds that flies made blind by the absence or abnormality of eyes are unable to mate. The genes producing this eflect are not lethal somatically; but they are lethal genetically. They must therefore be selected against, or as we may say, they have a negative survival value. Again this is an extreme case; but any mutation which impairs the faculties of its possessor must have a survival value reduced by an amount proportional to the impairment. Selection The principle of selection can now be seen in much greater breadth and depth than would have been possible at the beginning of this book. In the hrst place we can see it operating in different stages of development and at different levels of organization. The defective cell which arises by gain or loss of a chromosome is eliminated as readily as the defective individual. This cell also may be a spore or a fertihzed egg. In the Renner eflfect we sec the defective spore removed. Also we notice that it is removed owing to a defect 272 DARWINISM AND GENETICS which is by no means lethal in an absolute sense, but is fatal to it merely as a potential embryo-sac, and vis-a-vis a particular competitor. Selection, therefore, must favour the individual which fits the conditions of its environment better at all stages of develop- ment. Thus we can establish two important generalizations; first, that heritable differences occur between different individuals of any group; and secondly, that, as a consequence of these differences, the individuals enjoy different, yet characteristic, chances of surviving and leaving offspring under the conditions of incomplete survival, arisuig from both internal failure and external competition, that exist in nature. As Darwhi and Wallace pointed out, natural selection will work to eHminate some variants and to perpetuate others. The theory of evolution by natural selection relates the whole of evolution to this process. As conditions change and as new variants occur new types will arise, better fitted, or adapted, than their predecessors to survival in the reigning environment. By virtue of these new adaptations evolutionary change will constantly be going on : evolution is the sum of adaptation. Darwinistn and Genetics Genetics has strengthened and amplified Darwin's theory in both of its basic relations : to variation and to selection. Darwin was able to show that hereditary differences occurred between individuals on the scale required by his views, but he was never in possession of a theory of heredity capable of relating them to the action of natural selection. This lack of knowledge of the mechanism oi inlieritance placed Darwin in a dilemma about variation, to the solution of which the whole of his work on Animals and Plants under Domestication was unsuccessfully devoted. He thought that inlieritance was blending: that the contributions made by two parents to the hereditary endowment of their individual offspring blended like ink and water. Throughout posterity no separation was then possible of the maternal and paternal elements. Thus variation was always being lost, nay destroyed, to the extent of a half in every generation of a randomly breeding population. Either evolution Elements oj Gaiclks ^73 ^ SELECTION AND VARIABILITY must coiiic to a standstill, or the loss must be made good. Only by the production of new hereditary variation on a vast scale could this come about. It was to the means by which this new variation was produced that Darwin devoted his discussion. He supposed that the effects of domestication in altering plants and animals were direct effects of the changed environment. His difficulties, in fact, ultimately led him to adopt the ancient hypothesis of direct adapta- tion, the Lamarckian inlieritance of acquired characters, which he called pangenesis. While Darwin was wrestling with this problem, Mendel had already solved it: or rather had shown that it did not really exist. On the mendelian, or indeed any particulate theory of inheritance, as Fisher has pointed out, crossing does not destroy variation. When a tall pea is crossed with a short one, the parental difference vanishes in the F^. But the disappearance is only temporary: both parental types appear once more, side by side, in the Fg. Whatever differences disappear into a hybrid by crossing, reappear in its progeny by segregation. And since there is no permanent loss of variation by crossing there need be no production of new variation on a correspondingly large scale. Nor is there any large scale pro- duction of new variation, as Johannsen proved when he established his pure lines. Mendel's peas removed the need for postulating the rise of variation on a grand scale : Johannsen's beans showed that it did not occur. Darwin had been misled by not knowing enough about heredity. Mendel's experiments thus gave Darwinism the foundation it needed. Later experiments in genetics, by developing its particulate theory in the way we have seen, add to the strength of the joint structure. They show us that the most important changes in conditions, which lead to selective adjustment, are not, as was supposed, changes in the inanimate world: they are changes in heredity. One class of these, which we may mention in passing, are such as occur in other species, especially where the species live in the host-parasite relationship. All organisms are either hosts or parasites or both. Any genetic change in either is liable to affect the other, and a continual succession of mutual adaptations is the result. Each mutation to greater virulence by the parasite leads to seleaion of a mutation for greater resistance in the host. The system is shown 274 DARWINISM AND GENETICS diagrammatically in the relations of bacterium and bacteriophage described by Delbriick and Luria, and is also very simply demonstrated by the recent liistory of a wide range of crop plants. A second class of changes leading to selective adjustment are those in other genes of the same individual or race. Selection is a means of fitting the genes to one another. If one gene in a population is changed, all the others are exposed to new conditions of selection. Thus, as we saw, a culture of "eyeless" Drosophila becomes modified after a few generations. Polygenic modifications are selected which prevent the eyeless gene expressing itself so drastically. The environment for any one gene includes, therefore, the other genes. In this light we can see that the genie balance we have been discussing must always be the product of selection. Good balance will be one which has been exposed to selection under the established conditions and is consequently adapted to those conditions. Bad balance will be one which is not adapted to those conditions. Thus, while genetical principles enable us to see the answers to many questions of evolution, it is equally true that Darwin's principle of natural selection shows us how the genetical property of balance comes about. In the same way it shows us both the meaning and the origin of genetic systems. We have seen how the breeding system is genetically adjusted, and indeed Darwin was himself aware that the individuals of a species stood in a special adaptive relation to one another in reproduction. He was the first to show that the enforcement of inbreeding on plants which naturally outbreed resulted in a decline of vigour and fertility; but he was baffled by the general problem of these adaptations. He saw that the sex-ratio of a species must be selectively adjusted, but was unable to see how the selection could be achieved ; for he was unable to make out how adjustment of the sex-ratio, or indeed of any other property of the breeding system, could benefit the individual — which indeed it does not. This is the problem which we must now consider in order to see why some species outbreed and some inbreed; why there is sometimes a change from one to the other, or even the adoption of some more drastic device. We must compare these systems 275 Sni.rCTION AND VARIAIUIITY tlirough their effects on Darwinian fitness, the competitive power of the individuals comprising the species. How will inbred and outbred populations differ in the genotypes and phenotypes of their individuals? To answer this question we must look at the implications of elementary Mendclism, not now for experimental families, but for massed populations. The static must be translated into the dynamic. And in doing this, to understand crossbreeding and inbreeding, we shall fmd that we come to understand the organization of heredity in the wider sense of the interdependence of the relations between the genes in the nucleus and between the individuals in the population. The States of Variability Homozygotes and heterozygotes always differ in one respect, the respect by which they are recognized, i>iz. that heterozygotes give segregation in their progeny while homozygotes do not. If we breed a homozygote, or a group of like homozygotes, the offspring will be genetically and, apart from non-heritable fluctuations, phenotypically identical both with their parents and with one another. If, however, we breed heterozygotes (whether for one gene or for many), the offspring arc neither all alike nor all like their parents, genetically or somatically. Variation has appeared as a result of segregation. Now the germ of tliis variation must have existed in the heterozygotes, even though they themselves showed no variation. Thus we may distinguish between the latent or potential variability which heterozygotes themselves contain, and the variation or free variability which wiU make itself apparent in the phenotypes of their offspring when segregation has occurred. Let us consider a simple theoretical situation on a mendelian basis and, in the first place, in the absence of selection. A continued programme of inbreeding, by self-pollination or the mating of close relatives, when applied to the offspring of heterozygotes, will eventually result in the establishment of a population of homo- zygotes. Half of these will carry one (AA) and half the other {aa) of the two allelomorphs o(^ any gene for which the original ancestors were heterozygotes (Aa). The variability due to such a gene wiU then have changed from being entirely potential in the heterozygous 276 THE STATliS OF VAIUABILITY ancestors, to being entirely free in the homozygous descendants. Crossing two unlike homozygotes among these descendants [AA X aa) will restore the ancestral condition: the variability will once more be entirely potential. Variability is thus set free by segregation, and locked up by crossing unhke types. An inbreeding population consists completely of homozygotes, apart from the effects of mutation; but a crossbreeding population caimot consist only of heterozygotes, except in the special case where the gene happens itself to determine the breeding system. A gene such as that controlling incompatibility in cherries must always be in a heterozygous condition, because individual gametes alike in respect of it cannot be brought together. But a gene without this effect on the breeding system is in a different case. Maximum outbreeding in a large population can do no more than maintain such a gene in the states and frequencies given by random mating. With random mating, where the relative frequencies of the two allelomorphs {A and a) are u and v, the frequencies of individuals of the types AA, Aa and aa will clearly be : AA u" : Aa 2uv : aa v" and the maximum proportion of heterozygotes will be 0-5, achieved when u = v. With more than two allelomorphs the proportion of heterozygotes may be higher. Now, potential variability in respect of a single gene can be carried only by heterozygotes. A crossbreeding population will- therefore have some of its variability free and some potential. With two allelomorphs of equal frequencies, half the population will be Aa and half the variability must consequently be potential. Segregation will release half this potential variability in each generation, but at the same time inter-crossing of .4^4 and aa will be returning half the free variability to the potential state. These two processes thus balance one another, and the proportion of free to potential variability will remain constant. Nevertheless, because of the continual interchange of variability between the free and potential states, the situation cannot be a fixed one. There is, in fact, a. flow of variability: any particular variant, although present in the same proportions in different generations, appears in different families or lineages. The population may be, in a certain sense, 277 SELECTION AND VARIABILITY £xcd or Stable; but the genes are still moving and their combinations are changing. The flow of variability can be altered by selection; that is by the choice of certain types of individual as parents of the next generation. For example, we (or nature) can take as parents only the hetero- zygotes and one of the two types of homozygote [Aa and AA). We then effectively cut off the return of free variability to the potential state, by rendering impossible the cross AA X aa, through which this return comes about. At the same time we do not interfere with the reverse change, from potential to free, which depends on segregation amongst the progeny of hetcrozygotes. The net result of such a selection of parents is thus to lower the potential and to raise the free variability. The proportion of heterozygotes falls in the next generation; that of the homozygotes, of the two types taken together, increases. The joint increase is, however, due to increase of only one of these homozygous types, AA. In this process of selection the gene frequency has been changed. One allelomorph (A) is now more, and the other {a) less, common than formerly. The average phenory-pe will have moved in the direction of the more common type. If the selection is continued indefinitely the population will ultimately come to consist wholly of the one homozygote {AA), the other allelomorph of the gene having been eliminated. No variability will be left: it will have been expended on the change in average phenotype of the popula- tion. Thus selection of particular parents in each generation changes the mean phenotype, and this change is brought about by an alteration of the flow of variability in such a way that ultimately it will all pass into a. fixed state. Selection must then cease to produce any further change, because all the individuals have become genetically alike. Any differences they may show will be non- heritable. In a word, selection cannot produce changes unless free variability exists in the population. At the same time selection fixes, we may even in one sense say destroys, the variability on which the change depended. The alteration is permanent. A further aspect of this relation between variability and selection is seen when it is the heterozygotes which are used as the sole parents of the next generation. The flow from potential to free variability must go on, since (with the exception we have seen in 278 THE STATES OF VARIABILITY the complex hetero2ygote) segregation cannot be prevented. This segregation will be repeated regularly, and since the homozygotcs are eliminated as parents in each generation, the population will have the same composition each time after the first selection. It will always be ^ AA :\ Aa :\aa. Selection is not lowering the potential variability. No variability is being fixed, and selection is therefore ineffective in changing the average phenotype. With one gene difference in operation, as in the case we have been considering, there are only two states of variability. The heterozygotes contain all the potential variability, while the differ- ence between the phenotypes of the homozygotes always expresses the full action of the gene. There are, however, more possibihties where, as in polygenic variation, the genes have similar and supplementary effects on the phenotype. Even in the absence of dominance, different genotypes can, in such a case, give phenotypes differing only to the extent by which the genes do not correspond in effect. With genes of equal effect the same phenotype can be produced by a number of genotypes. In particular, genetically dissimilar homozygotes may, as we saw in Chapter 3, show like phenotypes. Thus with only two genes of equal effect, the homozygotes AAbh and aaBB will be alike, and will be intermediate betw^een AABB and aabh in their phenotype (Fig. 15). Yet these two inter- mediate homozygotes contain between them all the genetical material necessary for the production of the whole range of variation to which the two genes can give rise. They contain a new kind of variability, the potential variability of homozygotes, as opposed to that of heterozygotes. This new potential variability depends for its existence on the genes A and b, or a and B, balancing one another in action. Unlike the potential variability of heterozygotes, which not only can, but must, be partly freed by segregation in the next generation, the homozygotic potential will remain as such so long as cross- breeding is absent or at least restricted to like homozygotes. Thus, with close inbreeding, a population could be maintained which consisted of pure lines uniformly of the same phenotype, yet genetically of several kinds. The potential variability would be, as it were, frozen in such a population. Only by the crossing of unlike 279 SELECTION AND VAIUABILITY homozygotes could it be freed, and even then not ininiediately (Fig. 70). The first effect of crossing must be to produce hetero- zygotcsin which tlie variabihty is still potential. Only in the second, segregating, generation derived from these heterozygotes will the VARIABILITY GENOTYPES PHENOTYPES HOMOZYCOTIC UJ O 0.1 AAbb AND aaBB INTERCROSSED HETEROZYCOTIC PARTLY FREE 1 AaBb INBRED Segregating Family 1 I Fig. 70. — Part of the potential polygenic variability of heterozygotes is released, so that it shows as free variation in the phenotypes, by segregation in one generation. The potential variability existing in the differences between homozygotes, which are genetically unlike but having a similar balance and hence similar phenotypes, camiot be released in one generation. It must first be converted into heterozygotic potential by intercrossing. For the purpose of exposition in the diagram the genes A-a and B-b are assumed to be alike and additive in their action, and also to show no dominance. Thus AAbb, aaBB and AaBb have the same phenotypc (neglecting non-heritable variation), but the segregating family includes five phenotypes, in the frequencies shown also by Fig. 15 (after Mather, 1943). more divergent phenotypes be produced and the variability become partly free — only partly free because the balanced homozygotes will themselves reappear as part of the segregating generation. The heterozygotic potential state is therefore an essential inter- mediate step in the freeing of homozygotic potential variability. But on the other hand, the crossing of the phcnotypically extreme AABB and aahh gives AciBh from which AAbb and nnBB must 280 THE STATES OF VARIABILITY segregate in the second generation. The heterozygotic potential is thus equally the intermediate step in the opposite process — the transference of free variability to the honiozygotic store. Where more than two genes arc concerned the same principles apply : there is merely a greater range of possible balanced homo- zygotes. hi the case of four genes having equal and supplementary NEW POTENTIAL FREE FIXED MUTANT i HETEROZYGOTIC i t PHENOTYPIC SELECTED HOMOZYGOTIC Fig. 71. — The states of polygenic variability. There is a constant flow between the free and potential states, the heterozygotic being distributed by segregation to the free and homozygotic states in proportions depending on the linkage relations of the polygenic system, and the free and homozygotic variability passing into the heterozygotic state by crossing. There is no direct flow between free and homo- zygotic potential. Free variability is the raw material for selection, which is con- stantly using up or fixing a portion of it. This loss will be balanced over long periods of time by the rise of new variability through mutation; but the balance will not generally be struck accurately over short periods of time. The thinner arrows indicate that the loss by selection and gain from mutation are slower than the flow between the other three states (after Mather, 1943). effects, for example, six homozygotes exist of the general type AABBcah^, and four each of the less balanced general types AABBCCdd and AAhhccdd. The more the genes, the greater the variety, and hence importance, of the homozygotic potential state. Since the variability must all go through the heterozygotic potential state before it can be redistributed between the homo- zygotic states, free and potential, the proportion of heterozygotes in a population will govern the rate of flow of variability from one state to another (Fig. 71). Heterozygotes can, of course, arise from the inbreeding of pre-existing heterozygotes, but under such a system they will be present in a proportion which decreases from 281 Sni.ECTION AND VARIABILITY generation to generation. A fixed proportion of heterozygotcs in the population can be maintained only by means of crossing or outbreeding, and any given amount of outbreeding will maintain a corresponding proportion of heterozygotcs. The breeding system, in determining the proportion of heterozygotcs, detcrniincs the rate of flow of variability between the different states. Random mating gives virtually the maximum outbreeding and the maximum flow, while close inbreeding gives no flow at all. Outbreeding means genetical lability, inbreeding genetical fixity. Fitness and Flexibility The importance of the difference in effect between inbreeding and crossbreeding becomes clear when we consider what natural selection is doing. If we observe a character in which a population is freely and continuously variable, as for example stature in man, we find that most individuals show it to an intermediate degree and the extreme expressions are rare. As Galton put it, the majority are mediocre. The significance of this is shown by Bumpus' observations. He found that a number of characters, bone lengths, wing spread and skull conformation, varied in a sample of sparrows in the same way as stature in man, the mediocre again being the most common. Now these sparrows had been disabled in a rainstorm, but some of them later recovered and flew away. The birds which thus overcame the ill effects of the storm proved to be those which approximated most closely to the form characterized by means of the various measurements. Those that died were the more extreme individuals, regardless of the direction, positive or negative, in which they were extreme. Natural selection was operating in favour of the mean, and against departure, whatever its direction, from the common type. So far as the variation of these sparrows was heritable (and while there is no evidence on this point in regard to the sparrows, it is the general rule derived from a wide variety of organisms that a portion of such continuous variation is polygenically determined), the effect of selection was to favour the more balanced combinations of genes. Natural selection has been observed in operation in this way on 282 FITNESS AND FLEXIBILITY very few occasions, but the favouring of the mean type is in agree- ment with expectation. If one extreme were favoured consistently, the mean of the population would move in that direction, in so far, of course, as the variation was heritable. And it would continue to move as rapidly as the available variability permitted until it approximated to the optimum phenotype, at least sufficiently well for the environment not to favour departure preponderantly in one particular direction. In the third case, where both extremes are favoured at the expense of the mean, a new state of affairs arises, one we shall consider in the next chapter. A character such as fertihty might be regarded as being in a different situation, for one might expect greater fertihty to be favoured almost without limit. It must be remembered, however, that fertility is itself the expression of a number of sub-characters, and these must be balanced against one another. To take an example, man has a smaller number of offspring at a birth, or in a lifetime, than the pig. But we could hardly regard an increase to the pig's litter size as likely to increase the expectation of posterity in man, because the success of each child requires an expenditure of parental care and training which would thereby be rendered impossible. Too many offspring would be as bad, though in a different way, as too few. The adverse effect of too large a litter can be seen even in the pig itself. The mortality between birth and the age of three weeks becomes so great in litters of 14 and more that the number of pigs surviving to this age is somewhat lower in the bigger litters than it is in those of 14 and 15. At six weeks the disadvantage of too large a litter is still more striking and the size of litter which gives the maximum average number of survivors is even lower than at three weeks (Fig. 72). In the same way, other tilings being equal, the excessive production of eggs or seed by any animal or plant would mean a crippling reduction in the food supply with which each was endowed. Thus with fertility, too, the principle ot the optimum must apply. What will be the effect of this principle that the average is favoured at the expense of the extremes? With the inbreeding system, where the population consists entirely or very largely of homozygotes, the effect o{ selection in favour of the intermediate 283 SELECTION AND VARIAIHIITY classes must be mainly to favour the more balanced homozygotes at the expense of the less balanced. The favoured individuals wiU produce offspring genetically like themselves; and in so far as the environment is stable, the population must show high agreement with the optimum phenotype. Should the environment change 2 4 6 8 10 12 SIZE OF LITTER Fig. 72. — The average numbers of young surviving for 3 and 6 weeks from litters of various sizes at birth in pigs. The straight "birth" Hue is the line of no loss. The loss becomes disproportionately greater as the size of litter increases. The maximum survival to 3 or 6 v^ceks old is given by litters of interniediate size at birth. (Data for 3 weeks (as dots) from Johansson, 193 1, and for 6 weeks (as crosses) from Menzies-Kitchen, 1937.) permanently, however (as sooner or later it presumably must) the population camiot change genetically in the way necessary to give a new adjustment: the variability is frozen in the homozygotic potential form. Such a population, therefore, shows high immediate fitness but no flexibility. The inbreeding system, advantageous so long as the environment is stable, becomes a handicap when the environment changes. A population witli an outbreeding system is in the other case. 284 THE EFFECT OF LINKAGE Balanced genotypes of intermediate phenotype are favoured ; but owing to the flow of variability, which arises from the outbreeding, the extreme types can never be lost. They will constantly reappear by segregation. Thus the population must always vary around the optimum, and fitness can never be so high as in an inbreeding group. The flow of variability ensures, however, that should the environ- ment change, and thereby shift the optimum phenotype, the population will be able to achieve a corresponding adjustment by utilization of the free variability which is always available, hidividuals approximating to the new optimum will be present and, being favoured by the new conditions, will contribute more to the next generation. Thus the genetical constitution of the population can, and will, change to meet the demands of the new conditions. The system does not maintain the maximum immediate fitness, but it is flexible. As compared with inbreeding it is certainly at a disadvantage for the moment; but it confers on an organism a much greater prospect of leaving descendants in a changing world. The Effect Oj Linkage The disadvantage of high variability under the outbreeding system may be mitigated, and the opposing needs of fitness and flexibihty be partly reconciled, in a way which has been revealed by selection experiments. One of these, described by Sismanidis, was concerned with the number of bristles borne on the scutellum of Drosophila melanogaster. This number is almost always 4, but in some mass-bred stocks occasional flies, mainly females, are found with 5. Commencing with such females mated to normal males, Sismanidis endeavoured to raise the average bristle number by selective breeding, hi this he was successful. Two of his selection lines, maintained by brother-sister mating, are shown in Fig. 73. After 23 generations of selection the average was raised in females from below 4-1 to over 5 '2, but the advance had not been smooth. There was a large change from just below 4*2 to over 4*5 between generations 2 and 3 in one line, and between generations 6 and 7 in the other. In the next 1 1 generations in the one and 7 in the other the advance was only o*i, but between generations 14 and 17 a 285 SELECTION AND VARIABILITY second quick change of nearly o-6 occurred in both, to be followed by another period of near stability until the end of the experiment. The responses to the selection shown by tlie males were smaller, but conformed to the same pattern. Tests were made to determine the effects of the three major chromosomes on bristle number at generations o, 13 and 21, 5-5 50 4-5 40^ RESPONSE DUE TO CHANCE IN IT CHROMOSOME II,in 10 15 GENERATIONS OF SELECTION Fig. 73. — The effects of selection for increased number of scutellar bristles in Drosophila mclatiogaster. The average number of scutellar chaetae is plotted against generations of selection, all matings being of brothers and sisters. The two selection lines came from the same parents in generation i, and gave parallel results except that the first major response occurred four generations later in one than the other, and the second major response perhaps one generation later. The responses were nevertheless of equal or nearly equal size, and due to change in the same chromosomes, in the two lines (based on Sismanidis, 1942). i.e. before selection commenced, after the first chief advance, and after the second. They showed that the first advance in both lines was due entirely to an increase in bristle-producing power of chromosome II; while again in both lines the later advance also involved chromosome III. The X chromosome appeared not to have changed in either line during the experiment. Such regular changes can hardly be attributed to new variability arising by mutation. To what are they due ? Selection, as we have seen, is effective in changing the mean 286 THE EFFECT OF LINKAGE phcnotype oiily in so far as there is free phenotypic variability, which becomes fixed as a result of the selection. Since the method of selection was constant throughout Sismanidis' experiment, we must suppose that free variability was low or absent during the periods of little advance, but that it was present when the two chief responses to selection occurred. That one of these came after genera- tion 14 shows that variability was present during the preceding generations; but since the response was delayed, the variability must have been largely in the potential state. Now potential variability is freed by segregation and recombina- tion. In the present case the chromosome tests showed that the changes occurred within whole chromosomes. In other words, the effective recombination must have been of linked genes, as a result of crossing-over between them. Originally in the form of balanced linked combinations of polygenes, crossing-over must have led to the production of less well balanced genotypes, giving more extreme pheno types, i.e. to the freeing of variability which must have been present in the group of parents with which the experiment began. Linkage must have the general effect of tending to maintain genes in the same combinations even in cross-bred and heterozygous populations. If we cross AABB and aahh, where the genes are linked, the combinations aB and Ah wiU be produced in the segregating generation only so far as crossing-over gives rise to recombination. If the linkage is close, they wiU be uncommon for some time. The combinations AB and ab will be released equally slowly where the parents are AAhb and aaBB. Thus, while the proportion of heterozygotes, and hence of heterozygotic potential variability, wiU depend solely on the breeding system, the rate of flow of free variabiUty to the homozygotic potential state and back wiU be governed by linkage as well. The flow will be at its maximum when recombination is free, for a double heterozygote then produces equal numbers of AB, ah, Ah and aB combinations no matter what its origin may have been. But at the other extreme, with close linkage, the interchange of variability between the free and homozygotic potential states wiU be small: after passing into the heterozygotic pool, free variability wiU mostly emerge as free variability, and homozygotic potential as homozygotic potential. 287 SELECTION AND VARIABILITY Where natural selection is penalizing the less well balanced combinations of genes in a chromosome, genes of opposing or balancing effects must tend to become tied together by the linkage. The population will then show a smaller spread round the optimum plienotypc than it would in the absence ot linkage. Fitness will consequently be higher, as it is with an inbreeding system. But the more extreme combinations will still be produced on occasion by crossing-over ; and, even though less common, they will still afford the material for selective adjustment of the genotype to changing environment. In fact they will be just as effective in this way as if they were released with the unlinked frequencies. Flexibility will therefore be fully maintained at the same time that fitness is increased. In a large measure, linkage can reconcile their rival needs. Tlie Control of Recombination We may now ask ourselves: how tight is linkage in practice? How much crossing-over actually takes place between paired chromosomes e The chiasmata have shown us that all chromosomes in all species of sexually reproducing organism undergo crossing- over, and that the position of crossing-over in any particular pair varies from cell to cell and therefore, obviously, from generation to generation. It sometimes happens, for example in Viciafaha and in many species of lilies, tiiat each pair of chromosomes forms an average of five or more chiasmata — far more than the bare minimum number needed to hold them together at the first metaphase of meiosis. But this is rare. In general, what we find is a close restriction of the number of chiasmata to this mechanical need. Some variation in number is doubtless required if variation in position is to be maintained, and two-armed chromosome pairs frequently form two chiasmata. The adjustment of chiasmata to a minimum has been neatly shown in maize. An "asynaptic" gene, in the homozygous state, reduces the frequency of chiasmata at meiosis. Indeed in extreme cells of extreme plants it causes the chromosomes to fall apart at the end of pachytene without any formation of chiasmata at all, so that they are all unpaired at diakinesis and metaphase. Typical samples are shown in Table 25. 288 THE CONTROL OF RECOMBINATION TABLE 25 CHIASMA FORMATION AND CHROMOSOME PAIRING IN NORMAL AND ASYNAPTIC MAIZE (BEADLE, 1933) Number of cells Numbers of bivalents with dift'erent numbers of chiasmata 0 1 2 3 Average bivalents per cell Average chiasmata per bivalent Normal . . Asynaptic, moderate . . Asynaptic, extreme 10 27 58 0 27 72 1 50 49 171 0 552 28 0 0 10-0 8-1 0-5 1-7 14 005 When the asynaptic plants are bred, one might expect their progeny to show a great reduction in the frequency of crossing-over. The reduction, however, is not uniform. On the female side, in fact, there is no reduction at all. The explanation is instructive in several ways. Either the action of the gene in upsetting meiosis is different on the female side. Or the selected sample of the products of meiosis is different. The germ cells which are effective in breeding are those with a complete set of chromosomes. Complete sets are pro- duced only when the mother cell, at least in the large female cell where unpaired chromosomes are always lost, has had complete pairing. And complete pairing has been attained in those cells only in which the chromosomes had a normal, or nearly normal, frequency of chiasmata. In the male cells incomplete pairing will sometimes give complete sets by chance. And on the male side some reduction of crossing-over is found. The results are : normal, 24 per cent recom- bination between the genes shrunken and waxy; female asynaptic, 24 per cent; male asynaptic, 13 per cent. Apart from low-frequency adjustment there are two other methods of reducing the effective frequency of recombination. One is by locaHzation. In a greater or less degree aU organisms have some localization of chiasmata. This localization seems to be produced by a delay in pairing, so that the later pairing produces no coiling strain or is even totally inliibited. Crossing-over is then confined to the region where pairing began. In some species, such as the grasshopper Afecoifef//;/-? grossus or the lily, Fritillaria meleagris, the crossing-over is localized near the centromere. In others, a more numerous congregation, it is localized near the ends, for example in Elements 0} Genetics 289 T SELECTION AND VARIABILITY Tradescantia virginiana, the iicwt Triton pahnatus and, as we saw, in the species of Oenothera. Localization of cither kind of course, does something more than merely reduce recombination. It divides the genes of each chromosome into two regions, one with high and the other with low recombination or none at all. It is interesting, if disappointing, to discover that although the heterochromatin falls in the low group in Paris it occurs in Fritillaria chiefly in species without localization and in regions where crossing-over occurs. The third method of reducing recombination is found only in animals. It consists in nothing less than the abolition of crossing-over in the heterozygous sex. A new method of holding the chromosomes together at meiosis is found in the males of certain Diptera and Orthoptcra. The attraction between chromatids is extended from twos to fours, and the early repulsion of centromeres is suppressed. Crossing-over is retained with a normal meiosis in the homozygous sex. This means that a chromosome passing, for example, from one male Drosophila to another may avoid crossing-over generation after generation. But this may also happen with an ordinary low frequency of crossing-over in plants. For, with a single chiasma, only two of the chromatids are cross-overs, and from each bivalent two of the four germ cells have unchanged chromosomes. So far as the species is concerned, therefore, the genetical result of the abolition of crossing-over in one sex is quite indistinguishable from a reduction of chiasma-frequency to one half in both sexes, itself an unworkable arrangement since, as we saw, that frequency is usually already at a working minimum. These variations show that, in the development of the genetic systems of plants and animals, natural selection has paid a great deal of attention to this problem of recombination. Close linkage is nearly always encouraged. This we can understand if the tendency is for variability to exist in the form of balanced linked combina- tions, genetically unlike but of similar effect on the phenotype. In such cases, the amount of recombination favoured over any period will depend on the balance of advantage of fitness and flexibility, and hence indirectly on the rate of change of the environment during that period. Since only occasional recombination is required to maintain flexibility, the frequency of crossing-over which is favoured will generally be low. 290 BALANCE IN HOMOZYCOTES AND IIETEROZ YC OTES Balance in Homozygotes and Heterozygotes While inbreeding can maintain a state of uniform homozygosity apart from the effects of mutation, outbreeding, as we have seen' cannot of itself maintain a state of uniform and high heterozygosity, except for such genes, or super-genes, or differential segments, as control the breeding system itself The maximum proportion of heterozygotes for any one gene with two allelomorphs will not exceed one half in a large population. Yet, when all the genes carried by a zygote are taken into account, clearly the chance of its being completely homozygous is remote. Each genotype will be a mixture of the heterozygous and the homozygous. The average proportions of the mixture will depend on three factors, the breeding system, the number of allelomorphs of each gene, and the relative frequencies of these allelomorphs. This dual condition of the genotype has two consequences. At any given time some of the combinations of genes are being exposed to the test of natural selection in the homozygous condition. Linkage wiU therefore be favoured in the way that we have already seen. The whole set of genes, on the other hand, even those of one chromosome, will virtually never be homozygous simultaneously. The combinations of genes will therefore be adjusted or balanced to perform their task always in a partly heterozygous condition. Now where more than one gene is involved, and each gene shows dominance, the phenotype of a heterozygote will show no predictable relation to those of the corresponding homozygotes. Even with only two genes, the phenotype o(AaBh may fall between those of AAhb and aaBB or may transgress their range in either direction, according to the directions and strengths of the dominance properties of A-a and B-h. Thus, where a polygenic system is concerned, the favouring and balancing of partly heterozygous genotypes by natural selection can offer no guarantee that the corresponding homozygotes wiU be similarly balanced. Indeed in view of the multipHcity of genotypes, all more homozygous than the heterozygote from which they arise by segregation and recombination, there is no reason why any one of them should be automatically balanced. Smaller gene combinations forming parts of the genotype may themselves be balanced as a result of 291 Sni.nCTION AND VAUIAIUIITY previous exposure to natural selection in the homozygous state; but the wholes, made up of these parts, are unlikely to be adjusted. This is the more so because even the parts themselves must tend to change in the flow of variability. Thus, forcing an unnatural inbreeding on an outbreeding species will lead to the exposure of genotypes which, through the rarity of their occurrence under the original system, will not have been subjected to adjustment, by natural selection, in their action on the phenotype. The result is maladjustment, or inbreeding depression. It is contingent on inbreeding and must vanish when outbreeding is resumed. Even where a heterozygote was made up from gene combinations which were balanced when fully homozygous, inbreeding depression must soon become a property of its descendants. Segregation and recombination would ensure that, of the homozygotes which could be extracted from such a heterozygote, those which went to its making, or indeed any balanced combinations, would form but a small fraction. The greater the number oi variable genes concerned, the sooner and the more completely would this scrambling of the genotype lead to the loss of the property of balance in homozygous derivatives. Heterozygosity must obviously shelter recessive genes from natural selection. Not only does this lead to the lack of balance of polygenic combinations, liable to segregate as homozygotes, in the way we have seen; deleterious mutant allelomorphs of major genes, genes of drastic effect, should also survive under this shelter. Plants and animals collected in the wild have remarkably often proved to carry such genes when tested in the laboratory. Indeed, on the basis of tests with Drosophila pseudoobscura, Dob- zhansky estimates that at least one wild fly in four carries a mutation causing lethality, or near lethality, when homozygous. The segregation of such sheltered recessive allelomorphs of major genes will always follow inbreeding, but it is not, as has often been supposed, the cause of the characteristic inbreeding depression. Inbreeding does not produce a rising proportion of the more deleterious of two alternative phenotypes in succeeding generations, so giving an average decline only when viewed over the whole generation. Rather it gives a steady and progressive decline of all 292 BALANCE IN IIOMOZYCOTES AND IIETEROZ Y GOTES members of the population. Such a steady decline is to be expected from the increasing homozygosity for numerous polygenic combinations within each individual. The averaging process is within the individual rather than within the population. Inbreeding an outbreeder consistently leads to inbreeding depression: complete homozygotes are poor. Outbreeding an inbreeder has no such effect. The heterozygotes are seldom very different in vigour and fertility from their homozygous parents. Indeed, crossing different lines has not uncommonly given heterozygotes of somewhat greater vigour than either parent, for example, in the diploid cultivated tomato, and in the tetraploid wild Galeopsis tetrahit. The reason for this difference is apparent immediately we consider the flow of variability. In an outbreeder this flow is strong and, even were a heterozygote to be made from combinations balanced in the homozygous condition, these would soon be destroyed by the constant crossing, segregation and recombination. In an inbreeder, on the other hand, the flow of variability is weak or non-existent. Combinations, once established, remain in being, apart, of course, from mutation. If, when they were established, these combinations gave an adequately balanced heterozygote, as well as adequately balanced homozygotes, they would retain this property. Now the tomato is known to be derived from outbreeding ancestors in Peru. Inbreeding is similarly a recent imiovation in Galeopsis tetrahit, whose diploid ancestors were outbreeders. It is therefore to be expected that the combinations, now homozygous in the tomato and in G. tetrahit, will have retained the capacity for givmg balanced heterozygotes. There is a reduction of advantage in vigour and fertility of heterozygote over homozygote. But this is due to the grading up of the homozygotes, by the selection ot balanced homozygous combinations, not to the grading down of heterozygotes, by loss of balance through shelter from selection. Heterozygous or relational balance can only be produced by the action of selection in an outbreeder. It is nevertheless retained in the absence of selection when inbreeding freezes, as we may say, the flow of variability. Balance presumably can be lost in such a case through mutation; but loss by nmtation must be a slow process 293 SELECTION AND VARIABILITY compared with loss by redistribution of variability, as we can sec from observations such as those on the tomato and Gakopsis. Selection ami the Reservoir of Variability The sheltering effect of heterozygosity and dominance depends on the fact that selection discriminates primarily between phenotypes, and hence between genotypes only to tlie extent that they give different phenotypes. The phcnotype is, as it were, the organ by which the genotype is selected. Yet it is upon the favouring of particular genotypes that response to selection, as it is expressed in the phenotypes of succeeding generations, must depend. A lag of at least one generation must consequently intervene between the action of selection and the response which it produces. Thus, changes under selection can be adjustive only to the extent that the changes in environment, which produce them, are permanent. Not all changes in the environments to which succeeding generations are subjected can be of this kind, hi an ephemeral organism, such as a fruit fly or a chickweed, the different generations live at different times of the year, and hence are subjected to a series of environments whose main changes are cyclical rather than permanent. The changes of environment in which successive generations of ammal organisms find themselves are, if not cyclical, at least erratic rather than permanent. Only with longer-lived species will the vagaries of environmental change tend to even themselves out. Non-permanent fluctuations of environment must always be occurring, though their significance to the organism may not be constant. It will depend on the relative magnitude of any permanent changes which may be going on at the same time and which may be more rapid at one period than another. When, for example, the first birds evolved, they had a whole new field to themselves. As they multiplied and filled it, competition must liave increased rapidly, and with it the environment of each bird must have changed in the direction o{ becoming permanently more exacting. With the attainment of something approaching maximum numbers the environment, as determined by this competition, must have become more stable, though non-permanent changes would still 294 SELECTION AND THE RESERVOIR OF VARIABILITY occur according to chance fluctuations in numbers and other contributory circumstances. The non-permanent changes, originally of lesser importance, must have come to mask the permanent. Now the organism has no means of distinguishing permanent from non-permanent changes in its circumstances. If it is sufficiently flexible genetically to respond to permanent changes, it must also respond to the non-permanent ones. Cyclical changes in the environment may be accommodated by cyclical changes in the 100 ^ fi 80 CO 40 ^ 20 WO 80 60 *» IV X t930 IV X If X 1933 /V X /93^ IV X /S35 Fig. 74. — The percentages of the population of the ladybird Adalia punctata with black and red ground colours in April (IV) and October (X) over a period of six years in Buch, Germany. There is a regular seasonal change in the frequencies of these genetically controlled alternatives, red being commoner in spring than it is in autumn (after TimofeefF-Ressovsky, 1940). relative frequencies of allelomorphs of genes or super-genes govern- ing polymorphism in an ephemeral (Fig. 74); but this is a special case. Permanent adaptation must generally demand the irreversible fixation of genes, and if this can be brought about by permanent changes, so it can by temporary ones too. In responding thus to non-permanent changes, the stock will gain nothing in fitness, and will lose some of the variability on which prospective adaptation must depend, by the profitless fixation of free variation under the erratic selection. This loss must be made good in the long run by new variability from mutation (Fig. 71), as indeed must any reduction in variability whatever its cause, if the species is to survive. In fact the amount of variability, when at equihbrium in a popula- tion, will be such that the loss, itself proportional to the free and 295 SELECTION AND VARIABILITY hence to the total variabihty, is equal to the increase by mutation, an increase which is obviously independent equally of both kinds of variability. When loss exceeds gain, the total variability will diminish until an equilibrium is reached, and vice versa . Loss and gain of variability must balance in the long run, but they are unlikely to do so over short periods. Mutation might be expected to over-compensate for loss when this arises solely from erratic changes in environment, but to fall short of doing so when rapid permanent change is also causing the expenditure of variability. The importance of the reservoir of potential variability lies in its smoothing effect, in its ability to permit compensation of rapid expenditure over short periods by a steady trickle of gains over a longer time. All selective changes must ultimately depend upon mutation; and the capacity for mutation, itself apparently under some selective control, represents the final reserve of variability. But it is a reserve which is released at a more nearly constant rate. The immediate reserve of potential variability represents the storage of these mutations by the genotype, in such a way as to permit more rapid response to selection than mutation could give directly. Change of Genetic Systems: Inertia We can now consider the properties to be expected of genetic systems in the light of these principles. The genetic system seen in any stock or race of organisms today, with its special properties of balance, storage and release of variability, is that which has enabled the ancestral line of the organisms concerned not merely to become, but to be continue to be, adequately adapted to its changing environ- ment throughout the course of its history. The stock has survived, and with it the genetic system upon which survival has depended. Continuation of this system must depend on the extent to which the j^enetic system enables its possessor to meet the exigencies of future change. So long as the advantage lies with the maintenance of a highly uniform and constant phenotype, the inbreeder will be well endowed by its genetic system for success. But whenever the advantage lies less with high uniformity than with easy change of phenotype, the outbreeder will be the more successful. If the environment changes in its demands, the genetic system will cease 296 CORRELATED RESPONSE to be advantageous to its possessor except in so far as it can change too. Since the genetic system must ahvays be under its own control, an outbreeding system will be able to change in the direction of inbreeding by virtue of the flexibility which is its special property. The more rigid inbreeding system will be less likely to show change towards outbreeding. Evidently inbreeding systems must generally be in evolutionary dead ends, doomed by inflexibility to extinction when a crisis arises, but always being thrown off by the continuous stream of outbreeding systems. We have already seen that the evidence from stratification of breeding systems fully accords with this expectation. This evidence of stratification in the breeding systems of, for example, wheat and Pediculopsis, can leave no doubt that changes of system do occur. Nor can the comparison of the breeding systems of relatives such as wheat and rye, or the Galeopsis species. Yet there are obstacles to change inherent in the systems themselves : obstacles which must slow the change-over even where they do not prevent it. With the inbreeding type the obstacle, a fatal one, is, as we have seen, its own inherent rigidity. With the outbreeding system it is the less serious one of lack of balance of the more homozygous genotypes which increased inbreeding produces in greater numbers. Each system is internally consistent: change introduces a measure of discordance. They show what we must describe sls genetic inertia. Correlated Response : Capital and Subordinate Characters Genetic inertia is due to integration, the building up of an adaptive system providing the very properties which have been responsible for its past success. This inertia of adaptation can also be seen at work in other ways than in the retardation of change in the breeding system. In his experiment on the scutellar bristles of Drosophila, Sismanidis records that of seven selection lines with brother-sister mating, three died out — through infertility. This may, as we saw earher, have been the simple effect of inbreeding. But in other such experiments with flies, and also with fishes, a similar loss of fertility has regularly been found to accompany the change of a somatic character under selection. It has done so even where out- breeding was kept at a maximum. Furthermore, in an experiment 297 SELECTION AND V A K I A HI I IT Y with flics, selection for change in chaeta number not merely robbed, the stock of its fertility: it also changed the pattern of pigmentation, the mating discrimination and the number of spermathecac. This last change was especially marked : instead of the normal two, as in the parents, selected females had from none to five spermathecac. Correlated responses to selection such as these may be due, in part, to one gene affecting several characters, the pleiotropy which we discussed earlier. But there is also another agency at work. Chaeta numbers showing the effects of selection and at first associated with a lower fertility have, later in the same experiment, become asso- ciated with a higher fertility. The two effects must therefore be due to different genes, and the cause of the correlated response must be linkage. Crossing-over within a chromosome will bring about recombination, not merely within the polygenic combination affecting the character in which we are interested, but also within the other polygenic combinations which are intermingled with it along the chromosome. So in selecting for changed combinations of one set of genes, change in the combinations of sets affecting other characters, for which no selection is practised, will also be brought about (Fig. 75). And if these changes cause a sufficiently large unbalance, their consequences will appear as an alteration in the expression of the character they control; an alteration for which there is no direct selective cause. When this has happened, however, continued recombination of the gene sets can eventually lead to a reassociation of the characters, of the kind we see when the flies with changed chaeta number regain their fertility. Even pleiotropy may, as we saw, merely express complete linkage. But, with pleiotropy, the reassociation of characters would have been out of the question. Although the response must go in a fixed direction for the character on which selection is primarily acting, the correlated responses in other characters need not do so. The correlated changes will therefore often be deleterious. Correlated response should in consequence act as a brake on the primary response to selection, both in nature and in experiment. Decline and death is characteristic of selection lines in experiment and bears witness to the truth of this principle. The better adjustment in one character must be paid for by the worse adjustment, at least for a time, of others. The 298 CORRELATED RESPONSE price of linkage is therefore that, apart from the primary character, the immediate overall response to selection is poorer. Time is At Bi A2 ba Ol bl 02 B2 CROSSING -OVER A, bl 02 Bz 1 1 1 Az b; Ai bl 02 bz 01 Bl A2 B2 Ai bl A2 bz Ol Bl 02 B2 Fig. 75. — The mechanism of correlated response to selection. The two A-a genes represent members of one polygenic system and the two B-b genes of another all carried on the same chromosome. Both systems are balanced in the sense that each of the original chromosomes carry genes of opposing tendencies, as indicated by capital and small letters. Selection will pick out the chromosomes which have lost this balance by recombination bringing together two genes of reinforcing tendency either in the direction indicated by capitals or that by small letters. According to the position of the crossing-over, upon which recombination depends, (i) the A system may be unbalanced, the B sys- tem remains balanced (left); (ii) the B system may be unbalanced, the A system remaining balanced (right), or (iii) both systems may be unbalanced (centre). Then in (i) selection c^n be effectively practised for the character controlled by the A sys- tem without that controlled by B changing, or (ii) selection can be effective for the B character without the A character changing. But in (iii) effective selection for either character must also result in change in the other, solely by virtue of the linkage of the genes. This is correlated response, and it may be in either direction for the subor- dinate character, again according to the precise linkage arrangement. luccded for recombination to permit the attainment of a full new adjustment in the primary character without deleterious effects on 299 SELECTION AND VARlAlilLITY Others. Again wc see that, although a change can be achieved, there are obstacles to be overcome first. The very linkage v^hich is so powerful in reconciling the needs of flexibility and fitness when displayed between genes of similar action, results in an inertia when displayed between genes of divergent action. The extent of this inertia will be dependent on the degree of selective disadvantage resulting from the correlated response as compared with advantage arising from the primary response. It the correlated change should occur in a character of minor impor- tance to the organism's fitness, it could result in the fixation of a new expression of this character under the action of an unrelated selective force, hi other words, subordinate characters may be pushed about by the selective forces acting on capital characters. Selection can in this way produce a change from which no selective advantages could arise, and which therefore will show no trace of the agency which caused it. Subordinate selection is seen where the character in question was once of great moment for the organism but has lost all its importance. Such a character, for example, is sight in cave animals. Once the selective advantage of adequate sight is removed by the adoption of cave life, selection for other characters, such as sense of touch, is able, through correlated responses, to bring about the breakdown and atrophy of the visual mechanism which has been degraded to the rank of a subordinate character. We are now in a position to look back at the situation as it w^as left by Darwin. To Darwin, variation was what he saw in the members of a living species. He discovered the great problems of inbreeding and outbreeding, of adaptation and selection; but, in terms of the variation that he saw, these problems could not be solved. Now we realise that underlying the visible variation arc organisations of genes and chromosomes which exist largely as means of suppressing the appearance of variation. They contain the variability and reveal it in ways which the principles of Mendclism and Morganism enable us to understand. The operation of these principles is, of course, complex and in a sense abstract. But it is capable at every stage of being submitted to experimental test, and of yielding the predictions that are necessary both to the theory ot evolution and the practice of plant and animal breeding. 300 Srr.ECTION AND VARIABILITY REFERENCES BEADLE, G. w. 193 3. Further studies of as)Tiaptic maize. Cytologia, 4: 269-287. BUMPUS, H. c. 1899. The ehmination of the unfit as ilkistratcd by the introduced sparrow. Biol. Led. Woods Hole, 1898: 209. DARWIN, c. 1868. The Variation of Animals and Plants under Domestication. London. DARWIN, c. 1 871. The Descent of Man and Selection in Relation to Sex. London. DARWIN, c. 1876. The Effects oj Cross and Self-Fertilization in the Vegetable Kingdom. London. DOBZHANSKY, T. 1 941. Genet ics and the Origin of Species. 2nd ed. New York. FISHER, R. A. 1930. The Genetical Theory oj Natural Selection. Oxford. JOHANSSON, I. 193 1. Breeding pigs for high prohficacy. Pig Breeder's Annual, 11: 80-87. LURIA, s. E. 1945. Mutation of bacterial viruses affecting their host range. Genetics, 30: 84-99. MATHER, K. 1943 . Polygenic inheritance and natural selection. Biol. Revs., i8: 32-64. MATHER, K. 1943- Polygenic balance and the canalization of development. Nature, 151: 68-71. MATHER, K., and HARRISON, B. J. 1949. The manifold effects of selection. Heredity (in the press). MENZiES-KiTCHEN, A. w. 1937- Fertility, mortality and growth rate in pigs. J. Agr. Sci., 27: 611-625. MUNTZING, A. 1945. Hybrid vigour in crosses between pure lines of Galeopsis tetrahit. Hcreditas, 31: 391-398. RENDEL, J. M. 1945. Genetics and cytology of Drosophila suhohscura. II Normal and selective mating in Drosophila suhohscura. J. Genet., 46: 287-302. SISMANIDIS, A. 1942. Selection for an almost invariable character in Drosophila, J. Genet., 44: 204-215. TiMOFEEFF-RESSOVSKY, N. w. 1 940. Zur Analyse des Polymorphismus bei Adalia hipunctata. Biol. Zhl., 60: 130. VAViLOV, N. I. 1914. Immunit)^ to fungous diseases as a physiological test in genetics and systematics, exemplified in cereals. J. Genet., 4: 49-65 3OT CHAPTER 14 THE BREAKDOWN OF CONTINUITY The Mating Coiitimmm Restrictive Practices The Internal Origins of Isolation Floating and Fixed Discontinuity Restriction and Flexibility The Traces of Aticestry Selection, as we have seen, distinguishes only between phcnotypic differences. It is, therefore, effective in changing the genetic constitution of a population, or group of individuals, only in so far as the genetic differences of the group are, or can become, expressed in the phenotypcs, whether of cells, individuals or populations. Now the release of potential variability into the free state depends on segregation from heterozygotes. Hence, as we have seen, the potential variability of an inbreeding species is excluded from providing the material for selective change. In such species, change under selection will be confmed to the sorting out of such differences as appear immediately in the phenotype: it will soon be over and will be small in effect. Only groups with outbreeding systems can respond by extensive change to the continued action of selection, whether natural or artificial. And this is as true of the production of change in the genetic system itself, a property of the species, as it is of change in the phenotype of the individual, since the one as much as the other depends on change in the genotype. It is, therefore, to the properties of outbreeding that we must turn in order to see how changes in genetic systems come about, and in particular how groups initially unitary in their breeding come to break up. The Mating Continuum Under an outbreeding system an individual receives its genes from two parents which are always, or at least nearly always, genetically different. The same will have been true of the two parents in their turn. Each individual can therefore be regarded as representing the fusion of a number of lines of descent. In the same 302 THE MATING CONTINUUM way, each individual will pass on some of its genes to, on the average, two genetically distinct offspring and so may be regarded as initiating a number of lines of descent. The lines of descent, of which any individual represents either the culmination or the initiation, are obviously not independent of those which are represented by other individuals of the same generation. Sooner or later, if we trace the ancestry of the members of a population at one time, or if wc trace their descendants, we shall find the lines merging. In this sense an outbreeding group represents a mating continuum: a continuum within which any member may have received genetic materials from over the whole area which the group occupies. Looked at in another way, from the standpoint of the distribution of the group, the various sub-groups into which it may be divided spatially, or perhaps in other ways, can be regarded as exchanging chromosomes or exchanging genes. The individuals in one sub-group will have lines of ancestry tracing to other sub-groups, and will in turn leave lines of descent in these others; though the number of lines joining sub-groups will be less than those staying within any one of them. In so far as the members of a continuum cover an area of territory, they must be occupying a variety of ecological slots or niches, distinguished by altitude, nutrition, moisture and so on. Each individual, or each local sub-group of individuals, must be under the action of selective forces tending to adjust its genotype to the local environment (Fig. 76); and to the extent that these adjust- ments are to different environments, exchange of genes between the local sub-groups must lead to maladjustment. A mating continuum, therefore, is genetically flexible, but it does not permit the best local adaptation where differences occur within the common environment. Local maladjustments of an outbreeding species in a differentiated environment will always tend to increase, for the relations between environment and genotype are not constant. No environment can be stable in the long run, and almost any change must be for the worse in respect of an existing organism. Furthermore the environ- ment will not only be worsening for each individual: it will be worsening in different ways for individuals in different ecological circumstances. The need for local adjustment will not only be growing : it will be growing in different directions in different parts 303 THE BREAKDOWN OF CONTINUITY of the mating continuum and must be met by different genetical adjustments. The mating continuum of any group in a differentiated environment therefore has an iiilierent instabihty. How is this instability resolved ? The instability arises from the exchange of genes between individuals and groups requiring different local adaptations. As fast as the local conditions select and separate more favourable combina- tions of genes, these will be broken down and brought back towards Fig. 76. — The distribution of a recessive mutant determining "simplex" teeth in the field mouse Microtus arvalis, according to the percentage of homozygous individuals (after Zimmermann, 1937). the common level by the intrusion, from some other part of the continuum, of other combinations which have been selected under different conditions and hence adjusted in a different way. As fast as advantageous combinations of genes arise by segregation and recombination, they will break down by the same processes. The flow of variability, which arises from segregation and recombination, is necessary for the production of new and superior combinations of genes. Once, however, these combinations have been achieved, the means of their origin immediately becomes not merely urmecessary; it becomes actively harmful. The value of recom- bination lies, in fact, in bringing together combinations of 304 RESTRICTIVE PRACTICES genes which are so desirable as to make further recombination undesirable — at least for the time being. Thus, wherever there exists an advantageous combination of genes, selection must favour any device which restricts the effective recombination of its constituents. These devices are of various kinds, and they do their work in various ways. Restrictive Practices Recombination is effective only to the extent that the individual is heterozygous. The evil effects of recombination will not therefore be felt if heterozygosity can be eliminated, or at least reduced so that it does not endanger the basic advantageous combinations. We have already seen a great deal of restriction of inbreeding : in fact outbreeding itself is, in one sense, the result of restricting inbreeding. We now have to meet the opposite — a restriction of outbreeding. Restriction of outbreeding helps to preserve favourable combinations of genes. It may arise from either environmental or genetic causes, and it may be either partial or complete. The partial restriction of an individual's freedom of outbreeding is described as isolation. Mating is unhampered within the group but prevented between isolated groups. This restriction may be imposed by geographical means, as in the case of the different species of finch which Darwin found on each of the Galapagos islands. Or it may be by genetic means, that is to say by the develop- ment of gene or chromosome differences preventing mating, pollination, or fertilization, such as we have already seen at work in Petunia species. Geographical isolation cannot, of course, be under the control of the organism, and it is therefore in a sense accidental. Nevertheless such accidental isolation, particularly where it is in- complete may, as we shall see, favour the rise of genetic isolation. In the last chapter we have seen that the balance of gene combinations is brought about by the action of selection. In wild populations the poor combinations are eliminated and the advantageous combinations favoured. Now, in outbreeding organ- isms, many combinations are possible for every one which is advantageous, and only the test of natural selection can lead to the differential ehmination of those that are less favourable. If they are ELmeiUsofOotelus 3^5 U THE BREAKDOWN OI- CONTINUITY not exposed to this test they will not be eliminated. One consequence of tliis we have seen. When we inbrced a normally outbreeding stock, combinations become exposed in the homozygous condition for the first time. Their internal balances have never been tested and refined by natural selection, and in consequence they are mainly poor, hibrccding depression is the result. In just the same way, by crossing individuals which would not normally interbreed, we can obtain combinations in an untried heterozygous relation. If they have been free to change since the lines of descent diverged to give the individuals that carry them, these combinations will generally not be capable of working adequately together. Their joint action will not have been kept up to an adequate level by natual selection: balance is not stable, except in inbreeders. It decays if it is not being constantly adjusted by selection. The complete prevention of crossbreeding by some geographical obstacle wiU therefore mean that the genie combinations of the separated groups wiU gradually lose their relational balance, provided that within at least one of them sufficient outbreeding occurs not to freeze the flow of variability and so prevent all genetic change. As time goes on, the hybrid made by artificial intercrossing of the groups will come to be inadequate genetically. It will show an abnormal character or reduced fertility, more often the latter. The groups will ultimately become separated by the bar of genetical inter- sterility. They will be separate species. The same effect may, of course, also be brought about by ecological or seasonal isolation. The isolation, and not the means by which it is brought about, is the cause of the decay in relational balance. Where isolation by environmental agencies is incomplete, the decay of relational balance will be slower until genetical bars to crossing also arise. Soon after crossing becomes restricted between groups, the product of such crossing will begin to show the effects of the restriction in the form of some measure of hybrid incapacity, some failure in growth or reproduction. Such progeny are a waste of reproductive effort, and the less the number that an individual produces, the greater its chance of contributing to posterity. Any gene, therefore, which further restricts the rate of crossing between groups in individuals which bear it, will confer a selective advantage 306 RESTRICTIVE PRACTICES (J? I/) o a: u o < CO >- U < o. < U z a: CQ >- EXTERNAL and ENVIRONMENTAL 1. GEOGRAPHICAL— separation in range of occurrence 2. ECOLOGICAL— separation in habitat occupied 3. SEASONAL— separation in time of occurrence or breeding INTERNAL and GENETIC 1. PREVENTION OF MATING OF ZYGOTES by (i) Discrimination in Mating (ii) Mechanical Inability (iii) Failure of an Essential Intermediary (as where insects fail to carry pollen between two species of plants) (iv) Prevention of all Crossing in Hermaphrodites (by devices securing self-mating) 2. INCOMPATIBILITY of (i) Gametes (ii) Male Gamete and Female Soma (owing to genetical unlikeness) FERTILIZATION UNBALANCE OF 1. F, ZYGOTE— leading to Inviability and Genotypic sterility 2. Fi GAMETES— resulting from Genotypic effect of F^ zygote or, in plants, from Segregational sterility 3. F2 ZYGOTES— resulting, in animals, from Segregational sterility Fig. 77. — Isolating mechanisms preventing the effective exchange of genetic materials between populations. They fall into two groups: Bars to Crossing acting before fertilization and capable (in so far as they are genetic) of being produced by the direct action of selection; and Hybrid Incapacity occurring after fertilization, and incapable of being produced by the direct action of selection. The two groups are not mutually exclusive: they tend to encourage one another's occurrence. 307 THE BREAKDOWN OF CONTINUITY on its possessors and so will increase in frequency. The environmental isolation will be reinforced by genetic means. And as the restriction on crossing becomes greater, the relational balance will get even worse. Hybrid incapacity will increase, and with it the advantage of gcnctical bars to crossing. The system must therefore be self- stimulating, and once started, isolation will sooner or later become complete. Furthermore, the bars to crossing which are thus stimulated indirectly by loss of relational balance, may be reinforced by the direct expression of differences in form and function which arise as expressions of the increasing divergence of the two groups (Fig. 77). Where divergence has followed the course just described, the bars to crossing will generally prevent, or at least make difficult, crossing by artificial means. This is so, for example, witli the AtitirrJiwwn species, tnajtis and orontinm. One of the bars to crossing between them is the prevention of growth in the style of one species, of pollen from the other. Such a bar operates against artificial as well as natural cross-pollination. The two species, majus and glutinoswn, however, show us that the bar to natural crossing need not prevent artificial hybridization. When grown together and allowed to pollinate naturally under English conditions, they show less than 3 per cent crossing. They can, nevertheless, be crossed readily by artificial means. There appears to be no barrier to cross- fertilization once cross-pollination has been achieved. Flere the isolating mechanism is found to consist in the failure of cross- pollination, consequent on the failure of pollinating insects, mainly bees, to visit flowers of the two species alternately when making their working flights on a mixed stand of plants. In these species o£ Antirrhinum the plants have, so to speak, taken advantage of the insects' power to discriminate between different flowers, and of their predilection for confining their visits on one run to flowers of the same type. We have, therefore, the remarkable, but possibly frequent, situation of isolation between two species depending on the habits of a third. Flowers are, of course, generally adapted in form and colour to the insect which pollinates them. Here, in turn, not only the form but the discontinuity in form of the flower depends on the habits of the insect. 308 THE INTERNAL ORIGINS Of ISOLATION The Internal Origins of Isolation Isolation, as we have been discussing it, arises from the external genetical relations of one species with others. It can also arise from purely internal causes in a variety of ways. Of these the simplest is provided by the mere habit of inbreeding itself. Isolation is complete, or virtually so, while artificial crossing is readily achieved, in many inbreeding organisms. In wheat or oats, for example, all the species, even the diploids, can be readily crossed. Yet, when grown together, the species show no natural crossing. Here, however, in contrast to the Antirrhinum species, the lack of crossing between plants of different species is only a special case of the general failure of the individuals to outcross even with others of their own species. They are habitual inbreeders, and their isolation is a concomitant of their inbreeding. Where crossing of any kind is absent, restrictions on crossing between groups, as opposed to crossing within them, are obviously unnecessary. Indeed, since inbreeding freezes the flow of variability, the production by recombination of groups of genes producing bars to crossing will be impossible: so likewise will be the divergence of genie combinations from which hybrid incapacity arises. It is not surprising, therefore, that there is an absence of all genetic bars to crossing other than the normal inbreeding mechanism. Nor is it surprising that the species hybrids, when artificially produced, are both fully vigorous and fully fertile; apart, of course, from the effects of the numerical hybridity which follows the crossing of species having different chromosome numbers. These principles are seen in their simple form in wheat, oats and tobacco. They are illustrated even more critically in the species of Oenothera, which happen by their own special device to be highly heterozygous. They are habitual inbreeders, and they have an unlimited capacity for crossing within the genus. Isolation and inbreeding represent restriction on crossing of different degrees and with different consequences. They are both achieved, however, by means within the limits of the sexual mechanism. Apomixis, on the other hand, resolves the instability of a mating continuum by the abolition of the normal sexual cycle. Fertilization is done away with, and meiosis either does not intervene, 309 THt BREAKDOWN OP CONTINUITY or does not run its normal course. Segregation, even from a hybrid, is thus cither avoided completely, so that reproduction is clonal in all but appearance, or is so severely restricted as to give something near the genetical uniformity of a clone. Apomixis, as we have already seen, is shown very commonly by individuals so hybrid, even in regard to chromosome number, that only by side-stepping sexual reproduction can they propagate themselves; but it is not confined to such hybrids. In the aphides it is shown by species which under other circumstances are sexual. The sexual phase is to be seen at those seasons of the year which keep their numbers small, and the parthenogenetic phase in the summer when populations are rapidly increasing in size. As we have earlier observed, the use of parthenogenesis during a stage of rapid expansion of the stock is a reproductive economy. We can now see that it is also something more, for it avoids the deterioration which would result from sexual recombination at a period of prosperity when natural selection is dangerously relaxed. Isolation and inbreeding on the one hand, and apomixis on the other, though differing sharply in their mechanisms, agree in avoiding recombination by the abolition of segregation. Isolation and inbreeding do so by avoiding heterozygosity for at least the critical gene combinations; apomixis does so by accepting hetero- zygosity but avoiding sexual reproduction altogether. Polyploidy offers a device which accepts both heterozygosity and sexual reproduction, yet still avoids segregation of the old differences. In an allopolyploid, or ampliidiploid [A1A1A2A2), the individual is heterozygous for the differences between the sets of chromosomes of the original parents [A^Ai and A2A2). Yet in so far as the two representatives of each set segregate from one another on the scheme of Primula kewensis {Ai from A^ and A2 from /Ig), rather than from the sets of the opposite parent [A^ from A2) these parental differences arc passed on as wholes through each gamete to each offspring. They never segregate and their parts never recombine, so that a polyploid is, as we saw, potentially a true breeding hybrid. So even when descended from cross-breeders, a polyploid species can be an inbreeder without becoming homozygous and hence without serious inbreeding depression. It offers, in fact, a short cut from the flexibility of outbreeding to the fitness of 310 THE INTERNAL ORIGINS OF ISOLATION inbreeding, besides combining the bases of variation from the two parents. Polyploidy may also offer other advantages. An autopolyploid, derived by the simple doubling of the chromosome number of an existing species, can show physiological properties different from those of its parent, while yet maintaining the balance, the capacity for working as an integrated whole, of that parent. The drawback of autopolyploidy, especially in aimuals, where seed production is needed for maintenance, is the reduced fertility which follows multivalent formation at meiosis. This may, however, be avoided by restriction of pachytene pairing or of chiasma formation, so that only bivalents occur, as is seen in the autotetraploids Lotus corniculatHS and Tulipa chrysantha. Allopolyploidy avoids recombination within whole sets of chromosomes. Interchange, in its turn, offers a means of avoiding recombination of different individual chromosomes within the set, so that two pairs have one linkage group. As we have already seen, the recombinant gametes from interchange heterozygotes are lethal in plants, because they show simultaneous duplication and deficiency. In animals the zygotes to which such gametes give rise are lethal for the same reason, except where, either by a rare chance or by deliberate experimentation, fusion is with another gamete showing complementary unbalance. Those gametes which, on the other hand, carry a parental combination of chromosomes do not suffer from this handicap, and consequently enjoy a greater chance of success. Thus recombination of the chromosomes involved in the interchange is rarely or never effected. In one sense the suppression of recombination in interchange heterozygotes may be regarded as due to the exaggeration of the iU-effects of all recombination. At the same time, in Oenothera species and their like, we can see how, by the additions of differ- entially balanced gametic lethals and of that competition between potential embryo-sacs which we call the Renner effect, the loss of fertiHty consequent on this suppression may be overcome. The mechanism is then adjusted to give a situation resembling that in polyploids, except that interchange abolishes recombination between any number of chromosomes from two up to the whole set. Thus interchange, like polyploidy, affords a means of attaining the 311 THE BREAKDOWN OF CONTINUITY uniformity which inbreeding can give without its depression of vigour and fertiUty. Interchange and polyploidy avoid gross recombination between chromosomes, and it may be, therefore, that their chief advantage lies in the short cut each offers to inbreeding. Inversions, on the other hand, limit recombination within chromosomes. They can tie together, or peg, the very combinations of genes which will have been built up by the normal process of linkage in the way we have discussed in the previous chapter. It is also known that inversion heterozygosity need not lead to any marked sterility on the female side, for the inviable cross-over chromatids may be confmed to those products of meiosis (spores or polar bodies) from which the egg never develops. Floating and Fixed Discontinuity Inversion, interchange and polyploidy have one property in common. They abolish the recombination of genes which would otherwise recombine more or less freely, and in so doing they give rise to super-genes, units of transmission greater than the gene itself They peg the combinations of genes which have been successful in leading to high adaptation in the past. This pegging is not, however, a fmal process since the avoidance of recombination is seldom quite complete. In polyploids, aberrant pairing occasionally leads to aberrant recombination, while rare double crossing-over in interchange and inversion heterozygotes can lead to some redistribution of the constituents of the super-gene and so to the origin of spurious mutations. The result is that some genie heterogeneity may occur within one structural type. And even without this recombination a similar result would come about in time by virtue of true mutation alone. The consequences of this development have been followed by Dobzhansky in DrosopJiila psendoohscura. In this fly the third chromosome (to a much greater extent than its fellows) exists in a number of sequences derived from one another by inversions as revealed by polytene chromosomes. These inversions overlap one another so that they cannot recombine as units, and it has consequently been possible to work out the phylogenetic tree of 312 FLOATING AND FIXED DISCONTINUITY the sequences themselves. This is shown in Fig. 78, from which it will be further seen that not merely are the inversions of this species relatable to one another, but that they must be descended from an ancestral type, now lost from pseudoohscura so far as is known, D.MIRANDA D. PSEUDOOBSCURA Olympic Oaxaca Estes Park Chiricahua IE -I Texas T Arrowhead Cuernavaca Pikes Peak D. PERSIMILIS Cowichan Standard Hypothetica Fig. 78. — The phylogenetic relationships of the third chromosome sequences of the Drosophila species pseudoohscura, pcrsimilis and miranda. The hypothetical sequence is closely resembled by that found in miranda. Standard is the only sequence which occurs in two species: with its derivatives it floats in pseudoohscura but is fixed in persimilis. The regional names associated with the sequences relate chiefly to the places along the Pacific coast of North America where they were first found (after Dobzhansky and Sturtevant, 1938). which was common also to the related species persimilis (once called D. pseudoohscura, race B) and miranda. Each of these sequences in pseudoohscura is itself heterogenic, that is to say it exists in genically diverse forms in nature, and the different combinations within the sequence display relational balance with respect to each other. Thus any one chromosome may give rise to a poor, even a lethal, individual when homozygous; but when heterozygous with another chromosome of the same structural type, the resulting individual will be perfectly viable 313 THE BREAKDOWN OF CONTINUITY and fertile. Yet there must be an overall super-balance between sequences, because selection tests have shown that flies heterory^gous for two alternative sequences have an advantage over others homo- zygous in this respect, but heterozygous for at least part of the genie differences that occur within the same sequence in a strain or in the species at large. This balance of advantage between sequences changes with temperature. In pseudoobscura the various sequences float in the population, partly in a heterozygous and partly in a homozygous condition. A number of them may be picked up at any time in any group of individuals. Evidently their super-balance in respect to one another is still more effective in leading to the adjustment of the phenotype, than is the relational balance of the combinations within any one sequence. This is to be expected in a cross-breeding population in the early history of sequential variation, for the inversions will be pegging the combinations which as wholes work well in the heterozygous condition. A further development may, however, be envisaged. When sufficient variation is present within sequences to give a good relational balance without sequential heterozygosity, genie adjustment to any permanent change in environmental circum- stances will go on within sequential types rather than between them. Within a sequential type the adjustment of combinations can go on by the recombination of their genes ; but different sequences are genetically isolated from one another (Fig. 79). Thus each sequence may become better fitted than its alternatives for a particular environment. Any further pressure of selection wiU therefore tend to establish a sequence in one or more populations. The sequence will cease to be floating in these populations, and will become, with its derivatives, fixed, uniformly and exclusively homozygous; though at the same time it may continue to float in still other populations. This process appears to be in progress today in pseudoobscura, for although several sequences may be found in any one population, different populations are characterized by particular groups of sequences. The three species we have mentioned show the last stage of the process. All three have third chromosomes descended from a common type, but the line of development of miranda differs from 314 FLOATING AND FIXED DISCONTINUITY that of the other two. Two sequences, the so-called Santa Cruz and Standard types, have floated (together, possibly, with others of which all trace has been lost) in the common ancestor of pseudoohscura and persimilis. They and their derivatives stiU float within pseudoohscura, but persimilis has neither Santa Cruz nor any of its derivates. The Standard type has been fixed in the populations ancestral to this species, even though it has remained floating in the populations ancestral to pseudoohscura. Since the time of this fixation, the lines of variation of Standard have, of course, separated in the INVERSION . ABCDEF >- AEDCBF ABCOeF ABcDeF AEdCbF Fig. 79. — ^The occurrence of an inversion divides the affected chromosomes into two lines, the old and the new, genetically isolated from one another in that recom- bination is prevented in those segments affected by the inversion. Genie heterozygotes which are also structural heterozygotes can give only the parental types; but genie heterozygotes which are structural homozygotes can give rise to new types by recombination. Thus progressive genetical adjustment must proceed independently in the two lines of chromosome descent distinguished by the inversion. two Species, so that although this sequence is itself common to them, each of its various derivatives occurs in only one of them (Fig. 78). The transition from the floating to the fixed condition is seen also in interchanges, and the plants which show us this transition enable us also to compare its progress in inbreeding and outbreeding forms. For example, in the inbreeding species. Datura stramonium, different interchange types have quickly become fixed, that is to say homogeneous and homozygous, in different parts of the world (Table 26). When plants from various places are crossed, rings of 4 and 6 chromosomes are produced at meiosis in the hybrids. The different frequencies of the several interchange types in different parts of the world where more than one type is found indicates that selection is favouring one type in one region and another in another. It is 315 THE BREAKDOWN OF CONTINUITY sorting them out in such a way as to produce the differentiation between races in respect of interchange which already occurs between the species stratnotiiHm and nictcloidcs, for example. TABLE 26 DISTRIBUTION OF INTERCHANGE TYPES IN NATURAL STRAINS OF DATURA STRAMONIUM AS REVEALED BY CROSSING WITH A STANDARD TYPE (PT2) (BLAKESLEE, et al., 1937) Region Basic type (PT2) Interchanges Total A (PTl) B (PT3) C (PT4) D (PT7) C + D E (PT87) F (PT88) Strains Locali- ties C. and S. America Asia Africa and Australia Hawaii . . N. America (general) E. U.S.A. Europe . . 20 26 13 9 124 15 7 4 39 163 8 54 1 30 23 3 13 8 14 1 2 1 1 9/ 33 31 4 39 217 164 65 23 23 2 31 126 82 Total . . 192 236 55 53 24 17 1 1 579 352 PT = Prime Type. In the cross-pollinating Campanula persicifolia, on the other hand, one basic type occurs throughout Europe, and the numerous interchanges occur singly as heterozygotes in individuals. By crossing six of these successively a ring of I2 chromosomes can be built up step by step. Hence it seems that interchanges which are immediately fcced in an inbreeder (or by inbreeding) can be pre- served floating for a great length of time in an outbreeder. With this comparison in mind we can understand what happened to Oenothera, for there is a transition from the outbreeding western species with floating interchanges, such as organensis of Arizona, to the inbreeding eastern species, such as novae-scotiae, in which the interchanges are fixed, but fixed as heterozygotes. It is evident that the transition from outbreeding to inbreeding has been combined with the fixation of the interchanges in the heterozygous condition. 316 FLOATING AND FIXED DISCONTINUITY And the result has been to maintain the same degree of hetero- zygosity at the end as there was at the beginning. Inbreeding has fixed the interchanges, but only by virtue of this fixation has the move towards inbreeding been successful. Moreover the inter- (1) Quercifolia ] ^ and ferox J stramonium 4 7 \ / 4+7 (2) Basic T— J r B C 8(2) (3) basic: 7(2) (4) (4) Oenothera (California ) or Campanula (Europe) (4)+(4) (6) (6) (6)+(4y (8) (14) (14) Oe. nutans (New York) Oe. muricata (.Europe) Fig. 8o. — Three types of interchange evolution, as seen in (i) Datura, (2) Campanula, and (3) Oenothera. mediate stages of incomplete ring formation are unstable. There are as many species with a ring of 14 chromosomes as there are with all the lower sizes of ring (Fig. 80). In Hypericum and Rhoeo this process has gone further and the incomplete ring types have already disappeared. It only remains to add that in animals fixed ring- formation is hindered by the difficulty of establishing fixed in- breeding. 317 THli BREAKDOWN OF CONTINUITY Restriction and Flexibility Recombination is important in discovering combinations of genes which remove the need for further recombination under existing conditions. The restriction of recombination is important, therefore, in stabihzing present fitness. Yet in the same paradoxical way, these restrictions reduce the recombination which is necessary for pros- pective readjustment when conditions change, as sooner or later they presumably must. Devices restricting recombination must there- fore be viewed not merely in the light of the immediate fitness which they will preserve. They must also be examined for their effects on genetic flexibility, for on these effects will depend the long-range success of the stocks or races which carry them; and simultaneously, of course, the success of the devices themselves in evolutionary liistory. Inbreeding and apomixis restrict recombination by the abolition of heterozygosity and of the sexual cycle respectively. They abolish the very means on which genetic adjustment depends, and as we have seen they are dead ends in evolution. The isolation of races, whether by geographical or genetic means, restricts recombination only between those genes which are of immediate importance in giving good local adaptation. Cross- breeding still occurs within each isolated group and the use of any residual heterogeneity, or any new heterogeneity arising by mutation, for adaptive adjustment is not precluded. The same principle of checking recombination while leaving the way open for later expansion applies with inversion, as we have seen, and also with interchange, provided that the system has not, as in Oenothera, become adapted to inbreeding. When an Oenothera stock has reached the top of the ladder with its ring of 14 chromo- somes it can go no further; but this need not be true of the floating interchange system of Campanula which has not become tied down by inbreeding. Polyploidy is in a similar case. If it has arisen as a short cut to a rigid inbreeding system, the polyploid must be doomed by this rigidity. Probably most polyploids have a relatively short life for this very reason. But, in so far as any crossbreeding has been retained, and in so far as there occurs mutation or recombination 318 RESTRICTION AND FLEXIBILITY between the genes of the parental sets, genetical flexibility wiD be maintained and prospective readaptation will remain a possibility. Even a new inbreeding polyploid can for many generations retain some flexibility, through the gradual unloading of the differences between its diploid ancestors. We can thus see that complete restriction of recombination is Regular cross-fertilization within a MATING CONTINUUM. Heterogeneity and heterozygosity due to floating changes (1) structural and (2) genie. (1) interfering with recombination of (2). Break-up into many INBREED- ING or obligatorily self-fertilizing units which are potential new species since no gene exchange occurs between them. Adaptive combination of specific genes with specific structural types now floating jointly. APOMIXIS by progressive or sudden suppression of recombination, i.e. un- conditional fixation. Fixation of heterozygotes with cumulative complex differences (XY and Oenothero). Fixation of homozygotes by splitting into pairs of species each a new mating continuum. Fig. 8i. — Scheme of genetic changes in relation to species formation. final and suicidal. As usual, it is the compromise solution, the partial and adjustable restriction, which is ultimately the most successfid. The desirable genie combinations are held together sufficiently well for present needs by the abolition, or near abolition, of breeding between populations and of recombination between structural types; but the possibility is retained o( further adjustment either within the population or within the structural type. Each population or each structural type is separated from its fellows, but remains itself as defining a new and reduced mating continuum. This must start 319 THE BREAKDOWN OF CONTINUITY small. In so far as it is successful, however, it will grow until its own inlierent instability once again becomes too great and new restrictions on recombination arise within it to give a further break-up of the continuum (Fig. 8i). This course of events is well illustrated by the three Drosophila species which we have already had cause to discuss. These are distinguished simultaneously by differences in chromosome sequence and in breeding behaviour, the differences being less between the more recently separated pseudoohscura and persimilis than between these taken together and miranda. Although they wiU cross, they prefer to mate within the species. Their hybrids are both cyto- logically erratic and genetically inadequate. Evidently their genetic architectures are different and the exchange of genes is at least largely prevented between them. In consequence they can overlap in territory without loss either of fitness or of specific distinction. It would appear that miranda has been the least successful, and pseudoohscura the most successful, derivative of their ancestral mating continuum. D. pseudoohscura, in particular, has become widespread and is now showing signs suggestive of instability. Itself one of the products of splitting in past continua, it appears hkely to be on the verge of another sphtting. Summing up, it appears that the only universal and permanent type of species is that which allows of regular outbreeding. This type of species is a group of individuals held together in a continuum by genetic recombination. Any change leading to the restriction or breakdown of this recombination leads to discon- tinuity, or the origin of new species. Whether the first step m the restriction or breakdown is external, e.g. geographical or ecological, or whether it is internal, e.g. structural or numerical change of the chromosomes, a bar to crossing wiU be estabhshed which wiU bring aU other forces favouring isolation into action. There is yet another type of internal change, a gene or chromosome change which establishes obligatory inbreeding or apomixis, which in itself breaks up the continuum into its individual items. In so doing it produces species which, however, are unlike the parental outbreeding type and have no evolutionary future. 320 THE TRACES OF ANCESTRY The Traces of Ancestry All forms of discontinuity give us a clue to the relationships of organisms through descent, in other words phylogeny. In so doing they both utiHze and confirm the conclusions we have reached on the origins of discontinuity. The chromosomes remain to mark many of the steps by which species, genera, and even families, have diverged. The changes that we have to use for the larger groups are no longer the inversions and interchanges that serve to trace relationships within a small and inter-fertile group. We have to use changes of number arising from polyploidy, reduplication, and the fragmentation and fusion of chromosomes. Polyploidy is in a special position. It can take place effectively only in one direction : the diploid must nearly always be the parent of the polyploid. Moreover, even as simple doubling, it determines a genetic change at the same time as it establishes a discontinuity by creating a new form which will not cross with the old. Finally, a single colonizing polyploid individual can unload a degree of variation that would be far beyond the reach of a solitary diploid. For this reason when sudden changes are needed and a sudden opportunity for colonization created, as after the retreat of the ice, the new polyploid steps in and quickly acquires a large range from which its diploid parent is excluded. In Paeonia the Mediterranean seems to have created a barrier that has favoured the appearance of tetraploids in Europe such as are found only once in Asia and not at all in America. An external discontinuity has called forth an internal one (Table 27). Changes of single numbers also require a word of explanation. Fragmentation can be effective in adding to the number of chromo- somes only when the centromere itself is split to give two new chromosomes with terminal centromeres. The result has been found as a fragmentation heterozygote in Campanula pcrsicifoUa. Each fragment pairs with the arm of the old chromosome with which it is homologous, so that a chain of three chromosomes is formed at meiosis {A— AB— B). Homozygotcs with 8 {AB— AB) and with 9 {A— A, B — B) pairs have been derived by segregation from the heterozygote (Darlington and La Cour, unpub.). Changes ascribed to fusion, and doubtless preceded by breakage, EhmailsofGauiiiS 321 X THE BREAKDOWN OI CONTINUITY TABLt 27 SPECIES IN PAEONIA (BARBER 1941. STERN 1946) Region Europe Asia N. America Mlokosewitchii - (Caucasus) daurica (Crimea, etc.) Clusii (Crete) Cambessedesii - 2 other localized species in Mediterranean 1 widespread in Ukraine Witmanniana and varieties (Cau- casus and Elburz) mascula, banatica (widespread) officinalis, mollis and varieties (N. Mediterranean) Russi and varieties (Western islands of Mediterranean) 3 other widespread species unrelated to any surviving diploids japonica (Japan) 8 other widespread diploids obomta and varieties (E. Asia) No other tetraploids 2 diploids No tetraploids have been inferred in grasshoppers. They likewise give novel configurations at meiosis. When any such new types arise as fragmentation or fusion in heterozygotes there is some loss of fertility through irregularity at meiosis. This will favour the segregation and separation of homozygotes of the two types : so soon, at least, as their genie homozygosity does not conflict too strongly with the hybridity optimum of the species. In other words a chromosome change of fusion or fragmentation may be expected to act, like one of inversion and interchange, as a focus of discontinuity in the species. This expectation is borne out when we discover that in making inferences from changes in chromosome numbers in the larger groups, we are able to apply the rules we derive from what happens to the smaller structural changes within the smaller groups, the races and species. In the first place the basis of adaptation of races and species lies in their genes. Changes of structure and number of chromosomes are, as a rule, merely the means of directly or indirectly preventing recombination of these genes. The chromosome discontinuities, 322 THE TRACES OF ANCESTRY such as inversions, interchanges, and fragmentations, float in the species until they happen to combine favourably with the gene discontinuities. Two consequences follow from this accidental character in the relationship. In the first place changes may take place in chromosome structure or number, or in gene content, without any obvious change in the phenotype. Two complementary instances are offered by Drosophila and the bug Thyanta. D. simulans and melanogaster are externally almost indistinguishable except to one another. Genetically they differ by an inversion. In a species of Thyanta, Wilson in 191 1 found two races, one with 7 and the other with 12 pairs of chromosomes. Obviously these two races, not previously distinguished, would be intersterile and they were indeed described (after the event) as distinct species. Thus great structural and polygenic changes can go on under the surface, as cryptic variation, without any differentiation of external form. It follows that similarity of chromosomes is some- times a worse guide to similarity of descent than is external form. It also follows that it is a matter of pure chance whether the newer type of gene effect is combined with the new kind of chromosome character or not. It may as well be that the primitive morphology is combined with the new chromosome number as the reverse. In the second place a particular structural difference that we observe now as distinguishing two groups may, as we saw in Drosophila, not be the one which actually helped to separate them. It may stiU have been present in both at the time they were separated by some other agency of which we can no longer uncover the traces. Let us not, however, suppose that the systematist may safely proceed unguided by breeding experiment or chromosome analysis. Too often he feels compelled on the one hand to regard as a hybrid an individual or a type which is intermediate between two other arbitrary types from a heterogeneous mating continuum. And, on the other hand, he feels compelled to take as a variety an individual or a type which represents merely a marker gene, whether freely floating in such a continuum, or attached to a super-gene or a chromosome variation or an ecological situation. These errors he can avoid by combining the rules to be derived from external form and geographical distribution with those to be derived from breeding behaviour and chromosome variation. This combination has yet to 323 THE BREAKDOWN OF CONTINUITY be brought about but even now we can reach a number of plausible conclusions about ancestry from chromosome numbers. When we come to apply these rules we fmd that an enormous variation exists in the stability of chromosome numbers in relation to the external form in different groups of plants and animals. In the flowering plants, where we can compare the numbers of some 10,000 species, the chief source of this variation becomes clear. In the shorter-lived herbaceous plants chromosome numbers usually vary within genera; in Crepisand Crocus^ for example, every haploid number occurs between 3 and 18. In the longer-lived woody plants, on the contrary, they remain constant sometimes for whole tribes and families. Take the Pomoideae with their constant 17 chromo- somes. This group presumably arose by an uneven or secondary polyploidy from a section of the Rosacea^ with 7 chromosomes in the Eocene period, since when no change save a renewed polyploidy has occurred and established itself, and very little even of that. It is, therefore, to the woody flowering plants (which themselves must have arisen at different times from herbaceous plants) that we can make the most far-reaching conjectures from chromosome numbers with regard to descent and relationship. The diagram (Fig. 82) shows how these principles work, in the first degree by polyploidy, in the second by losses and gains in a polyploid, and in the third by losses and gains in diploids. It shows 7 as the common ancestral chromosome number of flowering plants. From this origin 8, 9 and an increasing series have arisen on only a few occasions, whereas 14, with its diminishing series, has arisen very frequently. In this scries 12 has often been stabilized and from its addition to 7, 19 has appeared several times. One remarkable instance of this last step in the Magnoliales requires special comment. If, as is customary, we divide the genera with 19 chromosomes among 3 families we are implying that this number arose from the union of 12 and 7 on three occasions. Thus the morphologist's subdivisions of this group seem to be in conflict with the probabilities of chromosome evolution, a conflict which further study will readily resolve. 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Philadelphia. 438 INDEX (excluding the Appendices: pp. 375 sqq. and all References at ends of Chapters) Abutiloit, 208, 211, 217 acentrics, 100, 131 Acetabularia, 17, 168, 190, 228 Acridimn, 335 Adalia, igs adaption, 273, 322, 327 fermentative, 181 additive effects, 79, 80, 158 adjustment, levels of, 227 AcschIus, 138 agouti, 118, 160 Akerman, 19 albinism, 157, 169 alcaptonuria, 162 algae, 17, 29, 44 allelomorph, 40, 48, 106, 152 multiple, 114 sqq. allergy, 349 Allium, 30, 152 Allomyces, 240 allopolyploid (y), 122, 134, 310, 318 ambilinearity, 185 amorph, 152 anaemia, 194, 352 anaphase, 25, 131 Andersson-Kotto, 186, 190, 327 anthocyanin, 163 anthropology, 354 sqq. antibody, 154, 208 antigen, 154, 165, 213, 227 antimorph, 152 Antirrhinum, 84 sqq., no, 158, 259, 30S, 327 aphides, 263, 264, 310 apomixis, 263 sqq., 270, 309, 318, 344 apospory, 201 Apotettix, 335 apple, 96, 126, 218 armadillo, 16 Artemia, 100, 265 aryan language, 360 Ascaris, 97, 197 Aspergillus, 343 Astbury, 146 asynapsis, no, 288, 289 attraction, 30, 33 autopolyploidy, 311 autosexing, 50 autosome, 48, 347 autotetraploid, 135 back-cross, 40 bacteria, 146, 275 bacteriophage, 208 sqq., 275 balance, 96, 104, 123 sqq., 128, 275 change of, 141 in hybrids, 229 sqq. internal, 305 loss of, 293, 305 of polygenic system, 291, 292 relational, 293, 306 Barber, 198 Barigozzi, 265 barley, 240, 241 bars to crossing, 307, 308 basic number, 126 Bateson, 38, 41, 109, in, 155, 182 Baur, III, 211 Bawden, 209 Beadle, 162, 289 Belar, 197 Belling, 134 beetles, 344 Billingham, 216 biparental progenies, 89 birds, 52, 227, 253 bivalent, 30 Blair, 217 blastula, 197 blending inheritance, 273 blood cells, 194 blood group, 153, 349, 362 Bomhyx, 113 Bonnier, 365 Bounure, 197 Boveri, 18, 26, 35, 197 Boyd, 349 Brachet, 147 breakage of chromosomes, 100, 331 breeder, 94 breeding systems, 237, 239 sqq., 269, 354 Bridges, 43 Bumpus, 282 Burgeff, 327, 328 Campanula, 104, 126, 134, 262, 316 sqq. 439 INDEX cancer (tumour), 147, 210 sqq., 350 CapscUa, 252 carcinogen, 213 Caspcrsson, 147, 329 caste, 355 sqq. cat, 49, 346 cell, 16 sqq. competition, 198 lineage, 212 sqq. see also gradient and pollen centromere, 25 sqq., 44 sqq., 100 sqq. characters, 36 capital and subordinate, 300 chemical mutation, i'. mutation chemical tests, 145, 159 cherry, u. Primus chiasma, 30 sqq., 44 sqq., 121 frequency, 137, 234, 288 chimaera, iii Chlamydoinonas, 44 chloroplast, 44, 169 sqq. chromatid, 25, 30, 45 chromomere, 27, 33, 119, 147, 329 chromosome breakage, 100 chemistry, 145 coiling, 30 homology, 33, 102, 135 mitosis, 23 numbers, 324 orientation, 123 pairing, 27, 30, iio, 135 supernumerary, 147, 150, 329 theory, 45 Chuksanova, 123, 124 Cimex, 150 Claude, 207 cleavage, 197 cleistogamy, 241 cline, 351, 364 clone, 16 Cockayne, 347 colchicine, 95 colonization, 140 colour-blindness, 49 competition cell, 198 in sexual reproduction, 268 plasmagene, 179 pollen-tube, 253 precursor, 165 co-operation of genes, 158 of nuclei, 199 of viruses, 209 330 Coprinus, 248 corolla length, 70, 71 correlation coefficient, 61, 62 fraternal, 90 parent-offspring, 90 correlated response, 298 sqq. covariance, 60, 78, 82 sqq. Crane, iii Crataegomespilus, 228 Crcpis, 105, 123 sqq., 324 Crocus, 26, 324 cross-breeding, v. outbreeding crossing-over, 30 sqq., 45 sqq., 100 sqq., 10 J sqq., 121 reciprocal, 52, 132 suppression, 132 Culex, 346 Cytisus, 228 cytoplasm, 17, 149, 168, 189, 216 Dahlberg, 347 Dahlia, 164 Darwin, 242, 248, 273, 300, 305, 328, 360, 370 Datura, 96, 104, 126 sqq., 315, 316 dauermodification, 183 deficiency, 103, 152 Delbriick, 274 delinquency, 349 development, 16, 37, 112, 127, 1$'] sqq., 168 sqq., 189 sqq. De Vries, 62, 263 diakinesis, 30 dicentric, 132 Diehl, 349 differential segment, 52, 133, 346 differentiation, 16, 150, 189 diminution, 197 dioecy, 240, 243, 252, 258 diploid, 24, 29 diplotene, 30 Diptera, 290 disease resistance, 210, 349, 353, 358 Dobzhansky, 292, 312, 313, 337 dominance, 37, ?>0 sqq., 109, 11^ sqq., 156 Drosophila abdominal hairs (chaetae), 73 sqq., 298 Bar gene, loj sqq., 152 chromosome assays, 76 chromosomes, 26 crossing over, 46, 290 cubitus ititcrruptus gene, 160 cytoplasmic heredity, 215 development, 190, 202 440 INDEX Drosophila — continued discriminative mating, 253 eyeless gene, 159, 275 fertility, 105, 126 genes, 106, 119 gynandromorph, 112 intersexes, 258 linkage, 42, 47 meiosis, 46 Minute genes, 107 mutation, 152, 155, 327 Notch gene, 107 polytene, 27, 106, 119 scute gene, 106, 152, 331 scutellar bristles, 285, 297 sex chromosomes, 49, 151, 330 sex determination, 231, 341 sperm, 105 triploid, 96, 121 wild-type, 43, 152, 159, 161 Drosophila simulans, 131, 253, 323 azteca, 337. 338 miranda, 313 sqq., 320 persimilis, 232, 313 sqq., 320, 338 pseudoobscura, 230, 292, ^12 sqq., 335 sqq. subohscura, 193, 272 duplication, 103 East, 65, 70, 71, 245 echinoderm, 18, 168, 191 Echinus, 18 egg, 24, 28, 96 sqq., 113 embryology animal, 113, 116, 197 plant, 195, 200 sqq. environment, 15, 64, 359 and selection, 294 enzyme, 179 Ephestia, 193 Ephrussi, 166, 311 Epilachna, 69 Epilobimn, 175, 228 epistasy, 157, 163 equational separation, 31, 52 euchromatin, 32, 146, 329 evolution, 273, 328, 344 expressivity, 159 factor, 40 effective, 92 Fankliauser, 99 fatuoid, u. oats fern, 186, 189 fertility, 95 sqq., I2I, 134, 193, 283 allelomorph, 259 fertilization, 24, 28, 97 sqq. Festuca, 234 Filzer, 245 fmches, 305 fish, 50, 253, 297 Fisher, 118, 161, 274, 333, 336 fitness, 276, 284 sqq. in man, 353 sqq. Flemming, 25 flexibility, 284 sqq., 318 fowl, 49, 157, 213 Fragaria, 168 fragment chromosomes, 100, 197 fragmentation, 321, 323 frequency distribution, 56 Fritillaria, 30, 104, 200, 289 Funaria, 173 fungi, 29, 162 fusion, 322 Gakopsis, 293, 294, 297 I Galton, 60, 61, 93, 282 320, gamete, 28, 125, 129 I gametic differentiation, 239 gametophyte, 29 Gammarus, 166 Gar rod, 347 gene, 42, 327 sqq. action, time of, 191 balance, 105 complementary, 156 dosage, 105, 152 duplicate, 156 expression, 159 inert, 151 interaction, 154 5^;^. lethal, 106, 116, 157, 202 major, 152 reproduction, 146 switch, 258, 259, 341 generations alternation of, 189 genetic system, 237 change of, 296 genotype, 15 geographical variation, 364 germ cell, v. gamete, pollen, egg germ line, 197 Goldschmidt, 113, 166, 184 Gordon, 160 gradient cell, 194 sqq. population, u. clinc 441 INDEX graft, 213, 217, 227 graft-hybrid, iii, 227, 228 grasshoppers, 322 Griincbcrg, 116, 202 Gudjonsson, 266, 267 guinea pig, 216 gymnospcrm, 201 gynandromorph, 1 12 Habrobracon, 113 Hadom, 202 haemophilia, 49 Haldane, 19, 229 Hammerhng, 17, 190 haploid, 18, 24, 98 haploid generation, 105 Harrison, 73 Hartung, 269 Hemiptera, 52 heredity, 15, 149 ambilinear, 183 matrilinear, 170 herpes, 217 heterocaryon, 163, 342, 343 heterochromatin, 32, 47, 146, 195 sqq., 329 heterosis, 237 heterostyly, 248 sqq. heterothally, 240, 247, 248 heterozygote, 38, 102, no, 276 hexaploid, 140 Hieracium, 201 Hildebrand, 248, 255 Hinduism, 356 Hodson, 365 Hoffmann, 184 Hogben, 347 Holmes, 210 homology, v. chromosome homostyle, 259 homozygote, 38, 102, 276 Hordeum, 171 host-parasite relation, 274 Huskins, 339 Hyacinthus, 123, 127 hybrid, 18 incapacity, 307, 308 interchange, 133 mendelian, 36 numerical, 127 species, 168, 191 structural, 128 true-breeding, 260 twin, 261 161. hybridity optimum, 237, 259, 269 equilibrium, 241 hypcrgamy, 357 Hypericum, 130, 317 hypomorph, 152 Imai, 171 inbreeding, 275, 282, 283 sqq, 297, 318 and isolation, 309 depression, 235, 292 in man, 353 sqq. mechanisms, 241 incest, 252, 253, 355 incompatibility allelomorphs, 332, 334 self, 164, 174, 243 sqq., 255 sqq,. 357 individuality, 16, 34 inertia, 297, 300 inertness, v. gene infection, 207 sqq. inheritance, v. heredity insects, 227, 308 pollination, 242 interchange, 102, 128 sqq., 311, 315, 318, 323, 335 interference, 45 intergenic change, 106 intersexes, 258 inversion, 102, 131, 212 sqq., 318, 323, 331.335 isochromosome, 103, 330 isolating mechanisms, 307 isolation, 305 sqq., 356 Janssens, 34 Johannsen, 15, 42, 62 sqq., 171, 274, 373 Johansson, 284 Johnson, 218 Jollos, 183 Jones, D. F., 339, 340 Jones, Sir William, 360 Jorgensen, in Kallmann, 350 Karpechenko, 135 Koller, loi, 215, 348 Kiihn, 193 La Cour, 195, 196, 321 Lamarckian effects, 182, 211 inheritance, 274 language, 360 442 INDEX Lathyrus, 158, 218 Lavatcra, 211 Lawrence, 164, 254 Li'bistcs, 50, 336, 340, 341, 346 Lens, 168 Lcpidoptcra, 52 lethal, u. gene Levit, 366 Lewis, 248, 332 L'Herider, 215 life cycle, 24, 29, 189^^. Lilium, 139, 288 Limnea, 192 Lindegren, 180 linear order, 43 linkage, 41, 68, 85 sqq., 130, 299 and variability, 287 sqq. linkage map, no, 114, 134 Linum, 174 localization, 30, 289, 335 Lolium, 234 Lotus, 311 Luria, 210, 275 Lycopersicum, 95, 135, 242, 255, 293 Lymantria, 166, 258 Lythrum, 250 sqq. McCUntock, 330 Macklin, 350 McLennan, 354 Magnoliales, 324 Maheshwari, 200 maize v. Zea Mays male-sterility, v. sterility mammals, 49, 227, 253 man, 49, 51 sqq., 91, 153, 253, 282, 346 blood, 194 map, linkage, 45, 47 Marchantia, 327 marriage, 347 sqq. mating continuum, 302 sqq. discrimination, 252 sqq., 298 legitimate and illegitimate, 249 premature, 260 random, 277, 282 mean, 58, 78, 8i Mecostethus, 289 Medawar, 216 medical genetics, 352 meiosis, 24, 28, 53, 97, 121, 148, 290 cause of, 196 in polyploids, 122, 140 Melandrium, 52, 204, 256 Mendel, 36, 55, 93, 262, 274, 369 mendelian experiment, 36, 55, 132, 150, 346 inheritance, 55, 66, 68 method, 36 sqq., 66, 68 Menzies-Kitchen, 284 merogon, 18, 168 metaphasc, 23, 31 metastasis, 212 microsome, 207 mid-parent, 81 millet, V. Sorghum misdivision, 103, 104 mitochondria, 207 mitosis, 23 sqq., 145, 194 Moewus, 44 monoecy, 242 monosomic, 105 Morgan, 42, 112, 197 Morris, 219 mosquitoes, 339 mosses, 189 See also Funaria mother cell, 29, 45 mouse, 116, 118, 160, 213 Mucor, 240 Muller, 45, 119, 152, 210, 331 mustard gas, 100 mutafacience, 173, 211 mutant, 95, 327 in Oenothera, 263 mutation, 106, 114, 152, 272 chemical, 184, 212 lethal, 272 plastid, 171 sqq. rate, 155 somatic, no, 214, 227 theory of evolution, 263 virus, 210, 219 Nabours, 335 Narcissus, 251 natural selection, 273, 275, 282, 305, 306 in man, 359 neomorph, 152 Neurospora, 162, 342 Nicandra, 104 Nicotiana, 70, 71, 168, 174, 245, 256 Nilsson-Ehle, 62, 64, 65 Nishiyama, 338 non-disjunction, 128 normal curve, 57, 58 nuclear membrane, 203, 221 nucleic acid, 145, 155, 194. 207, 268 nucleoli, 23 sqq., 32, 146, 211, 330 nucleotide, 148 443 INDEX nucleus, 17, 28, 145 sqq., 182 nullisomic, 105 nutrition, 160 sqq. oats, 16, 64, 158 fatuoid, 338, 339 Oenothera, 30, 106, 130, 170, 246, 309, 316, 332, 341 complexes, 261 sqq. embryo sac, 201 orientation, 31, 123 Orthoptera, 52, 290 osmosis (social), 358 outbreeding, 282 sqq., 297 bias, 247 devices, 242 restriction of, 305 sqq. species, 320 Oxalis, 251 pachytene, 29, 122, 128 Paconia, 321, 322 Painter, 27, 35 pairing segment, 52, 133, 346 pangenesis, 274 Paramecium, 175 sqq., 191, 203 parameters, 56 Paratettix, 335, 336 Paris, 290 parthenogensis, 98 cyclical, 264 Pasteur, 210 Patau, 131 paternity, 351 pattern, 157 pea, V. Lathyrus, Pistim Pearson, 61, 62 Pedicuhpsis, 241, 260, 297 Pelargonium, iii penetrance, 160 Penicillium, 343 pepsin, 145 Peto, 234 Petunia, 174, 253, 255, 305, 332 Pharbitis, 164 Phaseolus, 62, 72, 184, 2x8, 239 phenocopy, 184 phenotypc, 15, 156, 204 optimum, 283 philology, 360 phonetics, 360 Pickford, 49 pigmentation, 155 sqq., 163, 193, 2i( pigs, 237, 283 pin and thrum, 249 piracy, 222 Pisum, 36, 55, 182, 216, 240 plasmagene, 173 sqq., 203, 216, 344 plastid, 149, 169 plastogcne, 169 plciotropy, 115 290, I Pneumococcus, 210 Poa, 268, 269 point mutation, no polar body, 97 pollen, 96, 105, 123 sqq., 139 differentiation, 195 sqq. gene action in, 191 mechanism, 241 polygamy, 358 polygenes, 66, 78, 150, 329, 330 polygenic system, 66 sqq., 279 polymeric genes, 68 polymerization, 145 polymitosis, 198, 212 polymorphism, 335 sqq. polypeptide, 145 polyploidy, 95, 121, 134, 321 in blood, 194 secondary, 98, 324 polysomy, 95 polytene, 26, 47, 106, 131 chemistry, 145 Pomoideae, 324 Pontecorvo, 343 populations, 153, 351, 364 randomly breeding, 89, 90 position effect, 106, 118, 152, 155, 330 potato. III, 218 poultry, V. fowl precursor, 162 sqq., 177 cells, 194 Preer, 177, 203 Prell, 245 presence and absence, 106 primrose, v. Primula Primula, 114, 135, 169, 231, 24gsqq., 310 prophase, 23 protandry, 242, 258 protein, 145 sqq., 207 sqq., 219, 268 protogyny, 242 protozoa, 29 provirus, 214 Prunus, 243, 247 pure line, 62 Pyrethrum, 200 Pyrus V. apple quadrivalent, 126, 137 quantitative effects, 151 444 INDEX rabies, 210 Race, R. R., 333 race theory, 353 Raphano-brassica, 135, 231, 232 rat, 202 ratio, mendelian, 37, 156 recessive characters, 347 reciprocal crosses, 48, 125, 126 recombination, 33, 41 sqq., 117, 132, 237, 304 sqq. and correlated response, 297 and natural selection, 290 reduction, 33 reductional separation, 31, 52 regeneration, 173, 189 regression coefficient, 61 Rendel, 272 Renner, 170, 201, 260 effect, 201, 268, 272, 311, 342 reproduction, v. sexual V. self-propagation Rhesus blood-groups, 333 Rhoeo, 130, 317 Rick, 99, 242 ring-formation, 129 breakdown of, 263 Rischkov, 208 Risley, 354 rodents, 157 rogue, 182 See also mutant root-cutting, iii Rosanoff, 349 Rous, 213 Rudbeckia, 159 rye, 237, 260, 297 Saccharomyccs, 179 Salaman, 218 salivary gland, v. polytene Sax, 72, 206 scales, 58, 78 sqq. tests of, 79 Schnarf, 201 Schrodinger, 371 Sciara, 193, 252 Scilla, igj Scolopendrium, 186, 190, 327 sea urchin, v. echinoderni Secale, v. rye segregation, 38, 45, 68, 127, 237 somatic, 187 Seiler, 264 selection, 272 sqq., 302 correlated response to, 298 selection — continued discriminative action of, 294 self-fcrtUization, 36 diploid, 240 haploid, 240 self-propagation, 28, 149, 203 serology, v. blood group sex-chromosomes (X and Y), 46 sqq. sex determination, 46 sqq., 166, 193, 204, 256, 339 ^I'J-, 346 sexes, homogametic and heterogametic, 49. 229 sex-linkage, 48 sqq. partial, 50, 346 sex-ratio, 275, 337 super-gene, 336, 337 sexual differentiation, v. dioecy sexual reproduction, 16, 24, 29, 99 breakdown of, 268 shibboleth, 360 Sismanidis, 285, 297 Smith, K. M., 209 snail, 191 Solanum, iii, 228 Somiebom, 175 Sorghum, 104, 151, 195 sparrows, 282 species crosses, 135, 191 speech, 360 speltoid, p. w^heat sperm, 24, 28, 51, 105, 216 spermathecae, 298 spider, 52 Spiegelman, 179 spindle, 23 spiraUzation, 146 spore, 29, 32, 44 sporophyte, 29 sport, p. mutation standard deviation, 59 sterility, p. fertility genotypic, 231 hybrid, 235 male, 174, 190 segregational, 232 Stem, C, 161 Stem, F. C, 322 Stizolobium, 134 strawberry, 218 Streptocarptis, 228, 253 structural change, 100 sqq. structural hybrid, 122 Sturtevant, 132, 313 subsexual reproduction, 266 sugar beet, 237 445 INDEX super-gene, 46, u 8, 133. 312, 335 sqq. supernumerary, i>. chromosome suppressivcncss, 179 suppressor, 158 syndrome, 348 syphilis, 358 systematist, 323 tabu, 253 Talacporia, 264 Taraxacum (dandeUon), 265 sqq., 344 telocentric, 103, 104 telophase, 25, 146 temperature, effect of, 95, 100, 161, 178, 195, 198, 216 template theory, 146, 211 tetrad, 32 tetraploid, 95, 122 sqq., 134-S'?^- Thomas, 212 Thyanta, 323 Timofccff-Ressovsky, 159, 295 tissue specificity, 202 tomato, V. Lycopcrsicum Torula, 212 Tradcscantia, 289 transformations, 80 translocation, 102, 106, 137 transplantation, 166, 191, 202, 212 See also graft Trichoniscus, 265 Trifolium, 246 triploid, 96, 265 trisomic, 96 in Oenothera, 263 Triticum, v. wheat Triton, 30, 99, 168, 290 Triturus, 99 trivalent, 121 tuberculosis, 349 Tulipa, 100, 125 sqq., 139, 208, 311 tumour, V. cancer twins, 269, 270, 349 unisexual brood, 252 unity of heredity, 13055^. See also gene univalent, 121, 137 unreduced germ cell, 34 Upcott, 125, I'iJ sqq. Uvularia, 198 vaccinia, 207 variability, 276 and mutation, 295, 296 fixed, 278 flow of, 277, 285 variability — continued free, 276 frozen, 284 potential 276, 279, 280, 287, 296 reservoir of, 296 states of, 281 variance, 59, 78, 82 sqq. variation, 34, 272, 276 continuous, 56, 61, 349 cryptic, 323 discontinuous, 55, 61 in man, 364 non-heritable, 65 spectrum of, 65, 66 variegation, 170, 186, 211 vector, V. virus vegetative propagation, 16 Verbascum, 328 Veronica, 245 versatile reproduction, 269 Verschuer, 349 Vicia, 168, 288 Vinca, 209 Viola, 241 virus, 146, 149, 207 vector, 208 sqq. vitamin, 160 sqq. Wallace, 273 wan, 214 Weismann, 29, 35 Wcttstein, 173 wheat, 64, 158, 259, 338 speltoid, 338 Whiting, 113 wild-type, v. Drosophila Wilson, 197 Winge, 205, 256, 340 Winkler, iii woman, 347 sqq. Wright, 203 X chromosome, 46, 258, 330, 341, 346 Xiphophorus, 51 X-rays, 100, 146, 152, 213, 327, 331. 332 Y chromosome, 46, 147, 151, 329. 33°! 340, 341, 346 yeast, 179, 212 Zea .Mays, 30, 45, 126, 147, 235, 242, 329, 330, 339. 340 asynaptic, 288, 289 embryo sac, 200 mutation, 155 pollen, 191, 198 zygote, 28, 145 446