Advances in Genetics Volume 4

ADVANCES IN GENETICS VOLUME IV Edited by

M. DEMEREC Carnegie Institution, Cold Spring Harbor, N . Y.

Editorial Board G . W. BEADLE WILLIAMC. BOYD TH. DOBZHANSKY L. C. DUNN MERLE T. J E N K I N S JAY L. LUSH

A LFRED MIRRKY 11. J. MULLER

J . T. PATTERSON M. M. RHOADER 14. .J. STADLER CURT STERN

1951

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CONTRIBUTORS TO VOLUME I V

A. CARVALHO, Instituto Agrm6mico, Campinas, SGo Paulo, Brazil SALOME GLUECKSOHN-WAELSCH, Department of Zoology, Columbia University, New Y m k , New York ERNSTIIADORN,Zoologisch-vergl. amtomisches Ircstitut, University of Zurich, LYwitzerland

ALOHAHANNAH,Department of Zoology, University of California, Berkeley, California

C. A. RRUG,Instituto Agrolzdmico, Campinas, SGo Paulo, Brazil

R. MATTHEY, Laboratoire de Zoologie, Universite' de Lausanne, Lausanne, Switzerland SEIJINNAGAO, Plant Breeding Institute, Hokkaido University, Sapporo, Japan T. R. RICHMOND, Texas AgriculturaJ Experiment Xtatim, Texas Agricultural and Mechanical College System, College Station, Texas, and U . S. Department of Agricultirre

S . G. STEPHENS, Department of A g r o w m y , North Carolina State College, Raleigh, North Carolina

M. J. D. WHITE,The University of Texas, Austin, Texas

Physiological Genetics of the Mouse SALOME GLUECKSOHN-WAELSCH Department of Zoology. Columbia Vniversity. New Pork. New Pork CONTENTS

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I Introduction I1 Analysis of the Yellow Lethal. a Classic Case of a Lethal Mutation I11 Analysis of Gene-Determined Pigment Characters 1 Autonomy of Pigmentation a Mutant Spots b Transplantation 2 Histological Studies of Pigmentation in Different Gene Substitutions 3 Biochemical Studies of Pigmentation in Different Genotypes 4 Correlation of Histological and Biochemical Results 5 Tyrosinase System in the Determination of Pigment Pattern I V Analysis of Gene-Determined Abnormalities of the Blood 1 Siderocytic Anemia in Flexed 2 Macrocytic Anemia in Dominant Spotting V Analysis of Mutat.ions Affecting the Skin and Its Derivatives 1 Transplantation Studies in Waved-2 2 Transplantation Studies in Rhino 3 Rhino and Vitamin A 4 Developmental Studies of Crinkled V I Developmental Studies of Mutations Affecting the Urogenital Sy.stem 1 Kidney Development in Myelencephalic Blebs 2 Kidney Development in Danforth’s Short Tail 3 Inductive Relationship between Ureter and Metanephros 4 Urogenital Syndrome V I I Developmental Studies of Mutations Affecting the Central Nervous System and Sensory Organs 1 Hydrocephalus 2 Congenital Hydrocephalus 3 Pseudencephaly 4 X-Ray-Induced Translocations and Pseudencephaly 5 Shaker Short a Central Nervous System b Ear 6 Effect of Kreisler 011 the Central Nervous System and Ear 7 Eyelees 8 Microphthalmus VIII Analysis of Endocrine Disturbances-Pituitary Dwarfism I X Analyeis of Mutations Affecting the Skeleton 1 Skull a.Harelip 1

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b Tooth Abnormalities i Grey-Lethal ii Screw Tail 2 Extremities a L u x M o n g e n i t a l Absence of Tibia b Luxate c Polydactyly i Development ii Polydactyly and Embryonic Blebs iii Polydactyly and Central Nervous System iv Polydactyly and Maternal Age d Grey-Lethal and Failure of Secondary Bone Absorption 3.Sternum a Screw Tail b Short Ear 4 Spinal Column a.Flexed b Shaker Short c. Screw Tail d Undulated e Danforth's Short Tail The Developmental Effects of Mutations in Chromosome I X of the Mouse 1 Brachy6ry-Heterozygous Effect of T 2 Taillessness T/tn 3 Homozygous Effect of T 4 t-Type Mutations-Developmental Effects of t", t', t' 5 Kink-Homozygous Effect of Ei 6. F u s e d 7 Abnormalities of t ' / P Embryos 8 Miscellaneous Abnormalities in Individuals Carrying Mutations of Chromosome I X 9 The Role of Chromosome I X in Embryonic Growth and Differentiation Developmental Changes Induced by X-Rays Concluding Remarks References

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I. INTRODUCTION A review article on the physiological genetics of the mouse might at first sight appear to have chosen too limited a n object for discussion . But there are a number of reasons why just the house mouse (Mus musculus L.) lends itself well to a discussion of problems of physiological genetics. It is true that microorganims. for example. provide a material more accessible to the analysis of biochemical gene effects. It also is true that Drosophila has been analyzed so thoroughly genetically that it might offer better material for the study of the physiological mecha-

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nisms of gene effects. On the other hand, the problem of gene effect and gene action in a vertebrate organism stands alone in many respects, both from the theoretical and the practical point of view. The vertebrate organism differs from lower organisms in so many obvious ways that they need hardly be enumerated here. I n its structure, physiology, metabolism, endocrinology, etc., gene-controlled mechanisms and processes are operative which in many respects offer a unique material for the physiological analysis of gene effects. The vertebrate organism is, of course, of particular interest from the point of view of the relationship between genes and processes of embryonic and cellular differentiation because of the epigenetic nature of its development, and by virtue of the character and interdependence of its developmental mechanisms which have been analyzed in such detail, particularly in amphibian embryos. But Amphibia do not lend themselves easily to a genetic analysis, so that it has been difficult to use that particular group of vertebrates for extensive studies of the role of genes in development and differentiation. I n the mouse we know of a considerable number of genes with effects on embryonic processes offering a material on which to study the relationship between genes and embryonic differentiation. I n many cases these same genes may actually serve as tools for the causal analysis of embryonic processes and mechanisms which would defy elucidation by other means. Frequently the creation of an abnormal situation is the prerequisite for the causal analysis of the normal mechanism. The mammalian embryo because of technical difficulties has not been a ready subject for an experimental approach similar to that used in other vertebrates. It is, therefore, fortunate that mutations have been discovered which produce abnormal situations in mouse embryos that resemble those observed in other vertebrates resulting from delicate experimental operations. The analysis of such changes in developmental patterns and the examination of the steps preceding the abnormalities frequently make it possible to discover causal connections between processes which could not have been revealed through a study of the normal alone. The mutations whose effects have been studied in detail and which will be reviewed in this article concern different organ systems of the mouse. These include among others, the skeleton, integument, nervous system, pigment-forming system, blood, and developmental systems of the embryo. Different levels of development are affected by the mutations to be discussed. There are those, just mentioned, which interfere with processes of early embryonic organization and with inductive relationships between different parts of the embryo severely enough to cause death of

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the embryo. I n other cases, the interference of a mutation with an inductive effect does not interfere with viability but leads to abnormalities of structure. Mutations will be discussed where an effect on enzyme systems can be demonstrated, such as those affecting pigmentation. Hemoglobin synthesis will be shown to be disturbed in different hematopoietic tissues and in different phases of development by the action of mutational changes with far-reaching effects. One syndrome of abnormalities will be discussed which will be shown to have arisen as the result of a mutational effect on a hormone. The developmental analysis of some abnormalities of the nervous system will demonstrate how the original effect of a mutation may show no direct relation to the eventual symptoms. The analysis of other mutations reveals that their effects are due to changed rates of growth and differentiation of certain tissues. I n general, developmental studies of the type to be discussed here demonstrate the way in which genes express themselves developmentally, where the term “developmental” is used in a broad sense and is not necessarily restricted to the embryonic phase of the organism. The developmental analysis of mutations will be shown frequently to group gene effects which had appeared a t first sight to have quite different manifestations, while separating, on the other hand, gene effects which had originally shown close resemblance. J ust as the analysis of biochemical mutations in microorganisms has contributed considerably to the general knowledge of biochemical mechanisms, so the analysis of mutations in mice has increased our knowledge of normal developmental processes of different types in mammals, and this aspect will be stressed particularly in this review. From the practical point of view there exists, of course, always the hope that the results obtained from the genetic studies on mice may eventually have some bearing on other mammals, particularly man. F or the purposes of this discussion a considerable degree of selection ha5 been practiced, and we have considered only those mutations and those aspects of a particular mutation whose analysis was thought to contribute to the “physiology of heredity’’ i n the sense of Goldschmidt. The problem is not so much what particular effects some mutation may have but rather how the effect is brought about, i.e., to attempt to trace the steps that lead from primary action of the gene to its eventual manifestation. Thus, many mutations will be omitted from the discussion which have only been described and analyzed genetically, while others are going to be discussed incompletely. Gruneberg’s book (1943a) on the genetics of the mouse contains a complete compilation of data on mouse genetics and is a n invaluable

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help to anybody working in this or related fields. More recent data from the field of mouse genetics are included in Gruneberg’s second book, Animal Genetics and Medicine (1947). Another book with useful information is the Biology of the Laboratory Mouse edited by Snell (1941). This contains a very good chapter on early mouse embryology. For more detailed descriptive information about early mouse development the reader is referred to a series of papers by Sobotta (1895, 1902, 1911).

11. ANALYSISOF

YELLOWLETHAL,A CLASSICCASEohi LETHAL MUTATION

THE

A

One of the classic cases of a lethal mutation is the so-called yellow lethal in the mouse. Its study also represents the earliest attempt of a n analysis of a n embryonic lethal effect in a mammal. The first abnormal symptoms of yellow homozygotes are recognizable a t the time of implantation in the uterus ; this is the earliest stage a t which detailed observations of effects of any mutation have ever been recorded. The yellow mutation at the agouti locus is dominant over all other members of the agouti series. The absence of yellow homozygotes from the offspring of matings between yellow parents was discovered and pointed out by Cuenot (1905). Cuthot assumed that a “yellow” egg could not be fertilized by a “yellow” spermatozoon. The lethal vharacter of the yellow homozygote was ascertained by Castle and Little (1910) who concluded from their breeding experiments of yellow mice “ t h a t a Mendelian class may be formed and afterwards be lost by failure to develop.” I t was demonstrated later by Kirkham (1919) and still later by Robertson (1942) that the yellow homozygous zygote actually does form but dies in early embryogeny. The earliest reports of the findings of remnants of yellow homozygotes came from Ibsen a n d Steigk d e r (1917) and Little (1919). Kirkham (1919), however, made the first more or less complete study of these homozygotes, and reported the first abnormalities to be the indistinct cell boundaries of embryos of’ the morula stage. Blastocysts with a shrunken appearance, small crowded cells and shrunken blastodermic vesicle were assumed to be yellow homozygotes on the basis of their numerical frequency. A more extended descriptive analysis as well as a n experimental attack on the problem of the yellow homozygote was undertaken by Robertson (1942) who examined embryos from matings of yellow by yellow from the cleavage stages on and could not confirm earlier reports of abnormalities in late cleavage stages. The yellow homozygous embryo develops normally through stages of cleavage and blastodermic vesicle formation. It

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becomes lodged in a decidual crypt and the uterine epithelium surrounding it changes its character from columnar to low cuboidal while the subepithelial uterine connective tissue cells proliferate to a great extent. But the cuboidal epithelial cells do not disappear as they do in the normal implantation process and the further development of the implantation site is arrested, The blastocyst cavity disappears in the abnormal embryos. “Since the resulting cell mass is so small and has no appearance of differentiation into the various blastocyst structures, it is concluded that upon contact with the uterine epithelium the trophectoderm of the homozygous yellow blastocyst collapses and then quickly degenerates, leaving only the inner cell mass the cells of which soon become abnormal. ” I n the next stage, the egg cylinder stage, the decidual crypts of the abnormal embryos.are small and contain only a few scattered cells : the remnants of homozygous yellow embryos. The uterine mucosa is in a progestational phase and uterine epithelial cells are present in cuboidal form, as on the previous day. Death of the homozygous yellow embryo is assumed to occur after the trophectoderm of the blastocyst has come in contact with the uterine epithelium. Robertson (1942) used a very interesting approach to study the development of homozygous yellow embryos in an environment other than that of the heterozygous yellow mother. He transplanted ovaries from yellow mothers to nonyellow agouti hosts whose own ovaries had been removed. About a week after the operation, the host female underwent a test mating. If yellow offspring appeared in a cross to a male not carrying yellow, the graft was considered successful and the host female was mated to a yellow male; embryos were obtained from the female a t different stages. I n cleavage and preimplantation stages no abnormalities are observed, as expected. The appearance of the abnormalities occurs a t the same stage as in the normal environment, i.e., in the yellow mother. But the homozygous yellow embryo in the agouti mother can be clearly distinguished from the homozygotes in the yellow mother by an advance in development which Robertson summarizes as follows: “1) the development of an ectoplacental cone; 2) the more complete differentiation of a n area of the cell mass corresponding to the embryonic region of the normal implanted embryo j 3 ) the further development of the implantation site as exhibited by the disintegration of the uterine epithelium and the relatively greater depth of the implantation crypt ; and 4) the development of a n extra-embryonic membrane which appears to be Reichert ’s membrane. ’ ’ The onset of lethal action in the yellow homozygote is supposed to occur a t the time of implantation and to be due to the absence of a n

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enzyme normally secreted by the blastocyst roof and responsible for the erosion of the uterine epithelium and successful implantation. The effect of yellow in homozygous condition may be directed on this enzyme. “Since the homozygous yellow mouse embryo develops further in the agouti uterus than in its usual uterine environment, it is evident that the developmental phenomena peculiar to this embryo are a result of the uterine environment of the heterozygous yellow mother as well as the hereditary factors inherent in the embryo itself.”

111. ANALYSISOF GENE-DETERMINED PIQMENT CHARACTERS One of the developmental systems in the mouse most thoroughly studied from the point of view of gene action is that of pigmentation. Pigmentation offers a number of advantages for the stcdy of the chain of events between gene and character. The development of pigmentation may be observed in situ and is a strictly localized process apparently not determined by circulating substances. That this is the case is evident from observations on mutant spots and from transplantation experiments. I n addition to the strict localization and the autonomous behavior of pigmentation there are other features which make the pigment system a most suitable subject for studies of gene expression and its development. Since hair is being shed and replaced frequently during the lifetime of the mouse, the developmental events leading to pigment formation repeat themselves and can thus be observed continuously within the same organism. The hair, with its differences in pigmentation along the hair shaft, is an image of the processes going on in the hair bulb during the formation of pigment as determined by the individual genes of the particular cell. It has been shown (Rawles, 1947) that the pigment-forming cells of the mouse originate from the neural crest and that they make their way into the developing hairs and bring about their pigmentation. The melanophores reach their definitive positions in the skin of the mouse embryo by the twelfth day after fertilization. Determination of pigment pattern is thus completed a t that stage. The deposition of pigment in the hair by melanophores was demonstrated in witro by Hardy (1949) with the help of tissue culture techniques. 1. Autonomy of Pigmentation

a. Mutant Xpots. On the basis of the generally accepted conclusion that the color reaction of a cell is determined chiefly by the cell’s genes, the color mosaicism of a male mouse has been interpreted as being due

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to the presence of a changed locus in each of the abnormal color spots (Dunn, 1934a). The same male showed gonadal mosaicism, and a mutation in the cell ancestral to the gonadal and the somatic tissue was assumed to have been responsible for the mosaicism observed. The distribution of the patches which originally arose from a common mutant cell was considered an indication of a complex series of differential growth rates or migrations of the epidermal tissues in different directions. The existence and persistence of the mutant spots is an argument in favor of the local determination of pigmentation as against the decisive influence of generally circulating substances. b. Transplantatim. The autonomous behavior of hair pigmentation in mice indicated by the existence of mutant spots has been studied more closely with the help of transplantation experiments. I n a series of papers (Reed, 1938a, b ; Reed and Henderson, 1940) transplantation of skin of newborn mice between genetically different individuals has been reported. In the case of albinism, agouti pigmentation, dominant spotting, and piebald, complete autonomy of the pigment pattern of the transplanted tissues was found which must thus have been determined before birth. In the case of the black-and-tan mutation there seems to be a difference between tissue and cell determination. Tissues from the black-and-tan mutant ( a W ) themselves are autonomous in respect to their dorsal or ventral organization ; however, the individual prospectively “black-and-tan” cells are capable of forming either black or tan pigment, depending on whether they find themselves in an environment, of dorsal or of ventral cells.

2. Histological Studies of Pigmentation in Different Gene Substitutions Since the gene dependency of pigmentation is thus established the next questions concern the way in which the genes exercise their control, and the mechanisms of their effects. The effect of genes on pigmentation seems to be fairly direct, and the chain of processes connecting gene action and expression rela,tively short, making genetic pigmentation differences a most suitable material for the study of gene physiology. A number of studies have attempted to analyze the effect of differ. ent genic substitutions on pigmentation. Dunn and Einsele (1938) investigated the changes in the dark granular melanins of the hair in different combinations of the albino series with black ( B ) and with brown ( b ) . They found a graded reduction in amount of pigment, as measured by weight, in combination with black ( B ) . The changes in quantity of pigment were shown to be due to a decrease in size of pig-

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ment granules. “Each mutant gene in the c series thus exerts a characteristic effect on granule size.” Systematic histological studies of pigment in the hair cells of the mouse in different gene substitutions were done first by Werneke (1916). Using the same approach E. S. Russell (1946, 1948, 1949a) tried to ascertain the basic natnre of gene action in five of the main allelic series of mouse coat-color genes : agouti, albino, black/brown, intense/pinkeyed dilution, intense/blue dilution. Thirty-six different genotypes were examined in a series of very careful and thorough studies, and a number of different attributes of pigment granules were observed; it could be shown that a large number of different variable pigmentation attributes contributed to the differences in appearance of coat color mutants. They are color, size, shape, number, arrangement of granules, and several others. A certain amount of interdependence among these different pigment attributes makes it possible to separate them into four groups controlled by four key pigment characteristics : nature of granule color, granule size, granular clumping, level of pigmentation. The eventual goal of these studies is to relate the basic action of each of five allelic series (agouti, albino, black/brown, intense/pink-eyed dilution, intense/blue dilution) to variations in these pigment attributes. The nature of the pigment produced at any particular level of hair (i.e., whether i t is of the xanthic or the eumelanotic type) is determined by genes of the agouti series, while the genes of the albino series determine quantitative changes, i.e., degrees of pigmentation, by altering shape, color intensity, number, size, and distal arrangement of pigment granules. The change from dominant B (black) to b (brown) expresses itself mainly in a change in the nature of eumelanin from black to brown, i.e., in a change from the process leading to pigments of the black-fuscous series to one leading to brown pigments, while the quantitative effect is only minor. Size of pigment granules and deposition of pigment are affected by the change from P (full color) to p (pink). I n animals homozygous for d (dilution), the amount of pigment is a t least as high as in DD, but it is disarranged into large granular clumps and it is distributed unevenly. 3.

Biochemical Studies of Pigwentation in Different Genotypes

The biochemical aspects of pigment formation in various genetic backgrounds were studied (Russell and Russell, 1948) with the aim of getting closer to the actual effect of the pigment genes. The behavior of the dopa reaction was examined on frozen sections of mouse skin, a technique which permits a histological localization of dopa-oxidase activity. This method has a number of advantages over others using tissue

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extracts: it avoids the inclusion of nonpigmented cells which may introduce inhibitors ; the activity of specified parts of a, cell can be determined ; and each section serves as its own control, since autoxidation of dopa would lead to uniform darkening of the entire section instead of differential darkening localized in the hair bulb as produced by enzymatic oxidation. The same genic substitutions were used which had been studied histologically, and the studies were done on animals of 6-7 days of age since it was found that a t that age the follicles produce that part of the hair which had been examined in the histological compaxison of genotypes. The biochemical results could thus be easily correlated with the histological data. It was found that there existed a close parallelism between the amount of yellow pigment as determined histologically and the activity of the dopa reaction in different gene substitutions ; when a gene mutation had a n effect on the natural yellow pigment it would have a parallel effect on the activity of the dopa reaction. This effect was found in the yellow as well as in the corresponding nonagouti types, and from this fact the authors conclude that “the dopa reaction measures some phase of the yellow producing system and that this system is present in sepias as well as in yellows.” The authors propose several interpretations of their results for the nature of the black-yellow differentiation, one of which postulates that ‘‘sepia-yellow differentiating genes act to produce different substrates. ” “ The discovery of another chromogen whose effect paralleled sepia both in yellow and sepia genotypes would prove the postulate that the sepia-yellow difference was one of substrates rather than enzyme systems, and the relation between this chromogen (presumably akin to the sepia substrate) and dopa could provide a. chemical clue as to the action of sepia-yellow differentiating genes.” 4.

Correlation of Histological artd Biochemical Results

Correlating the results of the histological studies with the biochemical results it appeaxs that the activity of the enzyme system measured by the dopa reaction is not affected by the genes of the agouti series. The genes of the albino series, on the other hand, affect the activity of the enzyme system which in turn is responsible for the intensity of the pigmentation reaction. The change of eumelanin from black to brown due to the genic substitution bb is not accompanied by any change in enzyme as measured by the dopa reaction activity. The effect of the pink substitution on size of granules and level of pigmentation appears to be correlated with a slight but probably significant decrease in dopa. oxidase activity. The dd substitution surprisingly results in a n increase of the

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reaction system, which runs parallel with some increase of natural pigment in dd individuals. Of the five allelic series studied, the genes a t the C and possibly a t the P locus seem to be involved in the control of the enzyme system measured by the dopa reaction which shows a correlation with the natural yellow pigment. 5. Tyrosinase System in the Determimtivn of Pigme& Pattern Some preliminary studies (Foster, 1951) deal with the role of tyrosinase activity in the determination of the pigment pattern of different genotypes in the mouse. The method used for measuring tyrosinase activity was that of determining oxygen consumption in a Warburg respirometer. The enzyme preparations consisted of homogenized skin, suspended in distilled water. Tyrosine was used as a substrate. The results of this study seem to demonstrate the significance of the tyrosinase system for the development of eumelanin pigment, and the presence of tyrosinase inhibitors in the skin of mice. The very slight amount of tyrosinase activity and the presence of a. tyrosinase inhibitor in yellow skin seem to indicate that the yellow pigment is independent of the tyrosinase system. Albino skin seems to be devoid of the enzyme as well as of its inhibitors. These preliminary results may indicate the decisive role of the tyrosinase system in the control of the black (sepia) pigment system which had been assumed on other grounds to differ from the yellow pigment system (cf. section 111-3). IV. ANALYSISOF GENE-DETERMINED ABNORMALITIES OF THE BLOOD There exist a number of well-analyzed genetic conditions in the mouse which involve the hematopoietic system ; the systematic investigation of the effects of these mutations and the embryogeny of the conditions caused by them has contributed much to our knowledge of the formation of the blood in the mouse. 1. Siderocytic Anemia in Flexed

One of the best-analyzed mutations affecting the blood is the flexed mutation ( f l ) . It was reported by Hunt and Permar (1928), and its effect in the blood system was studied by Kamenoff (1935) and by Griineberg (1942a, c). Flexed ( f l ) also has an effect on the tail and produces a belly spot. However, we want to concern ourselves here only with its effect on blood. The first observations on flexed showed that newborn fljl mice were anemic but that this anemia was transitory and

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the mice recovered soon. I n studies of the blood of flfl embryos it waa possible to place the onset of the anemia at a time before the fourteenth day after fertilization, i.e., preceding the beginning of the hematopoietic function of the bone marrow (16 days) ; later its beginning was traced hack to 13 days after fertilization. Actually, the anemia of the flp mice hegins to improve at the time when the bone marrow starts its normal hematopoietic function. The flexed gene is thus not concerned with the functioning of the bone marrow. This point was confirmed by red cell counts of adults which were practically normal. Apparently the flexed mutation causes some abnormality in the hematopoietic function of the liver, as indicated by the studies of Oruneberg (1942a, c). Bruneberg reports the abundance of siderocytes in flexed mice, i.e., red blood cells whose iron can be demonstrated in the form of granules by means of the Prussian Blue reaction. Oruneberg originally suggested that flexed anemics were unable to synthesize hemoglobin normally but produced only a hemoglobin precursor, the iron of which could be demonstrated. This inability to synthesize hemoglobin would be, however, restricted to the liver since only its hematopoietic function is affected by the flexed mutation. Lately, on the basis of some work indicating that siderocytes are actually aging cells, Qriineberg (1947) has taken the point of view that the anemia of flexed tailed mice arises from an increased hemoglobin breakdown. Of course, this might in turn be considered a consequence of abnormal hemoglobin synthesis. We thus cannot draw any definite conclusions from the embryonic study of the flexed anemia except that the normal allele of flexed must be involved in the control of either hemoglobin synthesis in the liver, or in its structural maintenance in the erythrocytes derived from the liver. The question remains open why the other hematopoietic systems of the animal should be affected mildly a t the most, and be capable either of normal hemoglobin synthesis, or of producing hemoglobin resistant to premature breakdown. One would perhaps expect this synthesis to be equally disturbed throughout the entire organism. A time factor does not seem to be involved since the first red blood cells originating from the yolk sac are fully hemoglobinized and probably normal. The effect of fl seems thus to consist of a localized disturbance of erythrocytes originating from the liver. If some general agent were operative in the flexed mice, one could expect the bone marrow red cells to be equally affected. 2. Mncrocytic Anemia in Dominant #potting A different step in the development of blood is interfered with by the W mutation. Mice homozygous for the W gene are anemic and in-

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viable; they die soon after birth. Their anemia is of the macrocytic type, i.e., these mice have fewer but larger cells than normal mice of the same age. DeAberle (1927) traced back the anemia to the sixteenth day of embryonic life by the observation of markedly pale embryos a t that stage, and Russell, Fondal, and Smith (1950) by establishing the erythrocyte level found WW embryos to be definitely anemic at 14 days after fertilization. There is some increase in the erythrocyte level of the anemics during embryonic life, so that the W anemia is a hypoplastic rather than a n aplastic anemia. The failure of WW to improve during the period when the bone marrow takes over its hematopoietic function clearly shows that the effect of W differs from that of fl. W apparently has a general effect on erythropoiesis of the entire organism and is considered by Russell et d.to cause a deficiency of a substance essential to erythropoiesis from earliest embryonic stages. The normal allele of W thus seems to be involved in the control of hemoglobin synthesis in general. There exists another allele of the W series, W" (Little and Cloudman, 1937)? which, when homozygous, produces a condition intermediate between that of normal and of W W mice. W"W" mice also have a macrocytic anemia but less severe than that of WW mice with a reduction to about one-half the normal number of erythrocytes; they are viable and actually improve their condition during their lifetime. Their anemia is definitely of the hypoplastic type and W" seems to affect hematopoiesis in the same qualitative manner as W , but to a lesser degree. The same substance essential for normal hemoglobin synthesis and presumed to be deficient in the case of WW would perhaps be less deficient in the case of WOW". A peculiar phenomenon is the existence of a mild anemia in the presence of one dose of W " :lower erythrocyte counts and hematocrit readings were established in W"heterozygotes by Griineberg (1942b), while animals heterozygous for W , which in homozygous condition produces a severe anemia, had a completely normal blood picture. Both alleles, W and W", were recognized first by their effects on pigmentation. When present in one dose, W produces a white belly spot, while W u heterozygotes, in addition t o the white belly spot, show a dilution effect of dorsal and ventral pigmentation. Both homozygotes have unpigmented f u r and black eyes. The dominance relationship of the two alleles in their effects on blood and pigment was analyzed most thoroughly by Russell (1949b) , who showed that W had no effect in one dose on either blood or pigment, but a severe effect in two doses. Wv had an effect on both systems in one dose, expressed in a decrease of granule number and size in the case of pigment, and in lower erythro-

14

SALOME GLUECKSOHN-WAELSCH

cyte counts in the blood ; but it had a milder effect than W on the blood in two doses, while the effect on the pigment equalled that of W. Russell points out the clear parallelism between the effects on the two different systems, blood and pigment : whenever one system is affected, the other one is affected too; in addition, there is a parallelism in degree of abnormality of blood and pigment. On the other hand, the reviewer feels that the divergence in the dosage effect of the two alleles should not be overlooked. One dose of W v is more effective than one dose of W, but two doses of W v are less effective than two doses of W. A hypothesis for the explanation of the simultaneous effects of mutations in mice on pigmentation and blood was put forward recently by Serra (1947), who supposes that an effect on copper may link the manifestations of these mutations in two systems. W and W” would, according to this hypothesis, exert an effect on copper compounds involved both in the formation of pigment and blood. I n the absence of any experimental proof, this idea can be regarded as a n interesting hypothesis only.

v.

ANALYSIS O F MUTATIONS AFFECTING THE

SKIN AND

ITSDERIVATIVES

The development of skin and hair in the mouse is dependent on the collaboration of a great many genes as evidenced by the existence of numerous different hair mutations. Quite a number of them have been reported and analyzed genetically (e.g., waved-1, waved-2, rex, naked, hairless, rhino, crinkled) ; we shall concern ourselves here only with those in which an attempt has been made of an analytical study of the mechanisms responsible for the expression of the individual mutations.

1. Transplantation Studies in Waved-2 Transplantations of skin between normal mice and those homozygous for wa-2, whose hair is waved instead of straight, seemed to show that nonwaved tissues or cells became organized to produce waved hair in the waved environment (Reed, 1 9 3 8 ~ ) . These results were interpreted as revealing not only the a.utonomous nature of waved-2 tissues and cells but also their ability to cause genetically nonwaved cells and tissues to form waved hair. The effect of wa-2 was thus assumed to be the following : “waved” cells (wa-2 wa-2) produce a growth-inhibiting substance which is responsible for the effect of wa-2 on body size and hair structure (wa-2 wa-2 mice are smaller than normal). The growth-inhibiting substance diffuses into the normal tissue and there causes nonwaved cells to produce waved hair. This interpretation has become highly doubtful on the basis of some incidental observations in a different series

PHYSIOLOQICAL GENETICS OF THE MOUSE

15

of transplantations between mice with normal hair structure (Fraser, 1946). Here it was noticed that “both graft and host hairs, when situated near the borders of the graft, were often curved or bent, like the hairs of genetically ‘waved’ mice.” The waviness of the hair was ascribed to a n antagonism between several physical factors acting on the growing hair in its abnormal position. Since this “waved” hair could be produced in transplantations between normal mice, it becomes highly doubtful whether the effect of waved on normal cells is actually due to the diffusion of a growth-inhibiting substance from waved into normal tissue.

2. Transplantation Studies in Rhino Another mutation affecting the hair of mice is rhino (hrrh)which produces hypotrichosis. The mode of action of the rhino gene which causes a follicular hyperkeratosis associated with depilation has been studied in transplantation experiments by Fraser (1946). These transplantations were performed with the aim of finding out whether a cellular condition was responsible for the expression of rhino or whether it was the result of some more general change (e.g., endocrine system) in the physiology of the animal. Grafts of normal skin to rhino hosts beha.ved autonomously and grew a normal coat of hair. But the reciprocal experiment gave a different result. Those parts of the rhino graft which were adjacent to the normal epidermis did not develop the rhino characteristics but had normal hair. It could be shown that these hairs were not invasion hairs of the host. Thus it seems that normal skin cells produce a diffusible substance which is “necessary for the normal maintenance of the cutaneous epithelium.” The effect of rhino might consist in the inability to produce this substance although rhino cells are capable of utilizing it when it is supplied from the outside. 3. Rhino and Vitamin. A Some alleviation of the skin defects associated with rhino, such as formation of cysts in the sebaceous glands and follicle ends of the rhino mouse, was obtained in rhino homozygotes by the administration of large doses of Vitamin A (Fraser, 1949). The conclusion was drawn that “the presence of the rhino gene in the homozygous condition is associated with a decreased ability of the skin cells to utilize some metabolite of Vitamin A necessary for their normal maintenance.” Thus it seems, as though perhaps the production of some substance as well as the ability to utilize it might be affected in cells homozygous for rhino.

16

BALOME OLUECKSOHN-WAELSOH

4.

Developmental #t&s

of Crinkled

A recent study of the effects and of the development of another mutation (crinkled) which affects coat texture has shed some light on the normal events taking place during the development of hair (Falconer Fraser, and King, 1951). Crinkled is a recessive mutation with a number of effects among which that on the fur of the animal is the most obvious. While the normal coat of a mouse consists of 3 types of hair, guard hairs, awls, zigzags, that of a crinkled mouse has one type of hair only, and they resemble awls, the short straight type of hair of the under fur. The question asked in the analysis of the mode of action of crinkled was whether crinkled merely inhibited the differentiation of a n as yet undifferentiated hair type into the three types and directed it into the channel of awl formation only without affecting the total number of hairs formed, or whether it suppressed the formation of guard hairs and zigzags leaving awl hairs only. I n the former case, one might suppose that the three hair types were not developmentally distinct entities, while in the second case the developmentally distinct chasacter of the three types would be strongly indicated. Normally there exist three periods of hair follicle formation in the embryo, which might correspond to the three types of hair in the coat. But while the study of normal hair development was not able to establish critical evidence for the connection between periods of hair formation and type of hair formed, the study of crinkled made it very probable that the formation of guard hairs, awls, and zigzags was linked u p with the first, second, third period of hair follicles formation, respectively. While the first hair follicles form at 14 days in the normal embryo, no follicles are found in the crinkled mouse until 17 days after fertilization when the crinkled embryo forms follicles resembling in general appearance those of the second period in normals. After birth, when the third wave of follicle formation starts in the normal, the crinkled mouse again does not form any follicles. I n the crinkled individual, therefore, the first and third wave of follicle formation seem to be suppressed. During the second wave, hairs are formed resembling awls. The suppression of first and third wave of follicle formation together with the absence of guard hairs and zigzags in crinkled mice supports the hypothesis derived from the study of normal development which postulates that first wave and guard hairs, and third wave and zigzags are connected; the activity of the second wave together with the existence of awls in crinkled mice is strongly in favor of the hypothesis that awls are derived from the second wave of follicle formation.

PHYSIOLOQICAL QENETICS OF THE MOUSE

17

VI. DEVELOPMENTAL STUDIES OF MUTATIONS AFFECTINQ THE UROQENITAL

SYSTEM

The main known genetic factors causing abnormalities of the urogenital system which have been studied from the developmental point of view are: (1) my, myelencephalic blebs; (2) 8d, Danforth’s short tail ; (3) UT, abnormality of the urogenital system. Each one of these three mutations has other effects in addition to those on the urogenital system, and some of these are described in other parts of the review. The embryonic study of two of these mutations haa contributed to our knowledge of the developmental mechanics of the mammalian kidney.

1. Kidney Development in Myelencephalic Blebs The development of kidney abnormalities in the “myelencephalic blebs” strain of mice was studied by Brown (1931) who concluded from her studies that “complete metanephric development is a response to a mutual influence and fusion of two healthy anlagen, the ureter anlage from the lower end of the Wolffian duct and the blastema of the posteriormost part of the nephrogenic mesenchyme. Either one of these anlagen or both may be retarded i n which case no functional kidney is developed and varying degrees of anomaly are produced.”

2. Kidney Development in Danforth’s Bhort Tail The probably decisive role of the ureter in inducing the metanephrogenic blastema to form the secretory elements of the kidney, i.e., secretory tubules and Bowman’s capsule, was revealed by studies of the development of the kidney system in mice heterozygous and homozygous for the dominant mutation Sd (Glluecksohn-Schoenheimer, 1945). Animals heterozygous for Sd show abnormalities of the kidneys which vary from reduction in size to complete absence of one or both kidneys, making the more extreme heterozygotes inviable. The homozygotes have no kidneys and die soon after birth. The extent of the kidney and ureter abnormalities depends on the residual genotype and modifiers play a decisive role in the determination of the anomalies of the urogenital system of both Sd heterozygotes and homozygotes. F o r the study of the development of the abnormal urogenital system and the causal analysis of the developmental processes taking place in normal development, that genotypic background was of course most desirable which permitted a good expression of the urogenital effects of fld. It was found that embryos both heterozygous or homozygous for 8d had

18

SALOME GILUECKSOHN-WAELSCH

a metanephrogenic blastema which in its histological appearance did not differ a t all from those of their normal littermates. On the other hand, a great variability in the condition of the ureters was observed. They were of varying length and the differentiation and branching of the ureter tip into cranial and caudal pole tubules was disturbed or inhibited entirely; sometimes the branching was retarded and fewer than normal pole tubules developed. There seemed to be a correlation between the condition of the pole tubules and that of the differentiation of the metanephrogenic blastema. Secretory elements were always found in conjunction with differentiating pole tubules ; the number of kidney elements and the eventual size of the kidney appeared dependent on the degree of branching of the ureter tip and the number of tubules formed. On the other hand, if the ureter tip did not make contact with the metanephric anlage or failed to branch, absence of the kidney resulted.

3. Inductive Relatiomhip between Ureter and Metanephros Since metanephric differentiation was never observed in the absence of differentiation of the ureter tip, the conclusion seems justified that the metanephros is dependent on the ureter for some inductive stimulus for its normal differentiation. A similar relationship between these two structures had been shown before to exist in other vertebrates, e.g., the chick, where the results of transplantation experiments had led to such a conclusion. Thus, one effect of the Sd mutation appears to be the inhibition of growth and differentiation of the ureter tip, resulting in the failure of a n inductive stimulus to reach the anlage of the metanephros. The existence of this inductive relationship and its significance in mammalian kidney development has been made very probable by the study of the embryonic effects of Sd. This, however, is only one of the effects of fld; its other effect, that on the axial skeleton, is discussed in another part of this review. 4. U r a g e d d flyndrorne The third mutation known to affect the urogenital system ur (Dunn and Oluecksohn-Schoenheimer, 1947) has not been investigated embryologically. Its effects, however, are quite different from those of Sd, cystic, and hydronephrotic kidneys being most prevalent among ur homozygotes ; it thus serves to emphasize the dependence of the development of a n organ on a great many genes all of which are involved in the control of different processes which contribute to the eventual goal of normally developed structure and function.

19

PHPSIOLOQICAL QENETICS OF THE MOUSE

VII.

DEVELOPMENTAL STUDIES OF MUTATIONS AFFECTINQ CENTRAL, NERVOUS SYSTEM AND SENSORY ORQANS

THE

The development of the nervous system offers a great many opportunities for the occurrence of deviations from normal. The complexity of gene-controlled morphological and physiological processes which have to proceed normally in order to give rise to a normally formed and normally functioning nervous system is so great that one is surprised a t the relatively small number of hereditary abnormalities of the central and peripheral nervous system actually observed in the mouse. The great power of regulatory mechanisms operating during vertebrate development is probably part of the explanation for the relative scarcity of nervous abnormalities; on the other hand some of the deviations from normal development are perhaps so drastic that they lead to early embryonic death of the zygote. Only special 'methods would detect such hereditary lethal factors. There exist (uite a number of hereditary functional nervous abnormalities in the house mouse for which a n organic basis has not been established. I n some cases no morphological or histological anomalies have been found in spite of careful search; the mechanism responsible for those abnormalities might be of a physiological or biochemical nature not amenable to investigation for lack of methods. It is of course difficult, if not impossible, to study the embryonic development of such nervous disorders or to use such studies for a causal analysis of normal development of the nervous system in the absence of any morphological manifestations of the anomalies in the adult. But, fortunately, at least some hereditary abnormalities of the nervous system have obvious morphological manifestations and several of these have been examined embryologically and will be discussed now. 1. Hydrocephalus

One phenomenon brought out repeatedly by the embryological study of hereditary abnormalities is the diversity of mechanisms which may lead to the same manifestation in the newborn or adult mouse. As pointed out in other parts of this review, this phenomenon emphasizes the dependence of normal form and function of a n organ or part of it on a great number of different processes for their development. Another example illustrating this situation is the study of two mutations both of which cause hydrocephalus in the house mouse. One of the two types of hydrocephalus has been studied embryologically by Bonnevie and by Brodal (1943, 1944, 1945, 1946). The condition is due to a recessive mutation (hy) and manifests itself a t or

20

SALOYE QLUECKSOHN-WAELSClH

after birth as a hydrocephalus of varying intensity. The mutation was first discovered and studied by Clark (1934)’ who reported an obstruction of the aqueduct of Sylvius as one of the anomalies of hydrocephalus. When Bonnevie studied the embryology of this type of hydrocephalus, she found a number of different abnormalities in various stages of emhryogeny. According to Bonnevie ‘(. . the manifestation of the hy-gene proves to be highly varying, a variation which is primarily attached to the fluid circulation of early embryonic stages.” Abnormalities of the embryo are traced from preimplantation stages, ((irregularities during the formation of the unilaminar trophoblast, ” to early implantation stages, “wounds)) in the trophoblast ((through which maternal material (tissue fluid, cells, blood) from the uterine endometrium will enter the embryonic yolk sac)’; the abnormal content of the yolk sac is supposed to lead to abnormal fluid circulation in the embryo, and an abnormally heavy fluid tension is indicated. The excess of fluid is directed into the hrain ventricles as soon as the activity of the choroid plexuses starts a t about 12 days after fertilization. It now follows the normal circulation of the cerebrospinal fluid, and hydrocephalic dilation of the ventricles is a consequence of the excess of fluid which fails to be reabsorbed. Finally a series of brain abnormalities results from the dilation of the ventricles. One wonders whether this causal connection of “irregularities ’)in the trophoblast and excessive fluid formation is really justified.

.

2. Congenital Hydrocephalus While Bonnevie thus traces the eventual hydrocephalus back to original abnormalities of the trophoblast in very early embryonic stages, Gtriineberg (1943b) finds a quite different “pedigree of causes” underlying another manifestation of hydrocephalus. His congenital hydrocephalus” (ch) is recessive and lethal a t birth. “The most obvious anomaly is a steeply bulging forehead consisting of bilateral protuberances which correspond to the cerebral hemispheres. These bulges are . not protected by flat skull bones; the bulges are somewhat flabby sacs, etc.” I n addition, the eyes are open a t birth, the nose is shortened, the sinus hairs are abnormal, and ossification of the sternum is retarded permanently. Gtriineberg was able to trace all these different anomalies back to a n abnormality of the early cartilage. “This condition seems to be of a transitory kind and does not seriously interfere with the development of the embryo, except a t the base of the skull” where the topographical relations are destroyed permanently so that a brain anomaly results which leads t9 the death of the ch-homozygote. The following figure from Briineberg (1943b) gives a picture of the ((

..

21

PHYSIOLOGICAL GENETICS OF THE MOUSE

“pedigree of causes” involved in the different abnormalities of ch-homozygotes which can all be traced back to an original anomaly of the cartilage consisting in a poorly developed matrix, areas of vacuolization and liquefaction, and retardation of cartilage growth. We thus see that the ch-mutation seems to have a general effect on cartilage development and we may conclude that its normal allele is concerned with the control of normal processes involved in cartilage formation ; another mutation, se (cf. section IX-3-b), was shown to afTect cartilage formation, but judging from its different effects, its normal allele seems to control different steps of the cartilage formation process than does the normal allele of ch.

CARTILAGE ANOMALY

Basicranial cartilage

I

Rletacarpals Metatarsals

Meckel’s cartilage

pituitary and Ganglion Class-

bra1 hemorrhages

Hydrocephalus

flat slrnll bones

of sinus hairs

eri

F’IG. I. Pedigree o f causes of congenital hydraeephalus. (1943b).

L

Death From Qriineberg

3. Pseudencephaly

Still another hereditary abnormality of the nervous system has been studied and traced back to abnormalities of early embryonic processes by Bonnevie (1936a). This is the so-called pseudencephaly, a deformity of the head which carries the dorsally open neural tube like a wig turned inside out. Pseudencephaly is probably due to a recessive let.hal mutation (ps). Embryos with this abnormality rarely survive iintil term; in addition to a certain degree of variability, they have a n i i m her of common characteristics, mainly the wiglike appearance of the brain which bulges out of the skull and whose two halves a.re turned

22

SALOME QLUECKSOHN-WAELSCH

inside out. Furthermore, the neural tube is crooked and shows a number of abnormal curvatures in the neck region. Two critical stages are supposed to occur during the development of pseudencephalic embryos ; the first stage occurs very early, embryos die according to Bonnevie “as gastrulae with unseparated germ layers, ” Those pseudencephalic homozygotes which survive this stage die at the end of the embryonic period after the manifestation of pseudencephaly has been completed. The complete eversion of part of the brain with the defective formation of the cranium and other consequences were all ascribed to a “purely mechanical brain catastrophy,” which in turn is supposed to be connected with the existence of abnormal wrinkles and curvatures in the neck region of the medullary tube. The origin of these curvatures is considered to lie in the fact that the embryonic neural tube is too big to find room in its normal sized mesodermal surrounding. Bonnevie considers an early shift of the growth balance of the otherwise normal anlagen the original manifestation of the ps-mutation, and a “delayed separation of the two primary germ layers” the cause of this shift. This latter failure may be severe enough to cause the death of the embryos in the first “critical phase.’’ The shift in growth balance results in a n increased growth rate of the neural folds which do not find room in the normally growing surrounding and are forced to curve and wrinkle. The connection between the early embryonic manifestation of p s and the later extensive malformations-if corroborated-would make this a particularly interesting case of developmental analysis of a mutation.

X-Ray-IitducedTranslocations and Pseudencephaly Pseudencephaly has also been demonstrated in embryos of mouse 4.

strains carrying X-ray-induced translocations (Snell, Bodeman, and Hollander, 1934; Snell and Picken, 1935). The abnormality was interpreted to be the consequence of chromosomal unbalance of the zygotes and not a result of the action of some specific factor or factors, since “nearly all translocations in mice give rise to rather similar types of abnormal embryos. ” Differences in modifying factors and in the intrauterine development are considered to be responsible for the numerous variations found among the abnormal embryos. The neural folds of the abnormal embryos fail to close anteriorly, and the ectoderm which normally forms the roof of the brain has turned outward, thus exposing the floor of the brain. The abnormal parts of the brain include telencephelon, diencephalon, mesencephalon, and the plexuses. The remarkable resemblance of pseudencephaly as determined either by a single recemive gene, or as the result of chromosomal unbalance, or as a consequence of disturbance of embryonic processes by the action of

PHYSIOLOGICAL GENETICS OF THE MOUSE

23

X-rays (cf. section XI) indicates the great sensitivity of the early developmental period of the neural folds as well as the dependence of their normal development on a great many different factors.

5 . Sh,aker Short a. Central Nervous Systeni. A very striking ahnormality of the nervous system with obvious morphological manifestations which has been examined embryologically is the so-called shaker-short mutation first described by Dunn (1934b). Shaker short is a recessive and the homozygotes show a shortening of the tail, choreic movements a n d deafness. The embryology of these animals has been studied by Bonnevie (1936b, 1940), who found brain hernias with far-reaching destruction of mesencephalon and metencephalon in homozygotes a t or shortly before birth. Bonnevie assumes that some explosive forces must have led to this brain destruction and that “the cooperation of various forces and organ systems is necessary to cause the explosive disturbance of the brain.” I n the search for these “forces and organ systems” Bonnevie reports an inhibition of the development of the brain roof and absence of the foramen of Magendie at around 9-11 days after fertilization. This stage is followed by one in which only rudimentary Plexus chorioidei are found, resulting in a deficiency of cerebrospinal fluid, which, in connection with the absence of the foramen of Magendie, leads to disturbances in the circulation of this fluid. Abnormalities of the meninges and the cranium are a consequence of the decreased distance between brain burface and the surrounding condensations of connective tissue. A sudden change of heart activity, determined from sections of embryonic hearts, is reported to set in at this stage and its slowing down is ascribed to a n abnormally high pressure on vagus and sympathetic nerve centers. Finally, brain hernias develop in the dorsal part of the mesencephalon and metencephalon. This entire complicated “pedigree of causes” was further traced back by Bonnevie to an “abnormal thickening of the epithelial parts of the early embryonic brain roof.” A t 8 days after fertilization Bonnevie reports to have observed a n abnormal thickening of the dorsal closure of the medullary tube in the form of a stringlike mass of cells in intimate contact with the medullary tissue underneath it. Moreover, even the observation of a n ectodermal hypertrophy in implantation stages prior to mesoderm formation has been reported. “The causal connection of this hypertrophy of embryos in stages of implantation with the previously described manifestation of all anomalies characteristic for the shaker-short mice may be considered certain. ” The embryonic abnormalities are striking, and their origin in a n ectodermal hypertrophy in premesodermal stages is so interesting

24

SALOME GLUECKSOHN-WAELSCH

from the point of view of developmental mechanics that one would like to see this study extended. One would, for example, like to see stages of archenteron and mesoderm formation studied and note the detailed effect of the ectodermal hypertrophy on other early embryonic formative processes. The reviewer, therefore, agrees with Criineberg (1947) in his criticism of Bmnevie’s analysis : “The resulting pedigree of causes has several weak points which require further study.” b. Ear. A lateral compression of the fourth ventricle of the brain and ensuing abnormal pressure conditions are considered to be decisive for the abnormal development of the ear vesicles of the shaker-short mouse which remain oval, laterally compressed, and practically undifferentiated. However, in analogy with Hertwig’s (1944) analysis of the “kreisler” mutation (cf. section VII-6), one might perhaps rather assume a disturbance in the developmental interrelationship between ear vesicles on the one hand and brain and surrounding mesenchyme on the other hand to be responsible for the failure of the shaker-short ears to differentiate normally. It is interesting to note that Bonnevie in her developmental analyses of abnormalities, consistently favors the disturbance of mechanical conditions as explanations for successive anomalies, rather than developmental interdependencies of different tissues and structures. Pseudencephaly, for example, according to Bonnevie arises as the result of a “purely mechanical brain catastrophy ” (cf. section VII-3), hydrocephalus is due to excessive fluid circulation (cf. section VII-1), and shakershort, to a deficiency of cerebrospinal fluid connected ultitnately with a n “explosive disturbance of the brain” (cf. section VII-5-a). Furthermore, Polydactyly in “Little and Bagg’s abnormal mouse tribe” (to be discussed later, cf. section IX-2-c-ii) is supposed to be due to localized disturbances of development by blebs originating from an abnormally high quantity of fluid expelled from the fourth ventricle through the foramen anterius and migrating eyer the surface of the embryo,

6 . Eflcct of Kreisler on the Central Nervous System and Ear I n contrast to these purely mechanical explanations of developmental abnormalities, Hertwig in her studies (1944) takes into consideration the epigenetic nature of vertebrate development, and developmental interrelationships. Whenever descriptive methods alone are used in the developmental analysis of a mutation, the causal relationship between a n early embryonic abnormality and the disturbance of a n inductive process can only be indicated ; transplantation or explantation experiments are needed for final proof of such relationships. Frequently, when working

PHYSIOLOQICAL QENETICS OF THE MOUSE

25

with an organism such as a mammal whose developmental mechanics are still largely unknown, the analysis of hereditary embryonic abnormalities may give the first strong indication of the very existence of an inductive relationship between two embryonic structures. A case which has been analyzed along these lines of thought is that of another behavior mutation in the mouse, the “ kreisler” mutation (Hertwig, 1944) where the chain of processes connecting the first visible embryonic abnormality with the eventual neurological symptoms of the young mouse has been worked out and the causal connection between a t least some links in the chain made most probable. The mutation kreisler (kr) is a n X-ray-induced recessive. “Kreislers” may be recognized 10-14 days after birth by their peculiar behavior-they tend to crawl in a circle. Older animals show behavior abnormalities such as dancing and head shaking when disturbed and are deaf ; their mortality is high. Pathological-anatomical examination revealed extensive brain defects. Cysts were demonstrated which arose from the rudimentary ductus cochlearis and extended into the subarachnoid space disturbing brain development. Deafness was shown to be the result of the absence of cochlea and the organ of Corti. The examination of “kreisler” embryos showed that a t 9 days after fertilization the invaginating ear vesicles of “kreislers’ ’ were shifted laterally so that their walls did not touch the neural tube, as is the case in the normal, but were separated from it by a layer of mesenchyme. At a slightly later stage, when the normal ear vesicle was pear-shaped and showed the anlage of the ductus endolymphaticus, no differentiation of the ear vesicle was visible in the abnormal embryos. Still older “kreisler” embryos were easily recognized by the absence of ductus and saccus endolymphaticus and by abnormalities in the semicircular canals. The separation of sacculus and utriculus remained incomplete and the ductus cochlearis did not form a spiral cochlea but remained a wide duct only coiled slightly. The defects of the membranous labyrinth resulted in underdevelopment of the periotic cartilage and consequent absence of part of the cerebellum. The absence of ductus endolymphaticus and thus of its efferent function is considered responsible for an increase in endolymphatic pressure. This in t urn gives rise to an evagination of the epithelium of the ductus cochlearis which forms cysts extending into the subarachnoid space. These cause serious defects in the rhombencephalon, the pons, and even the cerebral hemispheres. Only animals with small or unilateral cysts are able to survive. In search for the primary causes of the malformations of the membranous labyrinth Hertwig compares her descriptive results with data

26

SALOME QLUECKSOHN-WAELSCH

obtained in the analysis of the developmental mechanics of the amphibian ear. There a progressive determination of different parts of the differentiating ear vesicle and a strong dependence on normal surroundings for normal organogenesis have been demonstrated ; in analogy with it, the ear anlage of the mouse embryo is considered to be not fully determined a t the time of invagination and disturbed i n its further differentiation by the abnormal conditions of its surroundings. The greater distance of the neural tube keeps it from exerting its normal inductive influence on the ear anlage. Thus, the ear vesicle remains small and is not able to form endolymphatic duct and normal semicircular canals. The analysis of the developmental behavior of the “kreisler ” mutation has thus made very probable the existence of a developmental interrelationship in the mouse between neural tube, surrounding mesenchyme and ear vesicles j it has furthermore shown that ductus and saccus endolymphaticus are responsible for the regulation of the lymphatic pressure in the normal ear, and the efferent function of the ductus endolymphaticus. The motor disturbances could not be traced back to any definite defect. Of course, the question of the nature of the effect of the “kreisler” mutation in producing the origilral lateral shift of the ear vesicles remains open, and the developmental analysis, while showing a “pedigree of causes” and revealing a number of most interesting embryological phenomena, has not been able to find out anything about the primary action of the gene involved.

7. Eyeless Among a number of mutations which have been reported to affect the eye there is one with particularly good penetrance and expressivity, and this eyeless mutation, ey-I, has been made the subject of a careful embryological investigation (Chase and Chase, 1941). I n the anophthalmic strain studied 90% of all the animals homozygous for eyelessness lack the eyes, and the remaining 10% have abnormal eyes. The investigation of the embryonic development of the eyes of such homozygotes showed that the first evagination of the optic vesicle a t 9 days after fertilization was normal. At about 10 days a n inhibition of the eye vesicle set in which prevented it from growing sufficiently in order to come in contact with the ectoderm and induce a lens. Either no invagination a t all occurred to form an eye cup, or a small and irregular cup formed. The subsequent failure of the choroid fissure to close was considered to be either a direct result of the growth inhibition of optic vesicle and cup or an indirect result, due to the failure

PHPSIOLOQICAL GENETICS OF THE MOUSE

27

of lens formation; organogenesis of the eye did not proceed any further. At 13 days after fertilization, no eye was found in 90% of the mice of the anophthalmic strain, and no optic nerve was formed. Consequently, there was a n underdevelopment of the visual centers of the brain, and a number of abnormalities of the cranial nerves could be traced back to the absence of the eye. The absence of the eye, in turn, was not a result of degeneration but of “failure of definitive organization beyond the eye vesicle stage. ” The developmental study of this eyeless mutation seems to show that the mouse behaves developmentally like that group of species of Amphibia where the lens is completely dependent on the eye cup for its formation and where the prospective lens tissue lacks any potency for self-differentiation. Studies of the developmental mechanics of eye formation have demonstrated the principle of double assurance in the formation of the eye in some species of Amphibia, i.e., the eye cup is able to induce the lens, but the prospective lens tissue is also able to selfdifferentiate to a certain degree in the absence of a n inductive stimulus from the eye cup. I n other species, the lens is completely dependent upon the inductive stimulus from the eye cup for its development, and there exist gradations between these two extreme conditions in still other species of Amphibia. It would be very interesting if one could analyze the developmental potencies of the eye anlage of the mouse along the same lines a s done in Amphibia and thus confirm the conclusions obtained from the descriptive embryological study of “eyeless. ” While thus the abnormalities of eye formation in “eyeless” could be traced back to a primary inhibition of the optic vesicle, the effect of the eyeless mutation in producing this inhibition remains still unknown.

8. Micruphthalmus Another mutation affecting developmental processes in the formation of the eye, is “microphthalmus” ( m i ) which arose in an irradiated (X-ray) strain of mice. The homozygous animals have small eyes with colobomas. A very careful study of Miiller (1950) analyses the developmental steps leading to the eye abnormalities in this mutation and uses these findings for a n interpretation of the normal developmental mechanics of the eye. The first observable deviation from normal consists in shifts of the normal relationships of mitotic rates of nervous and pigment layers of the retina at about 10 days after fertilization. The result is a relatively increased growth rate of the pigment layer, resulting in an abnormally shaped eye cup and failure of the choroid fissure to close. Disturbances in nervous connections of eye and optic nerve follow, also eversions of the retina, abnormalities of the optic nerve, ab-

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normal pressure conditions in the eye chamber, lens deformities, and disturbances or complete absence of function. The successive appearance of a variety of abnormalities in the eye is utilized for a n analysis of the interdependence of different eye structures in normal development. A shift in growth rates of the different layers of the retina is thus considered to be the first observable effect of “mi” on eye development ; again, the mechanism by which the mutation produces this shift remains unknown.

VIII.

ANALYSISOF ENDOCRINE DISTURBANCES-PITUITARY DWARFISM

While the involvement of an endocrine gland was only indicated in the case of the abnormalities produced by the grey-lethal mutation (cf. section IX-2-d), there exists another mutation where the connection between endocrine disturbance and subsequent anomalies due to the effect of a mutational change could be clearly demonstrated. This is the mutation “pituitary dwarfism, ’’ a recessive analyzed genetically by Snell (1929). Dwarfs can be recognized at the age of 2 weeks when their growth and development cease; for the next 2 weeks they do not gain weight and occasionally even lose weight. After this period some animals start to grow again, others do not. Skeletal growth and differentiation are retarded. Male and female dwarfs are sterile, and their mortality is increased. These abnormalities of the dwarf mice seemed to simulate those of hypophysectomized rats, and furthermore, a histological study of the anterior lobe of the pituitary of dwarf mice showed complete absence of eosinophiles, a reduction in number of chromophobe cells and the appearance of a connective-tissue network. Smith and MacDowell (1930), therefore, tested the hypothesis that a defect of the anterior lobe of the pituitary was responsible for all abnormalities observed in the dwarf mouse. Fresh r a t anterior pituitary lobe was implanted daily into mouse dwarfs and gave positive results : “along with the striking resumption of growth, came a rapid loss of the dwarf traits.” Soon “the dwarfs could not be distinguished from normal mice,” aside from lower body weight. Males began to breed and females showed signs of a n estrous cycle. However, “the anterior lobe of the pituitary remains in the same defective condition as in the untreated dwarfs. ” All abnormalities of the mouse homozygous for pituitary dwarfism could thus be traced back to an effect of the mutated gene on the anterior lobe of the pituitary. Further studies were directed toward an analysis of the particular hor-

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mone effected by the mutational change, b u t for details of these studies the reader is referred to the individual papers (cf. Gruneberg, 1943a). OF MUTATIONS AFFECTINQ THE Ix. ANALYSIS

SKELETON

1. Skzcll a. Harelip. Throughout this review we have stressed repeatedly how different pathways may lead to the same end result, and that thus similarities of manifestation (lo not necessarily indicate similarities of disturbed development. A good illustration of this point is provided by some studies of harelip, a hereditary condition in mice. This condition has been studied by different authors in strains which were all derived from the same parent strain. The genetics of harelip is rather complicated, and it is not even certain whether harelip is due to one or several genes. The study of the embryonic development of harelip showed that apparently disturbances of different kinds may be responsible for the manifestation of the harelip condition. Reed (1933), in his studies, found that the clefts of the jaw were due to a failure of fusion of the lateral and of the medial nasal processes. The reason for this anomaly is sought for in a retarded growth rate of the maxillary processes whose lateral pressure is needed for the completion of fusion. Retardation of growth is also responsible for the failure of the palatine processes to unite. Tn a n examination of his strain of harelip mice, supposedly with the same genetic basis for harelip, Steiniger (1941) found, in addition to embryos which showed the nonfusion origin of harelip, others where the harelip was clearly the result of a break-through of embryonic cysts into the mouth and nasal cavity, and thus of secondary origin. In view of the fact that different disturbances in the same region seem to be responsible for the existence of the harelip condition in these mice, one might perhaps think of a n increased susceptibility of the mouth and nose region i n prospectively harelip mice. Due to a lowered threshold of resistance to different kinds of embryonic traumata, these embryos might react with retarded growth or with cyst formation in the mouth region to conditions which normally would pass unnoticed by the developing embryo. The case of harelip might be similar to the case of taillessness in the rat (Dunn e t al., 1942) where it was found that no single hereditary factor was responsible for the appearance of taillessness b u t where “the genetic constitution determines the threshold of a reaction which is subject to alteration by minor and more or less random accidents early i n development. ” b. Tooth Abnormalities. (i) Grey lethal. The tooth abnormalities of mice homozygous for the grey-lethal mutation (cf. Gruneberg, 1943a,

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for details) can be traced back to failure of secondary bone absorption which will be discussed in greater detail below (cf. section IX-2-d). (ii) Screw tail. Abnormalities of the teeth, the mandibles, and the skull are symptoms also of another, the so-called screw-tail, mutation. Mice homozygous for this recessive mutation were first recognized by their tail abnormality. The extensive malformations of the cranium, the mandibles, and the teeth have been described in detail (MacDowell et al., 1942).

2 . Extremities a. Lrc.ccc’--C‘o~~~ywzitul Abvencr of l’ibiil,. I h e to the relative paucity of mutations aflecting the extremities of the mouse not many studies have been made which contribute to the developmental analysis of limb structures. Nevertheless, one of the very first mutations studied from the embryological point of view is one affecting the hind limbs (Hovelacque and Noel, 1923). The mutation is a recessive called lux6 and expresses itself in congenital absence of the tibia. The examination of embryos from homozygous abnormal stock showed mesenchymal blastema in the hind limb bud resembling those which, in normals, would differentiate into two anlagen, that of the tibia, and that of the fibula, and become chondrified and ossified eventually. However, the hind limb buds of the embryos with congenital absence of tibia in spite of the presence of normal-appearing blastema show differentiation of only one cartilagenous structure which later ossifies, narriely, the fibula; the tibial anlagt? develops mostly fibrous tissue and only some cartilage in its upper portion which in comparison with the fibula is retarded in its development. The mutant gene thus affects specifically one part of a general anlage, i.e., the tibial portion of the limb bud mesenchyme, preventing it from developing along the normal path of chondrification and ossification while the neighboring anlage of the fibula in the same blastema shows no disturbance whatsoever of the identical processes. The ramifications of this situation for problems of cellular differentiation, such as the respective roles of inherent cell properties and the cell’s environment, were well recognized by the authors at the time of the embryological description of the condition. b. Luxate. Recently a new niutation has been reported (luxate, Carter, 1949, 1951) which in its edects strikingly resembles “1ux6, ” the so-called congenital absence of tibia discussed before. I n homozygous luxates the tibia is reduced, and in addition there are widespread defects of digits and other parts of the leg, prevalently on the preaxial side of the hind limbs. Heterozygotes are either normal, or show abnormalities of the digits also on the preaxial side of the hind feet. Since the original

PHYSIOLOCIICAL GENETICS OF THE MOUSE

31

“1uxQ” stock is extinct, genetic tests of the identity of the two stocks could not be made. Unpublished results of an embryological study (Carter, 1950, personal communication) indicate, however, the probable identity of lnx6 and luxate. The studies of luxate were able to tracr h w k the abnormality to a ninc.11 earlier stage of embryogeny. Abnormal shape of the hind limb buds was observed in luxate homozygotes of ll$$ days embryonic age ; at this stage the hind limb buds were narrower and more symmetrical than those of normal embryos. Differences in rate of development between preaxial and postaxial halves of the hind limb are considered to be the explanation for the differential effect of the mutation on tibia and fibula. c. Polydactyly. Another aspect of limb development that could be studied ,with the help of methods of developmental genetics is that of the regulation of toe pattern. The appearance of supernumerary toes is a rather frequent phenomenon in mice and the developmental study of those cases where this abnormal condition is determined genetically might reveal some of the mechanisms involved in the control of the normal developmental pattern of toe formation. ( i ) . Development. This point of view was kept in mind especially by Chang (1939) who studied the development of supernumerary toes in Fortuyn’s strain of polydactylous mice, where chiefly the first toe of the right hind foot was more or less completely doubled. Shape differences of the right hind footplate of embryos were found to be the first symptoms of polydactyly. The medial side of the abnormal footplate bulged out more sharply than that on the normal side. In addition to the excessive local growth of the medial side of the posterior limb buds there does not seem to be any abnormality of histogenesis. I n contrast to assumptions that some change in the epidermis might be responsible for subsequent abnormalities of the affected toe, Chang considers the excessive growth of both epidermis and mesenchyme to be caused by interaction of mesodermal and ectodermal factors. Such an interaction is operative in amphibian limb development, as shown by transplantation experiments. Chang’s analysis of polydactylism represents one of the first cases to illustrate the attempt to combine descriptive studies of the development of a mutation in mice with results from transplantation studies in Amphibia, with the aim of a causal analysis of mammalian development. (ii) Polyductyly and embryonic blebs. Different causes have been made responsible for the appearance of supernumerary toes by other workers. Bonnevie (1934), in an investigation of the embryogeny of polydactyly in “Little and Bagg ’s abnormal mouse tribe, ” discovered the presence of blebs migrating over the surface of the embryos where

32

SALOME QLUECKSOHN-WAELSCH

the bleb fluid was supposed to have originated from the medullary tube and to have been expelled through the foramen anterius in abnormally high quantity. The author considers these blebs to be responsible for localized disturbance of development, causing for example, as border blebs, the characteristic polydactyly. However, there are several phenomena wliicli could not be explained by such a n assumption. One of thein is the fact that the appearance of border blebs and original abnormalities of the footplate o m i r s a t least simultaneously, if not in reversed order, i.e., abnormalitiw of shape are recognizable before first appearance of blebs. (iii) Polydactyly and central n e r u m s system. The possibly causal relationship of polydactylism and hypertrophy of the ventral motor horn cell column a t the lnnibrosacral level was investigated by Tsang (1939). IIe fonntl that a correlation cxist,rtl between polydactyly and a hypertrophy of the retrodorsolateral cell column in the homolateral ventral horn of the spinal cord. Froin his results Tsang concluded that polydactyly was the immediate consequcnce of the neural hypertrophy. Ilowever, a reverse order of cause and effect seems more likely in view of experimental evidence in frogs and birds where hyperplasia of neural growth is produced as a consequence of implantation of supernumerary peripheral structures. Furthermore, Chang 's studies reveal, as pointed out by Griineberg (1943a), that the first abnormalities in shape of the footplate appear prior to the time of neural differentiation. (iv) Polydactyly and maternal age. That polydactylism may be influenced in its expression by a number of mutually interdependent developmental factors aside from genetic ones is apparent from studies which showed the effect of maternal age in the manifestation of the polydactyly gene, which decreased with increasing maternal age (Holt, 19-18). d. G'rey-T,ethal and Foilirve of Rccondary Bone Absorption. The extremities of mice show deviations from normal a s the result of the effects of still another mutation which produces a number of additional abnormalities. The mutation is the grey-lethal, discovered and very thoroughly analyzed by Griineberg (1936, 1937, 1938). One of the main abnormalities of mice homozygous for the grey-lethal mutation is the absence of secondary bone absorption which leads to changes in the shape of all the bones, including those of the extremities. No common marrow cavity exists in the long bones, and shape anomalies of the long bones develop. I n the absence of secondary bone absorption the skeleton of the grey-lethal mouse provides very good material for a comparison with the normal slieleton for the purpose of studying normal bone absorption.

PHYSIOLOGICAL GENETICS OF THE MOUSE

33

The cells which are responsible for bone absorption, i.e., osteoclasts, are found in normal number in the grey-lethal. The embryonic effects of the grey-lethal mutation have not been studied. However, reciprocal transplantation experiments between grey-lethal and normal mice (Barnicot, 1941) at least excluded some possibilities of explaining the effect of grey-lethal, without however revealing its actual nature. These reciprocal bone transplants did not behave autonomously, but grey-lethal bones, transplanted into normal hosts, approached normalcy while normal bones, transplanted to grey-lethal hosts, would sometimes assume grey-lethal characteristics although the results were not constant. On the basis of these experiments the effects in grey-lethal mice were ascribed to either the absence of an essential substance o r the presence of an inhibitory substance in the circulation and not to any factor inherent in the bone itself. Barnicot (1945) then attempted to answer the question of whether the grey-lethal affected some endocrine gland and caused a hormonal deficiency. One of the glands to be suspected in the grey-lethal mouse was the parathyroid, since it was known that parathyroid extracts caused bone absorption. The grey-lethal mice differed from normal mice in their reaction to the injection of parathyroid extracts; there was no evidence, however, that the treatment cured the faulty bone absorption, although the skeleton reacted to large doses of the hormone with considerable bone absorption. While the interpretation of these results remains speculative, there seems no question that the parathyroid, or its hormone, is somehow affected by the grey-lethal mutation and that the function of the gland or that of its hormone is disturbed in the grey-lethal mouse. I n addition to its effect on the skeleton, the grey-lethal mutation interferes with pigment formation : animals homozygous for grey-lethal have no yellow pigment in their fur. So far no physiological connection between these different effects of grey-lethal has been discovered.

3. Sternum a. Screw Tail. One of the very striking abnormalities of the screwtailed mouse (cf. section IX-l-b-ii) is the absence of segmentation of the sternum. I n contrast to the normal mouse, where the sternum consists of six separate segments, the sternum of the screw-tailed mutant is unsegmented, and while six centers of ossification exist in the normal, the screw sternum, except for the xiphoid process, arises from a single center of ossification and is considerably shorter than normal. I n addition, there are abnormalities of the ribs. It was hoped that a study of the development of the abnormal sternum of the screw-tailed mouse might reveal some of the mechanisms operating in the development of the nor-

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ma1 sternum, particularly in respect to the mutual relationship between ribs and sternum. Such a study was undertaken (Bryson, 1945) and indicated the decisive role which the ribs played in the determination of the ossification pattern of the sternum and thus its segmentation. According to the author, (‘the induence of costal cartilages upon the sternum is to delay ossification at the region of contact, both in the mutant and normal structure.” Thus ossification is normally inhibited in those regions where the “opposing zones of inhibition” have come in contact medially as a result of growth of the ribs. I n the screw-tail sternum, however, retarded growth of the ribs has kept the “paired zones of inhibition” apart so that ossification can proceed throughout the entire sternum without inhibition. The screw-tail gene is considered to have a general retarding effect on the axial skeleton of which the retardation of rib growth is only one symptom. I n normal development, ((th e determination of ossification pattern occurs after the union of ribs and sternum and is due to a n inductive effect.’’ b. Short Ear. Another aspect of the development of the sternum was studied by Green and Green (1942) who used the short-ear, se, mutation in the mouse as a material. Animals homozygous for this mutation show abnormalities of the ear, decreased body size, and a malformation of the xiphisternum. Depending on the residual genotype, the xiphisternum may be either reduced, practically absent, or bifurcated. The embryogeny of this abnormality was investigated and compared with normal development ; it was found that the sternal bands appeared shortened at about 13 days after fertilization so that the last rib frequently did not attach to the sternum. When the sternal bands moved together to form the sternum, it appeared that the xiphisternum was either reduced or completely absent in conformity with the shortening of the sternal bands. I n those cases where bifurcation of the xiphisternum occurred, the abnormality did not become apparent till slightly later in embryonic life-at about 15 days the posterior ends of the sternal bands of the animal with a prospectively bifurcated xiphisternum did not unite. Preceding this stage the sternal bands were not as clearly outlined a t their posterior ends as were the normal bands and the mesenchyme appeared less condensed. On the basis of their observations the authors conclude that (‘the condensation of mesenchyme leading to cartilage formation is certainly defective in the xiphoid region of the sternal bands, ” and that thus “defective precartilage is the actual forerunner of the adult departure from normality.’’ The short-ear gene thus seems to have a locally determined effect on certain regions of precartilage.

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35

A more recent paper of M. C. Green (1950) demonstrates a more widespread effect of the mutation in the bony skeleton as well as other cartilages. How f a r all these effects can be attributed to a single cause remains for future elucidation.

Spinal Colwmn The development of the spinal column is dependent on a great number of genetic factors for its normal morphogenesis. Many mutations have been found which frequently have effects on the vertebral column as well as on other organ systems. Actually, the spine and particularly its distal portion, the tail, serve as a good indicator for the detection of mutations in the mouse. As we shall see, a number of mutations with most interesting additional effects, have been recognized first by their effects on the tail. Not all the tail mutations known have been examined developmentally, and in this case the mechanisms responsible for their effects on spine and tail development are quite unknown. We shall not deal here with these mutations, but restrict our discussion to those in which a thorough investigation of the embryology of the tail abnormalities has been made. We shall see that most of the mutations to be discussed have made their appearance in other parts of this article because they all have effects in addition to those on the tail. Their pleiotropic nature is a characteristic phenomenon of these tail mutations. a. Flexed. One of the first tail mutations whose developmental effects were studied thoroughly is “flexed, ” a recessive mutation affecting both blood and skeletal system (Kamenoff, 1935). Flexed homozygotes have stiff segments i n different parts of the tail, and flexures which are due to unilateral fusions between two successive vertebrae. Typical asymmetrical fusions of vertebrae are also found in the thoracic, lumbar, and sacral region of the spine. The flexures are primarily abnormalities of the intervertebral disc where cartilaginous or bony tissue substitutes for the normal felted fibers. Vertebrae in the mouse, as in mammals in general, are formed in the following way. The posterior half of one sclerotome unites with the anterior half of the subsequent sclerotome to form the anlage of the centrum. The first evidence of the intervertebral disc is rapid proliferation of cells from the two half-sclerotomes adjacent to the intervertebral fissure, and subsequently the notochord shows bulgelike thickenings at the site of the discs. Shortly before birth the notochord is present only in the intervertebral masses as the anlage of the nucleus pulposus. The cartilagelike cells of the intervertebral disc lose their matrix, elongate, and gradually become more fibrous in their appearance ; this change proceeds from the periphery of the disc toward the center. At 4.

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birth, the normal intervertebral disc consists of a central nucleus pulposus surrounded by a mass of fiberlike cells which will give rise to the felted fibers of the adult disc. The first abnormality recognizable in flexed embryos appears a t 14 days after fertilization when the cartilaginous cells of the intervertebral discs fail to be modified into elongated fibrous cells. These cells produce a bridge of cartilage from one vertebra to the next. If this bridge is unilateral, a true flexure will result; if it is bilateral, a straight stiff joint will be found. I n all cases, the original abnormality consists in the failure of the early cartilage of intervertebral discs to differentiate into felted fibers. The effect of “flexed” on the blood has been discussed before (cf. section IV-1) ; flexed homozygotes stiffer from a. transitory anemia, the so-called siderocytic anemia. I n addition, the flexed mutation produces a growth retardation in early embryonic stages as evidenced by shorter lengths of vertebrae. Two hypotheses are advanced to connect the different effects of flexed: (1) that growth retardation is responsible for both anemia and abnormalities of intervertebral discs; (2) that the anemia produces, by reducing the oxygen supply, the more general retardation evident in the axial system. b. Shaker Short. The extensive effects of shaker short on the brain with ensuing abnormalities of hearing and sense of balance have been discussed in another part of this review (cf. section VII-5) ; a “pedigree of causes” traced all these anomalies back to a hypertrophy of the ectodermal germ layer in very early embryonic stages (Bonnevie, 1936a, 1940). The effect of shaker short by which the mutation was originally recognized has not been discussed yet. Homozygous shaker-short mice have short tails. I n her report on the embryogeny of shaker-short mice, Bonnevie refers only briefly to he; studies of the tail development of these mice : “. . u p to about 12 days, the tail of the embryo was quite normal. Now, however, simultaneously with the abortive formations of the plexus (chorioideus), its tip, on account of the blood tension, swells like a blister, resulting in an arrest of development and in a more or less long filamentous thinning out of this tip-known also from other mutations.” It seems thus that circulatory abnormalities may be made responsible for the abnormalities of the tail of the shaker-short mouse, although the details of the abnormal processes do not appear from Bonnevie’s description or illustrations. c. Screw Tail. Another mutation first recognized by its effects on the tail is the screw-tail mutation; its effects on the development of ribs and sternum were discussed before, and it was pointed out that the

.

PHYSIOLOGICAL GENETICS OF THE MOUSE

37

screw-tail gene was supposed to have a general retarding effect on the axial skeleton (section IX-3-a). The tail of the screw-tailed homozygote is coiled a t birth and straightens out later on (MacDowell et al., 1942). Some flexures remain in the caudal, lumbar, and thoracic region of the spine. The centra of some of the vertebrae are shortened, and two or three vertebrae are mising from the tip of the tail. The difference in tail length between normal and screw-tailed mice becomes establirhed a t about 12 days after fertilization and is not due to a decrease in number of soiriites but to a reduction in size of the individual vertebrae. The tail abnormafities are thus considered to be due to a general mesodermal growth deficiency of the axial skeleton. d. [Jnclulatecl. Recently the effects of a previously reported recessive mutation (undulated, Wright, 1947) producing abnormalities in spine and tail have been analyzed in some detail (Griineberg, 1950). Although the critical embryological evidence is still forthcoming, some interesting conclusions have been drawn on the basis of observations on animals between the ages of late fetuses and fully adult individuals. The tail of undulated individuals is shortened and has a number of kinks which are not due to fusions of the vertebrae but to irregularities in the distal ends of cauclal vertebrae. Kyphosis of the lower tlioracic and upper lumbar region may be ohserved. A niimber of anatomical abnormalities of the individual vertehrae of the spine have h e n described. The reduction in size of indivicliial vertebrae is more niarlretl distally than proximally. The undulated syndrome is explained on the following basis : “The mesenchymatous axial hlteletoii of the undulated mouse would differ from that of a normal mouse hy a reduction in size which would bring certain esposed parts below the threshold size necessary for chondrification while other parts, though reduced in size, remain above the threshold and hence chonclrify and ossify normally.’’ There is a n interesting similarity between the effects of screw tail and those of undulated on the asial skeleton, as concluded from the developmental analysis of both of these mutations. T t i both cases, a general mesodermal growth deficiency of the axial skeleton i s considered to h~ produced by the mutations. It wo~ild he interesting to esamine the genetic relationship between scww tail and undulated. This has not been done so far. e. Daiaforth’s Short Tail. The last mutation to be discussed in this group is a dominant mutation with striking effects on the tail and axial skeleton whose urogenital effects were analyzed above (cf. section VI-2) ; it is the Danforth’s short-tail mutation, Sd. Sd heterozygotes have a short tail o r no tail a t all, and sacral as well as remaining caudal verte-

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brae may be shaped abnormally and fused with each other (QluecksohnSchoenheimer, 1943). The abnormalities of the axial skeleton of the homozygotes are more striking; the tail is always absent, and caudal and sacral vertebrae are missing completely while the few lumbars present are abnormal. The development of the tail abnormalities was studied in heterozygotes and homozygotes (Oluecksohn-Schoenheimer, 1945) ; the tail of hetcrozygotes was found to be normal up to the age of 10 days after fertilization. At 10%-11 days small hematomata are found in the tip of the tail, the somites appear small and irregular, and a progressive degeneration of tail somites in distal-proximal direction may be noted. The hematomata increase in size and sometimes fill one-half to one-third of the entire tail, while all structures in the affected part of the tail such as neural tube, notochord, tail gut, and blood vessels, break down. Progressive cell necrosis may be observed in all affected parts of the tail even prior to the appearance of hematomata. The abnormalities of the tail of the homozygotes are similar in principle but more extensive. Pycnotic granules may be observed in earlier stages in all structures of the tail ; they increase in number as the degenerative process in the tail continues. Eventually all tail structures which had originally formed normally, such as neural tube, notochord, somites, tail gut break down, and, at the end of 12 days, only a filament is left with large hematomata inside. The effect of Sd on the axial skeleton in one and two doses seems to consist in the initiation of a cellular degeneration process which starts out in the mesenchyme of the tail and subsequently attacks notochord, neural tube, and somites. The appearance of hematomata in the abnormal tail is ascribed to a breakdown of the walls of the tail blood vessels rather than to a general circulatory defect in all distal parts of the embryo, because the extremities are always free of such hematomata. Since cell pycnosis is observed even in the absence of hemorrhages, it cannot be a consequence of breakdown of circulation. Cell degeneration is considered to be the first recognizable effect of the Xd-mutation in one or two doses.

EFFECTS OF MUTATIONS IN CHROMOSOME X. THE DEVELOPMENTAL IX OF THE MOUSE From the point of view of the experimental embryologist, the most interesting mutations in the mouse are a group located on chromosome IX. These mutations were first recognized by their effects on the tail of newborn and adult mice; but their main interest lies in their effects

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39

on early processes of embryonic growth and differentiation which, in most of the homozygotes of this series, are severe enough to lead to the death of the embryos in early stages. F or details about the genetic behavior of these mutations the reader is referred to earlier papers (cf. e.g., Dunn and Gluecksohn-Schoenheimer, 1950). Suffice it here to say that we are dealing with a group of three closely linked dominant mutations and a number of either allelic or pseudoallelic recessives. Most of these mutations are lethal when homozygous, and animals heterozygous for two of the lethals not showing recombination breed true as a result of a balanced lethal system. The study of the developmental effects of these genes has made it possible to trace the individual steps which lead to the manifestation of the eventual mutant conditions in the axial skeleton of the newborn or the adult. Furthermore, these mutations serve as tools for the study of the normal developmental mechanics of a mammalian organism since it is only through the comparison with the abnormal that we can analyze the processes and events of normal development. Finally, the developmental effects of these genes provide us with material to study the general relationship between genes and embryonic differentiation and thus at least certain aspects of gene action (cf. Gluecksohn-Schoenheimer, 1949b). I n heterozygous condition all the mutations in this group affect thc development of the axial skeleton, and we shall deal first with this aspect of their effects. 1. Brachyury-Heterozygozts

Effect of T

The first mutation whose enibryological effects were analyzed i n detail is a dominant, T, the so-called Brachyury mutation. Animals heterozygous for T have a short tail, and homozygotes die a t about 10 days after fertilization (Chesley, 1935). The tail of the heterozygous embryo is normal up to the age of 11 days after fertilization. At this time a sharp constriction appears in the tail, and the point of constriction determines the end of the tail of the newborn mouse. The part of the tail distal to the constriction becomes smaller and smaller and loses its segmentation. Eventually, only a short filament remains. Histologically, notochordal abnormalities are a characteristic feature of the heterozygote in stages even prior to the onset of external abnormalities. Irregularities of the neural tube are frequent but occur always in conjunction with irregularities of the notochord, indicating the primary character of the notochord anomalies.

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2. Taillessness, T / t n The abnormalities of the tail become more extreme when T is combined with one of the recessive mutations of the t-type also located in chromosome IX and behaving like alleles of T. Animals heterozygous for T and one of the t ’ s are usually completely tailless. The embryog eny of such prospectively tailless animals was studied, and it was found that the embryo formed a normal tail which elongated u p to the age of about 11 days after fertilization (Glnecksohn-Schoenheimer, 1938a). At that time a constriction appeared, always at the base of the tail, and subsequently the constricted tail became smaller, lost its segmentation, and eventually only a filament remained. Histologically, the prospectively tailless embryo could be recognized prior to the appearance of the constriction by the absence of a notochord. Neural tube, somites, and tail were formed normally, but were unable to persist and continue differentiation. Actually these striictures dedifferentiated and eventually only undifferentiated mesenchyme was found in their place. We thus see that in T/+ neural tube abnormalities seemed to follow those of the notochord and in T/tn the complete absence of the notochord from the tail preceded the subsequent dedifferentiation of the originally normal tail structures : neural tube, somites, tail gut. An original effect of T alone or in combination with t on the notochord or its precursor is thus one conclusion to be derived from the study of the developmental eflects of T and t, while on the other hand, the decisive role of the notochord in the normal development of the axial skeleton is strongly indicated. More information about the developmental effects of T and some of the t’s and also about the role of the notochord in the developmental mechanics of the mouse embryo was obtained from the study of embryos homozygous for T and for t. 3. Homozygous Effect of 1’

Chesley (1935) reports the absence of any gross abnormalities in the embryo homozygous for T before the age of 8y2 days. A t about 9 days, “small paired or unpaired blebs or small vesicles on either side of the midline” appear. A slight irregularity of the neural tube is observed and the somites are less clear and less regular than in normals. Shortly before death, 1 0 days after fertilization, the homozygous T-embryo shows the following characteristic external features : the posterior region of the body, including posterior limb buds, is missing completely. Normal segmentation is absent but remnants of somites may be recognized. The fore-limb buds are directed dorsad instead of ven-

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trad. The closure of the neural folds does not occur along one straight mid-dorsal line but deviates laterally and is crooked and twisted. Histologically, the somites are normal in early stages and reduced in number and abnormal in shape in later stages. They are, however, not fused medially. The neural tube shows its twisted shape on sections and is found to send out secondary branches. The notochord forming material appears a t first histologically normal, but the differentiation of the chorda dorsalis is abnormal. By the day during which death occurs, no traces of notochord are present. The notochord is thus the most severely affected structure in the T-homozygote. One of the most interesting conclusions to be drawn from the developmental studies of both T-homozygotes and T-heterozygotes is the one concerning the developmental relationship of notochord and neural tube : “Abnormalities of the neural tube have not been encountered in the absence of those of the notochord while in the lumbosacral region notochordal abnormalities without those of the neural tube are common.” “From the facts reported, supported by experimental evidence from other sources, it is concluded that there is strong indication that abnormality of the notochord is one of the more fundamental of the disorders involved and that the condition of the neural tube is either wholly or in part due to the abnormality of the notochord” (Chesley, 1935). The decisive role of the notochord for normal axial development of the mouse embryo, indicated by the studies of prospectively short-tail or tailless embryos, becomes thus even inore certain as a result of the observations of the development of 7’/T embryos. Ephrussi (1935) explanted tissues from embryos homozygous for T and found that the individual tissues were able to survive far beyond the stage a t which they would have died if left in the embryo. T is thus not a cell lethal and the death of T I T embryos is supposed to be caused by a “disturbance of the normal correlations” and to be due “exclusively to internal factors.” In a n attempt to develop a method by which mouse embryos could be grown under extra-uterine conditions and details of mutant development observed and perhaps attacked experimentally, TIT embryos were explanted into the extra-embryonic coelom of the chick in an early embryonic stage (Gluecksohn-Schoenheimer, 1944). It was shown that the abnormal development of T I T embryos was determined in early egg cylinder stages before becoming apparent morphologically. An incidental observation reported is a severe abnormality of the allantois which prevents the umbilical vessels from developing normally and is responsible for the failure of circulatory connection to become established between mother and embryo. The time of death a t 10 days thus is consid-

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SALOME QLUECKSOHN-WAELSCH

ered to be determined by the absence of nutrition of the embryo by way of the maternal circulation and the cause of death seems sufficiently explained without any additional assumptions. 4.

t-Type Mutations-Developmental

Effects of to, tl, t4

It has been estimated (Dunn and Gluecksohn-Schoenheimer, 1950) that a t least 14 changes of the t-type have occurred in chromosome I X

of the mouse, i.e., 14 different t-mutations have been detected. Five of these are known to be lethal when homozygous, three are viable, and the behavior of the others in bomozygous condition is not yet known. The first t-type mutation studied in its effects on development in homozygous condition is to (Qluecksohn-Schoenheimer, 1940). Pt0 embryos are reported to be normal u p to a stage soon after implantation in the uterus, the so-called egg cylinder stage. But while the normal embryo now enters a stage of rapid growth and morphogenesis, growth and development of the homozygous embryo cease. It remains in a stage where a n unorganized inner cell mass (=ectoderm) is surrounded by a layer of entoderm (this “inversion of germ layers” is a typical feature of many rodent embryos) until disintegration sets in and the embryo becomes necrotic and is resorbed. During a time when organization of the ectoderm and mesoderm formation characterizn +he development of normal 7 signs of these processes; embryos, the abnormal embryo fails to shou it survives for about 40 hours before it disintegrates. The failure of organization and mesoderm formation is a characteristic feature of totoembryos. Another mutation of the t-type (t’) has been reported to be lethal before implantation (Gluecksohn-Schoenheimer, 1938b). The details of the abnormalities of tltl are not known. Unpublished observations of abnormalities of t4-homozygotes ( Qluecksohn-Waelsch) indicate that the death of these embryos occurs at the age of 7-8 days after fertilization, i.e., a t a stage when embryonic growth and differentiation are most active. 5. Kink-Hmoizygous

Effect of Ki

Still another mutation located on chromosome IX seems to affect processes of early embryonic growth and differentiation. This is the dominant mutation Kink which in heterozygous condition is responsible for the rather typically kinky appearance of the tail. The embryonic events leading to the manifestation of Kink in heterozygous condition have not been analyzed as yet. But the homozygous embryos, which die at about 9 days after fertilization, have been studied in detail (Gluecksohn-Schoenheimer, 1949a). Their abnormalities are typical and strik-

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ing although varying considerably from one another individually. The feature common to all these abnormal embryos is the presence of duplications of different types and degrees. The duplications reported range from complete or partial duplication of the entire embryonic axis through duplications of organs or parts of them to the presence of only some excess growth in the form of a lump of tissue. On the basis of these observations comparisons are made with results obtained in experiments on amphibian embryos, where constrictions of eggs resulted in different types and degrees of duplications, comparable to those observed in K i / K i embryos. From the existence of these duplications it is concluded that ‘ ‘ the mouse embryo possesses a considerable amount of regulative property in early developmental stages, ’’ and ( ( t ha t organization phenomena and inductive interrelationships between different parts of the embryo known to function in amphibians and birds apparently exist in the mouse as well.’’ I n connection with the duplications in Kink homozygotes a genetically determined duplication of the posterior part of the body of the mouse described by Danforth (1930) is of interest. The details of the genetics of “posterior duplication” are not known; the morphology of the posterior doubling varies considerably from typical duplicitas posterior with ectopic viscera and two complete sets of pelvic organs and hind limbs to those with only a slight degree of doubling. A “pronounced thickening of the ventral tissues a t the posterior end of the embryo” a t 11 days after fertilization is reported as the first observed symptom of the abnormality. ((Th e hypertrophy seems to be initiated in the mesoderm,’’ and “there is evidence of some direct and determining influence of one region on another. ’’

6. Fused The development of Fused, the third dominant tail mutation in chromosome I X in heterozygous or homozygous condition has not been studied in detail but Reed (1937) reports that ( ( a n embryological study of Fused showed the first observable abnormalities to be poor alignment of the notochord and distinct curves and angles of the neural crests,” without, however, giving any evidence for his statement. I n preliminary studies of the embryology of Fused homozygotes we have observed a considerable number of embryos with brain abnormalities resembling pseudencephaly (cf. section VII-3). These embryos are apparently unable to survive until term and die in utero. The effect of Fused in homozygous condition on the development of the anterior part of the neural tube does not seem to be regular, but occurs only in a fraction of the embryos and depends apparently on the residual genotype.

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We may deal here with another example of the type of developmental reaction discussed above (cf. section IX-1-a). The head abnormalities in Pu/Pw embryos might be due to an increased susceptibility of the prospective head region, or in other words to a lowered threshold of resistance to embryonic traumata which normally would not leave any effect on the developing embryo. The lowered threshold of resistance might in t ur n be due to the presence of the Fused mutation in two doses.

7. A b n m d i t i e s of to/tl Embryos While the main effect of the “tail” mutations of the ninth chromosome of the house mouse a t least when heterozygous seems to be on the posterior part of the body, Fu-homozygotes are not the only individuals in this group of mutations which exhibit abnormalities of the head region. Genetic data from intercrosses of two tailless lines (A and 29) differing in the type of t ( T / t o X T / t l ) had shown that the combination of to with tl produced normal-tailed offspring. However, there seemed to be a deficiency in the number of young of this constitution (to/tl) at birth. Uteri of females supposed to carry to/tl embryos were therefore examined in a search f o r possibly abnormal P/tl embryos unable to survive until term (Dunn and Gluecksohn-Schoenheimer, 1943). Such embryos were discovered and found to have severe abnormalities of the head, such as microcephaly, microphthalmia and anencephaly. Control experiments excluded the possibility that these abnormals were not of the to/tl genotype. Just as in the case of Fused-homozygotes only a fraction of to/tl embryos developed head abnormalities and apparently again the residual genotype played a role in determining the variability of manifestation of the to/tl zygote. But there is no doubt that the anterior part of the axial system may also be affected by the action of these “tail” mutations.

8. Miscellaneous Abnormalities in IncEividuals Carrying Mutations of Chromoame I X A general fundamental disturbance of early embryonic development by mutations on chromosome IX is also indicated by another set of observations ( Gluecksohn-Schoenheimer and Dunn, 1945). Monsters with extreme malformations of the posterior region of the trunk and the extremities (sirenoid malformations), or with striking abnormalities of the head (aprosopi), o r with severe intestinal abnormalities (complete absence of large parts of the intestine) were found among newborn mice which carried different combinations of mutations of chromosome IX. Similar abnormalities had not been observed in numerous other strains at the same laboratory. While they cannot be ascribed to any par-

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45

ticular genotype, they seem to owe their origin to the presence of different mutations of chromosome IX. Although these abnormalities have not been traced back embryologically, they are assumed to be caused by severe disturbances of early processes of embryonic growth and differentiation. It seems that the presence of mutations in chromosome IX makes the developmental system of the mouse sufficiently labile so that i t will react with severe disturbances to some embryonic trauma which normally would leave the embryo unharmed. 9. The Role of Chromosome I X in Embryonic Growth and Differentiation Chromosome IX of the mouse thus seems to carry a group of genetic factors all of which are concerned with the control of processes of early embryonic growth and differentiation. No mutation with other effects has been located in this chromosome. This does not mean, o f , course, that chromosome IX plays a n exclusive role in the control of these processes. There is no doubt about the existence of many genetic factors which are involved in the control of the same processes of early growth and differentiation and are located in other chromosomes. The heterozygous effect of several of the tail mutations of chromosome IX has already been shown to be duplicated by tail mutations in other chromosomes of the mouse, as described above. The important point, however, is the grouping of mutations with similar effects in one chromosome regardless of the fact that other such mutations most certainly do exist in other chromosomes as well. The individual mutations in chromosome IX affect either different steps of early embryonic processes, or the same processes in different ways. Evidence for such a n assumption comes first from the fact that individuals heterozygous for two mutations, each of which causes death of the embryo when present in two doses, are viable and sometimes even normal. Furthermore, the embryonic study showed that the developmental changes produced by these mutations were clearly different in the case of the different mutations. Changes in most of these genetic factors lead to abnormalities of derivatives of the notochord-mesoderm material and to phenomena traceable to a n absence of functioning or to abnormal functioning of this same material. Any hypothesis trying to explain the close linkage of genes with closely related effects on embryonic processes has to take into account their peculiar genetic behavior which has been reported in a series of papers (for references cf. Dunn and Qluecksohn-Schoenheimer, 1950). Some speculations in this connection were advanced recently (Gtluecksohn-Schoenheimer, 1949b). The close linkage of mutations with

46

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similar embryonic effect was discussed first in the light of Goldschmidt ’s ( 1949) hypothesis about the relation of heterochromatic heredity to processes of early growth and differentiation, and then in connection with Pontecorvo’s ideas (1950) about. the relation between location of genes and the processes they control. The grouping of these genes seems of the greatest significance for problems of gene action and the relation of genes and cell differentiation. XI.

CHANQESINDUCED BY X-RAYS DEVELOPMENTAL

The developpental changes in the mouse embryo discussed so fa r were produced by changes in genes, i.e., mutations. But attempts have been made also to affect developmental processes directly, e.g., with the help of X-rays. Kaven (1938a,b) irradiated pregnant mice with X-rays in different fitages of pregnancy and studied the effect between 7 and 19 days after fertilization. The resulting disturbances of development consisted of brain hernias, hydrocephalus, tail abnormalities, lens turbidity, and fiterility. Since these abnormalities had also been observed as the result of the effect of mutations, it was interesting to compare the sensitive periods during which X-ray irradiation produced a certain type of developmental abnormality with its onset as a result of a mutational change. Kaven found a highly lethal effect as the result of X-ray treatment a t 7 clays, corresponding to the time of manifestation of lethality in embryos carrying mutations which produced changes in early processes of growth and differentiation. Brain abnormalities were induced at 8 days after fertilization, the age at which the first symptoms of genetically determined brain anomalies in mice were observed. X-ray treatment between 9 and 13 days resulted in tail abnormalities, and again it is noteworthy that this is the time at which the first recognizable deviations of tail development could be observed in animals carrying tail mutations. . Recently L. B. Russell (1950) has attacked the same problem on a broader basis, extending the stages of treatment to the very beginning of the gestation period. An analysis of the changes induced by x-ray treatment is expected to reveal “intrinsic patterns of sensitivity in the organism and the discove;y of sensitive periods in particular developmental processes. ’’ These sensitive periods were established throughout the embryonic period by determining prenatal mortality and abnormalities a t birth. A comparison is made which shows a certain degree of parallelism bet ween the abnormalities produced by irradiation and those described as the effects of mutations. However, “the entire set of changes char-

PHYSIOLOGICAL GENETICS OF THE MOUSE

47

acteristic of a particular stage-dose group is usually more inclusive than the set of features characteristic of any parallel mutant.’’ A number of abnormalities described as the result of gene action were not observed in “newborns as a result of irradiation of embryos.” This may, however, be due to the fact that embryos with such abnormalities were unable to survive to term. A combination of results obtained with this method with those arrived at from studies of developmental genetics might prove very fruitful “in bringing analysis of the development of the abnormal character even closer (chronologically) to the original gene action.”

XII. CONCLUDING REMARKS The mutations discussed i n this review have been shown to effect practically every organ system and thus have contributed to the analysis of a great number of different mechanisms involved in the development of form and function of a mammalian organism. A number of general points of interest have emerged from this discussion. When abnormalities observed in the newborn or adult organism are traced back into embryonic stages it frequently appears that the first deviation from normal recognizable in the embryo is not necessarily one in the primordium of the organ which becomes abnormal later on. Such a ease is illustrated, for example, by the analysis of congenital hydrocephalus by Griineberg (1943b) (cf. section VII-2) which could be traced back to an original abnormality of the cartilage and where the effect on the brain was thus quite indirect. Developmental studies of mutations are therefore a prerequisite for the detection of the first visible effects of a mutation before the problem of primary gene action can even be approached. The vast number of genes contributing to the normal development of any organ is reflected in the large number of different mutations which were found to effect any single organ system. Although, as pointed out i n the introduction, only a relatively small and selected number of mutations was chosen for discussion in this review, and actually many more exist, it is evident even so that, e.g., the nervous system, the skeleton, or the skin, are dependent on the collaboration of a great many hereditary factors for normal form arid function. One very interesting phenomenon emerging from developmental studies of mutational effects is the appearance of definite abnormalities as the result of a number of vastly different agencies. Pseudencephaly, for example, was shown to originate as the result of (1) a simple recessive mutation, (2) chromosomal translocations and ensuing chromosomal

48

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unbalance, (3) the presence of mutations which lower the threshold of embryonic resistance (cf. section X-6, F u l F u ) , (4 ) X-ray irradiation of embryos. The same syndrome of urogenital abnormalities and those of the posterior trunk region has been shown to result from the action of different mutations (cf. section X-8), and more examples could be cited to illustrate this. phenomenon. The existence of any particular syndrome of abnormalities does therefore not necessarily reflect an identity of hereditary changes or of developmental pathways disturbed. Different mutations and different developmental changes may lead to the same final picture, emphasizing again the interdependence of developmental processes of the vertebrate embryo. Of course all these phenomena cannot be separated from one of the most important problems of gene action, namely that of pleiotropism. Does the gene have one or more than one primary effect P Or are some of its effects subordinated to the primary effect, i.e., a consequence of it 1 The developmental analysis of mutational effects can sometimes help to eliminate the problem, by the discovery, for example, that a number of different adult effects can be traced back to one common embryonic change. However, as the result of such studies, the problem merely ceases to exist in particular cases while it is by no means solved as such. The problem of pleiotropism in a mammalian organism has been discussed repeatedly on the basis of the analysis of different mutations (cf. e.g., Briineberg, 1943b ; Gluecksohn-Schoenheimer, 1945 ; E. S. Russell, 1949b). Briineberg (1943b), as the result of developmental analyses, arrives a t the conclusion that genuine pleiotropism does not exist and that the primary action of the gene is always either cell specific or tissue specific. It has been pointed out before ( Bluecksohn-Schoenheimer, 1949b) that one cannot expect an elucidation of primary gene action from the study of morphological gene effects which in their complexity must be far removed from the original gene effect. The question of whether a gene has one o r more primary functions will have to be approached with different methods. Furthermore (Gluecksohn-Schoenheimer, 1945), it does not seem that we are justified to call the gene’s primary action cell or tissue specific just because the developmental analyses of the gene’s visible effects with the crude morphological methods a t our disposal seem to indicate such a specificity of effect. Russell in her discussion of different effects of W on blood and pigment cells (1949) points out that it is logically possible that “unity of gene action could exist without tissue or cell specificity” and that the same original gene product could be active in two types of cells. She considers the interesting “parallelism of the effects of the entire W

PHYSTOLOGICAL GENETICS OF THE MOUSE

49

series of genes upon the blood and pigment-producing tissues” a n indication of the very close original connection between the two different effects of W and its alleles (cf. section IV-2). It seems that the problem of pleiotropic gene effects will have to wait for further elucidation until a more direct approach to the problem of primary gene action is possible. While prbblems of primary gene action thus cannot be expected to be solved by developmental studies of gene effects of the type discussed here, one of the most significant contributions of such investigations lies in the discovery of normal interdependencies of developmental processes as revealed by the study of the abnormal. A number of such instances have been discussed. I shall only cite again the dependence of the metaneplrros on a n inductive stimulus from the ureter (cf. section VI-3), the dependence of ossification processes in the sternum on normal rib growth (cf. section IX-3-a), the developmental interrelationship between neural tube and ear vesicles (cf. section VIT-s), the significance of the notochord-mesodermal material in mammalian development (cf. section X-3 and 4 ) , the discovery of organizer type phenomena, and of the existence of regulation in early mammalian embryos (cf. section X-5). Most of the early embryonic changes reported concern disturbances of inductive phenomena, while degeneration processes leading to absence of structures are surprisingly rare. It seems that the study of the role of genes in early growth and differentiation should find the mouse embryo a promising object also for future research, due to the existence of organizer and inductive phenomena on the one hand, and of relatively frequent mutations, on the other hand, which interfere with these processes.

XIII. REFERENCES DeAberle, 8. B., 1927, Amer. J . Anat. 40, 219-249. Barnioot, N. A,, 1941, Amer. J . A n d . 68, 497-531. 1945, J . A m t . 79, 83-91. Bonnevie, K., 1934, J . exp. 2001. 67, 443-520. 1936a, Norske Vidsk.-Akad. Oslo Skr. K1. 1, No. 9. 193613, Genetica 18, 105-125. 1940, Handb. Erbbiol. Menschen 1, 73-180. 1943, Norske Viidsk.-Akad. Oslo Skr. K1. 1, No. 4. 1945, Norske Vidsk.-Akad. Oslo Skr. K1. 1, No. 10. Bonnevie, K., and Brodal, A., 1946, Norske 8idsk.-Akad. Oslo Skr. K1. 1, No. 4. Brodal, A., Bonnevie, K., and Harkmark. W., 1944, Norske Vidsk.-Akad. Oslo Skr. Kl. 1, No. 8. Brown, A. L., 1931, Amer. J . Anat. 47, 117-172. Bryson, V., 1945, Anat. Reo. 91, 119-141.

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Carter, T. C., 1949, Heredity 3, 378-379. 1951, J. Genet. 60, 277-299. Castle, W. E., and Little, C. C., 1910, Science 32, 868-870. Chang, T. K,, 1939, Peking nat. Hist. Bull. 14, 119-132 Chase, H. B., and Chase, E. B., 1941, J. Morph. 68, 279-301. Chesley, P., 1935, J. exp. 2002. 70, 429-459. Clark, F. H., 1934, Anat. Rec. 68, 225-233. CuBnot, L., 1905, Arch. 2001.exp. gSn. 4" series, 3, Nates et Rev. CBXIII-CXXXII. Danforth, C. H., 1930, Am. J. Anat. 46, 275-287. Dunn, L. C., 1934a, J. Genet. 29, 317-326. 1943b, Proc. nat. Acad. Sci., Wash. 20, 230-232. Dunn, L. C., and Einsele, W., 1938, J . Genet. 36, 145-152 Dunn, L. C., and Gluecksohn-Schaenheimer, S., 1943, Gmrtics, 28, 29-40. 1947, J. exp. 2001.104, 25-52. 1950, Proc. nat. Acad. Sci., Wash. 36, 233-237. Dunn, L. C., Oluecksohn-Schoenheimer, S., Curtis, M. R., and Dunning, W. F., 1942, J . Hered. 33, 65-67. Ephrussi, B., 1935, J. ezp. 2002.70, 197-204. Falconer, D. S., Fraser, A. S., and King, J. W. B., 1951, ,7. Genet. 60, 324-344. Foster, Morris, 1951, in press. Fraser, F. C., 1946, Conad. J. Res. D24, 10-25. 1949, Canad. J. Res. 27, 179-185. Gluecksohn-Schoenheimer, S., 1938a, Genetics 23, 573-584. 1938b, Proo. 800. exp. Biol.,N. Y. 39, 267-268. 1940, Genetics 25, 391-400. 1943, Genetws 28, 341-348. 1944, Proc. nat. Acad. Sct., Wash. SO, 134-140. 1945, Genetics, 30, 29-38. 1949a, J. exp. 2002.~110,47-76. 1949b, Growth IX, 163-176. Gluecksohn-Schoenheimer, S., and D u n , L. C., 1945, Anat. Rec. 92, 201-213. Goldschmidt, R. B., 1949, Proc. 8 t h int. Congr. Genet., Hereditas 244-255. Green, E. L., and Green, M. C., 1942, J. Morpb. 70, 1-19. Green, M. C., 1951, J. Morph. 88, 1-21. Griineberg, H., 1936, J. Hered. 27, 105-109. 1937, J. A n d . 71, 236-244. 1938, J. Genet. 36, 153-170. 1942a, J. Genet. 43, 45-68. 194213, J. Genet. 43, 285-293. 1942c, J. Genet. 44, 246-271. 1943a, The Genetics of the Mouse. Cambridge University Press. 1943b, J. Genet. 46, 22-28. 1947, Animal Genetics and Medicine. Paill R. Hoeber, New York. 1950, J. Genet. 60, 142-173. Hardy, M. H., 1949, J. Anat., 83, 364-384. Hertwig, P., 1944, 2. menschl. Vererbungs.-u. Konstitutionslehre 28, 327-354. Holt, S. B., 1948, Ann. of Eugenics 14, 144-157. Hovelacque, A., and Noel, R., 1923, Bull. Biol. France el Belg. 67, 133-142. Hunt, H. R., and Permar, D., 1928, Anat. Rec. 41, 117. Ibsen, R. L., and Steigleder, E., 1917, Amer. N u t . 61, 740-752.

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Kamenoff, R. J., 1935, J. Y o r p h . 68, 117-155. Kaven, A., 1938a, Z.menschl. Vererbungs.-u. Konstitutionslehre 22, 238-246. 1038b, Z. menschl. Vererbungs.-u. Konstitutionslehre 22, 247-257. Kirkham, W. B., 1919, J. exp. Zool. 28, 125-135. Little, C. C., 1919, Amer. Nat. 63, 185-187. Little, C. C., and Cloudman, A. M., 1937, Proc. nat. Acad. Sci., Wash. 23, 535-537. MacDowell, E. C., Potter, J. S., Laanes, T., and Ward, E. N., 1942, J . Hered. 33, 439-449. Miiller, G., 1950, 2. ntikrosk-anat. Forsch. 66, 520-558. Pontecorvo, G., 1950, Biochem. 800. Symposia N . 4, 40-50. Rawles, M. E., 1947, Physiol. Zo6l. XX, 248-266. Reed, S. C., 1933, Anat. Ilec. 66,101-110. 1937, Genetics 22, 1-13. 1938a, J . exp. Zool. 79, 331-336. 1938b, J. exp. Zool. 79, 337-346. 1938c, J . exp. Zool. 79, 347-354. Reed, S. C., and Henderson, J. M., 1940, J. exp. 2001.86, 409-418. Robertson, G. G., 1942, J . exp. 2002.89, 197-231. Russell, E. S., 1946, Genetics 31, 327-346. 1948, Genetics 33, 228-236. 1949a, Genetics 34, 133-166. 1949b, Genetics 34, 708-723. Russell, E. S., Fondal, E. L., and Smith, L. J., 1950, Rec. Genet. SOC. Amer. 122123. Russell, L. B., 1950, J. exp. Zool. 114, 545-602. Russell, L. B., and Russell, W. L., 1948, Genetics 33, 237-262. Serra, J. A., 1947, Nature, Lond. 169, 504-505. Smith, P. E., and MacDowell, E. C., 1930, Anat. Rec. 46, 249-257. Snell, Gc. D.,1929, Proc. laat. Acad. Sci. W s h . 15, 733-734. 1941, (Ed.) Biology of the Laboratory Mouse. The Blakiston Company, Philadelphia. Snell, G. D., Bodeman, E., and Hollander, W., 1934, J. esp. Zool. 67, 93-104. Snell, G. D.,and Picken, D. I., 1935, J. Genet. 31, 213-235. Sobotta, J., 1895, Arch. mikr. Anat. 46, 15-93. 1902, Arch. mikr. Anat. 61, 274-330. 1911, Arch. mibr. Anat. 78, 271-352. Steiniger, F., 1941, 2. menschl. Vererbungs.-u. Konstitutionslehre 26, 1-27. Tsang, P.-C., 1939, J. comp. Neuro. 70, 1-8. Werneke, F., 1916, Arch. EntwMech. 4 5 72-106. Wright, M. E., 1947, Heredity 1, 137-141.

Developmental Action of Lethal Factors in Drosophila ERNST HADORN * Zoologisrh-vtrgl. n n a t o n i i s c . 7 ~Institzit, ~ Uniiicrsity of Zitrich, S ’ d t z e r l n n d CONTENTS

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I. Introduction 11. Phase a n d Type of Action 1. Embryonic Lethals , 2. E/L-Boundary Lethals . 3. Larval Lethals . . . 4. L/Pr-Boundary Lethals , 5. Prepupal Lethrtls , 6. Pupal Lethals 7. P/I-Boundary Lethals , , 8. Imaginal Lethals . , 111. The Validity of the Concept of Phase-Specificity IV. The Development of “ P a t t e r n s of Damage” V. Information Gained from Transplantation Experiments VI. Physiological Properties of Lethals VII. Phenocopies of Patterns of Darnage . V I I I . Conditioned Lethals IX. Lethality Due to Combined Genic Action X. Effects of Lethals in Heterozygotes XI. Conclusions X I I . References

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Page 53 54 54 60 60 61 61 62 62 62 62 65 67 71 76 77 78 80 82 83

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I. INTRODUCTION Lethal factors are Mendelian units that cause the death of the individual a t some stage during its development, thereby preventing it from reaching the reproductive stage. The different types of lethals and the terms used for them have been discussed recently by Hadorn (1949b). It is well known that lethals are among the most common mutations, arising experimentally after treatment of germ cells with ionizing radiation or with chemicals as well as spontaneously in wild populations and laboratory stoclrs. They therefore play an essential role in all theoretical and practical problems connected with gene mutability. The action of lethal factors demonstrates how development can be * I should like t o express my gratitude t o my collaborators, Dr. H. Gloor, Dr. P. S. Chen, and M. Schnitter for their contributions and kind assistance with respect to this review. 53

54

ERNST HADORN

altered fundamentally as a consequence of change or loss of genic material. Thus each lethal mutant reveals some specific developmental action of the mutated chromosomal region. We visualize normal development as a teamwork of cell constituents, to which several thousand different gene loci contribute. Any information we can gain from study of the developmental action of a lethal factor will therefore be valuable and essential with regard to the ultimate goal of a science in which embryology and genetics ought to be united. The aim of this paper is to review the information as f a r as the development of Drosophila is concerned. It is a n amazing fact that relatively few data are available, although hundreds of thousands of lethal mutants have been registered by Drosophila workers and many hundreds of them have been maintained in laboratory stocks. We find for instance in the exhaustive report The Mutants of Drosophila melanogaster (Bridges and Brehme, 1944) a total of 557 lethal mutants listed; for only about 60 of these do we have information concerning developmental effects. I n the case of the great majority of lethal factors we do not even know whether they lead to embryonic, larval, or pupal death. As to lethals with known developmental effects, only a few have been thoroughly investigated ; and cases for which both morphological and physiological studies have been made are exceedingly rare. Thus the lethal mutants represent a vast material for exploitation in future research.

11. PHASE AND TYPEOF ACTION Without discussing all details we shall restrict ourselves in this section to stating for the different known lethals the developmental stage at which they lead to death and, if such information is available, the kind of abnormality that can be considered the immediate cause of lethality. It is possible to classify the great majority of the lethal mutants according to the stage at which their development comes to a halt and death occurs.

1. Embryonic Lethals ( E ) The embryonic period of development begins with the fertilization of the egg and ends when, within the egg membrane, a fully differentiated larva is ready to hatch. I n Drosophila melartogaster this development is completed in 22 to 25 hours at 25" C." There is in this group a "Most of the information on normal development used in this paper is based on the very detailed treatises by Bodenstein, Sonnenblick, and Poulson in Biology of Drosophila, edited by Demerec (1950).

ACTION OF LETHAL FACTORS IN DROSOPHILA

55

regrettable discrepancy between the tremendous number of lethals found by Drosophila workers and the small number of cases studied in detail. Poulson (1940, 1941, 1945) investigated a series of embryonic-lethal genotypes, in each of which development is interrupted in a specific way and a t a definite stage. These, together with the others for which ac(Inrate data are available, have been entered in Table 1. W e can draw the following conclusions from the data for these few lethal genotypes. a. Every important step in embryonic development may be interrupted or become abnormal through loss or mutational change of genic material. b. There seems to be a positive correlation between the extent of a deficiency and the severity of the developmental damage. Genotypes deficient for the whole X chromosome perform less developmental work than the half-X deficiencies, and the latter die earlier and show a more fundamental “ anormogenesis” than the smaller Notch o r white deficiencies. c. The work of I’oulson (1945) on the Notch and white lethals has shown, however, that identical patterns of damage ensue in deficiencies of many bands, deficiencies of one band only, and point-mutations that cause no visible change in the salivary chromosomes. This becomes intelligible if one assunies that the detrimental effect of a deficiency is not quantitative in nature, but a consequence of the loss of one particular gene locus. The decisive loci would be those that first enter into action in normal ontogeny. Loss of these loci, or mere inertness produced by point-mutation, would therefore lead to the same effect. The more extended the deficiency, the greater the probability that loci of fundamental importance for the earliest steps in development would be lost. This interpretation is based on the principle that as development prooeeds a gradually and steadily increasing numher o f genes enters into action (Hadorn, 1948h). I n the case of very large deficiencies (loss of whole chromosomes or whole chromosomal arms), however, we have to consider still another mechanism that can cause lethality. I n such genotypes the balance of the genic system is severely upset. Lethal effects resulting from disturbances in the quantitative relations of chromosomal materials are shown by the tetra-IV and the triplo-X genotypes. In the first of these, where two supernumerary fourth chromosomes are added t o a normal caryotype, development is interrupted in the egg or larval stage (Li, 1927). I n the second, two sets of autosomes have to counterbalance three X chromosomes. These genotypes infrequently hatch, as weak and sterile imagines (supcrfemales) ; but most of them die in a late larval or pupal stage (Brehme, 1937). Since duplications of single chromo-

56

ERNST HADORN

TABLE I Drosophila melanognster

I

I

Embryonic Lethals about Which Reliable Facts Are Known Df = deficiency ; I' = point miitants nitli iiormnl s:ilivnry-cliroinosome strnctnre ; - translocation

I . . _

Symbol, name, c h r o m -

Time c i f t w fertilization and stage of lethal action

liind of ub?inriiiality i7i hcmizygolcs (he) or ho niosygotes ( h o )

Hcfprciio s

1 hour; cleavage mitosis

he ; abnormal distribution of cleavage nuclei ; Illantoderrn not formed

Poulson, 1940

Df-XR Df (right, arm of X )

2 hours ; h1aatoder.m f or ma t ion

lie ; incomplete blosto-

I'ouIwii,

I I t,l'lll

1940

Df-XL Df (left a r m of X )

3 hours; pregastrular cell movements

he ; germ layers not separated

Poulson, 1940

Nn

6 hours; primary organogenesis

he ; incomplete separation of hypodermis front h y p e r t r o p h i c nervous system ; mesodcrmal organs incompletely differentiated ; midgnt inr.omplete

Poulson, 1945

x

ho11r.s; segmentation of hrad and trunk

ho; defects i n middlemost segmeiitn

Glool',

12 hours; Iieginning of final organogenesis

he; mesodermal tissues abnormal a n d degenera t i n g ; midgut remains sac-like

Poulson, 1945

20 hours;

he; no a i r in tracheae; m u ~ c u l a r 111 o v e in P 11 t iinptlirpd

Poulmn, 1941)

soma1 t y p e

NuUO-X Df (whole

X)

Notch-Df No* Notch-Df N**'-' Notch-Df

N"'-" Notch-Df N'"-'' Notch-Df N'"4 Notch-Df Notch-P N"'4' Notch-P Not~,l-l'~

Kr Kruppel-P

wla~-ll

wl10-1k

~

~

'

white-Df white-Df white-Df ~ - 4 ~

Df(1)so' scute-Df

t vaclieae fill

with air, larval diff wentiatiou :Ilmost ronipleted

1950

57

ACTION O F LETHAL FACTORS IN DROSOPHILA

TABLE 1-Continued ~~

Df(1)R60-2

20 hours; lie; n o air in tracheae; tracheae fill innscular m o v e m e n t with air, larval impaired tliff erentiatic~n almost completed

Kaliss, 1939

Px Plexate-Df

21 liours; larval h o ; unable t o hatcli differentiation completed

Li, 1927 Bridges,

M(2)Z Minute-Df

21 hours ; larval 110; unable to hatch ; differentintion thin chorion conipleted

Li, 1927

S Stw-P

21 liours ; l a r ~ n llio ; nn:il)lc to Iiatcli ; tlifferentiation black spot benclntli completed niieropyle

SivertzevDohzhansky, 1927

1937

somes may lead to lethal effects comparable to those produced by big deficiencies, one could interpret the effect of deficiency-mutants also on a quantitative basis. It is thus impossible to decide whether the developmental effects in the first three cases of Table 1 are caused by loss of one gene o r by a gross disturbance of genic balance. The first interpretation results from an extrapolation of data obtained on the Notch and white series of lethals ; the second is suggested in view of the duplication lethals. Apart from the mutants of Table 1, one finds in the literature many reports of the occiirrence of lethality in embryonic stages. They are based mostly on the mere observation that the lethal mutants concerned fail to hatch from the egg membrane. However, a glance a t Table 1 shows how differently embryonic lethals interrupt and change development. Therefore superficial statements furnish no useful material for tackling the problems that physiological genetics has to solve. We may only state that apparently most of the dominant lethals that have been induced by X-raying mature sperms are effective in embryonic stages (Demerec and Fano, 1941; Lea, 1946). This treatment produces gross chromosomal aberrations, which actually do not impair the capacity for fertilization but lead to severe cytological abnormalities in the developing zygote. Sonnenblick (1940) studied such abnormalities and found cleavage mitoses with isolated and clumped chromosomes as well as dicentric and acentric fragments. As a consequence of the ensuing un-

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ERNST HADORN

TABLE 2 Postembryonic Letlials of Drosophila melanogaster

E = embryonic; L = larval; Pr = prepupal; P = pupal; I = imaginal Symbol and term

1. E/L C y Curly

-

Locus

Stage of death K i n d of abnormality References chromosomal in hemisygotes ( h e ) type or homozygotes (ho) In(2L)Cy

E/L boundary ho ; inability of larvae to develop

SivertzevDobzhansky, 1927 Hadorn, unpublished

ClB

I n ( 1 ) C1

E / L boundary he; inability of larvae to develop

Brehme, 1937

M ( 3 ) FlaMinute

3-79,7

E/L boundary ho; inability of

Brehme, 1939

l(l)w

1-66 -C allele of

larvae to develop

E/L boundary he; inability of larvae to develop

Schubel, 1934

2-101.2

First instar

ho; inability to grow and pass first instar

Brehme, 1939

3-79,7

First instar

110;

inability to grow and pass first instar

Brehme, 1939

l(2)ax

2-106.9

First instar

110; inability to grow and pass first instar

Bridges, 1937

Bd Beaded

3-93.8

First instar

110;

inability t o grow and pass first instar

SivertzevDobzhansky, 1927

First iiistar

he; defective ring gland

Cullen, 1948

Larvae of 2 mm. length

ho; abnormal development of tracheal and other chitinized parts

Bridges, 1937

2-72 f

Third instar

ho; disturbed growth r at e; unable to form puparium

Hadorn and Schmid, 1947 Sclimitl, 1949

1-0.3

Third instar

he ; obliteration of gut lumen before 70 hours

Russell, 1940

bb

2.

L

M(2)l' Minute

1( 2 ) N S

l(2)me

meander

59

ACTION OF LETHAL FACTORS IN DROSOPHILA

TABLE 8-Continued

3. L/Pr l ( 2 ) g l lethal giant larvae

Df (2)P

4. Pr

5. P

6. P/I

7. I

PaleDeficiency l(3)blo-1 bloated larvae

1-0 2

Mostly i n prepupal stages

formation of pseudopupae

Df in 2

As larvae or pseudopupae

ho; shrunken body within puparium

110;

-

Hadorn, 1937b Hadorn, 1948a Gloor, 1943

Li,1927

ho; larvae trans- T. Y.Chen, parent; irregular 1929 growth of imaginal discs 3-20,7+ Early or late ho; defects in im- Hadorn, 1941 t(3)tr pupal life aginal differentia- Hadorn, 1949 translucida Gloor, 1949a tion 3Late pupal ho; tumorlike l(3)hd Morgan, life growth in head head Bridges, Sturtevant, defects 1925 3-Zn(3R)C Late pupal ho; unable to Schultz life hatch from pupal of. Bridges and Brehme, case 1944 2-55-C Late pupal Head hidden in Hadorn u. crc Gloor, 1943 life thorax cryptoGloor, 1945 cephal Schultz and Pm' 2Late pupal Very light eye Dobzhansky, Plum color life 1934 Ives, 1942 3-35.5 Late pupal Inability t o push eYg life open pupal case; eye gone eyes and head reduced Starck, 1922 1Early imaginal Weak, short-lived i(i)sa life flies 1-29.0 End of pupal Melanotic degen- Gowen, 1934 me stage eration i n legfocal melanosis junction 2Early imaginal Leg tumors Spencer, 1(2)W life 1937 leg-tumors 3-47,7 Early imaginal Oral lobes changed Bridges and Pb life to tarsus-like or Dobzhansky, probosciarista-like struc- 1933 pedia tures; inability to feed Transloca- Early imaginal Winga not ex- Dobzhansky, T(b;C)C tion life panded; flies die 1930 early

3-

Prepupae or early pupae

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ERNST HADORN

balanced nuclei, the cytoplasmic differentiation becomes hampered ; instead of progressive cell structures one observes syncytial masses. Thus the effect of dominant lethals is in many respects comparable to the pattern of damage that Poulson (1940) found in male embryos deficient for large parts of the X chromosome. Only a few dominant lethals become active in larval instars, and none of them seems to allow development up to the pupal stage (Demerec and Fano, 1944).

2. E/L-Boundary Lethals It is a characteristic feature of many lethal mutants that some of the individuals die within the egg membrane as fully differentiated larvae, whereas others are able to hatch but unable to grow further as larvae of the first instar. We may call this category “E/L-boundary lethals, ” indicating that the lethality crisis becomes manifest a t the boundary between the end of embryonic ( E ) and the beginning of larval ( L ) life. Whether one or the other stage can be reached may depend on different environmental factors such as breeding-temperature or moisture of the culture medium. Then, also, the genotypic milieu surrounding the lethal factor may play an important role. F o r instance, the author has found by testing the hatching rate of many different stocks all containing a Curly chromosome ( Z n (2L) Cy), that in some stocks the Cy homozygotes die before hatching, in others they die as larvae of the first instar, and in still others some of the individuals die within the chorion and the rest after hatching. Even in isozygotic stocks where all external conditions for all individuals are made as equal as possible, we may expect typical E/L-boundary lethals to show a certain amount of variability with regard to the stage at which death occurs. This phenomenon could be merely a consequence of the variability that is presumably inherent to any developing system. The existence of E/L-boundary cases does not interfere with the concept of phase-specificity of action (Hadorn, 1948a). There is hardly a significant difference of developmental progress between the E ’s and the L’s. A series of lethals which belong to the category under discussion are listed in Table 2. 3. Larval Lethals ( L ) Larval life begins after the hatching of the differentiated larva of the first instar and ends when the fully grown third-instar larva forms a puparium. I n Drosophila melanogaster this period lasts about 96 hours a t 25” C. A t 25 hours the first larval molt transforms the first instar into the second one, and at 48 hours the second molt marks the change from the second to the third instar.

ACTTON OF TiETHAL FACTORS IN DROSOPHTLA

61

A few mutants h a w been investigated (Table 2 ) which die in the first instar, apparently nnable to grow further and accomplish the first larval molt. Lethal factors that cause death in a phase-specific manner during the second instar are not known. I n some third-instar Iethals (Z(2)w~e, l ( 2 ) 7 ) abnormalities of growth ant1 differentiation have been observed. 4. L/Pr-Roiindary T~ethals

A t the beginning of metamorphosis the cuticle of the third-instar larva (L) becomes hardened and pigmented, forming A tough pupal case called the puparium. Aboiit four hours after the onset of this process, the first internal molt occiirs within the pupariuni. The epidermis separates from the old larval ciiticle and becomes snrroiindecl by the very thin prepiipal ciiticle. The prepupa ( P r ) itself is headless, withoiit any protruding extremities. F o u r hours later this prepnpa develops into a real pupa. The process of pupation is preceded by a second internal molt, in which the prepupal cuticle is shed and evagination of the imaginal discs of head and thorax takes place. It is characteristic of L/Pr-boundary lethals that their development comes to a standstill either immediately before or immediately after puparium formation. I n the latter case, puparia of more or less normal shape are formed, but within such " pseudopupae" no further development goes on. The l ( 2 ) y l mutant, for instance, never differentiates beyond the early prepnpal stage (I-Iadorn, 1937a, 1948a). Consequently, the difference between individuals dying in the L stage and those reaching the early Pr stage is by no means very great. We know that the formation of the puparium, which indicates the transition from L to Pr, is induced by the hormonal activity of the ring gland (Hadorn, 1937b). Individual variability in the quantity of hormone secreted, or variability in the sensitivity of the reacting systems, will decide whzther o r not the threshold from L to Pr is passed.

5. Yrepupal Lethals ( P r ) Almost no reliable facts are known about strictly phase-specific prepupal lethals. The Z(3)blo-1 mutant probably belongs to this category (Chen, 1929). Irregular growth and degeneration of the imaginal discs here prevent the formation of the pupa. The ensuing pattern of damage is in many respects similar tn that caused by the l ( 2 ) g L locns (Hatlorn, 1938, 1945).

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ERNST HADORN

6 . Pupal Lethals ( P ) The only genotypes that should be considered pupal lethals are those that develop at least beyond the prepupal stage and die later a t any period between pupation and emergence of the adult fly. During this main period of metamorphosis, which takes about 34 days (at 25” C.), breakdown of the larval organs and tissues and differentiation of the imaginal structures have to take place. Two distinctly different types of anormogenesis can be distinguished. I n the first type we observe an inability of imaginal cell differentiation. It may affect, as in l ( 3)tr lethals, either the whole pupal body or only parts of it (Hadorn, 1949a). I n the second type a completely differehtiated imago develops, but is unable to hatch from the pupal case. This impotency may be due to gross morphological malformations (crc, E(3)hd) or merely to physiological “weakness” ( l ( 3 ) e Y ) .

7. P/I-Boundary Lethals The act of emergence of the imago ( I ) from the pupa ( P ) , as well as the E/L or the L/Pr change, may depend on highly variable quantitative factors, On the basis of such an interpretation we can easily understand the appearance of mutants among which some individuals succeed while others fail in overcoming the P/I threshold. Usually the emerged flies of such mutants, being weak and short-lived, are unable to reproduce.

8. Imaginal Lethals ( I ) I n spite of some hereditary malformations in imaginal differentiation, these mutants are capable of emergence; but their early imaginal life is interfered with. Such individuals are unable to feed ( p b ) , or move (me, 2(2)2gt), and consequently die before their reproductive activity sets in. 111. THE VALIDITY OF

THE

CONCEPT OF PHASE-SPECIFICITY

T o what extent lethal factors act in a phase-specific way has been discussed by the author in an earlier paper (Hadorn, 1948a). It was then concluded that interruption of development and death occur in most Drosophila lethals specifically at a definite stage. The data reviewed in the foregoing section corroborate such a statement. Many mutants that a t first do not seem to show the phase-specific mode of action can be classified as belonging to the distinct categories of E/L-, L/Pr-, or P/L-boundary lethals.

ACTION OR’ LETHAL FACTORS IN DROSOPHILA

63

Nevertheless, we have to consider whether there are known cases that contradict the principle of phase-specificity. Brody (1940) investigated six different second chromosomes of Drosophila melanogaster that contained lethal factors and found early as well as late embryonic lethality in each of the homozygotes. It is difficult, however, to see how egg mortality due to failure of fertilization might be separated in his investigations from genuine early embryonic lethality caused by the zygotic constitution. Besides, we have to consider that the manifestation of a lethal factor can be influrnced by the “genotypic milieu.’’ Hadorn (1940) , for instance, succeeded by means of continuous selection in separating from a 1(2)gZ/Cy stock two genotypes that differed considerably with respect to the time of death of the l ( 2 ) g l homozygotes. I n one line almost all lethals died in a late embryonic stage, whereas in the other only a few individuals failed to develop into full-grown thirdinstar larvae. This difference, due to chromosomal factors modifying the l g l reaction, is also subject to a maternal effect through the egg cytoplasm. With increasing homozygosity of the genotype bearing the lethal factor, a more accentuated phase-specificity of action would certainly result in many of those cases where apparently the time of death in different individuals is spread over a wide range of ontogenic stages. We shall discuss later further details of how the onset of the lethal crisis is influenced by additional genic and environmental factors. We must now consider some other facts that a t first sight do not seem to sustain the concept of phase-specificity. Thoroughly studied mutants like 1 ( 2 ) g l , 1(3)tr, and l(2)me are no doubt representative of the phase-specific type. Nevertheless, if one counts the lethal larvae and pupae in mixed cultures, where normal genotypes also develop, one finds the proportion of lethals usually short of the expected Mendelian frequency. Such deficits are due to the fact that the lethals, more sensitive than their normal culture-mates, have a higher embryonic and earlylarval mortality. Apparently lethal genotypes suffer a general weakness even before they reach their locus-specific phase of lethality. The development of any genotype is endangered by various unfavorable influences, to which lethal mutants succumb i n higher percentages than nonlethals. I n this respect, larval and pupal lethals do not differ from many “visible ” mutants that are characterized by reduced viability during ontogeny. I n both cases the developmental weakness is inseparably connected with the mutated constitution. Such early mortality, which prevents in a varying number of individuals the manifestation of the locus-specific pattern, conceals the phase-specificity of gene action. While this general mortality in early stages prevents various percentages of individuals from reaching the specific lethality phase, an-

64

ERN ST HADORN

other pheiionienon shows that development may go beyond the critical stage. It is typical of many nonlethal mutants of Drosophila that the characters specific to the mutated locus do not develop in all individuals. The penetrance of the gene is not strong enough to affect every member of its Mendelian class. Such mutants overlap the normal wild type. The same phenomenon may happen in the case of lethal factors; various numbers of individuals survive the period of phase-specific action and are able to develop further. Such "break-thronghs" (IIadorn, 1945, 1948a) may either become normal adults or die later during a second sensitive period. I n certain selection stocks of the l(2)gl mutant, whose first lethality phase is in the late embryonic stage, we find that those genotypes that break through this stage became lethal only during the L/Pr phase. I n the case of the Z(3)tr mutant we observe break-throughs from the early-pupal lethality phase, which develop to partially metamorphosed imagines (IIadorn, 1949a). Especially impressive is the fact that in rare instances even Curly homozygotes manage to break through the E/L lethality phase. Once having passed through this almost invincible danger, they differentiate into adults. Genotypes in which adult break-throughs appear regularly are called semilethals. This category of mutants is a common one, and it appears in every large sample of mutants from untreated populations or from any experiment with mutagenic agents. I n spite of this abundance of material we have a t present only a very meager insight into the developmental physiology of semilethal factors. W e know only that in many bemilethal stocks the relative frequency of lethal versus nonlethal individuals varies to a large extent as a function of environmental and genic modifiers. But in most cases it is not known at what developmental stage the lethal quota dies, o r whether a locus-specific pattern of damage can be found. Therefore future investigations are needed to show to what degree phase-specificity of action is manifested in semilethal mutants. One further dificulty presents itself in connection with problems of phase-specificity. I n spite of the fact that all cells and organs within a lethal genotype have the same genetic coastitution, they may react in entirely different ways. This has been shown especially for the Z(2)gl mutant (Hadorn, 1945, 1948a). Some systems, like the male germ cells (Gloor., 1943) and the imaginal discs (Hadorn, 1938), begin to degenerate during the early larval stages. The female germ cells, on the other hand, and most of the larval organs develop normally, whereas in the fat-body and the salivary glands there is a mere reduction of growth rate without cellular degeneration. Another example of such organspecific differences is provided by the Z(3)tr mutant (Hadorn, 1949).

ACTION OF LETHAL FACTORS IN DROSOPHILA

65

Partially metamorphosed individuals of this lethal genotype develop imaginal structures to a smaller or greater extent only in the head and thorax region, whereas the abdominal integument remains in the early pupal stage. Thus different organs and tissues of one single lethal genotype may have their own respective phase-specificities. Valuable information concerning the phase-specificity of gene action can therefore be gained only by studying the developmental fate of the different cell and organ systems separately. OF “PATTERNS OF IV. THE DEVELOPMENT

DAMAQE”

The total of all the characters in which a lethal mutant differs from the normal constitutes its locus-specific “pattern of damage’’ (Hadorn, 1945, 1948a). I n producing it the mutated locus usually acts in a rather complicated pleiotropic way. A few examples may show how such patterns develop. In the hemizygous lethals of the Notch series (Poulson, 1945), segregation within the blastema of the embryonic ectoderm proceeds abnormally. Too many cells become presumptive nervous tissue, a t the expense of the epidermis and its derivatives. At the same time, differentiation of the mesodermal and entodermal anlagen never begins. From the experimental work of Bock (cf. Seidel, Bock, Krause, 1940) on the nenropterous insect Chrysopa, we know that the pattern of segregation accomplished in the ectoderm acts as an organizing principle on which the blastema segregation of the mesodermal material depends. It is possible that in Drosophila too the ectodermal pattern plays an inducing role in the further organization of the embryo. The ensuing abnormalities in the Notch lethals might thus be the consequence of initial distortion within the ectodermal blastema. As to the cause of distorted segregation in the embryonic ectoderm we can only conjecture. I n developmental systems where one common blastema becomes separated into two or more areas, each of which later is separately differentiated, some balancing factors may control the segregation of competing fields. Patterns of damage of the Notch type might disclose genes normally engaged as regulators of such processes. Disturbance of segregation, however, is apparently not the sole damaging effect of the mutation concerned here. Apart from abnormal organogenesis, we observe that the cells are unable to differentiate. I t is true that in normal development these two processes seem to be inseparably related. I n the following section we shall discuss lethals in which one or the other of them is hampered.

66

ERNST HADORN

Gloor (1950) studied a dominant mutant “Kriippel” ( K r ) , which leads in heterozygotes to malformations in the thoracic region (Fig. 1). IIomozygous K r are embryonic lethals and manifest a very uniform pattern of damage. During normal development contraction of the embryo and segmentation of the body take place a t the age of 10 hours. I n K r lethals the primary segmentation is abnormal in many respects. The number of externally visible segments, normally 9, is reduced to 5.

FIQ.l b c FIQ.1. ( a ) K r / + heterozygous imago, showing typical abnormalities in the thoracic region. ( b ) The nervous system of a Lir/Kr homozygous lethal embryo at 15 hours (above), as compared to the normal condition (below). ( 0 ) E r / E r homozygous lethal embryo (above), showing defective segmentation and fragmentary tracheae, in contrast to normal control (below). ( a , Gloor, unpublished ; b and a, Gloor, 1949.)

ACTION O F LETHAL FACTORS IN DROSOPHILA

67

I n embryos aged 15 hours the middle sections of the ventral chain of ganglia are i n a state of disintegration, which later prevents the formation of a single subesophageal ganglion. The embryonic tracheae are represented by several fragments, which do not join closely into a continuous system. Malpighian tubules are apparently never formed. Although all these fundamental aberrations of organogenesis become manifest a t such an early stage (10 hours), histological and cellular differentiation astonishingly proceed farther. Twelve hours later the tissues have reached the same degree of structure as in normal animals ready to hatch from the egg membrane. K r lethals even show the vigorous muscular activity that normally precedes hatching ; but the defects in segmentation and abnormalities i n position and relative proportion of the organs are so severe that hatching never becomes possible. The K r mutant is a representative of those lethals that fail in the process of morphogenesis of the whole organ-anlagen, without interfering with the differentiation of histological and cellular structures. Moreover, the first abnormalities are visible long before the death-stage is reached. The development of the very complicated pattern of damage characteristic of the l ( 2 ) g l mutant has been discussed in earlier reviews (Hadorn, 1948a, 1950). It was shown that lethal effects appear in different cell-systems at quite diff’erent stages during ontogeny. By means of the transplantation technique it was further proved that the pattern of damage contains primary as well as secondary phenes (characters). The first are due to factors intrinsic to the cells involved; the second are caused by precedent abnormalities in the surrounding tissues, which have been primarily affected. As a contrast to the K r case just discussed, one fact deserves particular attention. I n embryos and larvae of the l ( 2 ) g l mutant, morphogenesis is normal as far as location and size of the primordia are concerned. Here it is histogenesis that fails. The anlagen of the imaginal discs are present in a larva, but their cells are unable to differentiate into imaginal structures. The same is true for the germinal cells within the testis. The existence of patterns of damage of either the Kr or the l ( 2 ) g l type seems t o prove that organogenesis and histogenesis may have independent gene-conditioned bases. Thus a “dissociability ” (Needham, 1942) of integrated developmental mechanisms becomes evident as a consequence of gene mutation.

v.

INFORMATION

GAINEDFROM TRANSPLANTATION EXPERIMENTS

A detailed investigation of lethal genotypes a t the stage of death usually shows that the different tissues and organs of the developing

68

ERNST HADORN

organism react very diflerently to the mutated constitution. It has already been pointed out that besides abnormal and degenerate parts one may encounter cell-systems that appear completely healthy. What are the developmental potencies of these seemingly undamaged parts 7 Reliable information may be gained by isolating such parts from their decaying surroundings in time, and then testing their autonomous potencies in witro in a suitable medium. I n spite of numerous attempts, no sufficiently successful method for insect tissue culture has been worked out so far. Thus one has to resort to cultures in vivo. The primordia are transplanted into genetically normal hosts and their further development is observed. Such experiments have been made by the author and his collaborators with many different parts of the 1(2)91, 1(3)tr, and l(2)me mutants. The results, which were reviewed recently (Hadorn, 1950), are of three types : a. The transplant does not develop beyond the stage it would have reached as part of the lethal individual. Primordia belonging t o this category are : the main imaginal discs of l ( 2 ) g l (Hadorn, 1948a), the male germ cells (Gloor, 1943), and the anlage of the adult salivary gland of the same mutant (Grob, 1946). b. The transplant distinctly passes beyond the stage characteristic of the lethal as a whole, but, after a period of progressive development during which the transplant keeps pace with the host, the lethal crisis sets in. Transplanted ovaries of l ( 2 ) g l and l(2)me behave in this way (Hadorn, 1937a ; Gloor, 1943 ; Schmid, 1949). c. The transplant develops normally and produces within the host all the expected imaginal structures. Examples are : the primordium of the imaginal ring gland (Vogt, 1947) and somatic cells of the gonads (Gloor, 1943) of Z(2)gl; imaginal discs, salivary glands, and gonads of l(3)tr (Hadorn, 1948a; Sobels, 1950) ; and imaginal discs and testes of Z(2)me (Schmid, 1949). Medvedev (1938) transplanted imaginal discs of eyes, wings, and legs from a mutant whose lethality phase is in the late pupal stage. Development of the respective parts was found to be normal. Cullen (1948) studied a lethal deficiency, B26s-20,which does not develop beyond the first instar and shows no growth whatsoever. Tissues transplanted into normal larvae and into abdomens of adult males showed essentially normal development. I n this case it was conclusively shown that the lack of growth was due to a failure of the ring gland. How are we to explain the three divergent types of transplantation behavior P Some possible interpretations are discussed below. a. Of the first-described transplants it may be supposed that they behave autonomously. They suffer from a “direct pleiotropic effect”

ACTION OF IiETHAL FACTORS I N DROSOPHILA

69

of the mutation (Hadorn, 1945). The developmental failure is irreparable, and the ensuing damage is due to intrinsic cell factors. We have to consider a n alternative interpretation, however. Since exchange of transplants between embryos is technically impossible, the experiment must be made in the larval stage. It can therefore be argued t h a t liberation from the lethal surroundings comes too late, and that systems which are not primarily affected could already have suffered from extrinsic influences before being transferred to the normal milieu. I n most cases it is impossible to refute such a n objection, yet there are a few natural “ implantation experiments” that disprove it directly. Through somatic crossing over or as a consequence of nondisjnnction i n mitotic divisions, single cells may arise that are homozygous or hemizygous with respect to a lethal factor. The lethal systems are surrounded by genetically normal tissues from the very beginning of their existence. By w i n g marked chromosomes Ephrussi (1934), Demerec (1934), and Stern (1935) succeeded in proving that different lethal factors caused inviability even in single cells that were embedded in a normal blastema. Such intrinsic cell-lethality exists, for example, in epidermal mosaics having the Notch constitution. The developmental defect observed by Poulson within the embryonic ectoderm of Notch males is therefore to be interpreted as a primary phene, conditioned directly by intrinsic cell factors. I n order to test the possible effect on intrinsically nonaffected primordia of remaining too long within the lethal organism, Oloor (1943) implanted normal testes into l ( 2 ) g l larvae, exposing them for a period of 24 hours to the lethal surroundings. These testes, after having been reimplanted into normal hosts, developed normally in every respect. 6. Transplants behaving in the second way clearly demonstrate the fact, already discussed, that every one of the different cell systems of a lethal genotype has its own phase-specificity. I n l ( 2 ) g l the imaginal discs a n d the spermatogonia degenerate during larval life, t h a t is, before the prepupal death of the whole organism. Transplanted ovaries, on the other hand, become affected only a t the beginning of imaginal life, after a prosperous period that allows them to develop f a r beyond the prepupal stage. From the fact that they nevertheless do not escape final destruction, we have to conclude that their ultimate failure is conditioned by intrinsic factors. c. The successful development to normality of some transplants from lethal organisms may be attributed to two basically different mechanisms. ( g ) The cell system in question may lie completely outside the pleiotropic pattern of damage. I n this case the difference between the normal

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and mutated loci is irrelevant to its developmental physiology. It is self-evident that transplants of this category differentiate normally. It is true that their development is promoted by the hosts, which furnish them with nutritious material and hormones. Nevertheless, genetically, the differentiation of the transplanted cells is to be regarded as autonomous. They receive from the host only those substances on which the development of any primordium is dependent regardless of its genetic constitution. Aid from the host is therefore not locus-specific. It by no means supplies items in which the lethal cells are deficient on account of their mutated locus. Any developmental process within a normal system may be regarded as a chain-reaction, dependent on intrinsic as well as extrinsic links (Hadorn, 1950). As long as the host furnishes only extrinsic links, it is not admissible to postulate that lethality has been overcome by specific factors. (2) The transplanted cells may belong to those parts of the organism that are primarily affected by the lethal constitution. I n this case they can develop normally only provided the host can help them in a locus-specific way. I n other words, the host has to supply in the transplant an equivalent of the function of the mutated locus. The developiiient of the transplant toward normality is therefore nonautonomous. A n “intrinsic link” has to be replaced from outside, in order to render possible developmental performances that are no longer characteristic of the genetical constitution of the cells involved, Numerous experiments, however, based on material gathered from visible mutants, have shown that the great majority of Drosophila transplants develop autonomously in hosts of different genetic constitutions. I n view of these observations, a vitalization of lethal transplants through locus-specific action on the part of normal hosts seems rather improbable. It is difficult, if not impossible, to decide whether the normal development of a given lethal transplant is due to a mechanism of type (1) or type (2). As we have shown (Hadorn, 1950) that most of the transplants that develop normally do not seem t o be intrinsically affected, one therefore need not postulate a host-engendered “ l g l + or lme+ substance” in order to explain their normal differentiation. The sole case in which an intrinsic lethality may have been overcome through genuine locusspecific action from outside was reported by Neuhaus (1941). By Xraying he obtained two mutants of the cinnabar locus ( c n ) which behaved as recessive lethals. If the homozygotes were fed during early larval stages with extracts from cn+ larvae and pupae, a few of them developed normally. This remarkable and exceptional result can be explained, however, on the basis of the following facts. According to the experiments of Ephrussi and Beadle (1937), the

ACTION O F LETHAL FACTORS IN DROSOPHILA

71

eye discs of nonlethal c n mutants show nonautonomous development as transplants within a host providing cn+ substance (oxykynurenin), which they are unable to synthesize themselves. The same effect results from feeding this substance (Beadle, Tatum, and Clancy, 1938). The lethal mutants studied by Neuhaus might be interpreted as especially strong alleles of the c n ’locus, causing not only defects in pigment synthesis but also additional disturbances in fundamental metabolism. Though the extent of the pattern of damage would be increased, it would still be curable. Such nonautonomous behavior of a lethal constitution must be considered an exceptional case, however. It might be just as rare among the total of lethal mutants as visible mutants of the cn type are rare among the total of nonlethals.

VI. PmsIoLoaIcAL PROPERTIES OF LETHALS The pleiotropic pattern of damage of a lethal factor usually includes not only morphological but physiological phenes as well. Certain characteristics of respiration, osmotic regulation, nutrition metabolism, and endocrinology may deviate from the norm. I n most cases we do not know whether a particular abnormality should be interpreted as a direct cause of lethal development or only as a secondary effect of a more fundamental disturbance. It is nevertheless valuable to know the physiological phenes and to “collect” them as fragments that mill later become integrated in a general theory of physiological genetics. Boell and Poulson (1939) measured the oxygen consumption of lethal male embryos of the Nullo-X type. They found a reduced respiration rate, which became manifest as soon as the developmental abnormalities already described were visible. Furthermore, they discovered the remarkable fact that embryonic cell division still proceeded, although the oxygen uptake was reduced to 20% of the normal amount. My collaborator, P. S . Chen (unpublished) has made extensive comparative respiration measurements on the three mutants 1 ( 2 ) g l , 1(2)*me,and 1(3)tr, in an attempt to determine the developmental pattern of this physiological trait. Only a few of the results may be reviewed here. Figure 2 shows the changes in oxygen consumption during larval development. I n this period body size increases and consequently there is a rise in oxygen uptake per individual. If the values are computed per unit dry weight, however, a rapid decrease characterizes the normal pattern. This fact does not necessarily mean that older larvae are less active than younger ones. It can be assumed, however, that the closer development approaches to the end of larval life, the greater must be the relative amount of more o r less inactive materials, and that re-

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serve substances and cuticular structures may be responsible for the observed decline. The l(3)tr homozygotes form puparia with a delay of one day (Hadorn, 1949a). The physiological age of the larva 120 hours after oviposition corresponds to that of normal controls a t 96 hours, The translucida curve of Fig. 2 should be shifted correspondingly to the left. But even after such a correction the oxygen consumption of this lethal appears significantly higher than that of normal larvae. This may be

t

anL 7”

-

30 25 20 35 15 1 .0 5-

--

a

L. a

1

I

I

I

I

I

I

I

I

l?m 2. Changes in rates of oxygen coiisuiiiptioii for controls and lethals during larval development. Ordinate: rate of oxygen consumption in cubic millimeters per milligram dry weight per hour. Abscissa : larval age = Ztr/Ztr in hours after oviposition. 0 = controls ; lethals; A = lg3/1gl lethals; A = t m e / h e lethnls. (P.S. Chen, unpublished.)

due to a lower proportion of inactive parts (fat body) to total dry weight. On the other hand, there is ample evidence that in both the l ( 2 ) g l and E(2)me mutants the respiration rate of larval tissues is distinctly below normal. It falls gradually to zero in the “meander lethal” but remains constant at a low rate in “lethal giant larvae.” This difference becomes manifest in the period during which the lethals survive as overaged larvae. Correspondingly, l(2)me larvae never pupate, and die early; whereas most of the surviving Z(2)gl larvae finally form a puparium, sometimes even after a delay of one week. A high respiration

ACTION O F L E T H A L FACTORS IN DROSOPHILA

73

rate is apparently not an indispensable condition f o r puparium formation. Figure 3 (P. S. Chen, unpublished) indicates, first, that the respiration rate of the lethal mutants is distinctly reduced a t the moment of puparium formation. It also reveals four other interesting facts : ( a ) The lethal mutants resemble the controls in that their oxygen consumpt,ion drops sharply during the first day, but they differ from the normal by failing to show the upward swing represented by the second part of

FIG.3. Changes in rates of oxygen consumption f o r controls and lethals during pupal development. Ordinate : rate of oxygen consumption in cubic millimeters per milligram dry weight per hour. Abscism: pupal age in hours a f t e r puparium formation. 0= controls; 0 = partially = nonmetamorphosed ltr/ metamorphosed Ztr/Ztr lethals ; Ztr lethals; A = ZgZ/Z.qZ lethalu. (P. S. Chen, uiipuhlished.)

the IJ-shaped curve. ( b ) Although all progressive development comes to an end in the lethals, they survive for an almost unlimited period, during which respiration continues a t low rates. ( c ) During this period of survival, metabolic activity is highest in the l(3)tr pupae, which are partially metamorphosed in the head and thorax; lower in the nonmetamorphosed translucida individuals ; and lowest in the I ( 2 ) g l pseudopupae. ( d ) Although in the partially metamorphosed l(3)tr group about half the body cells differentiate into imaginal structures, there is no rise in oxygen consumption, corresponding to the ascending second

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ERNST HADORN

part of the normal curve. It therefore seems doubtful to what extent the differentiation process influences the course of a respiration curve. Since Drosophila larvae live in fermenting fruits, they must be considered aquatic animals that have to solve the problem of osmotic regulation. Gloor discovered (1949b) an “anal organ” that is possibly concerned with an exchange of ions between the body fluid and the milieu (Gloor and Chen, 1950). The function of this organ might be one reason that in Z(3)tr the hemolymph accumulates excessively. Gloor and Chen (unpublished) determined the chloride concentration in the body fluid of l(3)tr and l ( 2 ) g l larvae, and found the value to be about 0.11% in both lethals. This amounts to less than half that in wild-type larvae, where an average chloride concentration of 0.24% has been determined. Although no further conclusion should be drawn from such preliminary results, we see new possibilities for penetrating deeper into the physiological properties of lethal mutants. That abnormal osmo-regulative characters might be a part of the pattern of damage was indicated by the work of Kaliss (1939) on Df 260-2. The cuticle of the lethals shows an exceptionally high permeability f o r water. Therefore dechorionated animals are unable to escape dehydration in a hypertonic solution. On the basis of a theory developed by Sikes and Wigglesworth (1930), it seems probable that abnormal osmotic properties of the tracheal wall prevent replacement of the initial water content by air, a process that fails almost completely in these lethals. Very informative observations concerning metabolic properties have been made by Schmid (1949) on the Z(2)me mutant. I n this case, as will be pointed out later, the locus-specific pattern of damage can be reproduced as a phenocopy by means of timed starvation, since inability to use the available food material seems to play a decisive role in the nutritional physiology of this lethal. Schmid starved normal as well as lethal larvae a t the age of two days and observed a characteristic reduction of the f a t body, which, formerly of a milky appearance, became depleted and transparent. Later these larvae were fed with either pure albumen, sugar, or fat. Normal genotypes showed the ability to rebuild a normal fat body with any one of these nutritivc materials. The l(2)me larvae, however, although they could utilize either sugar or fat, were completely unable to resynthesize the building material of the fa t body when fed nothing but albumen. An essential part of the protein metabolism must be regarded as being fundamentally affected by the lethal constitution. There is also some preliminary evidence of abnormal protein metabolism in the Z(3)tr mutant. Gloor (1949a) found, for instance, that the

ACTION OF LETHAL FACTORS IN DROSOPHILA

75

percentage of globulin in the total protein content of the hemolymph in lethal larvae was strikingly low. A lethality due to starvation has been found in l ( l ) 7 larvae. Russell (1940) showed that in this genotype the midgut cells become abnormal a t the beginning of the third instar. By completely blocking the intestinal lumen, the affected cells prevent further uptake of food material. Thus in this mutant a localized morphological abnormality causes starvation, whereas in the l(2)me larvae a physiological block interrupts more directly the internal nutritional metabolism. According to Beadle, Tatum, and Clancy (1938), feeding up to a larval age of 70 hours is necessary f o r continued development and metamorphosis. Since in both the l(2)me and the l ( l ) 7 mutant starvation begins before this point, a failure of metamorphosis must ensue. Another factor on which the course of metamorphosis depends is the timed action of hormones. If one finds mutants that fail in metamorphic process, the question arises whether a n endocrine dysfunction may be involved. Such considerations led to the discovery of the ring gland, which functions as the center of hormone production in Diptera (Hadorn, 193713). Larvae homozygous for the l(2)gl gene either form the puparium with great delay o r do not form it at all. Scharrer and Hadorn (1938) found a corresponding retardation in the development of the ring gland. Implantation of normal glands induces pupariurn formation regularly and at the proper time in l(2)gl hosts. Thus the reacting system is not itself affected by the lethal constitution, and would develop normally provided the hormone were available; it suffers only secondarily. On the other hand, it would be erroneous to consider the failure of hormone synthesis as a primary effect of the mutated locus. The imaginal discs, for instance, have already degenerated before the ring-gland hormone is due to come into action. The damage to the ring gland appears rather to be secondarily conditioned ( Hadorn, 1948b). Nevertheless, insight into the hormone physiology of the l(2)gl genotype has been fruitful in many respects. Larvae of the l(2)me genotype never form a puparium. Schmid (1949) found that implantation of normal ring glands was ineffective in l(2)me hosts. Here, in contrast to l(2)gl the larval integument is apparently unable to react successfully to the hormone. Still another type of metamorphic disorder is manifested in the l(3)tr mutant. Whereas puparium formation and pupation proceed normally, imaginal differentiation either fails completely o r succeeds only in the head and thorax. F. H. Sobels (unpublished), keeping l ( 3 ) t r pupae in a medium of pure oxygen, found that there was a much higher percentage of metamorphosing individuals, and also that imag-

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ERNST HADORN

inal differentiation became possible in the abdomen. It seems as though some threshold reactions are affected in this genotype. Many more such fragmentary observations are needed before the gene-conditioned basis of metamorphosis can be understood. As to enzymatic activities in lethals, only one case has been investigated so far. Poulson and Boell (1946a,b) measured cholinesterase activity in several of the Notch-deficient embryos (see Table 1). They found the activity to be 2-3 times that present in normals a t the time of hatching. This increased activity is correlated with the hypertrophy of the nervous system of the deficient genotype.

VIT.

PIIENOCOPIES OF PATTERNS OF DAMAGE

The development of a normal genotype can be interrupted by various kinds of unfavorable environmental agents. We are here interested only in cases involving a distinct pattern of damage, more or less identical with a locus-specific pattern caused by a lethal factor. I n these, an external induence induces a “phenocopy l 1 of a known gene-conditioned abnormality. From the nature of the effective agent and the sensitive period at which it has to be administered, certain conclusions may be drawn with respect to the type and time of action of the mutative locus in question. Gloor (1944, 1945) succeeded in reproducing the pattern of damage characteristic of the mutant cryptocephal ( c r c ), by treating normal genotypes of known age either with heat (40’ C.) or with low temperature (-3’ C.). The sensitive period begins 6 hours after puparium formation and ends 5 hours later, that is, immediately before the imaginal anlagen of the head become evaginated by the process of pupation in normal development. I n the crc mutant and in the phenocopies, eversion of the head fails. Consequently the head capsule remains enclosed within the thorax. The crc pattern of damage is not restricted to this character, however. I n addition, the legs, wings, and bristles of crc individuals are reduced in size, whereas the abdomen is much longer than in normal pupae. All these phenes, which appear regularly in the pleiotropic crc pattern of damage, occur also in the phenocopied individuals. We may conclude from these results that the primary crc reaction must be of a rather simple nature, so that it can be easily induced by unspecific external agents in normal genotypes. Moreover, since the abnormal temperatures must be applied within a restricted sensitive period, this period ought to indicate the time in crc animals during which the lethal constitution causes its locus-specific pattern of damage.

ACTION OF LETHAL FACTORS IN DROSOPHILA

77

Another phenocopy of a lethal pattern of damage is that of the l(2)wt.e mutant, produced by Schmid (1949). A distorted larval growth pattern is characteristic of this genotype. Although the third instar is regularly reached, we find only a few organs, such as the pharyngeal apparatus, growing to full larval size. Most of the other parts show restriction in growth, which is clearly of an organ-specific degree. The subesophageal ganglion and the stomach reach about 80% of their normal size. More strongly affected are the tracheae (70%), the hemispheres of the brain (60% ) , and body length (55 % ) . The most extreme growth restriction is found in the salivary glands, which remain very small (30% of normal). A very close phenocopy of this pattern can be induced in nonmutated genotypes by starving normal larvae that have been kept under standard food conditions u p to an age of 48 to 60 hours (after oviposition). During the starvation period they develop a n organ-specific growth patt,ern that corresponds exactly to that of a “well-fed’’ Z(2)me mutant. It follows that the Z(2)me constitution causes a state of inanition during the second half of larval life. As was shown earlier, there is good reason to assume a fundamental disturbance of the protein metabolism. Theoretically, it should be possible to produce phenocopies of any gene-conditioned pattern of damage. Genes or gene products are regarded as specific catalysts of biochemical reactions. Hence substances that act as enzyme poisons may be potent phenocopying agents. Rapoport (1947) reported, for instance, that he produced lethal effects with strong concentrations of enzyme poisons such as sodium borate (1:600),and that in weaker concentration (1:750) the same substance caused phenocopies of visible loci. He pointed out that many cases are known of allelic series in which the weaker alleles cause visible effects whereas the stronger ones act as lethals. Since no detailed developmental studies have been made on the lethal phenocopies produced by Rapoport, it is inopportune to discuss the theory postulated by him. VIII.

CONDITIONED LETHALS

I n “conditioned lethals” (Hadorn, 1949b) the penetrance of lethality is dependent on additional genic or environmental factors. Dobzhansky (1946) reared a genotype of Drosophila pseudoobscura at different temperatures. Its viability was almost normal a t 16.5” C., whereas a t 21’ C. it behaved as a semilet.ha1 and at 25.5’ C. as a full lethal. The viable mutant “engrailed” of Drosophlila hydei (Spencer, 1942) show8 a manifold pattern of manifestation in differeut body re-

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gions. If the animals are raised a t a high temperature (30” C.) they die as pupal lethals. Direct proof that the degree of developmental abnormality caused by a lethal gene can be modified by different factors has been shown with the cryptocephal mutant (Hadorn and Gloor, 1943). By means of selection different crc stocks, which differed widely in penetrance and expressivity of the crc effect, were established. I n “strong” stocks almost all individuals died within the puparia as cryptocephalic imagines, the head remaining hidden in the thorax. Only a few animals showed a more or less everted head, and emerging “break-throughs” were still more rare. In “weak” stocks, on the other hand, a high percentage of homozygous crc flies developed normally into vigorous and fertile adults. Thus the genotypic milieu, whose constitution became altered by selection, changed the degree of lethality or even checked it completely. Gloor (1945) showed, furthermore, that the percentage of normally developing crc homozygotes varied greatly as a function of breeding temperature, inasmuch as lethality was increased by high temperature. There is a special type of conditioned lethality in which the degree of damage is influenced by sex. Gloor for instance found a much higher percentage of crc break-throughs in males than in females. A few cases are known in which lethality is strictly limited to one sex. Mohr and Sturtevant (1919) described a recessive autosomal gene i n Drosophila funebris that causes pupal lethality in females, whose abdomens become severely malformed. The homozygous males are scarcely or not at all changed, and develop into viable imagines. Another kind of sex-limited lethality was reported by Redfield (1926). Here, too, only the females succumb to the lethal effect. It seems as if the differences in tempo and mode of development that exist between the two sexes may influence the conditions under which lethal factors become manifest.

IX. LETHALITY DUE TO COMBINED GENICACTION Most of the lethals known in Drosophila, and all those so f a r discussed in the present review, are based on just one mutated or lost chromosomal region. I n crosses they therefore obey the laws of monofactorial segregation. Even if the penetrance of such lethals happens to be modified by additional genic loci, there is always one “main gene” responsible for the developmental disaster. We now have to consider genotypes in which lethality is caused by the combined action of two or more mutated loci. At least two of these are essential for the lethal effect ; one factor alone proves to be harmless.

79

ACTION OF LETHAL FACTORS IN DROSOPHILA

Clemente (1939) reported that double homozygotes ( p r l p r ey/ey ) for the two genes, purple (pr, 2-54.5) and eyeless (ey, 4-0.2) are invariably lethal, The single homozygotes ( p r / p r +/ey or pr/pr and pr/+ ey/ey or ey/ey) are viable and show either one or the other of the phenes characteristic of these well-known mutants. This interesting case deserves thorough study. The Minutes are dominant factors that arise through mutations a t many different loci in all the four chromosomes of Drosophila melanogaster. I n heterozygotes they produce very similar or even identical smallbristle effects. The hemizygous or homozygous individuals behave as E/L lethals. Schultz (1929) combined different Minutes and constructed in this way genotypes of the general constitution: M l / Me/ or M e / M,/ +, . etc. I n none of such hybrids was an exaggeration of the Minute characters or a lethality obtained. If one compares different doses of the same M gene in diploids and triploids, however, the following statements can be made :“ M l / + = viable Minute, M l / M l = lethal; Ml/+/+ = viable but not Minute, M l / M l / + = lethal. These and other experimental results led Schultz to the conclusion that the primary reaction of Minutes from different loci (All, M,,. * . M n ) must be qualitatively different and therefore not additive. By combining different Minutes with some alleles of either one of the dominant factors Delta (DZ, 3-66.2) or Jammed ( J , 2-41.0), one gets a series of late pupal lethals of the constitution M l / + D l / + or M , / + DZ/+,etc. The lethal effect would therefore be due to a n unfavorable combination of the Delta (or Jammed) action, with a secondary Minute reaction, which is considered as differing only in degree among different Minutes. Bauer (1943) found a new allele of the facet locus (facet notchoid, fano),which did not impair viability and in homozygous or hemizygous doses caused a typical Notch effect in the wings. I n combination with three different Notches (two of them deficiencies, the other a “point mutation”), genotypes of the supposed constitution fan0/+ N/+ were produced. They behaved as larval lethals. We can explain this combined effect by assuming that there exists in the facet region of wild flies a duplication. Two loci, fa+ and spZ+ (split), would be present, During evolution these loci, originally identical, became gradually different in some respects but retained other functions that might still be common to both. The Notch phenomenon would arise if any two of the four loci in females or either of the two loci in males were lost or inactivated. The graphic symbols of Table 3 are based on this interpretation. They show that the lethality mentioned above is effected in females when only one normal locus is present. Thus the lethal development in

+/+

+/+

+

..

+

+,

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ERNST HADORN

TABLE 3 Interaction of Facet-Notchoid (fano)and Notch ( N ) in Producing Lethality, According to the Interpretation of Bauer (1943)

= normal locus;

-0-

-.

--

= deficient

or inactivated loc,us

-

Chromosomal constitution

Genic constitution

Phenotype

ra*/fa+

Viable wild type

fa+/rano

Viable, occasionally wit,h slight Notch effects

fa+/N

Viable Notah type

f ano/jano

Viable Notch type

-. .....-

f ano/N

Lethal in larval stage

-0.-

ra+

Viable wild type

fane

Viable Notch type

N

Lethal in embryonic stage

s! .-0 .a-.a..__

-0.-

-.......-

-0.. -0.. -0..

6

-0..

-......-

...-

.-

the combination fa.0 with N results from a system of overlapping deletions (or overlapping inert regions), and consequently is not a genuine combined effect of independent loci. This shows how cautiously one has to interpret presumable cases of multifactorial lethality. A very interesting type of combined lethals was found by Dobxhansky (1946). Through crossing over between chromosomes that were normally viable in homozygotes he obtained “synthetic” lethal chromosomes. It is probable that here the inviability resulted from unfavorable recombination of multigenic chromosomal blocks.

X. EFFECTS OF LETHALS IN HETEROZYGOTES How do recessive lethal factors act in heteroxygot,eR? A t first sight,, two sharply different types seem dist,inguishable.

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ACTION OF LETHAL FACTORS IN DROSOPHILA

1. Recessive Lethals That Produce N o Visible Efects in Simgle Doses Many factors belonging to this category have been discussed in this review (Tables 1 and 2, all mutants with lower-case initials). Although in such cases the heterozygotes appear completely normal, their viability may be affected by the lethal locus. Stern and Novitski (1948) tested numerous X chromosomes containing different recessive lethal factors (Z1, lla;..Zn). They found that on the average the viability of heterozygotes ll/Muller - 5, Z,/Muller - 5, * * .l,/Muller -5 was reduced if they were cultured in competition with +,/Muller -5, +,/Muller - 5, * +,/ Muller -5, respectively (the Muller -5 chromosome contains only the nonlethal marker genes scB1 B Ins Wa sc8). Earlier investigations by Dobzhansky (1939) and Dubinin (l946), however, showed no reduction of viability in heterozygous carriers of recessive lethals from wild populations. According to Dubinin’s discussion (1946),no statement of general validity seems possible a t present, Even in those cases where recessive lethals influence viability, any kind of insight into the developmental physiology of the heterozygous genotypes is lacking. I n addition to investigations on large series of different lethals, which serve the purposes of population genetics, detailed studies of individual cases are urgently needed. Only if the viability characters with respect to various environmental conditions are known, and only after accurate control of the genotypic milieu has been established, will a fruitful discussion be possible of the very intricate problems involved. It is essential to test even such factors as the age of the mother. Hadorn and Zeller (1943)found that young females which were heterozygous for the two lethals l(2)gL and Cy produced eggs a t a n exceedingly high rate. But during the second week of imaginal life fecundity and embryonic viability decreased to a very low level. Compared with normal genotypes, the l g l / C y heterozygotes would appear first as “superviable,” later as subviable.

-

2. Recessive A e t h d s urlth Dmirtamt Visible Effects in Eletercrzggotes

In Drosophila nielanogasler there are over one hundred different known mutants that behave as dominants and produce in heterozygotes various visible effects. About 70% of them act as lethal factors when homozygous or hemizygous. The student of developmental physiology is interested primarily in finding out whether there is any relation between the pattern of damage in the lethal homozygotes and the pattern of manifestation in the viable heterozygotes. The work of Brehme (1938, 1941) has enabled us to compare the development of homozygous with heterozygous Minutes. The former

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die as E/L lethals or L lethals of the first, instar. They are not able to grow as larvae. I n heterozygous Minutes the rate of larval growth is distinctly reduced and the time of development correspondingly prolonged. The fact that in both genotypes the process of larval growth is affected may take on significance as soon as more is known about the Minute reactions. Whereas in the Minutes comparable characters of a physiological nature have been observed in homozygous and heterozygous individuals, Qloor (1950) found in the Kriippel mutant, a certain correspondence in the gross anatomy, as shown in Fig. 1. I n K r / K r individuals the middle segments are particularly abnormal. It is remarkable that in the fully developed Kr/+ heterozygotes there occur various kinds of abnormalities that are restricted to the thorax, while the head and abdomen are normal. I n other words, the main region of manifestation of the mutated constitution seems common to both genotypes. Unfortunately, no further data on the mutational pattern in either affected homozygotes or heterozygotes are available. One should know, for instance, whether or not dominant mutations that in heterozygotes change the morphology of the wings cause a special type of embryonic or larval lethality under homozygous conditions. Would the pattern of lethality of “wing loci” differ from that of “eye-color 1oci”P

XI. CONCLUSIONS It follows from all the facts reviewed here that many approaches to a developmental physiology of lethal genotypes have been worked out.

Although our knowledge is still very fragmentary, it forms a sufficient basis for the design of future studies. Hereafter our efforts should be concentrated on the following items. 1. A complete inventory of characters that are included in the pleiotropic pattern of damage at the climax of the lethal crisis. Morphological as well as physiological phenes will be found. I n addition, cell- and organ-specificity of action may often be ascertained. 2. Investigation of the developmental history of the pattern of damage. The deviations from normality should be followed from their beginnings, 3. Analysis of pattern of damage by means of transplantation experiments. This method will enable us to distinguish between primarily and secondarily conditioned phenes. 4. Attempts to produce in normal genotypes phenocopies that imitate the locus-specific patterns of lethal mutants. If this succeeds, then

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some conclusions will be possible with regard to the special nature and time of action characteristic of the corresponding lethal mutants. 5 . Tests of the penetrance and expressivity of lethal effects in relation t o the following factors : genotypic milieu, sex, temperature, nutritional conditions, and other environmental influences. 6. Determination of the nature of the primary gene effect. According to the concepts of modern gene physiology one must expect distinct biochemical abnormalities to arise after a mutational change a t a vital locus. I n none of the lethal factors of Drosophila is the specific “inborn metabolic error” exactly known a t present. 7. Comparison and integration of results obtained with many different lethal genotypes. Only after this is done will we understand development as a sequence of processes, each of which depends on the action of numerous gene loci.

XII. REFERENCES Bauer, H., 1943, 2. indukt. Abstamm.- u. VererbLehre 81, 374-390. Beadle, G. W., and Law, L. W., 1938, Proc. SOC.e x p . Bwl., N . Y. 37, 621-623. Beadle, G. W., Tatum, E. L., and Clancy, C. W., 1938, Biol. Bull. Wood’s Hole 76, 447-462. Boell, E. J., and Poulson, D. F., 1939, Anat. Rec. 76, 65-66. Brehme, I(. S., 1937, Amer. Nat. 71, 567-574. 1938, Genetics 23, 142. 1939, Genetics 24, 131-161. 1941, J . e x p . 2001.88, 135-160. Brody, G., 1940, Genetics 26, 645-650. Bridges, C. B., 1937, Cytologia, Pujii Jubil. Vol. 2, 745-755. Bridges, C. B., and Brehme, K. S., 1944, The Mutants of Drosophila melanogaster. Carneg. Inst. Publ. 566. Washington, D. C. 257 pp. Bridges, C. B., and Dobzhansky, Th., 1933, Arch. EntwMech. Org. 127, 575-590. Chen, T. Y.,1929, J . Morph. 47, 135-199. Clemente, L. S., 1936, Philipp. J . Sci. 69, 177-187. Cullen, Sister M. U., 1947, Anat. Rec. 99, 590-591. Demerec, M., 1934, Amer. Nat. 68, 165. 1941, Proc. 7 t h int. Congr. Genet. 99-103. 1950, Biology of Drosophila. John Wiley & Sons, Inc., New Pork. 632 pp. Demerec, M., and Fano, U., 1944, Geneties 29, 348-360. Demerec, M., and Hoover, M. E., 1936, J . Hered. 27, 206-212. Dobzhansky, Th., 1930, Biol. Z b l . 60, 671-685. 1939, Biol. Rev. 14, 339-368. 1946, Genetics 31, 269-290. Dobzhansky, Th., aiid Spassky, B., 1944, Genetics 29, 270-290. Dubinin, N. P., 1946, Genetics 31, 21-38. Ephrussi, B., 1934, Proc. nat. Acad. Sci., Wash. 20, 420-422. Ephrussi, B., and Beadle, G. W., 1937, Bull. biol. 71, 54-74. Qloor, H., 1943, Rev. szrisse 2001.60, 339-394.

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1944, Rev. subse 2001.61, 394-402. 1945, Arch. Klaus-Stift. Vererb-Forsrh. 20, 209-256. 1949a, Rev. suisse Zool. 66, 281-285. 1949b, D.Z.S. 23, 89. 1950, Arch. Klawr-Stift. Vererb-Forsch. 26, 38-44. Gloor, H., and Chen, P. S., 1950, Rev. suisse Zool. 67, 570-576. Gowen, J. W.,1934, Arch. Path. 17, 638-647. Grob, H., 1946, Arch. Rlaw-Stilt. Vererb-Borsch. 21, 342-346. Hadorn, E., 1937a, Proc. SOC.exp. B i d . N. P.36, 632-634. 1937b, Proc. rmt. Acad. Sci., Wash. 23, 478-484. 1938, Rev. m k s e Zool. 46, 425-429. 1940, Rev. sztisse Zool. 47, 167-176. 1945, Aroh. Klaus-Stift. Vererb-Forscli. Xrg6nfzzrng8bnnd zu Band 20, 82-95. 1948a, 8ymp. SOC.exp. BWl., Growth 11, 177-195. 1948b, Bolia Biotheoretica 3, 109-126. 1949a, Rev. m k s e 2001.66, 271-280. 194913, Arch. Klaus-Stift. Vererb-Forsch. 24, 105-113. 1950, Rev. suisse ZooZ. 67, 115-128. Hadorn, E., and Gloor, H., 1942, Rev. suisse 2001.49, 228-236. 1943, Rev. sukse 2001.60, 256-261. Hadorn, E., and Schmid, W., 1947, D.I.S. 21, 68. Hadorn, E., and Zeller, H., 1942, Arch. Klaus-Stift. Vererb-Fcrsch. 17, 440-444. 1943, Arch. EntwiKech. Org. 142, 276-300. Ives, P. T., 1942, D.I.S. 16, 48. Kaliss, N., 1939, Genetbs 24, 244-270. Lea, D. E., 1946, Actions of Radiations od Living Cells. Cambridge University Press. 402 pp. Li, J. Ch., 1927, Genetios 12, 1-58. Medvedev, N. N., 1938, C.R. Acad. Sci U.R.S.S. 20, 319-321. Mohr, 0.L., and Sturtevant, A. H., 1919, Proc. SOC.exp. B i d . N. P. 16, 95-96. Morgan, T. H., Bridges, C. B., and Sturtevant, A. H., 1925, Bibliogr. Genet. 2, 1-262. Needham, J., 1942, Biochemistry and Morphogenesis. Cambridge University Press. 787 QQ. Neuhaus,-M. E., 1941, C.R. Acaa. Sci. U.R.S.S.N.S. 30,238-241. Poulson, D. F., 1941, Proc. 7th int. Congr. Genet. 240-241. 1940, J. exp. 2001.83, 271-325. 1945, Amer. Nat. 79, 340-364. Poulson, D. F., and Boell, E. J., 1946a, BWZ. Bull. 91, 228. 1946b, Anat. Reo. 96, 508. Rapoport, J. A., 1947, Amer. Nat. 81, 30-37. RedfIeId, H., 1926, Genetics 11, 482-502. Russell, E. S., 1940, J . exp. ZooZ. 84, 363-385. Scharrer, B., and Hadorn, E., 1938, Proc. nat. Acad. Rai., Wash. 24, 236-242. Schmid, W.,1949, 2. indubt. Abstamm- u. VrrerbLrhre 83, 220-253.. Schubel, F., 1934, Amer. Nat. 68, 278-282. Sohultr, J., 1929, Genetios 14, 366-419. Sohultz, J., and Dobzhansky, Th., 1934, Genetics 19, 344-364. Seidel, F., Bock, E., and Krause, G., 1940, Natzcrwisseneohaften 28, 433-446. Sikes, E. K.,and Wiggelsworth, V. B., 1930, Quart. J. micr. Sci. 74, 165-192.

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Sivertzev-Dobzhansky, N. I?., 1927, Arch. EntuMech. Org. 109, 535-548. Sohels, 3’. H.,1950,Pxperientia 6, 139-143. Roiinenblick, B. P.,1938, O e w t i c s 23, 169. 1940, Genetics 26, 137. Spencer, W. P.,1937, D.Z.8. 7, 13. 1942, Amer. Nat. 76, 325-329. Starck, M. B., 1922, J . ezp. 2007..19, 531-558. Stern, C., 1935, Proc. nut. Acad. Sci., Wash. 21, 374-379. Stern, C., and Novitski, E., 1948, Science 108, 538-539. Vogt, M.,1947, 2. Naturf. 2b, 292-294.

85

Localization and Function of Heterochromatin in Drosophila Melanogaster ALOIIA HANNAH Department of Zoology. University of California. Berkeley. California

CONTENTS

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I Introduction I1 Cytological Characteristics of IIeterochromatin 1 Mitotic Chromosomes 2 Salivary Gland Chromosomes a Major Heterochromatic Regions b Intercalary Heterochroinatin I11 Genetic Effects of Heterochromatin 1 Position Effect and Variegation a The Euchromntic Break and Variegation b The Heterochromatic Break and Variegation (1) X Chromosome (2) Y Chromosome (3) Autosomes c Comparison of X, Y. and Autosomal Heterocliromatiii d Modification of Variegation 2 Loci in Major Heteroehromatic Regions 3 Intercalary Heterochromatin a Interaalary Heterochromatin and Position Effect b Intercalary Heterochromatin and Location of the Gene c Polygenic Activity IV Summary V References

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The function of heterochromatin is a question of much controversy I n part this is due to the historical concept that heterochromatin is genetically inert. in part to the contradictory evidence and resultant confusion of fact and speculation. and finally to the lack of critical experimental data . As a result it has been found necessary. i n many cases in this review. to point out the contradictions and speculations without being able to resolve any of the basic questions regarding the fiinction of heterochromatin The few mutants which are known to be located in major hetero-

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chromatic regions are phenotypically not distinguishable from euchromatic ones (Schultz, 1939). The greater part of the heterochromatin, however, seems to have no distinctly discernible genes in classical Mendelian concepts. On the other hand, in certain genetic backgrounds, loss of blocks of heterochromatin results in inviability (Schultz, 1939) or produces gross morphological abnormalities (Morgan, Schultz, ancl Ourry, 1941 ), ancl its hyperfunction is associated with neoplasia (Daylingtoil and Thomas, 1941 ; Caspersson ancl Santesson, 1942; Kollw, 1947). Reitz (1928) introduced the term heterochromatin to describe special regions of the mitotic chromosome which retain a dense and compact htrncture during interphase. He was able to show (1928-1935) that these regions differ from euchromatin in the degree to which they change from telophase to interphase, and pointed out, after a comparative study of mitotic and salivary gland chromosomes, that there must be two types of heterochromatin : a, which cannot be uncoiled, and p , which uncoils to exhibit a diffuse mesh. When Heitz introduced the term heterochromatin he was describing only one of the characteristics of heteropycnosis. It had long been known that certain chromosomes or chromosomal regions are nucleated to different degrees during particular stages of meiosis and mitosis, If they are undernucleated they have been called negatively heteropycnotic, if overnucleated positively heteropycnotic. Some heterochromatic regions have the property of reversibility, that is, they are positive during one stage of the cycle, and negative in another; other regions do not show reversibility, being positive throughout the cycle. For this reason White (1945) defines heterochromatin as any chromosomal region which becomes heteropycnotic a t some stage in its cycle. Bchnltz (1947), on the other hand, limits heterochromatin with genetic function to “chromosomal regions which have the specific property of remaining as blocks in the intermitotic state.” Very soon after IIeitz’s cytological analysis of heterochromatin, it was realized that the heterochromatic regions in Drosophila chromosomes could be roughly correlated with the so-called genetically inert regions (Muller, 1914, 1918 ; Muller and Painter, 1932) and thus differentiated from the genetically active regions. However, the original concept of inert or degenerate genes has been replaced by that of specialized function. Muller (Muller and Gershenson, 1935 ; Muller, Raffel, Gershenson, and Prokofyeva-Belgovslraya, 1937 ; Muller, 1947) believed that the inert regions are essentially of nongenic material derived from a few active genes. Pontecorvo (1944) postulated that each gene, having its own characteristic cycle of nucleoprotein metabolism, if duplicated tandemly would give rise to differentiated regions having more or less, as the case

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might be, nucleoprotein than average, which would account for the unique genetic properties of heterochromatin. Mather (1941, 1944) proposed that the heterochromatin is active in the sense that it has genes, but that these genes have similar and supplementary effects governing continuous variation. Goldschmidt (1949) attributed a specific genetic fimct ion to the intercalary heterochromatin while the chromocentric heterochromatin may act in a generalized quantitative way. Caspersson and Schultz (for reviews see Caspersson, 3947; Schnltz, 1939, 1947) suggested that the heterochromatic genes function primarily i n the nucleic acid metabolism, while Koller (1947) believed its function to be that of a regulator responsible for maintaining equilibrium between supply and demand for nucleic acid. I n agreement with this concept, Vanderlyn (1949) concluded that the heterochromatin is a region of transfer of energy and/or substances at the nucleolar and niiclear membranes. Finally, Resende (1945) postulated that hetwochromatin may play an important role in speciation. 11. CYTOLOGICAL CHARACTERISTICS OF HETEROCHROMATIN

With the development of cytogenetic techniques and the resultant distinction between different types of chroma tin, two problems have emerged : the relation of cytologically distinguishable parts of the chromosome to their genetic functions ; and the analysis of the role and significance of heterochromatin, as well as euchromatin, in the metabolism of the cell, and their relation to evolution of genetic systems. This in turn raised the question of whether all heterochromatin is the same in that it is genetically active, or whether some has genetic function and other only participates in chromosomal metabolism. As the cyclic staining reaction of a chromosome is generally attribnted to synthesis and breakdown of desoxyribose nucleic acid, heteropycnosis has been interpreted by some (Koller, 1936 ; Muller and Prokofyeva, 1935) in terms of spiralization and nucleination of the chromosome. Others ( G u h i n , 1948; Koller, 1936; Koller and Darlington, 1934) presumed the determining factor t o be associated with the solitary condition of the X (as in spermatocytes). White (1940) postulated that heteropycnosis is not a function entirely of spiralization, but that heterochromatin is essentially different from euchromatin and therefore has characteristic cytological features. Schultz (1947) believed that the “genetic” heterochromatin is that which appears as bloclrs in the interphase cell ant1 has specific staining properties in prophase and metsphase stages.

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1. Mitotic Chromosomes Although the morphology of a chromosome varies during the different stages of mitosis, an estimation of the sequence and distribution of euchromatic and heterochromatic segments in mitotic metaphase chromosomes may be obtained by using certain constant structures, the constrictions, as landmarks. The chromosomes of Dyosophila melanogaster exhibit, a t metaphase, nonstaining gaps or constrictions which are constant i n position (Bridges, 1927 ; Kaufmann, 1934). The ‘‘primary constrictions” are associated with the spindle-fiber attachment regions or centromeres (Fig. 1 ) . The “secondary constrictions” are of two types, the nucleolus organizer and the gap or indentation constrictions. The gap constrictions may represent long non-nucleated but coiled chromonemata (Kaufmann, 1934) or they may be the genes responsible for the heterochromatic blocks, which do not become as coiled as other parts of the chromosome (Muller and Prokofyeva, 1935). The indentations have no known function. The heterochromatin in general extends from (and may include) the gap constrictions to the centromeres. I n D . m e l m g a s t e r the Y chromosome is totally heterochromatic, and the proximal third of the X is heterochromatic (Fig. 1). But there must also be differences within the inert regions of both the X and Y chromosomes as shown by cytogenetic tests. Translocations between the Y and fourth chromosomes, giving cubitus-interruptus position effect and affecting fertility, always occur at the distal ends of the Y (Khvostwa , 1939 ; Neuhaus, 1939). Deletions involving the proximal portion of the X either have nearly all the inert region present, or almost all absent (Dobzhansky, 1932 ; Muller and Gershenson, 1935 ; Muller and Painter, 1929 ; Painter and Muller, 1929). If the heterochromatin were genetically identical the breaks would be a t randonl. Cytologically, chromosomes 2 and 3 arc heteropycnotic both to the left and right of the centromere for about one-fifth of the length of each arm, and this heterochromatin is probably delimited by secondary constrictions (Heitz, 1933, 1935). However, I-Iinton (1942) found that the heterochromatin of the left arm of the second chromosome is composed of at least two blocks, one consisting of the region from the secondary constriction to the centromere, and another also represented by one-fifth of the left arm distally from the constriction. Presumably this heterochromatin is not heteropycnotic. The fourth chromosome in mitotic cells does not show pronounced heteropycnotic regions (Heitz, 1933 ; Kaufmann, 1934), but this may be due only to the small size of the chromosome. The extent of heterochro-

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Y

0

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FIG. 1. Comparison of mitotic Chromosomes, salivary gland Chromosomes, and crossover map distance. The salivary map is reduced to 1/200 in comparison to the mitotic map. Analogous regions were determined by translocations.

matic regions in the salivary gland fourth chromosome has been variously reported by different investigators. Griffen and Stone (1940) believed that there is no heterochromatin present in either arm. Bridges (1935) postulated that there are a t least two inert regions, one a t the tip of the chromosome, resulting in its tendency to conjugate with heterochromatin of other chromosomes, and the other basal and often embedded within the chromocenter. Slizynski (1944) suggested that there may be a heterochromatic region in the proximity of the chromocenter as well as at both free ends, and pointed out that the fourth is almost devoid of the heavy capsules conspicuous in the other chromosomes. Genetic evidence points to the probability that some if not all of the genes in this chromosome have heterochromatic properties (Schultz, 1939 ; Stern, personal communication). Localization of genetic loci along the length of the metaphase chromosomes by means of translocations (Dobzhansky, 1929, 1930 a and b, 1931, 1932 a and b, 1936; Muller and Painter, 1932; Painter, 1931; Painter and Muller, 1929 ; Sivertzev-Dobzhansky and Dobzhansky, 1933) revealed that although the linear order was the same as for the crossover

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map, the distances were different (Fig. 1 ) . The distal half of the X cliromosomc contains all the sex-linked genes but bobbed, which is in the inert region, A similar concentration of genes in the distal portions is exhibited by the second and third chromosomes. Cytologically, the regions which have the fewest genes also show the phenomenon of heteropycnosis (Kaufmann, 1934). I n addition, heterochromatic sections may also be within the euchromatin, f o r certain regions condense into a few large chromomeres in prophase chromosomes (Heitz, 1935 ; Kaufmann, 1946).

2. Xalivayy Gland C h r m s m e s a. Major Heterochomatic Regions. Meiotic and mitotic chromosomes give only an idea of the gross structure and distribution of the different kinds of chromatin, for it is difficult to determine the limits of either euchromatin or heterochromatin. Likewise, it is not possible to localize small heterochromatic sections in the euchromatin, or vice versa, because, as yet, there are no precise methods for determining, genetically or cytochemically, active and inactive regions. The salivary gland chroinosomes have proved to be more useful for detailed studies, for there are at least four recognizable kinds of heterochromatin: (1) the heterochromatin of the Y chromosome and the large block near the centromere of the X (and presumably the autosomes), which become part of the chromocenter and/or retain no definite structure ; (2) the heterochromatin which retains some structure in the salivary chromosomes; ( 3 ) the nucleolus-associated heterochromatin ; (4) the intercalary heterochromatin. The length of the major heterochromatic regions in a salivary gland chromosome of D . nzelanogaster, as well as other Drosophila species, is relatively much shorter than in the somatic chromosomes (Dobzhansky, 1944; Heitz, 1934, 1935; Pavan, 1946). About one-third of the length of the metaphase X chromosome is heterochromatic, but the same region forms less than one-twentieth of the X chromosome in the salivary nucleus (Fig. 1 ) . This may be because the heterochromatic regions adjacent to the centromeres fuse together into a common mass in the chromocenter (Bridges, 1935 ; Heitz, 1934, 1935), or because relatively long sections are represented by single bands (Muller, 1947; Muller and Gershenson, 1935 ; Muller, Raffel, Gtershenson, and Prokofyeva-Belgovskaya, 1937). The Y chromosome, which is longer than the X in mitotic chromosomes, is reduced to about nine discs in the salivaries, these being homologous with the heterochromatin in section 20 of the X (ProkofyevaBelgovskaya, 1937). The heterochromatic segment, from the centromere l o the first constriction, in the left arm of the second chromosome is re-

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duced to a single band in the salivaries, the bulk of the salivary heterochromatin of this arm being derived from the heterochromatin beyond the constriction (IIinton, 1942). The second kind of heterochromatin is that which retains some strncture in the salivary chromosome and therefore can be identified more or less by its banded nature. I n comparison with euchromatic regions in which all chromomeres of a band conjugate intimately, the heterochromatic bands are not as distinct, f o r the heterocliromonieres are not joined together in a regular manner. A precise differentiation between the two types of heterochromatin is even more difficult, for they have variable and overlapping morphological properties in tlifferent environments. Type 2, for example, may he a part of tlw cahvomocenter yet retain some banded structure. I n general the eii~hromomeresand Iiete~oc~liromonirres appear to be morphologically different. I n Drosophila, as in Chironomoiis, the euchromomeres may be small, completely stained dots, and the heterochromomeres large dots stained only at the periphery (Bauer, 1936). Or the heterochromomeres may be only slightly larger than the euchromomeres, yesiculate, and stained faintly (Kaufmann, 1944 ; Muller and Prolrofyeva, 1935 ; Prokofyeva-Belgovskaya, 1947). The heterochromomeres vary considerably under different circumstances, genetic and environmental. They may he capsules aggregated into a diffuse mass or meshwork, o r bands disaggregnted into chromioles (Prokofyeva-Belgovskaya, 1935, 1947) ; or they may manifeht rather distinct banding. The last is usually the case in the heterochromatic bands near the euchromatin. Even the boundary between euchromatin and heterochromatin is variable, for certain bands may appear either euchromatic or heterochromatic depending on the preparation (Pontecorvo, 1944). Nor are the characteristics of either necessarily retained in rearrangements involving euchromatin and heterochromatin. Schultz (Morgan, Schultz, and Curry, 1939) found that in one of the variegated mutants, white-variega ted-D-3 (breaks : 335.6 and following lOlF), the amount of nucleic acid in certain bands is correlated with the amount of variegation. At 25" C., when the phenotypic variegation is slight, bands in 3F shorn an augmentation of neucleic acid; a t intermediate temperatures, and intermediate variegation, the augmentation is more pronounced ; and at 16' C., and the greatest variegation, the regular arrangement of bands is replaced by an irregularity similar to that of the heterochromatic regions. Sutton (Sutton, 1940; Cole and Sutton, 1941) , on the other hand, found little heterochromatization of the euchromatic band when next to heterochromatin, and high variability within a single band. But, as pointed out by Schultz (1947), unless

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whole bands are compared the local difference between points within a band can mask differences between bands. Prokofyeva-Belgovskaya (1947) showed by use of statistical analysis that when inert regions are inserted into the euchromatin the heterochromatization decreases, and that when active regions are brought near inert ones the heterochromatization spreads to the active regions. Even though two neighboring nuclei may differ sharply, the same section being euchromatic in one and completely heterochromatic in the other, the per cent of heterochromatization has a constant value characteristic for a given stock under a definite condition. The third type of heterochromatic structure, the nucleolus-organizing region (NOR), likewise shows no morphologically distinct banding in the salivaries, although it has been established that nucleoli are produced at specific loci i n the heterochromatin but not on any specific chromosome. I n D. melanogaster, for example, the NOR’S are in the long arm of the X, adjoining 20B-C (Kaufmann, 1942) and in the short arm of the Y, whereas in D . ananassae, a closely related species, nucleoli are associated with the Y and the fourth chromosomes (Kaufmann, 1937). Transpositions or translocations of the NOR to other chromosomes in D. melanogader have occurred experimentally (Hannah, 1949 ; Kaufmann, 1938), and if it is broken in two parts each portion retains its nucleolar-organizing properties, proving that it must consist of several units (Kaufmann, 1938). The role of the nucleolus is difficult to evaluate, but it appears to be concerned with synthesis of ribonucleic acid. It may possibly act as a storehouse for products produced by the chromosomes and used by the cytoplasm, or it may participate in an intermediary step between the chromosomes and the cytoplasm (Caspersson and Schultz, 1940 ; Schultz, Caspersson, and Aquilonius, 1940). Breakage in the heterochromatic regions appears to increase the proportion of ribonucleic acid in the nucleolus (Schultz, 1939, 1941, 1944, 1947). Although the evidence for a particular function of the NOR is not conclusive, it does point to a close relation between the chromosome, particularly the heterochromatin, and the nucleolus in cellular metabolism. The limits of the major heterochromatic regions have not been established with certainty, either genetically or cytologically. However, by making use of a property of heterochromatin-greater sensitivity to breakage during or after treatment with radiation-it is possible to demonstrate the presence of heterochromatin in regions as small as the lettered subdivisions. X-ray-induced breaks are not distributed a t random on the basis of length of the chromosome as represented by genetic maps (Patterson, Stone, Bedicheck, and Suche, 1934) or by the salivary

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gland (;liroitiosoiiies (lhiier, 1939 ; Raucr, Demercc, and Kaufmann, 1938 ; Kaiifmann, 1939, 1946). I n general, the distribution of breaks is at random in the euchromatin but disproportionately high in the heterochromatic sections. I n the X chromosome about one-fourth of all breaks are in division 20. If this relation always holds, wide departures from randomness of breakage frequency within short distances can be attribnted to the presence of heterochromatin (Kaufmann, 1939). Division 19, €or examplv, has a high breakage frcquency, but most of this is due to the 1)reaks in 19141 ( ICiiiiFinnnn, 1939, 1946 ; Proltofyeva-Belgovskaya and Khvostovtt, 1!):i9). ‘I’Iiiis the major heterochromatic regions of the Y niiist cxtcntl from 19E through 20. The distribiition of break frcqucncics in the autosomes, based on divisions only, ;irv random, except for the divisions adjacent to the centromeres (Bniier, 1039 ; Tlauer, Ikmerec, and Kaufmann, 1938). Therefore, thc major lirtcrochromatic regions are in sections 40 and 41 in the second and 80 ;ind 81 in the third chromosome. Tn the fourth chromosome, the brcalts occur inoat frequently near the centroniere, a n d especially in 101F (Stern, unpublished). F. I n t e r c u l a r y Hcteroclwomatin. The diffuse banding or vesiculate nature of the major heterochromatin is not a criterion of intercalary hctcrochromatin (Slizynski, 1945). On the other hand, certain regions, within the euchromatin, simulate the behavior of the major heterochromatin in that they have a high break frequency. I n the X chromsome they are found in divisions 1, 3, 4, 11, 12, and 19 (Kaufmann, 1939, 19 44, 1946). Thc l~ehaviorsuggests that these sections are heteroehroiiiatic in nature. Secondly, certain regions of the euchromatin also exhibit, the property of nonspecific pairing. As pairing of corresponding hands is taken as a criterion o€ their similarity, when two “nonhomologous” regions exhibit this property it is assumed that they have some homology. The fact that they also show a tendency to pair with proximal hetrrochroniatin (ectopic pairing [ Slizynski, 19451 o r terminal adhrsionq [ IIinton, 19-15] ) indicates that these regions are heteroehromatic. Sections 1-4, 7, 9, 11, 12, 17, 19, and 20 exhibit the property of ectopic pairing ( I Iinton, 1945 ; Kailfmann, 1946 ; Prokofyeva-Belgovskaya, 1939, 1941 ; Slizynski, 1943). Finally Kodani (1941, 1946) found t h a t the salivary X chromosome showed a differential response to alkali-urea treatment, the “chromatic” (euchromatic) regions reacting less to the treatment than the ‘ ‘achromatic” (heterochromatic) regions. On the basis of this interpretation the heterochromatic sections are found to be in 2-3, 4, 6, 10, 11-12, 13, 18. The distribution of heterochromatin to the lettered subdivisions, as determined for high-breakage coefficients by Kaufmann (1946) and Prokofyeva-Belgovskaya a n d Khvostova (1939),

96

ALOHA HANNAH

and lor ectopic pairing by Kaufmann (1946), Kaufmaiin and collaborators (1948), Prokofyeva-Belgovskaya (1941), and Slizynski (1945) is given in Fig. 2. The fairly high degree of coincidence between the subdivisions showing the properties of ectopic pairing and high breakage coefficients suggest that these sections are intercalary heterochromatin. Such an extensive analysis of the intercalary heterochromatin of the antosomes has not been made, but it is probable that it occurs in these vhromosomes as well, and in a distribution similar to that of the X chroiiiosomc. Iiauer (1939)) calculating the breakage coefficients in the X vhroriiowiii(1 on the basis of 315 breaks distributed in 19 divisions (without taking into consideration the number of bands in a division), concluded that breakage is random. Kaufmann (1946), on the basis of 1048 breaks in 114 subdivisions, showed that certain intercalary regions have a significantly higher break frequency than others. It is probable that the break distribution in the autosomes would likewise be shown to be nonrandom if the analysis could be in terms of units smaller than divisions. Even in respect to divisions, if the number of bands per division is taken into consideration, certain ones show higher breakage coefficients than others (Fig. 2 j breakage coemcients recalculated from Bauer's data). These results are substantiated by other evidence. Dubinin, Khvostova, and Mansurova (1941) reported that breaks occur more frequently in subdivisions 21D, 22A-B, 22D, 22F, 57F, and 59F in thc second cliromosome, and 61C-D, 61F, 62B, 64B, 89E-F, 93E-F, 99A-€3, and 99F in the third. Patterson, Stone, Redicheck, and Suchc (1934; also Patterson, 1940) pointed out that the majority of translocation breaks occur either a t the free end or near the spindle fiber region of a chromosome, but that in chromosome 3 a high proportion of the breaks are found, genetically, to be between ebony (70.7) and rough (91.1). I n terms of the salivary gland chromosomes, this would be between 91F and 97F. Studies of ectopic pairing of the X arid antosomes reveals that there are several sections in both the second and third chromosomes which have characteristics of intercalary heterochromatin ( Kaufmann and rd.tilw, wportcd in Kanfmann, hlcl)onald, Gay, Wilson, Wymari, and Okii(Ia, 1948). Kven these incomplete data show a fair correlation between the regions having a high breakage cocfficierit and those exhibiting ectopic pairing (Fig. 2). Sections 89, 94, and 98 in chromosome 3, particularly, have a tendency to pair with other regions. This general region is also unique in having a high incidence of Minutes: nine of the 20 Minute loci reported for chromosome 3 in Bridges and Brehme (1944) are beyond 70 in the crossover map (Fig. 2). The Minutes, a group of mutants with similar

-

HETEROCHROMATIN IN DROSOPHZLA B l E L A N O G A S T E R

f

/

I \ 11-15

--

- --

- I -

97

98

ALOHA HANNAH

phenotypic effects, are found scattered along the length of every chromosome, but are especially concentrated near the spindle-fiber regions. Of the 23 Minute loci (those showing “allelism” are considered as a single locus) in chromosome 2, nine are within five crossover units of the centromere and Minute-S-10 (and an allele, M-2-D) is known to remove a large block of the heterochromatin. In the third chromosome, one-third of all Minutes are located within five crossover units of the centromere. They are thought by some investigators to be heterochroinatic in nature, hy others to be expressions of deficiencies p e r se (c.f., Goldschmidt, 1949 ; Mohr, 1932 ; Morgan, Bridges, and Sturtevant, 1925 ; Schultz, 1929). The concentration of Minutes near the centromeres supports the hypothesis that they are heterochromatic, or somehow associated with heterochromatin. If this is the case, Minute loci in distal portions of the chromosomes may be in intercalary heterochromatin. This relationship is further suggested by evidence obtained in conjunction with other tests. Minute loci in the second chromosome, as determined by deficiency tests, are a t 21C1-2 (Lewis, 1945), between 24D and 24F (Morgan, Schultz, and Curry, 1939), between 41B and 42A (Morgan, Schultz, and Curry, 1940), and in 49 (Morgan, Bridges, and Schultz, 1938). All of them are in regions having characteristics of heterochromatin. However, further analysis will be necessary before it will be possible to determine the exact relation between the Minutes and heterochromatin. Certain intercalary regions, showing a marked increase in breakage and having irregular and disturbed pairing, also appear to be repeats (Kaufmann, 1939, 1941 ; Prokofyeva-Belgovskaya and Khvostova, 1939). The concept that repeats are heterochromatic stems from Bridges’ (1935) study of the ‘(loops and turnbacks” in the left arm of the second chromosome. They were interpreted as duplications which had become fixed during the process of evolution. Depending upon their origin, repeats can be either direct (abcabc) or reverse (abccba), and each type is distinguished by a characteristic morphology (Schultz, 1947). Both types have been found in normal chromosomes, and have arisen spontaneously and in irradiated material (C. B. Bridges, 1935, 1936, 1938; C. B. Bridges and P. N. Bridges, 1939; P. N. Bridges 1941 a and b, 1942; Demerec and Hoover, 1939 ; Kaufmann and Bate, 1938; Lewis, 1941; Morgan, Schultz, and Curry, 1941 ; Muller, 1941 ; Muller, ProkofyevaBelgovskaya, and Kossikov, 1936 ; Offerman, 1936). From cytological evidence, section 34F to 35C appears to be a direct repeat of section 32F and 33 ; section 38E to 39E is a repeat of, roughly, 37 (Bridges, 1935). The reverse repeats are of two types: those which form capsules or bulbs and those which center around a ‘(weak spot” in the chromosome. The turnback in 36 and the shield in 30A have re-

HETEROCHROYATIN I N D R O S O P H I L A MELANOGASTER

99

versed sections symmetrical about a center, and arc examples of the capsule form. The larger capsules, such as 25A and 66F, are probably also symmetrically reversed repeats, and 33B is probably a reverse repeat within the direct repeat 32-33 (Bridges, 1935). The “weak spot” type is found in regions 3C, l l A , 12D, 12E, and 19E (Bridges, 1935; Kaufmann, 1939 ; Kossikov, 1936). I n the 11-12 region, “Kossikov’s repeat,” 11A and 11B conjugate with 12D and 12E, but the bands lying between do not. The sections showing repeats as determined by C. B. Bridges (1935, 1937, 1938), C. B. and P. N. Bridges (1939), P. N. Bridges (1941 a and b, 1942), and Kaufmann (1946) are given in Fig. 2. The 3C section has been studied rather extensively (Panshin, 1941), for several well-known mutants occur in this region. The repeat includes the bands from 3C1 through 3C7 (and possibly 3B4 and 3C8), 3C1 (on the basis of the reverse-repeat concept) being a repeat of 3C7, and 3C2.3 a repeat of 3C5.6. The “buclde-shape,” assumedly due to partial synapsis of homologous bands, has been reported to disappear when chromosomes, each of which had one of the duplicate loci removed, were combined (Morgan, Bridges, and Schultz, 1937). This would be expected if the duplicate bands were still partially homologous. On the basis of their characteristic morphology, “doublets” may also be repeats (Bridges, 1937 ; Demerec, 1940). They have been grouped with the large repeats, but it is probable that they have quite different genetic properties. Of 5149 bands in the salivary chromosomes, 1299 are doublets (Bridges, 1942 ; Slizynski, 1944 ; White, 1948). These data would suggest that there may be a number of duplicate or semiduplicate loci. Griineberg (1937) and Serebrowsky (1938) have pointed out that genes with a similar phenotype could have occurred as repeats and thus a t the time of origin have had identical function, but have since developed along divergent lines. Genetically, mutants that are allelic and yet have crossing over between them have been interpreted as repeats. Several such mutant pairs are known to be located in or a t least very close to doublets. Salivary analysis of Star and asteroid placed them in a duplicate band 21E1 and 2 (Lewis, 1945) ; BithoraxoidD, bithorax, and bithorax8 (with presumably only two loci involved) are within or proximal to the 89E1 and 2 doublet; Stubble and stubbleoid2 are in the neighborhood of the 89B3 and 4 doublet (Lewis, 1948). The lozenge complex, a triplicate locus genetically (Oliver, 1940 ; Oliver and Green, 1944‘; Green and Green, 1949) with perhaps almondex as a fourth member of the group (Bridges, 1936), has been located tentatively in section eight. Achaete, Hairy-wing and scute are in a region of three doublets (1A5.6, 1B1.2, 1B3.4) (Sutton, 1943). I n the scute-8 inversion (break following 1B1.2), which leaves achaete in the left end of the

100

ALOHA HANNAH

chromosome and transfers scute to the right end, the achaete and scute “bands” tend to conjugate, suggesting a cytological homology as well as a genetic similarity (Serebrowsky, 1938). Other mutant pairs which may possibly represent repeats are : chlorotic (0.1 - ) and yellow (0.0) and possibly silver (O.O&) ; miniature (36.1) and dusky (36.2) ; pupal (51& ) and pads ( 5 5 h 3 ) ;blistered (107.3) and balloon (107.4) ; tetrapter (51.3) and bithorax (58.8); blistery (48.7) and Bubble ( 4 8 k ) (Griineberg, 1937). Another attack on the problem of repeats was by comparison of the number of loci with dominant “notch-like wing” phenotype having a “facet-like eye’’ locus next to them. Of seven wing mutants, five (Notch, vestigialD, Jagged, Serrate, and Lyra) have mutants involving disarrangement of the ommatidia to either the right or the left, while two (Beadex and Beaded) do not (Morgan, Schultz, and Curry, 1940). The distribution of these types of factors with respect to one another is therefore not a t random. One interpretation could be that the first five represent repeats of a double locus (ABAB) and subsequent translocation of one pair (AB) to another region, or another chromosome. Of the seventeen pairs mentioned above having characteristics of repeats, seven are in intercalary heterochromatin (using breakage frequency and ectopic pairing as the criterion), seven are probably in intercalary heterochromatin, two are in euchromatic segments, and one pair has one locus in heterochromatin and the other in euchromatin. There is no a priori reason why intercalary heterochromatic regions should be associated with repeats. Muller (1918, 1938) suggested that genes which had mutated either to a deleterious type or to inertness would have greater survival if they were in repeats. Since one-half of the genes in a repeat are not necessary for life, it would be possible for one gene to retain its function whereas the other could lose its specific properties. This seems to be the case in the 3C duplication, for deficiencies including 3C5.6, but not 3C2.3, do not show the white phenotypes, and deficiencies covering 3C1, but not 3C7, are not Notch (data summarized from Bridges and Brehme, 1944). I n the “Kossikov repeat,” conversely, the garnet “loci” seem to have retained their functional activity, for about one-third of the viable garnet mutations are subliminal (have no phenotypic expression) except in compounds with a visible garnet allele (Muller, 1947). If, however, the heterochromatic property is not an expression of degeneration of euchromatin, the relation between repeats and heterochromatin must have another explanation. The behavior of the repeats intimates that they must have arisen in phylogeny by inversions or duplications (Bridges, 1935 ; Kaufmann, 1939 ; Kaufmann and Bate, 1938 ;

BETEROOHROMA‘PIN IN DECOSOPHIZA M E Z A N O G A S T E R

101

Kossikov, 1936 ; Offermann, 1936). If the heterochromatic property is an expression of the linear order of chromomeres of similar function, as suggested by Pontecorvo (1944), then a heterochromatic segment should arise every time a euchromatic region undergoes duplication and the replicas remain adjacent to each other. O r the heterochromatic regions may have been present very early in phylogeny, with the duplications coming only secondarily and remaining because duplications or deficiencies in the heterochromatic regions are less subject to rigid selection. The age of several of the duplicated regions is very great, for both the bulb in section 2 and the 11-12 duplication are found in both D . sirnulam and D . melawguster (Offermann, 1936 ; Kossilrov, 1936). Even species as remote as D . rnelanogaster and D . pseudoobscura have similarly high concentrations of repeats in chromosomes which, on other grounds, are presumed to be homologous (Dobzhansky, 1941).

111. GENETICEFFECTS OF HETEROCHROMATIN The greater part of the proximal heterochromatin appears not to have a distinctively discernible genetic function, and the few mutants located within major heterochromatic regions (bobbed in the X, light, straw, and lightoid in the second, inturned and pink in the third, cubitusinterruptus and bent in the fourth) are not in any way remarkable for their phenotypes (Schultz, 1939 ; Sntton, 1942). The lack of discrete active genes in the Y chromosome led Muller (1914, 1918) to postulate that the accumulation of inactive genes, as well as losses or duplications of chromosomal parts through translocations, could not be harmful to the fly because of the hypomorphic condition of the Y. When it was shown that the X chromosome has a similar genetically inert region (Muller and Painter, 1932) and that there is crossing over between the X and Y (Kaufmann, 1933; Neuhaus, 1936; Philip, 1935; Stern and Doan, 1937) it became obvious that the inert region cannot be full of degenerate genes. As the heterochromatin is fundamentally different in certain properties from the enchromatin, it was suggested by Muller and Gershenson (1935) that inert regions are essentially of nongenic material. This theory is substantiated by the fact that the Y and “Block A” of the X are not necessary either for life or for a normal external phenotype (hluller, Raffel, Gershenson, and Prolrofyeva-Belgoi.sItaya, 1937). It has been pointed out, however (Darlington and Upcott, 1941; Mather, 1942, 1944; Pontecorvo, 1944), that if there was a full quota of genes, each having a small quantitative effect, mutational changes would be difficult to detect, and deficiencies or duplications would consequently be less harmful. Polygenes appear to be examples of genetic fnnction

102

ALOHA H A N N A H

of this type (Mather, 1944). But heterochromatin also has functions, possibly qualitative as well as quantitative, which do not fit into the category of polygenic activity.

1. Positim Effect and Variegation. When it was found that genic action could be modified by chromosomal breaks located in the neighborhood of a gene, it was realized that the original concept of genes as discrete independent entities was somewhat too simple. Of the two theories to explain this phenomenon-(1) the theory of gene mutation, genetic suppression, or a cytological deficiency accompanying but independent of the chromosomal rearrangement (Bridges, 1923; Demerec, 1939, 1941 ; Morgan, Bridges, and Sturtevant, 1925 ; Muller, 1932 ; Muller and Altenburg, 1930) ; and (2) the theory that the function of the gene depends upon its position in the sequence, so that its properties are altered when it is transferred to a new position (Dobzhansky, 1932, 1936 ; Sturtevant, 1925)-the second, the "position-effect " hypothesis, is accepted by most today. Experimental evidence substantiating it is as follows. Cytological reversion accompanying a genetic reversion proves that no change other than the chromosomal change has taken place (Griineberg, 1937). A gene close to a rearrangement and showing position effect reverts to normal when removed by crossing over (Dubinin and Sidorov, 1935 ; Panshin, 1935). Crossing over between a white-variegated mutant (wm4)and other notquite-identical inversions shows that the decisive factor in determining variegation is located at the junction between the white locus and the heterochromatin (Schultz, 1943). Position effect may occur as a result of (1) suppression or change in the activity of the gene, either by loss or change in its position (Demerec, 1939, 1940, 1941; Muller, 1932) ; or (2) change in interactions between gene products or competition between genes for a common substrate (Muller, 1935; Offermann, 1935; Stern, 1949) ; o r (3) reversible modification of the genes o r their structural regions due to abnormal pairing (Ephrussi and Sutton, 1944) ; or (4) production of the phenotype of a mutant locus by a break occurring in the neighborhood of the normal locus (Goldschmidt, 1946). The results of numerous investigations show that alleles having phenotypic similarities may be caused by different cytogenetic changes. F o r example, of 102 mutations in the white-Notch region, 28 were fully viable point mutations at the white locus, 30 were deficiencies, 3 were lethal with no cytological changes, 39 were chromosomal changes, and 2 were both deficiencies and chromosome changes (Demerec, 1939, 1940, 1941). The rearrangements--R (4) or position-effect mutants-ould

HETEROCHROMATIN IN D R O X O P E I L A Y E L A N O B A S T E R

103

be divided into those in which the recipient region was euchromatin (10) and those in which it was heterochromatin (29). I n the former the breaks were immediately to the left or right of the Notch band (3C7), whereas those which had the second break in the heterochromatin could have the first break as far as 50 bands from the locus. Therefore, the heterochromatin must be capable of exerting a n influence over a much longer distance than the euchromatin. However, the expression of the mutant phenotype in the majority of these cases may be a “Dubinin effect”; that is, the phenotype is expressed only when heterozygous with a recessive (point mutation) allele and is normal when in the homozygous or hemizygous state. The most striking genetic property of heterochromatin is the induction and modification of variegation in somatic cells. Variegation is characterized by mottling (mosaic pattern of several colors) of eye o r body color, or mosaic pattern of affected and nonaffected bristles, or variable expressivity of the wing pattern. Structural changes, involving one break in the euchromatin and one in the heterochromatin, characteristically give rise to this type of variegation (Demerec, 1939, 1941; Demerec and Slizynska, 1937 ; Gowen and Gay, 1933, 1934 ; Muller, 1930, 1935; Noujdin, 1936, 1938; Schultz, 1936, 1939, 1941, 1944, 1947). Following the terminology introduced by Stern (Stern and Heidenthal, 1944; Stern, Schaeffer, and Heidenthal, 1946) and Lewis (1950), R(+) will be used to designate rearrangements having a position effect; those which are variegated will be referred to a s V-type and those which are somatically stable as S-type. There is some evidence that gross structural changes are not always necessary for the induction of variegation. Muller (1946) reported that a recessive sex-linked mottler mutant near vermilion, in combination with alleles of the white locus, causes them to be variegated, resembling “many of the lighter mottled alleles of white that are caused by the juxtaposition of the white locus to heterochromatin.” Among five cut mutants showing variegation, three were gross chromosomal rearrangements involving breaks in the euchromatin a t the cut locus and heterochromatin, hut in two no cytological change was detectible although there was a tendency for the 7B region to show considerably more ectopic pairing than characteristic (Hannah, 1949). It is possible that variegation in these mutants is due to a minute insertion of heterochromatin in the neighborhood of the cut locus. I n terms of the position-effect hypothesis, variegation is due to euchromatic genes being brought within the range of influence of the heterochromatic regions. Thus, if the euchromatic break is close to the white locus the eye will appear mottled for several colors. But other

SO4

ALOHA HANNAH

loci close to white (roughest, facet, split, Notch, diminutive, and echinus) may be affected as well. Other examples-yellow, Hairy-wing, achaete, and/or scute-may show variegation as a result of breaks in section 1 ; or brown, minus, and abbreviated if the breaks are in 59D or E. Schultz (1939) has put forward the following general explanation to account for the mottling effect; the juxtaposition of euchromatin and heterochromatin may alter the nucleic acid metabolism of the bands nearest to the point of breakage, resulting sometimes in heterochromatization of the bands, sometimes in an actual loss. The process appears to be a sort of inactivation, with the point of breakage acting as a center from which the disturbance spreads. I n line with this concept, Demerec (1939)attributed the instability to either a reversible chemical change, or a reversible suppression of the activity of the gene. Offermann (1935) postulated that the activity of the gene is influenced by the environment ; therefore, if a rearrangement changes the environment a different expression of the gene will be invoked. If this change is great, the affected locus may be dominant, since there is nothing corresponding to the substance produced by the other chromosome, and therefore no allele to cover it. Or the supply of substance may fail to satisfy the requirenients of the cell, and therefore is expressed in the phenotype as an absence. Prokofyeva-Belgovskaya (1947) maintained that there is no actual loss, but heterochromatization. Heterochromatin shortens the metabolic cycle. Increased heterochromatization of an active section will therefore shift it toward recessive manifestation. I n extreme heterochromatization, the genes affected may be entirely eliminated from the developmental processes, and the result will simulate the loss of the gene. a. The Euchromntic Break a.nd Variegation. Numerous loci show variegation in mutant forms having juxtaposition of euchromatin and heterochromatin. The length of the sensitive region varies for the different loci. I n the X chromosome, for instance, the sensitive region of the white-Notch groups may be as much as 30 bands in the salivary gland chromosome, that of rugose-vesiculated from 20 to 80 bands, and yellow-scute about 10 bands ; whereas in cut the breaks are always within one or two bands of 7B (Demerec, 1940,1941; Hannah, 1949). Position effect on the brown locus in the second chromosome and curled locus in the third may likewise extend an appreciable distance along the chromosome (Dubinin, 1936 ; Panshin, 1935). The relation of position effect to the euchromatic break is best shown by the white-Notch variegated mutants. Of those which have been analyzed (data from Bridges and Brehme, 1944), 48 had one or both euchromatic breaks in apposition to heterochromatin (Table 1). I n general the expression of variegation does not depend on both the euchromatic

105

HETEROCHROMATIN IN DILOSOPHILA Y E L A N O Q A S T E R

TABLE 1 Relation of the Euchromatic Break of R ( + ) white-Notch variegated Mutants to the Phenotypic Expression of White ( w ) , roughest ( r s t ) , facet or split ( f a ) , and Notch ( N ) . (Data from Bridges and Brehme, 1944.)

Euchromatic break

Heterochromatic break

Phenotype

X chrom. 2nd chrom. 3rd chrorn. 4th chrom.

Between 3B2 & 3C5.6

3

4

3G9.10 Between 3C9.10 & 3F6

3

9

2

1

w , rst, or w-rst r8t-N fa-N

W-N

-

w , rat, or w-ret

Between 3C5.6 &

5

1

1 1 3

1

1 1

TSt-N fa-N W-N

2

w , rut, or w-rst TS1-N fa-N W-N

1 2

7

ends being next to heterochromatin, but there is some evidence that the degree of expression may. The number of adjacent loci showing position effect likewise appears, in general, to be dependent on the position of the euchromatic break. Twenty of the twenty-four V-type R ( + ) mutants with breaks close to white (3C2 and 3 ) show only white, roughest, or white-and-roughest variegation ; R ( +) mutants with breaks near Notch (3C7) may have only facet and Notch phenotypes or all the mutant phenotypes (white, roughest, facet or split, and diminutive) ; breaks beyond 3C9, in eleven out of twelve mutants, show variegation for all loci from white through diminutive. Thus, some have a strict correlation of expression of phenotype with the distance of the locus from the break and others do not. This may be due to circumscribed reaction of breaks at specific loci in euchromatin, but is more likely correlated with the position of the heterochromatic break (see below). The second characteristic is progressive weakening of the expression of a specific locus the farther it is from the break. Thus in white-variegated-258-18 and white-variegated-D-3, with breaks to the right of white, in compounds with w and rst or spl, rough facets may be wild type in color (Demerec and Slizynska, 1937 ; Morgan, Bridges, and Schultz, 1938; Schultz, 1939). The extent of mottling in white-mottled-1 is directly correlated with the extent of the Notch expression (Muller, 1930). Another white-variegated mutant, however, was reported (Patterson,

106

ALOHA HANNAH

1932) to have independent expression of mottling of the eye and notching of the wings. This mutant also showed different expression in different genetic environments. It was variegated for white in the homozygous female and hemizygous male; variegated for white, Notch, facet, and TABLE 2 Summary of Effects of Rearrangements, Involving One Break in a Heterochromatic R.egion and Another in a Euchromatic Region, 011 the Expression of Position Effect at Euchromatic and Heteroehromatic Loci. (Data from Bridges and Brehme, 1944; KMTostova 1939; Stern, unpublished.)

Position of heterochromatic break

Zltduced phenotype Of :

X Chromosome 18 19

E-F A-C D Euchromatic loci None Not variegated Variegated Heterochromatie loci Both loci Not variegated

1 1 1 I? 1

E

1

20 F A

B C D

4 2 1

1 3

3 2 2

2 2 1 2 2

E F 1 1

It. rt. bb

. bb

het.

2 11 2 9

3 3 7

1 1 Chromosome 2 40

Euchromatic loci None Not variegated Variegated Heterochromatic loci Both loci VaPiegated a

B C

D

1

1

1

1

E

F 3 1 2 1

1

41 A B 6

1

C D E 2 2

2 1

8 3

1

42 F A 2 L2 Rh et . 2 1 4 2 2 3 4 1 I? 5 6 4 1 1

3

1

3

~

Chromosome 3 79 80 81 82 E F A B C C-D D-F F A - - E F A 3L 3R het. cent, 9 Euchromatic loci None Not variegated Variegated Both loci Variegated

1

1

2

1

1

1

1 1 3

2 1

1 1

1

1

1

5 7 1 6 1

3 2 5

3

3 3 9

107

HETEROCHROMATIN IN DROSOPHILA M E L A N O f f A S T E R

TABLE 2-Continued Chromosome 4 101 A B

Euchromatic loci None Not variegated Variegated Heterochromatic loci Both loci Not variegated a Variegated a

C

DE F 1

1

102

A B C D E F 4L 4R het. cent. 9 2

1

4

116

5

6

7

6

1 2 1 1 2 2 3 2

10 13 1 3 1 5

1

2

1

2

1

48 1 4

22190 1 2

Abbreviations: lt.bL, left of bobbed : rt.bb, right of bobbed ; het., heterochromatin; cent., close to the centromere; 1, position of the break not known. 1111 most of these case8 position effect on loci close to the break was not tested. This applies to both euchromatic and heterochromatic loci. *Not variegated (or variegated) refers only to the euchromatic loci.

echinus in the heterozygous female; variegated for white but not for facet o r echinus in the hyperploid female with two white chromosomes. The fact that echinus was affected would suggest that the break was beyond 3C9, but as the cytology is not known this may represent a special case ; possibly there were several breaks near white. The data, taken as a whole, point to the probability that the expression of a locus in V-type R(+) mutants is determined to a degree by the distance of the break from the locus in question. I n respect to the phenotype, the closer the point of breakage to a specific locus, the more extensive is the variegation and the more extreme is the allelomorphism in those cases where allelomorphic series are distinguished (Schultz, 1939). b. The Heterochromatic Break a d Variegation. Several lines of evidence have shown that the heterochromatic regions of the X and the autosomes exert the same effect as the Y in the expression of certain R ( ) genotypes. Extra pieces of the X, if they include the inert region, have an effect similar to that of an extra Y (Noujdin, 1936) ; X chromosomes deficient for the inert region enhance the expression of variegated mutants, and translocations of the X and Y are similar in their action (Khvostova, 1939). Breaks in heterochromatin producing variegated white-Notch mutations may have specificity in some cases, or may give quite similar phenotypes in others (Demerec, 1939, 1941). Data (compiled from all available sources) summarized in Table 2 include the effect of heterochromatin on R mutants with breaks a t the yellow-scute, white-Notch, cut, brown, curled, and cubitus-interrup-

+

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tus loci as well as others. Some-eut, for example-in which variegation was not tested are listed under the heading “Not variegated” (S-type). In actuality, this group must be smaller than is shown in Table 2. “None” (no known phenotypic change) means, in the majority of cases, that loci at the euchromatic break have not been tested for position effect, In general, the heterochromatic regions of all the chromosomes produce variegation phenotypes in R ( +) mutants. However, certain sections appear to give a higher incidence of V-type R ( +) mutants than others; e.g., 208, B, and C in the X, 40F and 418 in the second, 80C and possibly 80D in the third, and lOlF in the fourth. In spite of the fact that the centromeric heterochromatin of all the chromosomes is sufficiently alike to form a chromocenter, and all give variegated phenotypes (Tables 1 and 2 ) , there is some genetic evidence that there may be qualitative as well as quantitative differences. Certain white-Notch mutants have special characteristics dependent upon the heterochromatic break (Demerec, 1939) ; in other words, some are S-type mutants. Three scute alleles, with identical left breaks, show slight differences due to the position of the right break (Raffel and Muller, 1940). It appears probable that different regions of the heterochromatin differ in their potentiality to produce position effect and particularly to affect the expression of variegation. (1) X chromosome. The data in Table 2 concerning the X-chromosome R ( + ) mutants, compiled on the basis of the position of the break in the heterochromatin, show the following. Among three with breaks in the 3B2-3C5.6 region, two are left of the NOR and have whitemottled and roughest-bifid-mottled phenotypes, respectively ; and one in the NOR shows variegation for roughest only. Of four with euchromatic breaks between 3C5.6 and 3C9.10, one, left of the NOR, shows variegation for facet, Notch, and diminutive; one in the NOR shows variegation for facet and Notch; and of two to the right of the NOR, one shows stable changes a t facet and Notch and one a stable change a t facet and variegation for Notch. One having a break beyond 3C9.10 also has the right break to the right of the NOR and shows stable changes a t the roughest and Notch loci. These data confirm Demerec’s finding (1939) that the position of the right break of the X chromosome is partially responsible for the expression of the phenotype. The dependence of the phenotype on the heterochromatic break is even more clearly expressed in the case of the roughest locus. Expression of roughestS variegation is dependent upon the proximity of the roughest locus to that portion of the X which includes the NOR and the centromere, and

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reversion always involves a secondary break a t the NOR (Kaufmann, 1942). ( 2 ) Y chromosome. It was long thought that the Y chromosome, which is completely heterochromatic, is genetically inert (Bridges, 1916 ; Muller, 1914, 1918), but several investigators have pointed out that this inertness is not complete. Although the Y chromosome of D. melamgaster is not absolutely necessary for life, since males lacking a Y are viable, it does have regions which insure male fertility (Bridges, 1916; Neuhaus, 1939; Stern, 1929), and in some species the Y has more generalized effects, and may influence the viability of the male (Sturtevant, 1937). On a priori grounds one would expect that any part of the chromosome set which was completely useless would have been lost in the course of phylogeny. The fact that the Y chromosome is preserved (only one Drosophila species is known to lack a Y ; see White, 1948), and that the inert regions in all the chromosomes retain their general position next to the centromere in so many species, makes it seem probable that heterochromatic regions have some function necessary for survial of the species (Dobzhansky, 1944). It may be that the genes of the Y were never like those of the X in their present form (Muller, 1932) and that the Y has a different genetic function from the X, which cannot be measured by our present standards. I n D . melanogaster it is possible to obtain in genetic experiments flies that have one or two Y’s in addition to the normal set, or males that lack the Y. I n normal genotypes, addition of the two extra Y’s to either the female or the male induces mottling of the eyes (Cooper, 1949). Addition of a Y to V-type R ( + ) mutants whose normal loci are in euchromatin tends t o suppress variegation, and removal of a Y (XOd) increases variegation ; likewise, addition or subtraction of an arm of the Y (-YL,Ps)influences the expression of variegation (Dubinin and Heptner, 1935; Gowen and Gay, 1933, 1934; Schultz, 1936, 1939, 1941, and 1947; Schultz and Dobzhansky, 1934). Thus, in the case of euchromatic V-type R(+) mutants, the effect of heterochromatin appears to be a matter of genetic balance; the higher the heterochromatin in proportion to euchromatin, the less the variegation, and vice versa (Schultz, 1939). In agreement with these results, the effect of the Y on V-type R ( + ) mutants whose normal loci are in heterochromatin is the reverse; the XO light-variegated male, for example, is more nearly normal, and the XYY and XYYY males have lighter eyes (Khvostova, 1939 ; Morgan and Schultz, 1942 ; Schultz, 1936). Besides modifying variegation phenotypes, changes in the Y -chromosome number may produce new morphological types when in combination with deficiencies of other heterochromatic regions. XO ;

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N(2)S-10 males have spread wings and coarser bristles than X Y ; N(2)S-lO males, and there is some evidence that the XYY condition intensifies the Minute effect (Morgan and Schultz, 1942). It may be that the Y and 2R heterochromatin are antagonistic in their action (Hinton, 1950). Increasing or decreasing the number of Y chromosomes may have other effects; the number of Y’s exerts a determining effect on the structure of the salivary gland chromosomes, particularly the ends, and on general size and staining capacity (Bridges, 1935; Schultz, 1936, 1947). X X Y females have more nucleotides than X X females (Caspersson and Schultz, 1938; Schultz, 1939; Schultz and Caspersson, 1949), although this finding has been questioned by Callan (1948). The Y has specific modifying effects on the expression of podoptera mutants, and in one of them, the podoptera phenotype cannot be expressed in the females unless a Y is present (Goldschmidt, Hannah, and KellenPiternick, 1951). The Y influences cell size of the wing and eye corneolae (Barigozzi, 1947), the length of the third longitudinal vein in the wing (Neuhaus, 1939), and the number of sternopleural bristles (Mather, 1944). Two variegation mutants, white-258-18 and Xc8, are lethal in XO males (Schultz and Catcheside, 1937 ; Schultz, 1939). The presence of extra Y’s in the male decreases X-ray-induced visible and sex-linked-lethal mutation (Kerschner, 1949). The most important function of the Y chromosome is the assurance of male fertility. XO males and males with some of the deficient ’Y’s are sterile (Bridges, 1916; Panshin, 1935; Stern, 1929). In 90% of the translocations of the Y with the fourth chromosome, the males are sterile (Muller and Qershenson, 1935). Of 46 Y-4 translocations, Neuhaus (1939) found only eight which gave fertile males ; and, of these, four had chromosomal rearrangements more complex than a reciprocal Y-4 translocation. By analysis of the 38 R ( + ) sterile mutants, he concluded that there are ten genes affecting fertility, located near the ends of the arms of the Y chromosome, The fact that deficient Y or complete loss of Y results in sterility indicates that sterility is due to the loss of fertility genes. But XYYY males are also sterile (Cooper, 1949; Schultz, 1947), and there is some evidence that the XYYYY condition may be lethal (Cooper, 1949). Therefore sterility must be due to a balance of factors as well as to individual fertility genes. The relationship may be even more complex than either a balance or loss, however, since males deficient for most of the X but with a normal Y are fertile (Qershenson, 1933; SivertzevDobzhansky and Dobzhansky, 1933) ; males with certain aberrant Y

FIG. 3. Cytogenetic map of the eentromeric region of the second chromosome, showing location of mutants, Minutes, and genetic and cytological deficiencies.

0 Q B al Y

h

Lu

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chromosomes are fertile ; but deficient-X, aberrant-Y males are completely sterile (Schulte, 1947). ( 3 ) Autosmes. It was shown, for the X chromosome that probably the number of loci affected-the length of the sensitive region-in the R ( + ) mutants of the white-Notch region is primarily dependent on the position of the euchromatic break (Table l ) , but that deviations occur because of location of the break in the heterochromatic regions (Table 2 ) . If it is assumed that the length of the sensitive region in the euchromatic section is due to specific influences of heterochromatin, the regional differences in heterochromatin may be determined. In respect to autosomal heterochromatin, when the breaks of white-Notch R (+) mutants are before 3C5.6 and after 3C9.10 the expected phenotypes occur, namely, white and/or roughest in the first case, and white-Notch in the second. But four of the eight mutants with breaks between 3C5.6 and 3C9.10 are variegated for white as well as facet and Notch, and one shows roughest but not white variegation. Thus the autosomal heterochromatin does appear to have specific properties, and that of chromosome 2 may be more effective in initiating position effect and variegation than that of either 3 or 4. The heterochromatin in the right limb of the second chromosome is advantageous material for the study of heterochromatic effects, for a number of mutants are in this region, as well as a group of deficiencies and chromosomal changes giving position effect (Fig. 3 ) . The section usually considered heterochromatic is from 41A to 42A, although only 41A and B, and rarely 41C and D, form part of the chromocenter and show ectopic pairing with other heterochromatic regions. I n respect to expression of variegation in 41A, three of the six R (+) mutants not showing position effect of euchromatic loci were Y-2 translocations, and one was a white-diminutive-variegated mutant with the breaks of the white locus attached to 57/58, and the heterochromatin of the X attached to the heterochromatin of 2R. Eight had variegation phenotypes : one white-Notch, one roughest-Notch, one white-roughest, and two white; two brown; one clot-pied. In 41B, one was not tested and the other two were variegated, one white-diminutive and the other brown. 41C had only two, neither of which was tested. One mutant, a scute, which probably was not variegated, had its second break in 41D. Two breaks in 41E with position effect on the cut locus were not tested for variegation. Among the four mutants with breaks in 41F, the forked and scute were not variegated, and cut was not tested; the white was variegated, but as it also had a break in 41A the variegation effects may have been due to that break. Tests made with deficiencies of the 41-42 region (most of which

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show the Minute character) in compound with variegated mutants white, brown, Revolute) reveal that Minute (2) S-10 and Minute (2) D have an effect equivalent to that of removal of the Y chromosome, i.e., they enhance the variegation effect; and that duplications of this region suppress the variegation (Morgan, Schultz, and Curry, 1941). The Y chromosome, on the other hand, intensifies the Minute effect of Minutes-10. Analysis of the chromosomes of this mutant discloses that the salivaries are deficient for, at the most, only several bands in 41A, whereas there is a loss of from one-fifth to one-third of the right arm in mitotic chromosomes. The fourth chromosome is not heterochromatic in cytological terms, for it is not heteropycnotic (Kaufmann, 1934), but it does seem to have heterochromatic genetic properties (Schultz, 1939). Among 24 translocations involving the fourth chromosome, which displayed position effect on non-fourth-chromosome loci, 23 had variegation phenotypes. These breaks were scattered over the greater length of the fourth chromosome, although the majority were in 101 F. Either the nonvariegated mutants are not viable, all breaks are preferentially in heterochromatin, or the fourth chromosome is primarily heterochromatic in genetic action. I n the case of “reversions” the effect of heterochromatin seems to be somewhat different. Hinton (1950) found that in “reversion-mutants” of In(2LR)40d1the degree of reversion of the phenotype is correlated with the quantitative effect of the heterochromatin, and not all regions are equally effective in changing the phenotype. The heterochromatin of section 80, for example, acts as a euchromatic region. Four types of heterochromatin are suggested by the differences in effect : ( 1 ) heterochromatin that is visible in the salivaries and has a n inhibiting effect; ( 2 ) heterochromatin not visible in the salivaries, and inhibiting; ( 3 ) heterochromatin visible but not inhibiting ; and (4) the Y heterochromatin, which is not visible and not inhibiting. c. Comparsm of X , Y , and dutosomd Heterochrmatim. Heterochromatic regions in all the chromosomes are equally effective in initiating variegation of position-effect mutants with one break in the euchromatin and one break in the heterochromatin. Some regions display a more pronounced effect on the phenotypic expression of individual mutants, whereas others manifest an action which is primarily concerned with the length of the sensitive region from the euchromatic break. A direct comparison of quantitative effects of the different heterochromatic regions upon typical variegation mutants has not been feasible because of the numerous variables involved. Such a comparison, however, has been made for a fourth-chromosome mutant (not associated

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with a n interchromosomal exchange) which is sensitive to changes in heterochromatin balance (L. V. Morgan, 1947). This mutant, sparkling (spa), shows variegation, not in a single fly but between sibs and between different cultures; and thus it may not be strictly comparable to variegation mutants. On the other hand, the phenotype is modified by addition or subtraction of heterochromatin, and is affected by the physical environment. Tests were made for correlation between the manifestation of expression and the relative amounts of euchromatin and heterochromatin, by combining the mutant chromosomes with chromosomes having deletions or duplications of heterochromatin. When heterochromatin of the X or Y is increased, the manifestation of spa is diminished, and when heterochromatin of the X, Y, or 2R is decreased spa is enhanced. The order of effect from (complete) suppression to extreme expression is given for each chromosome: (1) Y chromosome. ( X ) Y Y : (X)Y:Yst (deficient for the short arm of Y ) :Ybb (deficient for an unknown amount of the short arm of Y, including the wild-type allele of bobbed) : Ybb- (deficient for bobbed and lacking two-thirds of the short arm of Y) : ( X ) O in the male. (2) X chromosome. (XX)XDp (fragment consisting almost entirely of heterochromatin) :XX :X (deficient for an unknown portion of the proximal right arm of each&) :Xbb (may be deficient for bobbed; the extent is not known) : XDf-D(the X is two-thirds normal length in metaphase; to the right of and including bobbed; 20C-D) : XDf-G(threefourths as long as normal; to right and left of and including bobbed; 19F to right of 20C).. (3) Chwomosome 2. Normal 2 : M-S-10 (deficiency of from onethird to one-fifth of the right arm of the second chromosome). (4) Chmmosome 4. Haplo-4 and Dp (2 ; 4 ) B equally effective. d. Modification of Variegaticm. Variegation is modified by temperature, by the Y-chromosome balance, and by modifiers (Chen, 1948; Gersh, 1949 ; Gowen and Gay, 1933, 1934 ; Morgan, Bridges, and Schultz, 1937 ; Schultz, 1939). One of the most striking effects of modification is the change of variegation which occurs in combination with other rearrangements. These may be in the same chromosome or may be completely independent. Schultz (Morgan and Schultz, 1942 ; Morgan, Schultz, and Curry, 1941 ; Schultz, 1941) determined the distribution of enhancers and suppressors of white-mottled-4. Seven sex-linked enhancers were translocations involving centromeric heterochromatin. Several of the autosomal enhancers, which gave a more extreme effect than the loss of n Y, were deficiencies, probably in 41A. Dubinin (1936) found that rearrangements superimposed upon the original Plum inversion changed

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the expression of Plum as a result of the new position occupied by the genetic material. Panshin (1941 a and b ) obtained derivatives of whitemottled-11 in which the quantity of heterochromatin associated with the white locus was changed, in some by changes at the white locus superimposed upon the original translocation, in others by breaks independent of the locus. Regardless of the type of secondary changes, the degree of mottling was inversely proportional to the quantity of heterochromatin associated with the white locus. Hinton (1950) found that in reversions of the phenotypic change associated with In(2LR)40d produced by irradiation some were complete and some were partial and that “the degree of reversion is correlated with a quantitative effect of heterochromatin. ” Griffen and Stone (1940) showed that new association of the whitemottled-5 locus gave new variegation types as well as pure white and normal types. Their contention that mottling and heterochromatin are not directly related has been questioned (Hinton, 1950 ; Lewis, 1950). Variegation would be retained to some extent if a small amount of heterochromatin was transposed with the white locus, and it might be possible that cytologically invisible heterochromatin would still be in apposition to the white locus. Another possible explanation (Kaufmann, 1942) is that the derivatives retain or lose the variegated phenotype depending on whether they are transposed to interstitial heterochromatin or euchromatin. Even the incomplete criterion presented i n Fig. 2, a s a means of locating the two types of chromatin, displays remarkable correlation : eight of the nine derivative-whites of wm6having normal phenotypes were translocations to “euchromatin, ” whereas in eight out of the twelve showing variegation the new breaks were located in intercalary heterochromatin. 2. Loci in Major Heterochromatic Regions

The variegated mutants (having chromosomal rearrangements involving heterochromatin) may show position effect for a locus or loci a t the heterochromatic break as well as for those a t the euchromatic break (Schultz, 1936, 1939). Table 2 summarizes the number of times loci a t both breaks are affected, as well as the number of cases in which only the locus a t the heterochromatic break is affected. If variegation is diagnostic of heterochromatin, then the mutants at the heterochromatic break are in heterochromatin, unless the sensitive region, as in white-Notch, for example, is very long. However, this does not appear to be the case for most heterochromatic loci. I n one analysis two out of six variegation rearrangements with one break in the X heterochromatin had the bobbed locus affected ; three out of five, light, and possibly one out of six lightoid

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for the second chromosome ; one out of eight, pink, and one each inturned and radius incompletus for the third chromosome (Sutton, 1942). If it is true that, in R ( + ) mutants, variegation is a n expression of apposition of heterochromatin and euchromatin, it follows that if loci at the heterochromatic break are affected they are, with all probability, in heterochromatin. Thus, on the basis of position effect a t the heterochromatic break, the following loci are in heterochromatin : bobbed (66.0; 19F or 20A) in the X ; light (55.0; 40F), rolled (55.1 ; 41A), straw (55.1 ; 41A-B 7), and peyhaps apterous (55.4; 41F/42A) and lightoid (56.0 & ) in chromosome 2 ; inturned (47.0), radius incompletus (47.1), Deformed (47.5), proboscipedia (47.7), and pink (48.0) in the third; and cubitus-interruptus (0.0; 102B) in the fourth (data from Bridges and Brehme, 1944). Bent (0.0) in the fourth mag also be a heterochromatic mutant (Schultz, 1939). The phenotypes of these mutants in homozygous recessive condition have nothing to distinguish them from similar phenotypes of euchromatie loci. They affect eye color, bristle shape, wing pattern, etc. In compound with position-effect rearrangements, bobbed is expressed by reduction or loss of some but not all of the bristles, light is mottled, pinkp (an allele of pink) shows a uniform coloration intermediate between pink and normal, and lightoid is uniformly pigmented but lighter than normal (Sutton, 1942). A characteristic of heterochromatic R ( + ) mutants separating them from those in euchromatin is their reaction t o suppressors of variegation. Addition of an extra Y chromosome to light results in no change in the eye color. If light is transferred by rearrangement to euchromatin, the phenotype of the heterozygote is wild type in the XO male, variegated in the XY male, and light in the XYYY male (Khvostova, 1939 ; Morgan, Bridges, and Schultz, 1937 ; Morgan and Schultz, 1942; Schultz, 1936). Thus, as would be expected, the expression of heterochromatic genes is opposite to that of euchromatic genes. Position effect on rolled has not been found yet i n combination with position-effect mutants showing variegation. This may not be a typical heterochromatic locus, for addition of a Y to the females does not change the phenotype. It is possible that rolled, as well as some of the other loci, especially in 41, are in small euchromatic regions intercalated between heterochromatic regions (Morgan, Schultz, and Curry, 1939). But rolled when heterozygous with Minute-S-10 (a deficiency for 2R heterochromatin) has variable manifestation (Morgan and Schultz, 1942). Thus rolled has some characteristics of heterochromatic and some of euchromatic loci. These data suggest that there may be two different kinds of heterochromatin, each with a different genetic action :

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one having properties opposite to euchromatin, and the other possessing properties intermediate between euchromatin and heterochromatin. The expression of position effect of the cubitus-interruptus locus is determined by the position of the second break. Khvostova (1939) believed that only the “active” regions in the autosomes, the long arm of the Y chromosome, and a fragment of the inert region of the X chromosome, as well as certain euchromatic regions, are analogous in their action in the production of the cubitus-interruptus phenotype. Thus, cubitusinterruptus could be interpreted as a typical heterochromatic locus, if it is assumed that the heterochromatin of the X and Y has special properties influencing both heterochromatic an d euchromatic loci. Stern (personal communication) found that breaks in the euchromatin gave the most extreme R ( c i ) cubitus-interruptus phenotypes, breaks in centromeric heterochromatin produced less extreme phenotypes, and those in the heterochromatin more distal from the centromeres had even less effect. Like rolled, cubitus-interruptus may have properties of both euchromatin and heterochromatin.

3. Intercalary Heterochromatin The role of intercalary heterochromatin has not been studied as extensively a s that of centromeric heterochromatin. Intercalary heterochromatin in the X, determined by various methods, has been localized to rather specific regions (Fig. 2 ) . Evidence for the autosomes is not as complete. The breakage frequencies in lettered subdivisions have not been analyzed, but even on the basis of divisions certain regions appear to be more subject to breakage than others. A few sections have a tendency for nonspecific pairing, and certain regions, generally called euchromatin, have a high incidence of Minutes. Likewise there is a fair correlation between ectopic pairing, high breakage coeEcients, and cytological repeats, especially of the reverse type. I f it is assumed that these criteria are valid in determining intercalary heterochromatin, these regions can be plotted and their genetic function determined. In spite of the fact that localizations are gross, and therefore subject to revision with more refined methods, there is an amazing correlation between these regions and specific genetic phenomena. a. Intercdary Heterochrmath and Position Effect. As was pointed out before, the derivative white-mottled mutants of Griffen and Stone (1940) showed a good correlation between expression of phenotype and the position of the break, indicating that those having a normal phenotype were transferences of the white locus to “euchromatin” and those retaining a variegated phenotype might be due to breaks in intercalary heterochromatin. Khvostova (1939) interpreted all breaks in the distal

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segments of the autosomes giving position effect on cubitus-interruptus as being in euchromatin. On the basis 0: data shown in Fig. 2, breaks in intercalary heterochromatin appear to give position effect on cubitusinterruptus more frequently than those in regions not showing characteristics of heterochromatin. The euchromatic regions of the third chromosome are 1.9 times greater than the intercalary or centromeric heterochromatin (as measured by the numbered divisions in the salivary gland chromosome), yet 30% more breaks giving position effect on cubitus-interruptus are in intercalary heterochromatin. These results could be interpreted as showing either that there is a higher break frequency in the heterochromatic sections, or that the cubitus-interruptus position effect is more readily produced in intercalary heterochromatin. b. Intercalary Heterochrmntin and Location of the Gene. Attempts have been made by various investigators to relate heterochromatin, position of the gene, and mutation, some believing that mutations, and thus genic loci, occur only in euchromatin, others that they occur in heterochromatin as well. Prokofyeva-Belgovskaya (1941)pointed out that most of the frequently mutating genes of the X chromosome (yellow, white, cut, lozenge, forked, and bobbed) are located near the heterochromatic regions, and concluded that mutability-spontaneous as well as X-ray-induced-is somehow related to the position of the loci in respect to the inert regions. Conversely, Slizynska and Slizynski (1947) found that deficiencies (lethals) were more frequent in the nogenic, lightly staining parts of the chromosome. Data from Slizynska and Slizynski (1947) for lethals, and from Spencer and Stern (1948)for both lethals and visibles as determined by crossover values, were analyzed as nearly as possible, in terms of closeness to euchromatin or heterochromatin. Some regions having a high incidence of mutations are in or near heterochromatin, and some in euchromatin, although two regions, (genetic localization 0 and 5 6 ) , both in or close to heterochromatin, have the highest incidence. The visibles are about equally distributed in heterochromatin and euchromatin. Thus it seems that loci in intercalary heterochromatin are similar to those i n euchromatin. It is possible, however, th a t the differences in mutation frequency for the various loci may be attributed partly to their position in the chromosome. If heterochromatin has a higher break frequency, the loci in or close to heterochromatin may have a higher mutation frequency. c. Polygenic Activity. Mather (1941, 1942, 1944) was the first to claim a specific function of heterochromatin in heredity. By selection experiments he was able to obtain lines with high a n d low numbers of abdominal chaetae. Addition of Y chromosomes increased or decreased

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the number of chaetae, but always with continuous variation. From these data he concluded that heterochromatin carries polygenes which have similar, small, quantitative effects. Wigan (1949) showed that the X chromosome also has polygenes affecting the number of bristles, and that they are distributed in the regions near yellow (0.0-2.8), cut (10-31)) a nd wavy (3149.5). Polygenic activity is associated with regions having a high number of visible and lethal mutations, but, owing to the general distribution of intercalary heterochromatin, the connection between heterochromatin and polygenic activity could not be established. Since that time, Goldschmidt (Goldschmidt, Hannah, and KellenPiternick, 1951) have described certain phenotypes-designated as podoptera-which are characterized by a penetrance that is very low and quite variable. The podoptera effect is based, genetically, upon a number of different factors in all the chromosomes, which seem to have complex interactions rather than simple additive action. Modifying effects were found for the Y chromosome and for many dominants, especially the Minutes. From these data, Goldschmidt (1949) concluded that heterochromatin has a genetic action analogous to euchromatin, but that the action is concerned with early differentiation. Heterochromatic mutants act as a series of multiple factors with generalized unspecific effect, which results in interaction of a pseudoallelic type between different loci, but may also have some specificity as shown by the different phenotypes characteristic of the different podoptera lines. The heterochromatic action is rather sensitive to the presence of additional amounts of heteropycnotic heterochromatin, which may have unspecific generalized plus-or-minus action modifying all the specialized actions of the specific heterochroinatic genes.

IV. SUMMARY The function of heterochromatin and its distinction from eiichromatin are still a matter of speculation. The earlier view that the euchromatic genes have become inert through mutation to inactive and stable allelomorphs has largely been modified to include the concept that, genetically, the heterochromatin contains genes essentially different from euchromatic genes in that they show, in general, no specific clearcut Mendelizing allelomorphs, and that, physiologically, heterochromatin is concerned primarily with nucleic acid metabolism, Both genetical and cytological evidence have pointed to the fact that in Drosophila there are several types of chromatin; and it is possible that some day

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a direct connection will be found between the series of genetic manifestations and the different kinds of chromatin. Major heterochromatic regions have a nonrandom distribution in the chromosome. The principle regions are found near the centromeres. Major heterochromatin may be differentiated into several kinds, both genetically and cytologically. Certain regions, for example, the chromocentric heterochromatin, which has been claimed by some to be accessory nongenic chromatin, show a diffuse structure, or a t the most are represented by a single band in the salivary chromosome ; and others form the typical banded structure. The most striking genetical property of major heterochromatin is the induction and modification of variegation in somatic cells. I n mitotic chromosomes, its most characteristic property is heteropycnosis ; and in the salivary gland chromosomes, indiscriminate pairing with other heterochromatic regions, and high breakability. Intercalary heterochromatin is not cytologically differentiated from euchromatin, but appears to be distributed throughout the chromosomes, possibly as single bands or even long segments. Its presence is inferred because it has the properties, like centromeric heterochromatin, of high breakability, ectopic pairing, and possibly modification of variegation. Certain of these regions also appear to be repeats. Although the “Bridges repeats” may be completely different from the doublets in genetic properties, both have been given the same name. It is probable that too much stress has been laid on the antitheses between euchromatic and heterochromatic loci, and that actually they are the extremes of a series of genetic types that include a number of intermediates. Certain heterochromatic mutants have typical Mendelizing characteristics and high penetrance and expressivity, and so differ little from euchromatic mutants. However, position-effect mutants with variegated phenotypes differ considerably in their reaction to the addition of extra heterochromatin; addition of Y chromosomes to the “euchromatic” R(+) mutants has a tendency to normalize the phenotypic expression, whereas addition of extra heterochromatin to the “heterochromatic” R (+) mutants may make the expression of the phenotype more extreme. Between these two extremes there may be a number of loci which under some circumstances act a s euchromatic and in other circumstances as heterochromatic. Finally, there is the third type of genetic material, that having polygenic activity. According to Mather, polygenes are a repetitive series of genes, characteristic of heterochromatic material as found, for example, in the Y chromosome. The members of a polygenic system have small, similar, supplementary effects, because their products are less highly differentiated and their action is less elaborate. Thus their

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function is that of governing continuous quantitative variation. They do not show mutations of a sharp or specific kind. Both heterochromatin and euchromatin may contain both “major” genes and polygenes ; but the latter may be in segment,s too small to be distinguishable as &her enchromatin or heterochromatin.

V. REFERENCES Barigozzi, C., 1947, Proc. 6th int. Congr. Exp. Cyt. 149-152. Bauer, H.,1936, Proc. nat. Acad. Sci., Wash. 22, 216-222. 1939, ChT0~608Oma1, 343-390. Bauer, H.,Demerec, M., and Kaufmann, B. P., 1938, Genrtios 23, 610-630. Bridges, C. B., 1916, Genetics 1, 1-52,107-163. 1923, Anat. Rec. 24, 426-427. 1927, Biol. ZbZ. 47, 600-603. 1935, 6.Hered. 26, 60-64. 1935, Tramactionn mi Ihr rlyiinmicn of drwlopmrnt 10, 463-473. 1936, Scbnce 83, 210-211. 1937, Cytologia, Fujii .Tub. Vol., 745-755. 1938, J . Hered. 29, 11-13. Bridges, C. B., and Brehme, K. S., 1944,Publ. Carneg. Insln. 662, 1-258,vii. Bridges, C. B., and Bridges, P. N., 1939, J. Hered. 30, 475-476. Bridges, P. N., 1941a, J. Hered. 32, 64-65. 1941b, J . Hered. 32, 299-300. 1942, J . Hered. 33, 403-408. Callan, H. G., 1948, Nature, Lond. 161, 440. Caspersson, T., 1947, Symp. 80c. exp. biol. I. Nucleic Acids. Cambridge TJniversity Press, pp. 127-151. Caspersson, T., and Scliultz, J., 1938, Nature, Lond. 142, 294-295. 1940, Proc. nat. Acad. Sci., Wash. 26, 507-515. Caspersson, T., and Santesson, L., 1942, A d a radiol., Stockh. Suppl. 46. Chen, S. V., 1948, Bull. biol. 82, 114-129. Cole, P. A., and Sutton, E., 1941, Cold Spr. Hnrb. Synap. Quant. Biol. 9, 66-71. Cooper, K. W.,1949, J. Morph. 84, 81-122. Darlington, C. D., and Thomas, P. T., 1941, Proc. roy. Soc. B130, 127-150. Darlington, C. D., and Upeott, M. B., 1941, J . Genet. 41, 275-296. Demerec, M., 1939, Proc. 7 t h int. Congr. Genet. 99-103. 1940, Genetics 26, 618-627. 1941, Cytology, genetics, and evolution. Univ. Penn. Bicentennial Conf., 1-11. Demerec, M.,and Hoover, M., 1939, Genetics 24, 68. Demerec, M.,Kaufmann, B. P., Fano, U., Sutton, E., and Sansome, E., 1942, Pearb. Carneg. Znstn. 41, 190-199. Demerec, M., Kaufmann, B. P., and Hoover, M. E., 1938, Pearb. Carnsg. Znstn. 37, 40-47. Demerec, M.,and Slizynska, H., 1937, Genetics 22, 641-649. Dobzhansky, Th., 1929, BWZ. Zbl. 49, 408-419. 1930a, Biol. ZbZ., 60, 671-685. 1930b, Genetics 16, 347-399.

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1931, Genetics 18, 629-658. 1932a, B b l . Zbl. 62, 493-509. 1932b, Genetics 17, 369-392. 1936, BWZ. Rev. 11, 364-384. 1941, Genetics and the Origin of Species, 2nd rev. ed. Columbia University Press, New York. 1944, Amer. Nat. 78, 193-213. Dubinin, N. P., 1936, Biol. Zh. Mosk. 6, 851-874. Dubinin, N. P.,and Heptner, M. A., 1935, J . Genet. 30, 423-446. Dubinin, N. P., Khvostova, V. V., and Mansurova, V. V., 1941, C.R. Acad. Sci. U.R.S.S. 31, 387-389. Dubinin, N. P., and Sidorov, B. N., 1935, Bwl. Zh. Mosk. 4, 555-568. Ephrussi, B., and Sutton, E., 1944, Proc. nut. Acad. Sci., Wash. 30, 183-197. Gersh, E. S., 1949, Genetics 34, 701-707. Gershenson, 8. M.,1933, J. Genet. 28, 297-313. Goldschmidt, R., 1946, Experientia 2, 197-203, 250-256. 1949, Proc. 8th int. Congr. Genet., Suppl. Hereditas, 244-255. Goldschmidt, R., Hannah, A., and Kellen-Piternick, L., 1951, Uniu. Cal,if. Publ. 2001.66, 67-294. Gowen, J. W., and Gay, E. H., 1933, Proc. nat. Acad. Sci., Wash. 19, 122-126. 1934, Genetics 19, 189-208. Green, M. M.,and Green, K. C., 1949, Proc. nat. Acad. Sci., Wash. 36, 586-590. Griffen, A. B., and Stone, W. S., 1940, U&. Tezas Publ. 4082, 190-200,201-205. Griineberg, H., 1937, Nature, 140, 932. Guhin, H. A., 1948, J. Genet. 49, 23-36. Hannah, A,, 1949, Proc. 8th int. Congr. Genet., Supp. Hereditas, 588-589. Heitz, E., 1928, Jb. wiss. Bot. 69, 762-818. 1933, 2. Zellforsch. 20, 237-287. 1934, B k l . Zbl. 64, 588-609. 1935, Z. indukt. Abstamm.- u. VererbLehre 70, 402-447. Hinton, T., 1942, Genetics 27, 119-127. 1945, B W ~ .BUZZ. wood’s H O Z C aa, 144-165. 1950, Genetics 36, 188-205. Kaufmann, B. P., 1933, Proc. nat. Acad. Sci., Wash. 19, 830-838. 1934, J . MWph. 68, 125-155. 1937, Cytologia Fujii Jub. Pol. 1043-1055. 1938, 2. Zellforsch. 29, 1-11. 1939, Proc. 7th int. Congr. Genet. 172-173. 1941, Cold Spr. Earb. Symp. Quant. Biol. 9, 82-93. 1942, Genetics 27, 537-549. 1944, Yearb. Carneg. Inetn 43, 115-120. 1946, J. exp. 2001.102, 293-320. Kaufmann, B. P., and R. C. Bate, 1938, Proc. nat. Acad. Sci., Wask. 24, 368-371. Kanfmann, €3. p., McDonald, M. R., Grly, H., Wilson, K., Wyman, R., and Okuda, N., 1948, Yearb. Carneg. Instn. 47, 144-155. Kerschner, J., 1949, Proc. nat. Acad. Sci., Wash. 36, 647-652. Khvostova, V. V., 1936, BbZ. Zh. Mosk. 6, 875-880. 1939, Bull. Acad. Sci., U.R.S.S.5, 541-574. Kodani, M.,1941, J. Eered. 32, 147-156. 1946, Thesis deposited in the Univ. Calif. (Berkeley) Library.

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Koller, P. C., 1936, J . Genet. 32, 79-102. 1947, Symp. SOC. esp. biol. I . Nucleic Acids. Cambridge University Press, pp. 270-290. Koller, P. C., and Darlington, G. D., 1934, J . Genet. 29, 159-173. Kossikov, K. V., 1936, C.R. Acad. Sod. U.R.S.S.N.S. 3, 299-302. Lewis, E. B., 1941, Proc. nat. Acad. Sci., Wash. 27, 31-34. 1945, Genetics 30, 137-166. 1948, G e n e t h 33, 113. 1950, Advances in Genet. 3, 73-115. Mather, K., 1941, J . Genet. 41, 159-193. 1942, J . Genet. 43, 309-336. 1944, PTOC.TOY. SOC. B132, 308-332. Mohr, 0. L., 1932, Proc. 6th int. Congr. Genet., Part I , 190-212. Morgan, L. V., 1947, Genetiosl 32, 200-219. Morgan, T. H., Bridges, C. B., and Sturtevant, A. H., 1925, Bibliogr. Genet. 2, 1-262. Morgan, T. H., Bridges, C. B., and Schulte, J., 1937, Pearb. Carneg. Znstn. 56, 298305. 1938, Pearb. Carneg. Inst. 37, 304-309. Morgan, T. H., and Schulte, J., 1942, Pearb. Carneg. Znstn. 41, 242-245. Morgan, T. H., Schultz, J., and Curry V., 1939, Yearb. Carneg. Instn. 38, 273-277. 1940, Yearb. Carneg. Zmtn. 59, 251-255. 1941, Pearb. Carneg. Imtn. 40, 282-287. Muller, H. J., 1914, J . esp. Zool. 17, 325-336. 1918, Genetics 3, 422-499. 1930, J . Genet. 22, 299-334. 1932, Proc. 6 t h Int. Congr. Genet., P a r t I, 213-255. 1935, Summ. Commun. XV. inst. physiol. Congr. Lenigr. Yosb., 286.280; R o c . 15th int. physiol. Congr. (Leningr. Mosc.), 587-589. 1938, Collect. N e t (Wood's Hole), 13, 183-195. 1941, Cold Spr. Harb. Symp. Quant. Biol. 9, 290-308. 1946, D.I.S.20, 88-89. 1947, J. Hered. 38, 259-270. 1947, Genetics 32, 98-99. Muller, H. J., and Altenburg, E., 1930, Genetics 16, 283-311. Muller, H.J., and Ciershenson, 8. M., 1955, Proc. nat. Acad. So&, wash. !Zl, 69-75. Muller, H. J., and Painter, T. S., 1929, Amer. Nat. 63, 193-200. 1932, 2. indubt. Ab8tmm.- u. VererbLehre 62, 316-365. Muller, H. J., and Prokofyeva, A. A., 1935, C.R. Acad. Sci. U.R.S.S.N.S.1, 658-660. Muller, H. J., Prokofyeva-Belgovakaya, A. A., and Kossikov, K. V., 1936, C.B. A m d . Sci. U.R.S.S. N.S. 1, 87-88. Muller, H. J., Raffel, D., Ciershenson, 8. M., and Prokofyeva-Belgovskaya, A.A., 1937, Genetias 22, 87-93. Neuhaua, M., 1936, 2. indukt. A b s t a m m - u. VererbLehre 71, 265-275. 1939, J . Genet. 37, 229-254. Noujdin, N. I., 1936, Nature, Lond. 137, 319-320. 1938, Bull. Bwl. Med. exp. U.R.S.S.6, 548-551. Offermann, C. A., 1935, Bull. Acad. Sci. U.R.S.S.1, 129-152. 1936, J . Genet. 52, 103-116. Oliver, C. P., 1940, Proc. not. Acad. Sci., Wash. 26, 452-454.

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Oliver, C. P., and Green, M. M., 1944, Genetics 29, 331-347. Painter, T. S., 1931, Science 73, 647-648. Painter, T. S., and Muller, H. J., 1929, J. Hered. 20, 287-298. Panshin, I. B., 1935, C.R. Acad. Sci. U.R.8.S. N.8. 4, 85-88. 1938, BioZ. Zh. NO&. 7, 837-868. 1941, C.R. Acad. Sci. U.R.S.S. 30, 57-60. 1941, D.I.S. 16, 33-34. Patterson, J. T., 1932, Genetics 17, 38-59. 1940, UnC. Tesas Publ. 4032, 246 pp. Patterson, J. T., Stone, W. C., Bedicheck, S., and Suche, M., 1934, Amer. Nat. 68, 359-369. Pavan, C., 1946, Proc. nat. Acad. Sci., Wash. 32, 137-145. Philip, U.,1935, J. Genet. 31, 341-352. Pontecorvo, Q., 1944, Nature, Lo&. 163, 365-367. Prokofyeva-Belgovskaya, A. A., 1935, C.R. Acad. Bci. U.R.S.S.N.S. 2, 498-499. 1935, Cytologia, Tokyo 8, 438-443. 1937, Genetics 22, 94-103. 1939, BUZZ. Amd. Sci. U.R.S.S. 3, 362-370. 1941, D.Z.S. 16, 34-35. 1947, J. Geset. 48, 80-98. Prokofyeva-Belgovekaya, A. A., a i d Klivostova, V. V., 1939, C.R. h a d . Sci. U.R.S.S. 23, 269-271. RsfPel, D., and Muller, H. J., 1940, Genetics 26, 541-583. Besende, R., 1945, Port. Acta. BioZ. Al, 139-156. Sohulte, J., 1929, Genetics 14, 366-419. 1936, Proc. nat. Acad. Sci., Wash. 22, 27-33. 1939, Proc. 7th int. Congr. Genet. 257-262. 1941, Cold Spr. Harb. Symp. Quant. BioZ. 9, 55-65. 1943, Ann. Rev. PhysbZ. 6, 35-62. 1944, coizoia Chem. 6, 819-850. 1947, Cold Spr. Harb. Symp. Quant. BioZ. 12, 179-191. Schultz, J., and Bridgee, C. B., 1932, Amer. Nat. 66, 323-334. Sehultz, J., and Caspersson, T., 1949,Nature, Lond. 163, 66-67. Schultz, J., Caspersson, T., and Aquilonius, L., 1940, Proc. nat. Acad. Sci., Wash. 26, 515-517. Schultz, J., and Catcheside, D. Or., 1937, J . Genet. 36, 315-320. Schultz, J., and Dobzhansky, Th., 1934, Genetics 19, 344-364. Serebrowsky, A. S., 1938, C.R. Acad. Sci. U.R.S.S. 19, 77-81. Serebrowsky, A. S., and Dobzhansky, Th., 1934, Genetics 19, 344-364. Sivertzev-Dobzhansky, N. P., and Dobzhsnsky, Th., 1933, Genetics 18, 173-192. Slizynska, H., and Slizynaki, B. M., 1947, Proc. roy. Boo. Edinb. B62, 234-242. Slizynski, B. M.,1944, J. Hered. 36, 322-325. 1945, PToO. TOY 800. Edinb. B62, 114-119. Spencer, W. P., and Stern, C., 1948, Genetics 33, 43-74. Stern, C., 1929, Bid. ZbZ. 41, 718-735. 1949, Scienue 108, 616-621. Stem, C., and Doan, D., 1937, Proc. nat. Acad. Sci., Wash. 22, 649-654. Stern, C., and Heidenthal, Q., 1944, Proo. nat. Bead. Sci., Wash. 30, 197-204. Stern, C., Schaeffer, E. W., and Heidenthal, GI., 1946, Proc. nat. Acad. Sci., Wash. 32, 26-83.

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Sturtevrtnt, A. H., 1925, Genetics 10, 117-147. 1937, Proc. nat. Acad. Sci., Wash. 23, 360-362. Sutton, E.,1940, Genetics 26, 534-540. 1942, D.Z.X. 16, 68-69. 1943, Genetics 28, 210-217. 1943, Genetics 28, 97-107. Vanderlyn, L., 1949, Bot. Rev. 16, 507-582. White, M. J. D., 1940, J . Genet. 40, 67-82. 1948, Animal Cytology and Evolution. Camlwirlge University Press. Whittinghill, M., 1937, D.I.X. 8, 82-84. Wigan, 1,. G., 1949, Heredity S, 53-66.

125

The Genetics of Coffea C . A . KRUG

AND

A. CARVALHO

Instituto AgronGmico. Campinas. 860 Paulo. Braail CONTENTS

. . .

. . . . . . . . . . . . .. Morphology . . ....... . . . . . . . . . . . . . .

Puge

. . . . . . . . . . 128 . . . . . . . . . . 128 . . . . . . . . . . 132 . . . . . . . . . . 134 . . . . . . . . . . 136 . . . . . . . . . . 136 a. Chromosomal Mutations . . . . . . . . . . . . . . . 137 b . Gene Mutations . . . . . . . . . . . . . . . . . . 137 i. Angustifolia . . . . . . . . . . . . . . . . . . 137 ii . Anomala . . . . . . . . . . . . . . . . . . . . 138 iii. Anormalis . . . . . . . . . . . . . . . . . . . 138 iv. Bronze . . . . . . . . . . . . . . . . . . . . 140 v. Calycanthema . . . . . . . . . . . . . . . . . . 140 vi. Caturra . . . . . . . . . . . . . . . . . . . . 141 vii . Cera . . . . . . . . . . . . . . . . . . . . . 141 viii . Crespa . . . . . . . . . . . . . . . . . . . . . 142 ix. Erecta . . . . . . . . . . . . . . . . . . . . 142 x . Fasciata . . . . . . . . . . . . . . . . . . . . 143 xi. Laurina . . . . . . . . . . . . . . . . . . . . 144 xii . Maragogipe . . . . . . . . . . . . . . . . . . . 144 xiii . Mokka . . . . . . . . . . . . . . . . . . . . . 146 xiv . Murta . . . . . . . . . . . . . . . . . . . . . 147 xv. Purpurascens . . . . . . . . . . . . . . . . . . 148

. Introduction . . . . . . The Genus C o f e a . . . Chromosome Number and . Methods of Reproduction . Genetics . . . . . . 1. C o f e a arabica . . .

I I1 I11 IV V

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xvi Large Sepals xvii Semperflorens xviii Typica xix Xanthocarpa xx Independence of the Genes Br and X c xxi Other Mutations c Cytoplasmic Inheritance 2 Other Species of C o f e o 3 Interspeci5c Hybrids V I Importance of Genetic and Cytological Investigations for Coffee Breeding V I I Summary and Conclusions V I I I References

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......... 150 . . . . . . . . . . 151 . . . . . . . . . 151 . . . . . . . . . . 151 . . . . . . . . . . 152 . . . . . . . . . . 152 . . . . . . . . . . . 153 . . . . . . . . . . . 154 . . . . . . . . . . . 155 .......... 157

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I. INTRODUCTION Nearly all species of Coffea are native to Africa. The economically important Coffea arabica L. is of Abyssinian origin and was introduced into cultivation in Arabia, some 500 years ago. Today C.arabica is cultivated under tropical and subtropical conditions in nearly all Latin American countries and in several regions of Africa, Asia, and Oceania. The total number of coffee plants under cultivation has been estimated to be about 5 billions. I n 1947/18 the world production reached a n estiinatrd 28 million bags of 60 kg each or a total of 1,680,000 metric tons (Testa, 1949). Coffee is the most important crop plant of Brazil and particularly of the State of Sslo Paulo, where over one billion plants are under cultivation. I n spite of the fact that coffee is one of the main staple crops in many countries, relatively few investigations have been carried out on coffee genetics. The Genetics Division of the Instituto Agrondmico, Campinas, SBo Paulo, Brazil, began in 1933 a n extensive breeding program to improve locally grown coffee varieties (Krug, 1936; 1945a; 1945b). Essential basic investigation of taxonomy, flower biology, cytology, and particularly the genetics of Coffea were included as a necessary part of -the coffee variety improvement program. Investigations of the genetic characters of a woody perennial plant such as coffee require long periods of time, and involve considerable expense, labor, and space in a lathhouse and field plantings. These are important factors that have tended to limit the information available. It is the objective of this paper to present a general review on coffee genetics including some unpublished data.

11. THE GENUS Coffea Early referelices to the coffee plant are found in the botanical literature a t the end of the sixteenth century. It w&;y Linnaeus who first described the genus C o f e a in 1735, and eighteen years later (1753) he described the first species, which was the economically important C . arabicu. Following the introduction of coffee into the American continent, in 1713, its cultivation expanded rapidly under the favorable environment of these tropical and subtropical regions. Botanists then became interested in the study of other species of Coffea, and numerous representatives of this genus were described. A detailed study of the taxonomy of Coffea and its geographic distribution was carried out by Auguste Chevalier, noted French botanist, who has spent a good deal of his life exploring the flora of tropical

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Africa. In 1940,1942,and 1947 Chevalier published general revisions of the species of Coffea, grouping them into Sections and Subsections and reducing to about 60 the number of valid species. The majority of these were found to be indigenous to Central and West Africa and were distributed across this continent from the Atlantic coast to Abyssinia. The greatest concentration of species was found along the tributaries of the Congo River. A large group was observed to be native to Madagascar, and only six species were found in Asia and Oceania. No indigenous representatives of the genus Coffea have been found in the Americas, Australia, o r in Polynesia. A general review of the geographical distribution and botanical classification of the genus Coffeahas been published by Carvalho (1945,1946). The 60 valid species were grouped by Chevalier (1942) into five Sections : Paracoffea.; Argocoffea; Masmrocoffea; Eucoffea; and Mozambicoffea.. Section Eumffea comprises t>he RubsectioncJ Erythrocoffea; Parhyeoffea; Melawcoffea, and Nanocoffeu. The three best-known speciea of Caffea are classified under the Subsection Eryfhrocoffea and are Coffea arccbica L., Coffea cmgensis Froehner, and Coffea canephma Pierre ex Froehner. Other species frequently referred to in literature, but very little cultivated, are Coffea liberka Hiern and Coffea Dewevrei De Wild. and Th. Dur., of the Subsection Pachycoffea (Chevalier, 1942). The geographic distribution of C. Dewevrei is limited to the river basins of the Upper Shari and Ubangi of French Equatorial Africa. The varieties of this species are usually plants of considerable height, bearing large crops, but they are seldom cultivated, as the coffee beans produced are of inferior quality. The species C. liberica indigenous to Sierra Leone and Liberia on the west coast of Africa was studied by Dutch investigators because it was thought, at first, that this species was resistant to the rust caused by Hemileia vastat& Berk. and Broome, which causes a severe defoliation and limits the use of C. arabica in the Far East. It is now known that C. ZibericcG has little if any resistance to the disease and that it produces poor quality coffee. C . congensis grows wild in the region of the Congo River and its tributaries; its coffee is of inferior quality, and the species is not extensively cultivated. C. cancpkora is native to a very extensive area in Africa, from Gabon at sea level up to 1300 m at French Guinea and from the Ivory Coast, Gold Coast, Dahomey,,Cameroons, Angola, up to Uganda and the north of Lake Victoria. This species is resistant t o the Hemile& leaf disease and is now cultivated extensively in Java and has largely replaced the susceptible C . arabica species. The quality of the coffee beans

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produced is inferior to C . arabica, and the fact that C . canephora is selfsterile creates problems in both breeding and cultural practices (Ferwerda, 1948). C. arabicu, the most important cultivated species, seems to have originated on the mountain slopes and river valleys, 1000 to 3000 m altitude in South Abyssinia, 7 to 9 degrees latitude north of the equator. It is a polymorphic species with numerous distinct varieties. The typica variety is the most widely known and probably was the first introduced into cultivation not only in Arabia and Java but also on the American continent. Typica has been considered by Cramer (1913) to be the “type” of the species. The description of C. arabica given by Linnaeus corresponds to the typica variety. The picture of the wild ccrabica coffee plant native from Barbuk area (Anglo-Egyptian Sudan) presented by Thomas (1944) seems to be of the typiccs variety. C . arabica is a shrub 2 to 3 m high of almost cylindrical shape ; its primary lateral branches make an average angle of about 65 degrees with the main axis, and the extremities are slightly inclined downwards. Secondary and tertiary branches are abundant. The leaves (Fig. 1, a ) are persistent, dark green when mature, elliptical, slightly coriaceous, their blade and margins being somewhat undulated; the “domatia” (small cavities in the lower leaf surface a t the insertion region of the lateral veins) (Fig. 1, c ) have medium-sized openings of irregular shape on the lower side of the leaf, no hairs usually being present a t the margins of the opening. They are slightly protuberant on the upper leaf surface. The stipules, which are located between the insertion region of the petioles, are deltoid, acuminated with a tip of varying length (Fig. 1, b ) . The flowers have a white corolla and are grouped into glomerules protected by calicules. The number of flowers varies from 2 to 19 per leaf axil (Fig. 1, d - f ) . The ovary is biloculate or triloculate; the calyx is rudimentary, denticulate. The fruits (Fig. 1, g) have short pedicels, are oval-elliptical, red, of smooth surface, brilliant when mature ; they have a fleshy mesocarp and a fibrous endocarp (Fig. 1, h ) . The seeds are plane-convex, greenish, covered with a thin coat, called silver skin (Fig. 1, i). The endosperm is corneous, greenish, the embryo being located a t the base (Cramer, 1913; Krug, Mendes, Carvalho, 1939). A general revision of the genus Coffea,in the light of cytological and genetical investigations, would be desirable. First of all, a living collection as complete as possible, should be established to facilitate comparative morphological investigations. Only five species are represented at the Campinas collection ( C . Ziberica, C. canephwa, C . congemis, C. Dewevrei, and C . arabica), as introduction has so f a r been handicapped by quarantine regulations, chiefly because of the H e d e i a disease, which

FIQ.1. Main characteristics of C o f e a arabioa var. typica. All drawings natural size except c which i s enlarged SX; a, mature leaf; b , main shoot; G, domatia; d, flowers; e, gynaeceum; t, calicule containing one glomerule; g, ripe fruit and its cross sections; h, seed with endocarp; i, seeds.

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does not exist in America. The C . arabica collection a t Campinas is, however, very complete, nearly all varieties being represented. A total of 25 varieties and 4 forms has been described (Krug, Mendes, Carvalho, 1939, 1949). It has been proposed that all homozygous plants for new mutants not known before will be considered as new varieties of coffee. Recombinations of already known genes will not be considered as such. 111. CHROMOSOME NUMBER AND MORPHOLOGY All species of Cofea, so far examined, have shown great regularity with respect to chromosome number. C. arabica, has 44 somatic chromosomes (Fig. 2, a ) , the remaining species have 2n = 22 chromosomes (Fig. 2, c ) . Von Faber (1912) thought that 8 was the basic number, however Fagerlind (1934) and Krug (1934, 1937a, 1937c) found 11 as the basic haploid number for the genus and 2n = 44 for C. arabica. Homeyer (1933) has determined 2n = 22 for the same species. I n excelsa coffee (C. Dewevrei), A. J. T. Jfendes (1938) found that the somatic chromosomes can be divided into three classes according to their size, which varied between 1.0 and 3.3 microns. Three of the main chromosomes can be easily distinguished, one of them having a secondary constriction. Prophase chromosomes of C . canephova were studied by C. H. T. Mendes (1950), their morphology being difficult to observe a t pachytene stage (Fig. 2, d ) . The centromeres are visible in all chromosomes, heteropycnotic areas being detectable on each side of them. The ends of the chromosomes are not easily recognized in this phase. One or two chromosomes are attached to the nucleolus. At metaphase I in average, 8.4 bivalents occur with only one chiasma, 1.6 with two chiasmata, and 0.97 with three chiasmata. The varieties of C. arabica have 2n = 44 chromosomes with the exception of the bullata variety (CnjgPea arabica L. var. btcllata Cramer) which has two forms,'respectively, with 66 and 88 chromosomes (Kriig, 1937a) and the monosperma (Coffeaarabica L. var. monosperma Ottolander and Cramer) which is haploid with only 22 somatic chromosomes (A. J. T. Mendes and Bacchi, 1940). All commercial varieties have 44 chromosomes, as the 22-, 66-, and 88-chromosome types are practically sterile due t o abnormal meiosis. The morphological features of the chromosomes of C . arabica have been studied by A. J. T. Jlendes (1950) and Medina (1950) who found that they also do not stain well a t early prophase stages (Fig. 2, b ) . The centromere can be detected rather easily, being located between two heteropycnotic regions. The ehds of the pro-

W O . 2. Chromosomes of Coffea and emasculation technique ; a, somatic cliromosome8 of Coffea arabica (2n = 44) ( ~ 1 9 8 0 ;) b, early meiotic prophase of C. arabioa (~1430)( a and b courtesy of A. J. T. Yendes) ; c, somatic chromosomes of c. canephora ( 2 n = 2 2 ) (~1870);d, early meiotic prophase of c. canephora (~1070)(c and d courtesy of C. R. T. Mendes); e, emasculating coffee flowers; 1, scissors used for emasculation; g, flower buds ready for emasculation; h, same buds g after emasculation; i, flower buds one day after emasculation.

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phase chromosomes are not distinguishable and are lost in the cytoplasm. I n average, 9.2 bivalents occur with only one ohiasma, 10.8 with 2 chiasmata, and 2.0 with 3 chiasmata. Other phases of meiosis and gametogenesis were also studied (A. J. T. Mendes, 1950). Investigations to determine the origin of the nutritive tissue of the coffee seed were made by several authors. Von Faber (1912),who apparently published the first account on this subject, considered the bulk of the coffee seed to be normal endosperm, but Houk (1936, 1938) thought that it was perisperm. Several authors however disagreed with Houk; Mayne (1937),Leliveld (1938),and Fagerlind (1939)have presented evidence that the coffee seed has real endosperm. A genetic demonstration of the existence of true endosperm was presented by Krug and Carvalho (1939)based on the occurrence of a case of xenia. A. J. T. Mendes (1941)re-examined the situation, confirmed the existence of true endosperm, and also determined the time at which the zygote starts to develop (60 to 70 days after the flower opening). Through interspecific hybridization triploids were obtained which are sterile because of meiotic irregularities, the nature of which has been determined in several instances (Krug and A. J. T. Mendes, 1940). A special method was developed by A. J. T. Mendes (1947) for doubling the chromosomes with colchicine by treating scions before grafting. One hexaploid, obtained by doubling the chromosome number of a n interspecific hybrid (arabica X canephora), has furnished a rather heterogeneous progeny, due to slightly irregular meiosis. These occurrences in interspecific triploids and hexaploids indicate that C. arubka is most probably a n allo-polyploid. Chromosome duplications by means of colchicine, in the monosperma variety of C. urabicu, have resulted in the establishment of pure lines of tetraploid coffee plants. Also various aneuploid plants are under investigation, which are of special cytological and genetical interest (A. I. T. Mendes, 1946,1949). IV. METHODS OF REPRODUCTION Zimmermann (1928) published general information regarding the biology of the coffee flower. Taschdjian (1932) concluded that in C. arabica there is no self-pollination in the closed flower bud; that by bagging the flowers (paper bags) a reasonable good fruit set is observed and that both insects and the wind are agents which contribute to promote pollination. He was the first to use a genetic tester to determine the degree of “vicinismus” in coffee. Using for this purpose the purpurascens variety (recessive), Taschdjian obtained in his seedbeds be-

THE GENETICS OF C O m A

135

tween 39% and 93% of hybrid, nonpurpurascens seedlings, due to natural cross pollination. Based on these observations he concluded that C . arahkut is t o a large extent allogamous, in spite of being self-fertile. Krug (1945b) and Krug and Costa (1947) published further investigations on this subject, using the same genetic indicator; they concluded that natural cross pollination in this variety seemed to be around 40 to 50%. Based on more extensive investigations and by use of another genetic tester, a yellow endosperm mutant, Carvalho and Krug (1949a) reached the conclusion that in the cera variety of C . wabica, which is more similar to typica variety than the purprgmens, the percentage of hybrid seeds due to natural cross pollination is only 7 to 970. Based on these data C . arabka has been considered t o be predominantly autogamous. The different results obtained with cera and purpurascens mutants used as genetic testers may be explained in various ways. Using the cera mutant the amount of cross pollination is directly determined by counting the hybrid green seed; while using the purpurascens tester the amount of cross pollination is measured by the number of hybrid plants obtained in the seedbed. The first method is therefore more accurate than the other mentioned. Furthermore the flowers of the purpurascens are pink, instead of white, and occur less abundantly than on plants of the commercial varieties, including cera. These facts, besides possible differences in the amount of pollen produced, may account for the discrepancies encountered by using the two above-mentioned genetic testers (Carvalho and Krug, 1949a). In a study of several species of Cofea other than C. arabica, Cramer and Wolk (1923) and Zimmermann (1928) concluded that cross pollination was predominant, and that wind and insects were mainly responsible for pollen transmission. As all diploid species have subsequently been found to be self-sterile, cross pollination seems to be predominant in the genus Cofea. Using glass slides covered with a sticky substance and placed at various distances from a flowering coffee garden, Ferwerda could demonstrate that a comparatively large amount of coffee pollen floats in the air for distances up to 100 m. This pollen rises fairly high, a considerable amount of it being collected on slides, 8 m above the ground (Ferwerda, 1948). Based on observations made on the structure of the coffee flower, Krug (1935) developed a special technique for emasculation whereby a large number of flower buds can be emasculated in a relatively short time (Fig. 2, e - i ) . This technique is important, as flowering in general Occurs once o r twice a year and lasts only two to three days. Vegetative propagation of coffee is possible by grafting and by cut-

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C. A. gRUQ A N D A. CARVALHO

tings, but it must be kept in mind that coffee has both orthotropic and plagiotropic branches and that only the orthotropic branches, when used as scions and cuttings, give rise to normal upright growing plants.

V. GENETICS Until recently there were very few references in literature to the genetics of coffee. McClelland (1924) examined the progenies of murta plants and concluded that this variety was of hybrid constitution, &s the progenies were made u p of approximately 50% murta plants and 50% normal and dwarfed individuals in equal number ; no conclusions were drawn, concerning the genetic constitution of the murta variety. Stoffels (1936) presented some data Concerning the heredity of the color of young leaves in C. arabicu, without reaching a definite conclusion regarding dominance relations of the genes involved. This author reported that plants with dark bronze leaves are more easily adaptable to varying environmental conditions being also more resivtent to die-back. Ciferri (1937) mentioned several already known cofFee mutants without presenting any data concerning their hereditary mechanism. A t the Instituto Agrondrnico (Campinas) genetic investigations of C. arabica varieties began in 1933. The results of these studies have been published in a series of articles, a general summary having been presented a t the Eighth International Genetic Congress by the authors (1949). Knowledge of the genetic characters of the diploid coffee species is very limited. They are known to be self-sterile and therefore their genetic analysis is extremely difficult.

1. Cof'ea ambicu During the last eighteen years, about thirty C. uruhica mutants have been analyzed. Some mutants are now recognized as resulting from chromosomal reduction or duplication. The majority are known to have originated through mutation of one or more gehetic factors. Several instances of cytoplasmic inheritance, expressed as variegations, have been observed and have been related to the occurrence of abnormal plastids. I n order to establish the dominance relations of the genetic mutants, all of them were crossed to the t y p h or bourbon varieties for general comparisons. These two varieties were selected for this purpose, because typica is considered to be the primitive type of the species and hourbow is very closely related to the typica. Both are widely cultivated in all coffee growing regions,

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137

A greater number of mutants are known in the tetraploid C. arabka species than in the diploid C. canephora species. This may be due to the fact that C. canephwa is a self-sterile, cross-pollinated species, and frequently is propagated by grafting, whereas C . arabka is self-fertile and is propagated by seeds. Chances of finding new recessive mutants are therefore greater in C. arabica than in C . canephora. a. Chromosomal Mutatims. The most striking chromosomal mutations which have been found in several varieties as typica, bourbon, and maragogipe are the hexaploid and octoploid bullata forms and the diploid monosperma types. The occasional occurrence of unreduced gametes has been supposed to be responsible for the origin of the bullata types and the rare parthenogenetic development of a normally reduced egg cell seems to account for the appearance of the diploid monosperma plants (A. J . T. Mendes, 1946). The octoploid bullata differs from the hexaploid bullata in leaf character, the former having more coriaceous and somewhat smaller leaves. Both bullata types have thicker and broader leaves than normal tetraploids. The diploids, on the other hand, have characteristic .narrow and thin leaves, easily distinguishable from the leaves of the tetraploids. Stomata counts (Franco, 1939) showed that an inverse relation exists between the number of chromosomes and the number of stomata per unit area of the leaves of these coffee varieties. Several instances of somatic chromosomal mutations have been observed (Krug, 1937b). Tetraploid plants have been found to give rise to octoploid branches and, vice versa, diploid monosperma individuals have mutated to a tetraploid. However no case has been found of a tetraploid plant giving rise to a diploid branch. The bullata plants have no economic value, as their productivity is very low, due to abnormal meiosis. Only a small percentage of their gametes are viable and these give rise to 44-chromosome plants. The monosperma sets very little seed, usually only one in each fruit. b. Gem Mutations. Gene mutations in C. arabica are numerous. Many of the mutants here described have been found in the coffee nursery at the Central Experiment Station of the Instituto Agron8mico. The following discussion includes only those mutants which showed normal segregation. i. Alzgustifoliu. Angustifolia mutants occur rather frequently in coffee nurseries and have been described as Coffea arabica L. var. angustifolia (Roxb.) Miq. The shape of their leaves is their main differentiating character (Fig. 3, a ) ; they are elongated, narrow, with an angle characteristically narrow at the base; they are thicker than the leaves of the typica plants, their veins being also less conspicuous, usu-

138

C. A. KRUQ

AND A. UARVbLHO

ally having no domatia. Various genetic factors are responsible for the angustifolia phenotype (Krug, 1949;Krug and Carvalho, 1949). Up to now two recessive, independent genes, agl a g 1 and agz agz are known that give rise to the angustifolia phenotype. These genes have a pleiotropic effect, influencing also the size and shape of the plant. The factor agl conditions the abnormal development of numerous stems right from the base ; the branches are slender and easily breakable, and the yield is very low; agz has a similar effect, but the plants have only one main stem. By crossing these two genotypes, a normal F1 develops, and in F2 a ratio of 9 normal to 7 angustifolia plants is obtained. Various other angustifolia plants are being investigated which apparently differ in their genetic behavior from the two above mentioned. ii. Anomla. The anomala mutant was found in 1933 in a very old coffee plantation at the Central Experiment Station in Campinas. It represents an extremely rare mutation, as it has not been found again in spite of the fact that many hundred thousands of plants have been inspected for variants in the coffee nurseries and in numerous coffee plantations. The size of the anomala shrub is somewhat larger than that of normal coffee plants, and nearly all its vegetative and reproductive organs are abnormal. The anomala is a recessive mutation, one pair of genes, cun an being responsible for all its differentiating characters (Krug and Carvalho, 1945). The pleiotropic effect of this gene is remarkable, as it affects the plant growth, its type of branching, the shape and size of the leaves (Fig. 3, 6 ) and the morphology of flowers, fruits, and seeds. The number of fruits produced is very small, and sometimes even three to four seeds develop in the same fruit. Furthermore, frequently more than one ovule develops in each locule, resulting in the formation of abnormally shaped seeds, a phenomenon usually described as false polyembryony (Krug and J. E. T. Mendes, 1935). The F1 hybrids between this mutant and the normal C. arabica are almost completely normal, with the exception that occasionally pairs of leaves develop with slightly misshaped apex. It is supposed that the appearance of this abnormality in F1 is due t o special environmental conditions which allow the art gene to manifest itself. iii. Anormalis. The anormalis is also a very rare mutation, which was first found in the coffee nursery of the Central Experiment Station at Campinas. Later its occurrence was also noticed in an old coffee plantation at AvarB, in the southern part of the State of Sgo Paulo. This mutation affects the size of the plant, its branching habit and its leaves, flowers, fruits, and seeds. The internodes of the branches are sometimes very short and sometimes long. The leaves are extremely

Wo. 3. Distinctive features of various genotypes of Coffea arubica; a, repre; b, young leaves of anomalo (x%); sentative leaf of an angustifolia mutant (x%) c, adult leaf of anorlnalis (xs)j d, flower of calycanthema showing petaloid calyx (natural size); e, adult leaf of orespa (natural size); f, comparison between a normal plagiotropic and upright growing lateral branches of erecta; gl, fasciated main stem of a homozygous (FsFs) plant; g n and g,, fasciated flower and fruit (natural size) ; h, fruit and seed of lamha.

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C. A. KRUQ AND A. CARVALHO

abnormal (Fig. 3, c ) ; subdivided often almost palmate and of various sizes. The corolla has a variable number of lobes, the stamens are almost normal, but the style and stigma are very reduced and misshaped. The fruits are rather large and have a disc of variable size. The seeds have a slightly corrugated surface. A rather high percentage of the locules contain more than one ovule, a fact which is responsible for the occurrence of many abnormally shaped seeds. This mutant is due to the presence of one pair of genes Am Am, which shows incomplete dominance in F1 (Krug, 1949; Krug and Carvalho, 1949). The F1is intermediate in the expression of the abnormal characters, particularly with respect to the shape of the leaves and the type of branching. A rather high degree of variability is noticed among heterozygous individuals, making it somewhat difficult to classify F2 populations. iv. Brcmze. I n C . arabica, two colors are predominant in young leaves, light green and bronze. The bronze color shows u p only a t a certain stage of development of the leaf. Very young leaves are entirely green, developing the bronze color, under the influence of light, u p to a certain size; then the color gradually fades out, and the adult leaf is again entirely green. Light has a pronounced effect on both the intensity of the bronze color and the period of its maximum manifestation. The genetic analysis of the bronze, young leaf color character showed that only one pair of genes controls the development of the bronze color in young leaves (Krug and Carvalho, 1942a). Plants carrying the Br genes have dark bronze young leaves, the double recessive individuals producing light green ones. The heteroeygous plants (Br br) develop a light bronze color. Dark bronze is therefore incompletely dominant over green. A certain variation in the degree of the maximum intensity of the bronze color has been noticed among homozygous plants of different parentage. Either modifying factors are responsible for this variation, or various alleles occur at the same locus. v. Calycanthema. The calyx of the coffee flower is usually reduced to very small scales, which are hardly detectable. I n 1935 a coffee shrub was found in a commercial plantation, having flowers with large conspicuous petaloid calyx. This is the only mutation of this type found among numerous coffee plants ; its occurrence is therefore extremely rare. The size and shape of the shrub as well as its branches and leaves are normal and very similar to plants of the typka variety. The sepals are almost identical to the petals, giving the impression of a flower with two corollas (Fig. 3, d ) . The anthers are normal, and the pollen is viable; the style and stigma are, on the contrary, very abnormal so that the stigma sometimes lacks the typical papillose surface. The ovary is

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141

white colored; it is normally divided into two locules, each containing one ovule. The pedicel is also white and when the corolla dries, usually the whole flower, including the ovary, drops from the branch. The production of fruits and seeds is extremely rare. The few fruits so far examined were rather small and contained two abnormally developed seeds. This mutant is almost completely female sterile, which is a handicap for genetic investigations. By crossing this mutant with normal varieties of C. arabka, a n offspring was obtained which segregated into 50% normal and 50% plants having the petaloid calyx, all of the latter being again female sterile. So f a r it has not been possible to secure homozygous plants for the calycanthema characteristic. To this dominant gene the symbol C was given, derived from the word calycanthema, notma1 plants being double recessives for this gene (c c ) (Krug, 1949; Krug and Carvalho, 1949). vi. Caturra. The caturra is a mutant of great potential economic importance (Krug, J. E. T. Mendes, and Carvalho, 1949). It probably originated in the State of Minas Gerais, Brazil. The shrub is very similar to the bourbon variety, its size however being smaller, its internodes much shorter and its leaves a little broader. As it is also very productive, this mutant is being rapidly introduced into cultivation not only in the State of Siio Paulo, but also in various other coffee growing states of Brazil. By crossing caturra plants with normal individuals, 100% caturra-like plants were obtained in F1. The backcross to normal plants segregated into 50% normal and 50% caturra and the backcross to caturra gave 100% plants with caturra phenotype, which could not be separated into two groups. Dealing with a mutation affecting size of the plants, length of the internodes, etc., it is not possible to distinguish homozygous from heterozygous individuals as young seedlings in the lathhouse. Perhaps this distinction will possibly be made among adult plants in the field. These data show that the characteristics of the caturra mutant are controlled by one pair of genes which show almost complete dominance over the normal types of coffee. The symbol C t Ct was selected for this mutant, the name caturra meaning “small size” (Krug, 1949 ; Krug and Carvalho, 1949). vii. Cera. The endosperm of the seed of G. arobica is normally greenish, the color usually fading out when the seeds get old. Several other species of Cofen, however, have seeds of yellow endosperm. I n 1935 several coffee trees of C. a,rabica were found in private coffee plantations a t two different regions of the State of SBo Paulo, producing seeds with yellow endosperm. These mutants were called cera, meaning “wax coffee. ” This mutation apparently affects only the endosperm color, as the shape and size of the shrub, as well as the characters of the

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C.

A. KRUQ AND A. CARVALHO

branches, leaves, flowers, and fruits are identical t o the ones of the tyficn variety from which it probably originated. By crossing cera plants, using as male parent plants producing green seeds, green hybrid seeds are obtained (xenia), which indicates that the cera alleles (ce ce) are recessive t o one dose of the normal allele Ce. Further genetic analysis showed that endosperm of the constitution Ce oe ce, Ce Ce ce, and Ce Ce Ce are equally green (Carvalho and Krug, 1949b). Studies with this mutation furnished in 1939 the genetic proof of the existence of real endosperm in the coffee seed (Krug and Carvalho, 1939). Up to that time it had not been definitely established whether the bulk of the coffee seed was true endosperm. The cera mutant has become valuable as a genetic tester in the study of pollination mechanism in coffee. The cera coffee has certain commercial value, when completely free from green seeds. viii. Crespa. At two regional experiment stations of the Instituto Agron8mico two seedlings with very small crinkled leaves (Fig. 3, e ) were found in the coffee nurseries in 1936. It is presumed that they represent independent mutations at the same gene locus. Hybrids of these two plants with representative individuals of the typ-ca and bmrbon varieties gave offspring which segregated into 50% normal and 50% similar to the two original seedlings. These results indicated that plants with the crinkled leaves were heterozygous for one pair of factor, Cr cr (Krug, 1949 ;Krug and Carvalho, 1949). By selfing these plants, normal and crinkled leaf individuals were obtained. Some of the crinkled leaf plants are small in size and grow very slowly and are presumed t o be homozygous (Cr Cr) ; they have not flowered yet. This gene has a marked pleiotropic effect affecting the branches which are more slender than the branches of the ty&a variety. The number of flowers is small and fruit set is very scarce. When mature the fruits are slightly reddish. ix. Erecta. Coffee plants of almost all varieties, so far known, develop normal plagiotropic branches, which form an angle of about 65 degrees with the main stem. In 1913 a variety was described by Cramer in Java, which he named “erecta” and that develops orthotropic (upright growing) lateral branches (Fig. 3, f ) . These branches form at &heir base an angle Of only about 25 degrees. This mutation has been found to have a very specific effect, as none of the other plant characters appear to be influenced. Erecta plants have been found in the State of Ssio Paulo in two different regions, but it is not known whether they have the same origin.

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143

I n nurseries, among a great number of seedlings, this mutation has never been found, The genetic analysis revealed that one pair of completely dominant genes (E r E r ) is responsible for this striking modification of the branching habit. F1plants (normal X erecta) are indistinguishable from the homozygous erecta plants, a clear segregation of 3 erecta to 1 normal plant occurring in Fz. Furthermore the gene shows complete penetrance and constant expressivity (Carvalho and Krug, 1950). In spite of the radical change in the growth habit of the lateral branches, the dimorphism of the main and the lateral branches still persists in this mutant, as grafts made of lateral branches of the erecta develop only into low-growing individuals. I n order to obtain normal growing erecta grafts, it is necessary to use the tip of the main stem as scions or of some secondary upright growing shoot. This peculiar dimorphic behavior of grafts derived from the main stem and from lateral branches is well known in normal coffee and also in some other perennial plants. x. Fasciata. Two different types of fasciation are known in coffee. One type is nonhereditary, and sometimes occurs on young vigorous shoots that develop on plants which have been heavily pruned, specially when they are grown on rich, fertile soil. This type of fasciation soon disappears, the shoots becoming normal, a few nodes away from the base of the fasciated stem. The other type of fasciation is genetically conditioned, representing the main character of Cofea arabica L. var. p d y sperrna Burck. This mutation has been known for many years, having been described in detail by Cramer in 1913. The genetic analysis of this mutant revealed that fasciation is due to one pair of genetic factors, which show incomplete dominance in the heterozygous conditions (Fs fs) (Krug and Carvalho, 1940a). This 3’s gene has a n extraordinary pleiotropic effect, influencing almost all parts of the plant. I n plants homozygous for this gene, the main stem is extremely fasciated and becomes twisted to such a~degree that periodically the main growing point is unable to develop any further (Fig. 3, gl). New sprouts originate again at one or more of its leaf axils; they are slightly fasciated at the beginning, but become more and more fasciated, until upward growth is stopped. As the fasciation becomes more marked, the number of leaves per node also increases. The flowers are also influenced: the number of lobes of the corolla increases from 6 u p to 10 or 12; the number of anthers increases in direct proportion to the number of corolla lobes; the style is fasciated due to the intimate union of numerous single styles and the stigma lobes are numerous, instead of only two (Fig. 3,gS). The ovary is flat and has two concentric

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C. A. KRUO AND A. CARVALHO

series of locules ; the internal series usually does not produce seeds. The locules of the external series contain normal ovules which develop into abnormal wedge-shaped seeds of no commercial value (Fig. 3, g s ) . Occasionally slight irregularities are noted at meiosis, the 44 chromosomes not being distributed normally to the poles at first division. This irregularity in chromosome distribution conditions the appearance of a small percentage of abnormal pollen tetrads. The great majority of the pollen grains is normal. The heterozygous plants show an intermediate degree of fasciation and a certain amount of variation is noted among both homozygous and heterozygous individuals. This variation in fasciation is probably due to the action of modifiers. xi. Laurina. The luiwina variety, Coffen arabka L. var. laurine (Smeathman) DC, has been known for many years. It is a shrub of rather small size and conical shape ; its leaves are smaller than those of the typica variety, and its fruits and seeds (Fig. 3, I t ) are slightly pointed a t the base. These characteristics result from the action of one pair of recessive genes, lr lr, the hybrids (typica X laurima) being entirely normal and the F2 and the backcross to laurina segregating into distinct classes of normal and laurina (Krug, 1949 ; Krug and Carvalho, 1949). Classification of the plants in segregating populations, should be made when they are one to two years old. Plants of several progenies have been harvested separately a t several experiment stations of this Institute. At Campinas, after fourteen years of consecutive harvests, the plants have shown excellent yields and appreciable resistance to drought, but in other regions where tests have also been made, the yields have been rather low. The quality of the coffee produced is considered excellent. xii. Maragogipe. Maragogipe is a mutation that was first observed about 1870 in a private coffee plantation in the Maragogipe county, State of Bahia, Brazil. It represents a gigas form of C. aru&ica. All parts of the maragogipe plant are larger than the typica variety including height, length of internodes, leaves (Fig. 4, a), flowers, and fruits. 1.1 spite of being more vigorous than almost all other coffee varieties, its productivity is rather low. Its chromosome number is normal (2% = 44). The genetic analysis of maragogipe revealed that this mutant is due to the action of a dominant gene, Mg. The hybrid (mragogipe X t y p ica and maragogipe X bourbon) are indistinguishable in their general aspect from the homozygous maragogipe. The yield of the hybrids seems to be a little higher than of the pure maragogipe (Krug and Carvalho, 1 9 4 2 ~ ) . Various investigations have been carried out to determine the causes

FIG.4. Distinctive features of various genotypes of Cofea arabica; a, mature leaf of a maragogipe mutant ( x % ) ;b , and b,, mature leaf, fruit and seed of rnolcka; c, eight-year-old dwarf seedliiig of n a n n ; d, somatic mutations na + N a ; E, fruit of goiaba mutant (sdsd) (natural size) ; f, top view of young seedling with variegated cotyledones.

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of the low productivity of the maragogipe coffee. It has been found that a lower number of flowers per leaf axil and a reduction in the percentage of fruit setting in comparison with the typica and bourbon varieties are probably the main causes of its low yielding capacity (Carvalho, 1939). A special project was included i n the breeding program of the Qenetics Division of the Instituto Agronamico in 1935 with the objective of improvement of the yielding capacity of the maragogipe variety. As a part of this project, selections were made on the basis of high yield, from a mixed population of maragogipe and other varieties on a private plantation. From progenies whose plants have been harvested individually for the last twelve years it has been possible to select individuals which are much more productive than ordinary maragogipe plants. The majority of these plants, however, are heterozygous ( M g m g ) . Due to its vigor, the maragogipe mutant has been extensively used, not only as rootstock in grafting (J. E. T. Mendes, 1938), but also as a parent in crosses with several other commercial varieties. xiii. Mokka. No information is available as to the date and place of origin of Cufea arabica L, var. mokka Hort. ex Cramer. It has been known for a long time, and it is found in almost all coffee collections. The mokka variety is characterized by small habit of growth, conical shape of the shrub, and very small leaves possessing relatively very large domatia. (Fig. 4, 51). The flowers are somewhat smaller than those of typica variety, and the fruits and seeds are the smallest known to occur in the arabica species. (Fig. 4, b 2 ) . The quality of coffee is considered excellent. Because the fruits are small, the total production per plant is less than that of other commercial varieties of the same species. A genetic analysis of its characters was started in 1933 and may now be considered almost complete. Thousands of plants have been studied under lathhouse and field conditions. Its general growth aspects and also the shape and size of its seeds have been studied. I n order to collect accurate data on these characters it was necessary to maintain each plant during a period of several years and study F z and also F3 generations. Large Fz progenies of more than one hundred plants were selfed plant by plant in order to determine their genetic constitution. It was finally concluded that the characters of the mokka variety are controlled by two pairs of genetic factors. One of this gene lr (lauripza) is entirely recessive, and the other m o (mokka) has slightly incomplete dominance (Krug, 1949). True mokka plants are of the following constitution lrlr momo, thus they are closely related to the 1au.Urina. variety (lrlr M o M o ) . By crossing mokka with typica or bourbon the following genotypes occur in F1 and F2:

THE GENETICS OF COFFEA

PI: LrLr YoMo Fl:Lrlr Momo Fa:LrLr YoMo

(normal)

x

147

lrlr mom0 (mokka)

= iiormal LrLr Mom0 = like Fl LrLr mom0 = in adult stage very similar to mokka Lrlr MoMo = normal Lrlr Mom0 = similar to Fl LrZr mom0 = in adult stage very similar to mokka lrlr MoMo = laurina lrlr Mom0 = laurina bearing round (not pointed) seeds lrlr momo = mokka

The F, plants are normal in size although their leaves are somewhat smaller; their domatia are a little more protuberant than those of the typica variety. The F2plants of the constitutions L r L r mom0 and L r l r momo, when in the seedling stage, demonstrate a n abnormal type of branching. While on normal plants lateral branching starts from the ninth to the eleventh leaf axils of the main stem, on these genotypes plagiotropic branches only develop a t the axils of the twenty-fifth to the thirty-sixth pair of leaves. Due to this fact the main stem has no low side branches. Its leaves are a little larger than those of the true mokka, but the domatia are developed to an extreme degree. After transplanting the seedlings to the field, new orthotropic branches develop a t the base of the plant and normally develop lateral branches. Therefore the adult plants look very similar to the true mokka variety (lrlr momo), only exceeding it in size. Their fruits and seeds are almost identical to those of true molrka. F or this reason these two F2 genotypes have been classified as moklra. The genotype lrlr Mom0 is very similar to laurina (lrlr MoMo) but it has larger domatia on its leaves and its fruits and seeds are slightly rounder and larger than those of the true mokka. It has been found possible to “synthesize” the laurina by crossing mokka with typica or bourbon. On account of the similarity of the true mokka (lrlr momo) and the two other mokka genotypes, it is possible that representatives of this variety, found in some coffee variety collections, are not of the double recessive type. xiv. Murta. The first genetic study of coffee conducted a t Campinas was undertaken with the murta variety which is related to the economically important bourbon coffee. I n the selfed progeny of murta plants (Nama), three genotypes were obtained : bourbon, murta, and nana, in the proportion of 1:2 :l. Only one pair of factors is involved in this segregation. The nana plants vary in size, some of them becoming similar to murta type (Krug, 1939a).

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The murta plants have smaller leaves than those of the bourbon variety and the primary veins are rather conspicuous. The nana plants are extremely slow in their development (Fig. 4, c ) ; many do not flower at all, others flowering only after 8 to 9 years. The number of flowers is very small, but the fruits are normal in size and shape. The mna gene has been found to be unstable in the somatic tissues, frequently mutating to the dominant condition (Carvalho, 1941). Sometimes the mutation affects all tissues including the one responsible for the formation of gametes. In other instances it has been found that the germ layer has not been affected, a chimera resulting from this situation. I n consequence nana plants have produced branches of the murta (Nana) type, whose seeds furnished an offspring segregating in the proportion of 1 N d a : 2 Nana: 1 w n a . In other instances the mutant murta (Nana) branches which developed on nana plants, furnished progeny almost entirely consisting of nana plants (Carvalho and Krug, 1946). In one instance a nana seedling was found which developed into a mosaic plant and showed all sorts of tissue combinations with various appearances (bourbon, murta, and nana). On this plant, pairs of leaves might develop either two NaNa leaves or one of Nana and the opposite as nana. Sometimes only half the leaf is influenced by the mutation; when this happens the leaf becomes distorted as there is considerable difference in size of the leaves of the bourbon, murta and nana (Fig. 4, d ) . The W M gene also influences the yielding capacity of the plant. Murta plants of the same progeny produce about half as much as their sister bourbon plants. Dwarf plants produce almost nothing. In addition the murta plants are more susceptible to die-back (Krug and Carvalho, 1946b). xv. Purpwrascens. This mutant has been known for many years and has been described its Coffea urabka L. var. purprascens Cramer. The young leaves are dark purple, and the adult leaves show this purple coloration to a slight degree. The flowers are pink cdlored, and the fruits when young show purple streaks on a green background. When mature the fruits are dark red similar to those of the @pica variety. Its yielding capacity is low. By crossing this type with plants possessing young green leaves ( b r b r ) , the purpurascens behaves as recessive, the F1 individuals having green leaves. I n some crosses with green plants and under certain environmental conditions the Fl’s have been found to have slightly yellowish young leaves. This is possibly due to the effects of modifying factors. In crosses of purpurascem with plants possessing dark bronze young leaves ( B r B r ) , the F1 plants develop young leaves of somewhat darker bronze color. In Fz the following phenotypes occur : plants with

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a purple leaf color, plants with young green leaves, and plants which differ in the intensity of the bronze leaf color. I n view of the very small differences in color grades, it has not yet been possible to establish definite, numerical classes in the F z and backcross generations. It may be concluded that a primary pair of genes (prpr) is involved in the purple leaf color. This prpr gene is hypostatic in relation to brbr but its mode of interaction with the Br has not yet been definitely established (Krug, 1949 ; Krug and Carvalho, 1949). xvi. Large sepals. Normally coffee flowers develop only a very rudimentary calyx. The g o k b a variety, however, has a well-developed persistent calyx made of five sepals (Fig. 4, e ) The fruits of this variety of coffee are similar to small fruits of the guava plant (Psidium guajava L.) which also have a persistent calyx. This mutant apparently originated on a private farm close to Limeira in the State of S f o Paulo. The F1plants (normal x g o k b a ) have a calyx of intermediate size, the Fz generation segregating into three types : SdXd (normal), Sdsd (with intermediate calyx) and sdsd (with well-developed sepals) in the proportion of 1:2 :1 (Krug and Carvalho, 1946a). Slight irregularities in branching habit and chlorophyll formation develop on certain branches of the homozygous goiaba type plants. I n a former chapter a mutant, calycanthema, was described as also affecting the calyx. By crossing homozygous goiaba plants ( s d s d ) with individuals heterozygous for the cdycanthema gene ( C c ) , 50% calycanthema plants (Xdsd C c ) and 50% heterozygous goiaba plants (Sdsd cc) have been encountered. The C gene is therefore completely epistatic over sd, as even plants of the constitution sdsd Cc are of the calycanthema type (Krug, 1949). xvii. Semperflorem. Under normal environmental conditions coffee plants in the State of S f o Paulo flower two to four times in a period between the end of Ju ly and October of each year. Only rarely do they flower beyond these limits. The semperflorens variety, however, flowers several times and in almost any period of the year. Frequently one may find buds, open flowers, and fruits of various sizes a t the same time on its branches. The coffee yield of this variety is distributed throughout the year, but is more intensive during two periods, one of them corresponding with the normal coffee harvest, the other occurring in OctoberNovember. Its annual yield is of the same magnitude as that of the bourbon variety. The semperflorens could be profitably grown on small coffee farms (Krug, 1939b). The characteristics of the semperflorens variety are controlled by one pair of recessive factors (sfsf). This pair of genes also affects the shape of the plant as the lateral branches develop in a more upright direc-

.

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tion. The F1plants (semperflorens X normal) are entirely normal, the F2segregating into 3 normal and 1 semperflorens (Krug, 1949 j Krug and Clarvalho, 1949). This mutant originated from the bourbon variety. xviii. Typiea. Typicu variety is considered to be the primitive type of C. arubicu. Nearly all the mutants described in this paper have been crossed with this variety in order to determine their genetic relationship to it. By crossing this variety with the murta and nana genotypes some further information have been obtained concerning the genetic constitution of ty/pico. When na/na is crossed with typku the Fl plants are nearly normal, which, when selfed, give rise to a series of plants that can be classified as typica, bourbon, nana, murta, and a new type of murta having much larger leaves. If it is assumed that typica plants carry, in addition to the NuNu genes, another dominant gene TT and that the nana plants are double recessive for these t genes (nana t t ) , then one should get by selfing the F1 ( T t Nanu) plants the following classes in the Fa: TT NaNa = TT Nana = TT nam = T t NaNa = T t Nana = = Tt r m ~ t t NaNa = t t Nana = tt nana =

typica almost typica almost typica (very similar to bourbon) typica almost typica murts with larger leaves bourbon murta nana

The T gene is epistatic over na and N a is dominant over na in the presence of tt. This results in the appearance of two types of murta, that is, murta with large leaves and the common, small-leaved murta (Krug, 1949). By selfing the murta plants with the large leaves Tt nunn, the progeny has been found to include: normal plants TT nuna with even slightly larger leaves in addition to the murta individuals with large leaves T t nmu and the dwarf plants tt n w , in the approximate proportion of 1 :2 :l. On the basis of the above results it is evident that there is a genetic difference between bozirbon and typica varieties of coffee, the bourbon being double recessive ( t i ) and the typica carrying the dominant alleles of the T gene. All varieties of C. arabka can now be grouped into two classes, having either tt or TT. This recognized difference is helpful in deciding whether a given mutant has been derived from bourbon or from typica. It is now possible to state that the mutants catwrra, semperfEorens, laurina, and others are derived from bourbon, whereas the mara-

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goyipe, cera, goiaba, calycanthema, etc., originated from typica. The genetic tester used to decide this question is the common murta ( t t Nana) . If, as one believes, the typica is the primitive type, the mutation from T to t must be considered of utmost economic importance. The bourbon variety was obtained in this way and it has a much higher yield than the t y p k a variety. xix. Xanthocarpa. Up t o 1870 only red, ripe fruited varieties of coffee were known. By that time, in the Botucatii county (State of S l o Paulo, Brazil) a coffee plant was found which had yellow fruits. This new yellow-fruited coffee type which probably originated through mutation was rapidly multiplied and cultivated in many coffee regions of the State of SBo Paulo and was named “Amarelo de Botucatii,” Coffea urabica L. var. typica Cramer forma xanthocarpa (Caminho8) Krug. The mutation must have occurred on a typica plant. The “Amarelo de Botucatti” is similar to typica in all characteristics except in the color cif its fruits, earliness of maturing, and possibly in yield capacity. Yellow fruit color is controlled by one pair of genes, xanthocarpa ( x c x c ) . The red color XcXc is almost completely dominant over the yellow, the F1 hybrids producing red fruits of slightly lighter color, especially when not completely matured (K ru g and Carvalho, 1940b). The yellow fruits sometimes show sectors of light red color probably due to somatic mutation of the gene xc to Xc in the pericarp tissue. The same phenomenon is noted on fruits of hybrid plants which then possess dark red sectors, By crossing xant/zocarpa plants with puppzirascens, in F z , yurpurascens individuals with yellow fruits are obtained. These fruits are of slightly darker yellow color, resulting from an interaction between the purpurmcens ( p r p r ) and xanthocarpa genes ( x c x c ). It is believed that the zanthocarpa mutant contributes to increase the yield. This has been found to be true in the yellow bourbon, Coffea urccbicu b. var. Boitrbon (B.Rodr.) Choussy forriia zanthocarpa K.M.C. (J. E. T. Mendes, 1949) and the yellow caturra, C o f e a arabica L. var. caturra K.M.C. forma zanthocarpa K.M.C. The santhucorpa mutants are therefore of special interest in breeding work. xx. Independence of the genes Br- and X c - . Swamy (1940) reported that the Br and Xc genes seein to be linked. Data obtained by the writers do not confirm his views and revealed that the genes bronze and xanthocarpa are independently inherited (Krug and Carvalho, 1942b). xxi. Other mutations. Several other mutants are being submitted to genetic analysis: a mutant with dark green brilliant leaves; plants

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presenting abnormal branching; the San Ramon variety; two types of pendula; plants with mucronata leaves; a new type of laurina with seeds pointed a t the upper end; plants bearing fruit with larger disc; plants possessing trifid stigma, etc. So f a r no case of linkage has been found, probably due to the relatively small number of genes studied and also due to the relatively high number of chromosomes of C . arabica ( n = 2 2 ) . c. Cytoplasmic Inheritance. I n C . arabica cytoplasmic inheritance occurs to a certain extent in plants with variegated leaves which possess abnormabplastids in one or more layers of the leaf tissue. When these abnormal plastids occur only in one of these cell layers, areas of light green color are observed ; when these plastids are present in two or more such layers, yellow and even white areas can be noticed on the leaves (Krug, 1949; Krug and Carvalho, 1949). Variegated plants when selfed give origin to normal green and variegated plants in variable proportions. When one separates the selfed seeds produced on the axils of the normal green leaves from those that originated on the axils of variegated leaves of the same branch, different results are obtained-the former produce only green seedlings, whereas the latter sometimes give rise to 100% variegated seedlings, but sometimes a few green plants also occur. When branches from normal green plants are grafted upon variegated stocks, the leaves of the scion remain entirely green. The abnormal plastids are not transmitted through the pollen, as all hybrids obtained by using variegated plants as male parent are green. I n reciprocal crosses a few variegated hybrids are obtained. The selfed progeny of F1 (normal 0 X variegata S ) do not give rise to variegated plants, a fact which indicates that these variegated plants do not possess a genetic factor which determines the appearance of abnormal plastids.

2. Other Species of Coffea Very few species besides C . arabica are commercially cultivated in coffee growing countries. Among these species are C . canephora, C . liberica, C . Dewevrei and C . congensb. Practically nothing is lznown concerning the genetic constitution of the varieties of these species. They are self-sterile, so that their genetic analysis is particularly difficult. A t Campinas only a limited amount of information has been gathered in relation to some mutants of C. camephora (Krug, 1945b). Fasciation in C . canephoya is due to one pair of recessive genes fscfscwhereas in C . arabica fasciation is of incomplete dominance. It is not known yet whether the same gene occurs in both species. By cross-

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ing two plants of C . ca.nephora which are heterozygous for fasciation, a population is obtained which segregates in the proportion of 3 normals to 1 fasciated plant. As in C . arabica, the gene fs" is highly pleiotropic, conditioning fasciation of the stem, branches, and fldwer parts. In C . canephora another mutant also occurs characterized by dwarf plants. The seedlings develop very slowly, they flower, but only produce few fruits. By crossing two hybrid plants for this dwarf factor, a population is obtained which segregates clearly into 3 normals to 1 dwarf plant. The symbol naCnaOis proposed for these gene. The two genes fsc and nuo are independent, as an approximate segregation of 9 :3 :3 :1 is obtained by crossing two individuals heterozygous for both factors. Angustifolia and xanthocarpa mutants have also been found in the C . canephma species. I n C . congensis, angustifolia. seedlings have been found and also a plant with abnormal leaves and flowers. A type of C . Dewevrei with pink corolla has recently been found. The inheritance of this character is being studied.

3. Interspecific Hybrids Various crosses have been made between C . arabica and other species of Cofea. F r uit set in interspecific crosses is very low. It has been noticed that the best success can be obtained by using C . arabica as female parent. Probably this is related to chromosome number of the endosperm. The triploid hybrid grows very slowly requiring special attention. Various crosses have been made to obtain triploid hybrids which might be of special economic importance, after chromosome doubling. Other crosses have been made to study the behavior of certain arabica genes in another genetic background. A few examples of these interspecific crosses will be given below. Hybrids between C . canephora and C . arabica var. mokka have leaves of intermediate size, possessing rather large domatia, indicating that the moklra genes (morno) act with incomplete dominance. Crossing C . canephora with polysperma variety of C . arabica (FsFs)results in the development of an interspecific hybrid which shows e certain degree of fasciation. This indicates that the Ps gene has incomplete dominance even in this interspecific combination. I n one particular tetraploid hybrid which probably originated through the combination of two genomes of C . canephora with one genome of C . arabica the fasciation was less intense, indicating that two "normal" alleles of the fasciata gene of C . can,ephma reduced the expression of one dose of the fasciata gene of C . arabica (Krug and A. J. T. Mendes, 1943). Hybrid seeds obtained by crossing the green seeded bourbon variety

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of C. arabica as female plant with the yellow seeded C . Dewevrei var. excelsa were found to be yellow, indicating that one excelsa gene for yellow seed color was dominant to two doses of green endosperm of C. ambica. The leaves of the hybrid seedlings derived from these seeds were similar but slightly smaller than those of excelsa. Spontaneous interspecific hybrids between C . tiberica and C . arabica have been found in Java, which are self-compatible (Ferwerda, 1948). Recently a new tetraploid form of Coaea was found which probably originated through natural crossing between C . ara.bica and C. D e w e w e i , the latter having probably contributed with two sets of chromosomes. This tetraploid form has been extensively crossed with varioiis genotypes of C. arnbico, (A. d. T. Mendes, 1949; Krug et nl., 1950). Jt has been verified that, in general, all recessive mutants of C.arnbkca also behave as recessives in these interspecific hybrids. On the other hand, all dominant genes of C. arabka maintain their dominant expression when crossed with this new tetraploid form. OF GENETICAND CYTOLOGICAL INVESTIGATIONS FOR VI. IMPORTANCE COFFEERREE~ING

The results of all basic investigations of taxonomy, flower biology, genetics, and cytology have been used in the coffee breeding program conducted since 1932 at the lnstituto Agronamico. Some 50,000 plants have been tested. The accurate selection of thousands of individuals representing numerous economic varieties and their progenies growing at various regional experiment stations was possible only after a thorough study of their principal morphological characters. The technique of selfing and crossing was improved after a detailed study of the coffee flower and the mechanism of pollen transmission. It was found that C. arabica is predominantly an autogamous plant. About 7 to 9% of the seeds come from natural crossing under the environmental conditions in Campinas. These results indicate that it is not necessary to isolate multiplication plots, planting them too far away from other coffee plantations. The data so far collected concerning the genetics of C. arabica have been extremely useful in planning various breeding projects through hybridization. The results derived from cytological investigations explained the sterility of many interspecific hybrids and are of great importance for the synthesis of new types of coffee.

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VII. SUMMARY AND CONCLUSIONS Investigation of the genetic characters of a woody perennial plant such as coffee requires long periods of time and involves considerable expense. These are important factors that have tended to limit the information available related to the genetics of coffee. The genus Coffea is polymorphic. All species so f a r studied have been found to have 212 = 22 chromosomes with the exception of C . arabka which has 212 = 44 chromosomes. The haploid form of C. arabica occurs rather frequently and is relatively vigorous. The haploid forms have been used for artificial chromosome duplication and production of homozygous plants which are of special value for breeding and for study of mutation rates, both natural and artificially induced. The natural synthesis of C. arabica is supposed to have occurred long ago, as all genetic differences so far studied (more than 2 0 ) are due to single pairs of genes. No mutants conditioned by duplicate genes have so fa r been found. The morphology of the mitotic and meiotic chromosomes of C . canephora and C . arabica has been studied. At the beginning of the meiotic prophase the chromosomes of both species present heterochromatic, intensively stained regions around the centromeres and faintly stained euchromatic ends. All species so far studied are self-sterile except for C . arabica which is self-fertile. The results derived from the study of natural pollen transmission have shown that C. arabica is predominantly autogamous. The study of several generations resulting from artificial selfings of several varieties of C. arabica have also shown that inbreeding does not have a harmful effect on either plant development or yield. A greater number of mutants occur in the tetraploid C . arabicla than in the diploid C . canephora. The reason for this may be the fact that C . canephwa is self-sterile and therefore a cross-pollinated plant. I n contrast C. arabica is self-fertile and is usually propagated by seed and the chances of finding recessive mutants are greater. Both chromosomal and gene mutations have been found in C . arabica. The most striking chromosomal mutations are the hexaploid (2n = 66 chromosomes) and octoploid (2% = 88) bullata forms and the diploid (2% = 2 2 ) monosperma variety. Several instances of somatic chromosomal mutations have been found. General characteristics of the gene mutants so f a r studied in C . arabica may be described as follows : a. These mutants can be grouped into three, more or less, distinct and equally frequent classes, as approximately one-third of them are

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dominant, one-third show incomplete dominance, and one-third are recessive in relation to the typica variety, considered to be the wild representative of G. arabica. A rather high proportion of mutants are therefore dominant or show incomplete dominance. This tends to differentiate coffee from most other organisms whose genetic variants have been studied. 6. Several of the recessive mutants are very vigorous and some have exceptional economic importance ; the bozcrbm variety was probably derived from the t y p k variety through mutation of a single pair of main genetic factor T + t ; the laurina and semperflorens mutants, both having important economic characteristics were also probably derived through single recessive gene mutations from the barbola. c. Some dominant mutations do not offer any advantages over the typicca variety ; however others are of special economic interest, as the rnaragogipe and caturra varieties ; the caturra has been recently introduced into cultivation in Sgo Paulo. d. Another rather striking fact is that of the 20 genes so far studied, 14 have a pleiotropic effect, some of them affecting many plant characters as the anomala, anormalis, crespa, f asciata, maragogipe, and mokka genes; in contrast some genes have a very specific effect, as the erecta, cera, and bronze genes; the erecta factor has shown a complete penetrance and constant expressivity. e. Most of the genes studied show great stability; however, the nu factor has been found to be very unstable under certain circumstances resulting in the production of various types of genetic chimeras ; for instance, nana plants have produced branches which are phenotypically murta (Na ma) but which, following self-fertilization, have produced progenies of predominantly nana plants. f. I n spite of the fact that only a limited number of genes has been studied, various instances of epistasis and of factor interaction have been found. g. The results of the genetic studies have been of considerable help in the coffee breeding work, and some of the new gene combinations may prove to be of economic value. it. The data obtained from the study of gene mutations also allow a better insight into the origin of many of the cultivated coffee varieties. AOKNOWLE~MENTE

It is evident that a reaearch project such as the one reported in this chapter could not be carried out successfully without the help of various colleaguea. Special acknowledgments are due to J. E. T. Mendes, head of the Coffee Division, H. Antunes Filho and C. 8. Novae8 Antunes, of the Genetics Division of the Inatituto

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Agrontjmico, for the valuable help during field and laboratory work. Particularly to Dr. T. J. Grant and A. S. Costa thanks are due for the revision of the manuRcript. A11 drawings were made by Lino Dorelli.

VIII. REF’ERENCES Carvalho, A., 1939, Bol. tbc. Znst. Agr. Campinas 69, 1-45. 1941, Bragantia 1, 453-466. 1945, Bol. Superint. Sew. Caf6 Est. S6o Paulo 20, 1138-1146. 1946, Bol. Superint. Sew. Cafe Est. Siio Paulo 21, 6-10, 69-73, 127-130, 174-184. Carvalho, A., and Krug, C. A., 1946, Bragantia 6, 239-250. 1949a, Bragantia 9, 11-24. 1949b, Bragantia 0, 193-202. 1950, Bragantia 10 (in press). Chevalier, A., 1940, C. R. Acad. Sci. Paris 210, 357-361. 1942, Les cafbiera du globe. 11. Iconographie des cafbiers sauvages e t cultivbs et dee rubiaobes prises pour des cafbiers. Paul Lechevalier, Paris. 35 pp. 1947, Les cafbiers du globe. 111. Systbmatique des cafbiers et faux-cafbiers, maladies et insects nuisibles. Paul Lechevalier, Paris. 356 pp. Ciferri, R., 1937, L’Agricoltura colon. 31, 515-521. Cramer, P. J. S., 1913, Gegevens over de Variabiliteit van de in NederlandschIndiij verbouwde kofle-eoorten. G. Kolff & Co. Batavia. 696 pp. Cramer, P. J. S., and van der Wolk, P. C., 1923, Fruwirth, Handbuch der landwirtschaftlichen Pflanzenziichtung. Kaffee 6, 143-161, Paul Parey, Berlin. Faber, F. C. von, 1912, An. Jard. bot. Buitenzorg, Java, 2’ ser., 10 (= 25), 59-160. Fagerlind, F., 1934, Hereditas, 19, 223-232. 1939, Svensk bot. Tidskr. 33, 303-309. Ferwerda, F. P., 1948, Economic Botany 2, 258-272. Franco, C. M., 1939, Bot. Gag. 100, 817-827. Homeyer, H., 1933, Planta 18, 640. Houk, W. G., 1936, Science 83, 464-465. 1938, Amer. J . Bot. 26, 56-61. Krug, C. A,, 1934, Ziichter 6, 166-168. 1935, J. Hered. 26, 325-330. 1936, Bol. tbc. Znst. Agr. Campinas 26, 1-39. 1937a, Bol. tbc. Znst. Agr. Campinas 22, 1-5. 1937b, Rev. Agricultura Piracicaba 12, 101-109. 1937c, J. Genet. 34, 399-414. 1939a, J . Genet. 37, 41-50. 1939b, Rev. Inst. Cafb Est. Sdo Paul0 26, 858.861. 1945a, Plants and Plant Science in Latin America 16, 243-258, Chronica Botanica Company, Waltham, Mass. 1945b, Rev. Superint. Serv. Cafb Est. S6o Paulo 20, 863-872, 979-992. 1949, Bragantia 9, 1-10. Krug, C. A., and Carvalho, A., 1939, Nature, Lond. 144, 515. 194Oa, Bol. tbc. Znst. Agr. Campinas 81, 1-36. 1940b, Bol. tbc. Znst. Agr. Campinas 82, 1-16. 1942a, Bragantia 2, 199-220. 1942b, Bragantia 2, 221-230.

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1942c, Bragantia 2, 231-247. 1945, Bragantia 5, 781-792. 1946a, Bragantia 6, 251-264. 1946b, Bragantia 6, 547-8.57. 1949, Hereditas, Lund. Ruppl. Vnl. 611-612. Krug, C. A., and Costa, A. IJ., 1947, A Fmenda 42, 35, 46-47. Krug, C. A., and Mendes, A. J. T., 1940, J . Gentf. 39, 189-203. 1943, Rev. Bgricult ura Pirariraba 18, 399-408. Krug, C. A., and Menden, 5. E. T., 193.5, Rev. X n d . Cap6 8 8 1 . SBo Pnulo 19, 13291335. Krng, C. A., Menden, J. E. T., and Carmllio, A., 1939, Rol. tPc. Innt. Agr. Campinas 62, 1-57. 1949, Bragantia 9, 157-163. Krug, C. A., Mendes, J. E. T.,Carvolho, A., and Mendes, A. .J. T., 1950, Bragantin 10, 11-25, Leliveld, J. A., 1938, Avch. voor CFe Rofle-cultuur in Ned. Zndie 12, 127-164. McClelland, T. B., 1924, Bull. P. R. agrio. Exp. 8 f a . 30, 1-27. Mayne, W. Wilson, 1937, Bull, Mysore Cofre Exp. H a . 16, 1-16. Medins, Dixier M., 1950, Bragantin 10, 63-66. Mendes, A. J. T., 1938, Bol. tdc. Znnl. Agr. Cnmpinan 66, 1-10. 1941, Amer. J . Bot. 28, 784-789. 1946, Bragantia 6, 265-274. 1947, Bragantia 7, 221-230. 1949, Bragantia 9, 25-34. 1950, Bragantio 10, 79-84. Mendes, A. J, T., and Bacchi, Oewaldo, 1940, J. Agronomin Piracionba 3, 183-206. Mendes, C. H. T.,1950, Bragantia 10, 97-104. Mendes, J. E. T., 1938, Bol. t6c. In&. Agr. Campinar 39, 1-18. 1949, Bragantia D, 81-101. Stoffels, E., 1936, Bull. Inst. nat. pour l'etude agron. du Congo Belge Serie Soient. 11, 1-41. Swamy, R. L. N., 1940, Indian J. agric. Sci. 10, 414-421. Taschdjian, E., 1932, 2. Ziioht. A17, 341-354. Testa, J., 1949, Anuhrio Estatfstiro Superint. Serv. Cnf6 %t., SEo Panlo. pp. 136137. Thomas, A. IJ., 1944, Rmp. -7. rxp. Agrio. 12, 1-12. Zimmermnnn, A., 1928, Rnffee. RangertR Andand-Riieherei, Hamhiirg. 204 pp.

The Chromosomes of the Vertebrates *

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* This article was written in Frcnch nnd was translated into English b y Dr Erich Hirschberg. of the Department of Cancer Research. College of Physicians and Surgeons [Editor’s Note.]

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R. MATTHEY Laboratoire de Zoologie. Universitt? de Lausanne. Lausanne. Switzerland CONTENTS

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. . . . . . . . . . . . . . . . . . 159 1. Sex Chromosomes . . . . . . . . . . . . . . . . . . . 162 2. Comparative Chromosoma I Morphology . . . . . . . . . . . 163 3. Technique . . . . . . . . . . . . . . . . . . . . . . 164 T I. The Chromosomes of Agnatlia niid Pisces . . . . . . . . . . . . 165 1. Heterochromosomes . . . . . . . . . . . . . . . . . . . 165 2. Comparative Morphology . . . . . . . . . . . . . . . . . 166 I11. The Chromosomes of Amphibia and Reptilia . . . . . . . . . . . 167 1 General Considerations

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1 Amphibia a Heterochromosomes b Comparative Morphology 2 Reptilia a Heterochromosomes b Comparative Morphology 1V The Chromosomes of Aves 1 Heterochromosomes 2 Comparative Morphology V The Chromosomes of the Mammnlia 1 Heterocliromosomes 2 Compsrative Mmphology VI . References

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I. GENERALCONSIDERATIONS Chromosomal characteristics can render precious services in investigations of phylogenetic order . Our attempts to reconstruct the course of events have the better chance to approach reality the greater the number of criteria they are based upon . On this score. chromosomal morphology should not be neglected . Slnce. moreover. the chromosomes constitute the most important. if not the only. substrate of the genes. the evolution of chromosomal systems must necessarily be connected with the evolution of organic systems. i.e., of living beings I n other words. if evolution remains the

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R. YATTHEY

central problem of biology, chromosomal evolution must be understood. This comprehension may be reached by two methods : ( a ) investigations in the laboratory provide information on the nature of chromosome mutations appearing “spontaneously” or induced experimentally ; ( b ) a n analytical study of a great number of animals and a comparison of the cytological observations may, a posteriori, suggest a reconstruction of the historical processes of chromosomal evolution. ( a ) Modern research has provided us with the answer to the secret of a certain number of chromosome mutations. The problem of how to pass from the genome, i.e., the haploid assortment, of a species A to that of a species B admits of various solutions, which may be listed as follows, according to White (1945) and Muller (1940) : 1. Inversions (Iiomosomal alterations) A . Pericentric, i.e., involving the oentromeric segment (heterobracliial) B. Paracentric, excluding the centromere (homobracllbl) 2. Translocations (homosornal or heterosom:tl alterations) C. “Shift,” transfer into a new position of a segment of a chromosome broken in three (homosomal) D. Insertion: a segment of a chromosome broken in three interposes itself between the two segments of a nonhomologous cliromosome broken in two (heterosomal) E. Mutual or reciprocal : the terminal portions of two nonhomologous chromosomes are exchanged a f t e r rupture of the latter (heterosomal)

These various transformations, which have actually been observed or induced in the laboratory, fail to take into account sufficiently a process revealed by comparative chromosomal morphology and demonstrated by Robertson in 1916. Some preliminary explanations should aid the nonspecialists in understanding what is involved. If we adopt the terminology of White (1945), we can distinguish three types of chromosomes : (1) the metacentric type where the centromere is placed between two “arms” which may be of equal or uneqnnl length but which are always sufficiently developed SO that at metaphase the chromosome appears as a more or less symmetrical V ; ( 2 ) the acrocentric type where, since the centromere is very close to one end, there is one long arm and one short arm, the latter being often negligible ; the chromosome has the form of a J or a n I ; (3) the telocentric type with a terminal centromere; the existence of such chromosomes, with the form of an 1 and with a single arm, is very generally denied at the present time, perhaps without sufficient reasons. From a practical viewpoint, it is very dificult, in most instances, to demonstrate the “short arm” of acrocentric chromosomes, which therefore look like telocentric chromosomes. Robertson, in comparing the genomes of different species of leaping Orthoptera,

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THE CHROMOSOMES OF THE VERTEBRATES

finds that in a genus or a family the number of “long arms” is often constant; thig is to say that if a species M has, for example, a genome formed by 6V (metacentric) chromosomes representing 1 2 arms, then the species N, 0,P, Q, R, and B will exhibit (5V 21), (4V 41), (3V + 61), (2V S l ) , (1V 101), and (121) chromatin elements. The haploid numbers, 6,7,8,9,10,11, and 12, could be related by the hypothesis that in evolution two nonhomologous and acrocentric (or telocentric) chromosomes may unite to give a single metacentric one, or, inversely, that a metacentric chromosome may break into two acrocentric or telocentric elements. This hypothesis has been largely confirmed by numerous studies bearing on various groups. White (1945) gives the name “centric fusion” to the union of two acrocentric chromosomes to form a metacentric one, and the present author (1945) has designated as “fundamental number’’ (“nombre fondamental”) the number of arms which is constant for a given group, such as 12 in the above example. The mechanism of centric fusion is difficult to comprehend; if we admit that there is a single centromere in all chromosomes, we must assume the existence of reciprocal translocations, but this theoretically admissible explanation appears to me to be very improbable. In effect, reciprocal translocation, if we wish to pass from two acrocentric chromosomes to a metacentric one, involves a breakage a t the level of the short arm of the first chromosome and at the level of the long arm of the second. Since breakages occur “ a t random,’’ there is only a slight chance that they might involve a short arm which is often so rudimentary that it escapes direct observation ; such modifications then should be extremely rare, while comparative cytological studies have demonstrated that they occur very frequently. On the other hand, there is a principle of “homologous change” (White, 1945), according to which, in a given group, the pairs present successively the same type of change, particularly centric fusion. This involves the idea of a favored direction in chromosomal evolution, while mutual translocation depends on mere chance. It would be tempting to assume that the V is the result of the apical fusion of two telocentric chromosomes. According to this hypothesis, the V reshlting from this fusion would possess two centromeres and could, inversely, yield two telocentric elements. However, there axe two things wrong with this assumption, simple and attractive though it is: it has never been possible to demonstrate the existence of two centromeres in a metacentric chromosome, and even the existence of strictly telocentric chromosomes-disregarding the experimental findings of Upcott (1937)-is contested. Be that as it may, the phenomena of Rob-

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R. MATTHEY

ertsonian type have played an important role, but their mechanism has not been elucidated. From a n evolutionary point of view, chromosomal mutations which entail rearrangements in the genic sequences may have significant consequences because of “effects of position” the extent of which has been demonstrated by geneticists. As far as the vertebrates are concerned, the zoological interest of cytology is divided between two main subjects: (1) sex chromosomes; (2) comparative chromosomal morphology, which can more particularly be employed in investigations of phylogenetic order. Before we deal with these two subjects, we must emphasize the very incomplete state of our knowledge. All in all, we have trustworthy data on approximately 400 species, while the vertebrates encompass at least 30,000, to use a very conservative estimate ; in short, we are barely able to characterize cytologically one species in a hundred. Many highly interesting forms have not yet been studied, or have been investigated only very slightly. We know essentially nothing about the Cyclostomata, very little about the Elasmobranchii and nothing a t all about the Polyptera, about Lepidosteus, Amia, Latheria. While the various families of Amphibia and Reptilia have been surveyed rather actively, the study of avian cytology has been undertaken only recently and has remained the exclusive province of the Japanese school (Oguma, Yamashina) ; only the latter have perfected adequate techniques. Among mammals, the Monotremata, whose affinities have been so widely discussed, have been the subject of no more than fragmentary observations by White and Matthey. On the other hand, the Marsupialia are rather well known while, among the Eutheria, the Sirenia, Proboscidea, Hyracoidea, Baleopitheca, and Lemuroidea are still totally unexplored. We have only scant information on the monkeys, Cetacea and Pinnipedia. In view of this most limited documentation, the conclusions to which we may be led are fraught with uncertainties.

1. #ex Chrmosomes All vertebrates, with the exception of the metatherian and eutherian mammals, appear to lack sex chromosomes which are differentiated on the morphological level and therefore recognizable under the microscope. A t least, that is the opinion which I hold and which I shall attempt to document in the sections of this review devoted to the various classes. It is known that from the point of view of genetics, Pisces and Amphibia are heterozygotes, sometimes in the male and sometimes in the female sex ; the most rigorous demonstration of this heterozygosis has

T H I CHROMOSOMES O F THE VERTEBRATES

163

been made in the female axolotl (Humphrey, 1942) and the female toad (Ponse, 1924-1949). ‘‘ Sex-linked” inheritance is unknown among the Reptilia whereas among the Aves female heterozygosis is a general rule and contrasts with the male digamety of the mammals. Of course, the fact that genetic heterozygosis exists does not mean that there must be a morphologically demonstrable heterogamety ; it is possible to conceive of sex factors situated on the chromosomes of an apparently homomorphous pair. This plain fact appears to have been neglected for a long time; many cytologists allow themselves to be guided in their work by preconceived ideas. Actually, the classical criteria which permit heterochromosomes to be recognized are not easily applied to most of the vertebrates. The existence of this or that type of digamety may be of a certain zoological interest. If, for example, we knew the type of heterozygosis of the Monotremata, we would possess a valuable indication in favor of a predominantly avian (in the case of female heterozygosis) or of a predominantly mammalian parentage (in the case of male heterozygosis) . 2. Comparative Chrornosomal Mwphology Purely cytological methods cannot yield information on the real homologies which may exist between chromosomes or “arms” of different species. Thus, various Sauria belonging to the families of the Agamidae, Iguamidae, Gerrh osauridae, and Arnphisba,en/idae have exactly the same chromosomal formula, the genome comprising 6 large metacentric chromosomes and 1 2 small punctiform elements. Genetic analysis alone would be able to tell us about homologous genes and their relative positions. But, on the other hand, the fact that in four different families chromosomal evolution has taken place in the same fashion indicates that the formulae are not just accidents and that laws-still to be discovered --direct their elaboration. The study of several species of the same genus should be particularly enlightening since we then work on a less heterogeneous genetic material than when we compare representatives of hierarchically elevated systematic categories. The number of chromosomes and of arms and the general character of the karyokinetic figures also permit of certain cautious inductions : the Dipnoi have chromosomes and a karyokinetic type which separate them from all the other Pisces. On the other hand, they resemble the Amphibia in this respect, and their 38 chromosomes, all metacentric, correspond to 76 arms, the same number that is found among the most primitive Urodela, the Hynobiidae. Are we then not justified in concluding that the Dipnoi are very closely related to the Hylzobiidae? All the American species of Triturus (Fankhauser, 1945) have 22

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chromosomes ; the European ones have 24. Since the number 24 is certainly the most widely distributed among the Salamandridae, it is permissible to suppose that the formula of 22 was established after the geographic isolation of the American Tritonidae from the European ones and, moreover, that all the American Tritonidae have a common origin which occurred after this isolation. The very numerous and very thin chromosomes of the Monotremata are divided into two categories, macrochromosomes and microchromosomes ; these characteristics are found again, exactly alike, among the Aves and, to a smaller extent, among the Chelonia. On the other hand, the Marsupialia never have more than 28 chromosomes, and these are relatively short and thick. This fact alone tends to bring the Monotremata closer to the Sauropsida while separating them from all the other mammals. The application of the rule of Robertson is equally fruitful. However, it cannot be denied that while this rule is useful if the genome comprises only V’s and telocentric elements (at least in appearance), it is hard to apply if there are chromosomes that are acrocentrio with a relatively well-developed short arm or that are very asymmetrically metacentric: the fundamental number can then not be evaluated precisely. In short, as I wrote in 1931, “the chromosomal formula can furnish some information but this must always be related to the sum total of the Any classification is the more natural the greater characteristics the number of varied characteristics on which it is based: cytology, in all modesty, adds one unit to the list.”

...

3. Technique It may be asked why the vertebrates have for such a long time been neglected by cytologists. The answer to this question takes into account factors of a technical nature. It waB not until 1928 that Minouchi demonstrated the extraordinary results which could be obtained when osmium chromic solutions, without acetic acid, were used as fixatives. These solutions (types Flemming-Heitz or Champy) are perfectly adapted to the fixation of the chrombsomes of all the vertebrates. Their use has nevertheless remained the almost exclusive province of the Japanese school and of Matthey. The Flemming-acetic acid and the BouinAllen solutions are of very mediocre value ; the latter is still widely used by Anglo-Saxon authors. The other usual fixatives cannot be employed on any vertebrates with the exception of the Amphibia: with the latter, it is relatively easy to obtain well-preserved material, and solutions such as those of Bouin or Zenker bring about the proper fixation. Acetic

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165

carmine, an agreeable fixative because it is rapid, may be used only for a preliminary orientation. To attempt to carry out a study with this agent alone means to run the risk of serious errors. It is clear, then, that with the Amphibia i t is easy to obtain adequate fixation. With the Pisces, Reptilia, and Mammalia, the technique of Minouchi-and only this technique !-gives very good but always variable results. With the Aves, great difficulties are still to be solved and only two authors, Oguma (1938) and particularly Yamashina (19401946) have published figures which give evidence of a perfect mastery of avian material. Tlie chromosome count is affected adversely by the main difficulty which remains to be solved, namely the agglutination of chromosomes which occurs with snch deplorable ease. I n general, the proper fixation is the harder to achieve the higher the chromosome number; however, other factors also have some influence : for example, agglutination is a greater danger with the Amniota than with the Anamniota. The physicochemical and physiological state of the cells and of the animals also plays a significant role ; otherwise, it wonld be hard to explain why different individuals of the same species can give very different results and why, in one and the same individual, the different regions of an organ may be fixed either very well or very badly. A complete investigation, which is rarely possible, would be based on the study of the testicle and ovary in the adult, newborn, and embryo. Bone marrow smears often show splendid karyokineses derived from the myeloblasts. While malting his observations, the cytologist must guard carefully against any preconceived notions and must forget whateve he may know about genetics ! 11. THE CIIROMOSOMES OF AQNATHA AND PISCES

Very few papers on this subject are available and many of the data,, which were obtained from material subjected to insufficient fixation, are rather unreliable. The most outstanding investigations are those of Makino (1937-1939).

1. Heteruchromosomes No modern author has located sex chromosomes in fishes; therefore, the existence of male o r female heterozygosis can be demonstrated only by the genetic method. From this point of view, the most important investigations are those of Kosswig (1931-1941) and particularly those of Winge ; the latter author published an excellent summary of his work in 1947. The fact which is difficult to explain is the coexistence, in one and the same genus, of species exhibiting male heterozygosis and of oth-

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ers exhibiting female heterozygosis. Gordon (1946) has shed some light on this problem, with the demonstration that domestication of Cyprinodontidae involves modifications of the sex-determining mechanism, perhaps due to breeding with other species. According to Winge, sex is conditioned by a series of masculinizing and feminizing factors distributed on the autosomes, and by dominant sex genes localized on the chromosomes of an apparently homomorphic pair which, however, corresponds genetically to an X-y pair in the male and an X-X pair in the female. Normally, the male has the formula mfmfmfmf-2”-M and the female nifmfmfmf-F-F. By selection of autosoma1 masculinizing or feminizing factors, i t is possible t o arrive a t the following schematic results : 8 : mmmmmmmm-F-F; 9 : ffffffff-F-M, where the sum of the autosomal genes prevails over the inverse action of the dominant determinants of X and Y. Consequently, there will be $ 8 XX and 9 9 XY. Kosswig is of the opinion that the autosomal genes T, with masculinizing or feminizing tendencies, determine a genetic constitution that is labile with regard to external influences which orient the fundamentally bipotential organism toward one or the other sex. There are then no contradictions between cytological observations and conclusions drawn from genetic analysis. The “dominant genes” of Winge might be placed on any pair of chromosomes and it is a posteriori that the investigator designates as sex chromosomes those which are presumed to carry these genes.

2. Comparatise Morphology Agnatha. Here we have at our disposal only some very old investigations from which an indication of a diploid number close to 50 may be recalled. Elasmobranchii. Raja meerdervoortii has the highest number of chromosomes which has been found in a vertebrate : 2n = 104 ; the elements, which are all acrocentric, appear to represqnt a number of arms approximately equal to that of Squalus sucklei and of Scyllium catula (2n = 62). Ostcichthyes. The formula 2n = 104 is encountered again in Cyprinus carpio, where all the chromosomes are acrocentric. The smallest figures are found in the Gasterosteidae (2.n = 42) and the Cyprinodontidae ( 2 n = 46 to 4 8 ) . The experimental findings are so few in number and several of them are so questionable that any generalization would be premature. Dipnai. The three genera have been studied and have been shown to be very homogeneous cytologically. Lepidosiren paradoxus (Agar,

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167

1911) has 38 metacentric chromosomes corresponding to 76 arms. The morphology and cellular dimensions, the size and volume of the chromosomes, and the absence of a major meiotic spiral all contribute to bringing the Dipnoi nearer to the Urodela, particularly to the Hynobiidae. The same criteria remove the Dipnoi decidedly from the Osteichthyes and to a lesser extent from the Elasmobranchii.

111. THE CHROMOSOMES OF AMPHIBIA AND REPTILIA We have seen that there are no technical diEculties in working with the Amphibia. Furthermore, these animals possess voluminous cellular elements and the number of chromosomes is rarely very high. Conditions are therefore very favorable for cytological analysis and, for this reason, we have a larger number of observations at hand. The comparison of the genomes is, nevertheless, quite difficult to carry out : in most species, there are many chromosomes with a subterminal or submedian attachment, and this fact renders the precise determination of the number of arms arduous and rather arbitrary. This disadvantage is less marked in the Reptilia : the Sauria, in particular, exhibit chromosomal assortments in which the elements are typically metacentric or acrocentric and are easily identified. Besides, fixation in the Reptilia is successful only by the method of Minouchi and the cells, like the cells of all the Amniota, are relatively small. The number of chromosomes is high only in the Chelonia. Very frequently, there are two clearly distinct categories of macrochromosomes and microchromosomes (or m-chromosomes) ; at the metaphase, the macrochromosomes form a peripheral corona at the center of which the constellation of the m-chromosomes appears.

1. Amphibia Data are available on the great majority of families. However, the Sirenidae, Proteidae, Amphiumidae among the Urodela and the Brachycephalidae among the Anura have been studied very sketchily or not a t all. It is a very frequent occurrence among the Amphibia that the same “modal number” (White, 1945) characterizes all the species of a family. a. Heterochromosomes. It is well known that from the genetic point of view the question of sex heterozygosis is a very confused one: the analysis of haploid or polyploid Urodela and that of parthenogenetic ombryos or of the lineage of hermaphroditic individuals among the Anura lead to contradictory conclusions which suggest that the methods used are inadequate for the solution of the problem. The splendid investigations of Humphrey (1942-1945), on the other hand, have established

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irrefutably the female heterozygosis of the axolotl and the viability of the superfemales PP; the studies of Ponse, over a span of 25 years, have led to the conclusion that in toads the male is homozygotic. In those instances, the information appears to be definitive. From a cytological point of view, the question has long been under discussion; Makino (1947) using a Hynobius as a representative of the Urodela, was unable to find the slightest difference when he compared the different types of karyokinesis of the two sexes. This conclusion terminates a long controversy ; the absence of morphologically differentiated sex chromosomes is a certainty among the Caudata. With respect to the Anura, the problem is more complex: Witach’i has claimed since 1922 that in frogs and toads there is a heteromorphous pair X-Y which characterizes the males and which separates a t the second maturation division. Iriki (1930-1932) and Sato (1936) admit that the male sex is monogametic, but that the ‘two X chromosomes have a particular behavior which makes possible their recognition. These conclusions have been criticized : neither Stohler (1928) nor Wickbom (1945) have been able to verify the observations of Witschi; the assertions of the Japanese authors have been recognized as erroneous by Galgano (1933), Saez and his collaborators (1934-1936), and Wickbom (1945). A detailed analysis of the literature has led me to state (1949) that among the Anura as well as the Urodela sex heterozygosis does not find any morphological expression. b. Comparative Morphology. The phylogeny of the Amphibia has been firmly established. Fo r the Urodela, Noble (1931) developed the following sequence based upon more and more marked specialization, which is here reproduced with the addition of the number of chromosomes and the fundamental number ( P . N . ): Family Eymbiidae Cryptobranchidae Proteidae Amb yslomidae Salamandridae BmphZumidae Ptethodontidae

2n

F.N.

40-62 62-64 1 28 22-24

56 48

28

f 66

1

76 74

1

If it is recalled that the Dipnoi have a P.N. of 76, the following hypothesis may be advanced : there is a reduction in the number of chromosomes and of the F.N. in passing from the archaic to the more specialized forms. A comparison of the metaphases of Hynobiidae, where the corona of the macrochromosomes surrounds a large group of m-chromosomes, and those of the Salamandridw, where the spindle appears “empty” because the

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corona of macrochramosomes exists alone, suggests the elimination of the m-chromosomes which have become inert during evolution. There are intrafamilial variations only among the Hynohiidae, Cryptohranchidue, and Salamandridae, and these variations are very well explained, a t least for the first two families, by centric fusions. All the other families are characterized by a single modal number. A chromosomal discontinuity corresponds to the familial discontinuity of the systematicians ; within the families, the fundamental homologs of the chromosomes are respected ; there have been no great chromosome mutations but essentially gene mutations. The chromosomal formula gives a n objective value to the notion of family. Examination of the Anura leads to very similar conclusions. A table conforming to the classification of Noble and set u p like that for the Urodela may be prepared: Family Disooglossidae Pipidae Polypedatidae, Ranidae Brevioipitidae Hylidae Buf onidae

2n 38-42 36 26 28 22-24 22

F.N. 44-50 48

50 50 48 44

Increasing specialization is accompanied by a reduction of the diploid number but not of the P.N. The most advanced families have a single modal number ; m-chromosomes exist only in the archaic forms. Apoda. Two genera of Coecillidae have been studied : Ichthyophis has 42 chromosomes, Uraeotyphlus has 36, and the F.N. is 52 (Seshachar, 1936-1939). Centric fusion has therefore occurred and the F.N. indicates a very advanced specialization.

2. Reptilia All reptilian orders have been studied in recent investigations with the most reliable techniques ; however, much more information io available on the Sauria and Ophidia than on the Chelonia and particularly on the Crocodilia among which only one species has been studied. a. Heterochromosomes. Nothing is known on the genetics of the Reptilia; a priori, a female heterozygosis, as present in Aves, may be assumed, but this hypothesis is without basis of fact. Cytological examination had a very late start. Dalcq (1921) described a n X-0 chromosome in the males of the blindworm, and Painter (1921) found multiple X chromosomes (8 : X1-X2; 0 : X IX Z -X ~ - XZin) several Tej& and Iguamidae. These results were questioned by Nakamura (1927) and Matthey (1929) who, using the technique of Mi-

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nouchi, obtained preparations of previously unequalled quality. Male homogamety was solidly established during the following years by a series of investigations by the same authors. Does this male monogamety have as a corollary a female digamety which is expressed morphologically? The Japanese school answers this question in the affirmative: Oguma (1934-1936), Asana and Mahabale (1940-1941), Makino (1948), and Asana and Makino (1948) state with certainty that the females have an odd number of chromosomes, that is, a digamety of the type X-0. On the other hand, Matthey (1943) and Margot (1946) have been unable to find the slightest difference between masculine and feminine genomes. The most convincing instance is that of the chameleon, since it involves the reptile in which the chromosomal conditions are most favorable for observation; this saurian has 12 large V and 12 rn-chromosomes ; Matthey has found this assortment in 9 blastoderms of unknown sex, as well as in the gonads of young females and of males. Since the count never involves more than 12 elements (because macrochromosomes and microchromosomes can be examined separately), it would appear that all error is excluded ; this is not the case in the species having more complicated formulae which were studied by the Japanese cytologists. It may therefore be asserted that the Reptilia, like the Aves and the Anamniota, do not have heterochromosomes a t the morphological level. b. Comparathe Morphology. Among the Chelonia, the number of chromosomes is very high (50-64) and the P.N. varies from 54 to 70. Too few species have been studied cytologically to allow conclusions to be drawn. The general morphology of the karyokineses resembles that of the Aves and Monotremata. Alligator mississipiensis is the only crocodilian with a known chromosomal formula : 2n. = 32 ; P.N. = 42. Nakamura (1927-1935) and Matthey (1928-1938) have carried out the cytological analysis of about fifty species of Sauria belonging to 15 families. This extended survey has demonstrated the role of centric fusion which was all the more evident because in Reptilia the distinction between metacentric and acro- (or telo-) centric chromosomes is always easy to make. Matthey divided the Sauria into three groups according to chromosomal criteria : the Geckonoida, the Scinco-lacertoida, and the Iguanoida; in this latter group, the law of Robertson is applied most satisfactorily, with almost schematic strictness ; the m-chromosomes are most frequently 24 in number; with regard to the macrochromosomal genome, we may consider it to be constituted typically by 24 arms; there are, therefore, at the most, 24 acrocentric chromosomes and, at the very least, 12 metacentric ones. The following sequence of the Iguanoida may therefore be established, ignoring the m-chromosomes for the moment :

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THE CHROMOSOMES OF THE VERTEBRATES

Agamidae, Iguunidae, Chamaeleontidae, Gemhosauridae, acrodont Ampliiabaenidae : Helodermatidne : Varanidae : Xantueiidae : Anguidae :

12 v, 10 v, 4 8V, 8 6 V, 12 4 V, 16

I 1 I I

If the 24 m-chromosomes are added to the 24 macrochromosomal arms, we obtain a P.N. which is 48 in 12 species and 46 in 9 (of a total of 43). Rut if some complementary hypotheses suggested by analysis are taken into account, the P.N. of 48 is found 22 times in the 43 species. The same F.N. also characterizes the Sphenodon (Keenan, 1932). The Ophidia differ little from the Sauria: 22 species have been examined; the P.N. is 46 in 19 instances, 48 in two, and 50 in one. The situation is therefore very similar to that in the Sauria and the role of centric fusion is equally evident. From a n evolutionary point of view, it may be said that in the Reptilia we are dealing with a “hynobian” stage; the m-chromosomes are still present, but their genetic importance is probably weak ; for example, it is striking to find that while almost all the Lacertidae have 38 chromosomes, of which one pair are m-chromosomes, the latter pair is missing in Lacerta vivipm-a. Comparative chromosomal morphology has provided a further argument for the derivation of the chameleons from the Agamidae, and for the direct relationship between the Zguanidae and the Agamidae. The acrodont Amphisbaenidae originate from the Agamidae while the pleurodont ones are chromosomally related to the Tejidae. The polyphyletism of the Amphisbamidae, surmised by many systematicians, has thus been confirmed by the cytologists. The comparative study of orders tends to separate the Chelonia distinctly from the Ophidia, Sauria, Rhynchocephalia, and Crocodilia. This opinion is also shared by the paleontologists who believe that the Chelonia are derived from the eunotosaurian stock prematurely detached (at the beginning of the Permian period) from the Cotylosauria, while the four other living orders are thought to be of eosuchian (snakes, lizards, Xphenodon) o r archosaurian origin (crocodiles).

IV. THE CHROMOSOMES OF AVES With regard to the chromosomal apparatus, the Aves are the least known of all the vertebrates. This lack of information is due to the material itself. The chromosomes are very numerous (50-80))and we

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R. MATTHEY

have seen that the cytological fixation is all the more difficult the higher the number of chromatin elements; on the other hand, these chromosomes, many of which are very small (mchromosomes), tend to fuse under the influence of the chemical agents which constitute the fixatives. Finally, since the cells are of a particularly small size, the microscopic analysis even of well-fixed material is confronted by considerable difficulties. Although great progress has been made in cytological techniques, the fixative of choice still remains to be found: while Oguma (1927, 1937, 1938) has made excellent use of the solution of Hermann, the authors who have attempted to follow this method (cf. Matthey, 1939) have not been able to reproduce the results of the Japanese cytologist. In the same animal, the chicken, the number of chromosomes was originally greatly underestimated ; in ten years, thanks to a progressive 4 4 uncovering’’ of m-chromosomes, we have seen a rise of the diploid number from 28 to 661 The latter value, moreover, is not definitive since Suzuki (1930), after counting 74 chromatin elements in the rooster, finally estimated that this gallinacean has 78 such elements ! We must therefore attribute the regrettable lag in our knowledge and the uncertainty in which we are left even by the best investigations to the inadequacy of techniques and to the difficulties inherent in the counts: objective evidence is too slight and even the most honest investigator must judge subjectively.

1. Heterochromosomes Genetically, we know that in Aves the study of sex-linked heredity reveals a female digamety. Since this fact has been known for a long time, authors have striven to bring cytological findings into agreement with this experimental result. It is of interest to review the opinions which have been expressed one after the other about the chicken. This bird probably has 78 chromosomes, of which only the largest are accessible to analysis. The various authors are more or less agreed on the morphology and classification of these large elements, which number 12. In the male, all cytologists find that the chromosomes can be grouped in pairs, while in the female one of the pairs has been said to be represented either by a single element of heterochromosomal sigeificance (X-0), or by two dissimilar chromosomes (X-Y). According to some authors, the X chromosome belongs to the first pair, according to others to the fifth. I have shown (Matthey, 1939) that these assertions are entirely without basis in fact and that, at the present state of our knowledge, morphologically differentiated heterochromosomes do not exist in the Aves. However, this statement does not by any means exclude the existence of sex factors, of

173

THE CHROMOSOMES OF THE VERTEBRATES

heterozygotes in the female sex which suffice to account for the sex-linked type of inheritance. Meanwhile, in a series of remarkable investigations published in Ja pa n during World War 11, Yamashina (1940-1946) has made a considerable effort toward resolving the problem cytologically and claims to have succeeded. By analysis of the karyokineses in both sexes, the existence of a female digamety of the type X-0 is said t o have been proved, and i t is indeed the fifth pair of the chicken which is said to be heterochromosomal. Despite the superb technique of Yamashina, I do not believe that this demonstration is definitive; it rests in effect on two series of observations : ( a ) the diploid number in the male and in the female j ( b ) the existence, i n the diploid mitoses of the latter, of a small odd metacentric element. With regard to the first point, it is impossible, in my opinion, to arrive a t an objectively certain count of 77 or 78 chromosomes, whatever may be the quality of the material. The listing of chromatin elements and the recognition of pairs is plagued by considerable difficulties and the claim of the odd character of a little V is most hazardous. It cannot be excluded that Yamashina is right, but as long as the reductional divisions of the female have not been studied, final judgment must be reserved.

2. Comparative Morphology The karyokinetic type is very homogeneous. The large chromosomes form a peripheral corona inside which the microchromosomes arrange themselves. However, these two categories are not clearly separated but are connected by a gamut of elements of progressively decreasing size. There is little evidence of centric fusion: if, with Oguma (1937), we compare the chromosomal formulae of the eight species of Aves which have been studied, we arrive at the following table : Species Oceanodroma leucorrhoa Phalacrocorax carbo Sternula albifrons L a m s argentatus Brachyramphys marmoratus Lunda cirrhata Anas platyrhinca CoturnQ coturnix

2n 74 70 66 66 50

50 80

78

Formula 74 acroceutric 66 acru., 4 metacentric 60 acro., 6 meta. 60 acro., 6 meta. 40 acro., 10 meta. 40 acro., 10 meta. 7 6 acro., 4 meta. 72 acro., 6 meta.

F.N. 74 74 72 72 60 60 84 84

This table demonstrates that the phenomena of centric fusion alone have not intervened in the chromosomal evolution of the Aves. On the other hand, it indicates a fundamental number (P.N.) between 60 and 84. It is then of interest to compare this figure with that of the Reptilia

174

R. YATTHEY

where the fundamental number varies between 38 and 70, the highest values (54-70)having been found only in the Chelonia; the latter thus appear to be, among all the Reptilia, closest to the Aves from this as well as other points of view. The Sauria and Ophidia are actually characterized by a P.N.varying between 38 and 50 while in the alligator, the only species of Crocodilia studied u p to the present time (Matthey, 1947), this number is 42. The data available at present are entirely insufficient to permit a discussion of general problems. The relationships between the chromosomal formula and the systematic position, and the phylogenic inferences based on cytology would require an exact knowledge of each familial type. Right now there are scarcely a dozen species which we have information on. However, with the meager knowledge a t our disposal, it would already appear that the chromosomal conditions are very homogeneous in each class and that it is very doubtful that a more extensive investigation would prove to be fruitful for studies of systematic o r phyletic order. By analogy with the data obtained in the study of Reptilia and Mammalia, it is nevertheless permissible to state a hypothesis, i.e., the relation between a low chromosome number and a primitive systematic position. From this point of view, the Alom may be considered to be actually the most primitive of the Aves.

V. TEE CHROMOSOMES OF THE MAMMALIA Although from a cytological point of view the Lemures, Proboscidea, Hyracoidea, and Sirenia are completely unexplored and the Cetacea, Pinnipedia, and Monotremata almost entirely so, we have a t our disposal a great number of documents of very unequal value. Thus, the Anglo-Saxon investigations, even those of contemporary vintage, have been and are based on material fixed in Bouin-Allen, a treatment which gives greatly inferior results when compared with the fixatives of Champy or Flemming-Heitz. Nevertheless, sufficient fixation has been accomplished so that these investigations cannot be simply disregarded ; but in many instances they can be accepted only as a guide for further studies.

1. Heterochrmnosames The old discussion on the type of male digamety seems to have been definitely terminated. The overwhelming majority of the metatherian and eutherian mammals depend on the scheme X-Y. Only four exceptions are certain: Macropus tcalabatw (Agar, 1923) and Smex araneus (Bovey, 1949) have multiple sex chromosomes; the male has an odd

THE CHROMOSOMES OF THE VERTEBRATES

175

number of chromosomes and may be X1X2-Y or X-YlY2. Only the study of the females will permit a decision to be made between these two formulae, depending on whether the homogametic sex has 1 (XX) or 3 (XIX1-X2X2)chromosomes more than the male. Sharman, McIntosh, and Barber (1950) have shown that a species of rat-kangaroo, Potorous tridactylus, has the following formula: male, 2n = 10 X Y1 Y2; female, 2n = 10 2X. Microtus montebelli (Oguma, 1937) is the only authentic case of an X-0 digamety. The X and Y chromosomes vary greatly from one species to the next ; the Y chromosome is often the smallest of all the elements (1 p or less) while the acrocentric or metacentric X chromosome may be, most frequently, shorter than most of the autosomes but in certain instances has been found to be the largest of all the chromosomes. A very interesting finding involves Microtus agrestis (Matthey, 1949-1950). In this vole the heterochromosomes exhibit a veritable gigantism : the metacentric X chromosomes measures 7.5 p and the Y chromosome, of telocentric appearance, is 5 p long ; even the longest autosomes do not exceed 3 p. Since 1934, a discussion has been going on concerning a conception proposed initially by Darlington and Koller, developed by the latter in ti series of publications, and utilized finally by I-Ialdane to set u p a map of the X chromosome of man. This hypothesis postulates that originally the sex chromosomes are homomorphous and that their differences, which are purely genetic to begin with, are accentuated progressively. However, the X and Y chromosomes would always maintain a homologous segment, thus making possible a zygotenic coupling and in that way the formation of “crossing over ” and of chiasmata, followed by the correct ana+phasic separation. If this is so, one can foresee, from the relative positions of the centromeres and of the homologous and differential segments of the X and Y chromosomes, the behavior of the heterochromosomes during the first maturation division. This behavior is going to depend not only upon the factors which are mentioned below but also on the number of chiasmata that will be formed. If the first division separates the X from the Y chromosome, it is called “prereductional” ; if it directs one half of each of the sex chromosomes toward the two anaphasic poles, we speak of “ postreduction.” I n the rat, Darlington and Koller observed 10% postreductions and 90% prereductions ; in their opinion, this is proof of the existence of equal and differential segments. The question is important because the genes located on the homologous segment would be only partially sex-linked while those on the differential segment would be rigorously sex-linked. We have here a basis upon which one may thus construct a map of the sex chromo-

+

+ + +

176

R. MATTHEY

mmes using the genetic results bearing on the factors carried by these chromosomes. If the heterochromosomes are really endowed with equal segments, one may expect a more or less normal premeiotic behavior ; that is exactly what is claimed by the Anglo-Saxon authors who have observed a typical zygoteny. The Japanese school, particularly Malcino (1941-1943))has taken a position against this theory. I n the rat, a study of 312 auxocyte metaphases never showed anything but prereduction ; premeiotic evolution does not involve typical zygoteny, and the late association of the X and Y chromosomes is not due to chiasmata. Matthey (1938-1950) has concerned himself intensively with this problem j a priori, he would tend to agree with the Japanese cytologists whose experimental material is of incomparably better quality than that of Koller ; his own studies have demonstrated that typical zygoteny does not exist in mammals. The previously mentioned case of Microtus agrestis is very clear in this regard : when the leptotenic autosomes begin to unite in pairs, the X and Y chromosomes, strongly vacuolated, flow together in a great mass and there is no technique which succeeds in showing up despiralized chromonemata ; at the metaphase I , it is doubtful that the heterochromosomes, always united distally, are joined by chiasmata ; and the anaphase exhibits only prereduction. However, there are mammals where postreduction is the rule, Apodemus in particular. I n Arvicola shermam,, Matthey (1938) himself observed both prereduction and postreduction. Finally, the conception of Koller is theoretically so satisfying that one cannot reject it without a more extended investigation. One point is already certain, namely that the evolution of the heterochromosomes is accompanied by phenomena which are entirely special to them. Even in the females, where the two X chromosomes are evidently homologous, zygoteny is of a very special kind. I n Microtus agrestis the two X chromosomes, despite their great length, form only a single chiasma which is invariably located on the short arm. And in Crketus auratus, IIusted and collaborators (1945) have made similar findings. All this demonstrates that the problem of the mammalian heterochromosomes is most complex. I n promoting the conception of Koller to the status of a general theory and in basing on this theory a map of the human X chromosomes, the cytogeneticists have largely gone beyond the conclusions which may be drawn from the facts which have been firmly esablished.

T H E CHROMOSOMES OF T H E VERTEBRATES

177

2. Comparative Morphology I have had occasion to state how the type of attachment of the chromosomes is often difficult to establish in the Mammalia. Here is a n example illustrating this fact : in the brown rat, all modern authors have counted 42 acrocentric chromosomes ; however, Oubnin (1945)) in an analysis of the karyokineses found in the testicle of the newborn, a n extremely favorable material, demonstrated the existence of 9 pairs of metacentric chromosomes. The P . N . therefore changes from 42 to 60! I n a general fashion, the genome of the Mammalia shows, beside the typical acrocentric or metacentric elements, some chromosomes with more or less unequal arms; the establishment of the P.N. becomes a very delicate task and this value can be fixed only in a n approximate and often arbitrary manner. The three mammalian subclasses are cytologically well defined. The Monotremata have a high number of chromosomes (60-70, approximately) which may be divided into macrochromosomes, which are long and slender, and m-chromosomes ; they are much more like the Chelonia and especially the Aves than the other Mammalia, and their karyokinetic chromosomal type is more different from that of the Marsupiales than from that of the Eutheria. The Marsupials have few chromosomes (12-28) and their P.N. lies between 22 and 36. The chromosomes are voluminous, massive, easily stained. The Eutheria have a minimum of 23 (5bres 6) and a maximum of 86 chromosomes; the P.N. is never lower than 40 and usually much higher. The comparison between orders of Eutheria is not very fruitful, and it is very difficult to know which P.N. may be considered to be the primitive one. I n effect, in certain orders a high P.N. characterizes the primitive forms (Erinaceus : 212 = 48 ; P.N. = 90) while i n others it is a property of highly specialized species : for example, the hog, a rather archaic artiodactyl, has 40 chromosomes, with a P . N . between 50 and 60; the ox, a highly specialized animal, has 60 chromosomes (PN. = 6 0 ) and the reindeer has 7 2 ( P . N . = 74). I n summary, the P.N. remains more or less constant among the Artiodactyla and it is the diploid number which tends to increase with specialization. Among the Amphibia, we have seen that the diploid number and the P.N. tend to diminish in the course of evolution. On the evidence of comparative chromosomal morphology, Makino (1948) associates the Cetacea and the Ungulata; this conforms to our present ideas. The Cmida.e exhibit striking differences between the members of

178

R. MATTHEY

the vulpine series and those of the canine series. The foxes have few chromosomes (38 in Vulpes uulpes) whereas the dog has 78, like the wolf. It is doubtful that this difference is to be attributed solely to centric fusions, but it appears to demonstrate the impossibility of achieving the frequently discussed cross dog X fox; moreover, it confirms the existence of the two series of Cmidu.e which have been recognized by the systematicians. What is the reason for this variability in the diploid numbers of the C d a e , while all the Musteliihe (weasel, ferret, marten) and Pelidae (2 species) which have been studied u p to the present time have 38 chromosomes, like the fox? We know nothing about this, but there incontestably exist “modal numbers, ” the most frequent ones being 48 (recognized already by Painter in 1925), 54, and 60. Is polyploid evolution likely? It would not seem to be, although certain cases are disturbing: here, for example, we have the series of European Myoxidae studied by Renaud (1938). Species Dyromys nitedula Muscardinus avellanariur Eliomys quercinus Glis glis

2n 48 48 52 62

Approz. F.N. 78-84 72-76 72-76 96

The formula of the dormouse cannot be reduced to that of the three other species, and yet it would be desirable to understand such a type of chromosomal evolution. The explanatory domain of centric fusions thus appears to be rather limited. Nevertheless, when we have at our disposal reliable observations bearing on several species of the same order, the Robertsonian processes become evident, as shown by the results obtained by Bovey (1949) in a study of Chiroptera. Species Rhinolophus ferrum-equinum R. euryale R. hipposideros Mgotia myotis M. mystacinus M. emarginatus M . daubentoni Minioptems schreibersii Pipistrellus pipistrellus Plecotus auritus Barbastella barbastellus

2n 58 58 54 44 44 44 42 46 42 32 32

F.N. 62 62 60 54 52-54 62 54 52

B

54 54

T H E CHROMOSOMES OF THE VERTEBRATES

179

The F.N. of the Rhinolophidae thus lies between narrow limits (60-62), like that of the Vespertilionidae (52-54). It is therefore clear that the comparative morphology of chromosomes in the Mammalia poses a whole series of problems which are f a r from solved. In our opinion, an extensive study of a very abundant order is now required. If a pleiad of cytologists, employing all the same techniques, were to establish the chromosomal conditions of a thousand rodents, it is permissible to assume that this teamwork might open new horizons. It is a curious thing to recall that this wish was already expressed by Painter in 1925. VI. REFERENCES The reader will find listed below only references t h a t a r e not included in my book, Les chromosomes des Vertbbrds (Rouge, Lausanne, 1949, 356 pp.). This bibliography, rather than listing the works cited i n the article, complemeuts the one already published i n that book; both together now include all the references up to January 1, 1951. Creighton, M., 1938, Chromosome structure in Amblyatoma punctatum. Cytologia, Tokyo 8, 497-504. Cross, J. C., 1938, Chromosomes of the genus Peromyscus (Deer Mouse). Cytolopia, Tokyo 8, 408-419. Darlington, C. D., 1947-49, 38th, 39th, 40th Annual reports of the John Iniies Horticultural Institution, London. Drummond, F. H., 1938, Meiosis in Dasyurus viverrinus. Cytologia, Tokyo 8, 343352. Fankhauser, G., 1932, The role of the chromosomes in the early development of merogonic embryos in Triturus viridescens. Anal. Reo. 64, 73-74. Gordon, M., 1946, Interchanging genetic mechanisms f o r sex determination. J. Hered. 37, 307-320. Husted, L., Hopkins, J. T., Jr., a n d Moore, M. B., 1945, The X-bivalent of the golden Hamster. J . Hered. 36, 93-96. Koller, P. C., 1946, Control of nucleic acid charge on the X-chromosome of the hamster. Proc. roy. SOC.B133, 313-326. Kupka, E., 1950, Die Mitosen und Chromosomenverhaltnisse bei der grossen Schwebrenke, Coregonus wartmanni (Bloch), des Attersees. Oest. 2002.Z. 2, 606-623. La Cour, F. L., 1944, Mitosis a n d cell differentiation in the blood. Proc. roy. SOC. Edinb. B62, 73-85. Makino, S., 1948, Sex-Chromosomes and Sex-Determination in Vertebrates. Tokyo. 180 pp. 1949, A review on the chromosomes of domestic mammals. Jap. J. zootech. Sci. 19, 5-15. 1949, A chromosomal survey in some asiatic species of the Muridae, with special regard to the relationship of the chromosomes upon taxonomy. Cytologia, Tokyo 16, 153-160. 1950, Studies on murine chromosomes. VI. Morphology of the sex-chromosomes in two species of Microtus. B m o t . zool. jap. 23, 64-68,

180

B. MATTHEY

Makino, S., and Momma, E., 1949, An ideogram study of the chromosomes in some species of reptiles. Cytologia, Tokyo 16, 96-108. Matthey, R., 1950, Les chromosomes sexuels g6ants de Microtus agrestis L. Cenule 63, 163-184. 1951, La formule chromosomique de Yicrotus ormaensis Millais. Remarques m6thodologiques et discussion critique. Rev. suisse Zool. 68, 201-213. Momma, E, 1948, Chromosome numbers of some Reptiles. Zooh Mug. Tokyo 68, 1-3. Pomini, P., 1939, Fenotipi e genotipi nei Salmo italiani. sci. Genet. 1, 206-218. Sharman, G. B., MeIntosh, A. J., and Barber, H. N., 1950, Multiple sex chromosomes i n the marsupials. Nature, Zond. 166, 996. Slizynski, B. M., 1949, A preliminary pachytene chromosome map of the house mouse. J . Genet. 49, 242-245. Vols@e, H.,1944, Structure and seasonal variation of the male reproductive organs of Vipera berus. Spolia Zool. Mus. Hauniensis 6, 124-127. Wickbom, T., 1950, The chromosomes of Pi pa pipa. Hereditas 38, 363-366. 1950, The chromosomes of Ascaphus trzcei and the evolution of the anuran karyotypea. Hereditas 36, 406-418. Yosida, T., 1948, A preliminary note on the karyological study of Yoshida ascitesmrcoma in the white rat. Cyt. Oguma Commen. VoZ. 130-133.

Genic Analysis and Linkage Relationship of Characters in Rice S E I J I N NAGAO Plant Breeding Institute, Hobkaido University, Sapporo, Japan CONTENT8

Page

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I. Introduction 11. Color Inheritance 1. Genes for Apiculus Coloration 2. Color of Leaf and Leaf Sheatli 3. Color of Stem Node and Internode 4. Color of Seed Coat 111. Abnormal Mutant Characters and Their Inheritance IV. Linkage Studies 1. gl Linkage Group 2. P1 Linkage Group 3. lng Linkage Group 4. S p Linkage Group 5. General Considerations V. Conclusions VI. References

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192 195 203 204 205 206 207 208 208 .211

.

I. INTRODUCTION Because of its economic importance, rice has been used as material for genetical studies since the early days of genetics, and a considerable amount of data concerning the inheritance of various characters has been accumulated by many workers, especially in Japan, India, and the United States. Results have been reviewed by such authors as Ikeno (1927)) Yamaguchi (1927b)) Matsuura (1933), Sakai (1935), Nagao (1939), and Yasuda (1939). I n this article the writer intends to review the published data, with special attention to papers published in the Japanese language, which have had only limited circulation. In order to avoid confusion in terminology, brief descriptions of some of the terms used are given below. Empty glurnes. This term is used to refer to the two small outer gliimes a t the base of the spikelet. Lemma and palea. Refer to the two inner glumes, which enclose the stamens and pistil and later the seed as the ovary develops. Apiculus and awn. Apiculus denotes the apical part or tip of the 181

182

SEIJIN NAaAO

lemma and palea. In the awned varieties, the apex of the lemma extends and develops as an awn.. Floral gluine or glume. These terms are used for parts of the spike. let.

11. COLORINHERITANCE 1. Genes for Apiculus Coloration

Anthocyanin pigmentation occurs in various parts of the rice plant, such as the leaf blade, stem node, stigma, apiculus, and root (Kadam, 1935), showing a good many variations in color intensity as well as in localization of color, which form some of the most striking varietal characters. The color characteristics of the apiculus, including the awn and empty glumes, are particularly important in analyzing the mode of color inheritance, not only of the apiculus itself but also of other parts of the plant. The reasons for this are as follows. ( a ) Anthocyanin pigmentation develops in the vegetative organs only when anthocyanin color occurs in the apiculus. It is impossible to find a plant with purple leaf blades in which the apiculus is uncolored (green). ( 6 ) The genes concerned with apiculus color formation also determine the color of the other vegetative parts of a plant. The apiculus color genes may therefore be said to be the basic ones in connection with anthocyanin coloration in all parts of the rice plant. The anthocyanin color characters of the apiculus may be roughly classified in two types, colored and green, the latter becoming i n some varieties a rich brown color called tawny (Chao, 1928), whereas in other varieties the ripening color is yellowish white. Although the inheritance of apiculus color, as well as that of lemma and palea, has received considerable attention, the interpretations of various authors do not agree and have not been brought together under one general scheme. Recently Nagao and Takahashi (1947b) have made crosses between varieties differing in apiculus color, and have proposed an interpretation that agrees with all the data. According to this view, anthocyanin coloration is dependent on the complementary effect of the gene C, which is concerned with the formation of chromogen, and the gene Xp, which has to do with the formation of reducer for C. Pigmentation occurs primarily a t the apiculus and in the empty glumes as a result of the interaction of these genes and is located in the cell sap of the epidermis of these parts. Four alleles are known at the C locus, namely, CB>CBp>CBr>c, and three a t the S p locus, S p > 8 p d > s p . The expression of anthocvanin color characters of the apiculus is

ANALYSIS AND RELATIONSHIP OF CHARACTERS IN RICE

183

determined by the combination of alleles of the C and S p loci, as described below. CB. I n combination with S p , this gene gives dark violet color in apiculus, awn, empty glumes, and stigma, and purple lines on the internodes. With sp, it makes these parts green a t the time of flowering and tawny a t ripening. CBp. With Sp, makes the same parts a light violet or pansy purple ; the absence of purple lines on the internodes is the most conspicuous difference between CBXp and CBPSp. With sp, CBP gives green a t flowering and tawny at ripening. The tawny color is paler in CBP than in CB.

The genes Cn and C B p of the writer correspond to the genes Ty and H t , for tawny inner glumes, proposed by Chao (1928) and Jodon (1948), respectively. CBr. With Sp, produces pale red or rose red in apiculus, awn, and empty glumes, but green stigma and internodes. When CBr is combined with sp, these parts are green both at flowering and in the ripening stage, becoming straw-colored a t maturity. In the writer's opinion, the pale red coloration of CBrXpis due to a lack of sufficient chromogenic material to develop dense violet color. c. Recessive allele of C; no chromogen is formed. S p . I n combination with CB, C B p , or CBr,converts their respective chromogenic substances into anthocyanin pigment. #pd. I n combination with CB or C B p , gives pale red coloration. C B Xpd. Produces light red color of apiculus, awn, and enipty glumes in the flowering stage and tawny a t ripening. Internodes and stima are similarly colored. CBP Xpd. Same as above except that internodes are not colored. CBr Xpd. Produces no anthocyanin coloration, the apiculus and other parts remaining green at flowering time. Thus the phenotype is similar to that of a plant homozygous for c. Considering the nature of action of S p a from the physicochemical standpoint, one could assume that this gene is less potent than Xp, that is, can utilize in the formation of anthocyanin pigment only a fraction of the chromogenic substance produced. This mould account for the light red, rather than violet, color of the S p d phenotype, and also for the tawny color of the apiculus and other colored parts a t maturity, when the remaining quantity of unreduced chromogenic substance will be changed to brownish pigment. (Nagao and Takahashi, 1948c.) I n addition to the genes mentioned above, another gene, Rp, is known, which is responsible for distributing the pigment substance produced in the apiculus over the whole surface of the palea and lemma.

TABLE 1

CI

m I+

Summary of the Segregation of Anthocyanin Color Characters in Floral Glumes (From Nagao and Takahashi, 1947, with additional data)

Phenotypic Segregation in Fs l'ccrieties crossed

Genic combinations

It

II

111

CBSpRp CBPSpRp CBSp

1x 8

c-++

x C'SpBp

1

x 10

car++

x C%p+

9

x8

CBr++

x CB5pRp

9 x 10

CB.++

)( C'Sp+

6 x 11

Cs++

x CBWpBp

67

706

;

IV

IX

X

XI

CBpSp CBrSpRp CBRp CB'Sp+

9

9

3

9

233

233

81

2Gl

9

3

1097

335

9

27

CBPRp

XII

XIIZ

CB

CBP

27

393

9

167

CB'Spd CB:Rp CB' C'

--

B2

-

3

3

1

in2 3

1696

J

9

1920 4

,

126

154

9

J

3

1

166

64

63

27

9

154

3 46

216

9


0.8

s p pl

A piculus

Pale red

Violet

Leaf blade

Violet

Genotype

CB' s p P1

Observed Calculated

599 613.1

(9:3:4)

P

Grcen CB'

Green CB' s p P1 CB' s p pl

Total

232

259

1090

204.4

272.5

1090

sp pl

xs = 4.72 P

> 0.08

188

REIJIN NAGAO

Table 1 summarizes the experimental data obtained by Nagao and Takahashi (1947b) and segregation ratios expected from the genic assumption described above. Table 2 shows the results of two crosses involving C B and Spd alleles of the C and ij’p loci. 2. Colsr of Leaf aozd Leaf 8heath The normal color of the leaf and leaf sheath in most rice varieties is some shade of green ; in certain varieties, however, anthocyanin coloration can be observed. Nagao and Takahashi (1947b), who have reported the results of detailed studies on types of leaf color, their mode of inheritance, and the histological distribution of anthocyanin pigment, have distinguished the following three types of anthocyanin coloration of the leaf blade : a. Purple Leaf Blade. This character has also been designated “purple plant.’’ Gene symbol, PI. b. Purple Leaf Apex and Margin. The pigment appears mainly at the apex and margins, with scattered, irregular, fine purple stripes here and there on the leaf surface. Gene symbol, Pla. c. Purple Midrib. The pigment is found primarily in the midrib and is especially distinct on the lower surface of the leaf. Gene symbol, Plm. The occurrence of these three color characters depends on the action of the genes PI, Pla, and Plm, respectively, in cooperation with the genes C ( C B or CBP) and 8p. As to the relation among these three genes, it was inferred from the results of crossing experiments by Nagao and Takahashi (1947b), in which the segregation ratio of the F2was always found to be 12:3:1, that the gene PI is epistatic to both Pla and Plm, and the gene Pla is epistatic to Plm. Action. of the Plm allele. It is a generally known fact that types of rice with some purple in the leaf blade invariably have a colored leaf sheath, whereas the converse is not true. The gene Plm is responsible for the appearance of a purple line on the midrib of the leaf blade and purple lines on the leaf sheath. Histologically, the purple line of the midrib is due to pigment dissolved in the cell sap of the parenchyma adjacent to the large vascular bundle. This purple line extends to the leaf sheath, which is divided by a lacuna into an outer and an inner part; the purple line is due to the pigment of a single cell layer of the outer parenchyma tissue (Fig. 1). Action. of the P1 allele. The gene P1 is responsible for purple color in the leaf blade, leaf sheath, ligule, pulvinus, internodes, and stem node. The purple color of the leaf blade is due to the presence of pigment in the epidermal cells, motor cells, and sclerenchyma cells located outside

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the phloem. The purple appearance of the whole surface of the leaf blade is a result of the dense distribution of colored cells, though a considerable number of green cells are interspersed among them. The color of the leaf sheath is due to pigment in the single layer of parenchyma cells surrounding the bundle sheath and in the intervening epidermis of each bundle (Fig. 2).

F~ff.1. Cross section at midrib of leaf blade (R,K’) and of leaf sheath (Q), showing distribution of pigment with Plm. (Original.) R’, part of K enlarged ; c, assimilation tissue ; e, epidermis; lac, lacuna; ’m, motor cell; was, large vascular bundle ; miv, small vascular bundle ; p a r , parenchyma ; p h , phloem ; px, primary xylem; s, stereom.

Actim of the Pla allele. Purple coloration appears i n the ligule, auricle, pulvinus, and stem node. The purple line of the midrib in this case is due to the pigmentation of the cell sap of the stereom surrounding the phloem of the vascular bundles present in the lower side of the leaf. This is histologically quite different from the condition determined by the Plm allele, where the coloration occurs in parenchyma cells, although the appearance of the purple midrib is similar in the two cases. The purple line and stripe seen in leaf apex and margin are caused by pigment in the cell sap of the motor cells and epidermis (cf. Fig. 1 and Fig. 3 ) .

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SEIJIN NAGAO

The color of the leaf sheath is due to pigment in the epidermal cells. It appears in wide purple stripes, because the epidermis is intersected regularly by stereom, which remains green (cf. Fig. 1). Thus the distribution of the color pattern is alternative t o that determined by Plm. However, the leaf sheath is not fully purple even when both Pla and Plm'occur together, but shows the same distribution of pigment as when Pla alone is present. This agrees with the experiment,al data indicating that Pla is epistatic to Plm.

FIQ. 2. Barne ;in Fig, I, Rliowiug distribution of pigment with PI. (Original.) E',part of R enlarged; Q', part of Q enlarged.

The color of the vegetative organs, such as leaf blade, leaf sheath, stem node, internode, and so on, is dependent on the genetic constitution of apiculus coloring. For example, purple pigment is able to occur in the leaf blade when the PI gene is present in combination with either CB S p or C B p S p ; but when the apiculus color constitution is CBr S p , no pigment develops in leaf blade or leaf sheath. They remain green no matter what leaf-color genes are present. Furthermore, the purple leaf color becomes paler when P1 is combined with CB Spd or C B p Spd, appearing as greenish purple instead of dark purple. The experimental data

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191

concerning the relation of apiculus color and leaf-blade color are given in Table 3. To sum up, it should be emphasized that the occurrence and expression of anthocyanin pigment in leaf blade and leaf sheath are closely related to the genetic constitution of apiculus color. Kadam and Ramiah (1948) asaumed that leaf-sheath color is determined by a gene,

FIG3. Same as Fig. 1, showing distribution of pigment with Pla. (Original.)

Lsp; but it still seems uncertain whether the expression of such color depends on the action of a specific gene or on the pleiotropic effect of the apiculus color genes.

Color of Stem Node and Intermde Anthocyanin color in the stem node develops in the presence of the gene Pn. in combination with C Sp. The intensity of oolor varies greatly ; 3.

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SEIJIN NAQAO

the genetic mechanism responsible for this variability is still obscure, although it is known that the genes for apiculus color are partly responsible. The apiculus color genes have an effect here similar to their effect on the development of leaf-blade color; that is, stem-node color does not appear when the genetic constitution is CBr 8 p Pn. Many variations may be observed in internode coloration, and it is still obscure whether or not the occurrence of color depends on the action of a specific gene. Kadam and Ramiah (1943) suggested that a gene designated N t p is responsible for the development of internode color. However, it should be pointed out that the occurrence of pigment in the internode is invariably associated with the CB 8 p apiculus color type. The pigment in the internode is present in a single layer of the parenchyma cells adjacent to the bundle sheath of the large vascular bundle. Sometimes pigment is found in the parenchyma cells arranged in horseshoe form aroiincl t,he small vascular bundle adjacent t,o the hypodermis (Fig. 4 ) .

FIG.4. Cross section of internode (M,M'),showing distribution of pigment with CBSp. (Original.) M', part of Y enlarged; b8, bundle sheath; h, hypoderm; spv, spiral vessel. Other symbols, same as in Fig. 1.

4. Cdw of Bee& Coat Usually the seed coat of rice grain is white ; in some varieties, however, it is red or purple. Red-colored grain is commonly found in both wild and cultivated varieties, but purple rice is rather rare. The inheritance of red color in the rice seed coat has been studied by various investigators, who have reported that in most cases red is a simple dominant over white, giving monogenic segregation (cf. Matsuura, 1933 ; Jones, 1930; Hara, 1942a). However, Kato and Tshikawa (1923) explained their results by assuming the presence of three genes, A, B, and

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193

C, with ABC responsible for red grain color, ABc for yellowish brown, a.nd all other combinations for white. According to Nagao and Takahashi (1947b), the occurrence of red color in the grain is determined by the complementary effect of two genes, Re and Rd. Rc is responsible for production of the pigment in so-called gray-brown rice (Parnell et al., 1917; Kato and Ishikawa, 1923), which has dark-brown irregular speckles on a reddish brown background. Rd, when it is present along with Rc, is responsible for spreading the color of Rc, giving dark-red pericarp, or red rice. Rd in itself does not pro,duce any pigment. Thus the genic constitution for red rice is assumed to be Rc Rd, that for gray-brown rice Rc rd, and that for white rice either rc Rd or rc rd.

WQ.5. Cross section of pericarp of graybrown rice. (B,S’.) (Original.) Pe, pericarp ; en, endosperm ; ec, epicarp;

mc, mesocarp; crc, crossed cell; tuc, tube cell; sc, schlerenchyma; n, nucellus; al, aleuron grain; st, starch grain; S’,

enlarged cross section of speckles, showing the various kinds of surface conformation.

According to Kato and Ishilrawa (1923), the brown pigment produced by the action of the gene Rc (Nagao’s symbol) belongs to the protocyan group of pigment, which is different from the anthocyanin group. They also found that the color reaction of red rice to caustic soda solution is different from that of gray-brown rice. The pigment of the former is easily extracted by the caustic soda solution, giving a deep red color, whereas the pigment of the latter is extracted with difficulty and shows a yellow-brown color reaction. The gene Re belongs to the lng linkage group of the writer’s classification, whereas Rd belongs to the S p linkage group, indicating that these two genes responsible for the occurrence of red-pigmented rice grains are different both genetically and biochemically. The gray-brown coloring determined by the gene Re is due to the presence of pigment in the cells of the testa. In white rice the integu-

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SEIJIN NAGAO

ment or testa is compressed on ripening into a thin layer, but in the gray-brown rice it is thick. In the form of speckles and dots over the surface, the pigment occurs in chlorenchyma cells, in the mesocarp layer, and even at the epicarp. lJsually the surface of the seed is dented a t the sites of the speckles, which thus appear V-shaped or humped in cross section, but are sometimes flat (see Fig. 5). When the gene Rd is combined with Re, as stated already, the color of the whole grain is red, the pigment of the speckle being spread over the surface tissue, in chlorenchyma, mesocarp, and even the vicinity of the epicarp. The color is darkest in the testa (Nagao and Takahashi, unpublished data). In crosses between red-grained and white-grained, F2 ratios of 3 red to 1 white plant have been obtained by some authors and ratios of 9 red to 3 gray-brown to 4 white by other authors. The difference in these segregation ratios may be explained by the linkage between Rd and Sp. According to Nagao and Takahashi (1947a,b), these two genes are closely linked, with a recombination value at the most 0.3%. Thus monogenic segregation in the F2may be expected from crosses between red-grained and white-grained plants when both parents have anthocyanin pigmentation of the apiculus ; but digenic segregation, in the ratio of 9 to 3 to 4, is to be expected when red parent plant has anthocyanin-pigmented apiculus and the white parent plant has no anthocyanin pigment. Gray-brown rice is not found in plants with colored apiculus but is always found in plants having no anthocyanin coloration. Whether or not red rice occurs in plants having the green apiculus depends on the genetic constitution of the green parent. Specifically, red grain is found only in the plant$swith aritl.iocyanin-piginentedapiculus if the crosses were between C S p and C sp plants; whereas red rice can occur irrespective of the color of the apiculus in the cross C S p X c sp. This was experimentally established by Nagao and Takahashi (1947b). According to Jones (1930)) Colusa and C.I. 5346 have no colored organs, whereas Italian Red has purplish color in most of the organs, and red seed coats. I n crosses between Colusa and Italian Red and between C.I. 5346 and Italian Red, Jones obtained F2 segregation ratios of 3 red to 1 white for seed-coat color. I n the writer’s opinion, the genic constitution of the anthocyanin-colored parent in this case must have been C S p and that of the green parents c Sp. It may be concluded that the various data regarding the inheritance of seed-coat color can be explained consistently by taking into consideration the close linkage between the genes S p and Rd.

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195

111. ABNORMAL MUTANTCHARACTERS AND THEIR INHERITANCE Various abnormal mutant characters in rice have been recorded. In this section some of these aberrant types, either reported during the last ten years or observed in the writer's present work, will be described. Dwarfness (Pigs. 6 cwld 7'). Dwarfness is one of the most distinct aberrant characters of the rice plant, and comprises several types for which different and independent genes are responsible. The inheritance

FIG.6. Four kind8 of dwarf mutants. L e f t t o riglit: tillering dwarf, iiormal wicety, Ebisu dwarf, Daikaku dwarf, Koilaikoku dwa.rf. (Original.)

of the dwarf varieties has been analyzed by several authors, such as Parnell et d. (1922), Sugimoto (1923), Akemine (1925), Nagai (1926), Yamaguchi (1931), Jones (1933), Morinaga et al. (1942, 1943), and Nagao and Takahashi (1943, 1946). This character is known generally to be a simple recessive to normal. Only one exceptional case has been described, where a dwarf appeared as a mutant in a true-breeding strain isolated from a hybrid (Sugimoto, 1923) and was dominant to normal, producing in the F2 3 dwarf plants to 1 normal. I n addition to the monogenic segregation generally observed, Nagao and Takahashi (1943) have reported a case in which the Fz segregation ratio was 15 normal to 1 dwarf (actually, 1301 to 77). This dwarf was 62.1 cm. in average height and was chasacterized by many tillers (53 per plant) ; the normal variety, Akage, is 122.4 cm. high and has 6.75 tillers per plant. The dwarf was found i n 1937 in the experimental

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nursery of Hokkaido University, and was named Bunketsu-waito (meaning in Japanese “ a dwarf rice with much tillering”). In the cross Bunketsu-waito X Akage, the F1plants were normal in appearance and segregated in the Fa according to the 15 :1 ratio mentioned above.

FIO.7. Shin-Kodaikoku, a new dwarf segregated as a triple recessive from cross between tillering dwarf and Kodaikoku.

(Original.)

It may be pointed out that as a general rule a cross between two dwarf strains gives a normal plant in the Fl. The complementary action of two genes for dwarfness was first reported by Akemine (1925) and later by Jodon and Beachell (1943). According to Akemine, in a cross between the two dwarf strains Daikoku and Ebisu-both produced as

ANALYSIS AND RELATIONSHIP OF CHARACTERS IN RICE

197

mutants from the variety Akage-the F1plant was normal (Akage type) and the segregation in the Fz gave 9 normal t o 3 Daikoku to 3 Ebisu t o 1 new, very short dwarf type, which was named Kodaikoku. Akemine assumed that the genic constitutions were : normal (Akage), AABB; Ebisu, AAbb; Daikoku, aaBB; and Kodaikoku, aabb.

FIG.8. “Open spikelet,” showing lemma and palea that have opened and are unable to close again. (Original.)

Nagao and Takahashi (1943) crossed the dwarf variety Bunketsuwaito with Akemine’s dwarfs Daikoku, Ebisu, and Kodaikoku. The F1 plants were normal in all the crosses; and it was especially significant that a normal F1plant was obtained from the cross between Kodaikoku, which is a double-recessive segregant from the cross Daikoku X Ebisu,

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SEIJIN NAGAO

and Bunketsu-waito. On the basis of these results Nagao and Takahashi assumed the following genic constitutions : Akage (normal variety), D1D,D3D, (+) ; Daikoku, dlD,D,D4 (d,) ; Ebisu, DldaD,D4 (ap); Kodailrolru, dld,D,D4 (d,d,) ; Bunlretsu-waito, D,D,d3d, (d,d,) ; ShinIiodailroku, dlD,dsd4 (dldsd4). Thus a normal type has to have one or the other of the genes D , and D4, as well as the genes D , and Da. A new dwarf phenotype was produced in the Fz segregation of the cross Bunlretsu-waito x Daikoku.

FIQ.9. Spikelets from various mutants. Top (left to right) : tillering dwarf, tetraploid, double awn, open spikelet. Bottom (left to right) : long empty glume, claw-shaped hull, compact-panicle sterile, Daikoku dwarf. (Original.)

This was a triple-recessive segregant, and was named Shin-Kodaikoku (English name: “tillering Kodaikoku”). This dwarf is 29.6 em. high and has o n the average 26.6 tillers per plant, whereas the Kodaikoku dwarf is 31.25 em. high and has an average of 6.25 tillers. Nagao and Takahashi (1946) found and reported that in one cross between Bunketsu-waito and normal varieties the segregation in the Fz was in the ratio of 63 normal plants to 1Bunketsu-waito, which indicates that there are a t least three different genes (d,, d,, d S ) responsible for the formation of the Bunketsu-waito type. They also reported another

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199

type of dwarf, named “lop-leaved dwarf’’ (gene symbol d o ) ,which in crosses with other dwarf varieties produces normal types. Open spikelet (Fig. 8). This is a character in which the palea and lemma are unable to close again after they have opened a t blooming time. Two good mutants have been reported. One of them is not sterile (Kondo and Isshiki, 1933), but the other is sterile. Both cases are reported to be simple recessive to normal (Nagao and Hoshika, 1938). Lmg empty glume (Figs. 9 m d 1 1 ) . The empty glumes are nearly as long as the lemma and palea. This character is reported to be simple recessive to the common short empty glume (Jones, 1933; Morinaga and Fukushima, 1943). Claw-shaped hull (Fig. 9). The palea is under-grown, and the lemma is bent SO as to overlap it. Recessive to normal (Takahashi, 1950). 1)oiible own ( P i g . 9 ) . An awn develops on bath lemma and palea (Takahashi, 1950). TrianrJzdar hzcll (Fig. 1 0 ) . The spikelet appears triangular because it is of the shape under the lemma. The variety having such spikelets is known in Japan as Sankaku-Ine. It is recessive to normal (Morinaga and Fukushima, 1943). 8ha”ttering (Fig.11). I n certain varieties the grain is tightly held before and after maturity, whereas in others, including wild species, the grain shatters very easily even when carefully handled. Between these two extremes there probably are varieties representing all degrees of tightness. Kadam (1936) reported that shattering is dominant over nonshattering, producing in the Fz FIQ.10. A a segregation of 15 shattering to 1 nonshattering. Mor- spikelet of the inaga (1940) found, in the cross between the cultivated t r ian g u 1 a.r species Oryza sativa and a wild shattering species 0. hull mutant, minuta, that shattering is dominant over nonshattering ( S a n k a k u in the F1 plant. Similar results were reported by Hara Ine). (Original.) (1942a, b) for crosses between cultivated varieties and a wild race in Formosa. Izumi (1944) reported the results of crosses between nonshattering Japanese varieties and a native variety of Kbrea that shatters very easily. According to his results, nonshattering is completely dominant in one cross and intermediate o r incompletely dominant in two other crosses. I n the Fz populations, plants that shattered very easily occurred in proportions of 12, 18, and 35 per cent in the different crosses. Clqutering. Two or more spikelets, instead of one, appear a t each

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rachis. This character is reported as a single-gene dominant (Ramiah et al., 1931; Jodon, 1940). Hokarnuri (Fig. 2 1 ) . Hutchinson and Ramiah (1938) classified panicles in three types according to degree of exsertion : well exserted, exserted, and partly enclosed. The partly enclosed type is known in Japan as “hokamuri.” In this type the large part of the panicle is enclosed by a bract leaf, which develops, in addition to the ordinary boot leaf, a t the node immediately under the neck of the panicle.

FIG.11. Panicles from various mutants. Left to right: Ebisu dwarf, tetraploid, compact-panicle sterile, open spikelet, shattering, long empty glume, partly enclosed (hokamuri), mottled green. (Original.)

Morinaga and Fukushima (1943) reported that a cross between normal and Hokamuri resulted in normal, i.e., completely exserted, in the F1,and an Fz segregation of 559 normal to 175 Hokamuri. According to Nagao and Takahashi (unpublished), total segregants in the F2 of two crosses between normal plants and Hokamuri were 978 normal to 357 Hokamuri, or a ratio of nearly 3:l. Spreadhg (Figs. 22 and 2 3 ) . Each stem grows obliquely instead of straight, so that the young plant has a spread-apart appearance. In Japan the variety that shows this characteristic is named Motsure, which means “tangled.” There are various gradations between normal and extreme spreading. Morinaga and Fukushima (1943) reported that in

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201

a cross between normal and spreading, the F1 plants were normal or somewhat spreading, and the F2ratio was 3 normal to 1 spreading. Brittleness. In the Japanese variety Kama-Irasu, whose name means “no need f0r.a sickle in harvesting,” the leaves, culms, and panicles are so brittle in both green and mature stages that they break off a t the slightest pressure. Jones (1933) found first that brittleness is a simple recessive to normal (toughness). This observation was confirmed by Morinaga and Fukushima (1943) and Nagao and Takahashi

n o . 12. Comparison of a spreading (right) with a normal plant (Original.)

(left).

(unpublished). According to the last two authors 90 normal and 37 brittle plants were obtained in the Fz generation of a cross between normal and brittle. Glassy. Plant leaves have a glossy surface, to which water easily adheres. Morinaga and Fukushima (1943) reported that this character is a simple recessive to normal. Compact-panicle sterile (Figs. 9 and 2 1 ) . Found in 1946 in the population of a variety called Ebisu-Mochi, this mutant shows various types of aberrant spikelets, with deformation and excessive development of the floral organs, such as palea and lemma, pistils, anthers, lodicules, and ovules which result in a high degree of sterility. Seed sets in about

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20% of the panicles. It is recessive to normal (Takahashi and Tate, 1951). Chlorophyll deficiency. During the past ten years several reports have been published in America on the occurrence of chlorophyll mutations (Jodon, 1940 ; De la Houssaye, 1942 ; Comeaux, 1947). According to Jodon (1940), virescence that appeared as a recessive mutant in a n F4 progeny of the cross Kameji X Blue Rose was found to be linked with four other genes, namely, apiculus color, clustered, glutinous endosperm, and maturity.

FIG.13. Spreadiiig see11 froiri above.

(Original.)

In Japan, Hara (1944, 1946a,b) has reported the occurrence of various groups of chlorophyll deficiencies such as chlorina, xantha, and albino. I n the chlorina group, he described four types, all of which are simple Mendelian recessive to normal, and designated them chI, chIr, chrIr and chIv. He showed that ChI and ChII,and also Ch,I and ChIIr, are complementary for normal development of chlorophyll and behave independently of each other. He further reported (1946b) that two xantha genes, xuI and xaII (Hara’s symbol), behave as duplicate genes, and that the coexistence of three kinds of gene, Ch, Xa,and AZ (dominant allele for albino gene, Hara’s symbol), is necessary for the development of chlo-

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rophyll. The absence of one of them, as in ch X a Al, Ch xa Al, or Ch X u al, produces chlorina, xantha, or albino, respectively. Recently Takahashi (1950) has described the occurrence of the following four types of chlorophyll deficiency : Fine-striped ( f s ) . This is an X-ray-induced mutant. I n the seedling stage fine greenish white stripes appear distinctly at the tip and margin of the leaf blade. They disappear in the tillering stage. The character is simple Mendelian recessive to normal green. White-striped. A mutant type, found in the variety Hokko, in which white streaks appear in leaf blade, leaf sheath, stem, and spikelet through the whole green stage. It is simple Mendelian recessive to normal green, although in most crosses between green and striped the number of striped plants is less than the expected ratio. Xuntha. This type was found in the F3 progenies of crosses between Kokushoku-Ine and Hatsi~murasaki, and between Ebisu-Mochi and Akage. It is simple Mendelian recessive to normal green. Albho. Albino appeared first among the progeny of selfed plants of the variety Matsudawase. 1669 green plants and 514 albino ones were counted in the 22 strains that segregat,ed albino plants, showing it to be simple recessive to normal. IV. LINKAGE STUDIES As the haploid number of chromosomes in rice is 12, the genes of this plant should be arranged in twelve linkage groups; but u p to the present comparatively few cases of linkage relations have been established with certainty. Recently, in his “Summary of rice linkage data,” Jodon (1948) has summarized and arranged the known data on linkage into eight linkage groups. I n other words, it may be said that eight of the expected twelve groups have been established, although on certain points there may still be some question regarding Jodon’s grouping of genes. He says : “The best established case of linkage in rice, that between apiculus color and glutinous endosperm, remains as Group I. There are a t least three factors involved in the expression of apiculus color. Since in two cases linkage was found between apiculus color and characters not linked in Group I, it seems logical to designate these as Groups I1 and 111. Group I V is based on linkage between outer glume length and red pericarp ; Group V on linkage between liguleless and leaf blade color; Group V I on linkage between awned and a dwarf character; Group V I I on an association between awned and pubescence ; and Group

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VIII on apparent crossingover between reaction to Cercospora oryxae

race 1and the intermediate dwarf character.” In this paper, I will add some new data on linkage and comment on Jodon ’a conclusions.

1. gl Linkage Group The g l linkage group, provisionally named by the writer (Nagao and Takahashi, 1947a,b, 1948b), includes characters that show linkage to the endosperm character gl (glutinous). This group corresponds to Group I of Jodon’s system. The best-established case of linkage in this group is that between the apiculus-color gene and the glutinous-endosperm gene. Of the two complementary genes, C and Sp, that are responsible for the development of anthocyanin coloration in the apiculus, only C is linked with gl. The recombination value between C and gl amounts t,o about 23 per cent (Table 4). TABLE 4

FI of Croan between Hokkai-Mochi and Akage, Showing Linkage of C and gl (Coupling Phase) (Nagao and Taknhanhi, 19471)) Observed

INad -

C’P Gl 1001

0.1054

gl 154

CBP

c 81

c (I1

Tntnl

146

213

1514

Recombination value, 22.8%

This linkage relation suggests that the gene C is identical with genes described under different names by various other authors, for example, Ap (Jodon, 1948), purple apiculus; S (Yamaguchi, 1926), reddish color of apiculus; Ap, (Chao, 1928), colored apiculus; T y (Chao, 1928), tawny color of ripened apiculus, although some of these genes seem to differ in their actions. Besides the C gene, several others are known to be included in this linkage group. According to Nagao and Takahashi (1946) the gene for the “bunketsu-waito” dwarf character ( d s ) is linked with gl, with a recombination value of about 17 to 18 per cent in the repulsion phase. They also have reported (194713) that the gene Pla is linked with g l , giving about 44% recombinants. Hara (1946~) reported that the gene for a male-sterile mutant (Hara’s symbol sf) derived from the variety Fukukame is linked with genes C and gl, showing recombination values of 18.9% for sf and C, 5.3% for sf and gl, and 21.7% for gl and C. In addition to those mentioned above, the genes virescent, clustered,

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maturity, and floating habit are known to belong to the g l (or Group I ) liiikage group (Jodon 1940, 1947, 1948). 2. PI Liqtkage Group The PI linkage group, which corresponds to Jodon’s Group V, includes six genes, namely, purple leaf blade (PZ), liguleless ( l g ) , ebisu dwarf (&), color reaction to phenol ( P h ) ,red or purple palea and lemma (Rp), and a semisterile ( s k ) . Morinaga (1938) was first to report linkage between PI and Zg, with 21 to 22% recombination in the coupling phase. Similar results were obtained by Nagao and Takahashi and are given in Table 5. TABLE 5

P, Data from Crom between PI and lg, Showing Linkage (Coupling Phase) Observed

_ be aa -

PlLg 148 0.0959

PI lg 21

Pl Lg 23

p l lg 34

Total 226

Recombination value, 21.9%

IAI ter, Nagao and Takahashi (1946) reported linkage between P1 end da, with a recombination value of 38*5% for the coupling phase. They also determined that the recombination between genes d, and l g is nearly 50%. Hara ( 1 9 4 6 ~ )reported that the gene sk for semisterile rice, which was found as a mutant in the variety Kokuryomiyako, is linked with Pb, the recombination value being 6.8%. It is known that the fruit and husk of certain varieties of rice develop dark violet color when treated with phenol. According to Morinaga et al. (1943), this character behaves as a simple Mendelian dominant (gene symbol P h ) and is linked with the gene lg for liguleless, giving between 5 and 7% recombination. They also reported that the genes Ph and Zg are linked with the gene for apiculus color (Morinaga’s symbol A p ) as well as that for color of palea and lemma (Morinaga’s symbol Rbe). The observed recornbination values are 19% for Ph and Ap, 26% for lg and Ap, 5 to 7% for lg and Ph, 17% for Ph and Rbe, and 27% for lg and Rbe. I n the writer’s laboratory Izumiyama (graduation thesis, 1949), unpublished) found that the Japanese varieties Kokushoku-Ine and Kuromochi show positive color reaction to phenol, and studied the linkage relations between the gene PI and the genes lg and Rp. According to his results the recombination values are as follows: 7.4% for P h and lg, 24.9% for Ph and Rp, and 31.9% for Rp and lg, suggesting that the order of these genes is Rp-Ph-lg. When these results are compared with

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those of Morinaga et d.,it appears probable that his symbols Rbe and Ap are identical with the writer’s symbol Rp. A t present the PI linkage group contains six genes. The probable order of five of these is d,-Rp-PI-Ph-lg, while the position of the sixth ( s k ) has not yet been determined.

3. lng Linkage Group The lng linkage group includes three genes : lny, d,, and Ilc (Nagao and Takahashi, 1947a, 1948b). It is apparent from Table 6 (Nagao and Takahashi, unpublished) that hog is closely linked with d,. TABLE 6

F2of Crosses between d, and Ing, Showing Linkage (Repulsion Phase) D Lng 1945 1946

Total Taken a8 r:s = 1:20.0

D lng

doLng

d, lng

Total

116 128 244

68 77 145

63 52 115

0 0 0

247 257 504

252.3

125.7

125.7

0.3

504

The gene ds is also linked with the gene Rc, the recombination value between them being 30.7%. From this it follows that Re should be linked with lng. According to experimental results obtained by Nagao and Takahashi (Table 7 ) , the recombination value between Re and lng is 30.2%. One lag linkage group corresponds to Group IV of Jodon’s classification. I n his Summary, Jodon (1948) states : “ A close linkage was reported between spikelet length and outer glume length. Ctlume length appears to be linked with red pericarp with 38 per cent crossingover, but spikelet length and colored pericarp were reported to be independent in one instance. Red pericarp gave 36.5 per cent crossingover with purple internode and 22 per cent with a maturity factor.” It should be recalled in this connection that red pericarp represents the complementary action of the genes Re and Rd, and that these two genes belong to different linkage groups, Re to the lng linkage group. and Rd to the S p linkage group, to be described next.

207

ANALYSIS AND RELATIONSHIP O F CHARACTERS IN RICE

TABLE 7

F, of Crosses between Bc and a,, and between Rc an d lng, Sliowing Linkage Relations in Coupling Ro- 4

1943 1945 1946 Total Taken as r:s = 2.2:l

::

Rc D

RC a,

rc D

433 241 481 1155

73 55 121 249

97 57 86 240

71 50 108 229

674 403 796 1873

1157.8

246.9

246.9

221.3

1872.9

- --

0.22593

TC

a,

Total

Recombination value, 30.7%

Rc - lng (1946) RcLng Observed Taken as r:s = 2.3:l bc

467

Rclng 106

466.3

95.5

a = 0.2169 4.

rcZng

rclng

Total

86

90

749

95.5

91.7

749

Recombination value, 30.2%

Xp Linkage Group

Experiments of Nagao and Takahashi reported in Table 8 show that the recombination frequency between Xp and P.n is 17.2%. TABLE 8 F, of Cross between S p and Pn, Sliowing Linkage Relations in the Coupling Phase

S p Pn Observed Taken as r:s = 4.8:l

8 p Pn

sppn

Total

242

26

25

49

342

229.6

26.9

26.9

58.6

342

- -- 0.05481 be ad

SP Pn

Recornbination value, 17.2%

According to Nagao and Takahashi (1947a,b), the gene S p is also fouiid closely linked with Rd, the recornbination value being less than 0.3 %. Recently Morinaga and Kuriyama (1948) reported two instances of linkage that may be considered to belong in this group. According to them, the gene pa (Morinaga’s symbol for depressed palea, an abnormal recessive type of spikelet having undergrown palea) is linked with the

208

SEIJIN NAGAO

chromogen-reducing gene A (Morinaga’s symbol ; identical with S p of the writer), the recombination value being about 20%. They also found another case of linkage between the genes A and lax (Morinaga’s symbol for lax or sparse spikelet), with a recombination value of about 31%. Thus it appears probable a t present that five genes, namely, Sp, Pn, Rd, pa, and lax, are included in this linkage group.

5 . General Considerations I n the foregoing sections the writer has classified the available linkage data according to four linkage groups, and shown that three of these correspond to the first, fourth, and fifth linkage groups of Jodon. Further investigation should determine other linkage groups not yet established. It seems desirable, in carrying on linkage studies to obtain complete genetic analyses of characters, especially when dealing with those affecting coloration. Linkage work would be complicated by inadequate genetic knowledge of complex characters. For example, purple lines at the internode are due to a pleiotropic effect of CB Sp, and are not present in the C E p S p genotype; and when CEr S p is present, no anthocyanin coloration develops in vegetative parts such as leaf blade or stem node even though P1, Pn, and other genes responsible for pigmentation of these parts are present. I n the writer’s opinion linkage relations between the color characters included by Jodon in linkage groups I1 and 111 may require reexamination, if the genetic complexity of color type is taken into consideration.

V. CONCLUSIONS There are many characters of rice whose inheritance has been studied and reported, and there is now an extensive literature on the subject. However, it appears that several discrepancies exist with respect to the identification of genes, especially those for color characters. A critical study of the published papers on color inheritance reveals considerable confusion with regard to naming the characters or genes that are responsible for various color types (Nagao, 1935). I n the writer’s view, acceptance of the gene system for anthocyanin coloration proposed by Nagao and Takahashi (194713, 1948c) may help reconcile the various explanations under one general gene scheme. As for the question of gene symbolization, Kadam and Ramiah (1938, 1943) have proposed a set of symbols, which was later accepted by Jodon (1948). However, their fundamental principle for selection of symbols seems to differ from that which is widely accepted by Drosophila and maize geneticists as well as by workers using other organisms.

ANALYSIS AND RELATIONSHIP OF CHARACTERS I N RICE

209

According to the general usage, the letter symbolizing the adjective in the name of the character is written before the letter representing the noun, so that the symbol for “purple leaf blade,” for example, should be P1, rather than L p as written by Kadam and Ramiah. On the basis of these considerations, the writer wishes to propose a system of symbols for rice which conforms to the mode of symbolization used by geneticists working with other organisms. This is presented in Table 9. TABLE 9 List of Genetic Characters of Rice

Charactera Albino Awnedness Awned sterile Brittle culm Barren sterile Cliromogen (anthoeynnin) Chlorina Claw-shaped hull Clustered spikelets Complete sterile Compact-panicle sterile Ripening tawny Resistance to Cercospora nrgzae Dwarf (general) Daikoku dwarf Ebisu dwarf Tillering dwarf Lop-leaved dwarf Double awn Dense or compact panicle Dark furrows Exsertion of panicle (partly enclosed or hokamuri) Fragrant flower Floating habit Flowering period F’ine-striped Female-sterile Qrain length (short ws. long) Qlutinous endosperm Qlossy leaf blade (hairy va. glossy) Green-and-white-striped

Symbols recommended b y Kadarn and Ramiah

Tentatiwe modification by Nagao

2 LO

SEIJIN NAOAO TABLE 9-Continued

Characters Green-and-yellow-striped Resistance to Helminthosporium oryzae Inhibitor of chromogen Inhibitor of purple leaf blade Inhibitor of awning Inhibitor of dark gold Lazy or ageotropic Resistance to Leptoapoeria cattanei Lodging Liguleless Long empty glumes Long panicle Lutescent Lemon yellow (palea and lemma) Resistance to Melanosperma Mottled green (momigare) Minute spikelet Multiple pistil Male-sterile Sinuous neck Oval grain Open spikelet Open-spikelet sterile Anthocyanin coloration (general) Palcnceous sterile Positive phenol reaction Resistance to Piricularia oryzae Purple leaf blade Purple leaf apex and margin Purple midrib (purple leaf axil) Purple leaf sheath Purple node Purple internode Purple pericarp Purple root Purple stigma Ripening brown Brown pericarp (rhromogen or chromophclein of red rice) Red pericarp (distribution of pigment produced by R c ) Ripening gold Ripening black

Symbols recommended by Kadam and Ramiah

Em

-

IlP Ian Ih la Le La

k? 9

-

Tentative modijicatwn b y Nagao

Em IC IPl IAn IRg la Le La lg

w LP

lU

1%

Ho Me hm

LY Me mg Mi mP ms ne og

Mi

mp ms Re LS

OP

A fPE

Pi

LP

opr P pa.? Ph Pi P1 Pla Plm Pln Pn Pnt PP Pr PS

Rb Pbr Pr Hg Hb, H a

RC

Rd .Izg

R1

211

ANALYSIS AND RELATIONSHIP OF CHARACTERS IN RICE

TABLE 9-Continued Symbols reconmended by Kadam avrd Ramiah

Churucters Colored palea and lemma due to anthocyanin distribution Sterility (general) Spreading (motsure) Shattering Semisterile Complementary gene for colored apiculus Staminoidal sterile Twisted leaf (lack of midrib) Triangular hull Tough-shelling Virescent Xantlia (letlial yellow)

Tentative modification b y Nagao

HP

BP

Ee Ish

Sg Sh

r8

88

f

8

AP, GP let tw

SP, SPd

to

to

-

8tS

tw th

V

V

Y

xa

VI. REJPENENCES Akemine, Y., 1925, Rep. Jap. Ass. Adv. Sci. 1, 308-314. Chao, L. F., 1928, Genetic8 13, 133-169. Comeaux, D. J., 1947, Proc. Louisiana Acad. Sci. 10, 23-26. De la Houssaye, DeBlane A., 1942, Proc. Louisianu Acad. Sci. 7, 27-34. Hara, S.,1942a, Shokubutsu oyobi Dobuteu (Botany and Zoology) 10, 321-325. 1942b, Jap. J . Genet. 18, 183-184. 1944, Jap. J . Genet. 20, 15-19. 29468, Jap. J . Genet..21, 1-9. 1946b, Jap. J . Genet. 21, 15-21. 19460, Jap. J . Gentt. 21, 32. Hutchinson, J. B., and Ramiah, K., 1938, IllthIrk J . agric. Sci. 8, 567-616. lkeno, S., 1927, Bibliog. geiietica 3, 313-354. Izumi, Y.,1944, Agric. and Horticulture 19, 417-419. Jodon, N. E.,1940, J . Amer. 800. Agron. 32, 324-346. 1947, Proc. Louisiana Acad. Sci. 10, 32-34. 1948, Plant Industry Station, Beltsville, 1-32. (Mimeographed.) Jodon, N. E. and Beachell, H. N., 1943, J . Hered. 34. 155-160. Jones, J. W., 1930, J. agric. Iles. 40, 1105-1123. 1933, J . agric. Ree. 47, 771-782. Kadam, B. S., 1935, J . Indian Bot. Sci. 14, 173-178. 1936, Proc. Indian Acad. Sci. 4, 224-229. Kadam, B. S., and Ramiah, K,, 1938, I m p . Bur. Plant Breed. and Genet. (Mimeographed.) (Cited from Yasuda, 1939.) 1943, Indian J . Genet. and Plant Breed. 3, 7-27. (Cited from Jodon, 1948.) Kitto, S., and Ishikuwa, J., 1923, Jap. J. Genet. 1, 1-7. Kondo, M.,and Isshiki, S., 1933, Nogaku-benkyu (Agric. Rea.) 20, 135-153.

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Matsuura, H., 1933, A Bibliographical Monograph on Plant Genetics, 1900-1929. 2nd ed. Hokkaido University Press, Sapporo 787 pp. Morinaga, T., 1938, Jap. J . Bot. 9, 121-129. Morinaga, T., 1940, Jap. J. Bot. 11, 1-16. Morinaga, T., and Fukushima, E., 1943, Bull. Sci. Fakultato Terkult. Euskyu I m p . Uniw. 10, 301-339. Morinaga, T., and Kuriyama, H., 1948, Jap. J . Genet. 23, 33-34. Morinaga, T., Kuriyama, H., and Aoki, M., 1942, Jap. J . Genet. 18, 297-304. Morinaga, T., and Nagamatsu, S., 1942, Jap. J . Genet. 18, 197-200. Morinaga, T., Nagamatsu, T., and Kawahara, E., 1943, Jap. J. Genet. 19, 206-208. Nagai, I., 1926, Jap. J . Bot. 3, 25-66. Nagao, S., 1935, Agric. and Horticulture 10, 1391-1394. 1939, Genetics and Breeding in Rice, 2nd rev. ed. Yokendo, Tokyo. 225 pp. Nagao, 5. and Hoshika, Y., 1938, Agric. and Horticulture 13, 521-530. Nagao, S., and Takahashi, M., 1943, J . Sapporo SOC.Agric. and Forest 18, 1-9. 1946, Seibutsu (Biology) 1, 27-36. 1947a, Jap. J . Genet. 22, 22-23. 1947b, Jap. J . Genet. Suppl. Vol. 1, 1-27. 1948a, Oguma Commemoration Volume on Cytology and Genetice, 53-58. 1948b, Abstract in the 1st Annual Meeting of Bot. SOC. in Sapporo. 1948c, Agric. Sci. of the North Temperate Region 2, 281-288. Parnell, F. R., Rangaswami Ayyangar, G. N., and Ramiah, K., 1917, Y e m . Dept. Agric. I.ndia Bot. Ser. 9, 75-106. Parnell, F. R., Rangaswami Ayyangar, G. N., Ramiah, K., and Srinivasa Ayyangar, C. R., 1922, H e m . Dept. Agric. India, Bot. Ser. 11, 185-208. Ramiah, K., Jabitharaj, S., and Mudsliar, 8. D., 1931, Y e m . Dept. Agrio. India Bot. Ser. 18, 229-259. Sugimoto, 8.,1923, Jap. J. Genet. 2, 71-75. Sakai, K,, 1935, J. Sapporo SOC. Agric. and Forest 27, 153-170. Takahashi, M., 1950, Ikushu-kenkyu (Breed. Res.) 4, 33-42. Takahashi, M. and Tate, W.,-1951, Jap. J. Breed. 1 ( I n press). Yamaguchi, Y., 1926, Ber. Ohara In&. 3, 1-126. 1927a, Ber. bhara Inst. 3, 319-330. 1927b, 2. ind. Abst. Vererb. 46, 105-122. 1931, Ber. ahara Inat. 6, 1-56. Yasuda, S., 1939, Abstract of literature in : Genetics and Breeding in Rice, Yokendo, Tokyo. 216 pp.

Procedures and Methods of Cotton Bresding With Special Reference to American Cultivated Species '$ T. R. RICHMOND Texas Agricultural Experiment Station, Texas Agricultural and Mechanical College System, and 17. S . Department of Agriculture CONTENTS

Page

...................... 213 . . . . . . . . . . . 215 . . . . . . . . . . . . . . . 218 . . . . . . . . . . . . . . . . . 218 . . . . . . . . . . . . . . . . . . . . 221 . . . . . . . . . . . . . . . . . 221 . . . . . . . . . . . . . . . . . 222 . . . . . . . . . . . . . . . . . 223 . . . . . . . . . . . . . . . . . . 225 . . . . . . . . . . . . . . . . 226 . . . . . . . . . . . . . . . . 227 . . . . . . . . . . 230 . . . . . . . . . . 233 . . . . . . . . . . . . . . . . . . . 233 . . . . . . . . . . . . . . . . . . . 236 . . . . . . . . . . . . . . . . 238 . . . . . . . . . . . . 239

I. Introduction 11. Early History and the Pure Line Concept TII. Recent Methods and Refinements 1. The Breeding Problem 2. Type Selection 3. Replicated Progeny-Row 4. Mass-Pedigree Selection 5. Bulked Progeny Test 6. Backcross Methods 7. Intervarietal Hybridization 8. Interspecific Hybridization IV. Hybrid Vigor in Fl a n d Advahccd Generations V. Special Characters of Breeding Valiie . . . 1. Fiber Properties 2. Disease Resistance 3. Insect and Other Resistance 4. Adaptation to Mechanical Harvesting VI. References

.......................

242

I. INTRODUCTION All the cottons of the world, whether cultivated or wild, belong to the genus Gossgpium. For convenience, they may be divided into three main groups: (1) Old World or Asiatic cultivated ( n = 13), (2) New World or American cultivated ( n = 26), and (3) Wild ( n = 13, with one anomalous exception). Though the reported number of species of Gossypium varies widely, depending on the classification system employed and the inclination of the taxonomist, a. recent work by Hutchinson et d.(1947)recognizes twenty. These writers place the cultivated Contribution from the Texas Agricultural Experiment Station, Cotton I m provement Section, Department of Agronomy, College Station, Texas, in cooperation with the Division of Cotton and Other Fiber Crops and Diseases, Bureau of P l a n t Industry, Soils, a n d Agricultural Engineering, U. 8. Department of Agriculture.

218

214

T. R. RICHMOND

cottons under four species: G . arboreurn L., G . herbaceurn L., G . hirsuturn L., and G . barbadense L. The first two are designated as Asiatic and the last two as American cottons, and all bear spinable seed hairs, called lint, which distinguishes them from the wild cottons which do not have spinable lint. Without exception, the cultivatecl varieties of cotton in the TJnited States fall into the New World group which is characterized by 26 pairs of chromosomes. According to the theory advanced by Skovsted (1937) and confirmed independently by Beasley (1940) and Harland (1940) , New World cultivated cottons are allotetraploids which have arisen in nature by amphidiploidy from hybrids of Asiatic and American Wild parentage. Though amphidiploid in origin, they are now so well differentiated genetically and cytologically that, in many respects, they function as diploids (1’3rown, 1950). The cultivated American cottons a w of two species: (1) C. ?tirsuturtt, commonly called Upland cotton ant1 characterized as a n annual subshrub with few or no vegetative branches (Hutchinson et al., 1947) with short to medium long and coarse to medium fine fibers borne on seeds within relatively large, rounded, usually 4 to 5 loculed capsules (bolls). (2) G. barbadense, commonly referred to as American-Egyptian and Sea Island cotton. Agricultural varieties of this species are inherently perennial shrubs, but they usually behave as annuals under American Cotton Belt conditions. Relatively long, fine fibers are borne on seeds within small to medium, tapering, usually 3 to 4 loculed capsules. Agricultural varieties of G. hirsutum account for nearly all the cotton produced in the United States, the production in 1949 being in excess of 16 million, 500-pound bales as compared to approximately 3700 bales of the species G . barbadense. The United States’ average production of American Upland cotton for the past ten years was a little more than 12 million bales, while the total production of both American-Egyptian and Sea Island during the same period was only slightly more than 2500 bales. I n recent years most of the IJpland cotton production has been from the agricultural varieties, Acala, Deltapine, Stoneville, Coker, Delfos, Rowden, Mebane, Hi-Bred, and Half & Half. The American-Egyptian cotton production has been from varieties designated as Pima, SXP, and Amsak, while the Seabrook variety has accounted for almost all the Sea Island cotton production. The welfare of American cotton depends on its ability to compete in the world textile markets with foreign cottons and domestic and foreign synthetic fibers. The development of factual information on hereditary characteristics and processes is fundamental to the improvement of the

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compet,itive position of cotton. Through the employiiient of genetic and breeding techniques present economic cotton characters can be modified, their behavior standardized, and their range extended, in short, specialized types can be produced for specific purposes. This review will be confined largely to procedures and methods of breeding American cultivated cottons and, more particularly, to work with American Upland types.

11.

E A R L Y H I s w H Y A N D THE PURE L I N E cONC33PT

The cotton plant bears complete flowers. Cultivated varieties generally are placed in the “usually” self-fertilized category with respect to crossing habit under natural conditions, but the per cent of crossing may vary from less than 5 to approximately 50 (Kime and Tilley, 1947; SimI)son, 1948), the amount at any one location being proportional to the number of wild and domesticated bees which visit the field. Since the cottons of commerce are propagated by seeds, there is more or less of a tendency toward inbreeding, depending on the breeding methods employed, the isolation of increase plots, the precautions taken against mechanical mixtures, and many other factors. Vavilov (1927) has shown that stable populations of crop plants exist (or existed) in certain limited areas called “centers of origin,” that variability in such centers is high, and that variability diminishes toward the periphery of the distribution. It is still possible to distinguish two centers of origin for American cultivated species, one for American IJpland, C . hirsutum, in southern Mexico and Central America, and one for Sea Island and related types, G . barbadense, in the Andean region of Peru, Ecuador, and Colombia. These American species have proved to be remarkably plastic genetically, and it has been possible through selection to develop agricultural varieties of a significant range of types, many of which have been widely adapted geographically. Through the years no set breeding system has been followed by all or any considerable group of cotton breeders, and considering the goals to be reached, time elements involved, funds, and facilities available, and the steady increase in genetic knowledge, the responsible scientist would express the hope that no rigid system will ever be imposed. But a glance at the history and development of cotton breeding will reveal certain trends and cycles in the methods employed. When American Upland stocks were first introduced into the temperate zone of North America, a wide range of types were in evidence and, as the geographical area of cultivation was extended, certain character expressions were

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T. R. RICHMOND

easily and readily observed. It was a siniple matter to select “good” plants and to reject those plants which were obviously low in vigor and otherwise unsuited to a particular area and system of cultivation. It soon was recognized that certain types were adapted to certain conditions and the desired types were maintained through systems of roguing, mass selection, and single plant selection. During this early period the crop was damaged relatively little by insects and diseases and advantage was taken of thc full length of the growing season by selecting types which were indclerminate in growth and fruiting habit. Tn American Upland cotton, large bolls 11 ere associated with many such types and, particularly in ‘l’exas, the “big-holled ’’ cottons were quite i n vogue. The advent of the cotton boll weevil, Alztliowomus grandis, Boh., ambout1900 and its rapid spread over most of the Cotton Belt forced selection toward determinate, smaller bolled, rapid fruiting types which were adapted to insect control measures and which would produce an acceptable yield of cotton before the usuill late season build-up of large i tisect populations. The development of varieties with such characteristics, which a t the same time gave yields as high or higher than the indeterminate varieties they replaced, represents one of the most outstanding plant breeding achievements in history (Richmond, 1947). It is interesting to note that this period coincided with the rediscovery of Mendel’s work and the subsequent development of the science of genetics. Early in this period Johannsen set forth the “pure line” concept which established the basis for the progeny-row method of breeding. The first proponents of the progeny-row method among cotton breeders were Halls, in Egypt, and Kearney in the United States. This method, in which the best progenies of single plants are selected and the best plants are chosen from selected progenies to be grown again in progeny-rows, was clearly a n extension of earlier methods. Since the progeny-row method of breeding leads eventually to the establishment of pure or relatively homogeneous lines which, at least in theory, could be maintained indefinitely without further deterioration in yield, plant type, and fiber properties, etc., it was widely adopted by cotton breeders. Many of the varieties of cotton in commercial use today were developed by the progeny-row method or some slight modification thereof. Although the propagation of abnormal o r “freak” plants usually is to be avoided, a number of varieties owe their existence to selection of a single unusual plant in an otherwise uniform strain and such practices, whether by institutional breeders, private breeders, or farmers, should not be entirely discouraged. Pima, a variety of American-Egyptian (Kearney and Peebles, 1940), and Montserrat, a variety of Sea ISland cotton (Harland, 1949), are examples of superior agricultural

COTTON BREEDINQ

217

varieties resulting from “lucky finds” on the part of institutional breeders. The present Stoneville strains of American Upland cotton are said to trace back to a single distinctly different plant in a stock of Texas “big boll” cotton which was recognized and selected by a private breeder. Many “unusual types” are discovered by farmers who are, on the whole, keen observers. An abnormal boll type in which the locks of cotton are closely held in carpels which open only partially a t maturity was found in a field of Half & Half cotton by a farmer in the Lubbock, Texas, area (Lynn, 1949). This type supplies an extremely useful character for the development of strains with bolls which are adapted to harvesting by hand snapping or machine stripping. The observation that cotton varieties or strains often lose in vigor and productivity under a regimen of selfing or close inbreeding, such as that resulting from the usual progeny-row breeding system, has led many farmers and breeders to consider “running out” of strains to be characteristic of cotton. Excluding genetic abnormalities such as balanced lethals, homogeneity is a natural consequence of prolonged inbreeding. The observed fact of reduction in productivity following inbreeding in a given strain is attributable to a number of factors chief among them being: (1) the degree of heterogeneity of the original parent stock, (2) the mathematical probability against accumulating all (or even most) of the favorable genes for yield in one homozygoiis line, (3) mechanical mixtures and cross pollination with inferior varieties, and (4)selection for one (or a small number) of characters without regard to other characters which have an important function in the genetic complex. The latter factor is of utmost importance and often determines the success or failure of a breeding program. On theoretical grounds there is no reason to suspect that pure lines of cotton are different in behavior from pure lines of any other crop. The practical consideration of a pure line involves both uniformity and superior performance. Selection for high yield “on a broad genetic base,” which usually is necessary if decreases in production are to be avoided, involves the simultaneous handling of a number of characters over a relatively long period; ,and such a procedure does not lead rapidly to homogeneity. Failure to observe the “broad base” concept and the desire for rapid uniformity in one character at the expense of all others has taught many cotton breeders the severe lesson that the probability of obtaining a “uniformly bad” strain is much higher than that of obtaining a “uniformly good” one. Cook (1932) warned against separation of lines of descent merely for the sake of following a system and pointed out that “pedigrees are records of descent, but other evidences of superiority are required to give such records a practical meaning.”

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F or many years Pima and Montserrat were the leading varieties of

8. barbadense grown commercially in their respective areas of production. Both were propagated by inbreeding from the original single plant selection and neither of them showed measurable deterioration in yield or other characters over the entire production period (Harland, 1949; Kearney, 1922). Although there are notable exceptions, examples of varieties that are propagated by strict inbreeding methods and that maintain maximum performance over a wide area under competition with other commercial stocks are not common in American Upland cottons. However, the five or six commercial varieties that account €or more than 90% of the United States’ production, while probably not homogeneous, have reached such a state of development that reselection within them for higher yields is now considered to be a most unrewarcling task (Waddle and Richmond, 1950). There is no reason to believe that relatively pure lines cannot be established and maintained over long periods as readily in agricultural varieties of 8. hirsutum as in 0.barhadense. The small number of examples i n Upland cotton may be attributed in considerable part to the great emphasis placed on high yields which, in turn, has created a highly competitive situation in which the breeders dare not sacrifice yield for homogeneity and miist strive constantly for higher levels of performance. Resort has been made in a number of instances to crosses between agricultural varieties in an effort to obtain favorable combinations of yield a n d other economic characters. The Deltapine varieties (Ewing, 1948) afford an outstanding example of the development of superior agricultural varieties by the use of multiple varietal hybrids. I n agricultural varieties of 8. barbadense, length and fineness of fiber are economic characters which carry considerable weight in relation to other economic characters, but even in such specialized cases productivity is a major consideration. I n recent years the Pima variety has been replaced by varieties extracted from crosses of Pima and Egyptian Sakel; and Harland (1949) reports that the vigor of Montserrat Sea Island “is decidedly augmented by crossing with unrelated strains. ))

111. RECENTMETHODS AND REFINEMENTS

1. Th,e Rreediag Problem It has been shown that the germ plasm which constitutes the genetic reservoir from which present agricultural varieties have arisen has been remarkably plastic and has yielded a number of productive types which are well adapted to their respective geographic areas of growth. Par-

COTTON BREEDING

219

ticularly has this been true of American Upland stocks. Reselection within varieties, and even within the progeny of varietal hybrids, over a period of many years inevitably has rekulted in severe inbreeding and the elimination of many beneficial as well as deleterious genes which were present in the native stocks. Since American Upland varieties in the United States are all interrelated and probably descended from not more than a dozen original introductions, it is doubtful if future requirements of special fiber properties, disease, insect, and drought resistance, mechanical harvesting, and other specialized uses and properties can be met by the usual selection methods restricted to present cultivated varieties (Richmond, 1947). Two approaches or combinations of two approaches to further progress and improvement come to mind immediately: (1) development of more precision in the breeding program through refinements in method and design to provide more discriminatory statistical tests and the establishment of indices which will measure the genetically potential performance rather than the actual end-result behavior; (2) introduction of new germ plasm into the breeding material. I n the first, the idea is to determine the amount of genetia variability in the material and to distinguish this variability from the variability due to environment. I n the second approach, genetic variability is increased purposely by the addition of new genes. Genetic variability outside the range of that now present in current agricultural varieties is available from three principal sources : (1) obsolete agricultural varieties, (2) primitive stocks in or near the center of origin, and (3) the wild species of the world. I n the United States, under a regional project in cotton genetics made possible by the Research and Marketing Act of 1946,the Mississippi Agricultural Experiment Station has the major responsibility for collecting and maintaining stocks from the first source, and the Texas Agricultural Experiment Station is responsible for the stocks from the last two sources. The importance of genetic variability in the primary breeding material cannot be overemphasized, for it is axiomatic that the breeder cannot bring out through selection anything more than is inherent in the raw breeding material. This principle was well understood by Cook (1932) who said “Selection is a sifting process, not a mending or a making.’’ Modern breeding methods are designed to preserve and control genetic variability, to guard against serious loss of favorable genes through the restricted selection of only a few superior plants or progenies in any one season. It follows that such methods must keep genetic variability high in relation to environmental variability, particularly in the early stages of the breeding program. It is a waste of time to attempt further improvements when the genetic variability in a selected strain falls be-

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low the minimum that can be discriminated by the breeding method employed; such strains should br placed on a maintenance basis if they show merit or discarded if proved to be unacceptable. Mason (1938) has suggested that “secondary” selection (selection in the immediate progeny of single plants) has been considerably overemphasized a t the expense of “primary” selection (single plant selection in unselected populations, hybrid material, etc.) . I n true functional diploids, inbreeding leads rapidly to homozygosity. I f American Upland cotton, which is of amphidiploid origin, can be considered to function as a diploid, selection in inbred lines for five to ten generations should result in strains so homozygous that further selection for economic characters would be impractical. I n dealing with quantitative characters, yield, in particular, under these conditions, a high genetic segregation and recombination will be found in F2, and in many of the extracted lines further segregation for the character in question will be observed. To obtain superior strains, the main problem then would be to slow down secondary selection, after primary selection has been practiced in Fz, sufficiently to make it possible to test the extracted biotypes under the environmental conditions in which they are expected to be grown. As has been mentioned earlier, stocks which are mixtures of relatively inbred biotypes, from one or more primary selections in Fz, because of wider adaptation, might be expected to give a higher average performance over a period of years than pure lines from the same material. Recently evidence has been presented which suggests that American cultivated cottons do not always perform as ordinary diploids. Comstock and Manning (1950), working with hybrids and inbred lines of Sea Island cotton obtained results which, at face value, suggest that “selection pressure was responsible for an increase in genetic variance of traits considered in selection or at least for a slower decrease in variance as a result of inbreeding.” Although the results obtained were attributed to the polyploid condition of the original stocks, a specific explanation of the phenomena was admittedly difficult. The authors mentioned the possibility of preserving heterozygous loci through selection pressures, but were inclined to believe that “ a high rate of point mutation, chromosomal rearrangements associated with ‘illegitimate pairing’ or a combination of the two, under sufficient selection pressure, could have accounted for the results obtained.’’ From experiments in which the genetic variance within and among five ‘(well-bred” commercial varieties of American Upland cotton and certain of their hybrids waa determined, Waddle and Richmond (1950) showed that “selectable variation was found within Rogers Acala for

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all characters measured and within Rowden 41B for seed index and lint percent only.’’ Genetic differences were iiiost evident in segregating material involving hybrids of varieties which exhibited the greatest contrast for the characters in question. The writers concluded that “the genetic portion of the total variance, both within the varieties studied and the F2 populations involving the varieties, was such that selection by non-statistical methods is rendered doubtful. ” Waddle (1950))from the same data, has suggested that the vigor observed in certain selections may be an expression of heterosis attributable to heterozygous loci which have been maintained in the material through selection pressure. 2.

T y p e Beleetion Less than twenty years ago Cook (1932) advocated renewed emphasis on “type” as the prime consideration in cotton breeding and in the maintenance of seed stocks. Under his “type selection” system, groups of progenies) instead of single progenies, were propagated, and maintenance of the stock was carried out by reselection within the groups. Selection and testing of progenies under different conditions “as a means of preserving the adaptive characters of varieties which otherwise may be lost even without being recognized” was recommended. The value of stable mixtures of strains or varieties in providing “greater flexibility of response” was recognized by Hutchinson. In his genetic interpretation of plant breeding problems, Hutchinson (1939, 1940) observed the association of rapid degeneration with the more closely bred varieties and recommended that “the effort at present devoted to achieving purity may profitably be used to increase the efficiency of selection.” 3. Replicated Progeny-Row

Hutchinson and Panse (1937) introduced randomization and replication into the progeny-row system of breeding. The system, which has been designated as the I ‘ replicated progeny-row method, ” provides all the information on means of progenies and means of plants within progenies obtainable by earlier progeny-row methods, and a t the same time, makes possible additional valuable information. In the design employed by Hutchinson and Manning (1943) the progeny of each selected plant was tested in ten randomized blocks, each plot of which contained five plants. Where strains from a number of families were to be tested in one layout, “compact family blocks” were arranged within the main experiment to provide more precision in the interfamily comparisons. Not only does the design reduce the environmental contribution to the variance, but it makes possible the partitioning of the total variance into its genetic and environmental components, thus min.

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iriiizing (‘environmental fluctuations while maintaining genetic contrasts.” The writers point out that selection on progeny means is as many times more efficient than mass selection in the same material as there are plants per progeny. Stephens (1944) gives the following summary of the system : ((Th e replicated progeny-row technique representh a considerable advance in plant breeding method, since i t not only transfers emphasis of selection from the single plant to the mean performance of its progeny, but recognizes maintenance of variability as being only slightly less important than mean yield. Furthermore, if used effectively it enables the plant breeder to recognize the scope and limitations of his material. H e is able to distinguish between real and apparent progress in the achievement of his object and decide when he has exhausted the possibilities of further improvement. ” I n their breeding work with superfine cotton, Sea Island V 46, Hutchinson and Manning (1943) compared modern strains and obtained no response to selection within them. However, Comstoclr and Manning (1950),after applying more critical measurements to the selected progeny of the variety, reached the conclusion that “genetic variation was present in the material even though it had been self-fertilized for five generations a t the time deliberate selection for yield was initiated. ’’ There was evidence for genetic variation among sister progenies of the tenth generation of continuous self-fertilization, and for definite and continuing yield advance during the interval from the fifth to the tenth generation.

4. Mass-Pedigree Selection. Mention has been made elsewhere in this section of the importance of a definite objective in the breeding program and of flexibility in breeding methods to provide reasonable probability of success in the allotted time and with the materials and facilities available. Tanguis, a n agricultural variety which has been classified taxonomically as G. barbadense, is the principal cotton grown in Peru. It was developed from a n “exceptional” plant discovered in 1908 by a farmer in a field of Upland cotton (Harland, 1943). The fibers of Tanguis are quite unique in that they are both long and coarse. The variety was productive and resistant to the wilt disease prevalent in it,s area of production. Over the years Tanguis became mixed with other varieties to the point where the quality and performance of the stock was in grave doubt and consideration was given to discarding the entire stock. Beginning in 1940, Dr. 5. C. Harland was given the task of restoring the lost qualities and making certain improvements in as few years as possible. To avoid some of the dangers inherent in progeny-row breeding when rigid selection is practiced on relatively few characters, Harland (1943,1949) inaugurated a breeding

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system which he terms ‘‘mass-pedigree selection.” I n practice, the system simply involves ( 1 ) the growing of progeny of a large number of selected plants, (2) determining the mean of each progeny for the characters under consideration, ( 3 ) arraying the progeny means for each character, and selecting progenies whose means fall on a certain segment of the distribution curve (the segments to be chosen by the breeder on the basis of the relative importance of one character as compared to the others, to the original variability of the material, etc.), and finally, (4 ) massing all the selected lines to form a bulk planting from which another selection cycle may be started. According to Harland (1943, 1949) “continuous selection by this method for any measurable character tends to produce a system of gene frequencies resulting in the manifestation of the character a t a higher level through the elimination of alleles, the combinatory effects of which are ordinarily antagonistic to the standards laid down for the character.” Harland (1943, 1949) calls attention to the principle of the genotype as actually applied by plant breeders long before the birth of the science of genetics; it is the principle that “the breeding value of a plant can only be stated in terms of the mean characteristics of the offspring.” The “ mass-pedigree selection” system makes full use of the principle and a t the same time it is designed to preserve genetic variability through the use of a large number of lines and maintain a broad adaptation base by propagating massed lines under varying seasonal and other environmental conditions. Furthermore, the method would preserve certain genes for vigor as heterozygous loci, a condition which, in Harland’s view, would give the stock a n advantage over strains in which the same genes were homozygous. Used with considerable success in rehabilitating Tanguis cotton, he recommends the method for wider application and would use it instead of pureline selection systems. The system is similar in principle to the older “type selection” methods but recognizes, defines and measures the component characters of the type and provides a much more critical progeny test. Actually, the method is not a t odds with the progeny-row method as employed by many contemporary plant breeders in which a number of tested lines of generally similar characteristics are massed at certain stages in the testing procedure and the seed stock distributed as an agricultural variety. The “mass-pedigree selection ” method obviates detailed records of families and lines. 5 . Bulked Progeny Test

Even though considerable genetic variability may remain in certain ootton varieties, the greatest opportunity, within cultivated Upland types, for improvement of yield and other economic characters which are

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inherited quantitatively still appears to be provided by extensive selection in large Fz populations. When relatively distantly related parents or parents with widely contrasting characters are employed, the probability of success is enhanced. Progeny tests of primary F2 selections will establish relative performance levels, though the progeny itself is not, expected to be uniform with respect to all or even one of the characters on which selection is based. Whether genetic variation is maintained in the early stages of the breeding program by mixtures of relatively inbred biotypes, heterozygous loci, chromosomal rearrangements or other factors, the important problems are to keep it high enough for efficiency in seoondary selection and, in later stages, to preserve it in such a manner that the increases obtained can be maintained at an acceptable level of phenotypic nniformity. A system of breeding employed by workers a t the Texas Agricultural Experiment Station, dehcribed as the bulked progeny-test method of breeding, is designed to preserve genetic variance in the bulked progeny of primary selections in Fz until the performance of the families can be tested over a period of years and their adaptation to environmental conditions appraised. Whole families are discarded according to the dictates of the test results. Reselection may be practiced in any bulked progeny from F3 onward. Reselections are considered as new < & primary selections,” but may not be subjected to all the tests outlined below. The method differs essentially from ITarland’s mass-pedigreeselection method in that families established through primary selection in Fz usually are maintained separately though in bulk in subsequent generations. I n general, the procedures employed in the bulked progeny-test system of breeding are as follows : 1. Single plants are selected in F, and selfed- and open-pollinated seeds a r e harvested separately. The selfcd seeds are stored in surli :i manner as to preserve the germinating qualities over ii period of several ye:irs. 2. Dupliwte progeny rows i ~ ei grown from open-pollinatril seeds in randomized blocks in F,; “check” rowa of the best commercial variety f o r the area a r e grown at 8-10 row intervals throughout the plots a s n standard of comparison f o r productivity, maturity, etc. Progenies are grown in large numbers a n d selection is first made on the basis of comparison with the “check.” Only those progenies with good “scores” in both replicates a r e marked for further attention. After the scoring, a random sample of bolls is harvested from one of the “duplicate rows” of each progeny selected to supply material for other agronomic, a n d fiber and seed determinations; the random boll sample also supplies seed for testing in F,. 3. I n the F, generation the bulked seeds from Fa are planted in a randomiaetl Iilock design of 4-8 replications; two or more “check varieties” a r e entered iu the design on the same basis as the new strains to be tested. The plots are harvested f o r yield as in a n ordinary strain or variety test. and the yield of t h e “cheak”

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(or checks) is used as a standard of comparison. Agronomic and fiber data are obtained from random boll samples a s in ( 2 ) above, and the seeds of the strains that are selected are used ill another testing and selection cycle. 4. When the testing of raiidoni boll bulks has reached F, to Fa, a sufficient amount o f data will have beeu accumulated on the strains that have not been discarded earlier t o furnish a basis for decision on subsequent disposition. Presumably :ill the remaining strains will tlieii have acceptable means for yield and other characters under study. Depending on how well the random boll samples actually represented each family and also assuming less than 10% cross pollination, maximum heterogeneity of superior biotypes will be preserved in FB-Fabut the majority of individual plants will have approached practical homozygosity. The variability exhibited within each of the strains that reach the F,-Fa stage also will depend on the extent to which mean yield rather than uniformity is taken into account in retaining or discarding whole families from Fa onward. This poses a critical decision, for the breeder may make a mistake by either selecting for uniformity too soon or maintaining variability in the breeding material too long. 5. Usually i t will be desirable to reselect on an individual plant basis in the bulked material of families a t the end of the first testing cycle (FB-Fa), and through such selection it should be possible t o raise the mean performance of some of the reselected progeny over the mean of the parent family. The second cycle of testing should seldom proceed past the second or third generation before the selected second cycle family bulks are ready for multiplication as new agricultural strain8 or varieties. 6. Whenever possible, strains with similar characteristics are massed and carried in subsequent strain and variety tests as massed-line varieties. 7. I n areas where cross pollimtion is extremely high it may become necessary to resort to the stored selfed seeds of the F2plant corresponding to each family that persists a t the end of the first testing cycle. Increases from selfed seed, in such cases, should be made before first test cycle is concluded in order to save time.

6. Backcross Methods When the breeding objective is to transfer a character which is conditioned by one simple gene, or a small number of such genes, from one variety or type to another without deleteriously affecting the desirable characters of the latter, the backcross method of breeding is clearly indicated. Only small progenies are required in each backcross cycle when such characters are available. Unhappily, in cotton, only a very few such simply inherited and easily distinguished characters have been recognized. The genes controlling resistance to certain cotton diseases are the best examples, but more will doubtless be discovered as tl?e work of “sifting” and evaluating recent foreign introductions proceeds. The backcross method has been employed in varietal hybrids in attempts to transfer characters which are inherited according to the quantitative scheme. This approach should not be entirely discouraged but in its application it should be pointed out that 50% of the potential variability is lost in the very first generation of backcrossing, and the

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genotype of the recurrent parent is approached constantly, though in ever diminishing proportions, in each succeeding backcross. Moreover, to obtain reasonable probability of success, large populations would be required, and each plant would have to be subjected to critical analyrais, preferably by progeny test. Knight (1945) goes so f a r as to state that the backcross ‘ ‘system is valueless in intraspecific (varietal) crosses because such hybrids, by the first backcross, are normally very similar vegetatively to the backcross parent-( thus)-the hybrid may look like the backcross parent, but it still contains a large proportion of the donor genotype and is unlikely to breed true for the various qualitative and quantitative characters desired.” Certain modifications or adaptations of the backcross and the straight hybrid systems, or combinations of systems, may prove useful in American Upland cotton breeding. Richey ’s (1927) “convergent improvement” method is a case in point. The object in this system is the transference of dominant favorable genes from one line to another. The method involves double backcrossing to parallel lines in cyclic repetition of backcrossing to parallel extracted lines. Another method suggested by Sprague (1946) and recommended for use in certain cotton experiments by Richmond (194913) is now called recurrent selection. I n this method lines bearing the character under study, from as many diverse sources as possible or practical, are selected in F z and after isolating relatively good complexes, but without carrying on selection in each line to its ultimate conclusion (complete uniformity), the selected lines are immediately crossed in all possible combinations and carried to a new FZin bulk. Selection for the character is then practiced again and the cycle repeated until the level of acceptability is reached.

7. Intervarietal Hybridization Selective material of measurable genetic variability can be produced by crossing certain cultivated varieties, but considerably more variability can be obtained from primitive stocks collected in “center of origin” or from crosses of such stocks with modern strains. Considering the American Upland cottons, it may be said with confidence that there are few major structural differences among the chromosomes and that hybrids of both agronomic and taxonomic varieties are practically 100% fertile. Furthermore, the difficulties attributable to genetic unbalance (Harland, 1932, 1936) and small structural differences in chromosomes (Stephens, 1950) as found in species hybrids are not in evidence in varietal hybrids. Thus, while the total range of genetic variability in varietal hybrids is considerably less than that usually found in species hybrids, the combination or transference of those special char-

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acters which are present in the parent material, if possible at all, can be achieved in a relatively few generations and with less likelihood of deleterious effects on productivity and other economic characters. The American Upland cotton breeding program of the Agronomy Department’s Cotton Improvement Section a t the Texas Agricultural Experiment Station largely employs varietal hybrids. Because of the wide range of soil and climatic conditions in Texas, the breeding program must emphasize particularly the conservation and maintenance of genetic variability in progeny tests extending over a number of years. Probably in no other cotton growing region is the maintenance of a “broad adaptation base” so important. Breeders have taken renewed interest in the native American Upland cottons of southern Mexico and Central America as a source of new germ plasm. I n view of the close genetic relationship which exists between such native cottons and the present cultivated varieties which were derived from them many years ago, important new economic characters, if found in the present native stocks, should be relatively easily transferred to currently cultivated stocks. I n 1946,T. R. Richmond and C. W. Manning made a preliminary exploration trip to the area. The next year S. 0. Stephens, who was then employed by the Empire Cotton Qrowing Corporation, explored the area; and in 1948 J. 0. Ware and C. W. Manning made a rather extensive survey of a wider area, including parts of San Salvador, Guatemala, and Mexico. As a result of these recent expeditions, more than 640 stocks have been collected.

8. Interspecific Hybridizatior. By all odds, the greatest range in variability in cotton exists among the wild and cultivated taxonomic species which are fairly well distributed over the tropical and subtropical areas of the world. The two cultivated American species, G . kirsutum and Q. barbadense, cross readily and give fertile progeny, as do the two cultivated Asiatic species, G. arbmeum and G.herbaceurn. To the uninitiated, it would seem a simple matter to transfer desirable characters from one species to another or t o obtain increases or improvements in certain characters through “transgressive breeding’’ (Stephens, 1944) in which characters conditioned by many genes are intensified in a single line by the action of favorable genes from the two parents, one of which carries a certain complex of favorable genes ; many of the genes may occupy loci different from those of the other parent. Such types would be expected from species hybrids, if all the genes acted independently. Though literally thousands of attempts have been made; there has not as yet been selected from such a crosa a strain in which two quantitatively inherited char-

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acters have been combined in the full expression of their original parental form. In fact, i t is extremely difficult to find good examples of satisfactory intermediate expressions of quantitative characters. The development of a variety now called Sealand (Jenkins e t al., 1946), a type whose expression of such characters as yield, lint length, fiber fineness, and boll size is intermediate between those same characters in the hirsutum and barbadense parents, is worthy of note. The work of Harland (1936) has shown that stable complexes of interrelated genes are built up within each species, and when such species are crossed, the “genetic balance” is disrupted in the Fz generation, giving a veritable mess of abnormal and unbalanced types. Stephens (1950) presents evidence to show “that multiple gene substitution, such as that suggested by Harland, is not sufficient to explain the cytological, genetic, and breeding phenomena encountered in critical studies of fertile interspecific hybrids and their progenies in Gossypium” and cites “recent evidence from studies of amphidiploids which casts doubt on the validity of ‘normal’ chromosome pairing and hybrid fertility as indices of structural homology. ” The structural (cryptic) differences to which Stephens refers are considered to involve much smaller “pieces” of chromosome than those involved in the gross structural changes which may be recognized cytologically and which may cause partial or total sterility in the hybrid progeny. I n spite of the fact that the cotton breeding literature is well l i salted” with recommendations against the use of hybrids between species in practical breeding programs, and though the process will unquestionably be of long duration, future requirements of the cotton industry to meet competition in trade in textiles and to facilitate further technological advances i n production and processing are likely to be such that they can be met, if at all, only by the introduction of characters outside the range of present cultivated varieties. Not only does it seem important to continue research in those interspecific hybrids which cross readily, but methods of utilizing the wealth of new germ plasm in the wild species of the world should receive special attention. Until recently, crosses between many of the cotton species with 13 pairs of chromosomes and all the crosses of 13 by 26 paired species, if successful a t all, gave sterile hybrids. Little more than ten years ago Beasley (1942) induced fertility in a cross of Arizona Wild ( G . thu.rbei-i Tad. by Q. arboreurn) by doubling the chromosomes of the sterile hybrid by treatment with colchicine. The resulting amphidiploid ( n = 26) was partially fertile with American Upland ( G . hirsutum, n = 26), and a high degree of featility was reached after the first backcross to Upland. Thus was opened up a vast new field of cotton im-

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provenieiit as yet only barely explored. When this backcross was made it was found that fiber strength of some individuals increased materially over anything previously known in the cultivated Upland cottons. This, of course, was quite unforeseen, as the Asiatic parent was by no means outstanding in respect to fiber strength, and the American Wild parent had no spinable fibers a t all. Subsequent studies have shown that the fibers of the new hybrid have narrow cross-sectional areas (narrow perimeters), a character introduced from the apparently worthless wild cotton from Arizona. Hence, in species crosses, the apparent valuable characters available f o r transfer are probably only a fraction of the important qualities yet to be discovered. Breeding experiments with the so-called tri-species hybrid (Asiatic X American Wild (doubled) X American Upland) have been in progress a t the Texas and North Carolina Stations from the time of Beasley 's first induced amphidiploid. Stephens' cryptic structural differentiation theory inay explain, in part, the slow progress made to date in transferring high strength from the triple hybrid to high yielding strains. Certain generalizations with respect to the inheritance of a quantitative character such as lint strength can be made from the research a t the Texas Station (Richmond, 1949a). When selection is made for yield, the IJpland type results in fewer generations than expected from theoretical considerations ; furthermore, high fiber strength is almost always lost. On the other hand, if in backcross populations, selection for fiber strength is made on the basis of critical measurements, the phenotype, and presumably the genotype, of the 1Jpland pOrent is not recovered anything like as rapidly as expected on theoretical grounds. Comparative trials of extracted high fiber strength lines from the fifth, sixth, and seventh backcross generations have shown a strong negative correlation between boll size and fiber strength. Data obtained from the Texas experiments indicate clearly that fiber strength is inherited quantitatively. J u s t how many genes are involved cannot, as yet, be estimated with any degree of accuracy, but it is known that some of the genes in the fiber strength complex are dominant while others are recessive. It seems reasonable to expect that some of these genes will be located on certain of the small chromosome segments which are structurally differentiated with respect to the chromosomes in the Upland complex. Along with the high strength gene, or genes, on these cryptic segments will be other genes from the donor parent, many of which are deleterious. I n crosses with Upland, most crossovers involving the differentiated segments with the gene for fiber strength would also carry the deleterious genes from the donor parent. It should be pointed out that duplications and deletions, as a result

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of crossing over within a structurally differentiated chrosomome, might account for part of the deleterious effects observed. Eventual success in these and other studies involving species hybrids, appears to rest on the not altogether vain expectation that a favorable crossover in a given differential segment will occasionally occur. Knight’s (1946) transference of blackarm (bacterial blight) resistance from one species of cotton to another is sufficient to demonstrate the tremendous benefits t o be enjoyed when the “breaks” finally come. IV. HYBRIDVIQORIN F1 AND ADVANCED GENERATIONS From the foregoing discussion there can be little doubt that the maintenance and improvement of yield and vigor has been a major consideration in cotton breeding programs. Productive capacity must not be ignored in future experiments, and the renewal of interest in hybrid vigor in cotton is proof enough that increases in yield still are to be sought. However, as the points of “diminishing returns” are reached in yield potential within strains and varieties, and as the demands for “special types for specific end-uses” increase, attention must be turned more and more toward other improvements and refinements. The substantial increases in yield and improvement in Qther economic characters obtained in first generation and double crosses in corn and the.almost universal acceptance of hybrid seed as the propagating material for commercial corn production have led some cotton investigators to reexamine the possibilities of similar methods in cotton. As early as 1911 ( U . S . Dep. Agric., Bur. Plant. Znd. Circ. 96) increases in yield and improvement in fiber and other characters were observed in F1 hybrids of a. hirsutum and Q. barhadense, and methods of hybrid Beed production using bees as pollen carriers were proposed. Hutchinson et d.(1947) reviewed previous work on heterosis in cotton and showed that significant increases in most plant characters attributable to hybrid vigor, including yield, have been reported in interspecific, intraspecific and intervarietal crosses. Most of the early information in the literature relative to hybrid vigor in varietal hybrids of American Upland cotton was only incidental to other investigations, and as was to be expected, by no means all the F1 varietal hybrids reported exhibited heterosis. In recent years several experiments, designed primarily to study hybrid vigor, have been undertaken. Three inbred lines from varieties of American Upland cotton and the six possible F1 and Ft hybrids from the lines were studies by Kime and Tilley (1947) for a period of three years. The F, seed was produced by hand pollination and the F2 by

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selfing. Over the three-year period, significantly higher yields were reported for each of the hybrids as compared to its highest yielding parent, but the important economic consideration was the finding that by no means did all the hybrids excel significantly in the single years. Clearly, the significant increases obtained for the three-year means resulted, in all but one case, from the additive effects of small single year differences which were nearly always in favor of the hybrid. Significant increases in the yield of the advance generation (Fz) over the most productive parent were recorded for only two crosses and these occurred in only one year. An investigation of the heterosis exhibited in the progeny of varieties and strains propagated by open-pollinated seed from a production area in which natural cross pollination approximated 50% has been reported by Simpson (1948). Using a split plot experimental design, he tested the progenies of seven varieties, seed of which were produced under two conditions, (1) open pollination (crossed) in a 25-entry variety test and (2) in isolated blocks (inbred). He reported that the seven progenies from the “crossed” seed exceeded those of “inbred” seed by 5.7 to 44.2%, or an average of 15.4%. Evidences of hybrid vigor also were observed in seedling growth. Of the several agronomic characters measured, boil size and lint index particularly showed increases in the “crossed” progeny over the respective “inbred” parents. Critical examination of the data reveals that the greatest percentage increases were obtained from the “crossed” progeny of the varieties with the lcwest yields in the previous year’s performance test, and vice versa. The practical significance of the data lies not so much in the average increase of 15.4% attributed to hybrid vigor, but in a comparison of the “crossed” stocks with the highest yielding agronomic variety. When such comparisons are made, it is seen that significant yield differences in favor of the “crossed” stocks occur in only a n occasional instance. Simpson (1949) also conducted an experiment to measure, by the method just described, the amount of heterosis resulting from natural crossing in test plots at several locations in the Cotton Belt. In 106 out of a total of 132 paired coqparisons, the progenies from “crossed” seed gave higher yields than the “inbred” progeny of the corresponding variety. No significant difference in yield was found among the three varieties used, which was not surprising, in view of their presumed close relationship, and Simpson considered the apparent hybrid vigor observed in the “crossed” stocks t o be a measure of both the amount of cross pollination at the different locations from which seed was obtained and the genetic diversity of the stocks which might have contributed the pollen. Though the data are impressive, it is difficult

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to reconcile the increases in yield obtained with breeding experience a t a number of the “seed source” locations. It is not improbable that certain physiological factors and differential interactions of physiological factors and differential interactions of physiological responses with the genotype at the various “seed source location’’ played a part in the differences obtained in the test at Knoxville, Tennessee, a location somewhat beyond the northern limits of the presently accepted Cotton Belt. One is inclined to withhold unqualified acceptance of the interpretation until such time as progenies of both selfed- and open-pollinated material from a number of ‘(seed course” locations are compared, in a suitably designed test a t Knoxville or some other designated point. Regardless of whether the occurrence of hybrid vigor may or may not be as prevalent among varietal crosses as Kime, Simpson, Tilley, and others have presumed, there can be no doubt that acceptable combining ability is in evidence among cwtain lines, and further investigation will doubtless bring forth even more acceptable combinations. Furthermore, there would seem to be nothing to be gained by restricting the search for suitable lines to any given species unless seed production from advanced generations is contemplated The great handicap to the practical utilization of hybrid vigor in cotton is the difficulty of producing the hybrid seeds. Two methods have been proposed by Simpson (1948) : (1) production of a small quantity of F1 seed by emasculation and hand pollination between lines whirh have shown high combining ability in previous tests and increasing the F1 seed for two or three generations in an isolated area, where natural crossing is known to be high, to a sufficient quantity for commercial planting; (2) multiply, according to the procedure described in (l), a mechanical mixture of two or more inbred lines of proved combining ability. Granting that a significant amount of hybrid vigor could be maintained and the possibility that a sufficient amount of seed might he produced by these methods to plant a considerable part of the present c!ommercial cotton acreage, it is extremely doubtful, in this age of specialization, that farmers would be willing to accept any such (‘vintage” product so dependent on the caprice of a e honey bee. For certain conditions in India, Balasubrahmanyan and Narayanan (1948) have proposed vegetative cuttings as a method of propagating F1 cotton hybrids on a commercial scale. Probably the most hopeful method now on the horizon is the use of cytoplasmic male steriles as “mother” plants, as is now being done on a limited scale in onions and corn. The difficulty so far in cotton has been the discovery of a male sterile character which is cytoplasmically inherited. If such male sterile stocks were available, hybrids of

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known composition could be produced by interplanting them, in areas of high cross pollination, with normal lines which are capable of restoring fertility. Recently several apparently male sterile types have been studied and found to be only partially o r periodically so.

V. SPECIAL CHARACTERSOF BREEDING VALUE 1. Fiber Properties Cotton was first domesticated for its fibers and, though the seed has become increasingly important economically, the lint which is used for textile purposes continues to be by far the most valuable product of the cotton plant. The increasing competition of synthetic fibers, as well as foreign grown cotton, has made the improvement of fiber properties one of the most pressing problems in cotton research in the United States. Cotton breeders, fiber technologists, and textile engineers have long realized the need of machines and methods for the evaluation of the different fiber properties. The development of special machines and apparatutr in the United States dates back to the Webb-Suter machine for the determination of fiber length distribution and the Chandler device for measuring the fiber strength of raw cotton. Later, methods of measuring fiber fineness by weight per unit length and of evaluating maturity by measurements, of ribbon width and cross-sectional areas were introduced. Spinning test procedures for large samples (10-30 pounds of lint) were developed, and the demands from merchants, processors, breeders, seedsmen, and others for spinning test information soon became so great that appropriations of funds were made to the U. S. Department of Agriculture to finance the building and operation of fiber and spinning laboratories at Clemson College, South Carolina, and the A. and M. College of Texas. During the middle thirties the U. S. Department of Agriculture sponsored a regional cotton variety study in which a standardized variety layout was grown at some sixteen different locations throughout the Cotton Belt for a period of three years. Not only did the study furnish valuable agronomic data, but it also supplied a wealth of comparable material for studies on fiber and yarn properties. The study must be given much of the credit for the initial impetus which launched a period of technological advances in the evaluation of fiber properties which persists to the present date. As most of the early methods of fiber testing were time consuming, tedious and expensive, they were useful to the breeder only for the pnrpose of evaluating the fiber and spinning properties of advanced

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strains and varieties. The search was on for rapid inexpensive methods which could be used on small samples, even on single plant samples. Outstanding success in this endeavor can be reported. Sorters were devised which made it possible to determine the fiber length distribution of small samples, down to the lint on a single seed if desired (Pressley, 1933 ; McNamara and Stutts, 1935). More recently there has been perfected a more nearly automatic instrument, employing a photoelectric principle, for the determination of fiber length distribution, which is now in wide use in laboratories (Hertel, 1940). A relationship between strength of fibers and the orientation of the cellulose fibrils in the cotton fibers, as determined by X-ray. defraction patterns, has been demonstrated, and methods have been devised for using this relationship in the measurement of fiber strength (Berkley et al., 1948). Perhaps because of its simplicity and the speed with which results may be obtained, a fiber strength tester developed by Pressley (1942) has become a standard machine in practically every fiber laboratory, and Pressley units of strength measurement are in common use by cotton breeders and fiber technologists as well. Several new devices for measuring the fineness of fibers, including the air permeanmeter (Pfeiffenberger, 1946), arealometer (Sullivan and Hertel, 1940) and micronaire (Smith, 1947) are in various stages of development and use; i n general these instruments employ the principle of the resistance of a porous mass of known density (cotton plug) to the flow of a gas and, by the employment of certain scales, relate the resistance obtained to surface area of. the fibers or other measurements. The polarizing microscope (Grimes, 1945) is being used in several laboratories to determine the maturity of fibers. Many improvements and refinements have been made in spinning test methods; “long drafting” * has replaced the older and slower methods of drafting previously used; ‘,‘yarn appearance’’ has taken its place with “number” and “strength” as important yarn properties. A great deal of attention also is being paid to “spinnability,” or the economy with which cottons can be spun ; of particular importance in this regard are waste and the comparative numbers of “ends down” in the spinning process. Recent progress made in the development of techniques for spinning smaller samples has been such that in the near future cotton breeders in the United States may have at their disposal a method of * Definition of textile terms used in this paragraph: Long drafting, an improved method of processing raw cotton into yarn. Yarn number, the number of hanks (840 yd.) of yarn required to weigh 1 Ib. Waste, material lost or removed from raw stock during the spinning process. Ends down, breaks in yarn during the spinning process. Skein, 120 yd. of yarn. Neps, small messes of tangled fibers i n yarn.

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determining the spinning properties of small samples. Even now formulae have been devised from which the spinning properties of a given variety or strain can be predicted from certain of the determinations on raw fibers mentioned above (Barker and Berkley, 1946). Properly designed experiments have made it possible to separate the genetic (varietal) differences from the effects of environment in the evaluation of fiber properties as related to variety, year, and place of growth. When such a distinction is made, it is found, for instance, that certain fiber properties of new fibers may have a preponderant effect on skein (yarn) strength when the genetic phase of the variance is analyzed, but quite another raw cotton property, or properties, may be most influential in the environmental phase (Barker and Pope, 1948). For a comprehensive summary of the recently developed methods of fiber property evaluation and the relationship to and use in the improvement of cotton varieties the reader is referred to a Progress Report, entitled “Better Cottons,’’ prepared by scientists of the Division of Cotton and Other Fiber Crops and Diseases, Bureau of Plant Industry, Soils, and Agricultural Engineering, Agricultural Research Administration, U. 8. Department of Agriculture. The data from the regional variety study and regional and local studies of a similar nature that followed have pointed to one salient fact; that is, the variety (genetic constitution) is the s k g l e most important consideration 6n the determination of quality of cotton fibers. That considerable genetic variability for fiber properties must have been present in certain of the relatively modern varieties of American Upland cotton is evidenced by the fact that breeders have made significant improvements in fiber strength and related characters by selection within varieties and varietal hybrids. The development of the Acala 1517 variety and its derivatives for the irrigated cotton areas of New Mexico and Texas (Stroman, 1948) is a notable example. The fibers of this variety are significantly stronger than those of the Acala strains which it supplanted j furthermore, the ginned lint contains fewer neps and gives such superior spinning performance that it now commands a price premium in the market where earlier strains were penalized. George J. Harrison, working at the U. S. Cotton Field Station in California, used the Acala 1517 in crosses from which Acala 4-42, a variety with high fiber strength, was developed for the San Joaquin Valley. I n the main Cotton Belt, Stoneville 2B has long been known to have superior fiber quality, and more recently similar properties have been developed in the Deltapine and Coker 100 varieties by appropriate breeding methods. The value of the wild and primitive cottons of the world as a source

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of fiber properties which are outside the range of American Upland types already has been emphasized. Strains with fibers 20 to 30% stronger than the better Upland types have been extracted from species hybrids at the Texas Station, and continuing yield trials show that perceptible, though slow, progress is being made in the transference of high fiber strength to acceptable Upland stocks. A wealth of untried fiber characters remains in the base material.

2. Disease Resistance Several diseases cause great damage to cotton and result in substantial annual losses in income to farmers. The “mature plant” diseases of greatest importance are Texas root rot (Phymatotrichum omnivorum), wilt (Pusarium oxysporiunt, f. vasilzfectum and Verticillum al6o-atrum) and bacterial blight (Xanithommas malvaceurum). Goldsmith and Moore (1941) and Pressley (1950) have reported indications of resistance or tolerance for Texas root rot, but no significant heritable resistance has been established in an agricultural variety. Since the disease attacks such a wide range of plants, including practically all of the dicots, there seems little likelihood of finding practical resistance in any of the cottons. However, resistance at a rather high level, has been obtained in certain Gossypiurn species for both wilt and bacterial blight. Recently Sherbakoff (1949) reviewed in detail resistance to the Fusarium and Verticillium wilts. I n respect to the Cotton Belt of the United States, Fusarium wilt is generally prevalent from the Atlantic seaboard to the eastern part of Texas. Verticillium wilt is considered to be a disease of the western irrigated regions, though it has been found in localized areas in the old on cotton as fa r east as the Texas blacklands, and the alluvial area of Arkansas, Missouri, and Mississippi. Breeding for resistance to Fusarium wilt resistance in Upland cotton has been in progress for more than forty years and the performance of such relatively recent varieties as Coker 100 Wilt and Stonewilt, and the more recently released varieties, Empire, White Gold, and Pandora stand as evidence of the success of the program. According to Jenkins et d . (1939) resistance to Fusarium wilt is inherited as a dominant character which is fairly easily transferred in intervarietal crosses. The complications encountered when wilt resistant varieties are grown on soils infested with both wilt and root-knot (Heterodera moir’oni) and the problems involved in breeding for resistance have been discussed by Smith (1941), who observed that wilt resistance often broke down when complicated by mild to severe root-knot conditions. After finding a higher range of types of resistance to Fusarium wilt in strains

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of Asiatic cotton than in the TJplands, Stephens and Armstrong (1949) initiated studies of the inheritance of resistance in Asiatic crosses. The Verticillium wilt disease, as a major problem in cotton production, is relatively new. I n certain parts of the western irrigated cotton growing region during the past five years, the disease has become so destructive in Upland cottons that growers have, in a few cases, turned to varieties of American Egyptian (a. barbudense), which are highly resistant, if not immune. Effort is being concentrated on breeding programs to discover more potent characters for resistance in the Upland cottons and to devise practical method8 of transferring resistance from b o r b h m e and other species to present agricultural varieties. Acala 1517 is now considered to be highly tolerant of Verticillium wilt and, considering the many strains now reaching advanced stages of testing at several experiment stations in the west, varieties with considerably more resistance soon should be available to farmers. Bacterial blight or the blackarm disease, as it is called by British workers, is prevalent in cultivated varieties of American cultivated cottons, particularly so in the barhadense group, Sea Island and Egyptian, but the Asiatic cottons appear to be immune. Knight (1946a)was able to transfer to Egyptian cotton an acceptable amount of the resistance found in a stock of Upland. He found that resistance ww conditioned largely by a single dominant gene; two weak genes for resistance also were shown to be present. I n certain sections of the United States Cotton Belt, considerable damage has been sustained by secondary organhms which enter the unopened bolls through bacterial blight lesions and cause spotting and discoloration of the lint which results in lower commercial grades. It is the belief of many investigators that more reduction in yield than is realized by the layman is caused by the lesions on the leaves and stems which cause the leaves to shed and branchex to break before the bolls they bear reach maturity. A considerable amount of recent work on the bacterial blight disease has been done by American scientists. Simpson and Weindling (1946) report the isolation of a strain of Upland cotton, Stoneville 20, which is highly resistant to bacterial blight and show that resistance can be transferred in varietal crosses. Evidence that the resistance in Stoneville 20 is inherited as a single factor, with resistance recessive to susceptibility, has been presented by Blank (1949). With the view to providing more critical conditions for selecting and testing, considerable research has been done on methods of inoculation and on seedling tests. The fact that the Asiatic cottons are practically immune to bacterial blight poses the interesting problem of attempting to transfer this immunity of the Asiatic type to Upland varieties. Preliminary investi-

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gations a t the Texas Station, in which backcrosses of the synthetic amphidiploid (Asiatic X Upland, doubled) were made to American Upland, failed to give a single case of immunity such as that found in the Asiatic species ; in fact, no resistance was found which was manifestly greater than that already extracted from several different sources in Upland. The seedling diseases of cotton deserve more attention from plant breeders in the future than they have received in the past. There is abundant evidence to show that good stands, and vigorous, disease-free (as a result of fungicidal treatment) seedlings have a pronounced effect on the total acre yield obtained. Any genetic, improvements in seedling disease resistance would contribute materially to economic cotton production. 3. Insect amd Other Resistance Though insects annually take a toll of millions of dollars from the American cotton crop, scientists in this country have given scant attention to the extremely important problem of breeding for insect resistance. Evidence obtained to date from a number of sources supplies a basis for optimism as to the distinct possibilities for cotton improvement which lie in this long-neglected field. According to Knight (1946b), who referred to previous reports, “ C f . thurberi appears, at Shambat, to be immune to pink boll worm (Pectinophora gossypiella), and G. armourknwn itself shows very marked boll worm-resistance.” British cotton investigators have worked extensively on Jassids (Emposca ficialis) and have found resistance to be associated v i t h plant pilodty, the more resistant types showing the greatest degree of “hairiness” on the under side of the leaf (Parnell et d.,1949; Dunham, 1936, 1939) found that, under dry conditions, hairy varieties retained significantly more calcium arsenate dust than smooth ones in experiments to test the insecticidal properties of the dust; on undusted cotton, the aphid popuIation increased in direct proportion to the number of hairs on the lower leaf surfaces. Slight resistance to nematodes (root-knot) has been reported by Smith (1941). The nutrition of the insect as well as the nutrition of the cotton plant may, separately or together, have a bearing on problems of insect resistance and in this connection Raney (1948) concludes that “consideration of the changes in various constituents of the developing boll does in fact throw considerable light on the changes in susceptibility to pests and diseases occurring during the course of boll development.” Probably no more remunerative use can be made of the extensive collections of wild and primitive cottons now available to breeders than

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to subject them and certain of their hybrids to critical study for resistance to major cotton insect pests. The same material should prove useful as a source of new or unfamiliar genes for drought resistance, cold tolerance, high oil and low gossypol content of the seeds, and many other economic characters. 4. Adaptution t o Mechanical Harvesting

I n cotton production, as in other industries, “mechanization” describes the process of utilizing machines for work formerly performed by men and animals. The practical approach to the modern idea of “ complete mechanization ” of cotton production requires careful planing, execution, and coordination of all of the interrelated phases and operations of production from the preparation of the seed bed to the ginning of the harvest crop. Beginning with the cotton gin, which was invented toward the latter part of the eighteenth century and which mechanized the removal of the lint from the seed, suitable machines were developed for one production phase after another until now only the weeding and harvesting operations utilize any considerable amount of hand labor, and both of these operations are yielding to the machine. True enough, the machines for these operations may not be as eficient as a hand laborer in all phases of an operation, but under present conditions of scarcity of manpower for such seasonal tasks and high wages or piecework rates required to attract workers to employment of this sort, there seems little doubt that the strong trend toward full mechanization will continne. Present models will be vastly improved and, perhaps, machines employing entirely new principles will be invented. The harvesting operation is one of the most expensive cotton production operations currently performed by hand, and the recent development of machines which will perform this operation represents the greatest technological advance in the mechanization of cotton production since the invention of the cotton gin. The trend in the development of cotton harvesters has been to design machines to dmulate the operations as they are performed by hand. There are two principal methods of hand harvesting: (1) picking, or removal of the cotton and seed (seed cotton) from the mature (open) boll, a procedure in use in most of the American Cotton Belt, including the western irrigated regions, and (2) snapping (pulling) or the removal of the whole boll, including the seed aotton and dried carpel, a method practiced in the high and rolling plains area of Texas and in southwestern Oklahoma. Consequently, cotton harvesting machines are of two main types : (1) picker and (2) stripper. Machines of the picker type employ revolving spindles to remove the seed cotton from the boll and, as is the case with hand

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picking, two pickings a season usually are made, the first when one half to two thirds of the bolls on the plants are open, and the final picking (scrapping) when the remaining bolls are all open. Removal of the green leaves from the plants by application of certain chemicals facilitates the operation of the mechanical picker in the field. The stripper machine is designed to remove both the dried carpels (b u rr) and the seed cotton from the plant. Since the stripping also removes all green or unopened bolls, only one stripping per season can be made and, naturally, this operation is performed when all or most of the bolls are mature. The choice of the variety is a very important consideration in planning a mechanized production program. Fortunately, several of the modern, rapid fruiting, early maturing, American Tipland varieties are fairly well adapted to spindle type picking, particularly when grown under planned systems of spacing and culture. Future work in this field is being directed along two lines: (1) the achievement of more efficiency in mechanical picking, i.e., obtaining a higher percentage of the cotton harvested as compared to the total available open cotton, and ( 2 ) obtaining raw, lint cotton which is equal or superior in grade and other lint qualities to that of hand-picked cotton, i.e., freedom from leaf, stem, and bract trash and other foreign matter and with a minimum of discoloration and staining due to exposure to weather. Progress in both lines of investigation requires cooperative work between plant breeders and agricultural engineers. Varietal differences in the spindle type picking efficiency of I Jpland cottons have been observed and, as mentioned above, it has been possible to control partially the growth habits of certain varieties and thus adapt them to more efficient harvesting. The present “type ideal” for spindle picking seems to be a plant (1)that will grow in a more or less upright position but at the same time be early in fruiting habit and fairly determinate in growth habit, ( 2 ) that will set its fruit in a n evenly spaced manner all over the plant, but beginning well off the ground, ( 3 ) that will have bolls which will allow the cotton to fluff and a t the same time cause it to stick in the burr strongly enough for good storm resistance, ( 4 ) that will mature its fruit early and in a very short space of time, and ( 5 ) that will shed its leaves readily when the major portion of the bolls have matured. Even a casual consideration of such a n “ideal” plant will reveal several physiological and morphological antagonisms, the idea of obtaining early fruiting and a t the same time a set of bolls well off the ground, to mention one. The “ideal ” plant type for stripper machine harvesting is considerably different, Here, a dwarf or semi-dwarf plant with short to medium

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fruiting branches is desired. Early fruiting and maturity is desired and here, too, the bolls should be borne well off the ground to allow the “snout” of the machine to slip under and engage the bolls without picking up dirt and extraneous plant materials. The seed cotton must be closely held in the boll at maturity, as all or most of the bolls on the plant must be mature before the stripper enters the field; but the seed cotton need not fluff. I n fact, the more compactly held seed cotton is less likely to be lost due to weathering in the field, and less trash and extraneous matter will be intermixed with the unginned cotton during stripping and ginning processes. Reference has been made elsewhere in this paper to a mutant boll character in which the seed cotton is closely held in bolls which open only partially. According to Lynn (1949), this stormproof boll type is inherited aa a simple, incompletely dominant character. Observations on the behavior of the character in crosses with normal (open-bolled) types suggest a complex of modifiers that operate in connection with the main gene to cause varying expressions of the characters. Uniform lines have been extracted which range from the extreme mutant expression to types indistinguishable from the Fl. The stormproof boll character has been established in high yielding strains adapted to stripper type harvesting by D. L. Jones and co-workers a t the Texas Agricultural Experiment Station’s substation st Lubbock, Texas, and two named varieties are ready for release. Regardless of the type of mechanical harvester employed, any new character, or refinement of existing characters which would result in less foreign matter in the harvested cotton and cleaner samples of ginned lint, would be a valuable contribution to the problem of mechanical harvesting, Varieties with many large, spiny plant and leaf hairs give lower grades of ginned lint from machine-picked cottdn than those with fewer and shorter hairs. A variety, designated as Delta Smooth Leaf, which is almost free of leaf and stem hairs, has been developed by workers a t the Delta Branch Station in Mississippi. The variety gives significantly higher grades of machine-picked cotton, and work is under way a t several locations to improve its yield and other agronomic properties or to transfer the character to other varieties. The large-toothed bracts (bracteoles) , characteristic of Upland cotton, are known to contribute materially to sample trash. Attempts are being made through breeding to reduce the size of the bracts or to remove them altogether, Two sources of breeding material are worthy of mention: (1) the small, almost *entirebracts of the ma& galante group of Upland cottons and (2) a deciduous bract type, first studied at the Delta Experiment Station of Mississippi, in which the bracteoles fall from the boll a t maturity.

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Granted that much can be done by the plant breeder to adapt the cotton plant t o machine methods of production, it still should be remembered that cotton is biological material in which the characters of economic importance in the present agricultural varieties are in equilibrium or balance as a result of many years of selection and testing. Minor changes may be made without greatly disturbing this balance, but when drastic changes are attempted, the equilibrium is disrupted and a new balance, if attained at all, will require many years of careful breeding. A careful economic analysis is necessary to determine how much yield can be sacrificed for a given special character, but it is safe to say that the sacrifice cannot be great. No responsible plant breeder will promise to produce m y combination of characters desired by the farmers or implement manufacturers. Mr. E. C. Ewing, Chief Plant Breeder for the Delta and Pine Land Company of Mississippi, put it so nicely in his address at a Spinner-Breeder Conference in 1946 when he said: “Most of the literature and of the talks that have appeared, dealing with machine production, show that their authors are intrigued by the idea that the cotton plant can and must be modified to make it more adaptable to mechanical harvesting. They are thereby complimenting the breeder unduly because his powers in this direction are limited. The hope for better mechanical harvesting, as in the past, depends mainly on the engineers and inventors, who must adapt the machine to the plant.”

VI. REFERENCES Balasubrahmanyan, R., and Narayanan, N. G., 1948, Hybrid cotton. Indian Cotton Growing lieu. 2, 125-129. Barker, H. D., and Berkley, E. E., 1946, Fiber and spinning properties of cotton, with special reference to varietal and environmental effects. U. S. Dep. Agric. Tech. Bull. 931. Barker, H . D., and Pope, 0. A,, 1948, Fiber and spinning properties of cotton: a correlation study of the effect of variety and environment. U. S . Dep. Agric. Tech. Bull. 970. Beasley, J. O., 1940, The origin of American tetraploid Gossypium apecies. Amer. Nat. 74, 285-286. 1940. 1942, Meiotic chromosome behavior in species, species hybrids, and induced polyploids of Gossypium. Genetics 27, 25-54. Berkley, E. E., Woodyard, 0. C., Barker, H. D., Kerr, Thomas, and King, C. J., 1948, Structure, determined by x-ray and strength of cotton fiber. U. 8. D e p . Agria. Teoh. Bull. 949. Blank, L. M., 1949, Breeding for resistance to bacterial blight of cotton. Plrytopathology 39(6), 494-495. Brown, M. S., 1951, The occurrence of spontaneous amphiploidy in species hybrids of cotton. EvoEution 6 ( l ) ,25-41.

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Comstock, R. E., and Manning, H. L., 1950, Idanuseript. Cook, 0.F., 1932, Cotton improvement through type selection, with special reference to the Acala variety. U. S. Dep. Agric. Tech. Butt. 302 Dunham, E. W., 1936, Pilosity of the cotton plant. J . Econ. Ent. 29(6), 1085-1087. 1939, Cotton aphid in relation to pilosity of cotton leaves. J . Econ. Enl. 31(6), 663-666. Ewing, E. C., 1948, The story of cotton varieties. Proc. 6th Ann. Spinner-Breeder Conf. Goldsmith, G. W., and Moore, E. J., 1941, Field tests of the resistance of cotton to Phymatotrichum omnivorzlm. Phytopathology 31, 452-463. Grimes, M. A., 1946, Polarized light preferred for maturity tests. Text. World 96(2), 161-163,214, 216. Harland, 8. C., 1932, The genetics of Gossyphm. B i b h g e . ae?wtioa 9, 107-182. 1936, The genetical conception of the spedes. BioZ. Rev. 11, 83-112. 1940, New polyploids in cotton by the use of colchicine. Trop. Agrwzclture, Trinidad 17, 53-54. 1943, The selection experiment with Tanguis cotton. Boo. Nac. Agr., Imtitrte of Cotton Gen., Lima, Peru Bull. 1. 1949, Methods and results of selection experiments with Peruvian Tanguis cotton. Part I. A survey of present methods of cotton breeding and a deecription of the “mass-pedigreeJJ system. Emp. Cott. Gr. Rev. 26, 163-174. Hertel, K. L., 1940, A method of fiber-length analysis. Text. Research 10(12), 510520. Hutchinson, J. B., and Panse, V. G., 1937, Studies in plaht breeding technique. Indian J . agrio. Soi. 7 , 531-564. Hutchinson, J. B., 1939, Some problems in applied genetics. Chrowica Botonioa 6, 403-404. 1940, The application of genetics to plant breeding. I. The genetic interpretation of plant breeding problems. J . Genet. 40, 271-282. Hutchineon, J. B., and Manning, H. L., 1943, The eBCiency of progeny row breeding in cotton improvement. Y e m . Cott. Br. Sta., Trintdad A20. Empire Cotton Growing Corp., London. Hutchinson, J. B., Silow, R. A,, and Stephens, S. G., 1947, The Evolution of Gossypium and the Differentiation of the Cultivated Cottons. Oxford University Press, London. Jenkins, W. H., Hall, E. E., and Ware, J. O., 1939, Cooperative breeding, genetic and varietal studies of cotton. b2nd Ann. Rep. Sowth Carolina Ag&. Exp. Sta. Jenkins, W. H., Harrell, D. C., Bailey, R. S., and Hall, E. E., 1946, Cotton improvement and breeding. 63th A m . Rep. South Carolina Agrw. Ewp. Sta. Kearney, T. H., 1914, Mutation i n Egyptian cotton. J . Agrb. Research 2(4), 287302. 1922, The uniformity of Pima cotton. 27. S. Dep. Agric. Ciro. 247. Kearney, T. H., and Peebles, R. H., 1940, SXP cotton in comparison with Pima. U. 8. Dep. Agric. Circ. 660. Kime, P. H., and Tilley, R. H., 1947, Hybrid vigor i n cotton. J . Amer. Boa. Agron. 39(4), 308-317. Knight, R. L., 1945, The theory and application of the backcross technique in eotton breeding. J . Genet. 47, 76-86. 1946a, Breeding cotton resistant to blackarm disease. I m p . J . Agric. 14, 163174.

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194611, Genetics and Breeding (Shambat). Prog. Repts. from Exp. Sta. Empire Cotton Growing Corp. Page 55. Lynn, H. D., 1949, The inheritance of “stormproof” boll type in Gossypiuln hirsutum. Texas A. and M. College Library. Mason, T. G., 1938, A note on the technique of cotton breeding. Emp. Cott. Growing Rev. 16, 113. McNamara, H. C., and Stutts, R. T., 1935, A device for separating different lengths of fibers from seed cotton. U. S. Dep. Agric. Circ. 360. Parnell, H. E., King, H. E., and Ruston, D. F., 1949, Jassid resistance and hairness of the cotton plant. Research Memoir 7 . Empire Cotton Growing Corp., London. Pfeiffenberger, G. W., 1946, Determining fibei fineness by means of the air permeameter. Text. Resrarch J . 16(7), 338-343. Pressley, E. H., 1933, A new type of cotton sorter. J. Amer. SOC.Agron. 26(2), 89-98. 1942, A cotton fiber strength tester. Am. 8. T . M . Bull. 118. Presley, J. T., 1950, Personal communication. Raney, R. C., 1948, Observations on the development of the cotton boll, with special reference to changes in susceptibility to pests and diseases. Research Memoir 6. Empire Cotton Growing Corp., Londoii. Richey, IF. D., 1927, The convergent improvement of selfed lines of corn. Amer. Nat. 61, 430-449. Richmond, ’ T. R., 1947, Special cottons for specific end uses through research. Rayon. Text. Monthly 28(7), 373-375. 1949a, Methods of cotton breeding. Proc. 46th Ann. Conv. Ass. Southern Agric. Workers. 1949b, The genetics of certain factors responsible for lint quantity in American Upland cotton. Texas Agric. Exp. Sta. Bull. 716. Sherbakoff, C. D., 1949, Breeding f o r resistance to Fusarium and Verticillium wilts. Bot. Rev. 16(6), 387-393. Simpson, D. M., 1948, Hybrid vigor from natural crossing f o r improving cotton production. J . Amer. SOC.Agron. 40(11), 970-979. 1949, Further evidence of larger yields from naturally crossed seed. Proc. 46th Ann. Coav. Southern Agric. Workers. Simpson, D. M., and Weindling, R., 1946, Bacterial blight resistance in a strain of Stoneville cotton. J. Amer. SOC.Agron. 38(7), 630-635. Skovsted, A., 1937, Cytological studies in cotton IV. Chromosome conjugation in interspecific hybrids. J. Genet. 34, 97-134. Smith, A. L., 1941, The reaction of cotton varieties to Fusarium wilt an d root-knot nematode. Phytopathology 31 (12), 1099-1107. Smith, W.S., 1947, Air gauge measures fiber fineness. Text. Industry. 3(11), 86-88. Rprague, 0.F., 1946, The experimental basis for hybrid maize. Biol. Rev. 21(3), 101-120. Stephens, S. G., 1944, The application of genetics to plant breeding. Trop. Agriculture, Trin. 21, 126-129. Stephens, S. G., and Armstrong, G. M., 1949, Personal communication. 1950, The internal mechanism of speciation in Gossypium. Bot. Rev. 16(3), 115-149. Stroman, G. N., 1948, Improved strains o f cotton for New Mexico. N . Mex. Agric. Exp. Sto. Bull. 337.

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Sullivan, R. R., and Hertel, K. L., 1940. Surface per gram of cotton fibres as a measure of fibre fineness. Test. Research 11(1), 30-38. Vavilov, N. I., 1927, Botanical-geographical principles of selection. Bull. a p p l . Bot, Plant Breeding 16(2), 420-428. Waddle, B. A., 1950, A study of genetic variation found in the segregating populations following inter-varietal u o s m in American Upland cotton. Texas A. and M. College Library. Waddle, B. A., and Richmond, T. R., 1950a, Measurement of genetic variation in certain varieties of American Upland cotton and their hybrids. Manuscript.

Possible SigniAcance of Duplication in Evolution S. 0.STEPHENS Department of Agronomy, North Carolina State College, Raleigh, North Carolha

CONTENTS

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I. Introduction 11. The Problem 1. Suitable Material 2. Criteria f o r Distinguishing Duplicate from Nonhomologous Loci a. Duplicates Carried in Different Chromosomes b. Nonhomologous Loci in Different Chromosomes c. Duplicate Linked Loci (Tandem Repeats) d. Linked Nonhomologous Loci 111. Some Possible Cases of Divergence in Function of Duplicate Loci 1. “Duplicates” Carried on Different Chromosomes 2. “Duplicate” Linked Loci a. Tandem Repeats b. Pseudo-alleles c. Nonquantitative “Allelic” Series IV. Conclusions V. References

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I. INTRODUCTION The evolutionary process depends initially on a mechanism capable of providing a pool of heritable variations which constitutes the rpw materials upon which the forces of selection can operate. Such a mechanism must be universal in living organisms and active enough in each organism t o provide a degree of heritable variability if evolution of that organism is to continue. Only one such universal mechanism has as yet been demonstrated satisfactorily, namely, gene mutation and its accompanying phenomena of segregation and recombination. Nevertheless the assumption made by the majority of geneticists that most heritable variations are ultimately dependent on gene mutations can still only be tested critically a t the intraspecific level. Above this level-in the differentiation of species, genera, and higher categories-the mutation theory remains for the present a matter of rational inference and extrapolation, and not a matter which is easily subject to experimental proof. I n spite of this weakness most geneticists probably prefer to retain a theory which is a t least partially verifiable, rather than to discard 247

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it in favor of alternatives which are more highly speculative and less generally satisfying. A minority of geneticists, however, is of the opinion that variations resulting from mutation are not adequate to account for differentiation of major evolutionary significance and look for alternative mechanisms to explain the origin of species, genera, etc. Thus Lamprecht (1948) draws a distinction between intraspecific and interspecific genes without attempting to explain the origin of the latter. Goldschmidt (1940, 1946), taking a more extreme view, questions the very existeiicv of genes and would interpret, variations of evolutionary significanct>i r i terms of rather abriipt changes in chromosomal patterns. Since, however, the existence of a pattern presupposes a qualitative differentiation of its components, this interpretation still leaves unanswered the probleni of how these components were differentiated. Furthermore the rapid advances in the theory of gene structure during the last ten years have reduced the sharp distinction which appeared to exist between the orthodox concept of gene mutation and the changes in chromosomal pattern envisaged by Goldschmidt. I n sonie degree what were apparently radical divergences of opinion now appear to be matters of quantitative distinctions and differences in terminology. During the last ten years considerable progress has been made in the analysis of gene action in relation to enzymatic control and in the interpretation of gene structure in terms of “chromatin chemistry. ” Though these studies are still in their infancy it has already proved feasible to construct theoretical models (Pauling and Delbruck, 1940 ; lhiersoii, 1945) which satisfactorily fit the essential facts as a t present understood. Excellent reviews of what may be termed the modern gene concept have been presented by Cfulick (1944) and Beadle (1945). The bare essentials of this concept would seem to be that the gene is a nucleoprotein in nature and that its dual role as a self-duplicating entity and ultimate controller of highly specific chemical reactions is dependent on a highly complex but specific surface structure. Furthermore i t still seems likely that each gene controls only one primary process, though Beadle’s original “one gene : one enzyme” theory may be an oversimplification (Wagner, 1949). This concept, if correct, exerts an important limitation on the efficacy of the mutation theory. As long as the gene was considered as an abstract unit of inheritance, the possibilities of mutation were limited only by the imagination of the theoretical geneticist. B u t if the gene owes its properties to a specific surface structure it follows that a mutation implies a loss or deformation of that structure and a consequent loss or impairment of the original function-with or without the concomitant acquisition of a new function. From the evolutionary point

POSSIBLE SIQNIFICANCE OF DUPLICATION IN EVOLUTION

249

of view this would mean that mutation per se could not provide an unlimited source of variation; at best it could only replace a finite number of functions by an equal number of new ones and a t worst it could result in a net loss in the number of functions. From a priori reasoning it is difficult to regard such a mechanism (in which a new function could be attained only at the price of discarding an old one) as a n efficient method of effecting evolutionary progress from the simple to the complex. One might expect (still on a priori grounds) that a mechanism in which new functions could be added and the old ones retained would have considerable selective advantage. Within the bounds of the theory, the only likely manner of achieving this “improvement” would be by increasing the number of genetic loci, either by the synthesis of new loci from nongenic material or else by tke duplication and subsequent differentiation of existing loci. Theoretically this would make possible the retention of existing functions by genes a t one locus leaving the other free to develop new functions. Further, since one locus could retain its original function, the other would initially be subject to a reduced selection pressure. Present knowledge is quite inadequate to determine whether it is possible for new genetic loci to arise de novo, or, in fact, to test its occurrence if the possibility existed, but the alternative mode of origin (duplication) is a well-established phenomenon. It is known to occur as a direct consequence of polyploidy in plants, and of unequal crossing over in Drosophila (Bridges, 1936). Indirectly it results from chromosomal recombinations following certain types of translocation and inversion (Dobzhansky and Dobzhansky, 1933; Muller, 1935; McClintock, 1941) and when the mechanism of its origin is unknown its occurrence can be inferred with some confidence from detailed studies of salivary chromosomes (Metz, 1937 ; Lewis, 1945). The possibility that duplications may provide an important source of new genetic material has often been considered (see Haldane, 1932; Muller, 1935 ; Serebrowsky, 1938 ; Gulick, 1944 ; Beadle, 1945 ; Sturtevant, 1948; White, 1948) but as far as the writer is aware only Serebrowsky has attempted to formulate a hypothesis of the possible steps involved, based on his studies of the scute-achaete complex in Drosophila. Moreover his interpretation only considers the distribution of functions between the duplicate loci--a type of specialization which does not include the acquisition of new functions. The present situation is, therefore, that duplication is the only known mechanism which migllt account for the multiplication of genetic functions, but no serious attempt has been made to consider the expected consequences in detail, nor to devise methods of demonstrating them in suitable material.

250

S.

G. STBlPHENS

11. THE PROBLEM 1. Xuitable Material

It is clear that if duplication were a source of new genetic functions we should expect to find cases of genes and their mutants a t two or more loci which were neither strict duplicates nor clearly heterologous, but which represented a transitional situation. The most likely place to look for the supposed transitional types would be in amphidiploid plant species, particularly those species which were well analyzed genetically and which had living diploid relatives. It is a reasonable assumption that in newly formed amphidiploids numerous loci must be duplicated and that any subsequent disappearance of duplicate functions must be attributed to (a) loss or inactivation of one of the loci or ( b ) divergence in function. If genes could be transferred from the diploid relatives to the amphidiploid a comparison should be possible between the genetic constitution of four originally homologous chromosome sets. Further consideration reveals the unfortunate situation that few plant genera fulfil the requirements. Species which are well mapped genetically (e.g., Zea and Pbzcm) are not amphidiploids, and conversely those species whose amphidiploid origin has been well established cytologically are often comparatively unknown genetically. In practice the most suitable material i s to be found in cultivated species of crop plants (e.g., Triticum, Nhtiana, Qossypium) where both amphidiploid and diploid species are available, and where a beginning has been made in comparative genetic analysis. A second potential source of information is provided by Drosophila where duplicated regions can be identified by examination of salivary chromosomes and where comparison of the action of genes in the duplicated regions can sometimes be made. Finally there are certain miscellaneous cases in which evidence of duplication is not critical but whose anomalous behavior is diPBcult to interpret on any other hypothesis. In this rather mixed category fall (a) multiple allelic series whose members show unexpected interactions in combination, ( b) pseudoalleles (only distinguishable from ( a ) by the fact that the units are separable by crossing over), and ( c ) complementary genes whose interactions are difficult to interpret on any orthodox model. 2. Criteria for Distirbgubhhg Dwplicate and Nolzhamcrlogous Loci Before commencing a search for evidence of divergence of duplicates it is necessary to have a clear idea of the criteria which should

POSSIBLE SIQNIFICANCE OF DUPLICATION IN EVOLUTION

25 1

distinguish alleles and duplicates on the one hand from unrelated (nonhomologous) loci on the other. These may now be considered briefly. a. Duplicates Carried in Differewt Chromosomes. The vast majority of mutants which have been described are completely or partially recessive to the wild type, i.e., amorphic or hypomorphic in Muller’s (1932) terminology. This is to be expected if each locus is concerned with one specific primary function, and alleles only differ in the efficiency with which the function can be carried out. As a result of selection, the “wild type” allele will almost always be the most efficient. The results of duplicating such a locus will depend on whether its mutants are completely recessive or only partially so. I n the first case segregation following mutation will yield the typical 15 :1 Mendelian ratio in F2 and in the second case a graded quantitative series will be expected. But no matter what numerical variations are possible, the resulting phenotypes should only differ from one another quantitatively, since they are the results of different dosages of essentially the same type of gene action. b. Nonhomologozls Loci in Different Chromosomes. Here the type of interaction obtained will depend on whether they control ( i) qualitatively different but serial steps in a common chain of synthesis or (ii) links in independent synthetic processes. i. This case provides a model for the action of complementary genes as shown below.

-s

A

PI P ,

B

Two genes, A and B, a t independent loci control two essential steps in converting substrate (S) to product (P2). Mutations from A to a or B to b will block the production of P2,and when A alone mutates, B will be unable to operate. This situation implies that the genotypes, aabb and aaBB will be phenotypically indistinguishable, and that the corresponding mendelian ratio in F2 will be 9 :3 :4. If A alone produces no striking phenotypic effect, this ratio will often be scored superficially as 9:7. ii. I n this case the primary processes should operate quite independently and any deviations from the expected 9:3:3:1 ratio in F2 should be attributable to secondary interactions. Often such interactions are obvious; for example coat color in the adult mouse cannot be expressed in the presence of the hairless mutation, though the color-producing mechanism is intact and can be seen in the young animal before the hairless gene becomes effective (Gruneberg, 1943). c. Duplicate Linked Loci ( T a d e r n Repeats). In the absence of

252

S. G. STEPHENS

position efiect or of special techniques for determining crossing over, recombinations of mutants a t duplicate linked loci would be indistinguishable from single locus mutations. For example, types (A1 az) and (al Az) would be identical phenotypically and of the two rare recombinants expected, ( A1 A z ) would be regarded as a hypermorphic mutant, and (al h) as a simple amorphic or hypomorphic mutant. d. Linked N m h m w l o g m Loci. These would be expected to exhibit a syndrome of pleiotropic effects for which no common physiological basis was apparent. Dominance relationships a t one locus should be independent of dominance relationships a t the other, and it would usually not be possible to arrange the “mutants” in a single quantitative series. Without special techniques for determining crossing over, recombinations would be regarded as “mutations in different directions.” I n the special case where the two loci controlled serial processes in the same synthetic chain, recombination of “alleles” should produce complementary effects. It will be evident from the foregoing considerations that genetic analysis confined to the scoring of phenotypic effects would be quite inadequate to recognise transitional cases between duplicate and independent loci. Some knowledge of the nature of the processes involved and/or independent cytological evidence would be essential. At present, even in the most promising material, analysis has in no case proceeded far enough to provide critical data. However, a rather restricted number of cases are known which suggest that the problem is not insoluble and offer hope that a more extensive search will be profitable.

111. SOMEPOSSIBLE CASES OF DIVERQENCE IN FUNCTION OF DUPLICATE LOCI

1. “Duplicates” Carried .In Diferent Chromosomes One of the most interesting cases in this category is provided by the R-CZ linkage groups in Qossy+m, the main features of which were first described by Silow (1946). The American cultivated (New World) cottons are amphidiploids comprising a set of 13 pairs of A chromosomes and a set of 13 pairs of D chromosomes. Diploid relatives (A and D types) are also known. Anthocyanin pigmentation is controlled by a single R locus in both diploid types-in the amphidiploids two R loci occur. By appropriate genetic analysis Harland (1935) and I-Iarland and Atteck (1941) were able to show that one of the R loci in the amphidiploids was homologous with the R locus carried by A type diploids (R2)and the other homologous with the R locus in D type diploids (R1),Another

POSSIBLE SIGNIFICANCE OF DUPLICATION I N EVOLUTION

253

pair of “duplicate” loci (controlling the normal development of the flowering branches) occurs in the amphidiploids. I n the recessive mutant types, cZl (cluster) and cZ2 (short branch), the flowering branch is condensed and tends to be fasciated. It had been known for some time that, the R1 and Cll loci were linked, and Silow (1946) was able to show that R2 and C l , were also linked, Recently it has been found (TIutchinson, personal communication) that an R2-CZ2 linkage group is also present i n A type diploids. So f a r no CZ1 locus has been demonstrated in D type diploids, though it is an interesting fact that one of the diploid species in this group ( G . am‘dunz) has a greatly reduced flowering branch (Hutchinson et al., 1947). The foregoing data show quite clearly that the R-Cl linkage group is a n ancestral arrangement that has been retained in certain diploid species and become duplicated as a consequence of amphidiploidy in the American cultivated types. One might expect to find duplicate factor inheritance of both R and C1 in the amphidiploids. I n the case of the R1 and Rz loci, however, although these loci must originally have been duplicates, and although they are both concerned with the development of the same (i.e., chromatographically indistinguishable) anthocyanin pigment, yet no allele at the R1 locus has a strict counterpart a t the R2 locus (Silow, 1946) and segregation at the two loci is quite independent, giving 9 : 3 : 3 : 1 ratios in Fa. I n the case of the CZ1 and C12 loci, although the recessive mutants, cluster and short branch, are similar phenotypically and must originally have been duplicates, they now behave as complementaries restoring the normal flowering habit in coinbination. The normal allele at one locus cannot mask the mutant allele a t the other locus in either case, i.e., CZ1 CZ1 c b ch is the genotype of short branch, and CZI cZl Cl, CZ2 of cluster. (The double recessive has not yet been described.) It is difficult to explain the inability of either normal allele to mask a recessive mutant a t its duplicate locus, except on the assiitnption that the loci have diverged i i i fuaction. However, the apparent divergence may be a question of increased specificity, sincc Rhyne (unpublished) working in this laboratory finds that the hybrid, cll Ch/C12 Ck, obtained by crossing cluster with a n autotetraploid A type is phenotypically normal. It seems likely that the CZ2 normal allele carried by the A type is able to mask the mutant allele, cZl, whereas the homologous normal allele in the amphidiploid can not. At present, though, the possibility that this apparent difference in masking ability is a question of C12 dosage cannot be excluded. Another interesting case in Gossypium which does not appear to he explicable on any orthodox interpretation is provided by the corolla color loci in A type diploids. Silow (1941) showed as a result of

254

8. 0. STEPHENS

extensive analyses that full yellow color of the corolla required the simultaneous presence of three genes a t different loci, Ya Yb Y,. When any one of these Y genes was replaced by its corresponding recessive, yp,, YPb, or yp,, the corolla was pale yellow in color, Crossing any two different pale yellow types restored the full yellow color of the corolla. At first sight this would appear to be an orthodox example of complementary gene action, the appropriate model for which has been presented earlier in this paper. We should expect that Y,, Yb, and Y , control different steps in a common chain of synthesis leading to the production of the full yellow color, e.g.,

S (pale)

-Ya

PI

y b

Pz

yo ~

+Pa (full yellow)

If a block occurred in the first step ( Y , replaced by y p , in the above model) then any further block a t Yb or Y , would be without effect and y p , Yb Y,, y p , ypb Y , and y p G ypb y p , should have the same phenotype, pale yellow. This does not agree with Silow’s finding that the pale yellow types can be distinguished from one another and that as a result of selfing complementary full yellow types, mew-white types segregated Y c / y / m Yb Y ogave 9 full yellow ( Y , Y b Y , ) : out. Thus selfing Y , 3 pale yellow ( y p , Yb Y o ); 3 pale yellow ( Y O ’ypb Y , ) : 1 near-white ( y p , ypb Y , ) . His data are more in accordance with the segregation of independent loci than of complementary loci. However, three independent loci would imply that three independent chains of synthesis are involved in the production of yellow pigment, an implication which receives no support from examination of the pigments in detail. It is known that the yellow pigments in cotton flowers are flavonols, and that full yellow types contain a mixture of glycosides of three flavonols, gossypetin, quercetin, and herbacetin (Neelakantam and Seshadri, 1937). Quercetin and herbacetin are isomers differing only in the position of one hydroxyl group. The former has a hydroxyl substituted in the flavonol nucleus a t position 3’ while the latter has a hydroxyl a t position 8. Oossypetin has a hydroxyl substituted in both positions. Chromatographic analysis in progress in this laboratory suggests that gossypetin is present in all full yellow types and lacking in pale yellows and white. The relevant data which are available a t the present time are summarized in Table 1. While it will not be possible to interpret fully the interactions of genes a t the three loci until all their possible combinations are analyzed, it is already apparent that they do not fit into any simple complementary scheme. Neither can the actions of the three loci be considered independent since they apparently only affect the relative proportions of the three pigments and the end product is

POSSIBLE SIQNIFICANCE OF DUPLICATION IN EVOLUTION

255

the same. The close similarity in fact suggests that the loci may originally have been duplicates. It may be possible t o obtain independent TABLE 1 Pigme&

Amociatrd with Vnrioiis Coml~inations of Y Q ~ n e sin Crossypiwwi P h m o t y p a of corolla Pigments present in corolla Full yellow Qlycosides of gossypetin, quercetin, and herbacetin Glycosides of herbacetin and Pale yellow quercetin Pale yellow, nearGlycosides of quercetin white at edge Pale yellow Glycosides of quercet,in Full yellow Glycosides of gossypetin, quercetin, and herbacetin Not analyzed Full yellow Not analyzed Near white Not analyzed Near white

N.B. Qlycosidal constitution of the pigments not known. present, but if so they occur in minute quantities.

Other flavonol pigments may be

supporting evidence for this suggestion since diploid A types are suspected to be either secondary polyploids (Skovsted, 1933) or to include several segmental duplications (Beasley, 1942). Furthermore, four independent brown lint loci have been described (Silow, 1944, 1945) one of which is linked with the corolla color locus, Y,. It is clear that the establishment of similar linkage groups involving the other Y loci would provide strong supporting evidence for the duplication hypothesis. It is difficult to understand why the anomalous cases in Gossypium which have been described above seem to have no counterparts in other amphidiploid genera. Thus the duplicate loci found by Clausen and Cameron (1944, 1950) in Nicotiana tabmum are apparently of the usual type giving 15 : 1 ratios in Fz. I n Triticum, duplicate and triplicate loci are found in the hexaploid species (see Sears, 1948, for recent review) but here also the duplication seems to be orthodox except that incomplete dominance may lead to the production of cumulative series as in the seed color loci. However, one interesting case may repay further investigation. This involves linkages between glume color and glume pubescence. Three genes, black, red-brown and yellow-brown, a t independent loci determine glume color. Black is completely linked with a gene for glume pubescence, while red-brown is linked with a second pubescence locus. “ I n certain crosses the factor f o r black color (and associated pubescence) appears to be linked with the recessive alleles

256

S. Q. STEPHENS

of red-brown and the second pubescence factor, but in other crosses no association with them is indicated” (Sears, 1948). This suggests the existence of residual homologies between the two-color pubescence associations and a n attempt to clarify the chemical relation between the black and red-brown pigments might be well worth while. Voss (1938) has suggested that differences in the reactivity of the glumes to the phenol test may depend on differences in tyrosinase activity, and tyrosine is known to be involved in the synthesis of melanotic pigments (Beadle, 1945).

2. “Duplicate” Linked Loci a. Tandem Repeats. The best authenticated examples of this situation are provided by the cases of Bar (Bridges, 1936; Sturtevant, 1925) ; scnte/achaete (Serebrowsky, 1938) ; Star/asteroid (Lewis, 1945) ; and the lozenge series (Green and Green, 1949) in Drosophila rne2anogastw. The duplicate nature of Bar and Star/asteroid was established visually by their association with repeats in the salivary chromosomes and experimentally by the demonstration of crossing over within the complex. That scute/achaete was duplex was established visually by breakage between the two loci following inversion, and subsequent pairing between the separated loci in the salivary chromosomes. Their duplicate origin was confirmed by the fact that scute alleles and achaete alleles have similar effects on the bristle patterns of the fly; their divergence following duplication was indicated by the fact that they affected different groups of bristles. The triplicate nature of lozenge rests on the demonstration of crossing over within the complex in two different positions and of the occurrence of parallel series of mutants a t each locus, Since Bar results from unequal crossing over it represents a relatively unstable arrangement. Recombinations in Star/asteroid and lozenge involve equal crossovers, and Lewis (1945) has suggested that the former may consist of a tandem reverse repeat which would be expected to be more stable than a d k e c t repeat. More recently (1948) he has reported two other probable cases of tandem repeats in what were formerly considered to be single loci (bithorax/bithoraxoid and Stubble/stubbloid) These cases of duplication in Drosophila, which are particularly favorable for critical examination, are none the less very difficult to relate to probable cases of duplication in other material. This is due to the noncorrespondence of dosage with phenotypic expression which has been interpreted as a form of position effect. The difficulty will be evident from a comparison between the expected consequences of duplication (with and without differentiation of the ‘units) and the actual consequences observed in Drosophila (Table 2 ) . In this table the

.

POSSIBLE SIGNIFICANCE OF DUPLIOATION IN EVOLUTION

257

“+”

wild type allele is represented by and the recessive mutant by “m.” It can be seen (column 1) that if the loci are unchanged in the duplicate condition then any equally balanced arrangement of wild type and mutant alleles should produce a wild phenotype. If a change in function accompanies duplication (column 2 ) then wild type alleles should only cover mutant alleles in their respective loci. The actual situation (column 3) is consistent with neither interpretation since the wild phenotype is only produced when the wild type alleles are both present in the same chromosome. If this situation is the result of position effect, we might expect that separation of the two loci beyond the limits of their sensitive distance might sometimes result in a reversion to either one of the situations shown i n columns 1 and 2. Separations of this type have not yet proved possible or have not yet been studied from this point of view, but without such evidence there would seem to be no critical means of determining whether the loci are strict duplicates or have diverged in function. A different interpretation of these cases is presented by Ooldschmidt (1950) who prefers to regard the supposedly “repeated loci” as a single unit, possibly including more than one band in the salivary chromosome. It is suggested that any structural change within the limits of this unit will produce mutant effects of the same or similar type, On this interpretation zygotes of the type

-.m

+ and m erly written as -

m

+ + and + should be more

propm m m + While this would account for the different

phenotypic expressions of these zygotes, as found in the Drosophila cases cited, without the need of assuming a special position effect, it poses a further problem in the interpretation of apparently similar cases in other material. I n these cases, some of which are presented below,

+ + and

- are indistinguishable as regards m m m + their phenotypic expression. Clearly these cases would require a different interpretation from that offered by Goldschmidt as a general explanation. b. Pseudo-Alleles. In other material evidence for tandem repeats rests on the occurrence of pseudo-alleles (i.e., adjacent units governing similar processes but separable by crossing over). Since no independent cytological evidence is available as to the nature of their origin, it is d B c u l t to reject the alternative hypothesis that they represent closely linked but historically unrelated units which by chance happen to affect similar processes. The latter interpretation, however, becomes less likely with every new case which is discovered. Another difficulty occurs in zygotes of .the type

258

S.

a.

STEPHENS

TABLE 2

Thouretical Phenotypio Consequences of Tandem Repeats in Coiiipnrisoii with Those Actually Obtained in Drosophila Genotype

++ m m

+ m + m

+ m m +

Resulting phenotype (1) Strict duplication ( 2 ) Differentiation of loci Of zooi

( 3 ) Actual

Wild type

Wild type

Wild type

Wild typc

Mutant

Mutant

Wild type

Mutant

Mutant

Wild type

Wild type

Mutant

attempting to distiriguish rare crossovers from mutations a t mutable loci. As a consequence no single case of pseudo-allelism provides critical information as to the nature of its origin, though the pooled evidence from several cases amounts to strong circumstantial evidence that they represent duplicates which have diverged in function. The pale yellow/yellow-green series in corn represents the only case of pseudo-alleles in plants which has been established cytologically (McClintock, 1944). Here it is known that the pale yellow and yellowgreen regions occupy adjacent positions in the ninth chromosome. That the regions are qualitatively different is shown by the fact that in combination they produce the complementary normal green color. However, in the absence of chemical information on the nature of the steps which are blocked by the mutants it cannot be inferred that the two regions originated as a result of duplication. The normal development of chlorophyll in corn can be blocked in several qualitatively different ways (Demerec, 1925) so there is no a priori reason to suspect any close chemical relation between the steps which are blocked by the pale yellow and yellow-green mutants. The A (anthocyanin) complex in corn was formerly considered to be a single mutable locus involving a series of multiple alleles whose phenotypic effects could not be arranged in any simple quantitative sequence. Recently Laughnan (1949) using appropriate genetic markers has been able to show that the origin of certain mutants is associated with crossing over within the A “locus,” i.e., a t least two adjacent loci must be concerned with anthocyanin pigmentation. While it does not necessarily follow that all spontaneous mutants in the A complex are

POSSIBLE SIGNIFICANCE OF DUPLICATION IN EVOLUTION

259

the result of crossing over it is evident that the nonquantitative seriation of the mutants is a result of changes in a t least two qualitatively distinct but adjacent regions. Is the close association of these regions fortuitous or the result of divergence following duplication 7 The fact that both regions apparently control the relative proportions of brown and purple pigments in different parts of the plant would argue in favor of a duplicate origin. However, the chemical relation between the two elassee of pigments is not yet known and is apparently much more complex than was formerly supposed (Laughnan, 1950). A rather similar situation is found in the anthocyanin ( R 2 ) complex in Asiatic (i.e., A type) cottons. The numerous “alleles” a t this “locus” cannot be arranged in any simple quantitative series (Silow and Yu, 1942). The situation is clarified if one can assnine that the “lociis” is a compound of at least three adjacent loci (Yu and Chang, 1948). On this basis, two pseudo-alleles, G and S, neither capable individually of producing anthocyanin in the petal, give a complementary red spot in combination. Genes at the third locus ( N )are concerned with the production of anthocyanin on the petal margins and over the plant body in general. Variations in M seem to be independent of variations in G and S. Further, Yu and Chang were able to show that if the units were presumed to be arranged in one particular linear order, M-G-S, then the various “spontaneous mutations” which they encountered in their material could be interpreted as single crossovers within the complex. The difficulty of distinguishing between the results of single crossavers and single point mutations will be a t once apparent, but the latter interpretation is unlikely as it would require a minimum of six different types of mutation to account for the eight “spontaneous mutants” obtained in culture. Attempts have been made to correlate the actions of genes a t the M, G, and S loci with specific chemical processes (Stephens, 1948a, 1948b, and unpublished). Development of the red spot at the base of the petal was found to occur in two steps: (1) the replacement of anthoxanthin (flavonol) pigments by a leuco-substance, (2) the replacement of the leuco-substance by a n anthocyanin. The first step is controlled by G and the second step by X. Since only one anthocyanin pigment (a glycoside of cyanidin) has been found in cotton, and only one flavonol pigment (a glycoside of quercetin) is found universally in all colored flower types, further, since cyanidin and quercetin are structwal analogues, it was snggested that a two-step reduction was involved : Quercetin

Q

---t

Leuco-substance

B

+ Cyanidin

260

8. Q. STEPHENS

On this basis the steps controlled by cf and S would be similar but not identical, thus suggesting that the genes were duplicates which had diverged in function. More recent investigations do not support this hypothesis. In certain genetic types which have lately been synthesized the leuco-substance can be produced in the absence of a, and in such types ~!3 does not produce anthocyanin. This means that the leuco-substance can no longer be considered the immediate precursor of anthocyanin as postulated in the original hypothesis, and that as a consequence no similarity between the actions of G and S has been demonstrated (Stephens, unpublished). A third case of pseudo-allelism has been described in the mouse (Dunn and Caspari, 1945). Three members of the series, Brachg, Fused, and Kinky, are known to be Carrie6 at three neighboring loci as they are separable by crossing over. Two others ( t o and t l ) do not cross over themselves and reduce crossing over between Brachy, Fused, and Kinky and hence are regarded as sectional rearrangements. All the members effect in varying degree a complex syndrome of characters in which abnormal tail development is the most obvious external symptom, Comparative developmental studies of the mutants reveal the existence of intrinsic differences which are greater than would be expected from a study of the external features alone but the nature of the material is not suitable to decide if these differences could result from similar primary processes. In short, the existence of at least three neighboring loci with similar effect has been established, but the possibility of duplicate origin remains an open question. c. Nonqzuuntitative “Allelic” Series. In all the cases so far considered it may be reasonably concluded that two or more loci in neighboring positions are involved. Cases are also known in which the existence of more than one locus has never been demonstrated, but which nevertheless “mimic” the actions of pseudo-alleles in their phenotypic interactions. For example the R locus in corn is closely similar to the A complex in the same material. Both control anthocyanin development in different parts of the plant, both are rather highly mutable and the alternative units cannot be arranged in any simple quantitative series. Yet extensive investigations by Stadler (1946, 1948) have produced no evidence that mutation is associated with crossing over, nor, in fact, that anything more than a single locus is involved. On the other hand his finding that the same allele shows no correlation in rates of plant color and seed color mutation suggests some type of surface subdivision of the locus (i.e., if genic specificity is determined by surface structure). In Trifolium patense a complex of completely linked loci governs the development, position and color of the characteristic leaf markings,

POSSIBLE SIGNIPICANCE OF DUPLICATION IN EVOLUTION

261

and the association or nonassociation of anthocyanin with those markings (Williams, 1937). This situation is very similar to that in Ctossypium where presence or absence of petal spot, with or without the development of anthocyanin, is controlled by the same pseudo-allelic complex (Silow and Yu, 1942). It would be a remarkable coincidence if a chance association of independent loci had produced a similar complex in the two genera. A more likely explanation is that a basically similar mechanism is responsible for the development of the two complexes, a n d this is consistent with the hypothesis that both result from duplication, with subsequent divergence in function of the repeated regions. Tan (1946) has described an extremely interesting allelic series which controls fifteen alternative color patterns on the elytra of ladybeetles (Harmorcia spp.). The patterns consist of the development of black pigmented areas on a yellow background. Each allele is associated with the development of a specific pattern and operates quite independently of any other allele in the series (ie., in hybrids the pattern characteristic of one allele is superimposed on the pattern characteristic of the other allele). Tan recognized the analogy which exists between this series and the scute series in Drosophila, where each allele has its own characteristic bristle pattern, but also considered the possibility that the patterns might be interpreted physiologically in terms of “diffusion streams” as demonstrated by Goldschmidt (1938) in other material. The two approaches are, of course, not mutually exclusive. It is known that during development of the elytra, the yellow pigment develops first and that the black pigment spreads inward from the outer edges a t a later stage. Inspection of Tan’s illustrations suggests that the fifteen distinctive patterns can be grouped into three subclasses which may possibly depend on three different processes: (1) a spotting process, ( 2 ) diffusion inward from the anterior edge, ( 3 ) diffusion inward from the posterior edge. Quantitative differences in each of these processes and their various combinations would suffice as an explanation of the fifteen patterns, and the analogy with the scute/achaete pseudo-allelic system would be still more pronounced. Other evidence is available, however, which shows that arguments based 011 analogies alone are not sufficient to distinguish between allelic aiid pseudo-allelic series. The agouti series in mice (Gruneberg, 1943) consists of five members : Yellow, White-bellied agouti, Agouti, Black and tan, and Black. The various combinations of these alleles show normal dominance relationships, except in the case of the hybrid Black and tan (at) X Agouti ( A ) which is phenotypically indistinguishable from White-bellied Agouti (Aw). The complementary action of these types might suggest a pseudo-allelic basis but Little and Hummel (1947)

263

S. G . STEPHENS

obtained a n A” mutant from a hoinozygous recessive Black stock ( a a), a situation which is much more readily explained as the result of a single dominant mutation than a result of two simultaneous dominant mutations in neighboring loci. As long as nonquantitative allelic series of this type remain unexplained no critical comparison can be made with the gene-antigen-antibody systems which exist, for example in the human blood groups (Stern, 1949) and probably also in self-incompatibility mechanisms in plants (Lewis, 1949). In the case of the A-B-0 blood groups the alleles, Za and I h differ froiii Z0 in their capacity to produce a specific antigen, i.e., in genetic terms I” and I h are dominant over I O . B u t in combination I* and I b behave quite independently, each producing its own specific antigen. A similar independence is found in the M-N blood groups. There appears to be no question that the alleles are qualitatively different and independent in their actions ; the usual quantitative seriation which is typical of most allelic systems is lacking. This distinction is rendered more emphatic by the discovery (Fisher, 1947) that the alleles associated with the Rh blood groups are each concerned with the production of not one but three qualitatively different antigens. If the properties of a gene depend on a specific surface structure, it is difficult to see how it can control three different primary processes without some form of spat,ial subdivision of that surface. On the other hand, if not one locus but three linked loci are involved, as Fisher suggests, it is quite as difficult to account for the juxtaposition of those loci. To suggest as a third possibility that the three loci may be repeats which have developed their own specificities would be mere speculation a t this time, and would only be justified if it could be shown that the corresponding antigens had similar structures, for example, if they competed for the same antibody. The self -incompatibility mechanism in several plant species depends on the fact that pollen will not germinate on or alternatively will not grow down the styles of plants whose genotypes include the same “S” allele. This inhibition is so specific and involves so many interallelic reactions that it seems virtually certain that it depends on the prodiiction o f specific antigens. On this interpretation, each allele must control the production of a specific antigen in the pollen and a corresponding antibody in the style. This dual function is apparently separable into its components, since Lewis has been able to produce mutant types by irradiation whose pollen (#’a) is no longer inhibited on styles of the original type ( 8 6 ) but whose styles (g’6) can still inhibit the original type (&) pollen. It seems that the mutant allele has lost its power to produce the antigen but not its power to synthesize the corresponding antibody and it is theoretically possible that the reverse type of mutation could also

POSSIBLE SIGNIFICANCE OF DUPLICATION IN EVOLUTION

263

occur. As Lewis points out the latter would not be detected by his present technique. It is interesting to note that if the dual nature of the function is related to a dual structure of the gene, then it is likely that the complementary nature of antigen and antibody reflects a complementary relationship a t the genic level also, Chance juxtaposition of two unrelated loci would be quite inadequate to explain the facts.

IV. CONCLUSIONS The vast majority of allelic differences which have been studied are in accordance with the hypothesis that each locus is concerned with one primary function as a consequence of its highly specific surface structure. On this basis a mutation would imply a loss or impairment of the original function with or without the concomitant acquisition of a new function. Theoretically, duplication of loci would appear to offer a means of gaining a new function without losing the old one. I f this were so it might be possible to find loci of duplicate origin which now perform different functions. The criteria necessary to establish a situation of this type are three in number. First, it would have to be shown that two or more separable units are actually present. Second, the duplicate origin of the loci from an originally single locus would have to be established. Third, it would have t o be shown that the loci were qualitatively different i n respect to the processes they controlled. A survey of the available data reveals no single case which satisfies all three requirements. I n the case of tandem repeats in Drosophila and in pseudo-alleles in other material, the existence of separable units can be established satisfactorily. I n the former case also the duplicate origin of the loci is well supported, but the evidence for qualitative differentiation is unsatisfactory since it is obscured by position effects. A t present there seems to be no good way of deciding whether the differences between loci are entirely attributable to differences in relative position or whether they are the result of intrinsic changes within the loci. (In Gtoldschmidt 's view of course such a distinction would be meaningless.) I n the case of pseudo-alleles in other material an opposite difficulty is encountered, i.e., evidence of qualitative differences between the loci is available, but evidence of a duplicate origin is not convincing. Nevertheless it is difficult to believe that pseudo-allelic systems result from chance associations, and it is possible that a closer relationship may be established in those cases which are amenable to a biochemical approach. Finally cases are known which parallel exactly the situation in pseudoallelic series except that no independent evidence suggesting that more than one lociis is involved has been found. It is interesting to note that

264

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ail the cases of pseudo-alleles and some of the cases of tandem repeats which have been described were originally considered to be allelic series. For the present it must be concluded that the case for divergence of duplicates is not proved. I n the writer’s opinion this does not mean necessarily that it is incapable of proof. Although it is possible to hold the viewpoint that the cases considered are exceptional and hence of no general significance, it is also possible to consider them exceptional only in the fact that they are particularly suitable for detailed investigation. Clearly the numbers of cases which are suitable for testing only one of the three criteria listed above must be greatly restricted-to find material in which all three criteria can be tested requires a more careful search than has yet been made. The Red-cluster linkage group in cotton which has been described suggests that amphidiploids may provide a promising field for investigation since here the problem of distinguishing single from linked loci does not arise, and a comparison of originally homologoiis chromosomes at different stages of evolution is a n attractive possibility.

V. REFERENCES Beadle, G. W., 1945, Chem. Rev. 37, 15-96. Beasley, J. O., 1942, Genetics 27, 25-54. Bridges, C. B., 1936, Science 83, 210-211. Clausen, R. E., and Cameron, D. R., 1944, Genetics 29, 447-477. Clausen, R. E., and Cameron, D. R., 1950, Genetics 36, 4-10. Demerec, M., 1925, Genetics 10, 318-344. Dobshansky, N. P.,and Dobzhansky, Th., 1933, Genetics 18, 173-192. Emerson, S., 1945, Ann. M o . bot. Gdn. 32, 243-249. Fisher, R. A., 1947, Amer. Scientist 36, 95-103. Goldschmidt, R., 1938, Physiological Genetics. McGraw-Hill, New York and London. 375 pp. Goldschmidt, B., 1940, The material basie of evolution. Yale University Press, New Haven. 436 pp. Goldschmidt, R., 1946, E x p e r k n t h 2, 1-40. Goldschmidt, R., 1950, Proc. nat. Acad. Sci., Wash. 36, 365-368. Green, M. M.,and Green, K. C., 1949, Proc. nat. Acad. Sci., Wash. 35, 586-591. Gruneberg, H.,1943, Genetics of the Mouse. Cambridge University Press. 412 pp. Gulick, A., 1944, Adv. in Enzymology 4, 1-39. Haldane, J. B. S., 1932, The Causes of Evolution. Harper and Bros., New York and London. Harland, 8. C., 1935, J. Gcnet. 30, 465-47G. Harland, 8. C., and Atteak, 0.M., 1941, J . Genet. 42, 1-19. Hutchinson, J. B., Silow, R. A., and Stephens, S. G., 1947, The Evolution of Gos.~ypium. Oxford University Press. 160 pp. Lamprecht, H., 1948, Agric. Hortique Genetica 8, 83-86. Laughnan, J. R., 1949, Proo. nat. Acad. Sci., Wash. 36, 167-178. Laughnan, J. R., 1950, 23-00. nat. h a d . Sci., Wash.38, 312-318.

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Lewis, D., 1949, Bwl. Rev. 24, 472-496. Lewis, E. B., 1945, Genetics 30, 137-166. Lewis, E. B., 1948, Genetics 33, 113. Little, C. C., and Hummel, K. P., 1947, Proc. nat. h a d . Sci., Wmh. 33, 42-43. McClintock, B., 1941, Cold Spr. Barb. S y m p . Qzucnt. Bwl. 9, 72-81. McClintock, B., 1944, Genetics 29, 478-502. Metz, C. W.,1937, Genetics 22, 543-556. Muller, H. J., 1932, Proc. 6th h t . Congr. Genet. 1, 213-255. Muller, H. J., 1935, Genetics 17, 237-252. Neelakantam, E., and Seshadri, T. R., 1937, Proc. Indian Acad. Sci. AS, 357-364. Pauling, L., and Delbruck, M., 1940, Science 92, 77-79. Sears, E. R., 1948, Adv. in Genetics 2, 239-270. Serebrowsky, A. S., 1938, C. R. Acad. Sci. W.S.S.R. 19, 77-81. Silow, R. A., 1941, J. Genet. 42, 259-368. Silow, R. A., 1944, J. Genet. 46, 78-115. Silow, R. A., 1945, J. Hered. 36, 62-64. Silow, R. A., 1946, J. Genet. 47, 213-221. Silow, R. A., and Yu, C. P., 1942, J. Genet. 43, 249-284. Skovsted, A., 1933, Ann. Bot. Lond. 47, 227-251. Stadler, L. J., 1946, Genetics 31, 377-394. Stadler, L. J., 1948, Amer. N a t . 82, 289-314. Stephens, 5. G., 1948a, G e w t k s 33, 191-214. Stephens, S. G.,194813, Arch. Biochrm. 18, 449-459. Stern, C., 1949, Principles of Human Genetics. Freeman and Co., San Francisco. 617 pp. Sturtevant, A. H., 1925, Genetics 10, 117-147. Sturtevant, A. H., 1948, Amer. Scientist 36, 225-237. Tan, C. C., 1946, Genetics 31, 195-210. VOSS,J., 1938, Plant Breeding Abstr. 9, 698. Wagner, R. P., 1949, Proc. nut. Acad. Sci., Wash. 36, 185-189. White, M. J. D., 1948, Animal Cytology and Evolution. Cambridge University Press. 375 pp. Williams, R. D., 1937, Rep. 4th Qt. Grassland Congr. Aberystwyth, 238-251. Y u , C. P., and Chang, T. S., 1948, J. Genet. 49, 46-56.

Cytogenetics of Orthopteroid Insects M . J . D . WHITE The University of Tezas. Austin. Texas CONTENTS

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I Introduction I1 Systematic Review of the Cytology of the Orthopteroid Groups I11 Chromosomes a n d Taxonomy in the Orthopteroid Groups . . . 1 “Chromosomal Races ’’ or ‘Sibling Species” in Gryllotalpa gryllotalpa I V Problems of Chromosome Structure 1 Centromeres 2 Spiral Structure 3 Euchromatin and Heterochromatin 4 Chromomeres V Meiosis 1 Chiasma Frequency 2 Chiasma Localization 3 Chiasma Tnterference across the Centromere 4 Meiosis of Polyploid Cells in Grasshopper Spermatogenesis 5 Apparent Absence of Chiasmata in Certain Mantids and Roaches 6 Chromosomal Segregation in Gryllotalpa hezadactyla VI Sex Chromosomes and Sex Determination 1 Origin of XY:XX from X 0 : X X Sex Chromosome Mechanisms in Grasshoppers 2 The Sex Chromosome Mechanism in the Praying Mantids 3 Sex Chromosomes in Phasmids 4 The Sex Chromosome Mechanism of Eneoptera surinamensis 5 The case of Paratylotropidia brunneri 6 Anomalies of Sex Determination V I I Cytological Polymorphism in the Orthopteroid Insects 1 Chromosomal Polymorphism in the Trimerotropine Grasshoppers a Mutual Translocations b Supernumerary Chromosomes c Supernumerary Chromosome Regions d Centromere Shifts 2 Heterozygosity for Centric Fusions 3 Translocations i n Wild Populations of Grasshoppers 4 Supernumerary Clironlosomes 5 Unequal Autosomal Bivalent8 in Phasmids

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. . . . . . . . . . 316 1. Parthenogenesis and Polyploidy in Saga pedo . . . . . . . . 319 2. Parthenogenesis in the Acridoidea . . . . . . . . . . . . . 320 IX. Genetie Work . . . . . . . . . . . . . . . . . . . . . 321 X. Experimental Hybridization . . . . . . . . . . . . . . . . 825 XI. References . . . . . . . . . . . . . . . . . . . . . . 326

VIII. Parthenogenesis in the Orthopteroid Groups

I. INTRODUCTION The term Orthopteroid insects will be employed in this paper to designate the rather miscellaneous assemblage of forms commonly called " Orthoptera, " i.e., the Roaches, Praying Mantids, Walking Sticks, Crickets, and Grasshoppers, together with a number of groups which do not possess common names. Whether the Termites, Earwigs, and Embiids should be included in the Orthopteroidea is doubtful-for present purposes they will be excluded. Phylogenetically, the living Orthopteroid insects may be regarded as the more or less modified descendants of the palaeozoic Protoblattoidea and Protorthoptera (Zeuner, 3 939). Ever since the pioneer work of McClung and his associates, from the beginning of the century to the present time, Orthopteran material has furnished the basis for a large number of cytological investigations on chromosome structure, chromosome behavior, sex chromosomes, the mechanisms of mitosis and meiosis, cytological polymorphism in wild populations, the effects of irradiation upon chromosomes, and a variety of other problems too numerous to enumerate. The large size of the cellular elements and the fact that the chromosomes are, as a rule, relatively few in number renders the Orthopteroid insects especially suited for cytological work. Genetical studies, on the other hand, have been much less extensive, due, no doubt, to the fact that most species of Orthoptera have only one, or at most two or three generations per annum. While this is a very serious disadvantage for genetical work of the classical type, there can be no doubt that many species of Orthoptera constitute almost ideal material for investigations on the significance of genetical and cytological polymorphism in wild populations. Orthopteran material has also been used extensively in studies on the effects of irradiation, centrifugation or other types of experimental treatment on the cell, but we do not propose to deal with such work here, since it can only be understood in relation to similar work carried out on other types of animal and plant material.

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TABLE 1 Classification of the Orthopteroid Groups of Insects * Orthopteroiden Order 1. Grylloblattoidea

SUPERORDER

2. Blattoidea (Roaches) 3. Mantoidea (Praying Mnntids) 4. Phasmoidea (Walking Sticks)

5 . Saltatoria (Jumping Orthoptera)

Suborder 1. Ensifera Family 1. Prophrlangopsidae 2. Gryllidae (Crickets) 3. Gryllotalpidae (Mole Crickets) 4. Gryllacrididae * 5. Tettigoniidae (Long-horned grasshoppers)

Suborder 2. Acridoidea Family 1. Tetrigidae (Grouse Locusts) 2. Tridactylidae 3. Pneumoridae 4. Proscopiidae 5. Eiimastacidae 6. Acrididae (Short-horned grasshoppers, Locusts)

High mountains of Western North America and Asia Worldwide Worldwide in tropical and warm climates Worldwide i n tropical a n d warm climates Worldwide, except in Arctic and Antarctic Two genera, one North American, one Asiatic Worldwide Worldwide Widespread in warm climates Worldwide

Widespread Widespread, semi-aquatic South Africa South America Wiclrsprend in warni climates Worldwiile

1 Suborders and families of the flrst four orders not enumerated. S T h i s family probably deserves to be split u p into several smaller units of fanlily rank.

REVIEW OF 11. SYSTEMATIC

THE

CYTOLOGY OF

THE

ORTHOPTEROID GROUPS

I n the present section we shall attempt to review the cytological conditions in the Orthopteroid gronps from the general standpoint of chromosome numbers and chromosome shape, with special reference to the relationship between the cytological data and the taxonomy and systematics of the various forms. According to Makino’s (1950) list of chromosome numbers i n animals about 350 species of Orthopteroid insects have been investigated cytologically; b u t this list is seriously incomplete as f a r as work published in the last ten years is concerned, and we believe that the total number of species which have been studied by cytologists is now about

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500. The number of species in which the cytology has been studied in a detailed manner is, of course, very much smaller. Grylloblattoidea. The only species of this small and primitive group which has been examined cytologically is Cfalloisiana nipponensis, which has a n XY pair of sex chromosomes in the male and 2N d = 30 (Nakamura, 1944, 1946). Blattoidea. The amount of cytological work which has been carried out on the Roaches is not large. As can be seen from Table 2 there is a rather considerable range in chromosome numbers from 2N d = 23 to 73. I n part this is no doubt an expression of the systematic complexity of the Order Blattoidea, which includes a large number of families and subfamilies, many of which have probably been separated since paleozoic times. It is noteworthy, however, that even within the single genus Periplaneta we find one species with 2N cf = 27 and one with 2N d = 33. I n both these species and in Phyllodromin germarnica all the chromosomes are metacentric, but in most of the other species which have been investigated, the published figures do not enable one to be certain as to how many of the chromosomes are metacentric and how many acrocentric. All species of Roaches so far investigated are XO in the male, the sex chromosome being invariably metacentric. Mantoidea. The chromosome numbers (2N d ) of the thirty species of Praying Mantids which have been studied cytologically range from 15 in Acontiothespis sp. (White, 1941a) to 39 in Humbertiella indica TABLE 2 Chromosome Numbers in the Blattoidea

Loboptera decipiena Phyllodromia germanica Periplaneta australasiae P . amerioana Blatta orientalis Blabera Iusca Leucophaea maderae Pycnoscelus surinamensis

2N

(8)

33 23 27 33 47 73 23 37

Author Suomalainen, 1946 Suomalainen, 1946 Suomalainen, 1946 Suomalainen, 1946 Suomalainen, 1946 Suomalainen, 1946 Morse, 1909 Matthey, 1948a

(Oguma, 1946 ; Hughes-Schrader, 1948). There is a strong tendency for most or all of the chromosomes to be metacentric. The majority of the species which have been studied are XO in the male sex, but in one group of genera an XIXzY sex chromosome mechanism has been developed (see page 295). I n the genus Sphodromantis a surprising difference in chromosome number exists between the South African S. gastrica, which has 12 pairs of metacentric autosomes, and Algerian

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271

material of 8.viridis, in which there are only 10 pairs of metacentric autosomes (White, 1941a). Ph,asmoidea. There is a general tendency for the chromosome numbers in this group to be somewhat higher than in most of the other Orthopteroid orders. The lowest number on record is in Sipyloidea paweticus (subfamily Necrosciinae) and Carausius rotundato-lobatus (subfamily Lonchodinae), where 2N 8 = 21 (Favrelle, 1934). On the other hand, some of the parthenogenetic species of Carausius have very high chromosome numbers, different strains of C. furcillatus having 64 to 73 and 85 to 100 chromosomes in the diploid set according to Cappe de Baillon, Favrelle, and de Vichet (1935, 1938). There has been considerable discussion as to whether some of these parthenogenetic species of Walking Sticks with very high chromosome numbers are polyploid or not, but the question cannot be regarded as definitely settled. Such parthenogenetic forms as Bacillw rossii (2N ? = 36) and Carausius theiseni (2N 0 = 40 or 42), in which the chromosome number is relatively low, are almost certainly diploids, but we cannot exclude the possibility that parthenogenetic forms with higher chromosome numbers are polyploids. Cryllacvididne. We are following Zeuner (1939) in regarding the Stenopelmatinae, Gryllacrinae, Rhaphidophorinae, and Schizodactylinae as subfamilies of the Gryllacrididae, although the differences between these groups are so profound that they should perhaps be regarded as separate families, equivalent taxonomically to the Gryllidae, Gryllotalpidae, and Tettigoniidae. The Stenopelmatinae and Rhaphidophorinae have high chromosome numbers, while the Gryllacrinae and Schizodactylinae have relatively TABLE 3 Chromosome Numbers in Gryllacrididae 2i-f

s

Author

Stenopel itialinae Stenopelmatus sp.

47

Rhaphidophorinat: Diestrammena japonica D . marmorata Ceuthophilus sp. C. maculatus

Ma k i tio, 1931 Molir and Eker, 1934 57 37 ( 8 ) Stevens, 1912 37 or 39 Thompson, 1911

Gryllacrinae Eremus testaceus Gryllacris signifera

17 11

Ohmachi, 1935c Heberer, 1937

Schizodactylinae Schizodactylus monstrosus

14

McCluiig and Asana, 1933

Steveus, 1909

57

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M. J. D. WHITE

few chromosomes (Table 3). With the exception of Schizodactylus monstrosus, which is XY in the male (McClung and Asana, 1933), all the other forms which have been studied are XO. Prophalangopsidae. No cytological studies have been carried out on either of the two genera included in this archaic family. Gryllidae. Relatively little work has been carried out on the cytology of the Crickets, the main contributions being those of Baumgartner (1904, 1917, 1929), Brnnelli (1909) and various Japanese workers (Honda, 1926 ; Ohmachi, 1927, 1935a ; Momma, 1941, 1942). There is a considerable range of chromosome number in the family, from 2N d = 9 in Eneoptera surinnmensis (Piza, 1946) to 2N d = 29 i n several species of Gryllus (Baumgartner, 1904 ; Ohmachi, 1929, 1935 ; Tateishi, 1932). Most of the species with the lower chromosome numbers have metacentric chromosomes, and there is a corresponding tendency for those with the higher numbers to have most or all of their autosomes acrocentric, so that the variation in chromosome number is certainly in part due to the species with the lower numbers having acquired centric fusions in the course of their evolution. With the exception of one species of Tree Cricket, which is XY in the male and of Eneoptera surinarnensis, which has a peculiar X Y I Y ~ sex chromosome mechanism (see page 297), all the species which have been studied are XO in the male. The X chromosome is a large metacentric element except in Cyrtoxiphus ritsemae and Homseogryllus japoniczrs, in which it is an acrocentric chromosome (Ohmachi, 1935a). The Tree Cricket Oecaitthus longicauda is exceptional in possessing a very small Y chromosome in the male, which regularly pairs with the X to form a bivalent a t meiosis (Makino, 1932). Two other species of the same genus have XO males (Johnson, 1931; Kitada, 1948). I n all three species there are 9 pairs of autosomes. It is not easy to understand the relationship between the XY and the XO mechanisms in this genus; in all probability the Y of Oe. longicauda is a neo-Y, but there is nothing to indicate its precise mode of origin. Gryllotalpidm. Relatively few species of this small, specialized family have been studied cytologically, but some of the results are of exceptional interest (see pages 279 and 290). Most species of Oryllotalpa and Scapteriscus seem to have 2N d = 23, most or all of the chromosomes being metacentric ; however, certain forms currently included under the name Gryllotnlpa gryllotalpa L. have lower numbers. Tettigoniidae. The “Long-Horned Grasshoppers” or Katydids are a rather diversified family of Orthopterous insects, divisible into a large number of subfamilies. Some of these are rather well known cytologically, while for others cytological information is scanty or lacking. The

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chromosome numbers of about 70 species of Tettigoniidae which have been studied cytologically range from 2N 6 = 21 in Euconocephdus nasutus (Hareyama, 1937, 1941) and Xiphidim macdatum (Ohmachi and Sokame, 1935 ; Hareyama, 1941) to 2N 6 = 35 in various species of Conocephalus (White, 1941b), Jamaicana (Woolsey, 1915) a n d in Tattigonia orientalis ibuki (Hareyama, 1941). We do not include Xaga pedo, in which 2N ? = 68, here, since this parthenogenetic species is clearly a tetraploid (see p. 319). I n certain subfamilies the chromosome number is relatively constant ; for example, nearly all the Old World Decticinae seem to have 2N S = 31, all the chromosomes being acrocentric, but the North American Atlantkus pmhymerus has 2N 6 = 25, the X and two pairs of autosomes being metacentric (White, 1941b). I n general, however, it can be said that the chromosome sets of the Tettigoniidae vary far more from species to species than do those of the Short-Horned Grasshoppers ( Acrididae), where the vast majority of the species have 2N 6 = 23. As a rule the medium-sized and the smaller chromosomes of the Tettigoniidae are acrocentric, but it is not unusual for one or more of the larger pairs to be metacentric. Some of the cytological differences between related species may be due to centric fusions, as first pointed out by Robertson (1916), but not all of them are of this type. I n certain genera of Tettigoniidae there are rather considerable cytological differences between closely related forms. Thus in the genus Tettigonia, the European T. viridissima has 2N 6 = 29, there being a large metacentric X and a pair of metacentric autosomes (Mohr, 1916 ; White, 194lb). T . orientalis orientalis has 2N 6 = 33, there being likewise a metacentric X and a pair of metacentric autosomes, but T. orientalis ibuki has 2N 6 = 35 and no less than four pairs of autosomes are metacentric (Hareyama, 1941). I n the genus Xiphidion, X . gladiatzcm and X . chinensis have 2 N 6 = 33, two pairs of autosomes being metacentric, while X . maculatum has 2N 6 = 21, four pairs of autosomes being metacentric (Hareyama, 1941). Most workers on Orthopteran chromosomes have reported strict constancy of chromosome number throughout the germ-line apart from such special cases as the supernumerary chromosomes of Camnula (Carroll, 1920). On the other hand Hareyama (1941) claims to have found variations in chromosome number within the individual in all of 19 species of Tettigoniidae which he studied. These variations (A- 1-3 chromosomes) only occurred in a small percentage of cells and were observed a t both the spermatogonial and the meiotic divisions. Although Hareyama’s figures show that the material was well fixed, we are strongly inclined to suspect that most of the supposed variations in chromosome number were due to errors in observation or interpretation.

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The sex chromosome mechanism of all t h e Tettigoniidae which have been studied cytologically is of the simple X0:XX type, but there is a considerable amount of variation in the size and shape of the X, which may be either an acrocentric o r a metacentric element. Even within single subfamilies such as the Decticinae and the Phaneropterinae, certain species have acrocentric X’s while others have the sex chromosome metacentric (Asana, Makino, and Niiyama, 1938 ; White, 1941b). It seems clear that on a number of occasions in the phylogeny of the Tettigoniidae structural rearrangements within the X chromosome have led to changes in the position of the centromere. Tetrigidm. As far as known, all the Grouse Locusts have 2N = 1 3 (d), 14 ( 0 ) . At, least this is so for the common species of Acrydium, Apotettix, Tettigidea, Nomotettix, Choriphyllum and Paratettix (Robertson, 1915, 1916; Rayburn, 1917; Harman, 1915, 1920; Misra, 1937). The chromosomes of all the species which have been investigated were invariably acrocentric. TridactyZidae. This family of cricket-like insects is in all probability more closely related to the Tetrigidae than to any other group of the Orthoptera. Tridactylus japonicus, which has been studied cytologically by Ohmachi (1935a) has the same chromosome number as the Grouse Locusts, namely 2N ( 3 ) = 13, but the X and five of the six pairs of autosomes are metacentric. Ohmachi wrongly considered the family Tridactylidae as a subfamily of the true Crickets (Gryllidae) . Powers (1942) reports the chromosome number of Ripipteryx sp. as 2N 6 = 25. Eumastacidae. The only published information on the cytology of this interesting family is summarized in Table 4. TABLE 4 Chromosome Numbers of Eumastacidae, Determined by E. R. Helwig (from Relin, 1948) Subfamily Chorotypinae Teicophrynae Morseinae Eruciinae

Eusohmidtiinae

Species Erianthus sp. Teicophrys inopinata Morsea californica Erucius sp. Penichrotes rneridionalis Amatonga spicata

21 17 23 21 19

(3)

19

Presumably all these species are XO in the males. Rehn does not state whether the chromosomes are acrocentric in every case, or whether some of them are metacentric. Proscopiidae. Two species of this South American family were

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studied by Piza (1943, 1945). Both showed a chromosome number of 2N d = 17. Apparently all the chromosomes are acrocentric. A third species studied by de Castro (1946) has one more pair of autosomes, so that 2N d = 19. Pneumoridae. Nothing appears to have been published concerning the cytology of this peculiar family of South African grasshopper-like insects. Acdidae. The family Acrididae (True Urasshoppers or Locusts) has been subdivided by Roberts (1941) into two sections, the Chasmosacci (comprising the subfamilies Pamphaginae and Pyrgomorphinae) and the Cryptosacci (comprising the subfamilies Truxalinas, Oedipodinae, Romaleinae, Ommexechinae, and Cyrtacanthacrinae) . This distinction based on the structure of the male genital apparatus is also supported by the cytological data, since in the Cryptosacci the basic chromosome number is 2N = 23 ( b ) , 24 ( O ) , while in the Chasmosacci the basic number is 2N = 19 (d), 20 (0). I n the great majority of species in both groups all the chromosomes are acrocentric, and there can be little doubt that this was the primitive condition in all the various subfamilies. No species of grasshoppers are known in which the chromosome number exceeds those given above, i.e., 2N d = 19 seems to represent a n upper limit for the Chasmosacci and 2N d = 23 a n upper limit for the Cryptosacci. Reductions in chromosome number have arisen in several different ways in the Acrididae. In certain species originally acrocentric chromosomes may have undergone “centric fusion” to form metacentrics (two rod-shaped elements giving rise to a single V-shaped chromosome). A single such fusion is constantly present in the homozygous state in the Mexican species Aidenzma aztecn (Cyrtacanthacrinae) which accordingly possesses an acrocentric X, nine pairs of acrocentric autosomes and one pair of metacentrics (2N d = 21). The South American A l e w vitticollis (Cyrtacanthacrinae) has two such fusions, there being seven pairs of acrocentric autosomes and two pairs of metacentrics (2N d = 19) (Saez, 1932). Finally, in a whole series of genera belonging to the subfamily Truxalinae (Stenobothrus, Chorthippus, Stauroderus, Omocestus, Chloealtis, Gomphocerus, Chrysochraon, Napaia) there are three such fusions (2N d = 17). Finally, Philoclem anomalus (subfamily Cyrtacanthacrinae) possesses five centric fusions between autosomes and one between the X and an autosome, so that 2N # = 12 (Helwig, 1941). This case, and a number of others in which the X chromosome has become involved in a centric fusion, will be dealt with later (page 293). All species of the subfamily Painphaginae which have been studied cytologically possess 19 acrocentric chromosomes in the male (Granata,

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1910; Chen, 1937). I n the Pyrgomorphinae, Rao (1937) has shown that a number of Indian forms likewise have 19 acrocentric chromosomes in the male. According to McClung (1932) some (but not all) individuals of ij’phenarium rnexicanum, an American representative of this subfamily, possess a centric fusion in the homozygous condition, SO that 2N d = 17. The distribution of this centric fusion in the natural p o p lations of the species was not studied by McClung. It has generally been assumed that “centric fusions” have arisen by very unequal translocations, one acrocentric being broken just proximal to the centromere, the other one just distal to it, i.e., in the short arm. Such a translocation would give rise to one large metacentric chromosome and another minute one which might be lost in subsequent generations without affecting viability if it consisted entirely of heterochromatic material. This view has been disputed by Helwig (1942) who has claimed that Orthopteran metacentrics which have arisen by centric fusion possess two centromeres situated very close together. The evidence for the compound nature of such centromere regions is not entirely clear, but we believe it is not impossible that some metacentrics which have arisen by centric fusion do in fact have the structure which Helwig ascribes to them, the two centromeres of each daughter chromosome always passing to the same pole at anaphase. If this interpretation is correct, such metacentrics have probably arisen through a type of mutual translocation in which both of the original acrocentrics are broken through the short arm. There is no critical evidence to support the view held by some of the earlier cytologists that centric fusions might result from simple adhesion of chromosome ends without previous breakage. Apart from centric fusions, certain species of grasshoppers possess less than the “standard” number of chromosomes, due to the apparent loss of one or more members of the primitive chromosome set. Thus in the genera Miramella, Zubowskyia, Niitacacris, and Indopodisma 2N 6 = 21, but all the chromosomes are acrocentric (Helwig, 1942). It seems highly unlikely that in these cases a whole chromosome pair was actually “lost” in the course of evolution; most probably the greater part of its length was simply transferred, by translocation, to another member of the chromosome set, only the centromere and small heterochromatic regions being actually lost. This process seems to have occurred three times in the evolution of Dactylotum bicolor picturn (subfamily Cyrtacanthacrinae) in which three pairs of chromosomes have disappeared so that 2N d = 17, all the chromosomes being acrocentric (Helwig, 1942). We shall deal in a later section with the very anomalous chromosome sets which have arisen in some members of the tribe Trimerotropi of the

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subfamily Oedipodinae, as a result of centromere shifts which have converted a varying number of the originally acrocentric chromosomes into metacentric elements. It is worth pointing out here, however, that something of the same kind appears to have occurred in the S. African Oedaleiis nigrofasciatus, in which 2N d = 23, but four pairs of autosomes are metacentric (Nolte, 1939). I n two other species of this genus the chromosome number is the same but all the chromosomes are acrocentric. The systematic position of a number of grasshopper genera is still somewhat uncertain, some authorities including them in one subfamily, some in another. The genus Phrynotettix (“Toadhoppers”) has been included in the Romaleinae by Roberts (1941), while Uvarov (1943) and Tinkham (1948) regard it as a n American representative of the Pamphaginae. Since Phrynotettix has 2N d = 23, the cytological evidence supports the former view. A number of authors have been concerned with the detailed comp r i s o n of chromosome lengths in related species of Orthoptera, with a view to establishing definite homologies between the elements of the chromosome set in different species. Thus McClung (1928) claimed that a particular chromosome in Mecostethus grossus, M . lineatus, and M. gracilis, which regularly forms two chiasmata (the others normally form only one) was actually the same element in all three species. Carlson (1936) studied a “megameric” chromosome (i.e., one containing a number of conspicuous heterochromatic regions) which occurs in Chorthippus curtipennis, Euchorthippus pulvinatus, Xtenobathrus lineatus, Omocestus ventralis, Stauroderus biguttulzts,~Gomphocerus rufw, and Aeropedellus clavatus. He came to the conclusion that the megameric chromosome was actually homologous throughout, this series of rather closely related genera of the subfamily Truxalinae. It should be obvious that, in the absence of any genetical evidence, the concept of interspecific or intergeneric chromosomal homology lacks precision. Only in the case of the X chromosome can we be reasonably certain that we are actually dealing with the same element throughout a series of related forms. The “megameric” chromosome in the seven genera studied by Carlson m a y be the same element in all of them, b u t the actual number, size and distribution of the heterochromatic blocks was not the same in the different species, so that it is virtually certain that structural rearrangements must have taken place within the “megameric” element since their evolutionary separation. It is probable that most of these rearrangements were intrachromosomal, b u t we cannot exclude the possibility that some translocations between the megameric element and other members of the chromosome set may have occurred in evolution. The only valid criterion of chromosomal homology between

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different species is provided by a cytological study of hybrids between them. I n many instances the relative lengths of the members of the chromosome set are extremely suggestive of homology (e.g., all species of Trimerotropis and related genera have two pairs of chromosomes which are much shorter than the rest) but A mere comparison of lengths is never conclusive evidence, either of homology or non-homology. AND TAXONOMY IN THE ORTHOPTEROID INSECTS 111. CHROMOSOMES

I n general, visible cytological differences between closely related species are rare in the Orthopteroid groups. I n some large genera such as Melanoplus (Meek, 1913; Nowlin, 1908; Hearne and Huskins, 1935) it seems probable that all, or almost all, the species have chromosome complements which look indistinguishable at metaphase (although carefiil nieasurements of corresponding chromosomes might reveal differences). This situation contrasts with that found in Drosophila, where the gross morphology of the chromosome complement varies greatly from species to species, even among forms that are almost indistinguishable externally. In certain groups of Orthoptera, however, the visible cytological differences between closely related forms are considerable. Thus in the Crickets of the genus Nemobius the chromosome numbers (2N S) range from 11 to 19 (Honda, 1926; Ohmachi, 1935a, Favrelle, 1936) while in the Tettigoniid subfamily Conocephalinae the corresponding numbers vary from 21 in Euconocephaius nasutus to 35 in Cmcephalus dorsalis (Momma, 1941; Hareyama, 1941; McClung, 1914 ; Asana, Makino, and Niiyama, 1938; White, 1941b; Piza, 1945). To some extent these differences are due to the species with lower chromosome numbers having acquired centric fusions between originally acrocentric chromosomes, so as to produce metacentrics; but at least in such genera as Nemobiiis structural changes of this kind account for only a part of the cytological differences between the species. Rather striking chromosomal differences are also found between some closely related species of Walking Sticks. For example in the genus Isagoras, studied by Hughes-Schrader (1947), Z, subaquilus has 2N d = 27, while I. schraderi has 2N d = 34, and a n unidentified species shows 2N 6 = 47. We shall deal later (page 301) with the situation in certain of the Trimerotropine grasshoppers, where the existence of numeroils striictural rearrangements has led to a situation in which microgeographic races can be distinguished, on cytological grounds.

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1. “Chrontosomal tlwces” or “Sibling Hpecies” gryllotalpa

ZiYb

279

Gryllotalpa

Most of Europe, North Africa, and the countries bordering the Eastern Mediterranean are inhabited by Mole Crickets which are currently regarded by taxonomists as constituting a single species, Gryllotalpa gryllotalpa L. Cytologically, a number of forms can be recognized within this taxonomic entity, but in the absence of any breeding experiments or precise biometrical studies the exact status of these forms must remain doubtful for the present. Either G . gryllotwlpa is a species with a large number of “chromosomal races,” or more probably this name covers a number of “sibling species” which may have reached about the same degree of morphological differentiation as the species of the Anopheles maczclipcnnis species group inhabiting approximately the same area. The Mole Cricket of Central and Northern Europe (France, Belgium, Germany, and northern Italy) has 2N 6 = 12 (de Winiwarter, 1927, 1937) ; the X and all five pairs of autosomes are metacentric, but the form of the small Y chromosome is undetermined. The race from Rumania (Steopoe, 1939) has 2N 6 = 14; again, all the chromosomes are metacentric with the possible exception of the Y. There is a complication, however, since in some individuals a small supernumerary chromosome may be present, and one of the autosomal pairs may also consist of two unequal homologs, presumably due to the presence of a supernumerary chromosome region. I n southern Italy there is an XO race, in which 2N 6 =15; here again all the chromosomes are metacentric. The Italian peninsula is also inhabited by another form in which 2N 6 = 18 (Barigozzi, 1942). Finally, in Palestine there are two “races” of G . gryllotalpa, both of which are XO in the male (Kushnir, 1948). One, which is generally distributed, has 2N 6 = 19, while the other, which has been found only in the neighborhood of the Dead Sea, has 2N d = 23, the same number that occurs in some other species of Gryllotalpa ( G . hexndactyla, G. africana) and in the related genus Scapteriscits (Dreyfiis, 1942). In view of the very considerable c*ytologicaJ difierences betweeu the foriiis of “(2. gryllofalpa” it seems almost inconceivable that they would be fully interfertile when crossed. I n all probability we arc dealing with a group of “sibling species” which are difficult or impossible to distinguish by ordinary systematic characters ; further studies are obviously desirable and it would be interesting to know whether the different forms could be distinguished by their stridulation as in the case of the Yibling species of Nemobius studied by Fulton (1933).

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n. WHITE

IV. PROBLEMS OF CIIRONOSOME STRUCTURE The chromosomes of Orthoptera have furnished material for a number of fundamental studies on the spiralization-cycle, on the nature of heterochromatin and euchromatin, on the structure and functions of the centromere and on the disputed question of whether chromomeres actually exist or not. 1.

C‘enlronzercs

Witliou t exceptioii, the ceiitromeres of all oi%hopterous chroniosomes seem to be strictly localized structures. Two kinds of chroniosomal elements occur : (1) rod-shaped chromosomes (telomitic in the terminology of McClung, acrocentric in that of the present author, (2) J-shaped or V-shaped elements (atelomitic or metacentric) . In the case of the latter the centromere lies somewhere near the middle of the length of the chromosome, which is accordingly divided into two arms which are equal or subequal in length. I n the case of acrocentric chromosomes, most authorities are now agreed that the centromeres are not strictly terminal (White, 1935, 1936 ; Darlington, 1936 ; Coleman, 1943). Thus even acrocentric chromosomes are two-armed structures, but one arm is so short that it can usually only be seen under exceptionally favorable circumstances, in metaphase chromosomes which are elongated to their maximum, rather than contracted as they frequently are in poorly fixed material. This viewpoint has recently been challenged by Makino and Momma (1950),who, on the basis of observations on the chromosomes of Podisma and Miramella, treated before fixation in hot or cold air, argue in favor of strictly terminal centromeres. It should be emphasized that

FIG.1. First metaphase in Coiaozoa sulcifroics (Acrididae), showing the minute “second arms” on the ends of several of the bivalents.

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the observations of the authors previously cited on the spermatogonial chromosomes of several genera of both Acrididae and Tettigoniidae, conclusively demonstrate the subterminal position of the centromeres in the acrocentric chromosomes OP those forms. Failure to observe the minute “second arms” in the chromosomes of other species should not necessarily be regarded as proof that they do not exist, since even in those forms in which they undoubtedly occur they can only be seen in exceptionally well-fixed cells. Furthermore, there can be little doubt that the “second arms’’ are distinctly larger in some species than in others, and it may be that they are particularly small in the species investigated by Makino and Momma. I n some species where the “second arms” have not been seen in the spermatogonial divisions they can readily be observed a t meiosis, owing to the fact that the chromosomes are very much stretched out on the spindle (Fig. 1).

2. Xpirul Structure Grasshopper chromosomes, on account of their relatively large size, have provided material for a number of studies on the spiralization cycle of the chromosomes during mitosis and meiosis. Nevertheless, even the largest Orthopteran chromosomes are not nearly as favorable for demonstrating spiral structure as those of certain plants such as Trillium and Tradescantia. Wilson (1912) published figures of the spermatogonial chromosomes of Phrynotettix tschivavensis showing “relic spirals” (i.e., those of the previous division uncoiling in the prophase of the next division) and Mohr (1916) also illustrated relic spirals in the case of the X chromosome of Tettigonia viridissimn, while de Winiwarter (1931) did the same for Decticus dbifrms. White (1940a) extended these observations to a number of other species of Tettigoniidae, showed that there was no essential difference between the spiral structure of euchromatic and heterochromatic regions and demonstrated that the direction of coiling (rightor left-handed) was not constant in the case of the X chromosome. All the above observations were carried out on relic spirals. More recently, various workers (Makino, 1936 ; Coleman, 1943 ; Makino and Momma 1950), using various modifications of the squash technique, have studied the spiral structure of the meiotic chromosomes, both during the prophase stages and a t first metaphase. It now seems probable that the diakinesis and first metaphase chromosomes of grasshoppers have a “coiled coil” structure, the minor spiral representing an anticipation of the spiral of the second meiotic division.

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3. Ezcchromatin and Heterockromatin

I n all Orthopteran insects the chromosomes appear to be built u p of two types of material, euchromatin and heterochromatin. It is, however, certain that more than one type of heterochromatin exists, and it may well be that what we ordinarily refer to as euchromatin and heterochromatin are merely the two ends of a continuous series of different types of “chromatins, ” Typical heterochromatin is recognized cytologically by its property of exhibiting heteropycnosis, or differential thickening a t various stages of mitosis and meiosis. As compared with euchromatin, this differential behavior may be in either a positive or a negative direction, Le., the heterochromatin may either be thicker and stain more intensely (“positive heteropycnosis”) or thinner and weak-staining (“negative heteropycnosis ’’) The X chromosomes of the Orthopterous insects seem to be invariably heterochromatic throughout their entire length, except in those cases where we are dealing with a neo-X chromosome, i.e., one which has recently received part of an autosome, as a result of a mutual translocation (in which case the formerly autosomal portion may be euchromatic). I n the Acrididae it is usual for the X to exhibit negative heteropycnosis during the early spermatogonial divisions, i.e., it stains lightly and is very much thinner than the autosomes a t metaphase. This type of behavior is not manifested, or only to a slight extent, in the later spermatogonial divisions. During the prophase stages of meiosis, on the other hand, the X shows strong positive heteropycnosis, i.e., from leptotene to diakinesis it is thicker than the autosomes and stains much more intensely. At the first metaphase and anaphase there is frequently a tendency for the X to be once more negatively heteropycnotic, while during interkinesis and in the spermatid nuclei it is once more positively heteropycnotic. This type of cyclical behavior has been referred to as “reversal of heteropycnosis” (White, 1940a). I n the Tettigoniidae, on the other hand, the X never seems to show negative heteropycnosis, so that no reversal occurs. I n some species of Crickets, a definite reversal of heteropycnosis occurs, but in the Phasmids and Mantids it. is, a t any rate, not obvious in those species which have been investigated. On the basis of the rather numerous detailed studies of the meiotic prophases which have been carried out in the Orthopterous insects, it seems fairly certain that all autosomes contain some heterochromatic segments, usually located in the neighborhood of the centromere or a t the distal ends of the chromosomes. These heterochromatic segments are

.

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very distinct in some species such as Stauroderus scalaris (Corey, 1933)) but in other species are much less distinct, either because they are actual ly shorter, or because the differential behavior is less pronounced. Many species of grasshoppers have one pair of autosomes which are largely heterochromatic, having only short intercalary segments of euchromatin. Such a pair of chromosomes exists in the species of Mecostethus, where it forms a thick, dark-staining bivalent a t meiosis, which was referred to by Janssens (1924) as the “dyade compagnon de l’X,” since it frequently lies alongside and in close proximity to the sex chromosome during the diplotene and diakinesis stages. Chromosomes of the same general type have been referred to as “megamerk” by some authors since they appear to be built u p of large “chromomeres” or blocks of dark-staining material during the prophase stages of meiosis. It can be stated quite definitely that this autosomal heterochromatin never shows reversal of heteropycnosis in the Acrididae. Thus, in the early spermatogonial metaphases, when the X is negatively heteropycnotic, “megameric” autosomes are not distinguishable from other autosomes by any diflerence in thickness or affinity for stains. There is, accordingly, no reason to postulate any kind of homology between the X heterochromatin and the autosomal heterochromatin, and the behavior of the “dyade compagnon” in Mecostethus spp. should be regarded as a type of nonhomologous pairing rather than as evidence for any true genetic homology. Where a pair of megameric chromosomes are present, they usually bear nucleoli (Wenrich, 1916; Corey, 1940). The sex chromosomes of the Orthoptera seem, at least in the vast majority of cases, to lack any visible nucleolar formations.

4 . Chromoineres The earlier workers on Orthopteran chromosomes (Wenrich, 1916 ; Belar, 1929 ; Hearne and Huskins, 1934) observed a beaded structure in the chromosomes during the meiotic prophases, and gave the name chromomeres to the dark-staining granules. Thus the idea of the chromosome as a sequence of numerous chromomeres (each one probably representing a gene) joined together by interchromomeric connections developed. This concept has recently been challenged by Ris (1945) who claims that the larger “chromomeres” (e.g., those studied by Wenrich) are blocks of heterochromatin while the much more numerous smaller ones are “optical artefacts” based on a misinterpretation of a spiral structure. Some of Ris’s criticisms are undoubtedly valid: there can be no doubt that the term chromomere has been applied too loosely and that

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several different categories of Structure have been confused under this name. The chroniomeres studied by Wenrich (1926) in Phrynotettix, in particular, were certainly large blocks or segments of heterochromatin. Ris’s view that all chromomeres, including even the smallest ones, are artefacts is, however, open to question, since his claim that even the chromomeric bands of the Dipteran salivary gland chromosomes are based on a misinterpretation of a spiral structure has not gained general acceptance. The argument that the “chromomeres” must be gyres of a spiral rather than granules because they disappear when the chromosome is stretched seems to us fallacious; drastic stretching of a chromosome almost certainly disrupts its finer structure so that it is very doubtful what reliance should be placed on the apparent absence of chromomeres in such stretched chromosomes.

V. MEIOSIS The meiotic divisions of most of the Orthoptera follow what has been called the “typical” or “normal” course; that is to say the anomalous modifications of the meiotic divisions which occur in so many of the Diptera, Scale Insects, Lice and other orders of insects are not known to occur in the Orthopteroid groups. Most animal cytologists probably regard the meiotic divisions in the spermatogenesis of the grasshoppers as the norm o r standard to which all aberrant types of meiosis should be compared. Actnally, they are no more “normal” than those of many other groups of animals, but the large size of the cells and chromosomes renders the details of meiosis much easier to study, so that this attitude of mind is justifiable on purely practical grounds. The usual sequence of stages can be observed in the spermatogenesis of almost all the Orthoptera. Very little cytological work has, however, been carried out on oogenesis, on account of the technical difficulties inherent in making cytological preparations of yolky eggs. The observations of McNabb (1928) on the oogenesis of the grasshopper Circotettix verruculatus and those of Matthey (1945) on that of the Roach Pycnoscelus surinamensk, together with other less complete studies, indicate, however, that the general course of the meiotic divisions is the same in the two sexes. Orthopteran eggs are usually laid at the time when the nucleus is in first metaphase or anaphase, the second meiotic division always taking place after the egg has left the body. 1. Chiasrna Frequency

I n those species of Orthopterous insects in which all the chromosomes are acrocentric the chiasma frequency is, as a rule, only slightly

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higher than the number of bivalents, i.e., most bivalents have only one chiasma, a few of them having two. Where metacentric chromosomes are present they may show 1, 2, 3, or 4 chiasmata, but bivalents with more than 3 chiasmata are decidedly rare i n Orthopteran material, and many species never show more than two chiasmata in any of their bivalents. Thus in most species of Acridiclae, with eleven bivalents in the male, the chiasma frequencies per nucleus range from about 11.5 to about 19.8. The relation between temperature and chiasma frequency seems to be a highly complex one; a t 0-2' C. it fa119 to a low level in Chorthippus parallelus, Locztsta niigratoriu, and Schistocerca g r e g w i a ; a t 37-45' C . it reaches a maximum in the two former species, while in Schistocerca it once more falls to a low level (White, 1934).

2. Ckinsma Localization

It is, unfortunately, not possible to determine by cytological means the exact positions along the length of the chromosome a t which the chiasmata arise. Nevertheless, if the clironiosomes are sufficiently large and the details of the diplotene stage of meiosis sufficiently clear, one can obtain a general idea of the distribution of the chiasmata along the chromosome, that is to say the chiasma frequencies of the individual regions rather than of the chromosome as a whole. The classical case of strict chiasma localization among the grasshoppers is that of the genus Rlecostethus (more correctly Stethophyma). Three species, M . grossus, M. lineatus, and Jf. gracilis, have been investigated (Janssens, 1924 ; McClnng, 1914, 1927, 1928 ; White, 1936). The closely related Arcyptera corema shows an essentially identical condition (Chen, 1942). I n all these forms ten of the eleven bivalents ordinarily form a single chiasma, quite close to the centromere, the distal regions of the chromosomes hardly ever showing chiasmata. Whether the same degree of chiavnia localization exists in the female is not known. In all the above species thew is one hivalnit (probably the seventh in order of size) which freqiirritly foinis txw chiawnata, one proximal, the other distal. Although chiasma localization is most strikingly shown in Mecostethus and Arcyptera, there can be little doubt that some degree of localization is extremely widespread and perhaps universal in species of grasshoppers. Little is known, however, as to the causes of localization. Darlington (1940) has stated that in Mecostethus spp. synapsis begins a t the proximal ends of the chromosomes and that it is never completed in the distal regions, but this is not in agreement with our observations of pachytene bivalents of M. grossus in which pairing seems to be as intimate in the distal regions as in the proximal parts. It is noteworthy

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that all the chromosomes of Mecostethus spp. have large proximal heterochromatic segments, so that the chiasmata are formed close to the boundary between euchromatin and heterochromatin. As Callan and Montalenti (1947) express it, “the distance of the proximal chiasma from the centromere seems to be limited quite precisely by the length of heterochromatin adjacent to the centromere. The chiasma falls just 0111 side the heterochromatic region ; since the amount of heterochromatin varies between the bivalcnts of the complement, the differential distance (i.e., the mean distance between the centromere and the proximal chiasma) also varies, but is more or less constant for each individual bivalent.” Most of the autosomes of Mecostethus lack the distal heterochromatic segments which are present in so many species of grasshoppers, and they only form distal chiasmata in very rare instances (except for bivalent 7, which has a large distal heterochromatic segment and frequently forms a distal as well as a proximal chiasma). Altogether, it is difficult to escape the conclusion that heterochromatic regions in Mecostethns somehow determine the formation of chiasmata in the euchromatin immediately adjacent to them, and that this is the effective cause of localization (White, 1942).

3. Chiasnza Interference across the Centromare The acrocentric chromosomes of most species of grasshoppers do not form chiasmata in the minute “second arm,” but there are a few cases in which these small regions may have a surprisingly high chiasma frequency. Thus in the species of Mecostethus, McClung (1928) showed that in one bivalent (called by him the “ditactic” chromosome) the association between the homologs a t metaphase was frequently confined to the short arms, so that the long arms were orientated in the axial plane of the spindle, rather than in the equatorial plane. Where no chiasma is formed between the short arms one is formed in the proximal region,of the long arms, but apparently chiasmata are seldom or never formed in both positions in the same bivalent. One group of closely related species in the genus Trimerotropis ( T . p d l i d i p e n n i s , T. diversellus, and an undescribed species) also show a “ditactic chromosome which behaves in exactly the same way, i.e., a proximal chiasma is formed, either in the long arm or in the short arm, but never in both situations (Fig. 6 ) . It will be obvious that these “ditactic” chromosomes provide evidence of rather strong chiasma interference across the centromere region.

4. Meiosis of Polyploid Cells in Gmsshopper #permatogenesis I n most species of grasshoppers polyploid spermatocytes are occasionally found in the testis. They are not, as a rule, present in every

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testis, and one may have to examine material from a number of individuals before encountering such cells. When found, they may occur singly, i.e., in cysts which are otherwise composed of diploid cells (or haploid ones, in the case of secondary spermatogonia) Alternatively, they may occur in groups of two o r more. Occasionally a large part of a cyst, or even an entire cyst, may be polyploid in constitution. These polyploid cells are probably of no functional significance, there being no evidence that they ever give rise to functional sperms. They do, however, provide important information on some problems of chromosome behavior. Some polyploid spermatocytes are obviously pathological and undergo degeneration during or shortly after the meiotic divisions, but others appear to be physiologically normal and pass through meiosis without any apparent difficulty. We may distinguish two main types of polyploid spermatocytes in grasshoppers. The first type arise through failure of anaphase separation at one of the spermatogonial divisions. Such cells are always tetraploid and may occur singly or in groups of 2, 4, 8, 16 . . according to whether the failure to divide effectively occurred a t the last spermatogonial division, the one before the last, etc. The second type of polyploid cells arise through fusion of 2, 3, 4, 5, or more neighboring cells a t any stage in spermatogenesis. The tfio categories are, perhaps not always distinguishable, but in the second case we may have pentaploid, hexaploid, heptaploid, etc. cells, as well as the more common tetraploid type. Where fusion of the cells has taken place after zygotene the chromosomes of the originally distinct nuclei will have already undergone pairing before the establishment of polyploidy. Under such circumstances no associations of more than two chromosomes will be present at meiosis; in XO species all the X’s will be present as univalents and all the autosomes as bivalents. We illustrate in Fig. 2 a case of a decaploid first spermatocyte of this type-there are 5 X’s and 55 autosomal bival e n t ~ . This cell presumably arose through fusion of five spermatocytes, probably a t pachytene or diplotene. Where polyploidy was established before the zygotene stage, one may expect to find some trivalents, quadrivalents or higher associations of chromosomes at meiosis. I n Xchktocerca gregaria, Chrysochram dispar, and Tettigonia viridissima only the larger chromosomes form multivalents, the shorter ones invariably forming bivalents (White, 1933, 1945 ; Klingstedt, 1939). It is significant that in these tetraploid spermatocytes the two X’s never form a bivalent at diplotene-diakinesis, thus suggesting that strong positive heteropycnosis interferes either with synapsis or with chiasma formation. I n oogenesis, where the two X’s do form a bivalent (RlcNabb, 1928) they are not heteropycnotic.

.

.

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I n some tetraploid spermatocytes of NeopodisrnopsG abdominalis Rothfels (1950b) has reported a peculiar tendency of the centromeres in t,he quadrivalents to be closely paired at first meta.phase.

*

i

1

I

FIO.2. Peinidia fenestralis (Aerididae): a 10ploid firRt nietaphase, Rhowing 55 bivalent5 and 5 X ehromononiea ( i n ontline). From a squash preparation.

5. Apparent Absence of Chiasmata in Certain Mantids and Roaches In the majority of the Orthopteroid insects there is no difficulty in detecting the chiasmata as visible “exchanges of partner” during the prophase of the first meiotic division. At least this is so in spermatogenesis-very few observations have been made on the corresponding stages in oogenesis, but the observations of McNabb (1928) on the meiotic divisions in the egg of the grasshopper Circotettix verruculatus suggest that there are no great differences between the course of meiosis in the two sexes.

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Where difficulties in the detection of chiasmata occur, they may be due to the prophase chromosomes being very diffuse and weak-staining, as they are in the Mole Crickets and some other forms; or to the chiasinata undergoing “terminalization” shortly after they are formed, so that they are shifted to the ends of the chromosomes and are represented merely by “terminal associations.” Neither type of difficulty is present in certain Mantids and Roaches, in which it is nevertheless not possible to detect chiasmata at any stage. The question is whether chiasmata are actually absent or merely “concealed” in some way; in either case the type of meiosis present in these species raises in an acute form the whole question of the relationship of cytological chiasmata and genetical crossovers. The case that has been most fully worked out is that of the Mantid Callimmtis anstillarum from the West Indies. This species was first studied cytologically by White (1938), who pointed out that there was no trace of chiasmata at any stage, the metaphase bivalents consisting of homologous chromosomes which are paired throughout their length, except where they are being pulled apart a t the centromeres. The similarity of these bivalents to those of the male Drosophila (where there is ample genetic evidence that crossing over does not occur) was so great that it was concluded that crossing over was probably absent in the male Callimantis. Later investigations on other Mantids (White, 1941a) showed, however, that a whole series of forms exist, ranging from those in which chiasmata are clearly visible (Miomantis sp., Sphodromantis viridis) through forms in which they can only be seen occasionally (i.e., in some bivalents) to those like Callimantis, in which chiasmata are never seen. I n the light of these later observations it seems clear that the distinction between the meiosis of Callimantis and that of other Mantids is one of degree only. There is, in fact, a tendency in all Mantids for the falling-apart of the homologous chromosomes, which in most organisms takes place after the end of pachytene, to be delayed or very incompletely manifested. In Callimantis this tendency is carried to a n extreme, since no separation of the homologs takes place until they are torn apart a t first anaphase. Since it is this separation of the homologs which, in all organisms with a “normal” meiosis, causes the genetical points of crossing over to become visible as chiasmata, it is natural that in Callimantis chiasmata cannot be seen. Of course, only genetical evidence could establish definitely whether crossing over occurs in the male Callimantis; but it does not seem necessary to conclude in this special case that a lack of cytologically visible chiasmata precludes the existence of crossing over; on the other hand Callimantis does seem to constitute a significant exception to Darlington’s (1932,

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1937) view that chiasmata are “needed” in order to hold together the homologs of which the meiotic bivalents are composed (such a n exception does not, of course, necessarily invalidate the general principle). The view that the difference between the meiosis of Callimantis and that of the other Mantids is only one of degree is not accepted by HughesSchrader (1943a,b) who has reinvestigated the case and believes that a more fundamental difference exists. A similar situation occurs in the Roaches (Morse, 1909; SUOmalainen, 1946 ; Matthey, 1945) where certain species such as Loboptera decipiens and Phyllodromia germanica show clear chiasmata while in some others such as Blabera fusca the male meiosis approaches the Callimantis type. 6. Chromosomal Xegregation in Gryllotalpa hexadactyla

A very peculiar anomaly of chromosomal segregation which occurs in the North American Mole Cricket Gryllotalpa (or Neocurtilla) hexadactyla was first discovered by Payne (1912), who later (1916) described his observations in greater detail. Males of G . hexadactyla (referred to by Payne as G. borealis) have 22 autosomes and a metacentric, heterochromatic X which is clearly homologous to that of other species of the genus Gryllotalpa. Of the autosomes, ten pairs are not remarkable in any way, but the eleventh invariably consists of two homologs one of which is several times the size of the other. The female is homozygous for the larger member of this pair, i.e., the small chromosome is only present in the male. At the first meiotic division in spermatogenesis the unequal bivalent resembles a rather elongated pear, in which the “body” represents the larger chromosome, the “stalk” the smaller element. The most remarkable thing is that a t the first metaphase the larger end of the “pear” is invariably directed toward the same pole of the spindle as the X, even though there seems to be no direct physical connection between any part of the unequal bivalent and the X, which is usually attached to the spindle near one of the poles. Thus a t first anaphase the larger member of the unequal bivalent invariably passes to the same pole as the X, so that two kinds of sperms are formed, one containing the X and the larger member of the unequal bivalent, the other containing the smaller member of the unequal pair. This behavior explains why all females are homozygous for the larger member of the unequal pair. It will be obvious that this case, if correctly interpreted, constitutes a striking exception to the law of independent segregation of the chromosomes, since there is no actual pairing (at least a t metaphase) between the unequal pair and the X. We have referred to the unequal pair as

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if they were autosomes, but it. will be realized that it would be formally correct to describe i t as an X2Y bivalent, the univalent which we have referred to as the X (and which clearly corresponds to the X of other XY or XO members of the genus) being labeled XI. This question of terminology is, however, not of any great significance, and the genet.ic

FIQ. 3. Gryllotalpa hexadactyla: a, spermstogonial metaphase ; b, first met:iplisse in polar view; c , d, same in side view; e , second metaphaso (11 chromosomes) ; f, second metaphsse (12 chromosomes). I n all figures S is the smaller member of the unequal pair of chromosomes, L is the larger mcmber.

properties o f the mernbers of the u n ~ q n a lpair are, of coiirse, quite unknown. Payne’s account was in such flagrant disagreement with a f unclamental genetic law which a t that time was being so conclusively confirmed by the careful work of Carothers (1917, 1921) on the unequal or asymmetrical bivalents of various species of grasshoppers (which did

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segregate independently of the X ) that it is not surprising that it was received a t the time with scepticism. It is significant that it is nowhere referred to in such standard works as Wilson (1925)) Belar (1928), and Darlington (1932, 1937). As a consequence the case of Bryllotalpa hexadactyla seems to have been relegated to that limbo of forgotten claims, apparently irreconcilable with genetic theory and hence to be regarded as based on erroneous observation or faulty interpretation. The present author has, however, recently examined the meiotic divisions of three males of B. hexadactyla from Zapata Co., Texas (Payne's material was probably collected somewhere in Indiana). Our own observations confirm Payne 's account in every detail, without adding anything new to it. The unequal bivalent is definitely present in all the Zapata individuals and in several hundred first metaphases and anaphases the larger member was invariably orientated toward the same pole as the metacentric X. I n none of the cells did we observe any trace of pairing between the X and any part of the unequal bivalentin fact these two elements are often widely separated on the spindle. The prophases of meiosis are extremely difficult to study in all the Cfryllotalpidae, and we were not able to determine whether any pairing between the X and the unequal bivalent exists at pachytene, diplotene or diakhesis. Our observations thus prove that Payne was entirely correct in interpreting the case of Bryllotnlpa hexadactyla as a significant exception to the Law of Independent Segregation, and that the condition he described is characteristic of the species and not merely present in some individuals, or at a particular locality. Detailed study of the prophase stages of meiosis will have to be carried out before a causal explanation of this extraordinary case can be put forward.

VI. SEX CHROMOSOMES AND SEXDETERMINATION The overwhelming majority of the Orthopteroid insects are XO in the male sex, X X in the females, the X being a large heterochromatic element which passes undivided to one pole a t the first meiotic division in the male. A few species are X Y ( b ) : X X ( 0 ) ) some others are XIXzY (d) : XIXIXpX~(0) and one is apparently XYIYz ( S ) : XX ( 0 ) ; but we may be reasonably certain that all these mechanisms are secondary, having arisen in the course of evolution from the simple X 0 : X X mechanism which is the primitive one for the whole group. I n the Blattoidea and in those Mantoidea which are XO in the male the X is invariably metacentric, the arms being subequal in length. This is also the condition in most of the Phasmoidea and in many of the

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more primitive Saltatoria. I n the Acrididae and many of the Tettigoniidae, on the other hand, the X is acrocentric. I n all these cases the X chromosome is very obviously heterochromatic, showing intense positive heteropycnosis at various stages of mitosis and meiosis. 1. Origin of X Y : X X from X 0 : X X Sex Chromosome Mechanisms in Grasshoppers

I n a fairly large number of species of Acrididae, belonging to several different genera not closely related to one another, chromosomal fusions have occurred between the X and one member of a pair of acrocentric autosomes. The effect of such a fusion is, obviously, the inclusion of autosomal material i n a “new” X chromosome. The homolog which remains unfused becomes confined to the male line and may be referred to as a “neo-Y” although its genetic properties will be, at any rate in the beginning, no different from what they were before the fusion occurred. At meiosis the X and the neo-Y will form a bivalent and chiasma formation will occur between the homologous chromosome arms. McClung (1905, 1917) reported that fusions of this type existed in Hesperotettix viridis pratensis and H . speciosus, but not in H . viridis brevipennis. Conditions in H . viriclis viridis are particularly interesting in that, while most populations have the X free, some,individuals from Marathon, Texas, show an X autosome fusion and may also h a w 1 or 2 fusions between autosome present in either the homozygoiis or the heterozygous condition. The chromosome number (2N 8) consequently varies from 18 to 23, apart from supernnmary chromosomes, which may also be present. An extremely interesting series of forms exists in the genus Merniiria (Truxalinae) . I n M. texnna and M . neomexicana the X is free and unfused (McClung, 1917), while in M . bivittata, M . maculipennis mucclungi, and M . intertexta the original X has become fused with a n autosome, so that we have a neo-X, neo-Y system in the male (hlcClung, 1917 ; Helwig, 1929 ; King, 1950). The neo-X is a metacentric, in which one limb (“XL”) consists of the original X, while the other ( “ X R ” ) was formerly an ailtosome. Originally XR and the neo-Y inlist have been homologous throughout their entire length, but various changes seem to have taken place since the occurrence of the fusion; the effect of these changes has been to abolish the homology except for short pairing segments at the distal ends of XR and the neo-Y. I n M . intertexta the neo-Y is acrocentric, but is somewhat shorter than X R ; both its differential and pairing segments have become heterochromatic, or at any rate contain numerous heterochromatic segments, while in the case of

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XR only the pairing segment has undergone “heteroohromatinization,” the differential segment remaining euchromatic. I n M . bivittata there are said to be no visible differences between XR and the neo-Y in respect of heterochromatic segments (King, 1950) ; perhaps this only means that such differences are less obvious than in M . iiatertexta. On the other hand, the neo-Y is shorter than XR and is slightly J-shaped instead of being strictly acrocentric, i.e., it has ac-

FIQ.4. Sex chromosomes of Mermiria and Pnratylotropidia. a, b , the XY bivalent of M. intertesta; c, of M . maculipennis macclungi; d, of M . bivittata; e, f, the X,X,Y trivalent of Paratylotropidia brunneri. a, e, in diakinesis, the others in first metaphase. CX, CY, ete., the eentromeres; asterisks represent the positions of the chiasmata. Figures redrawn from Helwig (1929), King and Beams (1938) and King (1950).

quired an additional arm, either as a result of a pericentric inversion, centromere shift, o r translocation of some kind. Finally, in M . maculipennis ,rnucclunyi the neo-Y has become a metacentric element with two arms of approximately equal length. If heteropycnosis be regarded as a sure indicator of genetical inertness, the neo-Y’s of Mermiria show a very interesting series of stages in the “degeneration” of an originally active chromosome in the direction of inertness, while the differential segments of XR must contain a series of sex-linked genes which have become gradually adapted to function

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equally well in the haploid condition as the “degeneration” of the neo-Y proceeded. Probably the first step in the development of the differential segments was the suppression of crossing over between them, either as a result of an inversion or in some other way. Whether the “degeneration” of the differential segment of the neo-Y was the result of genemutation or of recurrent deletions of euchromatic segments and duplications of heterochromatic ones is quite uncertain. I n Hypochlora alba (Cyrtacanthacrinae) a condition essentially like that of M . intertexta exists, the neo-Y being largely heterochromatic and divisible into a proximal differential segment and a distal pairing segment which forms chiasmata with the corresponding pairing segment of XR a t meiosis (King, 1950). Several other cases of X autosome fusions have been reported by IIelwig (1941, 1942), e.g., in Machaerocera naexicana and Philocleon anomalus. Both these species also possess several fusions between autosomes. Perixerus sqzramipennis apparently resembles Hesperotettix uiridis viridis in that the X is fused in some individuals and free in others (Helwig, 1941, 1942). No detailed studies on pairing and differential segments have been made on any of these forms. 2. T h e Sex Chromosome Xechaniswi in the Praying Mantids The earlier workers on the cytology of this group (Oguma, 1921; King, 1931; Asana, 1934) discovered the existence of a complex sex chromosome mechanism in the genera Tenodera, Paratenodera, Mantis, Stagmomantis, and Hierodula, the males being X1Y2Y, the females X1XIXZX2. I n the males of these genera a peculiar sex trivalent is formed at meiosis, X1 being paired with one limb of the metacentric Y while X2 is paired with the other limb. Presumably, in the females two bivalents are formed, XlXl and X2X2, h u t this has never been actually confirmed. Later work (White, 1938, 1941a ; Hughes-Schrader 1943a,b, 1948, 1950 ; Matthey, 1949 ; Oguma, 1946) has shown that not all Praying Mantids possess this complex sex chromosome mechanism, the males of the genera Callimantis, Acontiothespis, Iris, Empusa, Gongylus, Ameles, Miomantis, Angela, Liturgousa, Apteromantis, Humbertiella, Aethalochloa, Creobroter, Didymocorypha, Schizocephala, and Toxomantis being simply XO. I n all these genera the X is a fairly large (sometimes very large) metacentric element which is heterochromatic a t various stages of meiosis. There are thus, from a cytological standpoint, two groups of mantids, a large one in which the males are XO and a somewhat smaller one in which they are X1XZY. Later work has shown that the genera

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Sphodromantis, Statillia, Melliera, Stagmatoptera, Tauromantis, Vates, Phyllovates, Antenna and Choeradodis also belong to the XlX2Y group. Tn all these genera the three sex chromosomes are invariably metacentric ; the sizes and shapes of all three elements vary to some extent throughout the series of forms which have been invextigatetl. These fourteen XIXzP genera, together with a number of obviously related genera such as Rhombodera, Rhomboderella, Polyspilota, and Epitenodera (which have not yet been studied cytologically) seem to form a natural close-knit group which we shall refer to as the Mantinae s e w stricto. This group, which contains several hundred species at the present time, is preponderantly Old World in distribution, but seven of the genera are American and Choeradodis (which haa until now been placed in a separate family or subfamily, probably on inadequate grounds) is represented in both the Neotropical and the Oriental regions. If we define the Mantinae sensu stm’cto as those mantids which possess an XlX2Y sex chromosome mechanism, this excludes such genera as Callimantis and Miomantis, currently included in the subfamily Mantinae by taxonomists, since these are XO in the males. There can be little doubt that the XlX2Y forms are a monophyletic group which must be descended from a single species in which the complex sex chromosome mechanism first arose from the more primitive XO condition (White, 1941a; Hughes-Schrader, 1950). This is, in fact, an almost unique case in which strict monophyletism (i.e., the origin of a large number of genera and species from a single ancestral species) can be regardkd as decisively proved on cytological evidence (supposedly analogous instances based on paleontological evidence are always open to the suspicion that the fossil record may be incomplete). Presumably the XIXzY mechanism arose through a structural rearrangement (or rearrangements) in a previously XO species, The simplest, and in our opinion most probable, explanation is to suppose that a single mutual translocation between a metacentric X and a metacentric autosome (both breakage points being close to the centromere) converted the original XO mechanism into the XIXZY mechanism without any intermediate steps. On this view, the Y chromosome represents the homolog of the autosome involved‘in the translocation which has become confined to the male line, and may be considered as a “neo-Y.” The X,X2Y mechanism of the Praying Mantids seems to be rather ineaiient, since failure of pairing and malomentation of the trivalent on the spindle lead not infrequently to nondiajunction (White, 1941a).

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3. flex Chromosomes in Phasmids

Hughes-Schrader (1947) lists 17 species of bisexual Phasmids which are XO in the male sex. With the exception of Parasosibia parva which has a n acrocentric X (Favrelle, 1934), the sex chromosome is always metacentric. Isagoras schraderi is unique in having an XY :XX sex chromosome mechanism (Hughes-Schrader, 1947). This presumably arose as a result of some type of structural rearrangement (or possibly several rearrangements) during the recent evolutionary history of the species, since two other members of the genus have XO males, which undoubtedly represents the primitive condition. It is not possible to state just how the XY mechanism of I . schraderi arose, but it is fairly clear that a pair of autosomes became included in the sex chromosome mechanism. Both the neo-X and the neo-Y are large metacentric elements. Observations on the prophase stages of meiosis suggest that each has one heterochromatic and one euchromatic arm: chiasma formation is apparently restricted to the euchromatic arms, which probably represept material which was originally autosomal. 4.

The flex Chromosome Mechanism of Eneoptera surinamensis

According to Piza (1946) the South American Cricket Eneoptera surhamensis possesses a multiple sex chromosome mechanism of the following type : XYlY2 (13): XX (?). Of the three sex chromosomes, the X and YI are large metaccntrics, Y2 being a rod-shaped element. There are only 3 pairs of autosomes, so that 2N = 9 (d),8 ( 0 ) . The most extraordinary feature of this case is that, according to Piza's account, no chiasmata are formed between the sex chromosomes in the meiosis of the male : nevertheless the two Y chromosomes regularly arrange themselves in the opposite half of the spindle to the X, so that they pass together to one pole a t anaphase, the X passing to the opposite pole. Until this case has been reinvestigated and related species examined, it is useless to speculate as to how this peculiar sex chromosome mechanism could have arisen. 5 . The Case of Paratylotropidia brunnem'

The North American genus Paratylotropidia includes three very rare species of grasshoppers related to the common Melanopli. Only one of these, P . brunneri, has been studied cytologically, by King and Beams (1938). According to their account, the sex chromosome mechanism is of the XIXzY ( S ) : XIXIXzXp( 0 ) type. The X1 and Y are

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metacentric elements, Xz being an acrocentric chromosome. In both sexes there is one pair of metacentric autosomes and seven pairs of acrocentrics, so that 2N(d) = 19 and 2N(?) = 20. As in the case of the Mantids mentioned earlier, the three sex chromosomes form a trivalent at meiosis in the male (Fig. 4). One limb of the XI, which presumably represents the original X chromosome, is heterochromatic and always unpaired a t meiosis. The other limb of XI pairs with one limb of the metacentric Y, the other limb of the Y being paired with XZ. Thus the trivalent consists of a chain of three chromosomes, in which the X’s form the two end members and regularly pass to the same pole, while the Y lies in the middle and passes to the opposite pole a t the first anaphase. It will be apparent that the number of chromosome limbs in P . brunneri (neglecting the minute “second arms” of the acrocentric elements) is 23 in the male and 24 in the female, i.e., the usual number in the Acrididae. The chromosome complement of P . brunneri has apparently arisen from the usual one in which all the chromosomes are acrocentric as a result of three centric fusions : (1) between two autosomes to give the metacentric autosomal pair, (2) between the original X and an acrocentric autosome which we may call A, (3) between the homolog of A and another acrocentric autosome which we may call B. The second fusion would have created XI, the third would have given rise to the neo-Y, composed of two originally acrocentric autosomes A and B. X2, in all probability, is simply the original B chromosome. We have no means of determining the sequence in which the three fusions occurred. As in the case of Mermiria intertexta and Hypoclhlma alba (see page 293) the neo-Y of P. brumem’ has become largely heterochromatic, a fact which was earlier regarded by us (White, 1940b) as fatal to the simple hypothesis that the X1X2Y mechanism had arisen through two centric fusions, but which need no longer be so regarded in view of King’s (1950) clear evidence in the case of Mermiria and Hypochlora that “ heterochromatinization” of the neo-Y has occurred since the establishment of the fusion in the species. XIR and Xzboth possess small distal heterochromatic segments which pair with the two ends of the neo-Y a t meiosis ; their long differential segments are apparently euchromatic. A cytological investigation of the other two species of the genus would certainly be of interest.

6 . Anomalies of Hex Determination Gynandromorphs have been described in the Tettigoniids Iltsara elegans (Rehn and Hebard, 1914) and in Amblycmypha r a t u d f d i a

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and A . oblongifolia (Pearson, 1929). I n the case of the last species two gynandromorphic individuals were found on the same shrub, suggesting that a genetic factor leading to the production of gynandromorphs may have been present in the population. I n the Acrididae single gynandromorphic individuals have been recorded in Oedalemdus phryneicus (Hebard, 1919), Podima sapporoensis (Natori, 1931), MelmopEus adelogyrus (Hubbell, 1932), M . mexicanus (Severin, 1943), Anacridium moestum (Potter, 19401, Camlzuln pellucida (Paul, 1941 ; Friauf, 1947), Parddophoru phoenicoptera (Friauf, 1947), and in a hybrid between Trimerotrqn‘s maritimn and T . citrina obtained by Carothers (1941). Most of these cases were not investigated cytologically, so that their exact mode of origin remains unknown. Pearson’s Amblycorypha gynandromorphs were definitely XX in the female tissues, XO in the male portions ; they could accordingly have arisen through double fertilization of a binucleate egg or through elimination of a n X during certain of the early cleavage divisions. We shall deal later (page 318) with the question whether some of the very rare “male” individuals which have been recorded in certain of the parthenogenetic species of Walking Sticks may not have been gynandromorphs rather than true males. The only extensive investigation on intersexuality in the Orthopterous insects is that of Ohmachi (1935b, 1940) on a strain of the cricket Homoeogtyllus japonicus which produced, in addition to normal males and females, genetically male individuals which showed a certain degree of “feminization,” particularly in the structure of the tegmina. The breeding results indicated that a sex-linked factor was responsible for the “feminization” of these males. VII.

CYTOLOQICAL POLYMORPHISM IN THE ORTHOPTEROID INSECTS

By cytological polymorphism we mean the existence within a population of two or more cytologically distinguishable types of individuals. Most types of cytological polymorphism are due to structural rearrangements of chromosome parts having taken place during the evolution of the species, but we also include under this general heading the existence of supernumerary chromosomes which may be present in certain individuals but absent in others. Apart from these supernumerary chromosomes, there are a number of cases on record where supernumerary chromosome regions may be present, attached to or included in one of the members of the normal chromosome set which may also be present without the “extra” region. Paracentric inversions, the type of structural rearrangement which is so characteristic of many species of Drosophila and some other Dip-

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tera, have only been recorded in two or three species of grasshoppers. I n the absence of “salivary gland chromosomes” of the Dipteran typf one can detect such inversions by studying the pairing of the chromosomes a t pachytene (an extremely laborious method) or by observing dicentric and acentric chromatids (the result of crossing over in mutually inverted segments) at the meiotic anaphases. Darlington (1936)studied the meiosis of five individuals of Chorthippus parccllelus and four of 8tauroderzls bicolor from southern England. Apparently, all but one individual of the latter species were heterozygous for paracentric inversions. Coleman (1947)has stated that paracentric inversions are common in grasshopper species, without giving any details. Our own experience, based on very ext,ensive studies of over 50 species of Acrididae does not support Coleman’s view, since we have never found unequivocal evidence of such inversions, “False bridges,” due to the sticking together of some of the chromatid ends at first anaphase, may look deceptively like true dicentric chromatids ; they are fairly common in some species, and may have been regarded as evidence for inversions by some workers. We are convinced, however, that paracentric inversions are, in fact, extremely rare in wild populations of grasshoppers, or at least that they are rare in those regions of the chromosomes where chiasmata are regularly formed (there is a possibility that in some species with strict localization of chiasmata, inversions may be present in the regions where chiasmata are seldom or never formed, in which case they would be undetectable by the “bridge and fragment” method). It seems fairly clear that if paracentric inversions occurred in grasshoppers and if chiasmata were formed with any high frequency in the inverted segments, a certain degree of sterility would result, those sperms which contained broken or acentric chromosomes being lethal to the eggs which they fertilized. It is thus not altogether surprising to find that such inversions are at any rate very rare in most species of grasshoppers. There is one remarkable case on record of a grasshopper which was heterozygous €or no less than four different structural rearrangements. The individual in question was a male of C h m t h i p p s longicornis, collected in the wild and studied by Coleman (1947). Three of these rearrangements were reciprocal translocations while one was a pericentric inversion. Other individuals from the same population were apparently cytologically normal. It is conceivable that there was present in this population a gene or genes leading to an extremely high frequency of chromosomal rearrangement. Since the individual with multiple rearrangements was used for cytological studies its fertility was not tested, but must surely have been much lower than normal.

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1. Chromosomal Polymorphism in the Trimerotropine Grasshoppers The term “ Trimerotropine grasshoppers” will be employed here to designate the members of the three closely related genera Trimerotropis (about 44 species), Circotettix (7 species) and Aerochoreutes ( 1 species). This taxonomic assemblage is largely confined to western North America, although a few species of Trimerotropis occur in the eastern United States and two are found in South America. Circotettix and Aerochoreutes have many morphological features in common with certain Palearctic genera such as Bryodema and Angaracris, whose cytology is entirely unknown. It seems certain, however, that the evolution of the Trimerotropine genera has taken place entirely in the New World : if it should eventually be shown that Bryodema, Angaracris, and their allies present the same type of cytological picture as Circotettix and Aerochoreutes, then we would have to regard these genera as evolutionary offshoots of an essentially American group. The cytological interest of the Trimerotropine grasshoppers resides in the fact that many species (although not all) exhibit a n extreme degree of cytological polymorphism in their natural populations. Certain of the species were utilized in the classical studies of Carothers (1917, 1921, 1931), King (1924)) and Helwig (1929), to whom credit for the discovery of cytological polymorphism in this group is due. Later work by Coleman (1948) and White (1949, 1950, 1951, and later unpub. lished data) has greatly enlarged our knowledge of the cytological polymorphism of the Trimerotropi so that it is now possible to present a general account. It will be convenient at this stage to describe the main types of cytological polymorphism observed in the different species : a. Mutual Translocations. An instance of a mutual translocation in a single individual of T9-imerotrop.s citrina was recorded by Carothers (1931). No other cases of this type of structural rearrangement have been recorded in this group of grasshoppers. b. Supernumerary Chromosomes. Several types of supernumerary chromosomes have been found in members of all three genera. Some of these are acrocentric, while others are metacentric and probably represent isochromosomes. The whole problem of supernumerary chromosomes in grasshopper populations is dealt with in a later section (p. 310). c. Supernumerary Chromosome Regims. I n certain species of Trimerotropine grasshoppers there are no supernumerary chromosomes as such, but in certain individuals members of the regular chromosome complement may contain supernumerary regions which are usually lacking. As an illustration of this state of affairs we may take a population of Trimerotropis bilobata which exists in the desert five miles

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Y. J. D. WHITE

north of Las Vegas, Nevada. This species always has 2N = 23 ( b ) , 24 ( O ) , all the chromosomes being acrocentric. One of the autosomes (tenth in order of size) exists in two alternative lengths, a large heterochromatic segment being present in the longer type of chromosome and lacking in the shorter type (White, 1949). Of 56 male individuals studied, 50 were homozygous for the shorter type of chromosome (lOS/lOS), 6 being heterozygotes (lOL/lOS) . No homozygotes for the 1OL chromosome were found in the sample examined. Structural heterozygosity of this type was first recorded by Carothers (1913) in the grasshoppers Rrachystola magna, Arphia simplex, and Dissosteira carolinn. It was later observed by Robertson (1915) in the arouse Locusts Tettigidea parvipennis and Acrydium granulatum, by Wenrich (1916) in Phryndettix tschivavensis, by McClung (1928) in Mecostethus gracilis, by Belar (1929) in Stenobothrus lineatus, by Hearne and Huskins (1935) in Melanopkts femur-rubrum, by Carothers (1931) in Trimerotropis citm‘na, Mecostethus gradis and Amphitornus bicolor and by Darlington (1936) in Stauroderus bicolor. “Unequal bivalents” are thus extremely widespread in the natural populations of many grasshopper species, but their significance in terms of the population dynamics of these forms has never been worked out. The supernumerary regions in the longer member of the pair are probably heterochromatic in all these cases and are hence likely to be of relatively slight genetic significance. We believe, however, that they probably possess some adaptive significance. Possibly a supernumerary region in one homolog raises the viability or fecundity slightly, while two such regions depress it to some extent; if this were so we should expect to find significant deviations between the frequencies of the three types of individuals actually found in the population and the frequencies to be expected on the basis of the Hardy-Weinberg equilibrium, g : 2p (1 -p ) : (1 - p ) 2. The population samples which have been studied were all f a r too small for determining whether such deviations do, i n fact, occur. Ch’en (1942) has reported that in &4rcyptera coreana, a grasshopper very closely related to Mecostethus spp., a bivalent may be “unequal” (S/L) in some cells and equal (L/L) in other cells of the same individual. Since no details are given, it is not possible to assess the significance of thi8 surprising observation. d. Centrmnere Shifts. The Trimerotropine grasshoppers belong to a subfamily (Oedipodinae) in which the chromosomes are almost invariably acrocentric. However, in a t least 12 species of Trimerotropis and in all the members of the other two genera which have been studied certain of the chromosomes have become metacentric through shifts or transpositions of the centromere. Where such structural rearrange-

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ments have become completely established in the species and the original acrocentric type of chromosome has become lost in the course of evolution, no chromosomal polymorphism exists, of course, although it may have been present a t some time in the past, before the extinction of the acrocentric type of chromosome. I n many cases, however, both types of chromosome (the original acrocentric and the derived metacentric) still exist a t the present time, so that a particular chromosome pair is represented by two acrocentrics in some individuals, by a n acrocentric and a metacentric in others and by two metacentrics in yet others. It is this type of chromosomal polymorphism which is developed to a unique degree in certain members of the Trimerotropi and has especially claimed the attention of investigators. I n many species structurally homozygous individuals are actually rare, the vast majority of those caught in nature being structurally heteroeygous in respect of one or more chromosome pairs. Studies on the meiotic pairing of the homologous chromosomes in structnrally heterozygous bivalents have shown that there are no loops present at pachytene, such as would be found if the centromere transpositions were due to pericentric inversions ; it is therefore probable that the evolutionary coaversions of acrocentric chromosomes to metacentrics came about through centromere shifts, i.e., through structural rearrangements in which the centromere (probably with a minute chromosomal region on either side) was “lifted” out of its original locus almost a t the end of the chromosome and “inserted” into a n interstitial position (Wenrich, 1917; Coleman, 1948). If we accept the current views on structural rearrangements, each such shift would involve three breaks. The simultaneous occurrence of three spontaneous breaks in a single chromosome may seem highly unlikely, even taking into account the millions of generations of Trimerotropine grasshoppers which must have existed in the past. Rut Rothfels (1950a) has shown that in ChZoeaEtis conspersa, a species of grasshopper belonging to an entirely different subfamily, the rate of occurrence of spontaneous interchanges is approximately 0.01 per cell in the primary spermatocytes, i.e., vastly greater than in Drosophila, Maize, and Tradescantia. Some observations of Klingstedt (1937) on Chrysochraon dispar may also be interpreted as indicating a high rate of spontaneous chromosome breakage in that species. It is thus possible that a similarly high rate of spontaneous breakage existed a t one period in the evolutionary history of the Trimerotropine grasshoppers, although there is no evidence that the rate of breakage is unusually high in any of the present-day species. Of approximately 46 species of Trimerotropis currently recognized by taxonomists, 34 have been studied cytologically. These fall into two

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well-marked groups. In 20 species all the chromosomes are invariably acrocentric ; these forms may consequently be regarded as constituting a primitive (‘section A” of the genus, in which no centromere-shifts have occurred. In the remaining 14 species a varying number of chromosomes have become metacentric ; these species belong to our (‘section B” of the genus. Section B seems to represent a natural grouping, but section A is probably a rather artificial assemblage of species, several of which should almost certainly be removed from the genus Trimero-

FIQ.5. Bpermatogonial metaphases of representative members of aections A and B of the genus Trimerotropis. a, T.pistrimria; b, T.sohaefferi.

tropis. The 5 species of Circotettix which have been studied, as well as the single species of Aerochoreutes, exhibit the same type of cytological picture as the members of section B of Trimerotropis; they may accordingly be regarded aa evolutionary derivatives of the same phylogenetic line. A list of the species included in the two sections of the genus Trimerotropis has recently been published (White, 1950) and will not be repeated here; later work indicates that T . acta and T . melanoptera may be added to section A and T . saxatil& to section B. Of the approximately 12 species of Trimerotropis not yet studied cytologically, we may predict that T . cristata, T . rebellis, T . californicu and T . huasteca probably belong in section A ; the relationships of most of the other species are not sufficiently known for us to be able to hazard a guess as to which section they belong to. Several species groups are still in a state of considerable taxonomic confusion, so that the exact number of genuine species is still uncertain. Apart from the supernumerary chromosomes recorded in T.latifasc b t a (White, 1949), the supernumerary chromosome regions which are present in T . bilobata and some other species, and the translocation recorded by Carothers (1931) for T . citrina, the members of section A do not exhibit chromosomal polymorphism. Within section B there are

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a few species which do not appear to show any cytological polymorphism, i.e., certain chromosomes are invariably metacentric, the remainder being invariably acrocentric, so that structurally heterozygous bivalents are not found. T. palbidipennis is one such species. It has a relatively enormous area of distribution, since it occurs from central Texas and Kansas to the Pacific coast and from the western provinces of Canada to the southern end of the Mexican central plateau; although absent from the whole of Central America, it reappears in Ecuador and extends from there to Bolivia, Paraguay and the Argentine. No cytological stiidies have been carried out on South American material of this species, but material from western North America invariably shows a metacentric X, three pairs of metacentric autosomes and eight pairs of acrocentricn (White, 1949). Supernumerary chromosomes, which are present in so many of the members of section R, have not heen found in T. paUidipennis. Other members of section B which do not seem to show any structural heterozygosity are T. saxntilis, an Eastern species which ranges from North Texas and Oklahoma to the Atlantic coast, T. diversellus, a species which is confined to a small area of Wyoming and Montana, T . schasfleri from the Gulf Coast of Texas and an apparently undescribed species from the vicinity of Beatty, Nevada. Supernumerary chromosomes have, however, been found in T . diversellus and the undescribed species. Except for T. schaefferi and T . saxatilis, which have four pairs of metacentric autosomes, the other species have three such pairs, as in T . pallidipennis. The cytologically polymorphic species belonging to group B are : T. sparsa (White, 1951), T . gracilis sordida (Coleman, 1948), T. gracilis g r u i l k (White, unpublished), T. suffusa (Carothers, 1917 ; White, unpublished), T. verrucdato (Carothers, 1921 ; Helwig, 1929)) T. thalassica (King, 1924), T . cyaneipennis and T . pseudofasciata (King, 1924; White, unpublished) and T. ochraeeipelznis from Chile (White, unpublished). T. sparsa, a form characteristic of certain types of alkali flats and “badlands” in the western United States, seems to be an unusually complex species, consisting of a multitude of microgeographic races which differ in the relative numbers of acrocentric and metacentric aiitosomes, the amount of structural heterozygosity and presence or absence of supernumerary chromosomes, as well as in many features of external morphology (size, color of hind wings, markings on the tegmina, etc.). I n some instances populations only 30 miles apart and not separated by any obvious geographical barrier may differ greatly in cytological characteristics (White, unpublished). The chromosome number is usually

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M. J. D. WHITE

2N d = 23, but material from northwestern Colorado shows only 21 chromosomes, a fusion between two originally acrocentric autosomes having led to a reduction in chromosome number. The X and three pairs of autosomes are invariably metacentric in both the 21 and the 23 chromosome race, but the former possesses a fourth pair of invariably metacentric autosomes, due to the fusion. Of the remaining chromosome pairs, as many as seven are heteromorphic (i.e., they may be either acrocentrio or metacentric) in certain populations. In some localities

e

f

FIO. 6. First metaphases, in side view, of varions trimerotropine grasshoppers. a, T . cyanecipannis (21 chromosome race); b. .'2 cyanecipennis (21 cliromosome race), c, 2'. suffusa (an individual from Truckee, California, with an acrocentric supernumerary) ; d, 2'. sparsa (from Pojoaque, N.M.) ; e, T. pallidipennis; f, Aerochorevtes carlinianus. H, structurally heterozygous bivalent8 ; €3, supernumerary chromosomes; D, "ditactic" bivalent with chiasma in the short arm.

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almost all the individuals of this species are structural heterozygotes, while in others over half are structurally homozygous. Thus the mean number of heterozygous bivalents per individual ranges from about 0.4 (Piceance Creek, Colorado) to about 3.0 (vicinity of Lovelock, Nevada). Similar conditions seem to exist in T. gracilis gracilis, a species whose distribution is coextensive with that of the sagebrush (Arternisia tridentata) in western North America. Here, however, a chromosomal fusion seems to be constantly present so that 2N d is always 21. I n the northern race T. gracilis sordida, on the other hand, 2N cf = 23 according to Coleman (1948). I n T . g . gracilis populations from the extreme eastern fringe of the range of the species (e.g., a t Villagrove, Colorado) show about 2.43 structurally heterozygous bivalents per individual, while in central Nevada (Humboldt Range) the corresponding figure is only about 2.0. One general feature of the members of section B is that the X is almost invariably metacentric. I n T. g . gracilis, however, we have found one population (in Mineral County, Nevada) in which the X is acrocentric in some individuals, its length being equal to the sum of the arms of the metacentric type. Whether this acrocentric X represents the unaltered primitive condition found in section A o r whether it has undergone a n “evolutionary reversion” from the metacentric condition invariably found in most populations of the species is quite uncertain. T . cyaneipennis is a species which ranges from West Texas through New Mexico, Arizona, Colorado, Utah, and Nevada to southern Oregon. Material from central New Mexico shows 2N d = 23, but in western New Mexico and throughout Arizona, Nevada, and Oregon a chromosomal fusion between two autosomal elements is invariably present, so that 2N 8 = 21. Most populations of this species seem to have three chromosome pairs heteromorphic. T. suffwsahas a distribution from the Rocky Mountains to the Californian Sierras and the Cascade Mountains of Oregon. Populations from southern Colorado show a very low degree of structural heterozygosity (about 0.28 heterozygous bivalents per individual in Archuleta County), while those from California show a n extreme development of heterozygosity (about 4.0 heterozygous bivalents per individual near Truclree, California). I n the Californian Sierras at least 6 of the 11 pairs of chromosomes are heteromorphic, while in southern Colorado only 2 or 3 chromosome pairs exhibit heteromorphism. Five populations of the boreal Trimerotropis verruculata (Circotetti$ verruculatus of most authors) from different localities in New England and Michigan were studied by Helwig (1929), the total number of individuals being 295. Each population showed three heteromorphic

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M. J. D. WHITEl

pairs of chromosomes, the relative frequencies of the acrocentric and metacentric chromosomes in the case of each pair being significantly different in the five populations. These differences were, however, f a r less pronounced than those between populations of the closely related T . sufusa from California and Colorado. T . verruculata thus seems to be a relatively “uniform” species, at least in the eastern part of its range, in contrast to such forms as T . sparsa, in which populations only 30 miles apart may be strikingly different in cytological composition. Helwig did not find any supernumerary chromosomes in T . verruculata, whereas in T . s u f w a they are present in nearly all populations. Our knowledge of the cytological conditions in the Californian T . tha,lassicn is due to King (1924) who investigated a sample of 25 individuals from Mt. Lowe. This species seems to exhibit an extreme degree of structural heterozygosity, to which as many as 9 pairs of chromosomes apparentIy contribute. Most of these chromosomes are present i n two alternative types, acrocentric and metacentric, but in some instances there is more than one metacentric type, due to the existence of supernumerary chromosomal regions which may be present in some metacentrics but not in others. The number of cytologically distinct types of individuals in this species (assuming them all to be viable) is hence a t least 2 x 38 ( = 39,366) and the mean number of heterozygous bivalents per individual is about 4.2 according to King’s data. I n the genus Circotettix, C . rabula, C . zlmdulatus, C . crotalurn, and C . coconiw all possess a centric fusion which has reduced 2N S to 21. All these species show some degree of structural heterozygosity, of the same type as that met with in section B of Trimerotropis, but in the very local species, crotalwm and cocmino, this heterozygosity is only developed to a relatively slight extent. Thus in crotdum (confined to the Sheep and Charleston ranges in southern Nevada) and coconino (pine forests of northern Arizona) only one pair OP chromosomes shows heteromorphism (White, 1949; Evans, 1950). I n the widespread forms rabula and zcndulatus, on the other hand, at least 3 pairs of chromosomes are heteromorphic. Aerochoreutes curlininnus has not been studied in detail, but shows the same type of structural heterozygosity. The structural heterozygosity of the members of Trimerotropis (B), Circotettix, and Aerochoreutes is developed to such a high degree and is so constantly present (no cytologically monomorphic population of any of the polymorphic species has ever been found) that it seems clear that it must play some important role in the population dynamics of these grasshoppers. Apparently heterozygosity for centromere shifts acts as an absolute suppressor of crossing over in the region between the centromere of one homolog and that of the other since dicentric and

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309

acentric chromatids are never formed. It is thus theoretically possible that these contromere shifts play a similar role in the Trimerotropine grasshoppers to that played by paracentric inversions in Drosophila pseudoobscura (Dobzhansky, 1947 ; Dobzhansky and Levene, 1948), namely that they hav6 provided the physical basis for the development of a n elaborate self-perpetuating mechanism of adaptive heterosis. AS yet, however, there is no definite evidence for this possibility, and it is in any case unlikely that there is anything more than a general resemblance between the genetic consequences of these two types of cytological polymorphism.

2. Hetcrozygosity for Centric F u s i m Although, on general grounds, we are inclined to believe that nearly all types of cytological polymorphism possess some kind of adaptive value, it is difficult to imagine what advantage a centric fusion could confer on an individual heterozygous for it. Several such cases have been reported in grasshoppers, the heterozygous individuals showing a characteristic trivalent (two acrocentrics paired with the corresponding limbs of a metacentric) at the first meiotic division. A classical case is that of Hesperotcttix viridis viriclis in the region of Marathon, Texas (McClung, 1917; Helwig, 1942), where the individuals may be either homozygous or heteroxygous for two different fusions. Evans (1950) has recently discovered a similar case in Circotettix undulatus, a species which normally possesses a centric fusion, so that 2N 8 = 21. I n the vicinity of Truckee, California, males with 23 and 22 chromosomes were found, the former lacking the fusion, while the latter are heterozygous for it. In a number of other species such as Chortophaga viridifasciata (McClung, 1914) and Sphennrium mexicanurn (McClung, 1932) individuals homozygous for a fusion which is normally lacking have been described, but it is not known in these cases whether heterozygotes occur in nature. I n Trimerotropis cyaneipennis and T . sparsa we have found geographic races distinguishable by the presence or absence of a fusion, and in the latter species at any rate individuals heterozygous for the fusion occur in certain populations. On a priori grounds one might suppose that the trivalent formed in such individuals would sometimes become malorientated a t the first meiotic division and thus give rise to nondisjunction and some degree of sterility.

3. Tramlocations in Wild Populations of Grasshoppers Apart from centric fusions (which probably result from very unequal translocations, in which the breakage points are very near the centromeres) , heterozygosity for mutual translocations has been observed

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in a number of species of grasshoppers. Unfortunately, almost nothing is known about the distribution of such translocations in the wild populations in which they occurred, and in most cases it is not even clear whether the translocation was systemic (i.e., present in all the cell8 of the individual in which it was observed) or confined to a ,small group of cells. Since the rate of spontaneous breakage seems to be extremely high in certain species of grasshoppers (see page 303), we must expect to find individual spermatocytes which are heteroxygous for translocations, even though the individual as a whole is not. Carothers (1931) observed a ring or chain of four autosomes in a single individual of Trirnerotropis citrina from Kingman, Kansas. This was clearly a translocation heterozygote, and since the association of four chromosomes seems to have been observed in numerous spermatocytes, it is quite probable that the whole individual was heterozygous for the translocation. On the other hand, Ray-Chaudhuri and Dutt (1947) describe a single spermatocyte in an individual of Phlaeoba sp., i n which an association of four chromosomes was present: here it seems probable that the translocation was not systemic. Associations of four chromosomes which were almost certainly due to translocations were also recorded in single individuals of Podisma sapporoewis and P . motodomariensis by Helwig (1942). We have already mentioned the individual of Chorthippus longicornis studied by Coleman (1947) which was heterozygous for a translocation as well as for several other structural aberrations. I n the Tettigoniidae, White (1 9 4 0 ~ )has recorded the presence of occasional chains of four chromosomes in the spermatogenesis of M e t r i q tera brachyptera. These were only found in a very small percentage of the spermatocytes, possibly because the regions involved in the translocation were short or have a very low chiasma frequency. This case would not have been regarded as a systemic translocation except that the condition was found in a number of individuals collected in the same locality, in 1933 and 1937. 4. Supernumerary Chromosomes I n certain species of Orthoptera there are present in some individuals chromosomes which are not homologous or a t any rate only partially homologous to the members of the regular set. Such chromosomes may be called supernumeraries; their occurrence is essentially inconstant, that is to say they may vary in number from 0 to 3 or 4 in different individuals of the population. Wherever adequate surveys of wild material have been made on species with supernumerary chromosomes, it has been found that their frequency exhibits geographical variation, i.e.,

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they may be common in certain populations of a species and rare or absent in other populations. Certain European authors use the term “accessory chromosomes ” for these elements, a terminology which seems unfortunate, since the same term was employed by some of the earlier cytologists to designate the sex chromosomes. Supernumerary chromosomes have been studied in the mole crickets Gryllatalpa gryllotalpa (Steopoe, 1939) and G. afrkamz (Makino, Niiyama, and Asana, 1938 ; Asana, Makino, and Niiyama, 1940) a n d in the grasshoppers Camnula pellucida (Carroll, 1920), Truxalis nasuta (Minouchi, 1934), Locusta migrntoria (Itoh, 1934), Neopodimnopsis abdominalis (Rothfels, 1950b) and in various species of Circotettix (Carothers, 1917 ; White, unpublished ; Evans unpublished) and Trimerotropis (Carothers, 1917 ; King, 1924 ; White, 1949, 1951). They are probably much more widely distributed than this meager list suggests, since even in those species i n which they occur they are usually not present in all populations and a considerable number of individuals frequently have to be studied before one is found in which supernumeraries are present. It is possible to distinguish between supernumeraries whose mitotic behavior is normal in the gonial divisions, so that all the first spermatocytes of a single individual contain the same number of extra elements and those which are liable to undergo mitotic nondisjunction in some of the spermatogonial divisions, so that the later cell generations in the testis show varying numbers of supernumeraries. The supernumeraries of Circotettix and Trimerotropis spp. seem to belong to the former category, while those of Camnula peZl?bcida (Carroll, 1920) fall in the latter category. I n the genus Trimerotropis, supernumerary chromosomes have been found in T . 1ntifascia.ta (section A ) and in six members of section B ( T . suffusn, T . gracilis, T . sparsa, T . diversellus, T . cyaneipennis, and an undescribed species from Nevada). I n the genus Circotettix, they occur in C . undulatus and C . rabula and they are also found i n the closely related Aerochoreutes curlirtianus. As far as is known a t present, supernumeraries do not occur in T . pnllidipennis, T . saxatilis, T. schaeferi, T . verru,czclata, T . caeruleipewnis, T . thalassica, C. crotalum, and C. GOc o i h $ o , but i t is quite possible that they may eventually be found in some of these species when more material is studied. On the basis of chromosome morphology, two types of supernumerary elements exist in the Trimerotropine grasshoppers, a n acrocentric type and a metacentric type. I n some species only one type has been found, but in such species as T . sparsa and T . suffusa both types occur, often in the same population. I n all cases these supernumeraries are

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heterochromatic and show intense positive heteropycnosis during the prophase stages of the first meiotic division, in spermatogenesis; on the other hand they are not heteropycnotic in the spermatogonial divisions, when the X chromosome shows negative heteropycnosis ; there is hence no reason to suppose that they are in any way homologous to any part of the X. The meiotic behavior of the acrocentric and metacentric elements differs considerably. A single acrocentric supernumerary appears as a stout rod in diplotene-dialrinesis, resembling the X a t that stage very closely. Where two acrocentric supernumeraries are present they normally form a bivalent, being united by a single chiasma (White, 1951). Univalent acrocentric supernumeraries usually pass undivided to one pole a t the first meiotic division and only divide in the second division ; where a supernumerary bivalent is present, however, it usually divides into its constituent chromosomes at first anaphase. The behavior OP the metacentric supernumeraries is entirely different. When only one of these elements is present it invariably appears as a n almost spherical body during the prophases of the first meiotic division. This appearance is produced by the pairing of its two arms with one another. It is thus fairly clear that such metacentric supernumeraries are actually isochromosomes, in which the two arms are homologous and undergo pairing a t zygotene. It is difficult to make out any details in the very condensed spherical supernumeraries seen a t diplotene, diakinesis, and first metaphase, but we believe that their arms are probably joined by an intrachromosomal chiasma which terminalizes. These spherical supernumeraries usually attach themselves to the spindle of the first meiotic division near one of the poles and do not divide until the second anaphase. Where two metacentric supernumeraries are present they usually do not pair, each one forming a spherical univalent; the segregation of such univalents a t first anaphase is entirely random, so that two of them pass to the same pole in 50% of cases. Occasionally, the two metacentric elements pair to form a bivalent and are united by one or two chiasmata a t first metaphase. I n this case the supernumerary bivalent always orients itself on the equator of the spindle, so that one chromosome passes to each pole at anaphase. I n those few individuals of T.sparsa, T.suffusa, and C.undulatus in which three rnetacentric supernumeraries were present these may either form three univalents, a bivalent and a univalent, or a single trivalent. As before, univalent supernumeraries always attach themselves to the first meiotic spindle near one of the poles, while bivalents and trivalents attach in the equatorial plane. Some idea of the frequency of supernumerary chromosomes in natu-

TABLE 5 Supernumerary Chromosomes in the Trimerotropine Graashoppers Pereentage of individuals having 1, 2, 3

1 Species

Locality

T . htifasciata

Roswell, N. M.

T . sparsa

Craig, Colo. Whitewater, Colo. Pojoaque, N. M.

T . suffusa T . diversellw

C. undulatus C. rabula

Truckee, Calif. Archuleta Co., Colo. Yellow&one Nat. Park Ashton, Idaho Cowles, N. M. Carbondale, Colo.

1Unpnblished data of Y. J. D. White,

W. L. Evans, and

2

3

supernumerary supernumeraries supernumeraries 11.54

....

....

6.85

3.48

....

....

12.23

9.52 9.30 10.70 17.91 13.04

....

d

. . . supernumeraries'

.... 1.06

....

.... 0.53 0.53

2.33

....

....

....

7.14 4.35

....

3.57

....

....

No. oy individwb

studied 26

30 B

32 0

m

0

r

73 86 188 189 43 56 28 23 25

Q. Kone.

w c1 w

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ral populations of the Trimerotropine grasshoppers can be gained from Table 5, in which no distinction is made between the different types of supernumerary, It will be seen that the percentage of individuals with supernumeraries ranges from about 10% to 30%. I n Bibracte b i m u Zeta (subfamily Cyrtacanthacrinae) , however, Powers (1942) has reported that, out of a sample of 18 individuals, 12 possessed a single supernumerary and 2 had two supernumeraries. The functions of the supernumeraries in the population dynamics of those species which possess them are still somewhat mysterious. The fact that they are almQst always heterochromatic suggests that their genetical activity is in all probability relatively slight. I n a species such as Trimerotropis sparsa the supernumeraries are clearly not necessary, either for the life of the individual or for that of the population, since most individuals and some populations lack supernumeraries completely. On a priori grounds we must suppose that if they were deleterious to viability or fertility they would be eliminated by natural selection and that, conversely, if they were beneficial, regardless of “dosage, ’ ’ then they would undergo an evolntionary increase in frequency until they were present in all individuals. The situation actually found in the Trimerotropi is consistent with the hypothesis that under most conditions a single supernumerary raises the viability or fertility of the individual slightly while two or more supernumeraries decrease fitness to a slight extent. Obviously we cannot consider the genetic properties of supernumeraries apart from (1) the genetic constitution of the individuals and populations in which they occur and (2) the environment which those individuals and populations inhabit. Thus the Whitewater, Colorado, population of Trimerotropis sparsa may lack supernumeraries by mere chance (“drift” or “Sewall Wright effect”) or because they are deleterious in view of either (I) the particular genetic composition of the Whitewater population or (2) the physical and biotic environment in which the Whitewater population lives. I n those populations in which the supernumeraries reach a high frequency (e.g., in the Ashton, Idaho, population of C. unrlulntus and the colony of Bibracte referred to by Powers) it is possible that the deleterious effect of the supernumeraries does not begin until three or more are pisesent in the same individual. I n a single individual of Neopodismop.sis abdominalis Rothfels (1950b) has described two types of supernumerary chromosome, a large heterochromatic type similar to the X chromosome in appearance but slightly smaller (“X,,”) and a small euchromatic type (F). The individual in question appears to have been basically of the constitution X,X,F, i.e., it had two of the large supernumeraries and one of the small ones, but some cysts were X,F or X,X,, due, in all probability, to mitotic

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nondisjunction of the supernumeraries in the divisions of the primordial spermatogonia. The two X,chromosomes frequently, but not invariably, form a bivalent during meiosis. Rothfels interprets the X, chromosome as having been derived from the X, through deletion of the distal end, but there is no conclusive evidence on this point, and no observations were made on early spermatogonial divisions to determine whether X, shows the “reversal of heteropycnosis” which seems to be diagnostic of X heterochromatin (as opposed to autosomal heterochromatin) in the Acrididae. The small euchromatic supernumerary may possibly have been derived from autosome 9, since in one cell it appeared to be paired with it. Robertson (1917) described a supernumerary chromosome in a Grouse Locust, Tettigidea parvipercnis, which resembled the larger category of supernumeraries studied by Rothfels in Neopodismopsis. He, also, regarded the supernumerary as an X chromosome from which some region or regions had been deleted (since it was somewhat shorter than the normal X ) . We are exceedingly doubtful if this assumption was correct, in view of the fact that the individual in which the supernumerary occurred was a normal male and not a n intersex. A particularly interesting case of a grasshopper with a n extra chromosome has been reported by Callan (1941) in Mecostethzts grossus. The individual in question was one of 30 males collected a t a locality in southern England. Callan interprets this individual as a trisomic, i n which the third largest chromosome is represented three times. Since no trivalents were observed and the meiotic prophases are not described, this interpretation is hypothetical and the possibility that the extra chromosome represented a supernumerary cannot be ruled out. I n half of the first meiotic divisions the extra chromosome passed undivided to one of the poles, while in the other half it lagged on the spindle; in the latter case the normal elongation of the spindle failed to take place a t telophase and a diploid restitution nucleus was formed. Thus approximately half of the sperms formed by this individual contained a diploid set of chromosonies together with the extra element. Outside of the Acridoidea, there are very few well-authenticated records of supernumerary chromosomes in the Orthopteroid insects but in the Mole Cricket Gryllotalpcc africa.na from India Malrino, Niiyama, and Asana (1938) and Asana, Dlakino, and Niiyama (1940) have recorded supernumerary chromosomes in some individuals. Out of a total of 49 males studied, 4 possessed one supernumerary and 3 had two. The supernumeraries of Q. africama are much smaller than the regular autosomes, but their mitotic behavior is regular. Where a single supernumerary was present it underwent segregation at the first meiotic division :

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when two were present. they regularly formed a bivalent which behaved normally at meiosis. It will be recalled that Steopoe (1939) also found supernumerary chromosomes in Rumanian material of a. gryllotdpa. 5.

tJnequal Autosomal Bivalennls in Phasmids De Sinhty (1901)long ago described in the spermatogenesis of the Walking Stick Leptynia attenuata a bivalent composed of two chromosomes of unequal size. This was interpreted as due to the fusion of the X with one member of a pair of autosomes, such as has occurred in a number of species of grasshoppers (see page 293). A reinvestigation of de Sinety’s material by Cappe de Baillon and de Vichet (1940) has shown, however, that the bivalent in question is an unequal autosomal bivalent, the X being an entirely separate chromosome. Unequal autosomal bivalents have also been described by HughesSchrader (1947) in three species of Phasmids from Central America, Isapras subaquilus, Isagoras sp., and Pseudophasma menius. I n the first and the last of these three species there is in some male individuals an unequal autosomal bivalent composed of an acrocentric and a metacentric element, the overall length of the metacentric being considerably greater than that of the acrocentric. I n the single individual of Isagoras sp. studied there were two unequal bivalents; one of these resembled the one found in I . subaquilus, while the other was composed of two metacentric elements of different sizes. Although the number of individuals studied by Hughes-Schrader was extremely small, it is clear that a number of species of Phasmids belonging to the subfamily Pseudophasmatinae have genetic systems involving structural heterozygosity for supernumerary chromosome regions in a considerable fraction of the population. It is uncertain whether these supernumerary regions are heterochromatic or euchromti tic, although the former seems likely, on general grounds. VIII.

PARTHENOGENESIS I N THE ORTHOPTEROID GROUPS

I n a few species of Orthopteroid insects parthenogenesis is the normal method of reproduction, males being either unknown or extremely rare. Apart from these cases, there are a number of instances on record i n which unfertilized eggs of normally bisexual species are known to have undergone development. We shall deal first with those instances in which parthenogenesis is the normal method of reproduction. Among the Roaches, Pycnoscelus Suriname?& is the only species in which the existence of parthenogenesis has dafinit.ely been confirmed. This tropicopolitan Xpecies is apparently

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bisexual in Malaya, but parthenogenetic in Africa, Central and South America, and in greenhouses in Europe and North America, where it has accidentally been introduced. The females have 38 chromosomes, and i n the eggs of the parthenogenetic “race” there are two maturation divisions, both mitotic in type, so that. no chromosomal reduction occurs (Matthey, 1945, 1948a). It is not quite clear whether the chromosotnes pair during the zygotene stage, but if so they fall apart soon after and no chiasmata are formed. A single male individual which appeared in Matthey’s cultures was XO (37 chromosomes) ; its meiosis was entirely normal, 18 autosomal bivalents being formed. There can thus be no reasonable doubt that P . surinamensis is a diploid species, in spite of Suomalainen’s earlier (1945) suggestion that it might be a triploid. Apparently, facultative parthenogenesis can occur, even in the bisexual “race” from Malaya, if virgin females are kept in isolation; the cytology of this facultative parthenogenesis has not been studied, but the details are probably different from those in the regularly parthenogenetic form, since pairing and chiasma formation presumably occur. Since P . surinamensis has an ameiotic type of parthenogenesis, all the offspring of a particular female should be genetically identical to one another and to their mother. Among the Mantids, Brwnneria borealis is the only species in which parthenogenesis is known to be the normal method of reproduction. No males of this species, which inhabits the southern states of the United States, have ever been recorded. The maturation divisions of the egg have not been studied, so that it is not known if any effective meiosis occurs. The species is definitely diploid, however (White, 1948) since its somatic chromosome number is no higher than in related forms, and since there are a pair of large metacentric chromosomes which are ohviously present in the diploid state. In the African Mimantis savignyi, Adair (1924) showed that nnfertilized eggs were capable of parthenogenetic reproduction, but this species is normally bisexual. Among the Crickets, parthenogenesis is known to be the normal method of reproduction in Myrmecophila acervorum, which lives in ants’ nests in Europe. No cytological studies have been carried out on this species. A number of other species of the genus are known to be bisexual. I n the Walking Sticks (Phasmoidea), facultative parthenogenesis is probably rather widespread, but it seems to be only in certain Old World genera such as Carausius, Bacillus, Clonopsis, Leptynia, and Eurycnema that parthenogenesis has become the normal and exclusive

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method of reproduction in some species (most of these genera also have some bisexual species as well). It is only in Carausks morosus, an Indian form which is frequently reared for experimental purposes in European laboratories, that the maturation divisions in the egg have been studied in detail. I n thi8 species, whose reproduction is entirely parthenogenetic, l’ehani (1926) showed that two meiotic divisions take place in the egg. During the first of these, pairing and chiasma formation apparently occur. The parthenogenesis of C. rrwrosus is thus of the meiotic type and a chromosomal reduction presumably occurs. Ju st how the chromosome number is restored, whether by a fusion of the products of the second maturation division or by a, fusion of the cleavage nuclei in pairs, is not known. There has been considerable discussion as to whether any of the parthenogenetic Walking Sticks are polyploid or not. The matter is complicated by the fact that a rather considerable range in chromosome number exists even among the bisexual species of this group (see page 271). I n a number of the parthenogenetic species a few males have been recorded, either in the wild, or in laboratory stocks. I n most cases, however, some doubt exists as to whether these individuals were true genetic males, XO in constitution. I n the case of Clonwpsis gallica we can be certain that paxthenogenesis is the normal and exclusive method of reproduction in nature. Since 1876 seven supposed males of this species have been recorded; but Cappe de Baillon and de Vichet (1935) have shown that these were probably all individuals showing a mixture of male and female anatomical characters-whether they should be regarded as gynandromorphs or intersexes is not clear from the evidence. The few rare “males” which have been found in laboratory stocks of Carausius morosus by various European workers were probably similar in nature (Cappe de Baillon, 1931). The same is probably true of another parthenogenetic species of Walking Stick, Leptynia hispanica. Here Cappe de Baillon and de Vichet (1940) studied the spermatogenesis of a supposed “male” which was found among a large number of female individuals. Meiosis was highly irregular and no functional sperms were formed, thus suggesting that the individual was an intersex. Spermatogenesis was apparently somewhat more normal in two “males” of Carausius morusus studied by Pehani (1925) ; but even here a number of chromosomes seemed to be present as unpaired univalents during the first meiotic division. It thus appears that, as a general rule, the supposed males in these parthenogen-

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etic species of Walking Sticks are probably genetic intersexes in which spermatogenesis is to some extent abnormal. 1. Parthenogenesis and Polyploidy in Saga pedo Saga pedo is a large wingless Tettigoniid, whose distribution ranges from Spain to southern Russia, by way of the south of France, Italy, Switzerland, Austria, and the Balkans. It is an extremely rare species which exists in small, isolated colonies, at least in the eastern part of its range. About 15 other species of the genus Saga are known from the Balkans, Turkey, Palestine, Transjordan, and Persia; as f a r as known, all these other species are bisexual. S. pedo has a f a r wider range than any other members of the genus and also extends over a vast, area where no other species of Saga exists, a t least a t the present time. Saga pedo has long been known to consist solely of females, reproduction being exclusively parthenogenetic (two doubtful records of supposed males exist in the older literature). The cytological picture has been worked out by Matthey (1941, 1946, 1948b), using material from Switzerland and the south of France. The somatic number is 68, there being 12 large metacentric chromosomes and 56 acrocentrics. I n the maturation of the eggs no pairing of the chromosomes takes place and there is only one maturation division, which is a simple mitosis, not involving any chromosomal reduction. The parthenogenesis of this species is consequently of the ameiotic type (White, 1945), no crossing over can occur, and all individuals receive exactly the same complement as their mother possessed. Sibs should be genetically identical, as in the case of monovular twins, except for newly arisen mutations. The chromosome number of 8. pedo is so much higher than that of all other Tettigoniids which have been studied that Matthey (1941) suggested that the species was probably a tetraploid. This hypothesis was later confirmed by a cytological study of three bisexual species of the genus (Matthey, 1946, 1948b, 1950; Goldschmidt, 1946). Males of Saga gracilipes and 8.cappadocica have 2N = 31 and those of 5. ephippigcra have 2N = 33. I n these three species all the autosomes are acrocentric, only the X being metacentric. Obviously the chromosome number of 8. pedo is exactly twice that of 8. ephippigera; however a “hypothetical ancestor” of the parthenogenetic species should have possessed two pairs of metacentric autosomes in addition to the metacentric X. We have previously pointed out (White, 1945) that in an ameiotic tetiaploid such a s S. pedo mutation will probably be of almost no sig-

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nificance, since all newly arisen mutations will be “balanced” by three doses of the original allele and there will be no chance of their becoming homozygous. The evolution of 8. pedo is probably “frozen” by its peculiar genetic system and adaptation to new environmental conditions is probably impossible. It may be for this reason that 5. pedo has the character of a “relic” species, being extrerncly rare even in the isolated colonies in which it apparently lives. Saga pedo is the only species of Tettigoniid which normally reproduces by parthenogenesis but virgin females of a number of other species (e.g., Leptophyes punctatissima) are capable of laying eggs that occasionally hatch (Cappe de Baillon, 1939). 2. Parthenogenesis

ilz

the AcridoicEea

I n the Acrididae, no species is known to be normally parthenogenetic, but several workers have found that virgin females will lay eggs and that these sometimes undergo development. King and Slifer (1934) obtained hundreds of such eggs from Melanoplus diferentialis, Romdea microptera, Arphk sulphurea, Dissosteira carolina, Pardalophora apkulata, and Trimerotrqn‘s maritima interior, but only about 20 adults were obtained, most of the embryos being highly abnormal and inviable. Cytological study showed that the maturation divisions took place normally in unfertilized eggs. Following on the second meiotic division, development was sometimes arrested, but frequently continued, the haploid female pronucleus undergoing a series of cleavage divisions. I n a few instances diploidy was restored before cleavage began ; such individuals were then capable of further development. Most of the abnormal embryos which died were probably mosaics of haploid and diploid tissue, the latter resulting from chromosomal doubling without cell division during certain of the cleavage mitoses. King and Slifer’s work suggests that haploid tissue is either inviable or at any rate has a very low viability in grasshoppers. The reason for this is not entirely clear; the authors consider Darlington’s (1932) suggestion that lethal genes might be responsible, but reject this explanation, because the parthenogenetic offspring of females which had been produced parthenogenetically (and which would have been lethal-free if they arose from haploid eggs in which diploidy had been restored by fusion of cleavage nuclei) showed the same low percentage of viability as the first generation produced by parthenogenesis. In Ghorthippus loltgkormis unfertilized eggs also initiate development, but the embryos usually die, according to Creighton and Robertson (1941). Here, also, the eggs go through both meiotic, divisions and

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henee start o u l wilh the haploid number of chromosomes, although diploidy may be restored in some of the cleavage nuclei a t a later stage (Creighton, 1938). No species of Grouse Locust is known to reproduce by obligatory parthenogenesis, but in Paratettix texanus, P . aztecus, Apotettix eurycephalus, and Tettigidea lateralis eggs which have not been fertilized (e.g., those of virgin females) may undergo parthenogenetic development (Nahours, 1919, 1925, 1929, 1930 ; Robertson, 1925, 2930, 1981). The offspring ilrr iilriiost, inviiriably Teiiiale ; parthenogenetic progenies show segregation in respect OP genetic factors for which the mother is heterozygous, bnt the individuals produced by parthenogenesis were almost invariably homozygous for all the factors studied by Nabours. Cytologically, the individual “parthenotes ” were diploid. According to Robertson, if the egg is not penetrated by a sperm, the second meiotic division is abortive. I n the early somatic divisions of these parthenotes there is a marked tendency for the homologous chromosomes to lie side by side; apparently in some cases the division of the centromere which should take place at the second meiotic division never occurs, so that in the cleavage divisions one finds ‘‘diplochromosomes’7 twice as broad as normal chromosomes. If Robertson’s interpretation is correct, there is an important difference between the method of parthenogenetic development in the Acrididae and the Tetrigidae, since in the former group the second meiotic division is completed, in the absence of the sperm, while in the latter it is abortive. The homozygosity of the parthenotes is explicable if all the genes studied by Nabours lie relatively near to the centromere, i.e., proximal to the first chiasma. It is more difficult to explain the very rare males which were found in a few of the parthenogenetic progenies, but they probably arose as a result of some kind of nondisjunction.

IX. GENETICWORK The ntirnber of strictly genetic investigations which have been carried out on the Orthopterous insects is not large. By f a r the most extensive series of studies are those carried out by Nabours and his rollaborators on certain species of Grouse Locusts (Tetrigidae) (general reviews by Nabours, 1929,1937,1950). The work was mainly concerned with the strikingly different color patterns found in the highly polymorphic populations of several species, but some other factors such as a lethal, a gene controlling the length of the wings in Tettigidea purvipennis and some X-ray-induced translocations were also studied. I n Parafettix texnnus a total of 26 color factors were identified in

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material froiii Hoiith Texas and Mexico and formed the basis f o r the genetic work. Nearly all these were doiniiiant to an overall gray color which is a universal recessive, but exhibit no dominance among one another, the patterns produced by two or more such factors in a single genotype being “superimposed” in the phenotype. All these patterns are diagrammatically clear and do not seem to be influenced by temperature, humidity or other environmental factors to any appreciable extent. The 26 factors appear to lie in three linkage groups, one of which contains 24 of them, while the other two each contain one factor. Of the 24 linked factors, 22 have never showri any crossing over between one another and iriay be multiple alleles at H single locus. The gene H m shows less than 1% crossing over frorn the multiple locus, while Theta shows 25.34% crossing over from the multiple locus in males and 47.58% crossing over in females. I n Apotettix eurycephdus 17 dominant patterns, one recessive pattern and a lethal factor were studied. All these appear to be located in a single chromosome, the smallest of the six pairs of autosomes. The dominant factors are distributed over five different loci, there being several series of multiple alleles. I n this species crossing over has only been observed in the female, all the seven loci exhibiting complete linkage in the male. Since chiasmata are undoubtedly formed during spermatogenesis, it is probable that the apparent absence of crossing over in the male Apotettix euryoephalus as well as the difference in cross over values between the two sexes in Paratettix texanus results from a different type of chiasma localization or a different overall chiasma frequency in the male and female. The wild populations of Paratettix and Apotettix consist mainly of individuals, the individuals carrying the various dominant factors forming about a quarter of the total population. Fisher (1930) first showed, on the basis of Nabours’ breeding results with Apotettix euryocphalus, that the heterozygous individuals of the constitution +/A (where represents the universal recessive and A any one of the dominant factors) possessed (under the environmental. conditions of the breeding cages) a selective advantage over the homozygous dominants A/A. It was a t first uncertain whether they were also a t a selective advantage over the homoeygous recessives (+/+),but later work of Nabours (1950) makes it clear that this is so. Even in the case of the lethal, individuals of the constitution le/+ are superior to those homozygous for the absence of the lethal. Assuming that the heterozygotes are also a t a selective advantage over both homozygous types in the wild, we have a n adequate explanation for the maintenance of genetic polymorphism in the natural pop-

+/+

+

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ulations of these grouse locusts. The position is, however, more coinplex than this, since in wild populations of Paratettix texanzis “double dominants” (i.e., compounds of two different dominant factors) are selectively eliminated a t a rate estimated at 40% in each generation (Fisher, 1939). I n A p o t e t t i x cwyreplialus the data from wild populations were unfortunately not very extensive, but two samples also showed a deficiency of double dominants. I n the breeding experiments of Nabours, on the other hand, no such deficiency occurred. Fisher thus concludes that heterozygosis versus homozygosis for a single factor leads to an inherent difference in viability which is manifested both in the wild and in culture, while the great deficiency of double dominants in nature is ascribed to some selective agency (possibly predators) which does not operate in breeding experiments. The whole problem obviously deserves much further study. Nabours and his collaborators also studied two X-ray-induced translocations in A p o t e t t i x eurycephalus. One of these was a mutual translocation between autosomes 1 and 4, while the second was between autosome 1 and the X Chromosome (Nabours and Robertson, 1933; Nabours, Stebbins, and Robertson, 1941 ; Nabours, 1950). I n the latter case Nabours, Stebbins, and Robertson believed that one of the translocation products was a dicentric chromosome with the centromere of the X a t one end and that of autosome 1 a t the other end ; this interpretation seems somewhat unlikely, as this chromosome passed unchanged through a number of generations. I n the Acrididae, Sansome and La Cour (1935) studied the genetics of a number of color patterns in Chorihippus parallelus, a European species which is normally polymorphic in nature. They identified 14 color factors, a number of which exhibited various types of factor interaction. With the exception of two cases of linkage the remaining factors appear to be unlinked, and no series of multiple alleles were encountered. This work has been criticized by Uvarov (1948) on the grounds that the possibility of environmental effects on the phenotype was not taken into account. Creighton and Robertson (1941) carried out a preliminary study of some color factors in the closely related American species, Chwthippu.s longicornis; they discovered a series of 4 multiple alleles, or a t any rate genes so closely linked that no crossing over between them was observed. Neither in the work of Nabours nor in that of Sansome and L a C o w were any sex-linked genes detected, I n view of the fact that the X chromosome in the Orthopteroid insects is invariably heterochromatic, it may well be that it is relatively “inert” except in so fa r as sex determination is concerned.

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Apart from .these instances which have been investigated genetical ly, numerous cases of phenotypic dimorphism and polymorphism are

Iillown in the Acrididae, and it is probable that in each case a fairly simple genetic basis is present. As examples we may cite the green and brown types of Dichromorpha viridis and Chortophaga viridifasciata, both very common species in eastern North America; the long-winged and short-winged individuals of the Post Oak Locust, Dendrotettix querand the almost bewildering variety of color patterns in the little western grasshoppers Psolnessn t exaru and P . debicatula, in which every locitl population consists of a large number of different types (Rehn, 1942). Dimorphism in regard l o wing length, however, does not always have a genetic basis in the Orthoptera. Thus, in the grouse locust Tettigidea parvipennis the long-winged condition is determined by a single dominant gene, but in Apotettix eurycephalus and Paratettix texanus long wings are produced when the nymphal development is rapid, as it usually is during the spring generation; while short wings result from a prolongation of the nymphal period, as usually happens in the fall generation (Nabours, 1950). Apart from these cases, there are a number of instances in the subfamily Oedipodinae where the wings may be either yellow or red in different individuals of the same species. This is the case in Psinidia fenestralis on the Atlantic Coast of the United States, in the little Derotmema haydenii, a common species of our western deserts, and in certain populations of Arphia aherralas in west Texas. Nothing is known as to the inheritance of these “color phases,” nor does the literature contain any detailed information as to their distribution in nature. A particularly interesting type of dimorphism exists in the two closely related species I’rimerotropis sparsa and T . gracilis. I n certain localities in Utah and Nevada where orange or golden-colored lichens are present in large quantities on the dead limbs of Sagebrush, Shadscale, and other desert shrubs a certain percentage of the individuals of both these species will be found to bear a series of golden patches on the sides of the pronotum and the hind femora. If this is a case of protective resemblance, it is very interesting that even where lichens are extremely abundant only a minority of the individuals show the orange patches. I n arem where lichens are absent or only present in small amount all individuals of T . gracilis and T . s p r s a lack the orange markings. Apart from cases such as those cited above, where the color pattern genes are characteristic features of the population dynamics of a species, a few mutant genes have been studied which are extremely rare and presumably play an entirely different role from those which contribute (01~s:

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to the natural polymorphism of the Tstrigidae, Chorthippus, etc. Thus, the Katydids of the genus Amblycwpha are normally bright green in color, but a rare pink mutant of A. oblmzgifdia is known, having been found in a number of different localities in eastern North America. Breeding experiments indicated that the pink color is inherited as a simple autosomal dominant (Hancock, 1916 ; Nabours, 1928). This case is interesting since pink Katydids are extremely conspicuous on a background of green foliage, and one would expect that selection by predators would eliminate the mutant from the species in a few generations. Possibly we are dealing here with a gene having a fairly high mutation rate from the green to the pink allele. Yellow individuals are also known, but have not been studied genetically.

X. EXPERIMENTAL HYBRIDIZATION Very few studies on the production of interspecific hybrids have been carried out on Orthopterous insects, and in only one case have the results been analyzed cytologically. In view of the ease with which many species can be reared and the favorable conditions for cytological study, it is obvious that further investigations would yield results of considerable interest. Cousin (1934,1941) reared many hybrids between the cricket species Gryllus bimaculatus and Q. campeatris; apparently this cross is fully fertile and the hybrids themselves do not show any sterility, so that their meiosis is presumably normal or almost so. In the grasshoppers, Carothers (1939,1941) was successful in raising more than a hundred F1 hybrids between Trimerotropis m a d i m a 0 X T . citrirto d. No report on the cytology of the F1 individuals has appeared, and it is not clear whether they were sterile or not. One of the hybrids was a gynandromorph, whose exact mode of origin is not clear. The only Orthopteran species cross for which full cytological details are available is that between Chorthippus b i c o l w and Ch. biguttulus (Klingstedt, 1939). Three male hybrids were studied, one of which was caught i n the wild while the other two were bred in the laboratory. The pairing of the chromosomes in the hybrid individuals took place normally (thus proving that each of the eight chromosomes of one parent species has a homolog in the other species), but there were various anomalies of spindle formation at the first meiotic division and the chromosomes appeared to stick together a t first anaphase, so that the second meiotic divisions frequently show the diploid number of chromosomes. As a result, many of the spermatids were diploid or tetraploid. No attempt was made to obtain offspring from these F1 hybrids, so that it is

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M. J. D. WHITE

not known whether they were fertile t o any extent, although this appears doubtful from the cytological data. Several other grasshopper species crosses are known to have been successfully carried out by various investigators, but no account of them has ever been published.

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Author Index Numbers In italics refer to pages on which the references are listed at the end of an article.

A Adair, E. W., 317, 326 Agar, W. E., 174 Akemine, Y., 195, 196, 211 Altenburg, E., 102, 123 Aoki, M.,195, 212 Aquilonius, L., 94, 124 Armstrong, G. M., 237, 844 Asana, J. J., 170, 268, 271, 272, 274, 278, 295, 311, 315, 326, 388 Atteck, 0.M.,252, 164

B Bacchi, Oswaldo, 132, 158 Bailey, R. S., 228, 245 Balasubrahmanyan, R., 232, 242 Barber, H.N., 175, 180 Barigozzi, C., 110, 112, 279, 326 Barker, H. D., 234, 235, 242 Barnicot, N. A., 33, 49 Bate, R. C., 98, 100, 192 Bauer, H.,79, 80, 83, 93, 95, 96, 121 Baumgartner, W. J., 272, 326 Beachell, H.N., 196, 211 Beadle, G. W., 70, 71, 75, 83, 248, 249, 256, 264 Beams, H. W., 294, 297, 327 Beasley, J. O., 214, 228, 229, 255, 264 Bedicheck, S., 94, 96, 124 Belar, K.,283, 292, 302, 326 Berkley, E. E., 234, 235, 242 Blank, L. M., 237, 242 Bock, E.,65, 84 Bodeman, E.,22, 51 Bodenstein, 54 B o d , E. J., 71, 76, 88, 84 Bonnevie, K., 19, 20, 21, 22, 23, 24, 31, 36, 49 Bovey, 174, 178 Brehme, K. S., 54, 55, 58, 59, 81, 83, 96, 100, 104, 105, 106, 116, 121 Bridges, C. B., 54, 57, 58, 59, 88, 84,

90, 91, 92, 96, 98, 99, 100, 102, 104, 105, 106, 109, 110, 114, 116, 121, 123, 124, 249, 256, 264 Bridges, P. N., 98, 99, 121 Brodal, A,, 19, 49 Brody, G., 63, 85 Brown, A. L., 17, 49 Brown, M. S., 214, $42 Brunelli, G., 272, 326 Bryson, V., 34, 49

U Callan, H. G., 110, 121, 286, 315, 326 Cameron, D. R., 255, 264 Cappe de Baillon, P., 271, 316, 318, 320, 386 Carlson, J. G., 277, 326 Carothers, E. E., 291, 299, 301, 302, 304, 305, 310, 311, 325, 326 Carroll, M., 273, 311, 326 Carter, T. C., 30, 31, 50 Carvalho, A., 129, 130, 132, 134, 135,

130, 138, 140, 141, 142, 143, 144, 146, 148, 149, 150, 151, 152, 154, 157, 158 Caspari, E. W., 260 Caspersson, T., 88, 89, 94, 110, 121, 124 Castle, W. E., 5, 50 Castro, Yone G.P. de, 275, 526 Catcheside, D. G., 110, 164 Chang, T. K,,31, 32, 50 Chang, T. S., 259, 265 Chao, L. F., 182, 183, 204, 811 Chase, E. B., 26, 60 Chase, H.B., 26, 50 Chen, P. S., 53, 71, 72, 73, 84 Chen, S., 276, 586 Ch’en, S. T., 285, 302, 886 Chen, S. V., 114, 122 Chen, T. Y., 59, 61, 74, 85 Chesley, P.,39, 40, 41, 50 Chevalier, A., 128, 129, 167 331

332

AUTHOR INDEX

Ciferri, R., 136, 157 Clancy, C. W., 71, 75, 89 Clark, 1'. H., 20, 50 Clausen, R. E., 255, 26'4 Clernente, L. S., 79, 83 Cloudman, A. M., 13, 51 Cole, P. A., 93, 121 Coleman, L. C., 280, 281, 300, 301, 303, 305, 307, 310, 366 Comeaux, D. J., 202, 211 Comstock, R. E., 220, 222, 243 Cook, 0. F., 217, 219, 221, 243 Cooper, K. W., 109, 110, 121 Corey, H. L., 283, 326 Costa, A. S., 135, 157, 158 Cousin, G., 325, 327 Cramer, P. J. S., 130, 132, 135, 142, 143, 146, 148, 151, 157 Creighton, M., 179, 320, 321, 323, 327 Cross, J. C., 179 Cubnot, L., 5, 50 Cullen, Sister M. U., 58, 68, 83 Curry, V., 88, 93, 98, 100, 113, 114, 116, 123

Curtis, M. R., 29, 60

D Daloq, A. M., 169 Danforth, C. R., 43, 50 Darlington, C. D., 88, 89, 101, 181, 123, 175, 179, 280, 285, 289, 292, 300, 302, 320, 327 DeAberle, S. B., 13, 49 De la Houssaye, DeBlaiic, A., 202, 611 Delbruck, M., 248, 665 Demerec, M., 57, 60, 69, 88, 95, 98, 99, 102, 103, 104, 105, 107, 108, 161, 258, 264 Doan, D., 101, 124 Dobshansky, N. P., 249, 664 Dobzhansky, Th., 59, 77, SO, 81, 89, 84, 90, 91, 92, 101, 102, 109, 110, 121, 124, 249, 164, 309, 327 Dreyfus, A., 279, 387 Drummond, F. H.,179 Dubinin, N. P.,81, 83, 96, 102, 103, 109, 114, 122 Dunham, E. W., 238, 243 Dunn, L.' C., 18, 23, 29, 39, 42, 44, 45, 60, 260

Dunning, W. F., 29, 50 Dutt, M. K., 310, 329

E Einuele, W., 8, 50 Eker, R., 271, 328 Emerson, S., 248, 664 Ephrussi, B., 41, 50, 69, 70, 83, 102, 168 Evans, W. L., 308, 309, 311, 813, 387 Ewing, E. C., 218, 242, 243

F Faber, F. C. von, 132, 134, 157 Fagerlind, F., 132, 134, 167 Falconer, D. S., 16, 50 Fankhauser, G., 163, 179 Fano, U., 57, 60, 83, 121 Favrelle, M., 271, 278, 966, 387 Yerwerda, F. P., 130, 135, 154, 157 Yiulier, R. A., 262, 664, 322, 323, 3.97 Fondal, E. L., 13, 51 Foster, Morris, 11, 50 Franco, C. M., 137, 157 Fraser, A. S., 16, 50 Fraser, F. C., 15, 50 Friauf, J. J., 299, 927 Fukushima, E., 195, 199, 200, 201, 205, dl2

Fulton, B. B., 279, 387

a Galgano, Id., 168 Gay, H., 96, 103, 109, 114,182 Gersh, E. S., 114, 128 Gershenson, 8. M., 88, 90, 92, 101, 110, 122, 123 Gloor, H., 53, 56, 59, 64, 66, 68, 69, 74, 76, 78, 82, 83, 84 Gluecksohn-Schoenheimer, S.,17, 18, 29, 38, 39, 40, 41, 42, 44, 45, 48, 60 Gluecksohn-Waelsch, S., 42 Goldschmidt, E., 319, 327 Goldschmidt, R. B., 4, 46, 60, 89, 98, 102, 110, 119, 122, 248, 257, 261, 263, 264 Goldsmith, 0. W.,236, 243 Gordon, M., 166, 179 Uoweii, J. W.,59, 84, 103, 109, 114, f 2 2

333

AUTHOR INDEX

Granata, L., 275, 327 Green, E.L., 34, 50 Green, I(.C., 99, 122, 256, 264 Green, M. C., 34, 35, 50 Green, M. M., 99, 122, 164, 256,264 Griffen, A. B., 91, 116, 117, I62 Grimes, M. A,, 234, 243 Grob, H., 68, 84 Griineberg, H., 4, 5, 11, 12, 13, 20, 24, 29, 32, 37, 47, 48, 50, 99, 100, 102, 122, 251, 261, 264 GuBnin, H. A., 89, 122, 177 Guliek, A., 248, 249, 664

H Hadorn, E., 53, 55, 58, 59, 60, 61, 62, 63, 64, 65, 67, 08, 09, 70, 72, 75, 77, 78, 81, 84 Haldane, J. B. S., 249, 664 Hall, E. E.,228, 236, 243 Hancock, J. L., 325, 327 Hannah, A., 94, 103, 110, 119, I22 Hara, S., 192, 199, 202, 204, 205, 811 Hardy, M. H., 7, 50 Hareyama, S.,273, 278, 567 Harkmark, W.,19, 49 Harland, 8. C., 214, 210, 218, 222, 223, 224, 226, 228, 643, 252, 664 Harman, M. T., 274, 327 Harrell, D. C., 228, 243 Harrison, 0. J., 235 Hearne, E. M., 278, 283, 302, 327 Hebsrd, M., 298, 299, 327, 369 Heberer, G., 271, 327 Heidenthal, G., 103, 124 Heitz, E., 88, 90, 92, 122 Helwig,. E. R., 274, 275, 276, 293, 294, 295, 301, 305, 307, 308, 309, 310, 327 Henderson, J. M., 8, 51 Reptner, M. A., 109, I22 Hermann, 172 Hertel, K. L., 234, 243, 245 Hertwig, P., 24, 25, 60 Hinton, T., 90, 93, 95, 110, 113, 115, 122 Hollander, W.,22, 51 Holt, 8. B., 32, 50 Homeyer, H.,132, 157 Honda, H.,272, 278, $87

Hoover, M., 98, 121 Hoover, M. E., 83 Jiopkins, J. T., Jr., 176, 170 Hodiika, Y., 199, 212 Houk, W. O., 134, 157 Hovelacque, A., 30, 50 Hubbell, T. H., 299, 327 Hughes-Schrader, S., 270, 278, 290, 296, 296, 297, 310, 327 Hummel, K. P., 261, 265 Humphrey, R. R., 163, 167 Hunt, H. R., 11, 60 Huskins, C. L., 278, 283, 302, 327 Husted, L.,176, 179 Hutchinaon, J. B., 200, 211, 213, 214, 221, 222, 230, 243, 253,264 I I)isen, H. L., 5, 60 Iddles, 96 Ikeno, S., 181, 811 Iriki, S. H., 168 Ishikawa, J., 192, 193, 111 Isshiki, S., 199, dl1 Itoh, H.,311, 327 Ives, P. T., 59, 84 Izumi, Y.,199, 811 Izumiyama, 205 J

.Jabitharaj, S., 200, 21.9 Janssens, F. A., 283, 285, 327 Jenkins, W. H.,228, 239, 243 Jodon, N. E., 183, 190, 200, 202, 203, 204, 205, 206, 208, 211 Johannsen, 216 Johnson, H. H., 272, 387 Jones, D. L.; 241 Jones, J. W., 192, 194, 195, 199, 201, 611

K Kadam, B. S., 182, 191, 192, 199, 208, 209, 310, 211, 811 Kaliss, N., 57, 74, 84 Kamenoff, R. .J., 11, 35, 51 Kato, S.,192, 193, 211 Kaufmaun, B. P., 90, 92, 93, 94, 98,

334

AUTHOR INDEX

96, 98, 99, 100, 101, 109, 113, 115, 121, 122 Kaven, A., 46, 51 Kawahara, E., 195, 205, 616 Kearney, T. H., 216, 218, 649 Keenan, R. D., 171 Kellen-Piternick, L., 110, 119, 182 Kerr, T.,234, 646 Kerschner, J., 110, 266 Khvostova, V. V., 90, 95, 96, 98, 106, 107, 109, 116, 117, 168, 164 Kime, P. H., 215, 230, 232, 643 King, C. J., 234, 846 King, H. E., 238, 844 King, J. W. B., 16, 50 King, R. L., 293, 294, 295, 297, 298, 301, 305, 308, 311, 320, $87 Kirkham, W. B., 5, 51 Kitada, 272, $87 Klingstedt, H.,287, 303, 325, 867 Knight, R. L., 226, 230, 237, 238, 6449 Kodani, M., 95,188 Koller, C. P., 88, 89, D3, 175, 176, 179 Kondo, M., 199, 811 Kone, G., 313 Kossikov, K. V., 98, 99, 100, 101, 16.9 Kosswig, C., 165, 166 Krause, G.,65, 84 Erug, C. A., 128, 130, 132, 134, 135, 136, 137, 138, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151, 152, 153, 154, 157, 158 Kupka, E., 179 Kuriyama, H., 195, 207, 116 Kushnir, T., 279, 367

L Laanes, T., 30, 37, 51 La Cour, F. L., 179 L a Cour, L., 323, 869 Lamprecht, H.,248, 664 Laughnan, J. R., 258, 259, 164 Law, L. W., 88 Lea, D. E., 57, 84 Leliveld, J.. A., 134, 158 Levene, H.,309, 327 Lewis, D., 262, 263, 665 Lewis, E. B., 98, 99, 103, 115, 183, 249, 256, 265

Li, J. Ch., 55, 57, 59, 84 Little, C. C., 5, 13, 60, 61, 261, 665 Lynn, H.D., 217, 241, 644

M McClelland, T. B., 136, 158 McClintock, B., 249, 258, 865 McClung, C. E., 268, 271, 272, 276, 277, 278, 280, 285, 286, 293, 302, 309, $88 McDonald, M. R., 96, 182 MacDowell, E. C., 28, 30, 37, 51 MeIntosh, A. J., 175, 280 McNabb, J. W., 284, 287, 288, 528 MeNamara, H.C., 234, 844 Mahabale, T. S., 170 Makino, S., 165, 168, 170, 176, 177, 179, 180, 269, 271, 272, 274, 278, 280, 281, 311, 315, 326, $,@8 Manning, H. L., 220, 221, 222, 227, 648 Mansurova, V. V., 96, 1 M Margot, A., 170 Mason, T. G., 220, 644 Mather, K,, 89, 101, 102, 110, 118, 120, 123

Matsuura, H., 181, 192, 618 Matthey, R., 161, 162, 164, 169, 170, 172, 174, 175, 176, 179, 180, 270, 284, 290, 295, 317, 319, 368 Mayne, W. Wilson, 134, 158 Medina, Dixier M., 132, 158 Medvedev, N. N., 84 Meek, C. F. V., 278, 588 Mendes, A. J. T., 132, 133, 134, 137, 153, 154, 158 Mendes, C. H. T., 132, 133, 158 Mendes, J. E. T., 130, 132, 138, 141, 146, 151, 154, 156, 158 Metz, C. W.,249, 865 Minouchi, O., 164, 165, 167, 169, 170, 311, 828 Misra, A. B., 274, $28 Mohr, 0. L., 78, 84, 98, 128, 271, 273, 3228

Momma, E., 280, 272, 278, 280, 281, 928 Montalenti, G., 286, 386 Moore, E. J., 236, 245

335

AUTHOR INDEX

Moore, M. B., 176, 179 Morgan, L. V., 98, 99, 102, 105, 114, 188 Morgan, T. H., 59, 84, 88, 93, 100, 109, 110, 113, 114, 116, 123 Morinaga, T., 195, 199, 200, 201, 205, 207, 208, 212 Morse, M., 270, 290, 828 Mudaliar, 8. D., 200, 212 Miiller, G., 27, 51 Muller, H. J., 88, 89, 90, 91, 92, 93, 98, 100, 101, 102, 103, 105, 108, 109, 110, 128, 124, 160, 249, 251, 265

N Nahours, R. K., 321, 322, 323, 324, 325, 328

Nagai, I., 195, 212 Nagamatsu, T., 195, 205, 818 Nagao, S., 181, 182, 183, 184, 187, 188, 193, 194, 195, 197, 198, 199, 201, 204, 205, 206, 207, 208, 211, 212 Nakamura, K., 270, 828 Nakamura, M., 169, 170 Narayanan, N. G., 232, 248 Natori, B., 299, 328 Needham, J., 67, 84 Neelakantam, K., 254, 266 Neuhaus, M. E., 70,84, 90, 101, 109, 110, 123 Niiyama, H., 274, 278, 311, 315, 326, 328

Noble, G. K., 168, 169 Noel, R., 30, 50 Nolte, D. J., 277, 328 Noujdin, N. 19, 103, 107, 188 Novitski, E., 81, 85 Nowlin, N., 278, 888 0

Offsrmann, C. A., 98, 101, 102, 128 Oguma, K., 162, 165, 170, 172, 173, 175, 270, 295, 828 Ohmachi, F., 271, 272, 829 Ohmachi, S., 273, 274, 278, 299, 329 Okuda, N., 96, 122 Oliver, C. P., 99, 188, 124

P Painter, T. S., 88, 90, 91, 101, 128, 124, 169, 178, 179 Panse, V. G., 221, 248 Panshin, I. B., 99, 102, 110, 115, 164 Parnell, F. R., 193, 195, 812 Parnell, H. E., 238, 244 Patterson, J. T., 94, 96, 105, 124 Paul, L. C., 299, 329 Pauling, L., 248, $65 Pavan, C., 92, 184 Payne, F., 290, 291, 292, 829 Pearson, N. E., 299, 829 Peebles, R. H.,216, ,943 Pehani, H., 318, 329 Permar, D., 11, 50 Pfeiffenberger, G. W., 234, 244 Philip, U., 101, 124 Picken, D. I., 22, 51 Piza, 8. de T., 272, 275, 278, 297, 329 Pomini, P., 180 Ponse, Kitty, 163, 168 Pontecorve, G., 46, 51, 88, 93, 101, 224 Pope, 0. A., 235, 242 Potter, E., 299, 829 Potter, J. S., 30, 37, 51 Poulson, D. F., 54, 55, 56, 60, 65, 69, 71, 76, 83, 84 Powers, P. B. A., 274, 314, 329 Pressley, E. H.,234, 244 Pressley, J. T., 236, 244 Prokofyeva, A. A., 89, 90, 93, 123 Prokofyeva-Belgovskaya, A. A., 88, 92, 93, 94, 95, 96, 98, 101, 128, 124

R Itaffel, D., 88, 92, 101, 108, 123, 124 Ramiah, K., 191, 192, 193, 195, 200, 208, 209, 210, 211, 211, 212 Raney, R. C., 238, 244 Rangaswami Ayyangar, G. N., 193, 195, 212 kao, T. R., 276, 329 Ragoport, J. A., 77, 84 Rawles, M. E., 7, 51 Rayburn, M. F., 274, 329 Ray-Chaudhuri, 8. P., 310, 329 Redfield, H., 78, 84 Reed, 8. C., 8, 14, 29, 43, 51

336

AUTHOR INDEX

Rehn, J. A. G., 274, 298, 324, 329 Edanaud, P., 178 Reaende, F.,89, 184 Rhyne, 253 Richey, F. D., 22q 844 Richmond, T. R., 216, 218, 219, 220, 226, 227, 229, 844,845 Rim, H., 283, 284, 389 Roberts, H.R., 275, 277, $89 Robertson, 160, 164, 170 Robertson, G. ff., 5, 6, 51, 160, 165 Robertson, W.R. B., 273, 274, 302, 315, 320, 321, 323, 387, 388, 389 Rothfels, K. H., 288, 303, 311, 314, 315, 3b9 RUEEell, E. S., 9, 13, 48, 61, 58, 75, 84 Russell, L. B.,9, 46, 51 Russell, w.L., 9, 14, 52 Ruston, D. F., 238, 844 S

Saez, F. A., 168, 275, 389 Sakai, K,, 181, dl8 Sansome, E.,181 Saneome, F. W., 323, 389 Santesson, L., 88, 181 Sato, D., 168 Schaeffer, E. W,,103, 184 Scharrer, B., 75, 84 Schmid, W.,58, 68, 74, 75, 77, 84 Schnitter, M..,53 Schubel, F,,58, 75, 84 Schultz, J., 59, 79, 84, 88, $9, 91, 93, 94, 98, 99, 100, 101, 102, 103, 104, 105, 107, 109, 110, 112, 113, 114, 115, 116, 181, ids, 184 Sears, E. R., 255, 256, 866 Seidel, F., 65, 84 Serebrowsky, A. S., 99, 100, 184, 249, 256, 866 Berra, J. A., 14, 51 Seehachar, B. R., 169 Seshadri, T. R., 254, 865 Severin, H. C., 299, 319 Shaman, G. B., 175, 180 Sherbakoff, C. D., 236, 844 Sidorov, B. N., 102, 188 Bikes, E. K., 74, 84 Silow, R. A., 213, 214, 230, 843, 252, 253, 255, 259, 261, 864, 166

Simpson, D. M., 215, 231, 232, 237, 844 Siir&y, R. de, 316, J2D Sivcrtaev-Dobzhansky, N. P., 57, 58, 8.5,91, 110, 124 Skovsted, A., 214, 244, 255, 665 Slifer, E. H., 320, 387 Slizynska, H.,103, 105, 118, 181, 184 Slizynski, B. M., 91, 95, 96, 99, 118, 184, 180 Smith, A. L., 236, 238, 644 Smith, L. J., 13, 51 Smith, P. E., 28, 51 Smith, W. S., 234, 844 Snell, G. D., 5, 22, 28, 51 Sobels, F. H., 68, 85 Sobotta, J., 5, 51 Sokame, C., 273, 889 Sonnenblick, B. P.,54, 57, 85 Apaasky, B., 83 Spencer, W. P., 59, 77, 85, 118, 184 Sprague, 0. F., 226, 844 Srinivasa Ayyangar, C. R., 195, 812 Stadler, L. J., 260, 865 Starck, M. B., 59, 85 Stebbins, F. M., 323, 388 Steigleder, E.,5, 50 Glteiniger, F., 29, 51 Steopoe, I., 279, 311, 316, 389 Stephens, 8. ff., 213, 214, 222, 226, 227, 228, 229, 230, 237, 843, 844, 253, 259, 260, 864, 665 Stern, C., 69, 81, 86, 91, 95, 101, 102, 103, 106, 109, 110, 118, 284, 262, 86.5 Stevens, N. M., 271, 389 Stoffels, E.,136, 158 Stohler,, R., 168 Stone, W. C., 94, 96, 184 Stone, W. S., 91, 115, 117, 188 Stroman, ff. N., 235, 844 Sturtevant, A. H., 59, 78, 84, 98, 102, 109, 125, 249, 256, 865 StUttE, R. T., 234, 844 Suche, M.,94, 96, 184 Sugimoto, S., 195, 818 Sullivan, R. R., 234, 846 Suomalainen, E., 270, 290, 317, 329 Sutton, E.,93, 99 101, 102, 116, 181, 128, 1.85 Suzuki, K.,172 Swamy, R. L. N., 151, 166

337

AUTHOR JNDEX

T Takahashi, M., 182, 183, 184, 187, 188, 193, 194, 195, 197, 198, 199, 201, 202, 203, 204, 205, 206, 207, 208, 31 d

Tan, C. C., 261, 365 Taschdjian, E.,134, 258 Tate, W.,202, 818 Tateishi, S., 272, 389 Tatum, E. L., 71, 76, 83 Testa, J., 128, 158 Thomas, A. S., 130,158 Thomas, P.T., 88, 181 Thompson, C., 271, 369 Tilley, R. H., 215, 230, 232, 843 Tiiikham, E. R., 277, 389 Tsang, Y. C., 32, 51

U Upcott, M. B., 101, 181, 161 Uvarov, B. P.,277, 323, 389

V Vanderlyn, L., 89, 1%5 van der Wolk, P. C., 135, 157 Vavilov, N. I., 216, 845 Vichet, G. de, 271, 316, 318, $66 Vogt, M., 68, 85 Volsge, H.,180 Voss, J., 256, 865

W Waddle, B. A., 218, 220, 221, 245 Wagner, R. P., 248, 665 Ward, E. N., 30, 37, 61

Ware, J. O., 227, 236, 845 Weindling, R., 237, 244 Wenrich, D. H., 283, 284, 302, 303, 329 Werneke, F., 9, 51 White, M. J. D., 88, 89, 99, 109, 125, 160, 161, 162, 167, 249, 365, 270, 271, 273, 274, 278, 280, 281, 282, 285, 286, 287, 289, 295, 296, 298, 301, 302, 304, 305, 308, 310, 311, 312, 313, 317, 319, 380 Whittinghill, M., 125 Wickbom, T., 168, 180 Wigan, L. G., 119, 185 Wigglesworth, V. B., 74, 84 Williams, R. D., 261, 866 Wilson, E. B., 281, 292, dJ0 Wilson, K.,96, 122 Winge, O., 165, 166 de Winiwarter, H.,279, 281, 330 Witschi, E.,168 Woodyard, 0.C., 234, 846 Woolsey, C. I., 273, 330 Wright, M. E., 37, 51 Wyman, R., 96, 168

Y Yamaguchi, Y.,181, 195, 204, d l t Yamashina, Y.,162, 165, 173 Yasuda, S., 181, d l d Yosida, T., 180 Yu, C. P., 259, 261, 665

z Zeller, H., 81, 84 Zaiiner, F. E.,268, 271, 330 Ximmermann, A., 134, 135, 158

Subject Index A Acrididae, cytology, 276, 277, 280-286, 288, 293, 297-302, 308, 320-321, 323-324 distributiofi, 301-302, 305-310, 317 Agnatha, chromosomes, 165 albinism, in mouse, 8 Amphibia, chromosomes, 167-177 a,mphidiploida, with reference to evolution, 250, 264 i n cotton, 252-253, 255, 264 anemia, embryonic, in mouse, 11-13 macrocytic, 12-13 siderocytic, in flexed, 11-12, 36 anthocyanin, in oorn, 258-260 in cotton, 252-253, 259-260 in rice, 182-188, 191, 193 in Trifolium, 261 AVBB,chromoRomes, 170, 172-174, 177

B backcross method, in cotton, 225-226, 228-229, 238 blood, in mouse, 11-14 blood groups, human, 262 U chiasma, in Orthoptera, 284-289, 293, 295, 300, 310, 312, 317-318, 321322 chlorophyll, in corn, 258 in rice, 202-203 chromatin chemistry, 248 chromosome breaks, in Drosophila, 9496, 102-108, 112-113, 115, 117, 118 in Orthoptera, 303, 309-310 in Vertebrates, 161 chromosome evolution, in Vertebrates, 159-161, 163, 169, 173, 176-178 chromosome fixation, in Vertebrates, 164165, 172, 174 chromosume morphology, in Vertebrates, 338

160, 162-163, 166-168, 170-171, 173, 179 chromosome mutations, i n Vertelmtes, 160, 162, 169 ahromosome IX, in mouse, 38, 42-45 chromosome number, in Coffen nml)ic:i, 132, 134, 137, 144, 155 ill Orthoptern, 269-279, 293, 299, 301302, 304-309, 317-319, 321, 325 in Vertebrates, 163-167, 170-175, 177178 chromosomes, in Agnatha, 165 in Amphibia, 167-177 in Aves, 170, 172-174, 177 in cotton, 214, 224, 226, 228-230 in Drosophila : meiotic, 92 mitotic, 90, 92 salivary gland, 88, 91-94, 96, 99, 104, 112, 120, 249-250, 256-257 X-chromosomes, 55, 90-92, 95-96, 101, 104, 107-109, 112, 114, 117119 Y-chromosomes, 90, 92, 94, 101, 107, 109-110, 113-114, 116-117, 119-120 in Fishes, 165 in Mammalia, 174, 176-177, 179 in Orthoptera, 270-280, 302-309 behavior, 287, 289 segregation, 290, 292 structure, 280-286, 302-303, 305, 308, 316 supernumerary, 299, 301-302, 305, 308, 310-316 in Reptilia, 167, 169-170, 173-174 i n Vertebrates, 165, 167, 169-170, 172174, 176-177, 179 with reference to evolution, 251 Coffea arabica, abnarmalities, 132, 140141, 152-103 biology, 134, 154 chromosome number, 132, 134, 137, 144, 155 colchicine treatment, 134 cultivation, 128, 130, 141

339

SUBJECT INDEX

cytology, 154 emasculation, 135 endosperm, 134-135,141-142,153 fasciation, 143-144,152-153 gametogenesis, 134 geographical distribution, 128-130 hybrids, 134, 144, 151-154 inheritance, cytoplasmic, 152 meiosis, 132, 134, 144 mutations, chromosomal, 136-137,I55 mutations, genetic, 136-156 origin, 128 pollination, 134-135,142 reproduction, 134-135 taxonomy, 128-129,154 Coffea species, 152-153 Colchicine treatment, in Cotton, 228 color, see pigmentation, pigment pattern corn, anthocyanin, 258, 260 chlorophyll, 258 crossing over, 258-260 duplications, 258 mutations, 260 pigmentation, 259-260 Cotton, American, abnormalities, 216-217 amphiploids, 252, 253 anthocyanin, 252-253 backcross method, 225-226, 228-229, 238 breeding, 213-242 breeding methods, 218-232,235 chromosomes, 214, 224, 226, 228-230, 252 Colchicine treatment, 228 crosses, 218, 226-232,235, 241, 253-256 crossing over, 229-230 disease resistance, 236-238 harvesting, 239-241 heterosis, see hybrid vigor history, 215 hybrids, 214, 218-221, 225-232, 235, 239, 253 hybrid vigor, 230-232 inbreeding, 215, 217-220,224, 230-232 inheritance, 214, 224-225,227, 229, 241 insect resistance, 238-239 linkage, 252-253,255 methods for evaluating properties, 233234 mutations, 220 pigmentation, 253-256

progeny-row method, 216-217,221-223 “pure line” concept, 216 selection, 216, 220-224, 226, 229, 235, 242 taxonomy, 213-214,222, 226-227 testing techniques, see breeding methods variability, 215, 219, 222-227,235 varieties, 216-219, 221-223, 226-228, 230-231,235, 237, 242 Cotton, Asiatic, 213-214 anthocyanin, 259-260 mutations, spontaneous, 259 Cotton boll weevil, 216 crosses in Cotton, 218, 226, 227-232,235, 241, 253-256 crossing over, in corn, 258-260 in cotton, 229-230 in Drosophila, 99, 101-102, 249, 256257 in evolution, 252 in mouse, 260 in Orthoptera, 289, 295, 300, 319, 322323 aytology, in Coffea arabica, 154 in Orthoptera, 269-278,289, 299-304 in Vertebrates, 161-162,164, 167, 169, 172-173,177

D disease resistance, in cotton, 236, 237,238 dopa reaction, in mouse, 9-11 Drosophila, abnormalities, 57, 61, 65-67, 71, 72, 88 chromosomes, see chromosomes in Drosophila chromosome breaks, see chromosome breaks in Drosophila crossing over, 99, 101-102,256-257 duplications, 98, 100-101,114, 250, 256257 embryonic development, 54 euchromatin, 88-89, 92-95, 100-101, 103-105,109, 112-121 heterochromatin, 87-96, 98, 100-105, 108-110,112-121 interphase, 88-89 lethals, 54-63, 65-68, 70-72, 77-78, 8081 meiosis, 88

340

SUBJECT INDEX

metabolism, of lethals, 74-75,77, 89 metaphase, 89-90,92 metamorphosis, in lethals, 75 mitosis, 88, 90 mutants, see mutants in Drosophila osmosis, in lethals, 74 phase specificity, 62-65,69 phenocopies, of lethale, 76, 77 physiology of lethals, 71-76 position effect, 102-104,106, 108, 112113, 115-120 prophase, 89 puparium, 61, 72-73,75-76,78 rearrangements, 102-104,106, 114-116 respiration, 71 duplications, in corn, 258-259 in cotton, 253, 255, 257 in Drosophila, 98, 100-101, 114, 250, 256 in Orthoptera, 295 significance of, in evolution, 249-51, 263 in Trifolium, 261

F famiation, in Coffea arabica, 143-144, 152-153 Fishes, chromosomes, 165

a gametogenesis, in Coffea arabica, 134 gene action, 248 genes, effect of, in pigmentation of mouse, 8-10 Clossypium, see Cotton

H

harvesting Cotton, 239-241 heterochromatin, in Drosophila, 87-96, 98, 100-105,108-110,112-121 cytology of, 89 mutants in, 87 in Orthoptera, 276-277, 280-284, 286, 290, 292, 293-295, 297-298, 302, 314-316,323 E hetcrochromosomes, in Vertebrates, 16.5embryo, in mouse 167, 169, 170, 172-176 abnormal, 5-6,19-26,34-36,39-45,47 heterosis, see hybrid vigor brachyury, 39 liyhrids, in Coffea arabica, 134, 144, 1.51hereditary factors, 7 154 homozygous yellow, 5-7 in Cotton, 214, 218-221,225-232, 235, hydrocephalus, 19 239, 253 melanophores, 7 in Ladybeetle, 261 pseudoencephaly, 21 in Orthoptera, 299, 325 endosperm, in Coffea arabica, 134-135, hybrid vigor, in Cotton, 230-232 141-142,153 in Orthoptera, 309 euchromatin, in Drosophila, 88-89,92-95, 100-101,103-105,109, 112-121 I in Orthoptera, 280-283, 286, 294-295, 297-298,314-316 evolution, in Orthoptera, 297, 299, 301, implantation, ovaries, in mouse, 6-7 in Drosophila, 75 303-304,307, 314 inbreeding, in Cotton, 215, 217-220,224, in relation to amphidiploids, 250, 264 230-232 chromosomes, 251 duplications, 249-251,253, 255, 257- inheritance, cytoplasmic, in C o f f ~ a a., 152 259, 261, 263 insect reRiRtance, in Cotton, 338-239 inheritance, 248 interphase, in Drosophila, 88-89 segregation, 247 intersexuality, in Orthoptera, 299 selection, 247, 249 variability, 247 inversions, in Drosophila, 249 variations, 247-249,251 in Orthoptera, 295, 299-300,303

341

SUBJECT INDEX

L

pigmentation, 7-11,13-14,251, 261 pigment pattern, 11 siderocytes, 11-12 transplantation, 8, 14-15,33 tyrosinase activity, 11 mutants, in Drosophila euchromatic, 88, 112, 118, 120 heterochromatic, 87, 101, 112, 115, 118120 let,ld, 54-55, 60, 62-63, 71, 73-74,8283, 310, 118-119 iionlethal, 63-64,70, 71 M podoptera, 110, 119 Mammalia, chromosomes, 174, 176-177, semilethal, 64 179 sterile, 110 meiosis, in Coffea arabica, 132, 134, 144 X-ray-induced, 110, 118 in Drosophila, 88 mutations, abnormal, in rice in Orthoptera, 281-290, 292-293, 295, inheritance, 195-203 297-298, 300, 309, 312, 315-316, crosses, 196-203 318, 320-321,325 Chlorophyll, 202, 203 metabolism, in Drosophila lethals, 74-75, X-ray-induced, 203 77 mutations, chromosomal, in Coffea arabmetamorphosis, in Drosophila lethals, 75 ica, 136-137,155 metaphase, in Drosophila, 89-90,92 genetic, in Coffea arabica, 136-156 in Orthoptera, 290-292,306, 312 in Cotton, 220 mitosis, in Drosophila, 88, 90 in mouse, affecting blood, 11, 14 in Orthoptera, 281-282, 298, 311, 314, central nervous system, 19 317, 319 chromosome IX,38, 42-45 mosaicism, in mouse, 7-8 hair, 14, 16, 251 in Orthoptera, 320 pigment, 14, 262 mouse, abnormalities, 5-6,11, 17, 19-44 sensory organs, 19 albinism, 8 skeleton, 29-35,37-38 anemia, 11-12 skin, 14 blood, 11-13 urogenital system, 17-18 dopa reaction, 9-11 brachyury, 39 embryo, homozygous yellow, 5-7 eyeless, 26-27 gene action, 9 flexed, 11-12,35-36 gene, effect of, 8-10 kreisler, 25, 26 hair mutations, 14, 16 lethal, 5, 32, 39-45 hereditary factors, embryonic, 7 microphthalmw, 27 histology, 9-10 pituitary dwarfism, 28 homozygote, yellow, 5-6 shaker short, 23 Iiypotrichosis, 15 taillessness, 40 implantation, 67 iiiutations with reference to evolution, lethal yellow, 5 247 252 macrocytes, 13-14 0 melanophores, 7 mosaicism, 7-8 organogenesis, abnormal, in Drosophila, mutations, 5, 11-12, 14, 16-19, 23, 2565, 67 30, 34-35, 37-39, 42, 45, 47, 251, Orthoptera, abnormalities, 290, 310, 319262 320, 325 Ladybeetle, hybrids, 261 pigmentation, 261 lethal yellow, in mouse, 5 lethals, in Drosophila, 54-63, 65-68, 7072, 77-79 sex-limited, 78, 81 linkage, in rice, 203-208 linkage groups, in Cotton, 252-253,255 in Orthoptera, 322-323

~

342

SUBJECT INDEX

chiasma, 284-289, 293, 295, 300, 310, 312, 317-318,321-322 chromosome number, 269-279,293, 299, 301-302,304-309,317-319,321,325 chromosomes, 270-316 C y t o l o ~269-278, , 289-304,317 euchromatin, 281-283, 286, 294-295, 297, 298, 314-316 evolution, 297, 299, 301, 303-304,307, 314 genetics, 321-325 heterochromatin, 276-277,280-284,286, 290, 293-295, 297-298, 302, 314316, 323 hybrids, 299, 325 intersexuality, 299 meiosis, 281-290, 292-293, 295, 297298, 300, 309, 312, 315-316, 318, 320-321,325 parthenogenesis, 271, 273, 299, 316321 polyploidy, 287 sex chromosomes, 292-298 spermatogenesis, 284, 286-288, 290, 312, 316, 322 taxonomy, 268, 269, 271, 278, 296, 301, 303-304

P

pleiotropism, in Coffea arabica, 138, 142143, 156 in Drosophila, 65, 68-69,71, 76, 82 in evolution, 252 in mouse, 48-49 pollination, in Coffea arabica, 134-135, 142 polyploidy, in Orthoptera, 287 position effect, in Drosophila, 102-104, 106, 108, 112-113, 115-118, 120, 257, 263 in evolution, 252 in Vertebrates, 162 progenyrow method, Cotton, 216-217, 221-223 prophase, in Drosophila, 89 in Orthoptera, 292, 312, 315 “pure line” concept, in Cotton, 216

B rearrangements, in Drosophila, 102-104, 106, 114-116 in mouse, 260 in Orthoptera, 274, 277-278, 296-297, 299- 302 recombinations, chromosomal, 247, 249 reproduction, in Coffea arabica, 134-135 Reptilia, chromosomes, 167, 169-170,173174 respiration, in Drosophila lethals, 71-73 rice, anthocyanin pigment, 182-188,191, 193 chlorophyll, 202-203 inheritance, 181-182,187-188,192, 194195, 203, 208 linkage, 203-208 mutations, 195-203 parenchyma, 188-189,192 pigmentation, 182-194

parthenogenesis, in Orthoptera, 271, 273, 299, 316-321 plienocopies, in Drosophila lethals, 7677, 82 physiology, in Drosophila, 71-74 enzymatic activity, 76 hormone, 75 pigmentation, in corn, 259-260 in Cotton, 253-256 in Ladybeetle, 261 in mouse, 7-8,13-14 biochemical aspect of, 9-11 of hair, 7-9,33 S in rice, 182-194 selection, in Cotton, 216, 220-224, 226, distribution, 183, 189-190 229, 235, 242 inheritance of, 182, 187-188,192,194 with reference to evolution, 247, 249 phenotypes, 186 sex chromosomes, in Or’thoptera, 292-298 pleiotropism, 191 in Vertebrates, 162-163,166, 168 variation, 182, 192 pigment pattern, in mouse, 11, 261 spermatogenesis, in Orthoptera, 284, 286in Orthoptera, 321-325 288, 290, 312, 316, 322

343

SWJECT INDEX

T taxonomy, in Coffea arabica, 128-129, 154 in Cotton, 213-214, 222, 226-227 in Orthoptera, 268-269, 271, 278, 296, 301, 303-304 translocations, chromosomal, in Drosophila, 90, 94, 100-101, 107, 110, 112, 114-115, 249 in mouse, 47 in Orthoptera, 276, 282, 296, 300-301, 304, 309-310, 321, 323 i i i Vertebrates, 161 X-ray-induced, in mouse, 22 transplantation, see also implantation in Drosophila, 67-70, 82 in mouse, 8, 33 of bones, 33 of skin, 8, 14-15 tyrosinase activity, in Cotton, 256 in pigment pattern of mouse, 11

V variability, in Cotton, 215, 219, 222-227, 235

variations, with reference to evolution, 247-249, 251 in Cotton, 259 varieties, in Cotton, 216-219, 221-223, 226-228, 230-231, 235, 237, 242 Vertebrates, cliromosomes, 161-177 breaks, 161 evolution, 159-161, 163, 169, 173, 176178 fixation, 164-165, 172, 174 morphology, 160, 162-163, 166-168, 170-171, 173, 177, 179 uiutations, 160, 162, 169 uumber, 163-167, 170-175, 177 178 cytology, 161-162, 164, 167, 172-173, 177 heterochromosomes, 165-167, 169-170, 172-176 position effect, 162 sex chromosomes, 162-163, 166, 168

X X-rays, effect of, in Drosophila, 57, 70 in mouse, 46, 48