174 Cytologia4

Contributions to the cytology of germ cells. I. Chromosomes in the spermatocyte divisions of five North American species of *

By Emil Witschi Zoology Laboratory, The State University of Iowa ReceivedNovember 17,1932 Because of their large size the amphibian germ cells have been a favored object of the earlier students of cell division and chromosome development. Figures from the classical papers of Flemming, Meves and Janssens adorn many of our textbooks. In recent years we have become more specifically interested in the fact that in this class of sex chromosomes are not easily de monstrable. Studies in the genetics of sex determination in Rana temporaria (Witschi, 1914, 1922, 1929) led to the conclusion that the homogametic female must have a pair of X chromosomes, each carrying a female determining sex factor F, while the digametic male has an uneven or XY pair of sex chromosomes with corresponding sex factors F and f. A series of geographical races of this was found with different quantitative variations of the gene f, all lower in value than the allelomorphic F factors (Witschi, 1929, 1930). Genetical data therefore called for a Y chromosome that is not "empty" and which cytologically should differ not very markedly from the X. The cy tological facts were found to agree with these conclusions (Witschi, 1924). Both sexes have a diploid number of 26 chromosomes. The XY pair of the male frog exhibits some particular traits during the spermatocyte divisions, though its members do not differ too obviously in size nor in shape. Makino (1932)can not establish any morphological difference between X and Y in the Japanese race of the same frog species.

* Aided by grants from the Committee for Research in Problems of Sex of the National Research Council. 1933 Contributions to the cytology of amphibian germ cells. I 175

This situation appears of great interest with respect to the phylo genetical evolution of the sex chromosome mechanism. There is a good possibility that a careful comparative study of a larger number of amphibian species may throw further light on this engaging problem. Among the better known none has a more pronounced tendency toward hermaphrodism than the common European , Bufo vulgaris (Witschi, paper in press). Stohier (1928) who on my sugges tion has made an investigation of this and two other toads (B. viridis and B. calamita) was not able to identify any sex chromosomes. How ever, Minouchi and Iriki (1931) in Bufo bufo japanicus and Makino (1932)in Bufo sachalinensis describe a peculiar tetrad in the first sper matocyte division that is "vertical-V-shaped" and lies always at the periphery of the plate. Iriki is inclined to consider it as the tetrad of a sex chromosome and Makino assumes that it "corresponds to the supposed sex chromosome already found in Rana temporaria" The only work on the chromosomes of North American toads seems to be that of H. D. King. In her paper of 1907 on the spermatogenesis of Bufo lentiginosus she discusses in great length the conjugation of the chromosomes and the reduction during the spermatocyte divisions, though she does not give any consideration to the problem of sex chro mosomes. She states, however, that the diploid chromosome number, somatic and spermatogonial, is 24 and that 12 chromosomes appear in the metaphase plates of both the first and the second spermatocyte divisions. During my studies on sex differentiation in Amphibians I have collected a large material that is well suited for cytological investiga tions. This first paper on the spermatocyte divisions in five American toads is based on a number of testes from adult males that were pre served either in Zenker's fluid or in one of the modifications of Bouin's solution. The sections are stained with hematoxylin or by Feulgen's reaction. Of the five species, the dwarf B. quereicus Holbrook, the Oak Toad from Florida, is most characteristically different from the rest of North American toads. Cope (1889) says that it is "the smallest known species of the genus Bufo." We are inclined also to consider the , B. canorus Camp as a good species, characterized by a striking sexual dimorphism in the adult color pattern. The remaining three species are considered by the taxonomists as closely related and sometimes are spoken of as the lentiginosus group. In our work we used Fowler's Toad, B. fowleri Putnam from Woods 176 E. WITSCHI Cytologia4

Hole, the Southern Toad B. lentiginosus Shaw, Syn. terrestris Bon naterre from. Florida and the American Toad, B. americanus Le Conte from Iowa, Michigan and Washington, D. C. The three species appear quite distinct not only in their external characteristics but also with respect to the anatomy of their sexual organs (Witschi, 1933). The comparative study of the spermatocyte divisions of these five species reveals a surprising uniformity in the number and the general morphology of the chromosomes. As in the European and the Japanese toads the haploid number is always eleven. The six large chromosomes are usually arranged at the periphery and the five small ones in the center region of the metaphasic plates (figs. 1-10). In comparing first and second spermatocyte figures one has to consider that once the polar view shows the chromosomes from the apex (figs. 1, 3, 5, 7, 9) and in the second instance from the side (figs. 2, 4, 6, 8, 10). Side views of the first spermatocyte chromosomes may be obtained in lateral views of the corresponding spindles (fig. 12). About five of the larger chromosomes appear in the form of highly condensed tetrad rings. The exact number of rings is hard to decide upon, since in any one spindle one can never expect to get a face view of all of them. The central opening in the rings is always very small at this stage. Miss King in her paper of 1907 reports a number of twelve chro mosomes in both the first and the second spermatocyte divisions. It is not quite clear whether her Bufo lentiginosus corresponds to Shaw's species of this name or to Le Conte's B. americanus. The latter seems more probable. In any case, her count must be considered as erroneous in view of the now established great uniformity of the chromosome equipment within the genus Bufo. Bufo canorus differs from the other four investigated species by the larger size of the spermatocytes. The chromosomes themselves are larger too, with the exception of the smallest one, which we may call the microchromosome. The latter is in fact smaller than in any other form and exhibits a further peculiarity during the first spermatocyte division, when a strand of chromatic material is left behind on the central fiber, during the anaphasic process (fig. 12, left). The cells of Bufo fowleri appear to be slightly larger than those of lentiginosus and americanus, though the chromosomes are practically identical. Bufo quercicus has the smallest spermatocytes and probably also the smallest chromosomes. Of greatest interest are the peculiarities exhibited by the fifth chromosome (if the chromosomes are numbered in the order of decreas 1933 Contributions to the cytology of amphibian germ cells . I 177

Figs. 1-10. Chromosomes in the metaphasic plates of first (1, 3, 5, 7, 9) and second (2,

4, 6, 8, 10) spermatocyte divisions of five American species of toads. •~2400. 178 E. WITSCHI Cytolggia4 ing size). Like the other large chromosomes it lies at the periphery of the spindle, except for rare cases such as represented in figure 18. In the anaphase of the first meiotic division this chromosome proceeds to the poles ahead of the others (figs. 11, 12, 14) and therefore comes to lie on top of the telophasic plates (fig, 15). In polar views one can some times bring the high and the low elements separately into focus (figs. 3, 18). In no case have I been able to make out a place of fiber attachment on this chromosome. In all other chromosomes one can readily see strong leader-fibers originating from near the minute centriole anp attaching at the hillock that marks the apex of each chromosome. No hillock has been found on the fifth chromosome nor has it been possible to identify leader-fibers in connection with this element.

Figs. 11, 12. Bufo canorus. Lateral views of dividing primary spermatocytes. At the right of fig. 12 three additional tetrads are pictured which are seen at a deeper focal plane. •~2400. Fig. 13. Bufo canorus. Late anaphase of the second spermatocyte division. •~2400. Fig. 14. Bufo fowleri. Early anaphase of the first spermatocyte division. •~2400. Fig. 15. Bufo americanus. Late anaphase of the first spermatocyte division. •~2400. Figs. 16, 17. Bufo vulgaris. Side view and polar view of first sper

matocyte division. •~2400. 1933 Contributions to the cytology of amphibian germ cells. I 179

Another remarkable feature is the bipartite shape of the fifth chromosome during the anaphase of the first division (figs. 11 and 12). The Japanese authors have suggested that it is due to an early opening of one side of a ring tetrad. The constriction around each element should therefore roughly represent the apical region. I do not find that the earlier history of this chromosome favors this view, though final judgment will have to be based on the study of the conjugation process. In properly differentiated iron-hematoxylin preparations this chromosome appears slightly lighter than the rest, during the late stages of the first spermatocyte division. Towards the end of the second division it seems to be the first one to develop its chromosomevesicle (fig. 13). It is Fig. 18. Bufo canorus. not too easy to point out the fifth chromosome in First spermatocyte divi the metaphasic plate of the second division. sion. The fifth chromo somes in the center of From the study of the figures 19 to 26 it seems to

the plate. •~2400. evolve that it assumes the shape of a V with two legs of uneven length. There is enough reason to assume that this fifth chromosome oz the toads is homologous to the fourth one of the frog, described by the writer in 1922, 1924 and considered as a sex chromosome of the XY type. Iriki (1930) and Makino (1932) identified the same peculiar

Figs. 19-26. Bufo canorus. Eight metaphasic plates of secondary spermatocytes. Five small chromosomes in black, five large chromosomes in white, the odd chromo some is stippled. •~2400. 180 E. WITSCHI Cytologia4 chromosome in the genera Hyla and Rhacophorus in addition to describ ing it for several Japanese toads and . Obviously, it escaped the attention of Stohler (1928) in his study of the European toads. My.re investigation of Bufo vulgaris however demonstrates clearly its pre sence also in this species (figs. 16, 17). The wide distribution of this odd chromosome among anurans is in support of its interpretation as a sex chromosome. Iriki speaks of an XX pair of sex chromosomes. This seems rather unwarranted, since these symbols generally are understood to indicate homozygous con dition of sex determining genes as well as morphological identity of the chromosomes. If the genetical experiment should bring definite proof that the male toad-like the frog-is digametic with respect to sex determination, we should not hesitate to refer this back to an XY mechanism of sex chromosomes, even though it seems impossible to distinguish X and Y morphologically.

Summary

The chromosomic constitution of five American species of toads is much the same as that described by previous authors for European and Japanese species of the same genus. The haploid set consists of 6 large and 5 small chromosomes. The fifth chromosome shows peculiari ties that bear out its homology with the supposed sex chromosome of Rana temporaria. The importance of comparative studies in amphibian chromosomes with respect to the problem of the evolution of the sex chromosome mechanism is emphasized.

Bibliography Iriki, S., 1930: Studies on Amphibian Chromosomes. I. On the Chromosomes of Hyla arborea japonica Guenther. Mem. Coll. Sci., Kyoto Imp. Univ. B, 5. King, H. D., 1907: The Spermatogenesis of Bufo lentiginosus. Amer. Jour. Anat. 7. Makino, S., 1930: On the Chromosomes of Bufo sachalinensis Nikolski. Zool. Mag. Tokyo, 42.- 1932: Notes on the Chromosomes of Rhacophorus schlegelii Guenther. Proc. Imp. Acad. 8. - 1932: Notes on the Chromosomes of Rana temporaria L. and Bufo sachalinensis Nikolski. Ibid. 8. Minouchi, O. and Iriki, S., 1931: Studies on Amphibian Chromosomes. II. On the Chromosomes of Bufo bufo japonicus Schiegel. Mem. Coll. Sci. Kyoto Imp. Univ. B, 6. Strohler, R., 1928: Cytologische Untersuchungen an den Keimdrusen mitteleuro paischer Kroten. Z. Zellf. u. mikr. Anat. 7. 1933 Contributions to the cytology of amphibian germ cells. I 181

Witschi, E., 1922: Vererbung und Zytologie des Geschlechts. Z. ind. Abst. Vererb. 29. - 1924: Die Entwicklung der Keimzellen der Rana temporaria L. Z. Zellf. u. mikr. Anat. 1. - 1929: Rudimentary Hermaphrodism and Y Chromosome in Rana temporaria. Jour. Exp. Zool. 54. - 1930: The Geographical Distribution of the Sex Races of the European Grass Frog (Rana temporaries L.) Jour. Exp. Zool. 56. - 1933: The Nature of Bidder's Organ in the Toad. Amer. Jour. Anat. (in press).