No. 10] Proc. Acad., 52 (1976) 567

153. Chromosomal Studies on Interspeci fic Hybrids of (Papilionidae, ). VII

Studies in Crosses among P. memnon, P. ascalaphus, P. polymnestor, and P. rumanzovia

By Kodo MAEKI* ) and Shigeru A. AE**)

(Communicated by Sajiro MAKINO, M. J. A., Dec. 13, 1976)

Since McClung (1908) suggested in a pioneer work on orthop- teran chromosomes the correlation that could be expected to exist between the chromosomes and the structural organization of organ- isms, the behavior and morphological changes of chromosomes during phylogenesis has called prime interest of biologists, particularly in the field of cytogenetics. We are intending, in a series of cytogenetic studies of the genus , to provide critical data essen- tial for understanding the interspecific relationships of these in terms of their genetic makeup, in an approach from hybridization experiments. Several reports along this line have been published to account for chromosomal mechanism in relation to species-differentia- tion or interspecific relationship, inquiring into the meiotic behavior of chromosomes in the following hybrids from crosses among P. polyctor, P. bianor, P. paris, P. maackii, P. polytes, P. helenus, P. protenor, P. nepheles, P. aegeus, P. f uscus, P. macilentus, and P. mem- non (Maeki and Ae 1966, 1970, 1975, 1976a, 1976b, 1976c). The present article presents further data on the meiotic features, particu- larly of chromosome pairing in male individuals, derived from the following hybrids : P, polymnestor x P. memnon, P. polymnestor x P. rumanzovia, P. memnon x P. rumanzovia, and P. memnon x P. ascalaphus. The hybrid specimens were obtained by means of arti- ficial fertilization by Ae (1967, 1968, 1971, 1974). Chromosomes were investigated by Maeki according to the routine paraffin method. Results. Four parental species of Papilio here concerned (P. polymnestor, P. rumanzovia, P. memnon, and P. ascalaphus) were characterized by a uniform n-number of 30, along with an apparently similar karyotype. In the following, the essential features of chromo- somes are described for each hybrid. la. ? (from Ceylon) X (from Japan). *' The Biological Laboratory , Faculty of Science, the Kwansei Gakuin University, Nishinomiya, Japan. **' The Biological Laboratory , the Nanzan University, Nagoya, Japan. 568 K. MAEKI and S. A. AE [vol. 52,

lb. Papilio memnon (from Japan) X Papilio pot ymnestor (from Ceylon). Chromosome counts were made in 129 cells at M-I and 40 cells at M-II based on five hybrid males from the above reciprocal crosses (Nos. 2, 9,12 and 13 from Brood Py-2 ; and No. 3 from Brood M-76). The chromosome number of the M-I cells in the F1 hybrids was obtained as uniformly 30 in all cells so far studied (Fig. 1) . Notice- able is the fact that all the hybrid chromosomes appeared in a com- pletely paired condition at the M-I metaphase. This suggests a close kinship between the two parental species. 2. Papilio polymnestor (from Ceylon) X (from Philippines). Fifty-eight cells at M-I coming from four hybrid males in the above cross (Nos. 1 and 3 from Brood Py-12 ; and Nos. 1 and 6 from Brood Py-20) were available for chromosome counts. The chromosomes of these hybrid specimens varied in num- ber from n, 30 to n, 32 (Fig. 2). The metaphase complement con- sisting of 30 bivalents was most frequent in occurrence, constitut- ing 97% of the observed cells. This feature indicates that there are a good many chromosomes which undergo pairing in the first meiosis. This is indicative of that the parental species are con- siderably closely related. The chromosome-number distributions in the M-I metaphase observed in the 58 cells are shown as follows : n, 30 ...... 56 cells n, 31 ...... 1 cell n,32 ...... l cell 3. Papilio memnon (from Japan) X Papilio rumanzovia a (from Philippines). In this cross, sixty-three cells at M-I coming from four hybrid males (Nos. 6, 11 and 15 from Brood M-109-14; and No. 1 from Brood M-109-2) allowed chromosome investigation. The chromosome-number of the M-I cells in the present hybrids ranged in variation from 30 to 32 (Fig. 3). The chromosome com- plement showing 30 bivalents was most frequent giving 92% of the observed cells. The chromosome-number distributions studied in the 63 M-I cells are as follows : n, 30 ...... 58 cells n, 31 ...... 4 cells n, 32 ...... 1 cell 4. Papilio memnon (from Japan) X a (from Celebes). Counts of chromosomes in the above cross were carried out in 22 cells at M-I which were derived from three hybrid males (Nos. 1, 5 and 7 from Brood M-52). The variation in the n-number was rather remarkable in this hybrid showing a range from 30 to 35 (Fig. 4). The variation showing 31 to 33 chromosomes No. 10] Chromosomes of Papilio Hybrids. VII 569

Figs. 1-4: Meiotic chromosomes of Papilio hybrids (M-I, x3000). Fig. 5: Normal male-gonad (8 follicles, x40). Fig. 6: Accessory gonad of hybrid male (arrow, x50). 1: P, polymnestorx P, mom- non, F1, n, 30. 2: P. polymnestor x P. rumanzovia, F1, n, 30. 3: P. memnon X P. rumanzovia, F1, n, 30. 4: P. memnon X P, ascal- aphus, F1, n, 33. was most frequent, showing 86% of the cells under study, with the modal number at 33. The chromosome-number distributions observed in the 22 M-I cells are as below : n, 30 ...... 1 cell n, 33 ...... 10 cells n, 31 ...... 6 cells n, 34 ...... 1 cell n, 32 ...... 3 cells n, 35 ...... 1 cell Remarks. The behavior of chromosomes in phylogenesis has been a project of utmost importance to cytogeneticists, and direct cytogenetic investigations have served as an efficient tool for analysis of basic problems of species-differentiation in both and plants. We have been dealing with species-differentiation and interspecies- relationship of Papilio, by inquiring into the meiotic pairing of chromosomes in interspecific hybrids. It was shown in four types of interspecific crosses carried out in the P. memnon subgroup that the cross between P. polymnestor and P. memnon gave rise to F1 in- dividuals of both sexes together with offspring in F2- and F3-genera- tions, while the crosses, P. polymnestor x P. rumanzovia, P. memnon x P. rumanzovia, and P, memnon x P, ascalaphus, yielded F1-males only. F1 males derived from the crosses, P. memnon x P. ruman- zovia and P, polymnestor x P. rumanzovia, showed a good fertility, but fertility of F1 males from the cross between P. memnon and P. ascalaphus was reduced. Ranges of the chromosome-number varia- tion and the number-mode obtained so far in the 4 kinds of hybrids 570 K. MAEKI and S. A. AE [Vol. 52,

Fig. 7. Diagram showing the relationship of four Papilio species, based on analysis of meiotic chromosomal pairing in hybrid specimens. here concerned were as below : Cross Number-variation Mode polymnestor x memnon 30 30 polymnestor x rumanzovia 30-32 30 memnon x rumanzovia 30-32 30 memnon x ascalaphus 30-35 33 From the results of the present investigation, the following statements can be allowed that P. polymnestor and P. memnon are most closely related, and that the relation between P. polymnestor and P. rumanzovia, and that between P. memnon and P. rumanzovia, and that between P. memnon and P. ascalaphus, rank in order, in view of genie homology among these species. On the basis of the features established in the foregoing studies (Maeki and Ae 1976b, 1976c), and also of those presented in this study, the interspecific relationship among the studied species may be shown as thus : P. memnon is closer to P. polymnestor, P. rumanzovia, and P. ascalaphns than P. helenus, P. protenor, and P. macilentus. Systematically, in the black Papilio group, P. memnon constitutes a subgroup together with these three species. All the four species under consideration are closely related with each other from the view of genie homology. But, there occurs a remarkable difference in the wing-pattern among them. Probably, such a wing-pattern difference may involve mimic genie phenomena. In the gonads of the male F1 individuals produced between closely related species, there was the occurrence of an accessory gonad characterized by a specially thin covering (Fig. 6). Such a gonadal structure seems to be one of important features specific to hybrids.