No. 7] Proc. Japan Acad., 5.1 (1975) 577

123. Chromosomal Studies on Interspeci ftic Hybrids o f (Papilionidcte, ) 3. bianor x Papilio Paris ~, Papilio Paris ? x Papilio maackii a, Papilio bianor x Papilio maackii ~, and Papilio polytes x Papilio maackii

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

(Comm. by Sajiro MAKINO, M. J. A., Sept. 12, 1975)

The literature refers to a number of cytogenetical studies on interspecific hybrids in moths (Heterocera) , our knowledge has been very limited on butterflies (Rhopalocera) , we have been engaging in chromosomal researches on interspecific hybrids of Papilio : Papilio polyctorxPapilio bianor, Papilio polyctorxPapilio maackii (Maeki and Ae, 1966) , Papilio polytes x Papilio helenus, Papilio polytes x Papilio protenon, and Papilio pololytes x Papilio macilentus (Maeki and Ae,1970) , with special interest in pairing of chromosomes at the first meiosis in males. The present study deals with the chromosomes of the following hybrids : P. bianor x P. paris, P. bianor x P. maackii, P. paris x P. maackii, and P. polytes x P. maackii. The hybrid speci- mens were produced artificially by Ae (1961, 1962, 1963, and 1965) at the Nanzan University. Living specimens of P. paris and P. polytes were obtained in Hong Kong by Ae on the way of his collecting trip in April, 1965, and they were reared in the laboratory of the Nanzan University. Soon after emergence, females of P. paris and P. polytes were hand-paired with local males of P. maackii. Only adult males were produced from the crosses : P. polytes x P. maackii, P. paris x P. maackii, and P. bianor x P. maackii, while many adult males and females were available for study from the crosses between P. paris and P. bianor. Gonads from each specimen were exclusively fixed in Allen's P. F. A.-3 solution. The sections, 10 micra thick, were made according to the routine paraffin-method and stained with Heidenhain's iron-haematoxylin with counter staining of light green. Results. The chromosomal features of interspecific hybrids under study are shown in photomicrographs. Four parental species of Papilio, P. bianor, P. paris, P. maackii and P. polytes, showed

*' Biological Laboratory , Faculty of Science, Kwansei Gakuin University, Nishinomiya, Japan. **' Biological Laboratory , Nanzan University, Yamazato-cho, Showa-ku, Nagoya, Japan. 578 K. MAEKI and S. A. An [Vol. 51, uniformly n, 30, with an apparently similar karyotype. In the follow- ing, some accounts are given for each hybrid. 1. Papilio bianor S~(Japan) X Papilio Paris ; (Hong Kong). Counts of chromosomes in hybrids were made in 66 nuclei in the primary spermatocytes and 17 nuclei in the secondary spermatocytes, based on testes coming from eight hybrid males (Nos. 1, 2, 9,10,13 and 14 individuals of Brood D-77-10 and Nos. 1 and 4 individuals of Brood D-77-11). The haploid number of chromosomes in both types of the spermatocytes of the hybrids were determined as 60 (Figs. 1, 2). The primary spermatocytes of the hybrids were two times as large as those of the parental species. The hybrid chromosomes of primary spermatocytes were regular in size having the appearance of bivalent chromosomes. Also, the head of hybrid spermatozoon was large-sized in comparison with that of the parental species. 2. Papilio Paris ~' (Hong Kong) X Papilio maackii a (Japan). Counts of chromosomes were made in 69 nuclei in the primary sper- matocytes based on the testes taken from two hybrid males (Pa-2-1 & Pa-2-2). The chromosomes of the hybrid spermatocytes showed a considerable variation in number, ranging from 38 to 56 (Figs. 3-6). The distribution frequency of the chromosome number is as follows :

It was shown that the nuclei having 38 chromosomes had 16 univalents resulted from non-pairing. Also, 46-chromosome-nuclei had 32 univalents, 47-chromosome-nuclei showed 34 univalents, and 56-chromosome-nuclei contained 52 univalents. The nuclei from n, 43 to n, 48 showed a frequency of .62%. 3. Papilio bianor 1?-(Japan) X Papilio maackii a (Japan). Sixty-two nuclei in the primary spermatocytes of the testes from four hybrid males (D-31-21-1, D-31-21-2, D-29-21-1 and K-32-19-3) of P. bianor ? x P. maackii and P, maackii ? x P, bianor a were available for chromosome counts. The spermatocyte chromosome- number of these reciprocal hybrids of the both crosses showed a variation with a rang from 33 to 54 (Figs. 7-10). The chromosome constitution consisting of 23 bivalents and 14 univalents was most No. 7] Chromosomes of Hybrids 579

Figs. 1-2: P. bianor X P. paris, F1. Figs. 3-6: P. Paris X P. maackii, F1. Figs. 7-10: P, bianorXP. maackii, F1. Figs. 11-12: P, polytes x P. maackii, F1. Fig. 1: n, 60 (I, all bivalents). Fig. 2: n, 60 (II). Fig. 3: n, 38 (I). Fig. 4: n, 43 (I). Fig. 5: n, 50 (I). Fig. 6: n, 56 (I). Fig. 7: n, 40 (I). Fig. 8: n, 41(I). Fig. 9: n, 45 (I). Fig. 10: n, 54 (I). Fig. 11: n, 60 (I, all univalents). Fig. 12: n, 60 (I). (I)=primary spermato- cyte. (II)= secondary spermatocyte. X 5000 X 2/3. frequent showing 32% of the observed cells. The chromosome- number distributions are shown as follows : 580 K. MAEKIand S. A. AE [Vol. 51,

4. Papilio polytes (Hong Kong) X Papilio maackii (Japan). Counts of chromosomes were performed in 38 primary spermato- cytes from 2 hybrid males (0-27-1, 0-27-3). The haploid chromo- some-number obtained in the above was 60 so far studied (Figs. 11- 12). It is thus evident that the number of chromosomes in F1 hybrids is the same as that of the total number of the haploid chromosomes of the parental species. The hybrids showed a complete lack of chromosomal pairing at the primary spermatocyte metaphase. Discussion. It seems probable that 60 chromosomes which observed in the first meiosis of F1 hybrid males from P, bianor x P. Paris may be bivalent in nature, because 17 nuclei so far observed in the second meiosis had 60 chromosomes without exception. Thus it appears that these F1 hybrid males may be tetraploid. The follow- ing facts may be supplemental for this conclusion. (1) Diameter of the primary spermatocyte of the F1 hybrid was 25 p, while those of P. bianor and P. paris were 2op. According to the formula *2rr3 the calculation values were found to be 8179 p3 and 4188 p3, respectively. Thus, the hybrid cells were two times as large as those of the parental species. (2) Chromosome-size of the first meiosis of the F1 hybrids was the same as the size of the bivalent chromosomes of the parental species. (3) The sperm-heads of the F1 hybrid, 12 p in length and 1p in wide, were considerably larger than those of the parental species which were 10p in length and 0.5-o.7p in width. The formation of the tetraploid hybrids in this cross is not clearly understood at the present status of knowledge. It is a subject of particular interest in future investigations that such a feature was observed only in the cross between P. bianor and P. paris, and that

Fig. 13. The distances in each species show the relationship among five species based on chromosomal pairing in hybrids. No. 7] Chromosomes of Butterfly Hybrids 581 many other Papilio crosses produced no such a picture (Maeki & Ae, 1966 & 1970). Among 5 forms of interspecific hybrids in the P. bianor group (Ae,1971) , the crosses of P. bianor x P. polyctor (Maeki & Ae,1966 ) and of P. bianor x P. paris produced adult hybrids of both sexes, while others yielded males only. This suggests that P. bianor and P. paris are closely related. On the other hand it is impossible to discuss, because of tetraploidy, the systematic relationship between P. bianor and P. paris on the basis of genic homology. Ranges of the chromosome-number variations and the number- modes obtained so far in 4 kinds of hybrids of the P. bianor group were as follows :

Variation of Cross chrom.-number Mode bianorX polyctor 30-31 30 po lyctor Xmaackii 33-42 34 bianor Xmaackii 33-54 37 paris Xmaackii 38-56 43, 45

Referring to the above feature and to the view of genic homo- logy, it may be stated that P. bianor and P. polyctor are most closely related, and the relations between P. polyctor and P. maackii, between P. bianor and P. maackii and between P, paris and P. maackii rank in order. The hybrids between P. maackii and P. polytes are products between a species of the P. bianor group and a species of the black Papilio group (Ae,1971) . In F1 males from this cross, no bivalent chromosome was formed at first meiosis. This may be an evidence indicating that the P. bianor group and the black Papilio group are remotely related. Summary. The specific relationships among Papilio paris, P. bianor, P. maackii and P. polytes are examined based on chromosome pairing at first meiosis of F1 hybrids. At the 1st meiosis of the F1 from P. bianor x P. paris, 60 bivalent chromosomes were observed. Chromosomally they may be tetraploid. In view of genie homology, the systematic relationships between P. bianor, P. polyctor, P. maackii, P. paris, and P, polytes are discussed. Acknowledgements. We are grateful to Emeritus Professor Dr. Saj iro Makino for reading through the manuscript with invalua- ble advice and criticism, and to Dr. Charles L. Remington for helpful suggestion. 582 K. MAEKI and S. A. AE [Vol. 51,

References

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