K. Maeki and S.A. Ae (1979)
A chromosomal studies in interspecific hybrids of butterflies. 14.
P. alphenor, P. polytes, P. helenus, P. protenor, P. memnon, P.nepheles.
Proc. Japan Acad. , Vol.55, Ser.B, (4):196-201 (1979).

In the present article are presented some aspects on the chromosomal features of Fl-meiosis with regard to the species relationship in the crosses between the following six species, Papilio alphenor, P. polytes, P. protenor, P. helenus, P. memnon, and P. nepheles. Each species was found to have uniformly n, 30 which was common through other species of Papilio. The hybridization experiments were responsible to Ae (1964, 1971, 1972, 1973, 1974), while Maeki engaged in the chromosome study. The studies were exclusively subjected to the routine paraffin section-method as done in the previous cases.

Results.

The feature of meiotic pairing of chromosomes in male Fl-hybrids was the subject mainly concerned in the following observations.

la) Papilio alphenor Š (from Philippines) x Papilio polytes ‰ (from Hong-Kong). Chromosome counts were made in 28 cells at M-1 derived from 4 hybrid males (Nos. 4, 5, 13 and 15 from Brood Le-33) . The chromosome-number distributions in the M-1 metaphase coming from the 28 cells are as follows :

n, 30 . . . . . . . 26 cells
n, 31 .. . . . . . . 2 cells

lb) Papilio alphenor Š (from Philippines) x Papilio polytes ‰ (from Japan) . Ic) Papilio polytes Š (from Japan) x Papilio alpheuor ‰ (from Philippines) . In this cross were observed 52 M-1 cells which came from 5 hybrid males in the above reciprocal crosses (Nos. 4, 6 and 7 from Brood Le-57-14, and Nos. 4 and 5 from Brood 0-40-7-2412) . The chromosome-number distributions studied in the 52 M-I cells are as below :

n, 30 . . . . . . .29 cells
n, 31 . . . . . . . 14 "
n, 32 . . . . . . . . 7 "
n,33 . . . . . . . 2 "

ld) Papilio polytes Š (from India) x Papilio alphenor ‰ (from Philippines) . Counts of chromosomes in the above eross were carried out in 26 M-1 cells which were derived from 3 hybrid males (Nos. 4, 6 and 12 from Brood 0-34-14) . The chromosome-number distributions observed in them are as follows :

n, 30 . . . . . . .17 cells
n, 31 . . . . . . . 7 "
n ,32 . . . . . . . 2 "

le) Papilio alphenorŠ (from Philippines) x Papilio polytes ‰ (from Formosa). The chromosomes were studied in 39 M-1 cells obtained in 4 hybrid males (Nos. 4, 17, 22 and 24 from Brood Le-30-14) . The chromosome-number distributions so far observed in those cells were obtained as below :

n, 30 . . . . . . . 17 cells
n, 31 . . . . . . .16 "
n, 32 . . . . . . . 6 "

The chromosomes of the 145 M-1 cells studied in the above 16 hybrid males coming from the crosses, 1a) - 1e), between Philippine P. alphenor and Hong-Kong, Japanese, Indian, and Formosan P. polytes, showed a variation in number ranging from 30 to 33 (Fig. 1). The metaphase complement showing 30 bivalents was most frequent in occurrence, constituting 62 % of the observed cells. This is indicative of that the parental species under study are nearly related with each other. The distribution of the chromosome-numbers obtained in them are summarized as follows :

n, 30 . . . . .89 cells
n, 31 . . . . .39 "
n, 32 . . . . 15 "
n, 33 . . . . . . . 2 "

2a) Papilio protenor Š (from Japan) x Papilio alphenor ‰ (from Philippines). 2b) Papilio alphenor Š (from Philippines) x Papilio protenor ‰ (from Japan). Chromosomes of F1- hybrids were studied in 67 cells at M-I derived from 7 hybrid males in the above reciprocal crosses (Nos. 4, 7, 13 and 14 from Brood R-i-184-15-14 ; Nos. 1, 2 and 7 from Brood Le-44). The chromosome-number of the M-1 cells in this cross ranged in variation from n, 32 to n, 39 (Figs. 2-4). The cells showing 36 chromosomes were most frequent, being 34 % of cells observed. The chromosome-number distributions obtained in 67 M-I metaphases are shown below :

n, 32 . . . . . . . 1 cell n, 36 . . . . . . . 23 cells
n, 33 ............. 12 cells n, 37 . . . . . . . 4 cells
n, 34 ............. 13 cells n, 39 . . . . . . . 1 cell
n, 35 ............. 13 cells

3) Papilio alphenor Š (from Philippines) x Papilio helenus ‰ (from Japan). In this cross, 3 hybrid males (Nos. 1, 2 and 5 from Brood Le-57-2-28-10-2) provided at M-I 28 cells which were available for chromosome analysis. The chromosomes of these cells showed a fluctuation in number ranging from n, 37 to ,n, 53 (Figs. 7-9). The cells having the numbers from 47 to 50 were most frequent, giving 50% of cells so far studied. The chromosome-number distributions in the M-I metaphases observed in them were obtained as follows :

n, 37 ..... . . . 1 cell n, 46 ..... . . . 1 cell
n, 40 ..... . . . 1 cell n, 47 . . . . . . . 3 cells
n, 41 ..... . . . 1 cell n, 48 .... . 3 cells
n, 42 ..... . . . 2 cells n, 49.... . 5 cells
n, 43 .... .... 1 cell n, 50 . . . .. .. . 3 cells
n, 44 ... .. .. . 1 cell n, 52 . .. . . .. . 1 cell
n, 45 ... .. ... 2 cells n, 53 .. .. . .. 3 cells

4) Papilio alphenor Š (from Philippines) x Papilio memnon ‰ (from Formosa). In this cross, 19 nuclei at M-I cells derived from a single hybrid-male (Brood Le-85-1) provided materials for chromosome study. The n number of the M-1 cells in the above cross showed a variation ranging from 40 to 51 (Figs. 5-6). The chromosome number distributions in the M-I metaphase based on these cells are as follows :

n, 40 . . . . . . . 1 cell n, 47 . . . . . . . . 2 cells
n, 43 . . . . . . . 3 cells n, 48 . . . . . . . 2 cells
n, 44 . . . . . . . 1 cell n, 49 . . . . . . .2 cells
n, 45 . . . . . . . 3 cells n, 50 . . . . . . . 3 cells
n, 46 . . . . . . . 1 cell n, 51 . . . . . . . cell
5) Papilio nepheles Š (from Formosa) x Papilio protenor ‰ (from Japan). Chromosome counts were possible in 16 nuclei at M-I cells which were obtained in a single male of hybrid (No. 1 from Brood Ch-1-3). The n-chromosomes of the 16 M-I cells fluctuated in number from 51 to 60 (Figs. l0 -12). The metaphase complements having 59 or 60 chromosomes were most frequent in incidence, being 44 % in the observed cells. This indicates the fact that P. nepheles and P. protenor are very remotely related with each other. The chromosome-number distributions in the 16 M-I nuclei are as follows :

n, 51 . . . . . . . . 1 cell n, 57 . . . . . . . 2 cells
n, 54 ... .. .. . 1 cell n, 58 . .. .. . . . 1 cell
n, 55 ... .. .. . 2 cells n 59 . .. .. . . . 4 cells
n, 56 .... .. 2 cells n, 60 ... ... 3 cells

Remarks. The variations in chromosome-number and the modal numbers here obtained in hybrid males from the above 5 series of crosses which were made between the members of the black-Papilio group were obtained as shown in the following :

Cross Number variation Mode
alphenor x polytes . . . . . . . 30-33 . . . . . . . 30
alphenor x protenor . . . . . . . 32-39 . . . . . . . 36
alphenor x helenus . . . . . . . 37-53 . . . . . . . 47-50
alphenor x memnon . . . . . . . 40-51 . . . . . . . -
nepheles x protenor . . . . . . . 51-60 . . . . . . . -

The above data may allow the following statements : Among the crosses examined here Papilio alphenor and P. polytes are related most closely, and the relation between P. alphenor and P. protenor, that between P. alphenor and P. helenus, that between P. alphenor and P. memnon, and that between P. nepheles and P. protenor rank in order, on the basis of genic homology so far observed in the 6 species of Papilio concerned here. A diagram (Fig. 13) is given to illustrate the above feature.

Fig. 13. Diagram illustrating the relationship of 6 species of Papilio here studied, based on the analysis of meiotic pairing of chromosomes in hybrids.

According to Shirozu (1960), Papilio helenus was noted as a species of the II-group, P. nepheles and P. memnon were a member of the III-group, P. protenor, P. alphenor, and P. polytes belonged to the IV-group, on the basis of the morphological resemblance of male genitalia. The relationships between each 2 species belonging to different groups on the basis of the genitalian morphology seemed to be remotely related in those of chromosomal pairing of hybrids rather than in those seen among the morphologically identical species. In the F1- hybrids, the metaphase features of chromosomes having n, 30 consisting all of bivalents (Fig. 1) and those having n, 60 all being of univalents (Fig. 12) are shown to have a distinct appearance from one another. The chromosomes of the above cases fundamentally differ in nature and morphology, while their behaviors are rather identical. It seems apparent that the important factor for the formation of the equatorial plate is due probably to the balance of chromosomes in relation to the spindle organization.

Acknowledgements. Our cordial thanks are expressed to Emeritus Professor Sajiro Makino, M. J. A., for revising the manuscript with expert criticism, and to Professor Charles L. Remington for his continuous encouragement.

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