Karyological differentiation between two closely related mustelids, the Japanese weasel Mustela itatsi and the Siberian weasel M.

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1 CARYOLOGIA Vol. 53, no. 3-4: , 2000 Karyological differentiation between two closely related mustelids, the Japanese weasel Mustela itatsi and the Siberian weasel M. sibirica NAOKO KUROSE 1, RYUICHI MASUDA 2 *, TOSHIKI AOI 3 and SHIGEKI WATANABE 4 1 Cytogenetics Laboratory, Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo , Japan. 2 Chromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo , Japan. 3 Tomakomai Experimental Forest, Faculty of Agriculture, Hokkaido University, Tomakomai , Japan. 4 Aomadai-Higashi , Minoo , Japan Abstract Chromosomes of the Japanese weasel Mustela itatsi endemic to Japanese main islands except Hokkaido were examined by conventional, G-, and C-stainings, and compared with those of the Siberian weasel M. sibirica which is widespread in eastern Asia and Siberia. The two species shared the same diploid chromosome number (2n=38). However, their fundamental numbers and G- and C-banding patterns were largely differentiated from each other. Judging from the karyological features as well as mitochondrial DNA data and morphological characters previously reported, M. itatsi should be regarded as an independent species from M. sibirica, in agreement with the first description. Key words: chromosomes, karyological features, Japanese weasel, Mustela itatsi, Mustela sibirica, Siberian weasel. INTRODUCTION The Japanese weasel Mustela itatsi is an endemic mustelid species to Japanese main islands (HONSHU, SHIKOKU and KYUSHU) excluding Hokkaido. This weasel was first recorded as a distinct species Mustela itatsi Temminck, 1884, however, afterward it was sometimes considered as one subspecies of the Siberian weasel M. sibirica Pallas, 1773, which is widespread in eastern Asia and Siberia (NOWAK 1991; WOZEN-CRAFT 1993). Based on the cranial characters, YOUNGMAN (1982) reported that M. itatsi was phylogenetically closer to. sibirica. Meanwhile, IMAIZUMI (1960) showed significant morphological differences between the two taxa: the ratios of the tail length to the body-head length are more than 50% for M. sibirica, and about 40% for M. itatsi; and M. sibirica has a larger body size and a lighter brownish coat 'Corresponding author: fax ; masudary@ees.hokudai. ac.jp color. However, he regarded M. itatsi as one subspecies of M. sibirica. ABRAMOV (2000) reported that cranial differences between the two taxa are greater than geographic variations among M. sibirica populations from Siberia and Far East. Moreover, MASUDA and YOSHIDA (1994) and KUROSE et al (2000) reported that genetic distance of mitochondrial cytochrome b between M. itatsi and M. sibirica was remarkably larger, compared with differences between other mustelids, although these two weasels are most closely related among Mustela species. Both morphological and mitochondrial DNA (mtdna) data support that M. itatsi is an independent species from M. sibirica. On the other hand, M. sibirica was reported to have been introduced from Korean Peninsula to Japanese islands by the following two ways: one is escape or release from animal fur farms in Japan; the other is invading from Korean Peninsula through ship-transportation of human activity (MIYASHITA 1963). After the introduction to Japan, M. sibirica populations adapted themselves to Japanese environments, and they are

2 270 KUROSE, MASUDA, AOI and WATANABE currently expanding the habitat in Kyushu, Shikoku, and western Honshu. From the viewpoint of species conservation, hybridization between M. sibirica and M. itatsi is feared in these areas, as two weasels have the same diploid chromosome number (GRAPHODATSKY et al. 1976; TSUCHIDA 1979; OBARA 1985). However, no detailed comparative studies of karyotypes between the two taxa have ever been done. In the present study, karyological features of M. itatsi and M. sibirica were examined, using conventional, G- and C-stainings. We discuss here their karyological differentiation, in addition to molecular phylogenetic data and morphologcal features in the previous reports. MATERIALS AND METHODS Two males of M. itatsi from Ohshima Island, Japan and one female of M. itatsi as well as two females of M. sibirica from Wakayama Prefecture, Japan, were examined for chromosome analysis (Table 1). Identification between M. itatsi and M. sibirica was done according to the ratio of the tail length to the head-body length: more than 50% form. sibirica and about 40% for M. itatsi. Using fresh tissues of lung, heart, and skin from these weasels, fibroblasts were cultured in the Eagle's minimum essential medium (MEM, Nissui) including 15% fetal bovine serum. Harvested fibroblasts were suspended in M KC1 and fixed in methanolglacial acetic acid (3:1). Chromosomes spread on slides were stained with Giemsa (MERCK) in the phosphate buffer, ph 6.8. G- and C-band stainings were performed according to the trypsin-giemsa technique of SEABRIGHT (1971) and the BSG technique of SUMNER (1972), respectively. RESULTS Karyological findings on Mustela itatsi and M. sibirica are summarized in Table 1. The diploid chromosome number (2n) of M. itatsi was 38 and identical with that of M. sibirica. However, two taxa were different in autosomal constitutions. The conventional karyotype of M. itatsi (Fig. la) was constituted by 36 autosomes consisting of seven pairs of metacentrics, seven pairs of submetacentrics or subtelocentrics, four pairs of acrocentrics, and two sex chromosomes (Table 1). The fundamental number (FN) was 64. The heteromorphic secondary constriction was seen on the long arm of No. 17 of a female individual (Fig. la). However, the other two males had no secondary constrictions. No other autosomal differences among the three individuals were observed. By contrast, the karyotype (2n=38) of M. sibirica (Fig. 1b) consisted of seven pairs of metacentrics, four pairs of submetacentrics or subtelocentrics, seven pairs of acrocentricsas, and two sex chromosomes (Table 1). The FN was 58. The homomorphic secondary constrictions were seen on the long arm of No. 16, and no karyological differences occurred between two females. G-banded karyotypes of the two taxa are shown in Fig. 2. tlomologous banding patterns between M. itatsi (Fig. 2a) and M. sibirica (Fig. 2b) were seen on chromosome Nos. 1,2, and X, while patterns on the other chromosomes were different between the two taxa. In C-banded karyotypes of M. itatsi (Fig. 3a), C-blocks were located on centromere regions of large metacentric chromosomes (Nos. 1-4), and on short arms of five submetacentric chromosomes (Nos. 9-11, 13, and 14) and four acrocentric chromosomes (Nos ). The whole region of the Y chromosome was positive for the C-block. Meanwhile, M. sibirica (Fig. 3b) had C-blocks on centromere regions of large metacentric chromosomes (Nos. 1-4), two submetacentric chromosomes (Nos. 8 and 9), and X chromosome, and on short arms of three acrocentric chromosomes (Nos. 12, 14, and 15). TABLE 1 Karyological data of the Japanese weasel Mustela itatsi and the Siberian weasel M. sibirica examined in the present study.

3 KARYOTYPES OF JAPANESE AND SIBERIAN WEASELS 271 Fig. 1 Conventional karyotypes of a male Japanese weasel Mustela itatsi (a) and a female Siberian weasel M. sibirica (b). Heteromorphic secondary constriction on the long arm of No. 17 from a different individual is shown in squeare (a). Arrows indicate secondary constrictions.

4 Fig. 2 G-banded karyotypes of a male Mustela itatsi (a) and a female M. sibirica (b).

5 KARYOTYPES OF JAPANESE AND SIBERIAN WEASELS 273 Fig. 3 C-banded karyotypes of a male Mustela itatsi (a) and a female M. sibirica (b). Metaphases are the same as those in Fig. 1.

6 274 KUROSE, MASUDA, AOI and WATANABE DISCUSSION In the present study, it was obviously demonstrated that the chromosomal constitution and G- and C-banded karyotypes of M. itatsi were different from those of M. sibirica. The diploid chromosome number of M. itatsi has been reported to be 38 (MAKING and MURAMOTO 1966; TSUCHIDA 1979; OBARA 1985). OBARA (1985) reported that polymorphic small secondary con strictions were observed in several individuals of the M. itatsi population. In the present study, it was obvious that a female M. itatsi had heteromorphic secondary constriction on the long arm of No. 17, and that the other individuals (two males) did not have it (Fig. la). Secondary constriction was also seen on the long arm of No. 16 of M. M. sibirica (Fig. 1b). This structure could have been obtained in the process of karyotypic evolution of the two taxa. As for the G-banded karyotype between two taxa, banding patterns were homologous on chromosome 1, 2 and X. However, patterns on the other chromosomes were different from each other. It was difficult to compare G- banded karyotypes between two taxa, because of considerably difference between their banding patterns. Judging from the differences of G-banding patterns and chromosomal consitution between two taxa, it is impossible to explain their karyotypic evolution based on the simple Robertsonian rearrangement. Karyotypic differentiation of two taxa could have appeared by complicated translocations and in versions. On the other hand, as for the C-banded karyotype, the number of C-blocks in M. itatsi was more than that of M. sibirica. In several mammals, the speciation with variations of constitutive heterochromatins was shown (e.g., PATTON and SHERWOOD 1982; ROBINSON et al. 1983; SVARTMAN et al. 1999). Chromosomal differentiation between M. itatsi and M. sibirica may have been influenced by positional and quantitative variations of C-blocks in the proc ess of karyotypic evolution. Meanwhile, conventional and G-banded karyotypes of M. sibirica reported by VOLOBUEV et al. (1975) and GRAPHODATSKY et al. (1976) were basically in concordance with the present data. GRAPHOD- ATSKY et al. (1979) showed some data of conventional, G- and C-banded karyotypes of M. itatsi and M. sibirica. In the present study, we could make a more detailed karyological comparison between the two taxa by Giemsa, G-, and C-stainings. IMAIZUMI (1960) regarded M. itatsi as one subspecies of M. sibirica, indicating the difference of the ratio of the tail length to the headbody length between the two taxa. However, YOUNGMAN (1982) investigated the difference of cranial morphology between the two taxa and suggested that M. itatsi is a distinct species. In addition, based on the large genetic distance of mtdna between the two taxa, MASUDA and YOSHIDA (1994) and KUROSE et al (2000) indicated that M. itatsi is distinct enough to merit the spesific rank. The present study revealed the clear karyological differentiation between the two taxa. Therefore, it is strongly supported that M. itatsi should be classified as a distinct species from M. sibirica, in agreement with the first description of Mustela M.itatsi Temminck, In Europe, hybridization between the European mink M. lutreola and the steppe polecat M. eversmanniwas reported (YOUNGMAN 1982). Both species have the same chromosome number (2n=38), but differ in chromosome constitutions. Considered this case, hybridiza tion between native M. itatsi and introduced M. sibirica in Japanese main islands has been threatened. If hybridization between them occurs, the gene pool in the native M. itatsi populations could be contaminated and disturbed. Fortunately, no evidence of hybridization between M. itatsi and M. sibirica in Japan has ever been revealed by morphological, karyological, and molecular genetical analyses. In the further genetic survey of these species, karyological findings obtained in the present study provide useful information in conjunction with DNA and morphological data. Acknowledgements We thank Y. Miyoshi (Ohshima Park), T. Hosoda (Tama Zoological Park) for supplying specimens. We are also grateful to Dr. T. Oshida and C. Umehara (Hokkaido University) for critical comments on the manuscript. This work was supported in part by Grants-in-Aid for the Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan. REFERENCES ABRAMOV A.V., 2000 The taxonomic status of the Japanese weasel, Mustela itatsi (Carnivora, Mustelidae). Zool. ]., 79: (in Russian with Eng lish abstract).

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