Vol. 53, no. 3-4: 269-275, 2000

Karyological differentiation between two closely relatedmustelids, the Japanese weasel Mustela itatsi and the Si-berian weasel M. sibiricaNAOKO KUROSE1, RYUICHI MASUDA2*, TOSHIKI AOI3 and SHIGEKI WATANABE4

1 Cytogenetics Laboratory, Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo060-0810, Japan. 2 Chromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan. 3 TomakomaiExperimental Forest, Faculty of Agriculture, Hokkaido University, Tomakomai 053-0035, Japan. 4 Aomadai-Higashi 2-24-5-215,Minoo 562-0022, Japan

Abstract — Chromosomes of the Japanese weasel Mustela itatsi endemic to Japanesemain islands except Hokkaido were examined by conventional, G-, and C-stainings,and compared with those of the Siberian weasel M. sibirica which is widespread ineastern Asia and Siberia. The two species shared the same diploid chromosomenumber (2n=38). However, their fundamental numbers and G- and C-bandingpatterns were largely differentiated from each other. Judging from the karyologicalfeatures as well as mitochondrial DNA data and morphological characters previouslyreported, M. itatsi should be regarded as an independent species from M. sibirica, inagreement 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 en-demic mustelid species to Japanese main islands(HONSHU, SHIKOKU and KYUSHU)excluding Hokkaido. This weasel was firstrecorded as a distinct species Mustela itatsiTemminck, 1884, however, afterward it wassometimes considered as one subspecies of theSiberian weasel M. sibirica Pallas, 1773, which iswidespread in eastern Asia and Siberia (NOWAK1991; WOZEN-CRAFT 1993). Based on the cranialcharacters, YOUNGMAN (1982) reported that M.itatsi was phylogenetically closer to . sibirica.Meanwhile, IMAIZUMI (1960) showed significantmorphological differences between the two taxa:the ratios of the tail length to the body-headlength are more than 50% for M. sibirica, andabout 40% for M. itatsi; and M. sibirica has alarger body size and a lighter brownish coat

•'Corresponding author: fax ++81-11-736-6304; e-mail:masudary@ees .hokudai. ac.jp

color. However, he regarded M. itatsi as onesubspecies of M. sibirica. ABRAMOV (2000) reportedthat cranial differences between the two taxa aregreater than geographic variations among M.sibirica populations from Siberia and Far East.Moreover, MASUDA and YOSHIDA (1994) andKUROSE et al (2000) reported that geneticdistance of mitochondrial cytochrome b betweenM. itatsi and M. sibirica was remarkably larger,compared with differences between othermustelids, although these two weasels are mostclosely related among Mustela species. Bothmorphological and mitochondrial DNA(mtDNA) data support that M. itatsi is an inde-pendent species from M. sibirica.

On the other hand, M. sibirica was reportedto have been introduced from Korean Peninsulato Japanese islands by the following two ways:one is escape or release from animal fur farms inJapan; the other is invading from Korean Penin-sula through ship-transportation of human ac-tivity (MIYASHITA 1963). After the introduction toJapan, M. sibirica populations adapted them-selves to Japanese environments, and they are

CARYOLOGIA

270 KUROSE, MASUDA, AOI and WATANABE

currently expanding the habitat in Kyushu,Shikoku, and western Honshu. From the view-point of species conservation, hybridization be-tween M. sibirica and M. itatsi is feared in theseareas, as two weasels have the same diploidchromosome number (GRAPHODATSKY et al. 1976;TSUCHIDA 1979; OBARA 1985). However, no detailedcomparative studies of karyotypes between thetwo taxa have ever been done.

In the present study, karyological features ofM. itatsi and M. sibirica were examined, usingconventional, G- and C-stainings. We discusshere their karyological differentiation, in addi-tion to molecular phylogenetic data and mor-phologcal features in the previous reports.

MATERIALS AND METHODS

Two males of M. itatsi from Ohshima Island, Japanand one female of M. itatsi as well as two females of M.sibirica from Wakayama Prefecture, Japan, wereexamined for chromosome analysis (Table 1).Identification between M. itatsi and M. sibirica wasdone according to the ratio of the tail length to thehead-body length: more than 50% forM. sibirica andabout 40% for M. itatsi. Using fresh tissues of lung,heart, and skin from these weasels, fibroblasts werecultured in the Eagle's minimum essential medium(MEM, Nissui) including 15% fetal bovine serum.Harvested fibroblasts were suspended in 0.075 MKC1 and fixed in methanoLglacial acetic acid (3:1).Chromosomes spread on slides were stained withGiemsa (MERCK) in the phosphate buffer, pH 6.8.G- and C-band stainings were performed according tothe trypsin-Giemsa technique of SEABRIGHT (1971) andthe BSG technique of SUMNER (1972), respectively.

RESULTS

Karyological findings on Mustela itatsi andM. sibirica are summarized in Table 1. The dip-

loid chromosome number (2n) of M. itatsi was38 and identical with that of M. sibirica. How-ever, two taxa were different in autosomal con-stitutions. The conventional karyotype of M.itatsi (Fig. la) was constituted by 36 autosomesconsisting of seven pairs of metacentrics, sevenpairs of submetacentrics or subtelocentrics, fourpairs of acrocentrics, and two sex chromosomes(Table 1). The fundamental number (FN) was64. The heteromorphic secondary constrictionwas seen on the long arm of No. 17 of a femaleindividual (Fig. la). However, the other twomales had no secondary constrictions. No otherautosomal differences among the threeindividuals were observed.

By contrast, the karyotype (2n=38) of M.sibirica (Fig. 1b) consisted of seven pairs ofmetacentrics, four pairs of submetacentrics orsubtelocentrics, seven pairs of acrocentricsas,and two sex chromosomes (Table 1). The FNwas 58. The homomorphic secondary constric-tions were seen on the long arm of No. 16, andno karyological differences occurred betweentwo females.

G-banded karyotypes of the two taxa areshown in Fig. 2. tlomologous banding patternsbetween M. itatsi (Fig. 2a) and M. sibirica (Fig.2b) were seen on chromosome Nos. 1,2, and X,while patterns on the other chromosomes weredifferent between the two taxa.

In C-banded karyotypes of M. itatsi (Fig. 3a),C-blocks were located on centromere regions oflarge metacentric chromosomes (Nos. 1-4), andon short arms of five submetacentricchromosomes (Nos. 9-11, 13, and 14) and fouracrocentric chromosomes (Nos. 15-18). Thewhole region of the Y chromosome was positivefor the C-block. Meanwhile, M. sibirica (Fig.3b) had C-blocks on centromere regions of largemetacentric chromosomes (Nos. 1-4), twosubmetacentric chromosomes (Nos. 8 and 9),and X chromosome, and on short arms of threeacrocentric 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.

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). Hetero-morphic secondary constriction on the long arm of No. 17 from a different individual is shown in squeare (a). Arrows indicate sec-ondary constrictions.

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

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.

KUROSE, MASUDA, AOI and WATANABE

DISCUSSION

In the present study, it was obviously demon-strated that the chromosomal constitution andG- and C-banded karyotypes of M. itatsi weredifferent from those of M. sibirica. The diploidchromosome number of M. itatsi has been re-ported to be 38 (MAKING and MURAMOTO 1966;TSUCHIDA 1979; OBARA 1985). OBARA (1985) reportedthat polymorphic small secondary constrictionswere observed in several individuals of the M.itatsi population. In the present study, it wasobvious that a female M. itatsi had hetero-morphic secondary constriction on the long armof No. 17, and that the other individuals (twomales) did not have it (Fig. la). Secondary con-striction was also seen on the long arm of No. 16of M. M. sibirica (Fig. 1b). This structure couldhave been obtained in the process of karyotypicevolution of the two taxa.

As for the G-banded karyotype between twotaxa, banding patterns were homologous onchromosome 1, 2 and X. However, patterns onthe other chromosomes were different fromeach other. It was difficult to compare G-banded karyotypes between two taxa, becauseof considerably difference between theirbanding patterns. Judging from the differencesof G-banding patterns and chromosomal consi-tution between two taxa, it is impossible to ex-plain their karyotypic evolution based on thesimple Robertsonian rearrangement. Karyotypicdifferentiation of two taxa could have appearedby complicated translocations and inversions.On the other hand, as for the C-bandedkaryotype, the number of C-blocks in M. itatsiwas more than that of M. sibirica. In severalmammals, the speciation with variations ofconstitutive heterochromatins was shown (e.g.,PATTON and SHERWOOD 1982; ROBINSON et al. 1983;SVARTMAN et al. 1999). Chromosomaldifferentiation between M. itatsi and M. sibiricamay have been influenced by positional andquantitative variations of C-blocks in the proc essof karyotypic evolution. Meanwhile, con-ventional and G-banded karyotypes of M.sibirica reported by VOLOBUEV et al. (1975) andGRAPHODATSKY et al. (1976) were basically inconcordance with the present data. GRAPHOD-ATSKY et al. (1979) showed some data of con-ventional, G- and C-banded karyotypes of M.itatsi and M. sibirica. In the present study, wecould make a more detailed karyological com-

parison between the two taxa by Giemsa, G-,and C-stainings.

IMAIZUMI (1960) regarded M. itatsi as onesubspecies of M. sibirica, indicating the differ-ence of the ratio of the tail length to the head-body length between the two taxa. However,YOUNGMAN (1982) investigated the difference ofcranial morphology between the two taxa andsuggested that M. itatsi is a distinct species. Inaddition, based on the large genetic distance ofmtDNA between the two taxa, MASUDA andYOSHIDA (1994) and KUROSE et al (2000) indicatedthat M. itatsi is distinct enough to merit thespesific rank. The present study revealed the clearkaryological differentiation between the twotaxa. Therefore, it is strongly supported that M.itatsi should be classified as a distinct species fromM. sibirica, in agreement with the first descriptionof Mustela M.itatsi Temminck, 1884.

In Europe, hybridization between the Euro-pean mink M. lutreola and the steppe polecat M.eversmanniwas reported (YOUNGMAN 1982). Bothspecies have the same chromosome number(2n=38), but differ in chromosomeconstitutions. Considered this case, hybridiza tionbetween native M. itatsi and introduced M.sibirica in Japanese main islands has beenthreatened. If hybridization between them oc-curs, the gene pool in the native M. itatsi popu-lations could be contaminated and disturbed.Fortunately, no evidence of hybridization be-tween M. itatsi and M. sibirica in Japan has everbeen revealed by morphological, karyological,and molecular genetical analyses. In the furthergenetic survey of these species, karyologicalfindings obtained in the present study provideuseful information in conjunction with DNAand 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 workwas supported in part by Grants-in-Aid for the Sci-entific Research from the Ministry of Education, Sci-ence, Sports, and Culture, Japan.

REFERENCESABRAMOV A.V., 2000 — The taxonomic status of the

Japanese weasel, Mustela itatsi (Carnivora, Mus-telidae). Zool. ]., 79: 80-88 (in Russian with Eng lishabstract).

274

KARYOTYPES OF JAPANESE AND SIBERIAN WEASELS

GRAPHODATSKY A.S., VOLOBUEV V.T., TERNOVSKY D.V.and RADJABLI S.I., 1976 — G-banding of thechromosomes in seven species of Mustelidae (Car-nivora). Zool. J., 11: 1704-1709 (in Russian withEnglish abstract).

GRAPHODATSKY A.S., TERNOVSKAYA Y.G., TERNOVSKYD.V., VORNOV G.A. and VORNOV V.G., 1979 — G- andC-banding patterns of chromo somes in the M.itatsi or Japanese mink Mustela itatsi (Carnivora,Mustelidae). Zool. J., 58: 1607-1608 (in Russianwith English abstract).

IMAIZUMI Y., 1960 — Coloured Illustrations of theMammals of Japan. Hoikusha, Osaka (in Japa-nese).

KUROSE N., ALEXEI V.A. and MASUDA R., 2000 —Intrageneric diversity of the cytochrome b geneand phylogeny of Eurasian species of the genusMustela (Mustelidae, Carnivora). Zool. Sci., 17:673-679.

MAKING S. and MURAMOTO J., 1966 — The chromosomes ofthe Japanese mink (Mustela itatsi itatsi). Mammal.Chromosome Newsletter, 21: 148.

MASUDA R. and YOSHIDA M.C., 1994 —A molecularPhylogeny of the family Mustelidae (Mammalia,Carnivora), based on comparison of mitochondrialCytochrome b nucleotide sequences. Zool. Sci., 11:605-612.

MIYASHITA K., 1963 — Introduced animals 5: Theirhistory and ecology. Shizen, 18: 69-75 (in Japa-nese).

NOWAK R.M., 1991 — Walker's Mammals of theWorld. 5th ed., Johns Hopkins University Press,Baltimore and London.

OBARA Y., 1985 — G-band homology and C-bandvariation in the Japanese mustelids, Mustela er-minea nippon and M. Sibirica M. itatsi. Genetica,68: 59-64.

PATTON J.L. and SHERWOOD S.W., 1982 — Genomeevolution in pocket gophers (genus Thomomys): I.Heterochromatin variation and speciation poten-tial. Chromosoma, 85: 149-62.

ROBINSON T.J., ELDER F.F. and CHAPMAN J.A., 1983 —Evolution of chromosomal variation in cottontails,genus Sylvilagus (Mammalia: Lagomorpha): S.aquaticus, S. floridanus, and S. transitionalis.Cytogenet. Cell Genet., 35: 216-22.

SEABRIGHT M., 1971 —A rapid banding technique forhuman chromosomes. Lancet, 2: 971.

SUMNER A.T., 1972 —A simple technique for demon-strating centromeric heterochromatin. Exp. CellRes., 75: 304-306.

SVARTMAN M., 1999 — Comparative genome analysis inAmerican marsupials: chromosome banding and in-situ hybridization. Chromosome Res., 7: 267-75.

TSUCHIYA K., 1979 —A contribution to the chromosomesstudy in Japanese mammals. Proc. Jpn.Acad.,55: 191-195.

VOLOBUEV V.T., GRAPHODATSKY A.S. and TERNOVSKYD.V., 1975 — Chromosome set of the siberianweasel (Mustela sibirica). Zool. J., 54: 146-147 (inRussian with English abstract).

WOZENCRAFT W.C., 1993 — Order Carnivora. In: D.E.Wilson and D.M. Reeder (Eds), "MammalSpecies of the World: A Taxonomic and Geo-graphic Reference", 2nd ed, pp. 279-348. Smith-sonian Institution Press, Washington.

YOUNGMAN P.M., 1982 — Distribution and systematics ofthe European mink Mustela lutreola Linnaeus,1761. Acta Zool. Fenn., 166: 1-48.

Received 18 August 2000; accepted 6 November 2000

275