Department of Biological Sciences, Faculty of Science, Kanagawa University, Kanagawa , Japan

Transcription

1 Mammal Study 33: (2008) the Mammalogical Society of Japan Molecular phylogeny and taxonomy of the genus Mustela (Mustelidae, Carnivora), inferred from mitochondrial DNA sequences: New perspectives on phylogenetic status of the back-striped weasel and American mink Naoko Kurose 1, Alexei V. Abramov 2 and Ryuichi Masuda 3,* 1 Department of Biological Sciences, Faculty of Science, Kanagawa University, Kanagawa , Japan 2 Zoological Institute, Russian Academy of Sciences, Saint-Petersburg , Russia 3 Creative Research Initiative Sousei, Hokkaido University, Sapporo , Japan Abstract. To further understand the phylogenetic relationships among the mustelid genus Mustela, we newly determined nucleotide sequences of the mitochondrial 12S rrna gene from 11 Eurasian species of Mustela, including the domestic ferret and the American mink. Phylogenetic relationships inferred from the 12S rrna sequences were similar to those based on previously reported mitochondrial cytochrome b data. Combined analyses of the two genes demonstrated that species of Mustela were divided into two primary clades, named the small weasel group and the large weasel group, and others. The Japanese weasel (Mustela itatsi) formerly classified as a subspecies of the Siberian weasel (M. sibirica), was genetically well-differentiated from M. sibirica, and the two species clustered with each other. The European mink (M. lutreola) was closely related to the ferret group (M. furo, M. putorius, and M. eversmanii). Both the American mink of North America and the back-striped weasel (M. strigidorsa) of Southeast Asia were more closely related to each other than to other species of Mustela, indicating that M. strigidorsa originated from an independent lineage that differs from other Eurasian weasels. Based on biochemical, cytogenetic, and molecular differences as well as morphological evidence, it is proposed that the American mink be elevated to a distinct mustelid genus, Neovison. Key words: American mink, mitochondrial DNA phylogeny, Mustela, Mustela strigidorsa, 12S rrna. The family Mustelidae, which consists of 59 species, is the most species rich family in the order Carnivora (Wozencraft 2005). In this family, the genus Mustela (mammalian group generally called weasels ) is a polytypic genus widely distributed in Europe, Northern Africa, Asia, North America, and northern parts of South America. These small or middle-sized weasels occur in diverse habitats from tropical rainforests to tundra and from steppe and desert to riparian biotopes and coastal waters. Mustela is the largest genus of Carnivora and is comprised 17 species (Abramov 2000a; Wozencraft 2005). In Eurasia, 12 species of Mustela are known: mountain weasel (M. altaica), ermine (M. erminea), steppe polecat (M. eversmanii), Japanese weasel (M. itatsi), yellow-bellied weasel (M. kathiah), European mink (M. lutreola), Indonesian weasel (M. lutreolina), least weasel (M. nivalis), Malaysian weasel (M. nudipes), European polecat (M. putorius), Siberian weasel (M. sibirica) and back-striped weasel (M. strigidorsa). Some of these species have a Holarctic (M. erminea and M. nivalis) or a Eurasian (M. eversmanii, M. putorius and M. lutreola) distribution. The distribution range of the other species is restricted to Asia (M. altaica, M. kathiah and M. sibirica), and some species have small (or insular) distribution areas (M. strigidorsa, M. nudipes, M. itatsi, M. lutreolina). The American mink (M. vison) was introduced to the Old World from North America, and naturalized in Eurasia: from the British Islands to Siberia, China, and the Japanese Islands. Relationships among the species of Mustela have not been fully clarified. The grouping of species within the genus differ in the classifications of different authors. *To whom correspondence should be addressed. masudary@ees.hokudai.ac.jp

2 26 Mammal Study 33 (2008) Some authors divided the genus Mustela into two (Ellerman and Morrison-Scott 1951; Heptner et al. 1967), four (Pavlinov et al. 1995), or five subgenera (Youngman 1982; Anderson 1989). Recently, Abramov (2000a) divided this genus to nine subgenera and regarded the American mink as a separate genus, Neovison. Many studies have been performed to understand the phylogenetic relationships within this group. These studies are based upon the analysis of morphological characters (Youngman 1982; Anderson 1989; Baryshnikov and Abramov 1997; Abramov 2000a), biochemical data (Belyaev et al. 1980; Taranin et al. 1991) and genetic data (Graphodatsky et al. 1976; Lushnikova et al. 1989; Masuda and Yoshida 1994a; Davison et al. 1999, 2000; Hosoda et al. 2000; Kurose et al. 2000a; Sato et al. 2003). However, the overall phylogeny of Mustela is still unresolved. In the present study, to further understand the phylogenetic relationships among Mustela species, we newly determined partial sequences (about 960 base-pairs, bp) of the 12S rrna gene of the mitochondrial DNA (mtdna) genome from 11 species of Mustela of Eurasia, including the domestic ferret and the American mink. The poorly studied back-striped weasel (M. strigidorsa) from Southeast Asia was included in the genetic study for the first time. Combining the data with previously reported cytochrome b gene data, we present here the molecular phylogeny of Eurasian representatives of Mustela and discuss evolutionary and taxonomic relationships among species. Materials and methods Samples and DNA extraction Species of Mustela examined are listed in Table 1. Muscle tissue from animals were preserved in % Table 1. Profiles of samples examined in the present study Species Common name Code (individual no.) Chromosome No. (2n) # Tissue Sampling locality if known Accession Number & 12S rrna Cytochrome b Mustela nivalis Least weasel MNI (5) 42 # muscle Hokkaido, Japan AB AB026106* (38 $ ) Mustela altaica Mountain weasel MAL (RMNG1) 44 # muscle Great Hingan Mts, AB AB026100* Mongolia (ZIN Mustela erminea Ermine MER (1) 44 # muscle Hokkaido, Japan AB AB026101* Mustela itatsi Japanese weasel MIT (MR1) 38 $ muscle Iwate, Japan AB AB026104* Mustela sibirica Siberian weasel MSI (KYO1) 38 # muscle Kyoto, Japan AB AB026108* Mustela eversmanii Steppe polecat MEV (RURA1) 38 # muscle Chelyabinsk Province, Russia (ZIN AB AB026102* AB AB026107* AB AB Mustela putorius European polecat MPU (RLEN1) 40 # muscle Leningrad Province, Russia (ZIN Mustela furo Ferret MFU (2) 40! hair Domestic AB AB026103* Mustela lutreola European mink MLU (RPSK1) 38 # muscle Pskov Province, Russia (ZIN AB AB026105* Mustela Back-striped weasel MST (1) muscle Vinh Phuc Province, strigidorsa Vietnam (ZIN Mustela vison American mink MVI (1) 30 # muscle Domestic AB AB026109* Martes melampus Japanese marten MME (1) 38 $ muscle Iwate, Japan AB AB012351* Martes zibellina Sable MZI (1) 38 + muscle Hokkaido, Japan AB AB012360* Meles anakuma Japanese badger MEL (K6) 44 $ muscle Kitakyushu, Japan AB AB049800** # Cited from Graphodatsky et al. (1976). $ Obara (1991) reported 38 chromosomes specific to the population of the Honshu Island (Japan). + Cited from Graphodatsky et al. (1977).! Cited from Fredga and Mandahl Specimen no. of Zoological Institute of Russian Academy of Sciences, St. Petersburg (ZIN). & The nucleotide sequence data reported in the present study will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with these accession numbers. * Cited from Kurose et al. (2000a). ** Cited from Kurose et al. (2001).

3 Kurose et al., Molecular phylogeny of Mustela 27 ethanol at room temperature until use. As outgroup, the Japanese marten (Martes melampus), sable (Martes zibellina) and Japanese badger (Meles anakuma) were analyzed in addition to cytochrome b (accession no. X82296) and 12S rrna (AY012149) sequences of the domestic cat (Felis catus). Total genomic DNAs were extracted using the phenol/proteinase K/sodium dodecyl sulfate method of Sambrook et al. (1989) with some simplified modifications as indicated by Masuda and Yoshida (1994b). DNA extracts of M. lutreola (muscle tissues preserved in 70% ethanol for about 30 years) and M. altaica (muscle tissues preserved in 70% ethanol for about 100 years) were concentrated to approximately 100 fold using Centricon-30 microconcentrators (Amicon), because these tissues contained fragmented DNAs. DNA from hairs was extracted from M. furo using the method of Walsh et al. (1991). An aliquot (1 10 µl) of each DNA extract was used as template for the subsequent polymerase chain reaction (PCR). PCR amplification and direct sequencing The sequences of the 12S rrna gene of all samples were PCR-amplified using two primers (L5' and H3') quoted from Ledje and Arnason (1996). Sequencing primers were newly designed as follows: M-12S-F2 5'-GCCACCGCGGTCATACGATTA-3'; M-12S-F3 5'- CCCTCTAGAGGAGCCTGTTCT-3'; M-12S-R2 5'-AT- TAACAGTAGCTTTTTACRGCCTTG-3'; M-12S-R3 5'- GGTTTGCTGAAGATGGCGGTAT-3'. The complete sequence of the cytochrome b gene of one sample of M. strigidorsa was PCR-amplified and sequenced using the method of Kurose et al. (2000a). Cytochrome b sequences of the other Mustela species were obtained from Kurose et al. (2000a) and the accession numbers are shown in Table 1. The PCR amplifications were performed in 50 µl reaction volumes. In cases of aged samples where PCR was inhibited, 20 µg of bovine serum albumin (Boehringer) was added to the reaction mixture. Thirty-five cycles of amplification were performed with the following programs using a DNA thermal cycler (PJ2000, Perkin-Elmer Cetus): denaturing 94 C for 1 min; annealing 50 C for 1 min; extension 72 C for 2 min, and then the reaction was completed at 72 C for 10 min. To check PCR amplification, 10 µl of the PCR product was electrophoresed on a 2% agarose gel, stained by ethidium bromide, and visualized under an ultraviolet illuminator. The remaining 40 µl of each PCR product was purified with the QIAquick purification kit (Qiagen). Purified PCR products were labeled using the cycle labeling system Catalyst (Perkin-Elmer Cetus) and sequenced using the ABI Prism TM 377 automated sequencer. Sequence analysis Sequence alignment was done using the GeneWorks computer software (Intelligenetics). Molecular phylogenetic analyses were perfomed using PAUP * version 4.0b10 (Swofford 2001). The phylogenetic trees were constructed by three methods: neighbor-joining (NJ: Saitou and Nei 1987) using Kimura s (1980) twoparameter distances, maximum parsimony (MP) and the maximum likelihood (ML). Because phylogenetic relationships of species were very similar among separate analyses of cytochrome b and 12S rrna as well as analyses in which the two genes were combined, we used the combined sequences (about 2,110 bp) and excluded insertions or deletions (indels) for analysis. The MP trees were obtained using the heuristic search option with random sequence addition and TBR (treebisection-reconnection) branch swapping. All sites were treated as unordered and equally weighted. For the ML analysis, we selected the best-fitting model of molecular evolution using the program Modeltest 3.06 (Posada and Crandall 1998). This test chose GTR (general time reversible; Tavaré 1986) model of substitution taking account of the proportion of invariable sites (0.4182) and following a gamma distribution shape parameter of (GTR + I + G). In the rate matrix of this substitution model, the rates of substitutions were estimated as for A-C, for A-G, for A-T, for C-G, for C-T, and for G-T. Bootstrap values (Felsenstein 1985) were derived from 1,000 replications in both MP and NJ, and 200 replications in ML to assess the confidence of the trees. Results Nucleotide sequences of the partial 12S rrna gene (about 960 bp) from the 14 species of Mustelidae and the complete cytochrome b gene (1,140 bp) from Mustela strigidorsa were obtained in the present study. Table 1 shows accession numbers of the newly generated nucleotide sequences. The sequence alignment of the 14 mustelid taxa showed that 231 sites in the 12S rrna gene and 370 sites in the cytochrome b gene were variable. Because phylogenetic trees reconstructed separately from cytochrome b and 12S rrna genes formed very similar topologies, we combined the two genes (2,111

4 28 Mammal Study 33 (2008) Fig. 1. Phylogenetic trees of combined sequences of 12S rrna and cytochrome b genes, constructed using the maximum likelihood method (ML). Numbers near internal branches were bootstrap values of ML (200 replications), the maximum parsimony method (MP; 1,000 replications), and the neighbor-joining method (NJ; 1,000 replications), respectively. Details of analysis conditions were described in the text. Mu, Mustela; Ma, Martes; Me, Meles. bp) for analysis. From the 2,111-bp sequences, 32 bp were excluded as indel sites, leaving 2,079 bp. Phylogenetic relationships from the combined analysis of the two genes are almost same among ML, MP, and NJ methods, so that we showed the ML tree as respresentative. Bootstrap values estimated from the three methods are indicated on the ML tree (Fig. 1). For the MP analysis, 394 of 2,079 sites were parsimoniously informative. The consistency index (CI) was , the retention index (RI) was , and the rescaled consistency index (RC) was The three genera of Mustela, Martes and Meles were clearly separated. In the genus Mustela, both M. vison and M. strigidorsa were first to split from the other Mustela species and clustered with 86/79/71% (ML/MP/ NJ) bootstrap values. The genetic distances between both M. vison + M. strigidorsa and the other Mustela species were higher than those among the rest of Mustela. Mustela erminea was the next split from the rest of the species. Mustela altaica and M. nivalis were clustered with 94/85/89% bootstrap values: the cluster was named the small weasel group (Fig. 1). The other species (eversmanii, putorius, furo, lutreola, sibirica, and itatsi) formed a clade (named the large weasel group ). Although, within this group, a clade of M. itatsi and M. sibirica was split in ML and MP, the bootstrap values to support the relationship were low (78% and 62%, respectively; Fig. 1). These two species were not clustered in the NJ tree. The genetic differentiation between M. itatsi and M. sibirica corresponded to those between other distinct species. In addition, M. lutreola and the ferret group (M. putorius, M. eversmanii, and M. furo) were closely related to each other and clustered with 99 or 100% bootstrap values having small genetic distances. The domestic ferret (M. furo) examined was closer to M. eversmanii than to M. putorius. Discussion Phylogeny of the large weasel group Morphological classification (Youngman 1982; Wozencraft 1989; Pavlinov et al. 1995) as well as karyotaxonomy (Graphodatsky et al. 1976) supported a close relationship between Mustela lutreola and M. sibirica. The comparative analysis of antigenic structures of the immunoglobulin chain in Mustelidae (Taranin et al. 1991) showed that M. lutreola was closer to the ferret group than to M. sibirica. According to Abramov (2000a), M. lutreola belongs to the separate subgenus Lutreola which is equidistant to polecats (subgenus

5 Kurose et al., Molecular phylogeny of Mustela 29 Putorius) and M. sibirica (subgenus Kolonokus). The molecular phylogeny of the present study suggests that M. lutreola is nested between the ferret group and the M. itatsi + M. sibirica lineage of the large weasel group. Davison et al. (1999, 2000) reported that M. lutreola was included in the ferret group cluster comprising M. furo, M. putorius, and M. eversmanii, judging from the molecular phylogeny of partial cytochrome b sequences (337 bp). The partial sequence (Accession No. AF068544) reported by Davison et al. (1999) was identical with the homologous region within the complete cytochrome b sequences obtained through our previous study (Kurose et al. 2000a). The difference of position of M. lutreola may be ascribed to the short uninformative sequence (337 bp) used by Davison et al. (1999). Parental species of the domestic ferret M. furo The domestic ferret (M. furo) is generally thought to be domesticated from M. putorius, or from its congener, M. eversmanii. The diploid chromosome number is 2n = 40 for M. putorius as well as M. furo, while 2n = 38 for M. eversmanii. The former two taxa have morphologically identical chromosome sets, but the karyotype of M. eversmanii differs from those of the formers by a single Robertsonian rearrangement (Volobuev et al. 1974). The traits of developmental biology of M. furo were reported to be more similar to M. putorius than to M. eversmanii (Ternovsky 1977). Meanwhile, Blandford (1987) suggested that M. eversmanii has a superficially more similar cranial morphology with the domestic ferret. Experimental hybridization among M. furo, M. putorius, and M. eversmanii was found to be possible, and all hybrids were fertile (Ternovsky 1977; Ternovsky and Ternovskaya 1994). Based on partial cytochrome b sequences and control region fragment analysis, Davison et al. (1999) investigated the phylogenetic relationships among M. putorius, M. furo and M. eversmanii in comparison to some species of Mustela. Two geographically distinct polecat lineages were found in Britain, where one may be ancestral to the British polecat, and the other to the domestic ferret. However, the wild source of the ferret remains obscure. Davison et al. (1999) suggest that the relatively recent speciation from M. lutreola and M. nigripes, and the effects of polecat-ferret hybridization result in an unresolved molecular phylogeny among these species. The present study revealed the close relationships among the three morphologically similar taxa, M. putorius, M. eversmanii, and M. furo (Fig. 1) supporting previous results that the parental species of the domestic ferret were these polecat species. The genetic differentiation was the same as the level of intra-specific variations of other mustelids. The closer relationship between M. eversmanii and M. furo obtained in the present study suggests that M. eversmanii is a possible ancestor of the domestication. Further analyses of karyotypes, mtdna and nuclear DNA, and morphology of the ferrets from different lineages as well as the wild populations of polecats would more precisely illuminate the history of the ferret s domestication. Phylogenetic positions of the Japanese weasel M. itatsi and the Siberian weasel M. sibirica The taxonomic relationship between M. itatsi and M. sibirica has been obscure for a long time. Usually M. itatsi is considered conspecific to M. sibirica (Ellerman and Morrison-Scott 1951; Youngman 1982; Wozencraft 1989; Corbet and Hill 1992; Pavlinov et al. 1995). Recently, Abramov (2000b) reported that cranial differences between these two taxa are greater than geographic variations among M. sibirica populations from Siberia, Russian Far East, and Japan. Masuda and Yoshida (1994b) and Kurose et al. (2000a) based on cytochrome b sequences revealed that there is a relatively large genetic distance between M. itatsi and M. sibirica. The karyotypical differences were found between the two weasels (Kurose et al. 2000b), although both shared the identical diploid chromosome number (2n = 38). In ML and MP trees, M. itatsi and M. sibirica were clustered with each other. In the NJ tree, however, these taxa show successive splittings, with M. itatsi was the first to split (Fig. 1). The transversional difference of the third codon positions (0.79%) between M. itatsi and M. sibirica refers to a divergence time of about 1.6 million years ago (Table 2) according to the transversional rate (0.5%/million years) of mammalian cytochrome b of Irwin et al. (1991). The ancestor of M. itatsi might have been derived from M. sibirica probably in continental Asia in the early Pleistocene. After that, a certain ancestral population of M. itatsi might have immigrated to the Japanese islands, and then it could have been isolated on the islands through the strait formation. The other ancestral populations remaining on the continent might have been extinct. Alternatively, from an ancestor common to M. sibirica, M. itatsi might have evolved independently on the Japanese islands after their separation from the continent.

6 30 Phylogeny of the small weasel group Most authors unite small weasels (M. altaica, M. erminea and M. nivalis) in the subgenus Mustela (Ellerman and Morrison-Scott 1951; Heptner et al. 1967; Youngman 1982; Nowak 1991; Pavlinov et al. 1995). Abramov (2000a) placed the ermines (M. erminea and North American M. frenata) in the subgenus Mustela, whereas other small weasels (M. altaica, M. nivalis, and M. kathiah) were placed in the separate subgenus Gale. In the present study, the divergence time between M. erminea and the other Mustela species (except M. vison) was estimated around 3 5 million years ago (Table 2). Thus, species diversification within Mustela might have started from the end of the Miocene to the Pliocene. The result that M. erminea first split from the other Mustela species is supported by the karyological study (Graphodatsky et al. 1976; Obara 1991) that M. erminea is the more ancestral form among Eurasian Mustela. The closer relationship between M. altaica and M. nivalis indicated in the present study was also supported by morphological classification (Heptner et al. 1967; Youngman 1982; Wozencraft 1989; Abramov 2000a) and karyotaxonomy (Graphodatsky et al. 1976) as well as molecular phylogeny: cytochrome b (Kurose et al. 2000a); cytochrome b and nucler DNA (Sato et al. 2003); and nuclear DNA (Sato et al. 2004, 2006). The bootstrap values supporting the two species clade in the present study were larger than those in the previous molecular studies mentioned above. The two species were estimated to have diverged > 5 million years ago (at the end of the Miocene) (Table 2). Phylogenetic positions of the American mink The phylogenetic and taxonomic position of the American mink has long been unclear. The phylogenetic relationships between the American mink and the European mink has been questioned for years, with many scientists believing them to be conspecific or at least closely related. The morphological resemblance and similar mode of life of American and European minks provide evidence to group them in the subgenus Lutreola (Walker 1964; Heptner et al. 1967; Ternovsky and Ternovskaya 1994). Most authors now place the American mink in the separate subgenus Vison Gray, 1865 of genus Mustela (Youngman 1982; Nowak 1991; Pavlinov et al. 1995; Baryshnikov and Abramov 1997; Lariviere 1999). According to the International Code of Zoological Nomenclature, this name is invalid because Mammal Study 33 (2008) the type specimen for this taxon was of the European mink Mustela lutreola. At present, the valid genus name is Neovison Barysnikov and Abramov, During the last decades, the presence of large differences in biochemical, cytogenetic, and molecular characteristics between the American mink and the other Mustela species have been reported. For example, the diploid chromosome number of the American mink is 2n = 30, whereas those of the other Mustela species ranges from 38 to 44. Graphodatsky et al. (1976) examined karyotaxonomy among seven species of Mustela, and considered that the American mink first split from the other Mustela species. Belyaev et al. (1980) provided a comparative immunochemical study of serum proteins for Mustela species such as M. lutreola, M. putorius, M. eversmanii, M. sibirica, M. altaica, M. nivalis, M. erminea, and M. vison. The American mink was sharply detached from the other species of Mustela and had closer immunological affinities to the sable Martes zibellina. These results were confirmed by Taranin et al. (1991) in comparative analysis of antigenic structures of immunoglobulins. A comparative study of chemical composition of anal sac secretion in mustelids (Brinck et al. 1983) showed that the American mink is separated from other Mustela species as much as other analyzed genera (Martes, Lutra and Meles). Lushnikova et al. (1989) examined the patterns of blot-hybridization of cloned BamHI repeats to genome DNAs for estimation of the phylogenetic relationships among some Mustela species (M. lutreola, M. putorius, M. sibirica, M. erminea, and M. vison). They found a distant position of the American mink from the other species as well as the marbled polecat Vormela peregusna. Recent studies of nucleotide sequences of the mtdna cytochrome b gene (Masuda and Yoshida 1994a; Davison et al. 1999; Hosoda et al. 2000; Kurose et al. 2000a) and of the nuclear interphotoreceptor retinoid binding protein gene (Sato et al. 2003, 2004) also showed the high level of divergence between the American mink and the other species of Mustela. On the composite super-tree for Mustelidae by Bininda-Emonds et al. (1999), the American mink was separate from all Mustela species. There are remarkable cranial differences between the American mink and all Mustela (Youngman 1982; Abramov 2000a). The American mink has a small incipient metaconid on M 1 (lower first molar), the wide talonid of M 1 with lingual rim, large M 1 (upper first molar) with the extended lingual lobe, and large tworooted Pm 2 (upper second premolar). The lateral part of

7 Kurose et al., Molecular phylogeny of Mustela 31 Table 2. Percentage differences of transversions at the third codon positions of the cytochrome b sequences (above diagonal) and the estimated divergence time (million years, below the diagonal) using the transversional substitution rate (0.5%/million years) at the third codon positions of mammalian cytochrome b reported by Irwin et al. (1991). Code* MAL MNI MER MPU MEV MFU MLU MIT MSI MST MVI MME MZI MEL FCA** * Codes refer to those in Table 1. ** FCA (Felis catus) was used for outgroup. auditory bullae near the meatus forms a structure resembling the meatal tube. Some of these cranial characters are typical for the genus Martes (Youngman 1982; Abramov 2000a) and probably are plesiomorphic for Caniformia (see Bryant et al. 1993). The molecular phylogeny in the present study also demonstrated that the American mink was separate from the other Mustela species. The genetic distances between the American mink and the other species of Mustela nearly corresponded to those between the Mustela species and other mustelid genera such as Martes and Meles (Fig. 1). Using the molecular clock of cytochrome b (Table 2), the American mink was estimated to have diverged from the other Mustela species approximately 8 11 million years ago as described in Kurose et al. (2000a). Based on the biochemical, cytogenetic, and molecular differences as well as morphological evidence, we therefore recommend that the American mink be classified in a distinct mustelid genus, Neovison. The divergence times within Mustela estimated in the present study are not discordant with the data of Sato et al. (2003). Phylogenetic position of the back-striped weasel M. strigidorsa The back-striped weasel (M. strigidorsa) is one of the rarest and most poorly studied species among Mustelidae in the world. Information on this elusive weasel exists only as sighting and distribution reports (Lekagul and McNeely 1988; Duckworth et al. 1999). This species is distributed in the Himalayas (from eastern Nepal to Assam), Burma, northern Thailand, southern China, Laos and Vietnam. According to Schreiber et al. (1989) only about 30 museum specimens exist of this rare species. Usually M. strigidorsa has been included in the subgenus Lutreola together with M. lutreola and M. sibirica (Youngman 1982; Nowak 1991; Pavlinov et al. 1995). Some authors united M. strigidorsa and M. nudipes in the subgenus Pocockictis (Gray 1865; Pocock 1921, 1941). According to Abramov (2000a), M. strigidorsa is one of the most morphologically differentiated species in Mustela (except the American mink). Based on morphological characters, he placed M. strigidorsa in the separate subgenus Cryptomustela. The present study reports the mtdna sequence data on M. strigidorsa for the first time and revealed a large genetic divergence between M. strigidorsa and other Mustela. This result leads to a possibility that two distinct lineages of Eurasian weasels exist, and M. strigidorsa belongs to the independent Southeast Asian lineage which differs from other Eurasian weasels. This taxon probably split early from the Mustela stock and evolved independently at the edge of distribution range of the genus. Unfortunately the fossil history of M. strigidorsa is absolutely unknown. This species differs from other Mustela in the unique coloration of the body

8 32 and baculum structure. However, as a whole, the skull characters (shape and size of cranium, mandible and dentition) of this species are similar to those of other largesized Mustela, in contrast to the American mink. Our analysis suggests the genus Mustela may be paraphyletic with respect to the American mink (as Neovison vison). This result would indicate the necessity of taxonomic revision with the inclusion of all Mustela species. To infer the phylogenetic relationship and the molecular evolution of M. strigidorsa, it is necessary to investigate the Southeast Asian weasels, especially the Malayan weasel M. nudipes. It is also important to include the South American weasels M. africana and M. felipei, for which no genetic data is available at present. The present study revealed the phylogenetic relationships among Eurasian species of the genus Mustela. The results provide not only invaluable insight to reconstruct the taxonomy of the Mustelidae, but also useful information to survey genetic characteristics and hybridization problems between native populations and introduced populations for considering species conservation. In addition, further study involving other Mustelidae species of Eurasia and both North and South America would illuminate the worldwide evolution of Mustelidae. Acknowledgements: We thank H. Abe (Hokkaido University), Y. Masuda (Shiretoko Museum), F. Sekiyama (Iwate Prefectual Museum), and S. Watanabe for supplying specimens, and S. Nakagome (Hokkaido University) for assistance on data analysis. We are grateful to the Institute of Low Temperature Science of Hokkaido University for technical support. This study was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and by the 21st Century COE Program Neo-Science of Natural History at Hokkaido University financed from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by the joint grant from the Japan Society for the Promotion of Science and the Russian Foundation for the Basic Research (No ). References Abramov, A. V. 2000a. A taxonomic review of the genus Mustela (Mammalia, Carnivora). Zoosystematica Rossica 8: Abramov, A. V. 2000b. The taxonomic status of the Japanese weasel, Mustela itatsi (Carnivora, Mustelidae). Zoologicheskii Zhurnal 79: (in Russian with English abstract). Anderson, E The phylogeny of mustelids and systematics of ferrets. In (U.S. Seal, ed.) Conservation Biology and the Black- Mammal Study 33 (2008) Footed Ferret. Pp Yale University Press, New Haven and London. Baryshnikov, G. F. and Abramov, A. 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