The gray red-backed vole, Clethrionomys rufocanus, from Far East Asia including Sakhalin and Hokkaido is known to harbor intraspecific morphological and cytogenetic variations. Here we analyzed geographic variation in the mitochondrial cytochrome b (Cytb) gene and Y chromosome specific Sry gene by a PCR direct sequencing technique. Determination of sequences in the Cytb gene (1140 bp) in 29 individuals provided 28 haplotypes and construction of a neighbor joining tree clearly indicated that they were grouped into four major lineages, which predominated in Primorskyi territory, Kamchatska-Magadan, Sakhalin, and Hokkaido-Kuril, with 0.02–0.04 sequence divergence (Kimura's distance, all substitutions, all codon positions). The sequences for the Sry region (336–366 bp) included a variable repetitious region of microsatellites such as TG(TC)2. In total five sequences were recognized which can be grouped into two forms, continental and insular (Sakhalin and Hokkaido) based on the presence or absence of a segment of TG(TC)3TG(TC)4. It was revealed that, therefore, the population of Sakhalin possessed their own type of mitochondrial DNA but the same Sry gene as Hokkaido. A similar trend can be seen in the continental populations. Our results suggested that the population of C. rufocanus in each of the geographic domains accumulated own genetic elements in part but genetic exchanges between neighboring populations occurred during the course of evolution. It is noteworthy that the insular domains, Sakhalin and Hokkaido, have played an important role in raising the amount of genetic diversity in small rodent species.
The gray red-backed vole, Clethrionomys rufocanus, which can be characterized by the specific enamel pattern of its upper third molar (Kaneko et al., 1998), occurs in northeastern Eurasia from Scandinavia in the west through Siberia and Primorskyi territory to Sakhalin and Hokkaido (and peripheral islands including the southern Kuril Islands) in the east (Corbet, 1978; Corbet and Hill, 1991; Kaneko et al., 1998). The species shows morphological variations among geographic regions. For example, the tail length is quite short in Magadan and Kamchatska and rather long in Hokkaido (Kaneko et al., 1998; Iwasa, unpublished data), and individuals from Hokkaido are darker in body color than those from Russia (Imaizumi, 1960; Kaneko et al., 1998). Then taxonomists tend to treat these geographic populations as subspecies: populations of Russia have been separated to several subspecies (Gromov and Polyakov, 1992; Kartavtseva et al., 1998) while those of Hokkaido have been regarded as C. rufocanus bedfordiae (Imaizumi, 1960; Gromov and Polyakov, 1992; Kaneko et al., 1998). Furthermore, populations of the small peripheral islands of Hokkaido are known to have some specific morphological characteristics (Imaizumi, 1960; Miyao, 1968). The population of Rishiri Island, for example, tends to have a larger body size than that of Hokkaido (Miyao, 1968).
Intraspecific karyological variation has been also investigated in C. rufocanus, and it has been revealed that the karyo type is essentially the same (Vorontsov et al., 1980; Sokolov et al., 1990; Kartavtseva et al., 1998). However, the size of the Y chromosome is known to differ between populations of the continent and Sakhalin-Hokkaido (Vorontsov et al., 1980; Tsuchiya, 1981; Kashiwabara and Onoyama, 1988; Kartavtseva et al., 1998). To expand our knowledge on the morphological and chromosomal variations underlying the evolution of the Clethrionomys species, several genetic approaches have been taken. Recently, molecular analyses have been attempted using microsatellites, a nuclear ribosomal RNA gene and mitochondrial DNA markers to clarify the genetic background of this species (Ishibashi et al., 1995, 1997a, b; Suzuki et al., 1999; Wakana et al., 1996). In addition, several studies on the phylogenetic relationships among related species of red-backed voles in East Asia, including C. rutilus and C. rex, have been made. Clethrionomys rufocanus is shown to have a close affinity with C. rex that inhabits Hokkaido and its neighboring islands (Imaizumi, 1971, 1972; Kaneko, 1994; Kaneko et al., 1998) rather than with C. rutilus that is distributed in a wide area of the northern hemisphere, based on karyological (Gamperl, 1982; Kashiwabara and Onoyama, 1988; Obara et al., 1995; Iwasa, 1998) and molecular phylogenetic aspects (Suzuki et al., 1999; Wakana et al., 1996).
To obtain a clear phylogenetic view of geographic populations, it is necessary to understand the genetic constitution as a whole, since multiple genetic exchanges among populations as well as random lineage sorting would have occurred during the course of populational differentiation (Harrison, 1989; Avise, 1991). To this end, the study of these geographic populations with multiple phylogenetic markers would be highly effective. However, to date, only variation in the mitochondrial DNA (mtDNA) in a small number of specimens from Primorskyi territory and Hokkaido has been reported (Suzuki et al., 1999; Wakana et al., 1996).
Here we conducted a more intensive and extensive study on the intraspecific variation of C. rufocanus collected from several geographic areas of Far East Asia, Primorskyi territory, Kamchatska, Sakhalin, Hokkaido, and Kuril Islands, with two molecular markers, one each for maternal and paternal lineages. Using a PCR direct sequencing technique, we determined sequences for the mitochondrial cytochrome b (Cytb) gene and the sex-determination gene, Sry. The Sry gene is omnipresent on the Y chromosome in mammalian species and the chromosomal site is the region un-paired with the X chromosome in mouse and human, implying that the gene is a useful marker for tracing paternal lineages without degradation by meiotic recombination events, as is mtDNA for maternal lineages (Harrison, 1989).
MATERIALS AND METHODS
Analyzed specimens of C. rufocanus (34 individuals) were captured from own localities as shown in Table 1 and Fig. 1. The species identification was performed according to Kaneko (1994) and Kaneko et al. (1998), mainly based on the enamel pattern of molar dentition. All specimens are preserved at the authors' laboratories.
Collecting localities, specimen numbers, and mitochondrial DNA (mtDNA) and Sry haplotypes of Clethrionomys rufocanus examined in this study.
Direct sequencing of the mitochondrial cytochrome b gene
The total DNA was extracted from liver tissues by the conventional phenol-chloroform method. Amplifications of a fragment of the mitochondrial Cytb gene were performed with universal primers (L14724 and H15915; Irwin et al., 1991) at 1st PCR, and followed by a specific 2nd PCR amplification with nested (N-) primers that were designed for vole specimens (N-L14724, N-L15135, N-L15561, NH15155, N-H15599 and N-H15919; Table 2). The reactions were carried out for 35 cycles each consisting of 30 sec at 96°C for denaturation, 30 sec at 50°C for annealing and 30 sec at 60°C for extension. These reaction mixtures (20 ml) contained 50 mM dNTPs, 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2 (1.25 mM at 2nd PCR), 50 nM primers and 0.5 units Ampli Taq polymerase (Perkin Elmer). Both DNA strands of the product of the second PCR were directly sequenced by an automated method using the Dye Primer Cycle Sequencing Kit (ABI) and an automated sequencer (model 373A, ABI).
PCR nested primers for the mitochondrial Cytb gene and the Sry gene.
Direct sequencing of the Y chromosomal Sry gene
Amplifications of a fragment of the Sry gene at 1st PCR were performed with primers designed from human and murine sequences (SRY286 and HMG777, Fig. 3; Sinclair et al., 1990; Suzuki et al., 1997), and followed by a 2nd PCR with nested primers of R-SRY306 and U-HMG597 (Table 2; Suzuki et al., 1997) to obtain an approximately 350 bp fragment of the flanking region of the HMG box. The reaction of the 1st PCR was identical to that for the Cytb gene. The reaction for the 2nd PCR was carried out for 35 cycles each consisting of 30 sec at 96°C for denaturation, 30 sec at 62°C for annealing and 30 sec at 72°C for extension. The products of the 2nd PCR were directly sequenced as for the Cytb gene.
Construction of a phylogenetic tree
We constructed a phylogenetic tree by the neighbor-joining (NJ) method (Saitou and Nei, 1987) and performed bootstrap analysis (1000 replications), with genetic distances computed by Kimura's two parameter method (Kimura, 1980), using CLUSTAL W program ver. 1.6 (Thompson et al., 1994) for Cytb sequences. We sequenced the same gene region for C. rutilus and C. rex from Hokkaido and used them as an outgroup.
The DNA sequence data of C. rufocanus, C. rutilus and C. rex, which were determined in this study will be placed in the GenBank, EMBL and DDBJ nucleotide sequence databases with the following accession numbers, AB031553-AB031582 (Cytb) and AB031858-AB031862 (Sry).
Sequence determination of the Cytb gene (1140 bp) in 29 individuals of C. rufocanus provided 28 haplotypes (Table 1). And pairwise sequence divergences among the haplotypes were calculated to be 0.001–0.040 by Kimura's two parameter method (Kimura, 1980) considering all substitutions at all codon positions (Table 3). In the comparisons of these sequences within C. rufocanus, saturation bias would not be expected since transition to transversion ratios were high throughout the analysis with a minimum value of 10.94. A neighbor joining tree was constructed with the sequence divergences, and indicated the presence of four prominent clusters that were essentially specific to each geographic domain, Primorskyi territory, Sakhalin, Kamchatska, and Hokkaido (Fig. 2). Each cluster was supported by high bootstrap values (>87%, Fig. 2). There was substantial sequence divergence between the four prominent lineages with a Kimura's distance of 0.02 – 0.04 (Table 3). However, the branching patterns of these four lineages were unclear: the lengths between the nodes were short and the bootstrap values that supported the branching were low (<68%, Fig. 2).
The members of each of the clusters showed a strict geographic preference. Exceptionally, a haplotype (VS-RS1) from Vladivostok, Primorskyi territory was incorporated into the cluster Kamchatska, while the other seven haplotypes from Primorskyi territory formed a unique cluster (Fig. 2, Table 1). Further subdivision within the clusters is possible but the extent of differentiation was small (Fig. 2). For example, the Hokkaido cluster was likely to be further subdivided into three clades (monophyletic subclusters) that reflected the geographic source of the haplotypes: Hokkaido (NM-HKs, KMHKs,TB-HK1, NG-HK1, and TK-HKs), Rishiri-Rebun Islands (RS-HK1 and RB-HK1), and Taraku (=Polonskogo) Island, southern Kuril Islands (TK-KR1). The sequence divergences between the three clades were approximately 0.01 of the Kimura's distance.
We determined the sequences of the Sry region (336–366 bp) in 14 males of C. rufocanus (Table 1). The region showed a variable state within species, which was largely due to the presence of several repeats of microsatellite-like elements such as TG(TC)2 (Fig. 3). From the status of the polymorphic region, we recognized five types of Sry gene in our samples (types A–E). In addition, types D and E differed from the others in one base substitution (position 50, marked with an asterisk; Fig. 3). These Sry gene types showed a certain geographic distribution, although the tendency was not consistent with that for the mtDNA (Table 1). Types A and B were observed in individuals from Sakhalin-Hokkaido and Rishiri Island, respectively, while the other three types were obtained from those of the continent. Types A and B differed slightly with respect to the repeat number of TG(TC)2 and both contained a segment TG(TC)3TG(TC)4, associated with the microsatellite region, whereas the continental types, C, D, and E, lacked the segment (Fig. 3). Then, taking into account that the constitution of the repetitious number of the TG(TC)2 motif is labile but insertion or deletion of TG(TC)3TG(TC)4 is not reversible, we grouped the Sry gene types into two, continental (C, D, and E) and insular (A and B) forms, respectively.
With markers for the Cytb and Sry genes we revealed that there was substantial intraspecific variation in the populations of C. rufocanus from Far East Asia and that each of the genes had its own geographic distribution (Figs. 2 and 3). The results obtained here, hence, would be useful not only for the evaluation of the phylogenetic relatedness among the geographic populations of C. rufocanus but also for learning how molecular markers with different modes of inheritance have behaved during the course of evolution. In addition, our data provide a clue as to the roles of several zoogeographic domains in Far East Asia, especially Hokkaido, in the mode of genetic diversity in small rodents.
Genetic relationships of the geographic populations from Far East Asia
The mtDNA data provides us a rationale for the subdivision of C. rufocanus and approximate discrimination of geographic boarders (Fig. 2). Accordingly, we can denote four geographic domains for C. rufocanus in the Far East; Primorskyi territory, Sakhalin, Kamchatska and Hokkaido. This notion is somewhat consistent with the morphological classification where the populations of Primorskyi territory, Kamchatska, and Sakhalin are regarded as different subspecies (e.g., Gromov and Polyakov, 1992).
The mtDNA tree showed that the divergences between the lineages for the four geographic domains were distinct as indicated by their long branch lengths (0.03–0.04 for pairwise sequence divergence, Table 3 and Fig. 2), implying that considerable time has passed since their ancestors colonized these geographic areas. The divergence time of the four branches is likely to be a few million years, taking account of the traditional assumption of the divergence rate in mitochondrial DNA (0.01–0.02 per million years per two lineages; Brown et al., 1979). However it is uncertain whether such an estimation is accurate since the evolutionary rate would vary in each organism (Zardoya and Meyer, 1996) and, probably, in each lineage of the clusters of the voles.
In contrast to the mtDNA variation, the Sry gene data indicate a populational demarcation of C. rufocanus in Far East Asia into essentially two groups, continental and insular, based on the constitution of repetitious elements (Fig. 3). The different modes of geographic differentiation between both genes examined would be attributable to differences in their inheritance. In the case of red-backed voles, it has been reported that male individuals travel further than females (Ishibashi et al., 1997b) during their lifetime. Thus it is reasonable that mtDNA tends not to be replaced with neighboring types irrespective of the presence of gene flow which mainly occurs through male individuals. Such a phenomenon is sometimes recognized as a “founder events” with respect to mtDNA (Harrison, 1989). Even though we can not specify the reason for the contradiction at this moment, our results suggest that ancestral populations communicated genetically within the continental populations and between the Sakhalin and Hokkaido populations. This notion is consistent with the geographic variation of Y chromosomes in C. rufocanus in Far East Asia where acrocentric Y is observed in Sakhalin and Hokkaido and metacentric Y observed at several continental localities (Vorontsov et al., 1980; Kartavtseva et al., 1998). It is also consistent with the morphological view that the populations of Sakhalin and Hokkaido are the same subspecies (Gromov and Polyakov, 1992; Kartavtseva et al., 1998). The data of mtDNA from Vladivostok that showed affinity with those of Kamchatska also support the idea of interpopulational gene flow as an evolutionary trend between neighboring geographic populations (Fig. 2).
The close relationships between Sakhalin and Hokkaido can be seen in other small mammals. For example, the soricine shrew species, Sorex gracillimus, with a similar habitat to C. rufocanus, shows close genetic relationships between Hokkaido and Sakhalin populations in Cytb gene sequence data (Ohdachi et al., 1997). In the Cytb gene variation of the East-Asiatic wood mice, Apodemus peninsulae, which is distributed across Far East Asia including Sakhalin and Hokkaido, a similar tendency can be seen (Serizawa et al., unpublished data). Therefore, it can be considered that the fauna of small mammals in Sakhalin and Hokkaido tends to have similar genetic constitutions that differ from those of the continent. Gene flow across land bridges on the Soya Strait (45 m in depth in present; Oshima, 1990) during the glacial periods would explain such genetic similarity.
Evolutionary aspects of the Hokkaido population
Clethrionomys rufocanus populations inhabiting small peripheral islands of Hokkaido, such as Rishiri Island, are known to have specific morphological characteristics (Miyao, 1968). These insular populations are sometimes classified as distinct species or subspecies (Imaizumi, 1960; Japanese Environmental Agency, 1993). The present data, however, indicated that the populations of Hokkaido and its offshore islands including the Taraku Islands, in the southern Kuril Islands, share similar phylogenetic elements (Figs. 2 and 3). Wakana et al. (1996) reported a close affinity between the Hokkaido population and island ones (Rishiri and Daikoku Islands) as determined from mtDNA analysis. These close relationships are easily explained by geological factors such as the narrow straits between the islands (Oshima, 1990). It is noteworthy that on Okushiri Island, near the southwestern part of Hokkaido (Fig. 1) which is separated from Hokkaido by deep strait (>400 m; Oshima, 1990), there is no extant population of C. rufocanus.
From our data, and cytogenetic and morphological data in the literature (Vorontsov et al., 1980; Gromov and Polyakov, 1992, Kaneko et al., 1998), the C. rufocanus populations of Hokkaido including neighboring small islands can be considered to be undergoing further differentiation from Russian continental populations through a certain kind of geographic isolation. We consider that such processes may have been involved in the establishment of C. rex which is now confined to Hokkaido and neighboring islands (Kaneko et al., 1998) and is phylogenetically very close to C. rufocanus among red-backed vole species (Wakana et al., 1996; Kaneko et al., 1998; Suzuki et al., 1999). It is possible that the common ancestor of C. rex and C. rufocanus was once distributed across Far East Asia including Hokkaido like C. rufocanus presently is and a branched lineage that had been retained in Hokkaido is now recognized as C. rex. This notion can be supported by several lines of evidence. Clethrionomys rex is known to bear primitive morphological characteristics compared to other species of Clethrionomys (Imaizumi, 1972; Gromov and Polyakov, 1992). This species now exhibits a fragmented geographic distribution in Hokkaido, probably due to the invasion of C. rufocanus with ecological superiority (Nakata, 1995). In addition, C. sikotanensis, which is presumed to be a synonym of C. rex, is retained in the southern Kuril islands, peripheral part of Hokkaido (Tokuda, 1935; Gromov and Polyakov, 1992) from morphological features (Iwasa, unpublished data), suggesting oldness of the lineage of C. rex in the Hokkaido area.
Therefore, Hokkaido can be characterized zoogeographically as a phylogenetically important area that raises the level of species and genetic diversity in small mammals. Finally it is worth noting that the two species of red-backed voles, C. rex and C. rufocanus, in Hokkaido could represent a high level of genic endemism among the mammalian fauna of Hokkaido, in contrast to the low levels of genetic endemism in the East-Asiatic wood mice A. peninsulae (Serizawa et al., unpublished data), red fox Vulpes vulpes (Tsuda et al., 1997), and sable Martes zibellina (Hosoda et al., 1999).
The authors are grateful to Dr Hisashi Abe for valuable suggestions. We also thank Dr Nikolai E Dokuchaev, and Messrs Katsuhiro Hirama, Hirofumi Asano and Miss Megumi Inamori for cooperation in collecting the animals. Special thanks are due to Drs Yukibumi Kaneko, Yoshiyuki Kimura, Yoshitaka Obara and Alexei P Kryukov for their encouragements throughout this study.