Molecular phylogeny and biogeography of Oriental voles: genus Eothenomys (Muridae, Mammalia)

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Abstract

Oriental voles of the genus Eothenomys are predominantly distributed along the Southeastern shoulder of the Qinghai-Tibetan Plateau. Based on phylogenetic analyses of the mitochondrial cytochrome b gene (1143 bp) obtained from 23 specimens (eight species) of Oriental voles collected from this area, together with nucleotide sequences from six specimens (two species) of Japanese red-backed voles (Eothenomys andersoni and Eothenomys smithii) and five species of the closely related genus Clethrionomys, we revised the systematic status of Eothenomys. We also tested if vicariance could explain the observed high species diversity in this area by correlating estimated divergence times to species distribution patterns and corresponding paleo-geographic events. Our results suggest that: (1) the eight species of Oriental voles form a monophyletic group with two distinct clades, and that these two clades should be considered as valid subgenera—Eothenomys and Anteliomys; (2) Eothenomys eleusis and Eothenomys miletus are not independent species; (3) Japanese red-backed voles are more closely related to the genus Clethrionomys than to continental Asian Eothenomys taxa; and (4) the genus Clethrionomys, as presently defined, is paraphyletic. In addition, the process of speciation of Oriental voles appears to be related to the Trans-Himalayan formation via three recent uplift events of the Qinghai-Tibetan Plateau within the last 3.6 million years, as well as to the effects of the mid-Quaternary ice age.

Introduction

Oriental voles are traditionally included in the genus Eothenomys (Muridae: Clethrionomyini), and inhabit the Trans-Himalayan Ranges of Southwest China, small parts of Northeast Burma and the Assam province in India (Fig. 1). According to the fossil record, this group is of recent origin, and most likely diversified during the late Pliocene (Zheng, 1993). It is assumed that speciation events within this group are linked to historical changes in the geography of their main distribution habitat, the Trans-Himalayan Ranges, which have been severely affected by several uplift events along the Qinghai-Tibetan Plateau. These geological processes have been considered to play a fundamental vicariant role in species divergence of many other vertebrates endemic to this region (Chen et al., 1998; Pang et al., 2003; Yu et al., 2000). Thus, we wanted to test whether the uplift of the Qinghai-Tibetan Plateau also facilitated speciation and adaptation processes of Oriental voles.

The taxonomy of the genus Eothenomys is under considerable debate, primarily due to the inherent morphological plasticity among members of this group and to subjectivity regarding the descriptions of some species. This is reflected by the contrasting definitions of the subgenera and genera ascribed to the group (Table 1). Indeed, 7–9 nominal species have been assigned to the genus Eothenomys under the subtribe Clethrionomyini based on morpho-anatomical characters or cytological data (Allen, 1940; Corbet, 1978; Ellerman and Morrison-Scott, 1951; Hinton, 1923, Hinton, 1926; Musser and Carleton, 1993; Wang and Li, 2000; Yang et al., 1998; see Table 1 for summary). Allen (1940) further classified the genus Eothenomys into three subgenera: Eothenomys, Anteliomys, and Caryomys. Under this classification scheme, the subgenus Eothenomys contains species with the first upper molars displaying three outer and four inner salient angles, and the last upper molars exhibiting three or four outer salient angles. The subgenus Anteliomys is comprised of species with the first upper molars possessing three outer and three inner salient angles. The subgenus Caryomys includes only two species, both of which have inter-bedded molar triangles in the first and second lower molars. Ma and Jiang (1996) revised the taxonomic status of the subgenus Caryomys and elevated it to genus rank based on its karyotype (2n = 54) compared to the karyotypes of other species in Eothenomys (2n = 56) (Chen et al., 1994; Yang et al., 1998). They left only two subgenera in Eothenomys, Eothenomys and Antelionomys, as was also suggested by Wang and Li (2000) (Table 1).

Early classification schemes generally subdivided the subtribe Clethrionomyini into two groups based on the morphology of the molars: Clethrionomys (where species have rooted molars) and the Eothenomys/Caryomys complex (where species have rootless molars). However, following this scheme, the position of Japanese red-backed voles was ambiguous since these species possess rooted molars that appear quite late in adult life. Consequently, Japanese red-backed voles, which were traditionally included by most authorities in Eothenomys, are now sometimes reassigned to their own genus, Phaulomys, Thomas (1905) (Musser and Carleton, 1993; Kawamura, 1988; Suzuki et al., 1999). Wang and Li (2000) accepted this designation and hypothesized that the subtribe Clethrionomyini includes four valid genera: Clethrionomys, Eothenomys, Caryomys, and Phaulomys. Yang et al. (1998) summarized all available karyotype data and discussed the putative evolutionary relationships among the main lineages of the Clethrionomyini. These species are diploids and generally possess chromosome numbers between 54 and 56 with a fundamental arm number between 54 and 60. However, cytological data sometimes provides discordant results. For example, Yang et al. (1998) reported that the karyotype of the Yulong vole (Eothenomys proditor) (distributed in Lijiang region, Northwest Yunnan of China) exhibit a dramatically different diploid chromosome number (2n = 32). In addition, these authors suggested that karyotype data do not provide enough convincing evidence to elucidate the phylogenetic relationships within this group. A comprehensive phylogeny based on unambiguous characters and appropriate phylogenetic reconstruction methods is still required to shed light on the classification and evolutionary history of this group. In this context, Cook et al. (2004) recently examined the molecular systematics of red-backed voles, and suggested that the genus Clethrionomys is paraphyletic with respect to both Eothenomys and Alticola. However, important taxa from the genus Eothenomys were not intensively sampled for this study, with only one Oriental vole species included. Thus, it is imperative to include additional species from the genus Eothenomys to better investigate the phylogenetic relationships among the subtribe Clethrionomyini.

The levels of genetic divergence typically found between sister species and their congeners are usually in the range in which the mitochondrial cytochrome b (cyt b) gene is phylogenetically informative. The cyt b gene is usually not affected by severe saturation effects involving superimposed nucleotide substitutions (Johns and Avise, 1998; Meyer, 1993; Moritz et al., 1987). Hence, it has often been used to reconstruct phylogenetic relationships within and among numerous vertebrate groups (Andrews et al., 1998; Irwin et al., 1991; Johns and Avise, 1998), including arvicolid rodents (Cook et al., 2004; Iwasa and Suzuki, 2002; Suzuki et al., 1999). To explore the molecular phylogenetic relationships of Oriental voles and their taxonomic affiliation with other members of the subtribe Clethrionomyini, we thus sequenced their mitochrondrial DNA cyt b gene. Drawing on this data, the goals of this study were: (1) to elucidate the phylogeny of Eothenomys from the Southeast border default region of the Qinghai-Tibetan Plateau; (2) to revise the taxonomic status of Oriental voles as well as other species in the subtribe Clethrionomyini with reference to the molecular phylogeny constructed; e.g., we wanted to test whether the rank of genus or subgenus assigned to groups such as Eothenomys, Anteliomys, and Phaulomys are valid; (3) to investigate if the divergence events within the group are correlated with recent uplift events of the Qinghai-Tibetan Plateau. To achieve this final goal, we compared divergence times inferred from cyt b data with the orogenic events and corresponding biogeographic distribution patterns of voles from this particular area.

Section snippets

Data collection

Voles were collected along the Southwestern shoulder of the Trans-Himalayan Ranges (Fig. 1). The voucher numbers and localities of the collected samples are listed in Table 2. Except for Eothenomys fidelis, specimens were identified based on external characteristics and skull morphology following the system of Wang and Li (2000) (see Table 1). E. fidelis was defined according to its unique cytological pattern (Yang et al., unpublished data). Twenty-three specimens comprising seven Oriental vole

Sequence variations and phylogenetic information

The entire coding region of the cyt b gene was sequenced from 23 Oriental voles (Table 2), and deposited in GenBank (Accession Nos. AY426678–AY426690). Including the start and stop codons, all sequences were 1143 bp—the same as other related mammalian groups (Irwin et al., 1991; Iwasa and Suzuki, 2002). A total of 252 nucleotide sites were variable, 53 of which were parsimony-informative. Seventeen haplotypes were identified from the 23 sequences. The following taxa shared the same haplotype:

Systematics of the subtribe Clethrionomysi: are Japanese red-backed voles more closely related to Oriental voles than to other species?

Miller (1896) first proposed the subgenus Eothenomys (which included Oriental and Japanese red-backed voles) and Hinton, 1923, Hinton, 1926 subsequently designated it as a valid genus. Contrary to this suggestion, and regardless of the tree reconstruction methods employed, our phylogenetic analyses consistently grouped all Oriental vole species from the genus Eothenomys into a monophyletic clade separate from Japanese red-backed voles (Fig. 3). In fact, Japanese red-backed voles (E. andersoni

Acknowledgments

We extend our sincerest gratitude to Drs. Walter Salzburger, Masahiro A. Iwasa, Yun-wu Zhang, Xue-mei Lu, and Ms. Chun-hua Wu for helpful and critical suggestions. We thank Li-hua Chen and Wei Zhou for figure drawing and data analysis. Technical support from workers in the lab of Y.P.Z. is gratefully acknowledged. This work was supported by the Chinese Academy of Sciences (KSCX2-1-05), the Program for Key International S & T Cooperation Project of P.R. China (2001CB711103), and the National

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