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Phylogeny of Oriental Voles (Rodentia: Muridae: Arvicolinae): Molecular andMorphological EvidenceAuthor(s): Shaoying Liu, Yang Liu, Peng Guo, Zhiyu Sun, Robert W. Murphy, Zhenxin Fan, JianrongFu and Yaping ZhangSource: Zoological Science, 29(9):610-622. 2012.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.29.610URL: http://www.bioone.org/doi/full/10.2108/zsj.29.610

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2012 Zoological Society of JapanZOOLOGICAL SCIENCE 29: 610–622 (2012)

Phylogeny of Oriental Voles (Rodentia: Muridae: Arvicolinae):

Molecular and Morphological Evidence

Shaoying Liu1*, Yang Liu1, Peng Guo2, Zhiyu Sun1, Robert W. Murphy3,4,

Zhenxin Fan5, Jianrong Fu1, and Yaping Zhang3

1Sichuan Academy of Forestry, Chengdu, Sichuan 610066, China2College of Life Sciences and Food Engineering, Yibin University, Yibin, Sichuan 644007, China

3Kunming Institute of Zoology, the Chinese Academy of Science, Kunming, Yunnan 650223, China4Centre for Biodiversity and Conservation Biology, Royal Ontario Museum,

100 Queen’s Park, Toronto, Ontario, M5S 2C6 Canada5College of Life Sciences, Sichuan University,

Chengdu, Sichuan 610051, China

The systematics of Oriental voles remains controversial despite numerous previous studies. In this

study, we explore the systematics of all species of Oriental voles, except Eothenomys wardi, using

a combination of DNA sequences and morphological data. Our molecular phylogeny, based on two

mitochondrial genes (COI and cyt b), resolves the Oriental voles as a monophyletic group with

strong support. Four distinct lineages are resolved: Eothenomys, Anteliomys, Caryomys, and the

new subgenus Ermites. Based on morphology, we consider Caryomys and Eothenomys to be valid

genera. Eothenomys, Anteliomys, and Ermites are subgenera of Eothenomys. The molecular phy-

logeny resolves subgenera Anteliomys and Ermites as sister taxa. Subgenus Eothenomys is sister

to the clade Anteliomys + Ermites. Caryomys is the sister group to genus Eothenomys. Further, the

subspecies E. custos hintoni and E. chinensis tarquinius do not cluster with E. custos custos and

E. chinensis chinensis, respectively, and the former two taxa are elevated to species level and

assigned to the new subgenus Ermites.

Key words: Anteliomys, Caryomys, Eothenomys, Ermites, DNA barcoding

INTRODUCTION

Eothenomys (sensu lato) is a genus of Oriental voles

that mainly occurs in the Hengduan Mountains of southwest-

ern China, northeastern Burma, and Assam, India. Initially

erected as a subgenus of Microtus (Miller, 1896), Hinton

(1923) elevated the taxon to the level of genus based on

morphology. This arrangement is widely accepted.

The intrageneric taxonomy and systematics of Oriental

voles is controversial. Hinton (1923), followed by Gromov

and Polyakov (1977), did not recognize any subgenera of

Eothenomys. Hinton (1923) proposed recognition of Caryomys

and Anteliomys and considered them to be closely related

genera. Later, Hinton (1926), followed by Ellerman (1941)

and Ellerman and Morrison-Scott (1951), recognized

Eothenomys and Anteliomys as valid genera, and he con-

sidered Caryomys to be immature individuals of the subspe-

cies Evotomys rufocanus shanseius. Ma and Jiang (1996)

reinstated Caryomys as a valid genus. This rearrangement

has been accepted by many systematists (Luo et al., 2000;

Wang, 2003; Musser and Carleton, 2005). The recognition

of subgenera also varies. Some systematists recognize

Anteliomys as a subgenus of Eothenomys (Ellerman and

Morrison-Scott, 1951; Ma and Jiang, 1996; Luo et al., 2000;

Ye et al., 2002; Wang, 2003; Musser and Carleton, 2005).

In contrast, Allen (1940) suggested that both Caryomys and

Anteliomys should be subgenera of Eothenomys, an

arrangement that is accepted by most systematists (Corbet,

1978; Honacki et al., 1982; Nowak and Paradiso, 1983;

Corbet and Hill, 1992; Musser and Carleton, 1993; Nowak,

1999).

Taxonomic uncertainty is common in Eothenomys (sensu

lato). To date, 25 nominal species have been included at

least once in this genus, yet today only between five and 12

species are recognized; others have been either regarded to

be synonyms or subspecies of other species, or transferred

to other genera (Table 1). The main controversies focus on

whether these are valid species and subspecies. For exam-

ple, Thomas (1921) described E. cachinus as a full species

and Luo et al. (2000) and Wang (2003) recognized it. How-

ever, most mammalogists consider the taxon to be a

subspecies or synonym of E. melanogaster (Hinton, 1923;

Ellerman, 1941; Ellerman and Morrison-Scott, 1951; Corbet,

1978; Musser and Carleton, 1993), or a synonym of E.

miletus (Corbet and Hill, 1992). Recently, Musser and

Carleton (2005) elevated it back to a species (Table 1). Sim-

ilarly, controversy exists as to whether E. miletus (Thomas,

* Corresponding author. Tel. : +86-28-83226632;

Fax : +86-28-83226547;

E-mail: shaoyliu@163.com

Supplemental material for this article is available online.

doi:10.2108/zsj.29.610

Phylogeny of Oriental Voles 611

Table 1. Species and variations in the genera and species of Eothenomys (sensu lato).

SpeciesHinton(1926)

Allen (1940)Ellerman(1941)

Ellerman andMorrison-Scott

(1951)

Corbet(1978)

Honackiet al (1982)

Corbet andHill (1986)

Corbet andHill (1992)

Musser andCarleton(1993)

Musser andCarleton(2005)

Nowak(1999)

Luo et al(2000)

Wang(2003)

Eothenomysalcinous(Thomas,1911a)

Immature ofEvotomysrufocanusshanseius

Eo.eva alcinous

Synonym ofClethrionomys

rafocanusshanseius

Synonym ofCl. rafocanus

shanseius

Synonym ofEo. eva

N N Synonym ofEo. eva

Synonym ofEo. eva

Synonym ofCaryomys

eva

N Eo. evaalcinous

Eo.eva alcinous

Eo. andersoni(Thomas,1905a)

Ev.rufocanus

smithii

N Synonym ofCl. rufocanus

smithii

Synonym ofCl. rufocanus

smithii

Cl. andersoni √ Cl.andersoni

N Phaulomysandersoni

Myodesandersoni

Ph.andersoni

Cl. andersoni N

Eo. aurora(Allen, 1912)

Eo.melanogaster

aurora

Eo.miletus aurora

Eo.melanogaster

aurora

Eo.melanogaster

aurora

Synonym ofEo.

melanogaster

N N Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

N Eo. eleusisaurora

Eo. eleusisaurora

Eo. bozno(Cabrera,

1922)

N Synonym ofEo.

melanogastercolurnus

Synonym ofEo.

melanogastercolurnus

Synonym ofEo.

melanogastercolurnus

Synonym ofEo.

melanogaster

N N Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

N Synonym ofEo.

melanogastercolurnus

Eo. cachinus(Thomas,

1921)

Eo.melanogaster

cachinus

N Eo.melanogaster

cachinus

Eo.melanogaster

cachinus

Synonym ofEo.

melanogaster

N N Synonym ofEo. miletus

Synonym ofEo.

melanogaster

√ N √ √

Eo. chinensis(Thomas, 1891)

Anteliomyschinensis

√ An. chinensis √ √ √ √ √ √ √ √ √ √

Eo. custos(Thomas,

1912)

An. custos √ An. custos √ √ √ √ √ √ √ √ √ √

Eo. eleusis(Thomas,1911a)

Eo.melanogaster

eleusis

√ Eo.melanogaster

eleusis

Eo.melanogaster

eleusis

Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

N Eo.melanogaster

eleusis

Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

N √ √

Eo. eva(Thomas,1911b)

Immature ofEv. rufocanus

shanseius

√ Synonym ofCl. rufocanus

shanseius

Synonym ofCl. rufocanus

shanseius

√ √ √ √ √ Ca. eva √ Ca. eva Ca. eva

Eo. fidelisHinton,1923

√ Synonym ofEo.

miletus miletus

√ Synonym ofEo.

melanogastermiletus

Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

N Synonym ofEo. miletus

Synonym ofEo.

melanogaster

Synonym ofEo.

miletus

N Synonym ofEo. miletus

√

Eo. inez(Thomas,1908a)

Immature ofEv. rufocanus

shanseius

√ Synonym ofCl. rufocanus

shanseius

Synonym ofCl. rufocanus

shanseius

√ √ √ √ √ Ca. inez √ Ca. inex Ca. inez

Eo. kageusImaizumi,

1957

N N N N Synonym ofEo. smithii

N N N Synonym ofPh. smithii

Synonym ofMy. smithii

N N N

Eo. kanoiTokuda,

1937

N N N Eo. melanogasterkanoi

Synonym ofEo.

melanogaster

N N Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

N Eo.melanogaster

kanoi

Eo.melanogaster

kanoi

Eo. lemminus(Miller, 1898)

Aschizomyslemminus

As. lemminus Cl. lemminus √ √ √ N Alticolalemminus

Al. lemminus Al.lemminus

N N

Eo. libonotesHinton, 1923

Eo.melanogaster

libonotes

N Eo.melanogaster

libonotes

Eo.melanogaster

libonotes

Synonym ofEo.

melanogaster

N N Eo.melanogaster

libonotes

Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

N Eo.melanogaster

libonotes

Eo.melanogaster

libonotes

Eo.melanogaster

(Milne-Edwards,

1871)

√ √ √ √ √ √ √ √ √ √ √ √ √

Eo. miletus(Thomas,

1914)

Eo.melanogaster

miletus

√ Eo.melanogaster

miletus

Eo.melanogaster

miletus

Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

N √ Synonym ofEo. melanogaster

√ √ √ √

Eo.mucronatusAllen, 1912

Eo.melanogastermucronatus

Synonym ofEo.

melanogaster

Eo.melanogastermucronatus

Eo.melanogastermucronatus

Synonym ofEo.

melanogaster

N N Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

Synonym ofEo.

melanogaster

N Eo.melanogastermucronatus

Eo.melanogastermucronatus

Eo. nux(Thomas,

1910)

Immature ofEv.

rufocanusshanseius

Eo. inez nux Synonym ofCl. rufocanus

shanseius

Synonym ofCl. rufocanus

shanseius

Synonym ofEo. inez

N N Synonym ofEo. inez

Synonym ofEo. inez

Synonym ofCa. inez

N Ca. inez nux Ca. inez nux

Eothenomysoliter

(Thomas,1911a)

√ √ √ √ √ √ √ √ √ √ √ √ √

Eo. proditorHinton, 1923

√ √ √ √ √ √ √ √ √ √ √ √ √

Eo. regulus(Thomas,

1907)

Ev. rufocanusregulus

Cl. rufocanusregulus

Cl. rufocanusregulus

Cl. rufocanusregulus

√ √ √ N √ My. regulus √ Cl. regulus N

Eo. shanseius(Thomas,1908b)

Ev. rufocanusshanseius

Cl. rufocanusshanseius

Cl. rufocanusshanseius

Cl. rufocanusshanseius

√ √ √ N √ My. shanseius √ Cl.rufocanusshanseius

Cl.rufocanusshanseius

Eo. smithii(Thomas,1905b)

Ev. rufocanussmithii

N Cl. rufocanussmithii

Cl. rufocanussmithii

√ √ √ N Ph. smithii My. smithii Ph. smithii Cl. smithii N

Eo. wardi(Thomas,

1912)

An. wardi Eo.chinensis

wardi

An. wardi Eo. chinensiswardi

Synonym ofEo. chinensis

N N √ Synonym ofEo. chinensis

√ √ √ √

Tatal species 7 9 7 5 11 12 11 9 9 10 11 9 12

*: “√” maens the author accepting the species; Eothenomys (sensu lato) includes Eotnenomys, Anteliomys and Caryomys; “N” not mentioned; Eo. = Eothenomys; Cl. = Clethrionomys; Ev. = Evotomys; Ca. = Caryomys; Ph. = Phaulomys; My. = Myodes; An. = Anteliomys; As. = Aschizomys

S. Liu et al.612

1914) is a subspecies or synonym of E. melanogaster

(Hinton, 1926; Ellerman and Morrison-Scott, 1951; Corbet,

1978; Honacki, 1982; Musser and Carleton, 1993), or a full

species (Allen, 1940; Corbet and Hill, 1992; Nowak, 1999;

Luo et al., 2000; Wang, 2003; Musser and Carleton, 2005)

(Table 1). Of course, these issues skirt the question of

whether or not any subspecies are valid taxa (Frost and

Hillis, 1990; Burbrink et al., 2000).

Most previous studies on the systematics of Eothenomys

(sensu lato) have focused on morphological comparisons,

particularly external morphology. Molecular analyses of the

group are rare. Yang et al. (1998) summarized all available

karyological data and discussed the putative evolutionary

relationships among the main lineages of the Clethrionomyini.

All taxa are diploid, generally with 54–56 chromosomes,

having a fundamental arm-number between 54 and 60

(excluding E. proditor). However, analyses of cytological

data provide discordant results, and no analyses unambigu-

ously elucidate the phylogenetic relationships within this

group. Luo et al. (2004) first explored the phylogeny of

Eothenomys (sensu lato) using nucleotide sequence data

from the mitochondrial DNA (mtDNA) cytochrome b gene

(cyt b). Their results suggest that Oriental voles (eight species

included) form a monophyletic group with two distinct mater-

nal lineages, the subgenera Eothenomys and Anteliomys.

Because Caryomys was not included in their study, the

validity of the genus, as well as the relationships among

Anteliomys, Caryomys, and Eothenomys, remain unre-

solved. Some recent molecular phylogenies include speci-

mens of Eothenomys (sensu lato), but they have not

focused on the genus itself (Buzan et al., 2008; Robovský

et al., 2008).

Herein we use nucleotide sequences from two mtDNA

genes—cytochrome oxidase subunit I (COI) and cytochrome

b (cyt b)—to explore the

evolutionary history and

systematics of Eothenomys

(sensu lato). We include a

morphological comparison

that includes all putative

species other than E.

wardi. Our aims are to: (1)

test whether Caryomys and

Anteliomys are monophyl-

etic genera; (2) hypothe-

size the phylogenetic rela-

tionships of species within

Caryomys, Anteliomys, and

Eothenomys; (3) test

whether cryptic taxa occur

in this group; and (4) test

the efficacy of DNA barcod-

ing for the group.

MATERIALS AND

METHODS

Samples and data collection

A total of 458 specimens

representing 11 species

(including 15 subspecies) and

two unidentified forms of

Eothenomys (sensu lato) were collected. All specimens have been

deposited in Sichuan Academy of Forestry. Morphological data

were obtained from 426 specimens (Supplementary Text S1

online). We sequenced 110 specimens for cyt b and the standard

DNA barcoding gene COI. The sampling localities were indicated in

Fig. 1 and detailed in Supplementary Table S1 online; eight repre-

sentatives of the allied genera (Myodes, Alticola, and Microtus) in

Cricetidae were included for de novo sequencing to evaluate the

monophyly of the genera.

DNA extraction, amplification, and sequencing

Total DNA was extracted from 95% ethanol-preserved liver or

muscle tissue using the standard proteinase K and phenol/chloroform

method (Sambrook and Russell, 2002). The entire cyt b and COI

genes were amplified using primers described by Burbrink et al.

(2000) and Delisle and Strobeck (2002), respectively. For cyt b, we

followed the cycling sequence protocol of Burbrink et al. (2000) but

with an annealing temperature of 56°C. Prior to sequencing, PCR

products were purified using a commercial kit (Watson BioMedical).

Double-stranded PCR products were directly sequenced from both

directions using an ABI 3730 Genetic Analyzer (Applied Biosystems)

following the manufacturer’s protocols.

Alignment of cyt b and COI was straightforward because indels

did not occur. The inadvertent amplification of pseudogenes (Zhang

and Hewitt, 1996) was checked by translating the sequences into

amino acids using MEGA 5 (Tamura et al., 2011) and aligning them

with published sequences. Kimura’s (1980) two-parameter genetic

distances (K2P) were calculated for the cyt b and COI data using

MEGA 5.

Matrilineal genealogy reconstruction

We used multiple methods to infer phylogenetic (matrilineal)

relationships, including maximum parsimony (MP), maximum likeli-

hood (ML), and Bayesian inference (BI). MP trees were obtained

using PAUP* 4.0b10 (Swofford, 2003) with a heuristic search using

1,000 random sequence addition replicates and TBR (tree bisection-

reconnection) branch swapping. Bootstrap support values (BS) for

lineages were calculated from 1,000 pseudoreplicates (Felsenstein,

Fig. 1. The collection localities of samples of voles sequenced in this work.

Phylogeny of Oriental Voles 613

1985). ML analyses were implemented in RAxML (Stamatakis et al.,

2008). For BI, the nucleotide sequences were partitioned by codon

position. Six partitions were given, one each for codon positions 1,

2, and 3 independently for both cyt b and COI. The best-fit models

of evolution for each partition were chosen using MrModeltest 2.3

(Huelsenbeck and Crandall, 1997; Posada and Crandall, 1998,

2001). BI was implemented using MrBayes 3.1 (Huelsenbeck and

Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Three runs

were performed with four Markov chains (three heated chains and

one cold chain) starting from a random tree. Each of these runs was

conducted with a total of 5 million generations, and sampled every

100 generations. Stationarity was confirmed by inspecting plots of ln

(L) against generations using Tracer 1.3 (Rambaut and Drummond,

2003), and the first 1,000 generations were discarded as burn-in.

The frequencies of nodal resolution in the consensus tree were

termed posterior probabilities (PP).

As only 68 samples had sequence data from both genes, we

reconstructed matrilineal relationships using three different data-

sets: COI, cyt b, and a combined dataset (COI + cyt b). When mul-

tiple samples were sequenced from the same species, only unique

haplotypes were used to construct the trees.

Morphological analysis

A total of 426 individuals representing 11 species and two

unidentified forms of Eothenomys (sensu lato), including 15 subspe-

cies, were examined morphologically from the following taxa:

Eothenomys cachinus, E. chinensis chinensis, E. ch. tarquinius, E.

custos custos, E. cu. hintoni, E. cu. rubellus, E. eleusis eleusis, E.

el. aurora, E. eva eva, E. ev. alcinous, E. fidelis, E. inez inez, E. i.

nux, E. melanogaster chengduenus, E. me. melanogaster, E. miletus

miletus, E. olitor hypolitor, and E. proditor (Supplementary Text S1

online). Morphological structures of the teeth rows were scored.

Head plus body length, tail length, hind foot length, and greatest

length of skull were measured. External measurements were taken

in the field on freshly captured specimens and recorded to the near-

est 0.5 mm. Cranial measurements were taken with a vernier cali-

per to the nearest 0.02 mm. Morphological measurements followed

the protocol described by Liu et al. (2007). Measurements were

abbreviated as follows: HBL, head and body length, measuring from

snout to anus; TL, tail length; HFL, hind foot length excluding claws;

and SGL, skull greatest length.

Statistical analyses of the morphological data were performed

using SPSS v.12.0 for Windows (SPSS Inc., 1999). Descriptive sta-

tistics (mean, standard deviation, and observed range) were com-

puted for each species. Principal component analysis (PCA) was

used to obtain a general view of variation. We projected the most

informative factors of individuals to detect differentiation of morpho-

logical measurements between genera, subgenera, and species.

When the groups could not be clearly distinguished, t-tests were

used to determine if they differed statistically significantly while

assuming a priori a P < 0.05 to be significant.

Fieldwork was conducted following the animal care and use

guidelines of the American Society of Mammalogists (Gannon et al.,

2007).

RESULTS

Molecular data analysis

After alignment, a fragment of 1,539 bp was obtained for

COI from a total of 88 samples of Oriental voles. The frag-

ment was larger than the standard used for DNA barcoding

(Hebert et al., 2003). Seventy-five haplotypes were

screened de novo and 59 complete sequences were used

to hypothesize matrilineal relationships. Partial sequences

only were used in the combined datasets. Eight haplotypes

from the allied genera were also sequenced de novo. These

included Alticola stracheyi (one haplotype), Microtus fortis

(one), Myodes rufocanus (three), My. rutilis (two), and

Neodon irene (one). The following five additional sequences

were retrieved from GenBank as a part of outgroup based

on the study of Steppane et al. (2004): Hylaeamys yunganus

(Cricetidae: Sigmodontinae), Mesocricetus auratus

(Cricetidae: Cricetinae), Neacomys guianae (Cricetidae:

Sigmodontinae), Rattus exulans (Muridae: Murinae), and

Tatera indica (Muridae: Gerbillinae). For cyt b, up to a total

of 1,140 bp were obtained from each of the 99 samples suc-

cessfully sequenced de novo, and 81 unique haplotypes

were resolved, of which 69 complete sequences were used

to reconstruct matrilineal relationships. The remaining partial

fragments were used in the combined datasets only. Five

unique haplotypes of Myodes were also sequenced de

novo. From GenBank, we retrieved 16 fragments from

Oriental voles, 20 from the allied genera, and five from the

outgroup taxa. For the combined data, 68 Oriental voles had

both cyt b and COI gene sequences (de novo). Sequences

from either cyt b or COI only were available from 28 speci-

mens (de novo). Of six specimens of the allied genera

(Myodes and Alticola), five specimens of Myodes had

sequences from both cyt b and COI (de novo); one speci-

men of Alticola stracheyi had sequence data from COI only

(de novo). Sequences of cyt b only were retrieved from

GenBank for 20 samples of the allied genera. Two complete

outgroup sequences from the Muridae were retrieved from

GenBank. In our de novo sequences, no deletions, inser-

tions, or stop codons were found in either gene, indicating

that paralogous nuclear insertions had not been amplified.

Novel sequences have been deposited in GenBank

(GenBank accession numbers as follows: COI, HM165276–

HM165358; and cyt b, HM165359–HM165444) (Supple-

mentary Table S1 online).

Sequence divergence

Average K2P genetic distances for the standard barcod-

ing gene, COI, were generally sufficient to diagnose species

(Table S2) and lineages (not given in table). Within

Eothenomys, average interspecific divergences ranged from

0.011 to 0.086 and inter-individual differences averaged

0.056. Levels of divergence were greater than those values

within a new subgenus (erected below), where they ranged

from 0.020 to 0.052 and inter-individual differences aver-

aged 0.027. Clade Anteliomys had the highest average level

of interspecific divergence (0.106–0.134) and inter-individual

differences averaged 0.087. Within Eothenomys, the bar-

codes did not always unambiguously identify morphological

taxa. In contrast, sequence divergence for COI between

unambiguously identified the two species of Caryomys

(average divergence 0.066) and species within Myodes

(average divergence 0.134). Levels of divergence for cyt b

were generally equivalent to those of COI (Table S2).

Phylogenetic/matrilineal analyses

The average base frequencies for COI were as follows:

T = 26.6%, C = 29.4%, A = 34.6%, and G = 9.4%; for cyt b

T = 23.5%, C = 33.6%, A = 36.6%, and G = 6.3%. A total of

502 nucleotide sites (33%) were variable in COI, of which 454

(29%) were potentially parsimony-informative when including

the outgroup taxa. For cyt b, 445 sites (39%) were variable

and 393 (34%) were potentially parsimony-informative when

S. Liu et al.614

including the outgroup taxa.

MP analysis of the COI sequences produced two most

parsimonious trees of 1,595 steps (CI = 0.433, RI = 0.859,

RC = 0.372). MrModeltest was employed to identify the

following models of sequence evolution in the BI and ML

analyses of the partitioned data under the AIC criterion:

GTR + I + Г for the first codon position, HKY for the second,

and GTR + Г for the third.

For cyt b, MP resolved 45133 most parsimonious trees

of 2,783 steps with CI = 0.309, RI = 0.767, RC = 0.237. For

Fig. 2. Fifty percent majority rule consensus tree from a Bayesian analysis of cyt b nucleotide sequence data. Numbers represent nodal sup-

port of key nodes inferred from Bayesian posterior probability, ML bootstrap, and MP bootstrap analysis, respectively. Asterisks indicate nodes not supported by the MP analysis (< 50). Morphological correlates are as follows: triangles of the first lower molar of Caryomys interlaced; trian-gles of the first lower molar of the Eothenomys fuse transversely; the first upper molar of subgenus Eothenomys has four inner angles; the first upper molar of subgenera Anteliomys and Ermites has three inner angles; structure of the third upper molar of subgenus Anteliomys non-variable; and structure of the third upper molar of subgenus Ermites multivariable.

Phylogeny of Oriental Voles 615

the BI and ML analyses, the models SYM + I + Г, HKY + I,

and GTR + I + Г were selected for three codon positions,

respectively, under the AIC criterion.

Despite the different datasets and methods of tree con-

struction, all generated trees were largely congruent. Minor

disagreements were associated with poorly supported

nodes (Figs. 2–4).

Analyses of the sequence data always resolved a mono-

phyletic Eothenomys (sensu lato) and frequently with strong

support. In the BI analyses of the cyt b and combined data-

sets, PP = 100% and PP = 97%, respectively. The ML anal-

yses for COI and the combined datasets obtained BS = 77

and BS = 94, respectively. In the MP tree for COI, BS = 90.

Four maternal lineages of Oriental voles were recovered.

Three of those were accompanied with high support values

in all analyses, corresponding to Eothenomys, Caryomys,

and a new taxon. The fourth lineage, Anteliomys, was

strongly supported in the BI analyses. However, for this lin-

eage, the ML and MP analyses for COI had very low support

values (BS < 50%), the ML analysis for cyt b obtained mod-

erate support (BS = 74%), and the MP analysis for the com-

bined dataset had low support (BS = 64%) (Figs. 2–4).

The sister-group relationship between the new taxon

and Anteliomys was strongly supported with the exception of

the MP tree for the combined dataset (BS = 70; Fig. 4). The

sister-group relationship of Eothenomys with the new taxon

and Anteliomys was highly supported in both the BI and ML

trees for COI data (PP = 100%; BS = 93%) and the com-

bined dataset (PP = 99%; BS = 98%), but the results from

cyt b showed low support values in all three tree construc-

tion methods (Fig. 2).

The major lineages contained the same species

irrespective of method of analysis and dataset. In most

cases, the sublineages, including samples from the same

Fig. 3. Fifty percent majority rule consensus tree from a Bayesian analysis of COI nucleotide sequence data. Numbers are nodal support val-

ues for key nodes inferred from Bayesian posterior probability, ML bootstrap, and MP bootstrap analyses, respectively. Asterisks indicate

nodes not supported by the MP analysis (< 50).

S. Liu et al.616

Fig. 4. Fifty percent majority rule consensus tree from the Bayesian analysis of the combined data. Numbers represent nodal support inferred from

Bayesian inference, ML bootstrap, and MP bootstrap analyses, respectively. Asterisks indicate nodes not supported by the MP analysis (< 50).

Phylogeny of Oriental Voles 617

species, received strong support values. Most of the mor-

phologically identified subspecies correctly clustered in their

species. However, E. eleusis eleusis, E. el. aurora and E. el.

yinyjiangensis did not cluster together. The new taxon con-

tained two putative subspecies (E. chinensis tarquinius and

E. custos hintoni) and two undescribed forms. The former

two subspecies were distantly related to their congeners E.

ch. chinensis and E. cu. custos, respectively. Anteliomys

contained E. proditor, E. custos, E. chinensis, and E. olitor.

Eothenomys contained five species: Eothenomys cachinus,

E. eleusis, E. fidelis, E. melanogaster, and E.

miletus. Notably, three species (E. cachinus,

E. eleusis, and E. miletus) nested together,

but these were not resolved as monophyl-

etic (monogenealogical sensu Murphy and

Méndez de la Cruz, 2010) species. Mono-

phyletic Caryomys was comprised of E. inez

and E. eva.

Morphological groups

The Oriental voles were divided into

several morphological groups. (1) They

formed two groups based on the condition of

the first lower molar: Caryomys (Fig. 5A)

was distinguished from Eothenomys,

Anteliomys, and the new taxon by having

triangles of the first lower molar arranged

alternately; the triangles of the first lower

molar were confluent transversely in

Eothenomys, Anteliomys, and the new taxon

(Fig. 5B). (2) The first upper molar of

Eothenomys had four inner angles (Fig. 5C),

whereas that of Anteliomys and the new

taxon had three inner angles (Fig. 5D). (3)

The third upper molar of the new taxon

(including two previously described subspe-

cies, E. chinensis tarquinius and E. custos

hintoni, as well as E. sp. 1 and E. sp. 2) was

complex and variable (Fig. 5E1–3, F1–3, G1–

4), whereas that of Anteliomys was either

complex or simple, but without variation (Fig. 5H–K). (4)

Three species-groups were defined based on the proportion

of TL to HBL. In the first group, the tail was more than half

of the HBL. This group included Eothenomys chinensis

chinensis, E. ch. tarquinius, E. custos hintoni, and E. eva. The

second group consisted of Eothenomys custos, E. eleusis, E.

fidelis, E. inez, E. melanogaster, E. miletus, E. olitor, and E.

proditor. Here, TL was less than half of HBL. The last group

included the two unidentified forms. The TL was equal to half

of HBL (Table 2). (5) Three taxa (E. chinensis chinensis, E.

Fig. 5. Teeth from the right side of Oriental voles. (A) first lower molar of Caryomys (C.

eva); (B) first lower molar of genus Eothenomys (E. melanogaster); (C) first upper molar of

subgenus Eothenomys (E. melanogaster); (D) first upper molar of subgenera Anteliomys

and Ermites (E. custos); (E1–3) third upper molar of E. tarquinius; (F1–3) third upper

molar of E. hintoni; (G1–4) third upper molar of E. sp. 1; (H) third upper molar of E.

chinensis; (I) third upper molar of E. custos; (J) third upper molar of E. proditor; (K) third

upper molar of E. olitor.

Table 2. Morphological comparison of Oriental voles. Length values are shown as mean with range in parentheses (mm).

genera subgenera species HBL TL HTL TL/HBL SGL n 1st lower molar1st upper

molar3rd upper molar

CaryomysC. eva 87.73 (75–98) 51.61 (43–61) 16.36 (15–19) 0.59 22.98 (21.00–25.70) 33 Triangles of the

first lower rangealternately

With 3 innerand 3 outer

angles

With 3 innerand 3

outer anglesC. inez 92.14 (80–99) 34.86 (30–38) 15.46 (13–18) 0.38 23.79 (22.10–25.40) 14

Eothenomys

Eothenomys

E. melanogaster 97.92 (80–110) 37.10 (28.5–52)16.16 (14–19) 0.38 24.51 (21.76–26.08) 55

Triangles of thefirst lower molarwere confluenttransversely

With 4 innerangles

with 3–4 inner and3–4 outer angles.

Structures ofintraspecies

steady

E. fidelis 110.22 (98–121) 42.39 (38–49) 17.94 (15–19) 0.38 27.47 (26.56–28.30) 18

E. miletus 103.33 (91–115) 42.17 (36–51) 17.06 (15–19) 0.41 26.06 (24.42–28.42) 25

E. cachinus 97.67 (78–112) 39.08 (34–46) 17.79 (15–20) 0.40 25.17 (24.42–26.18) 13

E. eleusis 99.42 (82–112) 42.16 (36–50) 17.20 (13–21) 0.42 24.75 (22.30–28.28) 19

Anteliomys

E. chinensis 121.66 (102–134)64.12 (57–71) 22.41 (21–25) 0.53 29.92 (27.52–31.14) 82

With 3 innerangles

with 3–5 inner and3–5 outer angles,

structure ofintraspeciesmolar steady

E. custos 99.04 (79–110) 37.42 (32–50) 17.06 (15–20) 0.38 24.43 (22.76–25.96) 26

E. proditor 96.57 (90–110) 36.57 (35–40) 18.00 (15–20) 0.38 25.77 (24.62–27.46) 7

E. olitor 86.75 (86–88) 27.50 (26–29) 14.25 (14–15) 0.32 20.09 (21.78–22.40) 4

Ermites

E. tarquinius 114.31 (104–125)66.63 (62–75) 22.40 (18–24) 0.58 27.37 (26.10–28.72) 48

With 3inner angles

With 4–6 innerand 4–6 outer

angles,structure ofintraspeciescomplex andmultivariate

E. hintoni 98.79 (92–105) 56.11 (52–60) 18.00 (16–19) 0.57 24.39 (23.52–25.62) 19

E. sp. 1 96.27 (86–110) 46.21 (42–53) 17.02 (16–20) 0.48 22.91 (21.74–24.40) 42

E.sp. 2 97.19 (87–110) 47.24 (39–55) 17.38 (16–19) 0.49 23.44 (22.56–24.40) 22

HBL: head and body length; TL: tail length; HFL: hind foot length; SGL: skull greatest length; n: numbers of specimens.

S. Liu et al.618

ch. tarquinius, and E. fidelis) had

the largest HBL (> 110 mm),

nine taxa (E. cachinus, E. custos

hintoni, E. custos [E. cu. custos

and E. cu. rubellus], E. eleusis,

E. melanogaster, E. miletus, E.

proditor, and the two unidenti-

fied forms) had medium HBLs

(averages range from 95–105

mm), and the remaining taxa had

HBL < 95 mm (E. eva, E. olitor,

and E. inez). A detailed compar-

ison of the morphology was given

in Table 2. Based on morpholog-

ical characteristics, Eothenomys

chinensis chinensis, E. ch.

tarquinius, E. custos custos, E.

cu. hintoni, E. eva, E. inez, E.

olitor, and the two unidentified

forms were identified unambigu-

ously. All other subspecies were

correctly identified to their spe-

cies. However, Eothenomys

eleusis, E. fidelis, and E. miletus

could be distinguished by SGL

measurements only.

A principal component analy-

sis (PCA) was conducted to eval-

uate morphological variation in 17

measurements (ABL, CBL, EL,

HBL, HFL, IOW, LIL, LM, LMbT,

LMxT, MB, M-M, SBL, SGL, SH,

TL, and ZB) between species in

the subclades Eothenomys,

Anteliomys, and the new taxon of

Eothenomys and the clade Cary-

omys (Fig. 6). Clade Eothenomys

was not clearly distinguished

from Caryomys (Fig. 6A) in the

three component-factors PCA.

However, the t-test obtained

statistically significant differ-

ences in 14 morphometric char-

acters but not for IOW, M-M, and

ZB. The new taxon, which com-

prises Eothenomys chinensis

tarquinius, E.custos hintoni, and

two unidentified forms, differed

markedly from the subclades

Eothenomys and Anteliomys in

the three component-factors

PCA. However, subclades Eoth-

enomys and Anteliomys were

not clearly distinguished (Fig.

6B) yet the t-test detected statis-

tically significant differences in

all 17 measurements.

Many species were clearly

discernible, but not all. Two spe-

cies of Caryomys were clearly

distinguished in the three com-

A B

C D

E F

Fig. 6. Results of PCA analysis based on 17 morphological measurements. (A) Three component-

factors PCA analysis between genera Eothenomys and Caryomys; (B) Three component-factors

PCA analysis among subgenera Eothenomys, Anteliomys and Ermites; (C) Three component-factors

PCA analysis between Caryomys eva and C. inez; (D) Three component-factors PCA analysis

among interspecies of subgenus Eotheomys; (E) Two component-factors PCA analysis among inter-

species of subgenus Anteliomys; (F) Two component-factors PCA analysis among interspecies of

subgenus Ermites.

Phylogeny of Oriental Voles 619

ponent-factors PCA (Fig. 6C). In contrast, within subclade

Eothenomys, five species were not clearly distinguished in

the three component-factors PCA (Fig. 6D). The two com-

ponent-factors PCA clearly distinguished the following four

species in subclade Anteliomys: Eothenomys chinensis, E.

custos, E. olitor, and E. proditor (Fig. 6E). Within the new

taxon, Eothenomys chinensis tarquinius was clearly distin-

guished from the others. Eothenomys custos hintoni and the

other two unidentified forms were slightly differentiated by

two component-factors in the PCA (Fig. 6F). The two

unidentified forms differed statistically significantly by five

measurements: EL, LM, M-M, SH, and ZB.

The molecular and morphological results yielded two pri-

mary conclusions. First, Cayomys and Eothenomys are valid

genera based on the condition of the lower molar and the

molecular phylogeny. The latter genus included three sub-

genera: Eothenomys, Anteliomys, and a new subgenus.

Second, although matrilineal genealogies did not reject the

null hypothesis of panmixia, the historical relationships sug-

gest the existence of species. Our trees suggested that

Eothenomys chinensis tarquinius and E. custos hintoni were

valid taxa based on historical relationships and the extent of

lineage divergence. Our morphological results clearly diag-

nosed these taxa, and suggest the absence of gene flow.

Taken together, the genealogy and morphological analyses

served to reject the null hypothesis of conspecificity of E.

chinensis tarquinius and E. ch. chinensis as well as E.

custos hintoni and E. cu. custos. Therefore, we regarded E.

tarquinius and E. hintoni as distinct species. Further, the two

unidentified forms appeared to be undescribed species. The

lineage that included E. hintoni, E. tarquinius, and two

unidentified species formed a morphologically distinct mono-

phyletic group. These taxa could have been assigned to the

subgenus Anteliomys yet recognition of a new subgenus for-

mally united these taxa and the corresponding taxonomy

more finely reflected phyletic history. Thus, we found it

desirable to erect a new subgenus, whose diagnostic traits

and content are as follows:

Systematic account

Family: Cricetidae

Subfamily: Arvicolinae

Genus: Eothenomys

Subgenus: Ermites Liu, Liu, Guo, Sun, Murphy, Fan, Fu

and Zhang subgen. nov.

Type species: Eothenomys hintoni (as published in the

trinomen Eothenomys custos hintoni Osgood, 1932; herein

elevated to the rank of species)

Etymology.—The subgeneric name, masculine in gen-

der, is a patronym honoring Professor and Academician

Ermi Zhao, an internationally recognized herpetologist and

our teacher.

Diagnosis.—The palate is typical of Eothenomys. The

teeth are slender. Average skull length is less than 27.5 mm,

average head and body length is less than 115 mm. The tri-

angle of the first lower molar is confluent transversely. The

second upper molar lacks a second inner triangle. The third

upper molar is very complex, multivariate, and has more

than four inner and four outer angles. The tail is longer than

or equal to half of the body and head length.

This subgenus includes four species: Eothenomys

hintoni (= Eothenomys custos hintoni), E. tarquinius (=

Eothenomys chinensis tarquinius), and two undescribed

species (Figs. 2–4; E. sp. 1 and E. sp. 2).

Systematics of Oriental voles

Combined, the morphology of the first lower molar and

the matrilineal genealogy resolved the taxonomy of Oriental

voles as follows:

Caryomys Thomas, 1911

Caryomys inez (Thomas, 1908) [type species]

Caryomys eva (Thomas, 1911)

Eothenomys Miller, 1896

E. (Anteliomys)

E. (A.) chinensis (Thomas, 1891) [type species]

E. (A.) olitor (Thomas, 1911)

E. (A.) custos (Thomas, 1912)

E. (A.) proditor Hinton, 1923

E. (Ermites) subgen. nov.

E. (Er.) tarquinius (Thomas, 1912)

E. (Er.) hintoni Osgood, 1932 [type species]

E. (Er.) sp. 1

E. (Er.) sp. 2

E. (Eothenomys) Miller, 1896

E. (Eo.) melanogaster (Milne-Edwards, 1871) [type

species]

E. (Eo.) eleusis (Thomas, 1911) [species inquirenda]

E. (Eo.) miletus (Thomas, 1914) [species inquirenda]

E. (Eo.) cachinus (Thomas, 1921) [species inquirenda]

E. (Eo.) fidelis Hinton, 1923 [species inquirenda]

Because of a lack of specimens, the status of Eothenomys

wardi was not investigated.

DISCUSSION

The different datasets and methods of tree construction

yield a highly resolved, robust matrilineal genealogy and we

take this to be reflective of the phylogeny. Most nodes are

highly supported. In most analyses, the Oriental voles clus-

ter together with strong support. Morphological structure cor-

responds with the matrilineal genealogy.

Historically, Oriental and Japanese red-backed voles

are placed in the genus Eothenomys (sensu lato) (Corbet,

1978; Corbet and Hill, 1986). Using a molecular phylogeny,

Luo et al. (2004) suggested that Japanese red-backed voles

should be removed from Eothenomys and placed in the

genus Phaulomys, as initially defined by Thomas (1905b).

Subsequently, Musser and Carleton (2005) assigned

Japanese red-backed voles to Myodes. A detailed discus-

sion of the classification of Japanese red-backed voles is

beyond the scope of our work, but our results strongly sup-

port the exclusion of Japanese red-backed voles from the

Oriental voles. Additionally, our molecular phylogeny sug-

gests that the genus Myodes, as defined presently, is not a

monophyletic group. Here too, further work is needed.

The systematics of Oriental voles is fraught with differing

opinions. Validity of the genera and subgenera remains con-

tentious. Luo et al. (2004) considered Eothenomys and

Anteliomys as subgenera of genus Eothenomys. Although

their work did not include Caryomys, they recognized the

genus and suggested it might be the sister-group of

Eothenomys. Our analysis, with more extensive samples

and data from two genes, resolves the historical associa-

S. Liu et al.620

tions of the Oriental voles and confirms their prediction. The

group contains four distinct lineages and the nodes generally

receive high support values. These four lineages correspond

to the genera Caryomys and Eothenomys (sensu stricto), the

latter with the subgenera Eothenomys, Anteliomys, and

Ermites. Morphologically, all species of Caryomys possess

inter-bedded molar triangles on the first and second lower

molars; in Eothenomys, triangles on the first lower molar are

confluent transversely. The upper molars of subgenus

Eothenomys have three outer and four inner salient angles

and the first upper molars of subgenus Anteliomys have

three outer and three inner salient angles. In both subgen-

era, TL is less than half of HBL, except for E. chinensis. In

this exception, TL is greater than 60 mm, more than half of

the HBL, and the skull length averages more than 29 mm.

Finally, subgenus Ermites is diagnosed by the following

characteristics: first upper molars have three outer and three

inner salient angles, but the third upper molar is variable but

has more than four inner and four outer angles; TL is equal

to or longer than half of HBL; average skull length is less

than 27.5 mm; and HBL averages less than 115 mm (Table

2).

The interspecific relationships of subgenus Ermites are

well resolved. Traditionally, E. hintoni and E. tarquinius are

assigned to subgenus Anteliomys, but as subspecies of

either Eothenomys custos or E. chinensis (Ellerman and

Morrison-Scott, 1951; Corbet, 1978; Honacki et al., 1982;

Corbet and Hill, 1986; Musser and Carleton, 1993, 2005;

Nowak, 1999; Luo et al., 2000; Wang, 2003). However, our

genealogy does not cluster E. hintoni and E. tarquinius

together with E. custos and E. chinensis, respectively, and

they appear in different lineages (Figs. 2–4). Consequently,

we recognize E. hintoni and E. tarquinius as valid species

and assign them to subgenus Ermites.

Subgenus Anteliomys has a strongly supported sister

relationship with Ermites, in most cases. Anteliomys con-

tains four species. Individuals of the same species cluster

together with strong support and the interspecific relation-

ships are also well resolved. Yang et al. (1998) concluded

that karyologically the Yulong vole (E. proditor) substantially

differs from allied voles and they suggested further study

was required to determine the status of E. olitor and E.

proditor. Our molecular analyses obtain very high support

values for the group. Traditionally, E. wardi is placed in sub-

genus Anteliomys. Some systematists recognize this taxon

as a subspecies of E. chinensis (Allen, 1940; Ellerman and

Morrison-Scott, 1951; Corbet, 1978; Musser and Carleton,

1993), but others regard it as a valid species (Hinton, 1926;

Ellerman, 1941; Corbet and Hill, 1992; Kaneko, 1996;

Nowak, 1999; Luo et al., 2000; Wang, 2003; Musser and

Carleton, 2005). Unfortunately, the absence of samples of E.

wardi precludes us from making any taxonomic inference.

Subgenus Eothenomys is the most complex group of

Oriental voles. Often, this subgenus is considered to contain

five species: E. melanogaster, E. cachinus, E. eleusis, E.

fidelis, and E. miletus. Frequently the latter four taxa are

considered to be subspecies of E. melanogaster (Ellerman

and Morrison-Scott, 1951; Corbet, 1978; Honacki et al.,

1982; Corbet and Hill, 1986; Musser and Carleton, 1993).

Nowak (1999) accorded E. melanogaster and E. miletus

species status. Musser and Carleton (2005) recognized four

species, and Wang (2003) accepted five species. Our molec-

ular analyses unite four species (E. cachinus, E. eleusis, E.

fidelis, and E. miletus) exclusive of E. melanogaster.

Whereas all individuals of E. melanogaster and E. fidelis

cluster together as discrete lineages, and while noting that

E. fidelis has a very small sample size, individuals of E.

cachinus, E. eleusis, and E. miletus are intermixed in the

matrilineal genealogy (Figs. 2–4). Incomplete lineage sort-

ing, historical hybridization, and potential conspecificity all

provide possible explanations for the pattern. In order to test

these possibilities, further study involving more intensive

sampling and nuclear genes is required. Irrespective of the

above, our study does not support the proposals of Musser

and Carleton (2005), which recognize eleusis as a subspe-

cies of E. melanogaster.

Genus Caryomys is comprised of two species: C. inez

and C. eva. Recognition of these two species is well sup-

ported.

DNA barcoding reflects the morphological results. Spe-

cies in the subgenera Ermites and Anteliomys, and genus

Caryomys, are unambiguously identified. Within subgenus

Eothenomys, DNA barcoding also fails to segregate E.

cachinus and E. eleusis; the inability to sequence multiple

specimens of E. miletus precludes a comparison of the COI

and cyt b data, in which the latter gene fails to diagnose this

taxon. Regardless, DNA barcoding appears to be an effi-

cient approach to identifying unambiguous species as well

as being an efficient way to discern where additional

research is required.

In China, most species of Oriental voles are endemic to

Yunnan and Sichuan. They occur in the southern part of the

Hengduan Mountains. This region was severely affected by

several orogenic events associated with the Qinghai-Tibetan

Plateau (Luo et al., 2004). The most intense and frequent

orogenic events, known as the Qingzang Movement (Zheng

et al., 2000; An et al., 2001), are dated at between 3.6 and

1.7 million years ago (Ma). Subsequent events, termed the

Kun-Huang Movement (1.2–0.6 Ma; Jing et al., 2007), are

responsible for further shaping the geomorphology of this

area. These geological events likely isolated populations of

voles and in doing so drove speciation. Geological pro-

cesses greatly influence the divergence of species endemic

to this region (Luo et al., 2004; Yang et al., 2006; Zhang and

Jiang, 2006; Jin et al., 2008; Fan et al., 2009, 2011; Zhang et

al., 2010). Among Oriental voles, the subgenera Eothenomys

and Ermites occur in this region only. The Gonggashan

Mountains—the southern Hengduan Mountains in western

Sichuan: highest peak 7556 m—has the greatest concentra-

tion of Oriental voles with six species: Eothenomys

melanogaster, E. cachinus, E. eleusis, E. miletus, E. hintoni,

and E. tarquinius.

The oldest fossil of E. melanogaster is dated at 2.03 Ma

(Zheng, 1993), E. chinensis is dated at 1.8 Ma (Zheng,

1993), and Caryomys eva and C. inez are dated at 0.5 Ma

(Middle or Late Pleistocene) (Li and Xue, 1996). Caryomys

roots at the base of the genealogy yet the fossils are far

more recent than those of Eothenomys. Zheng (1993) pro-

poses that E. melanogaster lay at the center of diversifica-

tion of Oriental voles because extant specimens appear to

be identical to those from the Pleistocene. Our results do not

support his proposal because Oriental voles form the sister-

Phylogeny of Oriental Voles 621

group of Caryomys. However, the absence of fossil series

precludes a determination of the initial date of origin.

In order to identify the Oriental voles, a key to the gen-

era and subgenera is given as follows:

A. Triangles of the first lower molar ranged alternately .

........................................................................ Caryomys

B. Triangles of the first lower molar are confluent trans-

versely ..........................................................Eothenomys

a. The first upper molar with four inner triangles .....

.............................................. Subgenus Eothenomys

b. The first upper molar with three inner triangles

(a) Tail shorter or longer than half of the head and

body length (if tail is longer than half of the head and body

length, the body is very large, average head and body

length 120 mm, average skull length larger than 29 mm),

structure of the third upper molar non-variable ....................

............................................................ Subgenus Anteliomys

(b) Tail equal to or longer than half of the head and

body length (if tail is longer than half of the head and body

length, the body is medium, average head and body length

less than 115 mm, average skull length smaller than 27.5 mm),

structure of the third upper molar complex and variable......

..................................................................Subgenus Ermites

ACKNOWLEDGMENTS

This research was funded by the National Natural Science

Foundation of China (NSFC 30970330) and National Basic

Research Program of China (973 Project: 2007 CB109106) and to

R.W.M. by a Visiting Professorship for Senior International Scientists

from the Chinese Academy of Sciences and a NSERC Discovery

Grant (3148). R. Johnston (University of California, Los Angeles)

provided editorial assistance and R. Liao and J. Zhao assisted in

fieldwork. We thank S. Huang (Huangshan University) for help in

analyzing molecular data. Special thanks to J. H. Bai for drawing

the skulls and X. L. Jiang (Kunming Institute of Zoology) for provid-

ing some specimens.

REFERENCES

Allen GM (1912) Some Chinese vertebrates. Mammalia. Mem Mus

Comp Zool Harvard College 59: 1–14

Allen GM (1924) Microtinae collection by the American Museum

Asian Expeditions. Amer Mus Novi 133: 1–13

Allen GM (1940) The Mammals of China and Mongolia. Natural

History of Central Asia. Vol. XI. The American Museum of Natu-

ral History, New York

An Z, John EK, Warren LP, Stephen CP (2001) Evolution of Asian

monsoons and phased uplift of the Himalaya-Tibetan plateau

since Late Miocene times. Nature 411: 62–66

Burbrink FT, Lawson R, Slowinski JB (2000) Mitochondrial DNA

phylogeography of the polytypic North American rat snake

(Elaphe obsoleta): a critique of the subspecies concept. Evolu-

tion 54: 2107–2118

Buzan EV, Krystufek B, Hänfling B, Hutchinson WF (2008) Mito-

chondrial phylogeny of Arvicolinae using comprehensive taxo-

nomic sampling yields new insights. Biol J Linn Soc 94: 825–

835

Cabrera A (1922) Sobre algunos mammiferos de la China oriental.

Bol R Soc Esp Hist Nat 22: 162–170

Corbet GB (1978) The Mammals of the Palaearctic Region: A Taxo-

nomic Review. British Museum (Natural History), London and

Cornell University Press, Ithaca, NY

Corbet GB, Hill JE (1986) A world list of mammalian species. Sec-

ond edition. British Museum (Natural History), London

Corbet GB, Hill JE (1992) Mammals of Indomalayan Region. A Sys-

tematic Review. Oxford University Press, Oxford

Delisle I, Strobeck C (2002) Conserved primers for rapid sequenc-

ing of the complete mitochondrial genome from carnivores,

applied to three species of bears. Mol Biol Evol 19: 358–361

Ellerman JR (1941) The Families and Genera of Living Rodents.

British Museum (Natural History), London

Ellerman JR, Morrison-Scott TCS (1951) Checklist of Palaearctic

and Indian Mammals 1758 to 1946. British Museum (Natural

History), London

Fan ZX, Liu SY, Liu Y, Zeng B, Zhang XY, Cuo C, et al. (2009)

Molecular phylogeny and taxonomic reconsideration of the sub-

family Zapodiane (Rodentia: Dipodidae), with an emphasis on

Chinese species. Mol Phylogenet Evol 51: 447–453

Fan ZX, Liu SY, Liu Y, Zhang XY, Yue BS (2011) How Quaternary

geologic and climatic events in the southeastern margin of the

Tibetan Plateau influence the genetic structure of small mam-

mals: inferences from phylogeography of two rodents, Neodon

irene and Apodemus latronum. Genetica 139: 339–351

Felsenstein J (1985) Confidence limits on phylogenies: an approach

using the bootstrap. Evolution 39: 783–791

Frost DR, Hillis DM (1990) Species concepts in practice: herpetolog-

ical applications. Herpetologica 46: 115–128

Gannon WL, Sikes RS, the Animal Care and Use Committee of the

American Society of Mammalogists (2007) Guidelines of the

American Society of Mammalogists for the use of wild mam-

mals in research. J Mammal 88: 809–823

Gromov IM, Polyakov IYa (1977) Fauna of the USSR Mammals,

Vol. III, No. 8, Voles (Microtinae). English Edition: 1992, Douglas

Siegel-Causey and Robert S. Hoffmann. Smithsonian Institution

Libraries and the National Science Foundation, Washington DC

Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological

identifications through DNA barcodes. Proc Royal Soc London

B 270: 313–721

Hinton MAC (1923) On the voles collected by Mr. G. Forrest in

Yunnan, with remarks upon the genera Eothenomys and

Neodon and upon their allies. Ann Mag Nat Hist, Ser. 9, 6: 146–

163

Hinton MAC (1926) Monograph of the Voles and Lemmings

(Microtinae) Living and Extinct. British Museum (Natural History),

London

Honacki JH, Kinman KE, Koeppl JW (1982) Mammal Species of the

World. Published as a Joint Venture of Allen Press, Inc. and the

Association of Systematics Collection, Lawrence, Kansas

Huelsenbeck JP, Crandall KA (1997) Phylogeny estimation and

hypothesis testing using maximum likelihood. Ann Rev Ecol

Syst 28: 437–466

Huelsenbeck JP, Ronquist FR (2001) MrBayes: Bayesian inference

of phylogenetic trees. Bioinformatics 17: 754–755

Imaizumi Y (1957) Taxonomic studies on the red-backed vole from

Japan. Pt. 1, major divisions of the vole and description of

Eothenomys with a new species. Bull Nat Sci Mus 3: 195–216

Jin YT, Brown RP, Liu NF (2008) Cladogenesis and phylogeography

of the lizard Phrynocephalus vlangalii (Agamidae) on the

Tibetan plateau. Mol Ecol 17: 1971–1982

Jing MD, Yu HT, Wu SH, Wang W, Zheng XG (2007) Phylogenetic

relationships in genus Niviventer (Rodentia: Muridae) in China

inferred from complete mitochondrial cytochrome b gene. Mol

Phylogenet Evol 44: 521–529

Kaneko Y (1996) Morphological variation, and latitudinal and altitudi-

nal distribution of Eothenomys chinensis, E. wardi, E. custos, E.

proditor, and E. olitor (Rodentia, Arvicolidae) in China. Mam

Study 21: 89–114

Kimura M (1980) A simple method for estimating evolutionary rates

of base substitutions through comparative studies of nucleotide

sequences. J Mol Evol 16: 111–120

Li CL, Xue XX (1996) Biogeography and the age of the fossil

rodents fauna from Xishuidong Nantian, Shaanxi. Vertebrata

S. Liu et al.622

Pal Asiatica 34: 156–162

Liu SY, Sun ZY, Zeng ZY, Zhao EM (2007) A new species

(Proedomys: Aricolinae: Murida) from Sichuan province, China.

J Mammal 88: 1170–1178

Luo J, Yang DM, Hitoshi S, Wang YX, Chen WJ, Campbell KL, et al.

(2004) Molecular phylogeny and biogeography of Oriental voles:

genus Eothenomys (Muridae, Mammalia). Mol Phylogenet Evol

33: 349–362

Luo ZX, Chen W, Guo B (2000) Fauna Sinica. Mammalia, Vol. 6,

Rodentia, Part III: Cricetidae. Scientific Publisher, Beijing

Ma Y, Jiang JQ (1996) The reinstatement of the status of genus

Caryomys (Rodentia: Microtinae). Acta Zootax Sinica 21: 493–

497

Miller GS (1896) Genera and subgenera of voles and lemmings. N

Amer Fauna 12: 1–86

Miller GS (1898) Description of a new genus and species of micro-

tine rodent from Siberia. Proc Acad Nat Sci Philadelphia 50:

368–371

Milne-Edwards A (1871) Note sur une nouvelle espece de semnopi-

theque. Nouv Arch Mus Hist Paris Bull 7: 93

Murphy RW, Méndez de la Cruz FR (2010) The herpetofauna of

Baja California and its associated islands: a conservation

assessment and priorities. In “Conservation of Mesoamerican

Amphibians and Reptiles” Ed by LD Wilson, JH Townsend and

JD Johnson, Eagle Mountain Publishing, LC, Eagle Mountain,

Utah, pp 238–273

Musser GG, Carleton MD (1993) Family Muridae. In “Mammal Spe-

cies of the World: a Taxonomic and Geographic Reference” Ed

by DE Wilson and DM Reeder, 2nd ed, Smithsonian Institution

Press, Washington, DC, pp 501–535

Musser GG, Carleton MD (2005) Family Cricetidae. In “Mammal

Species of the World: a Taxonomic and Geographic Reference”

Ed by DE Wilson and DM Reeder, 3rd ed, The Johns Hopkins

Press, Baltimore, MD, pp 955–1039

Nowak RM (1999) Walker’s Mammals of the World. 6th ed, The

Johns Hopkins University Press, Baltimore and London

Nowak RM, Paradiso JL (1983) Walker’s Mammals of the World. 4th

ed, The Johns Hopkins University Press, Baltimore and London

Osgood WH (1932) Mammals of the Kelley-Roosevelts and Dela-

cour Asiatic Expeditions. Field Mus Nat Hist Zool 18: 193–339

Posada D, Crandall KA (1998) ModelTest: testing the model of DNA

substitution. Bioinformatics 14: 817–818

Posada D, Crandall KA (2001) Selecting the best-fit model of nucle-

otide substitution. Syst Biol 50: 580–601

Rambaut A, Drummond AJ (2003) Tracer v1.3. Available from http://

evolve.zoo.ox.ac.uk

Robovský J, Ra Ánková V, Zrzavý J (2008) Phylogeny of Arvicolinae

(Mammalia, Cricetidae): utility of morphological and molecular

datasets in a recently radiating clade. Zool Scripta 37: 571–590

Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylo-

genetic inference under mixed models. Bioinformatics 19:

1572–1574

Sambrook J, Russell DW (2002) Molecular Cloning: A Laboratory

Manual. 3rd ed, Cold Spring Harbor Laboratory Press

Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap

algorithm for the RAxML web-servers. Syst Biol 75: 758–771

Steppan SJ, Adkins RM, Anderson J (2004) Phylogeny and diver-

gence-date estimates of rapid radiations in muroid rodents

based on multiple nuclear genes. Syst Biol 53: 533–553

Swofford D (2003) PAUP*: Phylogenetic Analysis Using Parsimony

and Other Methods. version 4.0, Sinauer, Sunderland,

Massachusetts

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S

(2011) MEGA5: Molecular Evolutionary Genetics Analysis

using maximum likelihood, evolutionary distance, and maxi-

mum parsimony methods. Mol Phylogenet Evol 28: 2731–2739

Thomas O (1891) Description of a new vole from China. Ann Mag

Nat Hist Ser 6, 8: 117–118

Thomas O (1905a) The Duke of Bedford’s zoological exploration in

eastern Asia.—1. List of mammals obtained by Mr. M. P.

Anderson in Japan. Proc Zool Soc Lond 1906 (2): 331–363

Thomas O (1905b) On some new Japanese mammals presented to

the British Museum by Mr. R. Gordon Smith. Ann Mag Nat Hist

Ser 7, 15: 487–495

Thomas O (1907) The Duke of Bedford’s zoological expedition in

eastern Asia. List of small mammals from Korea and Quelpart.

Proc Zool Soc Lond (1906): 863–865

Thomas O (1908a) Mammals collected in the provinces of Shan-si

and Shen-si, Northern China. Abstr Proc Zool Soc Lond 63: 44–

45

Thomas O (1908b) List of mammals from the provinces Chih-li and

Shan-si northern China. Proc Zool Soc Lond 1908: 635–646

Thomas O (1910) Exhibition of mammals from China. Abstr Proc

Zool Soc Lond 83: 25–26

Thomas O (1911a) Mammals in west of China. Abstr Proc Zool Soc

Lond 100: 50

Thomas O (1911b) Mammals collected in the province of Kan-su

and Sze-chwan, western China, by Mr. Malcolm Anderson, for

the Duke of Bedford’s exploration of Eastern Asia. Abstr Proc

Zool Soc Lond 90: 3–5

Thomas O (1912) On insectivores and rodents collected by Mr. F.

Kingdom Ward in Northwest Yunnan. Ann Mag Nat Hist Ser 8,

9: 513–519

Thomas O (1914) Second list of small mammals from western Yunnan

collected by Mr. F. Kingdom Ward. Ann Mag Nat Hist, Ser. 8,

14: 472–475

Thomas O (1921) On small mammals from the Kachin Province,

northern Burma. J Bombay Nat Hist Soc 27: 499–505

Tokuda M (1937) The alpine Muridae from Formosa. Bot Zool Tokyo

5: 1115–1122

Wang YX (2003) A Complete Checklist of Mammal Species and

Subspecies in China: A Taxonomic and Geographical Refer-

ence. China Forestry Publishing House, Beijing

Yang DM, Liu RQ, Zhang YP, Chen ZP, Wang YX (1998) Chromo-

some study of Yulong vole (Eothenomys proditor). Cytologia

65: 435–440

Yang SJ, Yin ZH, Ma XM, Lei FM (2006) Phylogeography of ground

tit (Pseudopodoces humilis) based on mtDNA: Evidence of

past fragmentation on the Tibetan Plateau. Mol Phylogenet

Evol 41: 257–265

Ye XD, Ma Y, Zhang JS, Wang CL, Wang ZK (2002) A summary of

Eothenomi (Rodentia: Cricetidae: Microtinae). Acta Zootax

Sinica 27: 173–182

Zhang DR, Chen MY, Murphy RW, Che J, Pang JF, JS Hu, et al.

(2010) Genealogy and palaeodrainage basins in Yunnan Prov-

ince: phylogeography of the Yunnan spiny frog, Nanorana

yunnanensis (Dicroglossidae). Mol Ecol 19: 3406–3420

Zhang DX, Hewitt GM (1996) Nuclear integrations: challenges for

mitochondrial DNA markers. Trends Ecol Evol 11: 247–251

Zhang FF, Jiang ZG (2006) Mitochondrial phylogeography and

genetic diversity of Tibetan gazelle (Procapra picticaudata):

implications for conservation. Mol Phylogenet Evol 41: 313–321

Zheng HB, Powell CM, An ZS, Dong GR (2000) Pliocene uplift of the

northern Tibet Plateau. Geology 8: 715–718

Zheng SH (1993) Quaternary Rodents of Sichuan and Guizhou

Area, China. Science Press, Beijing

(Received April 4, 2011 / Accepted February 28, 2012)