Evolutionary History of Field Mice (Murinae: Apodemus), With

Zoological Journal of the Linnean Society, 2019, 187, 518–534. With 6 figures.

Evolutionary history of field mice (Murinae: Apodemus), Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 with emphasis on morphological variation among species in China and description of a new species

DEYAN GE1, , ANDERSON FEIJÓ1, , JILONG CHENG1, , LIANG LU2, RONGRONG LIU2, ALEXEI V. ABRAMOV3,4, LIN XIA1, ZHIXIN WEN1, WEIYONG ZHANG5, LEI SHI5 and QISEN YANG1*

1Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing 100101, China 2State Key Laboratory for Infectious Diseases Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206, China 3Zoological Institute, Russian Academy of Sciences, Saint Petersburg 199034, Russia 4Joint Russian–Vietnamese Tropical Research and Technological Centre, Hanoi, Vietnam 5Fanjingshan National Nature Reserve, Tongren, 554400, China

Received 22 January 2019; accepted for publication 25 March 2019

Mice of the genus Apodemus are widely distributed across Eurasia. Several species of this genus are hosts of important zoonotic diseases and parasites. The evolutionary history and dispersal routes of these mice remain unclear and the distribution of these species in China was poorly explored in previous studies. We here investigate the divergence times and historical geographical evolution of Apodemus and study the taxonomy of species in China by integrating molecular and morphological data. The crown age of this genus is dated to the Late Miocene, approximately 9.84 Mya. Western and Central Asia were inferred as the most likely ancestral area of this genus. Moreover, we recognize nine living species of Apodemus in China: Apodemus uralensis, A. agrarius, A. chevrieri, A. latronum, A. peninsulae, A. draco, A. ilex, A. semotus and A. nigrus sp. nov., the last from the highlands (elevation > 1984 m) of Fanjing Mountain in Guizhou Province and Jinfo Mountain in Chongqing Province. This new species diverged from A. draco, A. semotus and A. ilex approximately 4.53 Mya. The discovery of A. nigrus highlights the importance of high mountains as refugia and ‘isolated ecological islands’ for temperate species in south-eastern China.

KEYWORDS: divergence times – hantavirus – Ljungan virus – molecular clock – molecular phylogeny – mountains – new species – pathogen vectors – refugia.

INTRODUCTION A. sylvaticus (Linnaeus, 1758) in northern Africa (Libois et al., 2001) and A. agrarius (Pallas, 1771) Apodemus Kaup, 1829 is widely distributed across on the Danish islands Lolland and Faster (Andersen Eurasia and extends into small areas of the et al., 2017), which is not typical for mice of the genus northernmost part of Africa (Musser et al., 1996; Apodemus. More than 100 species or subspecies of this Musser & Carleton, 2005). The recent distributional genus have been established in the historical literature range expansion of several species in this genus is a (Thomas, 1922; Allen, 1938; Ellerman & Morrison- result of anthropogenic introductions. For example, Scott, 1951; Xia, 1984; Musser et al., 1996; Musser & Carleton, 2005). Species of Apodemus are most *Corresponding author. Email: [email protected] abundant in the broadleaf forests of Palaearctic and [Version of Record, published online 9 July 2019; Oriental areas. Several of these species host multiple http://zoobank.org/urn:lsid:zoobank.org:pub:0122DEEF- human pathogens (Klein et al., 2015; Ma et al., 2015), 3F68-4D2F-A119-378D8C4CA5CF] notably hantavirus (Guzzetta et al., 2017; Tian et al.,

2017) and Ljungan virus (Hauffe et al., 2010). A study variability of field mice was found in the Mediterranean on the evolutionary history and dispersal of this peninsulas than in northern Europe (Michaux et al., genus is, therefore, important for public health and 2003). Moreover, a large number of fossil species from biodiversity conservation in Eurasia and North Africa. Europe were described that were absent from glacial Rapid radiation of Apodemus during the Late assemblages, but are always present in interglacial Miocene is thought to have been associated with global assemblages (Martin Suarez & Mein, 1998; Knitlova Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 forest changes, when the flora changed from tropical & Horacek, 2017a, b). However, it is unclear how the to temperate during the Late Miocene (Serizawa et al., evolutionary history of Apodemus formed its current 2000). The earliest fossil records of this genus and its distributional pattern. sister genera were dated to the early Late Miocene, Faunas in many regions of China remain relatively approximately 11 Mya (Freudenthal, 1976; Martin poorly explored, impeding a comprehensive Suarez & Mein, 1998; Kimura et al., 2017), but based understanding of interspecific differentiation. on molecular data, the earliest divergence time of this Allen (1938) recognized five species of Apodemus genus was estimated to be approximately 6–8 Mya in China and Xia (1984) listed six: A. sylvaticus, A. (Michaux et al., 2002; Liu et al., 2004; Suzuki et al., 2008; draco, A. peninsulae, A. latronum, A. chevrieri and Darvish et al., 2015). Three crucial evolutionary events A. agrarius. Zheng (1993) reported fossil occurrences of this genus were recognized in previous studies: (1) of A. chevrieri, A. agrarius, A. sylvaticus and A. cf. initial broad dispersal and radiation approximately peninsulae that were dated to the Late Pleistocene in 6 Mya, (2) regional radiation in Europe and China Chongqing and Guizhou Provinces. Based on a large approximately 2 Mya and (3) westward dispersal number of Apodemus specimens from China, Musser of A. agrarius to Europe in the Quaternary (Suzuki et al. (1996) recognized seven species, A. agrarius, et al., 2008). These conclusions were proposed without A. chevrieri, A. peninsulae, A. latronum, A. draco, A. evidence from fossil information or robust statistical semotus and A. uralensis, but excluded A. sylvaticus. analyses. A comprehensive study including extant Smith & Xie (2008) included one more species, A. and fossil species is important for understanding the pallipes (Barrett-Hamilton, 1900), than the study of evolutionary history of this genus. Musser et al. (1996), but this number was not followed According to the most comprehensive mammalian by Wilson et al. (2016), detailed information are given checklists, the composition of Apodemus is still in Table 1. In recent studies, A. ilex is also considered disputable. Mammal species of the world and the a distinct species (Liu et al., 2004, 2012, 2017). Handbook of the mammals of the world listed 20 A previous study based on a wider range of sampling, species of Apodemus (Musser & Carleton, 2005; Wilson and using cytochrome oxidase subunit I (Cox1) et al., 2016). Traditional taxonomy divided Apodemus recognized A. agrarius, A. chevrieri, A. peninsulae, into three groups: the Apodemus group, the Sylvaemus A. latronum, A. ilex, A. draco and A. uralensis, and group and the Alsomys group (Zimmermann, 1962) identified a distinct genetic lineage from Guizhou or the Argenteus group (Musser et al., 1996). Studies Province (Liu et al., 2017). However, it is unclear how based on complete mitochondrial cytochrome b (Cytb) to identify these species based on morphology and a sorted Apodemus species into four groups or subgenera: detailed description of the distinct genetic lineage the Sylvaemus group [A. uralensis (Pallas, 1811), is lacking. An integrative study on the phylogeny of A. flavicolllis (Melchior, 1834), A. alpicola Heinrich, Apodemus and taxonomy of species in this genus in 1952, A. sylvaticus, A. mystacinus (Danford & Alston, China is, therefore, warranted. 1877), A. hermonensis (Filippucci et al., 1989)], the In recent years, we collected a large number Apodemus group [(A. agrarius, A. chevrieri (Milne- of samples of the genus Apodemus in China and Edwards, 1868), A. speciosus (Temminck, 1844), A. examined collections in several museums that draco (Barrett-Hamilton, 1900), A. ilex Thomas, 1922, preserve Chinese specimens. Here, our aim is to (1) A. semotus Thomas, 1908, A. latronum Thomas, 1911 investigate the evolutionary history of this taxon by and A. peninsulae (Thomas, 1907)] and A. argenteus integrating fossil occurrences and extant species, (2) (Temminck, 1844) and A. gurkha Thomas, 1924, use the broadest geographic coverage of Apodemus constitute two other distinct groups (Serizawa et al., to date, to assess the phylogenetic relationships and 2000; Liu et al., 2004; Suzuki et al., 2008). Generally, morphological variation of all species of this genus different aspects of species in Europe and eastern Asia recorded in China and (3) test whether the newly have been well studied. For example, genetic variation discovered genetic lineage in our previous study among species using protein electrophoresis suggested represents a distinct species. By integrating molecular a recent separation of members of the subgenus and morphological data, we established a new species Sylvaemus from a common ancestor, followed by rapid of the genus Apodemus from high mountains in south- radiation (Filippucci et al., 2002), and higher genetic eastern China (27.83ºN, 108.76ºE).

MATERIAL AND METHODS (Perkin-Elmer, Waltham, Massachusetts, USA) using an ABi PRISM BigDye Terminator Cycle Sequencing Compiling fossil occurrences Ready Reaction Kit with AmpliTaq DNA polymerase Fossil occurrences of Apodemus and its most closely (Applied Biosystems, Foster City, California, USA). related sister genus Parapodemus (Kimura et al.,

2017) were compiled from different resources. First, Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 we searched the Paleobiology database (PBDB, https:// Phylogenetic analyses of molecular data paleobiodb.org/classic), New and Old Worlds database We established three databases for phylogenetic of fossil mammals (NOW, http://www.helsinki.fi/ analyses. The first database included 206 individuals science/now/) and National infrastructure of mineral of Apodemus. Cytb, Cox1 and the D-loop (mitochondrial rock and fossil resources for science and technology DNA, mtDNA) were included in this analysis. Acomys of China (http://www.nimrf.net.cn/pub/msbz.jsp) to cahirinus (É. Geoffrey, 1803), Micromys minutus obtain basic fossil records. Second, we searched the (Pallas, 1771), Bandicota indica (Bechstein, 1800), Zoological Record database (from 1864 to 2018) of Rattus norvegicus (Berkenhout, 1769), Berylmys the Institute for Scientific Information (ISI) provided berdmorei (Blyth, 1851), Lenothrix canus Miller, 1903, by the Thomson Reuters Web of Knowledge (http:// Niviventer andersoni (Thomas, 1911) and N. fulvescens login.webofknowledge.com/) to add information (Gray, 1847) were used as outgroup taxa in this dataset. that was missing in PBDB. Martin Suarez & Mein The second database included 214 sequences of Rbp3, (1998) comprehensively revised both fossil and and four sequences from the genus Rattus, three extant species of Apodemus and closely related sister sequences from the genus Mus and one sequence each genera, providing a guideline with which to compile from Micromys and Tokudaia were used as outgroups. fossil occurrences of this genus. Moreover, Kawamura The third dataset was derived from the first dataset (1989) compiled a review on the fossil occurrences of but included only one individual for each species Apodemus in China and Japan, which greatly helped of Apodemus. Information for all samples in these us review historical occurrences of this genus in Asia. datasets is provided in Supporting Information, Table Duplicate occurrences of the same species from the S1. same locality in the same historic period were removed All three datasets were aligned by MUSCLE as from this database. implemented in MEGA6 (Tamura et al., 2013). To reduce the occupation of computing resources, we used DAMBE v.6.4.47 (Xia, 2017) to delete duplicated Sampling and sequencing of extant species sequences. We used PartitionFinder v.2.1.1 (Lanfear In the past ten years, we collected more than 2000 et al., 2017) to select the best subset settings in individuals of the genus Apodemus in China. These Bayesian inferences. We performed Bayesian field studies were conducted by the Research Group of phylogenetic analyses of the dataset using MrBayes Mammalogy, Key Laboratory of Zoological Systematics v.3.1.2 (Ronquist et al., 2012) with four independent and Evolution, Institute of Zoology, Chinese Academy runs of each dataset by one cold and three heated of Sciences (IOZCAS) and the National Institute for Markov chains of 80 million steps. Trees were sampled Communicable Disease Control and Prevention, every 1000 steps, and the first 25% were discarded as Chinese Center for Disease Control and Prevention a burn-in. Sequence divergence between species in (ICDC). We selected 174 individuals to represent China was calculated as the uncorrected p-distance different species of the genus Apodemus in China based on Cytb and Cox1 sequences in MEGA6, since and sequenced four DNA fragments from these these two DNA fragments have frequently been used samples: Cytb, Cox1, the D-loop of the mitochondrial as DNA barcodes in mammals. genome (D-loop) and the first exon of the nuclear interphotoreceptor retinoid binding protein (Rbp3). The total genomic DNA of these samples was isolated Inferring divergence time among species from muscle or liver tissue using a Tiangen blood, We estimated the divergence times of species in tissue and cell DNA isolation kit (Tiangen Biotech Apodemus using the Bayesian phylogenetic approach Co., Ltd, Beijing). Primer sequences used for PCR in BEAST v.1.8.2 (Drummond & Rambaut, 2007). For and sequencing and their original references were as this analysis, we used the combined dataset of three follows: L14723 and H15915 for Cytb (Irwin et al., 1991), mitochondrial markers, which included almost all BatL5310 and R6036R for Cox1 (Robins et al., 2007), extant species of Apodemus. Two fossil calibration EGL4L and RJ3R for the D-loop (Robins et al., 2007) points were used: (1) the basal split in Murinae (12 and IRBP_F and IRBP_R for Rbp3 (Ge et al., 2018a). Mya), which has frequently been used as a fossil All PCR products were directly sequenced in both calibration point in studies of mammals (Johnson et al., directions with an ABi 3730XL automatic sequencer 1985; Barry et al., 2002) and (2) a calibration point for

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 518–534 EVOLUTIONARY HISTORY OF FIELD MICE 521 the Apodemus/Tokudaia split (10.5 Mya), based on length (BL), basilar length (BSL), palatal length (PL), the recent study of Kimura et al. (2017). A Yule prior incisive foramen length (IFL), width of the incisive was used for the tree, and an uncorrelated lognormal foramen (WIF), greatest palatal breadth (GPB), relaxed clock was used to model rate variation among length of the tympanic bulla (LTB), length of the lineages (Drummond et al. 2006). We estimated maxillary molars (ULMM), length of the maxillary posterior distributions of parameters by Markov chain diastema (ULMD), mandibular length (ML), length Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 Monte Carlo (MCMC) sampling, with samples drawn of the mandibular molars (LLMM) and length of the every 1000 steps over 80 million steps. We ran four mandibular diastema (LLMD), were obtained by using chains and discarded the first 25% of the samples a Vernier calliper. The locations of these CMs are as a burn-in. Sufficient sampling was confirmed in illustrated in the Supporting Information (Fig. S1). TRACER v.1.5 (Drummond & Rambaut, 2007). Craniodental differences among taxa were assessed by principal component analysis (PCA) and linear discriminant analysis (LDA) of the original linear Ancient distribution reconstruction measurements. Multivariate analyses of variance To avoid unbalanced sampling among species, we (MANOVAs) were conducted with the PCA and LDA kept one individual for each species of Apodemus scores to test the overall morphological differentiation and ran separate Bayesian inferences by using the among species. All statistical analyses were conducted concatenated dataset of mtDNA (the third dataset). in Paleontological Statistics (PAST) software v.3.16 The current distribution of Apodemus was divided (Hammer et al., 2001). into (a) South Asia, (b) North-east Asia, (c) Europe and (d) Western and Central Asia, as illustrated in Fig. 3B. Widespread species were coded as present in RESULTS multiple regions. Analyses of the potential ancestral distribution of Apodemus were implemented in Fossil occurrences of Apodemus and Reconstruct Ancestral State in Phylogenies (RASP) Parapodemus v.3.2 (Yu et al., 2015). Trees obtained from the Bayesian Almost all fossils of Parapodemus were dated to the analyses of the third dataset mentioned above were Late Miocene, with those discovered in the Nagri used as phylogenetic uncertainties. Bayesian MCMC Formation, Siwalik Group and Potwar Plateau, analyses were performed to test the accuracy and Pakistan, considered the oldest in most recent studies stability of our inferences. (Kimura et al., 2017). Based on the data from the PBDB, one fossil occurrence of A. gudrunae Van De Weerd, 1976 unearthed from Casablanca M, Castellon, Spain, was Morphological comparison and statistical dated to 23.5 Mya. However, this date was dramatically analyses older than that obtained in all former studies. Multiple We compared external and craniodental morphology fossils of Apodemus unearthed from Casablanca among genetic lineages recovered from the above- M, Castellon, Spain, were dated to approximately described phylogenetic analyses. External morphology 11.06 Mya (Supporting Information, Table S2). This included pelage coloration, body weight (BW), head and dating appears more reasonable. Fossils of this genus body length (HBL), tail length (TL), hind foot length (HFL) unearthed from southern Europe and central China and ear length (EL). We tested pairwise morphological were mainly estimated to be from the Late Miocene differentiation in BW and external measurements (EMs) (Fig. 1A). A clear trend of northward expansion during by using analyses of variance (ANOVAs). the Pliocene is detected (Fig. 1B). This trend is clearer We examined holotypes of A. semotus (NHM 8.4.1.48), from the Pliocene to the Pleistocene and Holocene. The A. draco (NHM 98.11.1.20), A. ilex (NHM 22.9.1.122) range expanded to islands in northern Europe, North and A. peninsulae (NHM 6.12.6.45), which are Africa, Syria and Japan, and Korea in northern Asia. preserved in the Natural History Museum of London. Detailed information for literature that reported fossil Moreover, we examined large numbers of molecular occurrences of the genera Parapodemus and Apodemus voucher specimens for the present study, which are is provided in the Supporting Information (File S1). preserved in the mammalian collection, IOZCAS, Beijing. In total, 117 intact adult specimens were selected to represent species of the genus Apodemus Phylogeny, divergence time and ancestral in China. Eighteen craniodental measurements (CMs), centre of Apodemus namely, the total length of the cranium (TLC), nasal Bayesian analyses of the concatenated dataset of length (NL), greatest width of the ‘snout’ (GWS), mtDNA clusters our newly sequenced samples from shortest distance between orbits (SDO), zygomatic China into eight monophyletic groups (Fig. 2), among breadth (ZB), greatest mastoid breadth (GMB), basal which A. uralensis is placed close to A. agrarius.

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Figure 1. Fossil occurrences of Apodemus and Parapodemus and the current distribution of extant species. A, fossils of Parapodemus. B, fossils of Apodemus dated in the Miocene. C, fossils of Apodemus dated in the Pliocene (purple) and from the Pleistocene to recent (yellow).

Apodemus chevrieri clusters with A. speciosus to samples from the Fanjing and Jinfo mountains were form a well-supported clade. Apodemus latronum, A. collected above 2000 m elevation. The phylogenetic peninsulae, A. draco, A. ilex, A. semotus and samples structure of mtDNA is generally supported by Rbp3 from Fanjing Mountain, Jiangkou, Guizhou Province (Supporting Information, Fig. S2). However, A. draco, and Jinfo Mountain, Chongqing Province (A. nigrus sp. A. semotus and A. ilex form a paraphyletic lineage, and nov.; see taxonomic account in the following section), A. agrarius and A. chevrieri form another paraphyletic form another clade. Interestingly, A. nigrus appears to lineage based on this nuclear gene. The genetic be associated with high elevation, because most of the distances between species ranges from 0.167 ± 0.013 to

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Figure 2. Phylogeny and divergence time of the genus Apodemus reconstructed by using mtDNA. Posterior probabilities are given as node labels. Species distributed in China are marked by stars.

0.074 ± 0.007 based on Cytb and from 0.168 ± 0.014 to approximately 9.84 Mya [95% highest posterior density 0.069 ± 0.009 based on Cox1 (Table 2). confidence interval (HPD CI) = 11.23–8.50] (Fig. 2). Molecular dating based on mtDNA estimates the This result is generally in accordance with the earliest crown age of Apodemus to be during the Late Miocene, fossil records of this genus. Apodemus nigrus split from

Table 1. Known species and subspecies of the genus Apodemus in China

Species listed in Wilson Included subspecies or Type locality Current status et al., 2016 synonyms from China

A. agrarius (Pallas, 1771) A. a. agrarius (Pallas, 1771) Russia, Ulianovsk Obl., middle Volga distinct species Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 River, Ulianovsk A. a. pallidior Thomas, 1908 China, near Chefoo, Shantung (Shan- dong, Yantai) A. mantchuricus (Thomas, China, Manchuria 1898) A. a. ningpoensis (Swinhoe, China, Ningpo, Chekiang (Ningbo, Zhe- 1870) jiang) A. a. harti (Thomas, 1898) China, Fukien, Kuatun (Fujian, Guadun) A. insulaemus Tokuda, 1941 Formosa (Taiwan) A. chevrieri (Milne- A. chevrieri (Milne-Edwards, China, Sichuan, Moupin (Baoxing) distinct species Edwards, 1868) 1868) A. fergussoni Thomas, 1911 China, Wen-hsien, S. Kansu (Wenxian, Gansu) A. draco (Barrett- A. d. draco (Barrett-Hamilton, China, Fujian, Kuatun (Guadun) distinct species Hamilton, 1900) 1900) A. ilex Thomas 1922, China, Yunnan, Mekong–Salween distinct species synonym of A. draco divide A. restes Thomas, 1911 China, Sichuan, Omi (Emeishan) A. latronum Thomas, 1911 A. l. latronum Thomas, 1911 China, Sichuan, Tatsienlu (Kangding) distinct species A. l. lijiangensis Li and Liu China, Yunnan, Lijiang, Weixi 2014 A. peninsulae (Thomas, A. p. peninsulae (Thomas, 1907) Korea, 110 mi SE of Seoul, Mingyoung. distinct species 1907) A. p. praetor Miller, 1914 China, Manchuria, Sungari River, 60 mi. SW Kirin A. p.qinghaiensis Feng, Zheng China, Ledu, Qinghai and Wu, 1983 A. sowerbyi Jones, 1956 China, northern Shansi, 30 miles west of Kuei-hua-cheng, 7000 ft. A uralensis (Pallas, 1811) A. u. uralensis (Pallas, 1811) Russia, south Ural Mtns. distinct species A. pallipes (Barrett-Hamilton Afghanistan, Surhad Wahkan, Turk- 1900) estan (Badakhshan Province) A. semotus Thomas, 1908 No subspecies or synonyms Taiwan distinct species - A. nigrus Ge, Feijó and Yang, China, Guizhou, Fanjing Mountain distinct species 2019

A. draco, A. semotus and A. ilex approximately 4.53 Information, Tables S3 and S4. Apodemus chevrieri Mya (95% HPD CI = 5.31–3.78) Fig. 2. Reconstructing is the largest species in China (BW = 36.3 ± 6.8, the ancient distribution of Apodemus reveals Western HBL = 102.7 ± 12.2), while A. uralensis is the smallest and Central Asia (98.72%) as the most likely centre of (BW = 19.5 ± 3.2, HBL = 85.3 ± 7.1). The first three origin of this genus (Fig. 3). axes of the PCA captures 59.62%, 7.05% and 6.58% of the overall morphological differentiation, respectively, and the first three axes of the LDA captures 40.2%, Morphological differentiation among Chinese 28.0% and 16.5% of the total variation. PCA and LDA species plots of the CMs clearly differentiate most species pairs The skin, cranium and tooth morphologies of major (Supporting Information, Fig. S3). MANOVAs of PCA species from China are given in Figures 4–6. Descriptive and LDA scores reveal significant divergence between values of EMs and CMs for different species are given species. Molecular voucher specimens of A. nigrus in Table 3 and the original data are given in Supporting have a distinct morphology when compared with all

Table 2. Genetic differentiation among Apodemus in China. Estimates of evolutionary divergence over sequence pairs between species (with standard errors) using Cytb are given in the lower diagonal, and those of Cox1 are given in the upper diagonal

AN AA AC AD AI AL AP AU Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 AN — 0.143 ± 0.014 0.146 ± 0.014 0.076 ± 0.009 0.084 ± 0.010 0.104 ± 0.011 0.138 ± 0.013 0.159 ± 0.014 AA 0.158 ± 0.012 — 0.069 ± 0.009 0.139 ± 0.013 0.131 ± 0.012 0.146 ± 0.013 0.138 ± 0.013 0.166 ± 0.014 AC 0.159 ± 0.012 0.074 ± 0.007 — 0.148 ± 0.013 0.149 ± 0.013 0.144 ± 0.013 0.132 ± 0.012 0.150 ± 0.013 AD 0.118 ± 0.010 0.155 ± 0.012 0.152 ± 0.012 — 0.073 ± 0.009 0.105 ± 0.010 0.131 ± 0.012 0.163 ± 0.014 AI 0.116 ± 0.010 0.146 ± 0.012 0.146 ± 0.011 0.082 ± 0.008 — 0.107 ± 0.011 0.137 ± 0.012 0.157 ± 0.013 AL 0.123 ± 0.010 0.151 ± 0.012 0.155 ± 0.012 0.125 ± 0.011 0.121 ± 0.010 — 0.137 ± 0.012 0.168 ± 0.014 AP 0.147 ± 0.011 0.152 ± 0.011 0.149 ± 0.012 0.140 ± 0.010 0.141 ± 0.011 0.144 ± 0.010 — 0.153 ± 0.013 AU 0.150 ± 0.012 0.150 ± 0.012 0.152 ± 0.012 0.158 ± 0.012 0.148 ± 0.012 0.164 ± 0.012 0.167 ± 0.013 — AS 0.117 ± 0.011 0.155 ± 0.013 0.144 ± 0.012 0.093 ± 0.009 0.085 ± 0.009 0.123 ± 0.011 0.143 ± 0.011 0.147 ± 0.012

Abbreviations for species: AN, A. nigrus; AA, A. agrarius; AC, A. chevrieri; AD, A. draco; AI, A. ilex; AL, A. latronum; AP, A. peninsulae; AU, A. uralensis; AS, A. semotus. known species from China (Supporting Information, from the Late Miocene to the Pliocene, replacement Table S5; Figs 4–6). of forest mammals by species adapted to open habitat occurred in western Asia. The distribution of Apodemus in Europe did not change greatly from the Late Miocene to the Pliocene, but it likely expanded DISCUSSION to northern China and Mongolia in the Late Pliocene. Origin and migration of Apodemus Connections between mainland Asia and nearby The divergence time of Apodemus we estimate here islands appeared at least twice in the Late Pleistocene, was earlier than that in previous studies (Liu et al., in approximately marine isotope stage 16 (MIS16) (0.6 2004; Suzuki et al., 2008), but agrees with fossil Mya) and MIS12 (0.43 Mya) (Yoshikawa et al., 2007). occurrences. The eastern Mediterranean region and A land bridge between mainland Asia and Japan south-western Asia, particularly Iran, harbours a appeared in the Pleistocene when the sea level fell high diversity of Apodemus (Darvish et al., 2015). The sufficiently, which allowed the arrival of A. argenteus present study identified western and central Asia as and A. speciosus, two endemic species in Japan, at the centre of origin of Apodemus, which agrees with the separate times. Most fossils of Apodemus from Japan proposal of Wessels (1955), who considered south-west are dated to the Late Pleistocene to Holocene. The drop Asia, including Pakistan, to be an important centre of in sea level also facilitated the dispersal of A. agrarius rodent evolution. A tendency towards cooler conditions from mainland Asia to Japan and Taiwan. However, occurred in the late Middle Miocene and Late Miocene, the arrival of A. agrarius in Europe likely occurred which drove the southward dispersal of this genus much later, dated to the post-Neolithic age confirmed into southern Asia and southern Europe (Holbourn by fossil occurrences (Knitlova & Horacek, 2017b) and et al., 2018). The climate of the Middle to Late Miocene phylogeographic patterns revealed by genetic data was characterized by global cooling compared to that (Bugarski-Stanojevic et al., 2011; Koh et al., 2014). of the Early to Middle Miocene, but the temperature Human-facilitated range expansion likely occurred in was higher than it is today, and subtropical to warm A. agrarius (Andersen et al., 2017) and A. sylvaticus temperate forests covered large areas of the Northern (Libois et al., 2001; Lalis et al., 2016), with the former Hemisphere during this period. This forest expansion occupying the widest range of all members of the drove the northward expansion of Apodemus from genus. central Asia to northern Europe and north Asia (Fig. 1A). European species of Apodemus diverged early from the species from Asia (Figs 2–3), which implies early Differentiation of Apodemus in China and the ecological and climatic differentiation in these two origin of A. nigrus regions in the Late Miocene. The climate became The landscape of China is a complex mosaic, increasingly arid from approximately 5.68 Mya (Chang characterized by high-plateau mountain steppes in et al., 2017). Large areas of the continents underwent the west, temperate dry steppes and deserts in the drying, enhanced seasonality and reconstruction of north, a complex of mountain ranges harbouring dense terrestrial plant and animal communities (Herbert rainforest in the centre and subtropical, low-elevation et al., 2016; Holbourn et al., 2018). During the transition forests on the east coast, the so-called three levels of

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Figure 3. Reconstructing the ancient distribution of Apodemus. A, geographical evolutionary history inferred based on Bayesian-DIVA. B, the current distribution of Apodemus (yellow), original file downloaded from The IUCN Red List of Threatened Species (IUCN, 2018) and the predefined distribution area for analyses. terrain in China. Such habitat heterogeneity appears to approximately 3.26 Mya. Based on the locations of to favour the diversification of Apodemus species. The molecular voucher specimens, A. agrarius is widely large genetic distances and significant morphological distributed in the north to the south-east of China, A. variations among these species probably resulted peninsulae occurs along the ‘Hu Huanyong Line’ (Hu, from a long history of differentiation. For example, 1935) or ‘the 400 mm annual precipitation line’ from the split of A. draco and A. ilex, the youngest sister north-east to south-west China, A. uralensis inhabits species of this genus distributed in China, was dated the north-west Qinghai–Tibet plateau and Xinjiang,

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Figure 5. Skulls of Apodemus species. A1 and B1, A. nigrus, IOZCASGZ14081 (holotype); A2 and B2, A. draco, Figure 4. Skin specimens of Apodemus nigrus. A, dorsal IOZCASTMS 14023; A3 and B3, A. agrarius, IOZCASDX010; view of the type series. B, lateral view of the type series. C, A4 and B4, A. chevrieri, IOZCASBBD030; A5 and B5, A. ilex, ventral view of the type series of. Top: IOZCAS GZ14049 IOZCASAYC506; A6 and B6, A. latronum, IOZCASAZ048; (paratype); middle: IOZCASGZ 14081 (holotype); bottom: A7 and B7, A. peninsulae, IOZCAS32277; A8 and B8, A IOZCASGZ 14065 (paratype). uralensis, IOZCASXD028. and A. semotus is endemic to Taiwan (Liu et al., still overlooked biodiversity in China. The discovery of 2017). The remaining species, A. draco, A. chevrieri, the new species A. nigrus from the Wuling Mountain A. latronum, A. ilex and A. nigrus are endemic to the chain in Guizhou and Congqing Provinces reinforces mountainous region of southern China. This region this trend. harbours a high diversity of species and reflects The Wuling Mountains are located in central China, a marked heterogeneous habitat zonation along which is a transitional zone connecting the lowest mountain slopes, in some cases mirroring tropical– and highest of the three levels of terrain in China, temperate forest transitions. Several new mammal with a mean elevation of approximately 1000 m. This species were described from this zone in recent years region constitutes one of the major components of the (e.g. Fan et al., 2017; Ge et al., 2018b), suggesting a Jiangnan orogenic belt in southern China. The climate

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Figure 6. Occlusal views of left upper and lower molar rows. A, Apodemus nigrus (GZ14081, holotype); B, A. nigrus (GZ14065, paratype); C, A. nigrus (GZ14044, paratype); D, A. agrarius (IOZCAS 12393–5); E, A. chevrieri (IOZCAS PQ 039); F, A. draco (IOZCAS TMS14023); G, A. ilex (IOZCAS JD097); H, A. latronum (IOZCAS AZ 066); I, A. peninsulae (IOZCAS 14ML003); J, A. uralensis (IOZCAS XD028). of the Pliocene here was characterized by a warm karst areas by its high elevation, and it was selected as period from the Early to the Middle Pliocene (2–3°C a World Heritage Site in 2018. It is an ecological island higher than today) and a transition from relatively that hosts the endangered Guizhou golden monkey warm climates to the prevailing cooler climates of the (Rhinopithecus brelichi Thomas, 1903), the Chinese Pleistocene. The greatest warming appears to have dove tree (Davidia involucrata Baill.), various alpine occurred at higher latitudes, where temperatures were rhododendrons (Rhododendron spp.) and many other often sufficiently elevated to allow species of animals rare animals and plants. With an elevation of 2238 m, and plants to exist at higher latitudes than their closest Jinfo Mountain is the highest mountain in Chongqing modern relatives. Historical exploration rarely paid Province, and it is famous for its high biodiversity. attention to the Wuling Mountains, which is evident The split of A. nigrus from its sister taxa likely occurred today by sparse material for different taxa from this during the Middle Pliocene, approximately 4 Mya. region in museums. Climate warming drove its preferred habitat upwards, The new species A. nigrus is found on the Fanjing which caused this species to inhabit higher elevations. and Jinfo Mountains, both belonging to the Wuling However, this region was likely less affected by climatic Mountain chain. Fanjing Mountain (or Fanjingshan) oscillations in the Late Quaternary than other regions is the highest mountain in the Wuling Mountains. The of the world, for example, the stable demographic peak of this mountain is 2570 m above sea level. The history of Eothenomys melanogaster (Milne-Edwards, mountain acts as an ‘isolated ecological island’ with a 1871) and the survival of five Chinese giant salamander high degree of endemism of plants and animals. Fanjing species that diverged over four million years ago in the Mountain is unique in its geological history, landforms, same region (Lv et al., 2018; Yan et al., 2018). Subsequent geographical location and climatic conditions, and all global cooling allowed this species to migrate slightly to of these factors have created a terrestrial island with lower elevations, but the dependence of the species on a specific ecological environment. The main peak of local vegetation and climate warming at lower latitudes Fanjing Mountain is separated from the surrounding likely impeded widespread dispersal.

Table 3. External and craniodental morphology of Apodemus in China

AN AA AC AD AI AL AP AU AS

EM N = 11 N = 20 N = 31 N = 25 N = 25 N = 23 N = 19 N = 13 N = 1

BW 26 ± 5.3 24 ± 6.9 36.3 ± 6.8 27.1 ± 6.4 26.1 ± 3.9 28 ± 6.3 31 ± 10.5 19.5 ± 3.2 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 20–33 14–36 25–50 21–52.8 20–32 20–47 16–52 16–24 HBL 94.9 ± 7.2 94.5 ± 9 102.7 ± 12.2 94.9 ± 10.4 97.2 ± 7.2 101.2 ± 7.6 100.6 ± 8.6 85.3 ± 7.1 103 82–107 78–108 88–132 79–135 78–110 84–115 87–120 76–99 TL 103. 8 ± 6.8 75.5 ± 9.4 95.5 ± 10.9 103.1 ± 16.4 101.7 ± 7.3 104.5 ± 11.5 92.3 ± 8.3 79.5 ± 6.4 114 87–114 56–93 78–122 82–161 87–118 90–148 78–108 69–95 HFL 23.2 ± 1.0 19.0 ± 1.4 23.2 ± 2.4 22.8 ± 1.5 21.5 ± 0.8 23.3 ± 1.9 22.4 ± 1.2 19.8 ± 1.4 24 22–25 17–22 16–28 20–27 20–23 21–27 21–25 16–22 EL 17.5 ± 1.2 13.0 ± 1.5 16.7 ± 2.1 16.7 ± 3.0 17.8 ± 1.1 19.2 ± 1.4 16.5 ± 1.9 13.8 ± 2.4 16 16–19 10–16 14–26 11–24 15–20 16–22 14–21 10–17 CM N = 12 N = 14 N = 11 N = 12 N = 15 N = 13 N = 24 N = 12 N = 4 TLC 26.1 ± 1.0 24.5 ± 1.5 28.4 ± 1.2 26.6 ± 1.5 27.1 ± 0.8 27.1 ± 0.9 27.0 ± 1.1 24.3 ± 1.0 27.0 ± 0.8 23.7–27.5 21.8–27.3 27.1–31.4 24.8–29.7 25.9–28.8 25.8–28.5 25.1–29.1 21.9–25.9 NL 10.0 ± 0.7 9.3 ± 0.9 11.1 ± 0.8 10.1 ± 1.2 10.6 ± 0.7 10.5 ± 0.5 10.6 ± 0.9 9.5 ± 0.8 26.3–27.9 8.2–10.7 7.44–10.9 9.96–12.4 8.2–12.4 8.8–12.0 9.5–11.6 8.9–12.5 7.9–11.3 GWS 4.5 ± 0.4 4.4 ± 0.3 5.1 ± 0.4 4.4 ± 0.3 4.8 ± 0.3 4.3 ± 0.3 4.8 ± 0.5 4.3 ± 0.3 10.4 ± 0.9 3.7–5.0 3.9–4.7 4.3–5.7 3.9–4.7 4.0–5.3 3.7–4.8 4.1–5.9 3.8–4.8 SDO 4.9 ± 0.1 4.3 ± 0.1 4.7 ± 0.2 4.6 ± 0.2 4.7 ± 0.1 4.3 ± 0.2 4.5 ± 0.2 4.1 ± 0.1 9.5–11.8 4.7–5.2 4.1 ± 4.6 4.4–5.4 4.3–5.1 4.5–4.9 4.01–4.6 4.13–4.81 4.0–4.3 ZB 11.6 ± 0.8 11.0 ± 0.9 13.0 ± 0.8 12.1 ± 1.1 12.3 ± 0.6 12.3 ± 0.7 12.4 ± 0.9 10.8 ± -.4 4.3 ± 0.1 10.2–12.7 9.3–12.8 11.1–14.1 10.8–13.0 11.1–13.0 11.08–14.1 11.1–14.1 9.8–11.4 GMB 10.5 ± 0.4 10.0 ± 0.5 11.5 ± 0.7 10.8 ± 0.5 10.7 ± 0.5 11.1 ± 0.3 10.8 ± 0.5 10.2 ± 0.5 4.1–4.4 9.8–11.0 9.3–10.8 10.4–12.7 10.2–11.6 9.8–11.4 10.6–11.6 9.7–11.6 8.9–10.8 BL 21.7 ± 1.1 20.8 ± 1.2 24.2 ± 0.9 21.9 ± 1.6 22.1 ± 1.1 22.4 ± 1.1 22.6 ± 1.4 20.3 ± 0.8 4.7 ± 0.1 19.5–24.0 18.9–23.1 23.2–25.5 20.2–25.0 20.2–23.8 21.0–23.8 20.2–25.2 18.7–21.7 BSL 19.2 ± 0.9 18.5 ± 1.28 22.1 ± 1.0 18.9 ± 1.22 19.5 ± 0.9 19.7 ± 1.2 20.3 ± 1.3 17.6 ± 0.5 4.6–4.8 17.6–21.0 16.6–21.1 20.1–23.3 17.2–21.2 17.5–21.1 18.1–21.4 18.4–22.7 16.7–18.7 PL 12.7 ± 0.6 12.3 ± 0.6 14.4 ± 0.6 13.2 ± 0.9 13.4 ± 0.4 13.5 ± 0.6 13.7 ± 0.9 11.9 ± 0.4 12.7 ± 0.3 11.6–13.8 11.2–13.3 13.5–15.3 12.0–15.0 12.6–14.0 12.6–14.5 12.4–15.3 11.2–12.3 IFL 5.0 ± 0.4 4.6 ± 0.4 5.1 ± 0.3 4.6 ± 0.5 5.1 ± 0.7 5.5 ± 0.3 4.9 ± 0.4 4.1 ± 0.4 12.2–13.0 4.3–5.6 4.2–5.6 4.6–5.6 3.7–5.4 3.6–5.8 4.9–6.1 4.1–5.6 3.6–4.7 WIF 1.8 ± 0.1 1.8 ± 0.2 2.0 ± 0.1 1.9 ± 0.1 1.9 ± 0.1 1.9 ± 0.1 1.9 ± 0.1 1.5 ± 0.2 11.1 ± 0.6 1.5–2.0 1.5–2.5 1.8–2.3 1.60–2.2 1.8–2.0 1.7–2.0 1.7–2.2 1.3–1.8 GPB 5.0 ± 0.1 5.1 ± 0.2 5.8 ± 0.3 5.2 ± 0.3 5.4 ± 0.2 5.5 ± 0.1 5.4 ± 0.2 5.2 ± 0.1 10.5–12.0 4.8–5.3 4.7–5.5 5.2–6.6 4.8–5.7 4.8–5.6 5.4–5.7 5.1–5.9 5.0–5.6 LTB 4.6 ± 0.2 4.7 ± 0.4 5.3 ± 0.3 4.9 ± 0.4 4.7 ± 0.2 5.0 ± 0.1 5.2 ± 0.5 4.5 ± 0.3 22.1 ± 0.8 4.1–4.8 3.8–5.3 4.8–5.8 4.5–6.0 4.5–5.0 4.7–5.2 4.4–6.5 4.0–4.9 ULMD 3.9 ± 0.1 3.8 ± 0.2 4.3 ± 0.4 4.1 ± 0.1 4.1 ± 0.1 4.4 ± 0.2 4.1 ± 0.2 3.7 ± 0.1 21.4–23.1 3.7–4.0 3.5–4.2 4.0–5.4 3.6–4.4 3.9–4.3 4.0–4.9 3.7–4.8 3.5–3.9 ULMM 6.7 ± 0.4 6.8 ± 0.4 7.9 ± 0.4 6.9 ± 0.6 7.2 ± 0.2 7.1 ± 0.4 7.7 ± 0.6 6.2 ± 0.2 19.2 ± 1.1 6.2–7.4 6.1–7.5 7.2–8.6 6.1–8.3 6.8–7.7 6.7–7.7 6.9–8.8 5.8–6.5 ML 11.7 ± 0.9 11.1 ± 0.9 12.8 ± 0.8 12.3 ± 1.0 12.0 ± 0.8 12.4 ± 0.7 12.3 ± 0.9 11.0 ± 0.5 18.2–20.7 9.8–12.7 9.8–12.8 11.6–14.2 11.1–14.5 11.1–14.2 11.5–13.6 10.8–13.9 10.4–11.8 LLMM 4.0 ± 0.1 3.9 ± 0.3 4.3 ± 0.2 4.2 ± 0.3 4.1 ± 0.2 4.5 ± 0.2 4.1 ± 0.2 3.7 ± 0.2 13.8 ± 0.7 3.8–4.4 3.3–4.3 4.0–4.7 3.8–4.7 3.8–4.5 4.1–5.0 3.8–4.6 3.5–4.0 LLMD 3.4 ± 0.4 3.5 ± 0.5 4.2 ± 0.4 3.5 ± 0.4 3.5 ± 0.4 3.8 ± 0.4 4.0 ± 0.4 3.2 ± 0.3 12.9–14.5 2.9–4.2 2.7–4.4 3.4–4.8 2.9–4.1 2.9–4.1 3.2–4.3 3.3–4.8 2.8–3.7

Abbreviations for species are the same as Table 2. ‘N’ gives the total number of intact adult specimens included in analyses. BM = body mass, EM = external measurements, HBL = head and body length, TL = tail length, HFL = hind foot length, EL = ear length, CM = craniodental measurements, TLC = total length of the cranium, NL = nasal length, GWS = greatest width of the ‘snout’, SDO = shortest distance between orbits, ZB = zygomatic breadth, GMB = greatest mastoid breadth, BL = basal length, BSL = basilar length, PL = palatal length, IFL = incisive foramen length, WIF = width of the incisive foramen, GPB = greatest palatal breadth, LTB = length of the tympanic bulla, ULMM = length of the maxillary molars, ULMD = length of the maxillary diastema, ML = mandibular length, LLMM = length of the mandibular molars, LLMD = length of the mandibular diastema.

Table 4. Diagnostic features on the occlusal surface of teeth row

Species 1st upper molar 2nd upper 3rd upper

A. nigrus Posterior cingulum absent or Cusp t3 absent Larger than A. agrarius Three rows of cusps

rudimentar and A. chevrieri Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 A. agrarius Posterior cingulum absent or Cusp t3 absent Smaller than M1 and M2 Two rows of cusps (most rudimentar of specimens) A. chevrieri Posterior cingulum absent Cusp t3 absent Smaller than M1 and M2 Two rows of cusps (most of specimens) A. draco Posterior cingulum well- Cusp t3 present Larger than A. agrarius Three rows of cusps developed. and A. chevrieri A. ilex Posterior cingulum well- de- Cusp t3 present Larger than A. agrarius Three rows of cusps veloped. and A. chevrieri A. latronum Posterior cingulum well- Cusp t3 present Larger than A. agrarius Three rows of cusps developed and A. chevrieri A. peninsulae Posterior cingulum Cusp t3 present Larger than A. agrarius Three rows of cusps rudimentar and A. chevrieri A. uralensis Posterior cingulum absent Cusp t3 present Larger than A. agrarius Three rows of cusps and A. chevrieri

Taxonomic account m tall that form a continuous canopy during the rainy Order: Rodentia season. The vegetation is denser and more humid in Family: Muridae the valleys, even during the dry season. Subfamily: Murinae Apodemus Kaup, 1829. Paratypes: IOZCAS GZ14049 and IOZCAS GZ14065, two adult males preserved as dry skin and a skull, collected in the same site as the holotype by Deyan Apodemus nigrus Deyan Ge, Anderson Feijó & Ge, Jilong Cheng, Xue Lv, Zhengxing Huang and Qisen Yang, sp. nov. Fangyuan Yang on 2 August 2014. The EMs are as follows: BW = 30, 28; HBL = 92, 102; TL = 110, (Figs 4, 5A1, B1, 6A, B, C) 107; HFL = 25, 24; and EL = 18, 19. The CMs are as follows: TLC = 26.24, 26.77; NL = 10.06, 10.41; LSID:urn:lsid:zoobank. GWS = 4.55, 4.87; SDO = 5.22, 4.96; ZB = 11.56, org:pub:38D8C511-CC28-44FD-9422-4B7B5F8AB564. 12.16; GMB = 10.61, 10.7; BL = 21.68, 22.12; Common name: black field mouse. Holotype: BSL = 19.93, 19.47; PL = 12.65, 12.59; IFL = 4.86, 4.48; GZ14081, an adult male preserved as dry skin and WIF = 1.89, 1.73; GPB = 5.17, 5.13; LTB = 4.65, 4.55; a skull, deposited at the IOZCAS (Figs 4, 5A1, 5B1, ULMM = 3.96, 3.97; ULMD = 6.48, 6.73; ML = 11.95, 6A). This specimen was collected on 3 August 2014 11.34, LLMM = 4.11, 4.03 and LLMD = 2.97, 3.2. by Deyan Ge and Jilong Cheng. The Cytb (1.131 bp), Cox1 (676 bp), D-loop (536 bp) and Rbp3 (1209 bp) Additional specimens: Nine other specimens sequences of the holotype are archived in GenBank were collected at the same site as the holotype and with accession numbers MK329461, MK329600, paratypes and are preserved as dry skin and a MK329739 and MK329885, respectively. The EMs are skull. One adult male (IOZCAS GZ14063) and four as follows: BW = 33, HBL = 107, TL = 93, HFL = 23, adult females (IOZCAS GZ14058, IOZCAS GZ14073, and EL = 18. The CMs are as follows: TLC = 27.46, IOZCAS GZ14076, IOZCAS GZ14098) were NL = 10.1, GWS = 4.98, SDO = 5.03, ZB = 12.73, collected by Deyan Ge, Jilong Cheng, Xue Lv, GMB = 10.67, BL = 23.93, BSL = 21.01, PL = 13.76, Zhengxing Huang and Fangyuan Yang during IFL = 5.59, WIF = 1.92, GPB = 5.16, LTB = 4.77, August 2014. One male (IOZCAS GZ15214) and ULMM = 3.8, ULMD = 7.44, ML = 12.65, LLMM = 4.40 three females (IOZCAS GZ15193, IOZCAS GZ15208, and LLMD = 3.21. IOZCAS GZ15219) were collected by Qisen Yang, Jilong Cheng, Zhixin Wen and Xue Lv in April Type locality: Fanjing Mountain National Nature 2015. Fourteen specimens were collected in Jinfo Reserve, Guizhou Province, China (N27.70, E108.83, Mountain, Chongqing Province (N29.03, E107.19, elevation: 2078 m). This area is a complex of hills and elevation: 2130 m), by Deyan Ge, Anderson Feijó, valleys characterized by large deciduous trees 10–12 Alexei V. Abramov, Yanqun Wang and Jian Sun at

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 518–534 EVOLUTIONARY HISTORY OF FIELD MICE 531 the end of September 2018. These specimens include and broad, extending to close to the anterior margin seven males (IOZCAS CQ18091, IOZCAS CQ18098, of the toothrow. The interparietal is wide and short in IOZCAS CQ18100, IOZCAS CQ18101, the anterior–posterior plane. The mesopterygoid fossa IOZCAS CQ18102, IOZCAS CQ18141, is very narrow with parallel sides. First upper molar is IOZCAS CQ18143), six females (IOZCAS CQ18087, as long as the other upper molars. Posterior cingulum IOZCAS CQ18099, IOZCAS CQ18103, of the first upper molar (M1) is small and tends to Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 IOZCAS CQ18104, IOZCAS CQ18144, merge with the ridge connecting cusps t8 and t9 in IOZCAS CQ18145) and one individual of undetermined old individuals. Cusp t7 of M1 is slightly smaller than sex that was badly destroyed by other animal. lingual cusps t1 and t4. M2 lacks a posterior cingulum and cusp t3 is reduced or absent. M3 has three rows of Sequenced molecular voucher cusps. Anterocentral cusp of the first lower molar is well specimens: IOZCAS GZ14049, IOZCAS GZ14058, developed and posterior cingulum is present. Anterior, IOZCAS GZ14063, IOZCAS GZ14065, middle and posterior labial cusplets are well developed IOZCAS GZ14073, IOZCAS GZ14076, on the first lower molar m1. Anterolabial cusps of the IOZCAS GZ14081, IOZCAS GZ14098, second lower molar m2 are well marked (Table 4). IOZCAS CQ18087, IOZCAS CQ18091, IOZCAS CQ18098–IOZCAS CQ18104, and Diagnosis: Apodemus nigrus is easily distinguished IOZCAS CQ18141–IOZCAS CQ18145. Accession from other Chinese congeners by the general dorsal numbers are provided in the Supporting Information, black fur and well-developed anterior, middle and Table S1. posterior labial cusplets on the first lower molar. The species can be further differentiated from A. chevrieri External and craniodental descriptive statistics: and A. agrarius by supraorbital ridges restricted BW = 26 ± 5.3 (20–33, N = 11). EMs of 11 adult to the lateral margins of the frontal, not extending specimens: HBL = 94.9 ± 7.2, TL = 103.8 ± 6.8, along the parietal, and three rows of cusps on the last HFL = 23.2 ± 1.0 and EL = 17.5 ± 1.2. CMs from 12 intact upper molar. A developed cusp t7 on M1 is present adult specimens: TLC = 26.1 ± 1.0, NL = 10.0 ± 0.7, in A. nigrus but reduced in A. peninsulae. Cusp t3 is GWS = 4.5 ± 0.4, SDO = 4.9 ± 0.1, ZB = 11.6 ± 0.8, absent on M2 in A. nigrus and present in A. latronum, GMB = 10.5 ± 0.4, BL = 21.7 ± 1.1, BSL = 19.2 ± 0.9, A. draco, A. ilex and A. uralensis. Posterior cingulum PL = 12.7 ± 0.6, IFL = 5.0 ± 0.4, WIF = 1.8 ± 0.1, of M1 is reduced in A. nigrus and tends to merge with GPB = 5.0 ± 0.1, LTB = 4.6 ± 0.2, ULMM = 3.9 ± 0.1, cusps t8 and t9 in adult individuals, while it is evident ULMD = 6.7 ± 0.4, ML = 11.7 ± 0.9, LLMM = 4.0 ± 0.1 in A. ilex and A. draco. and LLMD = 3.4 ± 0.4.

Description: Apodemus nigrus is a small species with Distribution: We found this species only at high elevations soft fur and delicate spines. Upper parts are entirely (1984–2078 m) in the Fanjing Mountain National Nature black and finely sprinkled with brown. Dorsal spines Reserve in Guizhou Province and in the highlands of are blackish with brownish tips. Ventral pelage has the Jinfo Mountain National Nature Reserve (2130 m) a general greyish-white colour, contrasting sharply in Chongqing Province. Both mountains belong to the with upper parts in adult individuals. Ventral spines Wuling Mountain range in China. are uniformly whitish. Ears are long and black with black fur covering the base. Mystacial vibrissae are Etymology: The epithet nigrus (from Latin niger, long, extending to the posterior margin of the ear. black) refers to the black dorsal fur that is characteristic Hands and feet are covered with short whitish hairs, of this new species. clearly contrasting with the black legs and arms. The tail is unicoloured blackish brown, slightly longer than the HBL and covered with very short Comments: The occurrence of melanism in Apodemus hairs. Tail scales are arranged in an annular pattern was reported in A. sylvaticus, which was thought to be and approximately 0.5 mm in length. Mammae associated with black peat soil (Hussen, 1954; Green, 1/1 + 2/2 = 6. 1977). The colouration of the new species described in The skull has a relatively short rostrum and rounded the present study is different from the melanism of braincase. Its general dorsal profile is nearly flat. The A. sylvaticus, because all specimens of the new species dorsolateral margins of the interorbit are delimited by have black pelage. The original forests of Fanjing supraorbital ridges that are restricted to the lateral Mountain and Jinfo Mountain are well preserved, margin of the frontal. Zygomatic arches are slightly most likely because of the steep topography in this expanded laterally and their maxillary root lies region. The vegetation of the localities from which anterior to the toothrow. Incisive foramina are long we collected specimens for this new species includes

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, 187, 518–534 532 D. GE ET AL. alpine brushes, with alpine rhododendron and bamboo mid-latitude westerlies and their influence on Asian as the dominant plants. monsoon as constrained by the K/Al ratio record from drill core Ls2 in the Tarim Basin. CATENA 153: 75–82. Sympatric species: We found that A. agrarius inhabits Darvish J, Mohammadi Z, Ghorbani F, Mahmoudi A, the same region as A. nigrus, but A. agrarius was Dubey S. 2015. Phylogenetic relationships of Apodemus present only in lower-elevation localities near farms. Kaup, 1829 (Rodentia: Muridae) species in the Eastern Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 Mediterranean inferred from mitochondrial DNA, with emphasis on Iranian species. Journal of Mammalian Evolution 4: 583–595. ACKNOWLEDGEMENTS Drummond AJ, Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary We greatly appreciate Roberto Portela Miguez, Paula Biology 7: 214. Jenkins and Louise Tomsett from NHM (London, UK); Drummond AJ, Ho SY, Phillips MJ, Rambaut A. 2006. Xichao Zhu and Yang Yang from IOZCAS (Beijing, Relaxed phylogenetics and dating with confidence. PLoS China) for their assistance with the specimens Biology 4: e88. preserved under their supervision. We appreciate Ellerman JR, Morrison-Scott TCS. 1951. Checklist of comments from two anonymous reviewers and the Palaearctic and Indian mammals 1758 to 1946. London: associate editor for improving this manuscript. British Museum (Natural History). Yongbin Chang, Xue Lv, Zhengxing Huang, Fangyuan Fan PF, He K, Chen X, Ortiz A, Zhang B, Zhao C, Li YQ, Yang, Yanqun Wang and Jian Sun provided assistance Zhang HB, Kimock C, Wang WZ, Groves C, Turvey ST, in field work. Deyan Ge is sponsored by the Newton Roos C, Helgen KM, Jiang XL. 2017. Description of a new Advanced Fellowship of the Royal Society, United species of Hoolock gibbon (Primates: Hylobatidae) based on Kingdom (NA150142) and a grant from the National integrative taxonomy. American Journal of Primatology 79: Nature Science Fund of China (31872958). This e22631. research is also sponsored by National Special Fund Filippucci MG, Macholan M, Michaux JR. 2002. Genetic on Basic Research of Science and Technology in China variation and evolution in the genus Apodemus (Muridae: (2014FY210200, 2014FY110100), a grant from the Key Rodentia). Biological Journal of the Linnean Society 75: Laboratory of Zoological Systematics and Evolution 395–419. of the Chinese Academy of Sciences (Y229YX5105). Freudenthal M. 1976. Rodent stratigraphy of some Miocene Alexei V. Abramov is sponsored by International fissure fillings in Gargano (Prov. Foggia, Italy). Scripta Fellowship for Distinguished Scientists, Chinese Geology 37: 1–23. Academy of Sciences (Ref. 2017VBA0027). Anderson Ge DY, Lu L, Abramov AV, Wen ZX, Cheng JL, Xia L, Vogler AP, Yang QS. 2018a. Coalescence models reveal Feijó is supported by the Chinese Academy of Sciences the rise of the white-bellied rat (Niviventer confucianus) President’s International Fellowship Initiative following the loss of Asian megafauna. Journal of Mammalian (2018PB0040). Evolution Doi: 10.1007/s10914-018-9428-y. Ge DY, Lu L, Xia L, Du YB, Wen ZX, Cheng JL, Abramov AV, REFERENCES Yang QS. 2018b Molecular phylogeny, morphological diversity, and systematic revision of a species complex of Allen GM. 1938. The mammals of China and Mongolia. New common wild rat species in China (Rodentia, Murinae). York: American Museum of Natural History, xxvi + 620. Journal of Mammalogy 99: 1350–1374. Andersen LW, Jacobsen M, Vedel-Smith C, Jensen TS. Green RE. 1977. Melanism in the wood mouse, Apodemus 2017. Mice as stowaways? Colonization history of Danish sylvaticus. Journal of Zoology 182: 157–159. striped field mice. Biology Letters 13: 20170064. Guzzetta G, Tagliapietra V, Perkins SE, Hauffe HC, Barry JC, Morgan ME, Flynn LJ, Pilbeam D, Poletti P, Merler S, Rizzoli A. 2017. Population dynamics Behrensmeyer AK, Raza SM, Khan I A, Badgley C, of wild rodents induce stochastic fadeouts of a zoonotic Hicks J, Kelley J. 2002. Faunal and environmental pathogen. Journal of Animal Ecology 86: 451–459. change in the late Miocene Siwaliks of northern Pakistan. Hammer O, Harper DAT, Ryan PD. 2001. PAST: Paleobiology 28: 1–71. paleontological statistics software package for education and Bugarski-Stanojevic V, Blagojevic J, Stamenkovic G, data analysis. Palaeontologia Electronica 4: [unpaginated]. Adnadevic T, Giagia-Athanasopoulou EB, Hauffe HC, Niklasson B, Olsson T, Bianchi A, Rizzoli A, Vujosevic M. 2011. Comparative study of the phylogenetic Klitz W. 2010. Ljungan virus detected in bank voles structure in six Apodemus species (Mammalia, Rodentia) (Myodes glareolus) and yellow-necked mice (Apodemus inferred from ISSR-PCR data. Systematics and Biodiversity flavicollis) from Northern Italy. Journal of Wildlife Diseases 1: 95–106. 46: 262–266. Chang H, An Z, Wu F, Song Y, Qiang X, Li L. 2017. Herbert TD, Lawrence KT, Tzanova A, Peterson LC, Late Miocene–Early Pleistocene climate change in the Caballero-Gill R, Kelly CS. 2016. Late Miocene global

cooling and the rise of modern ecosystems. Nature Geoscience African wood mouse (Apodemus sylvaticus) populations: a 9: 843–847. comparative study of mtDNA restriction patterns. Canadian Holbourn AE, Kuhnt W, Clemens SC, Kochhann KGD, Journal of Zoology 79: 1503–1511. Jöhnck J, Lübbers J, Andersen N. 2018. Late Miocene Liu Q, Chen P, He K, Kilpatrick CW, Liu SY, Yu FH, climate cooling and intensification of southeast Asian winter Jiang XL. 2012. Phylogeographic study of Apodemus ilex

monsoon. Nature Communications 9: 1584. (Rodentia: Muridae) in Southwest China. PLoS ONE 7: Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 Hu HY. 1935. The distribution of population in China. Acta e31453. Oceanologica Sinica 2: 32–74. Liu X, Wei F, Li M, Jiang X, Feng Z, Hu J. 2004. Molecular Hussen AM. 1954. On a melanistic specimen of the long-tailed phylogeny and taxonomy of wood mice (genus Apodemus field mouse Apodemus sylvaticus sylvaticus (L.). Mammalia Kaup, 1829) based on complete mtDNA cytochrome b 18: 329–330. sequences, with emphasis on Chinese species. Molecular Irwin DM, Kocher TD, Wilson AC. 1991. Evolution of Phylogenetics and Evolution 33: 1–15. the cytochrome b gene of mammals. Journal of Molecular Liu RR, Ge DY, Lu L, Xia L, Liu QY, Yang QS. 2017. Evolution 32: 128–144. Identification and distribution of Apodemus species with IUCN. 2018. The IUCN red list of threatened species. Version DNA barcoding in China. Chinese Journal of Vector Biology 2018-2. Available at: http://www.iucnredlist.org (accessed and Control 28: 97–103. October 15). Lv X, Cheng JL, Meng Y, Chang YB, Wen ZX, Xia L, Ge DY, Johnson NM, Stix J, Tauxe LF, Cerveny PF, Liu SY, Yang QS. 2018. Disjunct distribution and distinct Tahirkheli RAK. 1985. Paleomagnetic chronology, fluvial intraspecific diversification of Eothenomys melanogaster in processes, and tectonic implications of the Siwalik deposits South China. BMC Evolutionary Biology 15:50. near Chinji Village. Pakistan Journal of Geology 93: 27–40. Ma CF, Yu PB, Wu R, Chen Hl, Cai ZH, Lu XL, Wang JJ, Kawamura Y. 1989. Quaternary rodents faunas in the Li HX. 2015. Spatial and temporal distribution of hosts Japanese islands (part 2). Memoirs of the Faculty of Science carrying hantaviruses. Chinese Journal of Zoonoses 31: Kyoto University Series of Geology and Mineralogy 54: 1–235. 26–29. Kimura Y, Flynn LJ, Jacobs LL. 2017. Early Late Miocene Martin Suarez E, Mein P. 1998. Revision of the genera murine rodents from the upper part of the Nagri formation, Parapodemus, Apodemus, Rhagamys and Rhagapodemus Siwalik group, Pakistan, with a new fossil calibration point (Rodentia, Mammalia). Geobios 31: 87–97. for the tribe Apodemurini (Apodemus/Tokudaia). Fossil Michaux JR, Chevret P, Filippucci MG, Macholan M. Imprint 73: 197–212. 2002. Phylogeny of the genus Apodemus with a special Klein TA, Kim HC, Chong ST, Kim JA, Lee SY, Kim WK, emphasis on the subgenus Sylvaemus using the nuclear Nunn PV, Song JW. 2015. Hantaan virus surveillance IRBP gene and two mitochondrial markers: cytochrome b targeting small mammals at nightmare range, a high and 12S rRNA. Molecular Phylogenetics and Evolution 23: elevation military training area, Gyeonggi Province, Republic 123–136. of Korea. PLoS ONE 10: e0118483. Michaux JR, Magnanou E, Paradis E, Nieberding C, Knitlova M, Horacek I. 2017a. Genus Apodemus in the Libois R. 2003. Mitochondrial phylogeography of the Pleistocene of Central Europe: when did the extant taxa woodmouse (Apodemus sylvaticus) in the Western Palearctic appear? Fossil Imprint 73: 460–481. region. Molecular Ecology 12: 685–697. Knitlová M, Horáček I. 2017b. Late Pleistocene–Holocene Musser GG, Carleton MD. 2005. Superfamily Muroidea. In: paleobiogeography of the genus Apodemus in Central Wilson DE & Reeder DM, eds. Mammal species of the world: Europe. PLoS ONE 12: e0173668. a taxonomic and geographic reference. Baltimore: Johns Koh HS, Shaner PJ, Csorba G, Wang YC, Jang KH, Hopkins University Press, 894–1531. Lee JH. 2014. Comparative genetics of Apodemus agrarius Musser GG, Brothers EM, Carleton MD, Hutterer R. (Rodentia: Mammalia) from insular and continental 1996. Taxonomy and distributional records of Oriental Eurasian populations: cytochrome b sequence analyses. Acta and European Apodemus, with a review of the Apodemus– Zoologica Academiae Scientiarum Hungaricae 60: 73–84. Sylvaemus problem. Bonner Zoologische Beiträge 46: Lalis A, Leblois R, Liefried S, Ouarour A, Beeravolu CR, 143–190. Michaux J, Hamani A, Denys C, Nicolas V. 2016. New Robins JH, Hingston M, Matisoo-Smith E, Ross HA. molecular data favour an anthropogenic introduction of the 2007. Identifying Rattus species using mitochondrial DNA. wood mouse (Apodemus sylvaticus) in North Africa. Journal Molecular Ecology Notes 7: 717–729. of Zoological Systematics and Evolutionary Research 54: Ronquist F, Teslenko M, van der Mark P, Ayres DL, 1–12. Darling A, Höhna S, Larget B, Liu L, Suchard MA, Lanfear R, Frandsen PB, Wright AM, Senfeld T, Huelsenbeck JP. 2012. MrBayes 3.2: efficient Bayesian Calcott B. 2017. PartitionFinder 2: new methods for phylogenetic inference and model choice across a large model selecting partitioned models of evolution for molecular and space. Systematic Biology 61: 539–542. morphological phylogenetic analyses. Molecular Biology and Serizawa K, Suzuki H, Tsuchiya K. 2000. A phylogenetic Evolution 34: 772–773. view on species radiation in Apodemus inferred from Libois RM, Michaux JR, Ramalhinho MG, Maurois C, variation of nuclear and mitochondrial genes. Biochemical Sara M. 2001. On the origin and systematics of the northern Genetics 38: 27–40.

Smith AT, Xie Y. 2008. A guide to the mammals of China. Wilson DE, Mittermeier RA, Lacher TE, Jr., eds. 2016. Princeton & Oxford: Princeton University Press. Handbook of the mammals of the world, Vol. 6. Lagomorphs Suzuki H, Filippucci MG, Chelomina GN, Sato JJ, and rodents 1. Barcelona: Lynx Edicions. Serizawa K, Nevo E. 2008. A biogeographic view of Xia W. 1984. A study on Chinese Apodemus with a discussion of its Apodemus in Asia and Europe inferred from nuclear and relations to Japanese species. Acta Theriologica Sinica 4: 93–98.

mitochondrial gene sequences. Biochemical Genetics 46: Xia X. 2017. DAMBE6: new tools for microbial genomics, Downloaded from https://academic.oup.com/zoolinnean/article-abstract/187/2/518/5530313/ by Institute of Zoology, CAS user on 04 October 2019 329–346. phylogenetics, and molecular evolution. Journal of Heredity Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 108: 431–437. 2013. MEGA6: molecular evolutionary genetics analysis Yan F, Lu J, Zhang B, Yuan Z, Zhao H, Huang S, Wei G, version 6.0. Molecular Biology and Evolution 30: Mi X, Zou D, Xu W, Xiao H, Liang Z, Jin J, Wu S, Xu C, 2725–2729. Tapley B, Turvey ST, Papenfuss TJ, Cunningham AA, Thomas O. 1922. On mammals from the Yunnan highlands Murphy RW, Zhang YP, Che J. 2018. The Chinese giant collected by Mr. George Forrest and presented to the British salamander exemplifies the hidden extinction of cryptic Museum by Col. Stephenson R. Clarke D.S.O. Annals & species. Current Biology 28:R590-R592.. Magazine of Natural History 10: 391–406. Yoshikawa S, Kawamura Y, Taruno H. 2007. Land bridge Tian H, Yu P, Cazelles B, Xu L, Tan H, Yang J, Huang S, formation and proboscidean immigration into the Japanese Xu B, Cai J, Ma C, Wei J, Li S, Qu J, Laine M, Wang J, islands during the Quaternary. Journal of Geosciences, Tong S, Stenseth NC, Xu B. 2017. Interannual cycles of Osaka City University 50: 1. Hantaan virus outbreaks at the human-animal interface in Yu Y, Harris AJ, Blair C, He X. 2015. RASP (Reconstruct Central China are controlled by temperature and rainfall. Ancestral State in Phylogenies): a tool for historical biogeography. Proceedings of the National Academy of Sciences of the Molecular Phylogenetics and Evolution 87: 46–49. United States of America 114: 8041–8046. Zheng SH. 1993. Quaternary rodents of Sichuan-Guizhou Wessels W. 1955. Miocene rodent evolution and migration area, China. Beijing: Science Press. (Muroidea from Pakistan, Turkey and Northern Africa). Zimmermann K. 1962. Die Untergattungen der Gattung Utrecht: Faculty of Geosciences, Utrecht University. Apodemus Kaup. Bonner Zoologische Beiträge 13: 198–208.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher's web-site. Table S1. Samples used in molecular analyses, the GenBank accessions of DNA fragments. Table S2. Fossil occurrences of Parapodemus and Apodemus from palaeobiology databases and original literature. Table S3. EMs of specimens used in the present study. Table S4. CMs of specimens used in the present study. Table S5. Morphological differences between Apodemus species in China as determined by LSD tests. Fig. S1. Illustrating the locations of CMs used in the present study. Abbreviations for measurements are the same as that of Tables 3 and 4. Fig. S2. Phylogeny of Apodemus reconstructed by using Rbp3. Fig. S3. Morphological variation of skull among species within the genus Apodemus. The first three axes of PCA scores (A) and discriminant function (B) are used to illustrate morphological differences among nine species. File S1. References cited in Tables S1 and S2.