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Home , Bamboo rat, Banma County, Cricetidae, Eospalax, Myospalax, Platacanthomyidae

Journal of Genetics (2020)99:38 Ó Indian Academy of Sciences

https://doi.org/10.1007/s12041-020-01200-2 (0123456789().,-volV)(0123456789().,-volV)

RESEARCH ARTICLE

Taxonomy and phylogenetic relationship of

YAO ZOU1, MIAO XU1, SHIEN REN1, NANNAN LIANG1, CHONGXUAN HAN1*, XIAONING NAN1* and JIANNING SHI2

1Key Laboratory of National Forestry and Grassland Administration on Management of Western Forest Bio-Disaster, Northwest Agriculture and Forestry University, Yangling 712100, People’s Republic of 2Ningxia Hui Autonomous Region Forest Disease and Pest Control Quarantine Station, Yinchuan 750001, People’s Republic of China *For correspondence. E-mail: Chongxuan Han, [email protected]; Xiaoning Nan, [email protected].

Received 24 October 2019; revised 19 February 2020; accepted 2 March 2020

Abstract. (Myospalacinae) is one of the subterranean , endemic to . Due to the convergent and parallel evolution induced by its special lifestyles, the controversies in morphological classification of zokor appeared at the level of family and . To resolve these controversies about and phylogeny, the phylogenetic relationships of 20 of and six species of zokors were studied based on complete mitochondrial genome and mitochondrial Cytb gene, respectively. Phylogeny analysis of 20 species of Muroidea indicated that the zokor belonged to the family , and it was closer to mole rather than rat. Besides, by investigating the phylogenetic relationships of six species of zokors, the status of two genera of and was affirmed because the two clades differentiated in phylogenetic tree represented two types of zokors, convex occiput type and flat occiput type, respectively. In addition, the two origins in Eospalax were found diverged at 3.71 million years ago (Ma) based on estimation of divergence time. It is suggested that the climate and ecology changes caused by the -Tibet Plateau uplift event in 3.6 Ma led to the inner divergence of Eospalax. The intraspecific phylogenetic relationships of partial zokors were well resolved, the two clades of Eospalax cansus represented two geographical populations, respectively, and the divergent pattern of Eospalax baileyi was characterized by allopatric divergence spatially. In this study, we explored the taxonomic status and phylogenetic relationships of Myospalacinae at the molecular level. These works would be significant to understanding the evolutionary process and to clarify the mechanism of differentiation of Myospalacinae.

Keywords. Myospalacinae; Spalacidae; taxonomy; phylogenetic relationships.

Introduction and Bell 1997). However, according to the morphological traits, some studies regarded zokor as a subfamily of Zokor (Myospalacinae) is one of the subterranean rodents (Luo et al. 2000; Pavlinov 2003; Wang 2003). By endemic to east Asia which lives most of its live under- collecting and morphological information, zokor was ground (Fan and Shi 1982). Due to the insufficient infor- believed to be of a single family Myospalacidae (Zheng mation of morphological features, and the convergent and 1994, 1997; Pavlinov and Lissovsky 2012). In addition, parallel evolution induced by its special lifestyles, the con- some current researches based on molecular evidences troversies in morphological classification of zokor appeared have suggested that zokor belonged to the family at the level of family and genus. The phylogenetic and Spalacidae (Miller and Gidley 1918; Allen 1940; Norris taxonomic status of Myospalacinae have been extensively et al. 2004). studied for several decades (Allen 1940; Ellerman and The phylogenetic relationships among Myospalacinae, Morrison-Scott 1953; Norris et al. 2004). Zokor was first Spalacinae and Rhyzomyinae of Spalacidae have also been classified into the family in 1876 by Alston because questioned in several studies. By analysing the mitochon- of its molars like , subtriangular infraorbital foramen drial gene Cytb and 12srRNA of three subfamilies, the and rudimentary ear conch, which was supported by related Myospalacinae was found to be a clade of early differenti- studies (Thomas 1896; Carleton and Musser 1984; McKenna ation, the other two subfamilies were closer in phylogeny 38 Page 2 of 10 Yao Zou et al.

(Norris et al. 2004). Studies based on nuclear gene IRBP Due to the limitation of morphology and insufficient showed that mole rat differentiated earlier, and samples, the taxonomic status and phylogeny of zokor have and zokor were most closely related (Jansa and Weksler not been elucidated clearly. As a result, it is necessary to 2004). However, by analysing concatenated gene of IRBP collect more samples and molecular data to conduct com- and GHR in later studies, it was found that bamboo rat was parative analysis with previous research data. In this study, the ancient clade, zokor and mole rat grouped together later the phylogenetic relationships of 20 species of Muroidea of (Jansa et al. 2009). Later researches based on skull mor- Rodentia and of six species of zokors were investigated phology and fossil evidence have shown that zokor and based on complete mitochondrial genome and mitochondrial bamboo rat were more closely related to each other than Cytb gene. The taxonomic status and phylogenetic rela- mole rat (Flynn and Lawrence 2009). tionships of Myospalacinae were explored at the molecular According to the difference of occipital morphology, level. These works would be significant to understand the zokors can be divided into three types: sunk occiput, flat evolutionary process and to clarify the mechanism of dif- occiput and convex occiput. The sunk occiput type including ferentiation of Myospalacinae. the Dingshi zokor (Myospalax gospalaxtingi) is already extinct. The flat occiput type includes M. psilurus, M. aspalax and M. myospalax. The convex occiput type Materials and methods includes Eospalax fontanieri, E. cansus, E. baileyi, E. rufescens, E. rothschilidi and E. smithi. It was believed Specimen collection that zokor has undergone an evolutionary process from convex to sunk, and then to flat occiput (Zheng 1994, 1997). We collected 55 specimens including M. aspalax, E. cansus This point of view has laid the foundation for understanding and E. rothschilidi from five locations of Hebei, Ningxia and the phylogenetic relationships of Myospalacinae. There have Shaanxi . The details of sampling location been disputed subjects about the taxonomic status of convex are listed in table 1. Zokors were captured by living trap and flat occiput type. Allen (1940) suggested that these two underground between March and November 2017. The types should be regarded as two subgenera Eospalax and muscle tissues of the zokors were obtained with scissors and Myospalax, which were supported by later studies (Song tweezers. Tissues were immediately frozen in liquid nitro- 1986; Corbet and Hill 1991; Lawrence 1991; Wang 1993; gen, and then stored in a refrigerator at -80°C in the Luo et al. 2000). However, from the other perspective, it was laboratory. believed that two types should be considered as two genera Eospalax and Myospalax (Zheng 1994; Norris et al. 2004; Smith and Xie 2009). DNA extraction and primer synthesis The phylogenetic tree of Eospalax have been constructed in previous studies based on fossil and morphological traits, Total genomic DNA of zokors was extracted with a Blood/ it was believed that the species of Eospalax differentiated Cell/Tissue Genome DNA Extraction kit (Tiangen) from the along three directions: E. rothschilidi represents a southward muscle tissues following the manufacturer’s protocol. The clade adapted to subtropical forest; E. smithi and E. baileyi primers were synthesized by referring to primer sequences spread to the westward to alpine environment; E. rufescens designed in literature (Zhou et al. 2004). The Cytb gene and E. cansus represent a clade that evolved northward to sequence was amplified with primers F0: CGAAGCTTGA the habitat of the Loess Plateau (Li and Wang 1996). TATGAAAAACCATCGTTG, R0:CGGAATTCCATTTTGG However, some phylogenetic trees constructed based on TTTACAAG. molecular data were different. For example, the phylogenetic relationships among four species of zokors were investigated based on D-loop and ND4 sequences, it was shown that PCR amplification and sequencing E. cansus formed a monophyletic group, E. baileyi and E. smithi gathered into a sister group and then grouped All polymerase chain reaction (PCR) were carried out in a together with E. rufescens (Su et al. 2011). The analysis of PCR amplifier (Applied Biosystems, Foster City, USA). phylogeny based on Cytb gene showed that E. baileyi was With 25 lL reaction volume, including 0.5 lL dNTPs mix the first differentiated species, E. cansus, E. rufescens and (10 mM concentration of each dNTP), 2.5 mL of 109 buffer E. smithi grouped into one clade afterwards (Yang et al. (20 mM Mg2?), 1.0 mL of each primer (6.25 pmolÁlL-1) 2009). It remained to be questioned which phylogenetic tree and 0.5 lL Taq polymerase (500 U), with sterile distilled could truly reflect evolutionary relationships among species. water added up to 25 mL volume. Amplification conditions Moreover, although partial species of zokors have been were 5 min at 95°C followed by 35 cycles of 95°C for 40 s, studied on intraspecific phylogeny, it is difficult to collect 53°C for 1 min, 72°C for 90 s, and a final extension at 72°C comprehensive samples of some species like E. cansus due for 7 min. PCR products were detected with a gel extraction to their wide distributions. The phylogenetic relationships kit (Takara) and then purified and sequenced by Shanghai within species were still unclear so far. Personal Biotechnology. Taxonomy and phylogenetic relationship of zokors Page 3 of 10 38

Table 1. Information of sampling location of zokors.

Longitude Latitude Altitude Sample Accession Sampling location Species (°E) (°N) (m) size number

Yueliangshan forest of Haiyuan county in Ningxia E. cansus 105.62 36.20 2339 9 MN518084– MN518092 Zhangjiawa forest of Xiji county in Ningxia E. cansus 105.56 36.10 2224 7 MN518093- MN518099 Chazi of Weichang county in Hebei M. aspalax 117.35 42.14 1528 30 MN518109– MN518138 Small mountain forest of Zhenba county in E. rothschilidi 108.02 32.50 1624 6 MN518100- Shaanxi MN518103; MN518105; MN518108 Baiyan villagr of Xixiang county in Shaanxi E. rothschilidi 107.56 32.82 1460 3 MN518104; MN518106; MN518107

Figure 1. The phylogenetic tree based on 13 protein-coding genes of mitochondrial genome of Muroidea (ML). The branch in bold represents Spalacidae. The branch of bamboo rat is in green, mole rat in red and zokor in blue, respectively.

Data analyses were from suborder , one sequence of suborder (Pteromys volans) and one of suborder Hys- Complete mitochondrial genome sequences of 20 species of tricomorpha (Thryonomys swinderianus) were chosen as Muroidea of order Rodentia were obtained from GenBank outgroups. PartitionFinder2 was used to the best-fit schemes (accession number are shown in figure 1). 20 sequences and models of evolution for 13 protein-codon genes of 38 Page 4 of 10 Yao Zou et al.

Table 2. Information of Cytb sequences from GenBank.

Species Accession number Location Sample size

E. cansus KM103797; KM103799 Datong county in Qinghai 8 KM103854–KM103857 Minhe county in Qinghai EF530738; EF530739 Tianzhu county in Gansu E. baileyi KM103774–KM103776 Banma county in Qinghai 25 KM103778-KM103783 Chengduo county in Qinghai KM103784; KM103786–KM103788; Datong county in Qinghai KM103790 KM103816-KM103819 in Qinghai KM103899–KM103904; Zhuoni county in Gansu KM103908 E. rufescens EF530742 Tanchang county in Gansu 2 AF326269 Ningshan county in Shaanxi E. smithi KM103905-KM103909 Zhuoni county in Gansu 4 M. aspalax KY754052 Unknown 1

Figure 2. The phylogenetic tree based on 13 protein-codon genes of mitochondrial genome of Muroidea (BI). The branch in bold represents Spalacidae. The branch of bamboo rat is in green, mole rat in red and zokor in blue, respectively. mitochondrial genome (Lanfear et al. 2017). Maximum- MEGA X. The pairwise genetic distances among species likelihood (ML) tree of Muroidea was inferred by IQ-TREE were estimated based on Kimura 2-parameter (K2P) model 2.0 (Nguyen et al. 2015), and Bayesian inference (BI) tree (Kimura 1980). The best-fit model was tested by jMo- was constructed by MrBayes 3.1.2 software (Ronquist and delTest2 (Darriba et al. 2012) and PartitionFinder2 (Lanfear Huelsenbeck 2003). et al. 2017). The ML and BI methods were used to construct Forty Cytb sequences of five species were obtained interspecific phylogenetic trees of Myospalacinae by from GenBank (table 2) including E. cansus, E. baileyi, RAxML 7.0.3 (Alexandros 2006) and MrBayes 3.1.2 soft- E. rufescens, E. smithi and M. aspalax. These sequences ware, respectively (Ronquist and Huelsenbeck 2003). were combined with the 55 Cytb sequences sequenced in this Besides, the divergence time within Eospalax was esti- experiment for analysis. All sequences were aligned using mated by BEAST v1.10.1 (Drummond and Rambaut 2007; Clustal X 1.81 (Jeanmougin et al. 1998). The base compo- Bouckaert et al. 2014) based on the calibration time of fossil sition and the numbers of variable sites were calculated on record. By investigating the classification and evolutionary Taxonomy and phylogenetic relationship of zokors Page 5 of 10 38

Table 3. The genetic distances among zokors based on Cytb gene.

Species E. rothschilidi E. cansus E. rufescens E. baileyi E. smithi

E. rothschilidi E. cansus 0.1267 E. rufescens 0.1364 0.1321 E. baileyi 0.1269 0.1199 0.1294 E. smithi 0.1465 0.1291 0.1347 0.1253 M. aspalax 0.1838 0.1752 0.1983 0.1832 0.1525

Figure 3. Phylogenetic tree of six species of zokors based on Cytb (ML). The name of species consists of name ? accession number. MA, M. aspalax (blue), EB, E. baileyi (red); EC, E. cansus (black); ER, E. rothschildi (green); RU, E. rufescens (purple); ES, E. smithi (orange).

relationships of zokor, Zheng (1994) suggested that the Monte Carlo (MCMC) as the search strategy, and uncorre- divergence time between Eospalax and Myospalax was lated lognormal relaxed clock as model. The option of tree about 4 million years ago (Ma), from which time distinctly prior was Yule process, and TN93 was the model of base differentiated of the two genera are known. This time substitution. The credibility of the results was tested with node was used as a calibration node to estimate the diver- Tracerv1.7.1 software (If ESS[200, the results were credi- gence time. The major parameters taken were: Markov chain ble) (Drummond and Rambaut 2007). 38 Page 6 of 10 Yao Zou et al.

Figure 4. Phylogenetic tree of six species of zokors based on Cytb (BI). The name of species consists of name ? accession number. MA, M. aspalax (blue); EB, E. baileyi (red); EC, E. cansus (black); ER, E. rothschildi (green); RU, E. rufescens (purple); ES, E. smithi (orange).

Results (accession number are shown in table 1). The base content of A, T, G and C were: 30.8%, 30.5%, 26.5% and 12.2%, The taxonomic and phylogenetic status of Myospalacinae respectively. The content of T?A(61.3%)ofCytb gene was higher The best partitioning scheme was specified by Parti- than that of G?C. Types of variation including inser- tionFinder2, and 29 subsets were included. As shown in tion, deletion, transformation and transversion were figures 1 and 2, the species of Muroidea in phylogenetic tree detected by MEGA X. Transformation is the main were divided into three clades based on 13 protein-codon variation type. Two hundred and ninety-five variable genes of mitochondrial genome. The species of family sites, 255 parsimony informative sites and 38 SNP sites and Spalacidae formed their own were detected. monophyletic group respectively, and the family Cricetidae Ninety-five Cytb gene sequences were obtained by and Muridae clustered into one clade. Within the family combining 55 Cytb sequences sequenced in this study Spalacidae, the bamboo rat formed a basal clade, the zokor with 40 Cytb sequences downloaded from GenBank. The and mole rat were grouped together. estimation of genetic distance among zokors based on K2P model is shown in table 3. It was suggested that the genetic distances among five species in Eospalax were Analysis of Cytb sequences and genetic distance smaller, ranging from 0.1199 to 0.1465. However, the genetic distances between zokors of Eospalax and M. Fifty five complete Cytb (1140 bp) sequences of three aspalax of Myospalax were larger, ranging from 0.1525 to species were obtained and analysed in this study 0.1983. Taxonomy and phylogenetic relationship of zokors Page 7 of 10 38

The interspecific and intraspecific phylogenetic relationships zokor and its closely related species were clarified based on of zokor complete mitochondrial genome of 20 species of Muroidea. Three major clades were differentiated in phylogenetic tree, ML and BI trees based on Cytb gene are shown in figures 3 the family Spalacidae formed one monophyletic clade con- & 4, respectively. One subset of GTR?G model was spec- sisting of the subfamily Myospalacinae, Spalacinae and ified by jModelTest and PartitionFinder2, and the topologi- Rhyzomyinae. Within the family Spalacidae, the Rhy- cal structure of the two trees were basically consistent based zomyinae formed a basal clade, the Myospalacinae and on GTR?G model. Most clades were well supported. The Spalacinae clustered together. Therefore, it was reconfirmed whole phylogenetic tree was divided into two major clades, that the zokor belong to the family Spalacidae and it is closer one was M. aspalax from Myospalax, the other was from to mole rat rather than bamboo rat. Eospalax including species of E. cansus, E. rufescens, The species of the family Spalacidae which belongs to E. smithi, E. rothschilidi and E. baileyi. There were two suborder Myomorpha were typically subterranean rodents clades within Eospalax, E. baileyi was an early differentiated with a long evolutionary history. The earliest fossil record of clade, the other four species grouped together as the other this family is dated to the late , and the species of clade. Among the other four species in ML tree, E. rufescens this family was earlier differentiated in Muroidea (Wilson and E. smithi clustered as a sister group, afterwards grouped and Reeder 2005). Zokor retained an underground lifestyle with E. rothschilidi, and finally grouped with E. cansus. in the family Spalacidae, but it differed from the species of Most clades were well supported, but other small clades the other two subfamilies within Spalacidae. Zokor digs within species were not. The phylogenetic relationships tunnels with its developed forelimbs, while mole rat uses among zokors were well resolved. protruding upper incisors for excavation because their fore- Some clades within species were well supported. As limbs are undeveloped (Lacey et al. 2001). Bamboo rat use shown in figure 4, the samples of E. cansus were distinctly both forelimbs and upper incisors (Norris et al. 2004). Zoker divided into two clades. One represented the samples col- share more similarities with bamboo morphologically (Flynn lected in this study from southern Ningxia, and the other and Lawrence 2009). represented the samples downloaded from GenBank from Before the emergence of molecular biology technology, Qinghai and Gansu provinces. Besides, the phylogenetic species were often classified and identified using morpho- trees constructed by ML and BI methods were well sup- logical traits and fossil evidences. The morphological char- ported within E. baileyi. The samples of E. baileyi were acteristics of zokor shared similarities with , such as distinctly divided into two clades, the samples from long claws, small eyes and ears, cutinized nose, strong Chengduo county of Qinghai province formed a mono- zygomatic arch and elongated olecranon (Tullberg 1899; phyletic clade of early differentiation, the other clade was a Lawrence 1991). Debates have been raised about the taxo- cluster of samples from Banma county, Datong county and nomic status of zokor based on morphology (Pavlinov Guinan county of Qinghai province and Zhuoni county of 2003). However, there are still limitations in morphological Gansu province. classification, especially for rodents like zokor living underground tunnels, who have adapted convergently underground lifestyles at different levels. Recently, with the Divergence time estimation rapid development of molecular biology technology, DNA molecular technology has shown good reliability in defining Using the time node as a calibration node, the divergence intraspecific and interspecific relations, and revealing the time to the most recent common ancestor of the two clades genetic structure of species (Marshall 2005). Similar within Eospalax was estimated at 3.71 Ma (3.43–4.00 Ma, researches have been conducted by some studies. For 95% HPD). As a result, it is speculated that the differenti- example, studies based on concatenated nuclear gene IRBP ation of two clades within Eospalax occurred in the late and GHR showed that bamboo rat differentiated earlier, mole . rat and zokor were most closely related (Jansa et al. 2009). Besides, by analysing the fossil morphology of Spalacinae and Rhyzomyinae, Perdiguero et al. (2015) suggested that Discussion bamboo rat and mole rat were not from a common ancestor, and they were differentiated at late Oligocene. These views The taxonomic and phylogenetic status of Myospalacinae were also supported by the phylogenetic analyses in this study. In this study, for the first time complete mitochondrial As mentioned previously, there were still controversies in genome was used to investigate the phylogenetic status of taxonomic and phylogenetic status of Myospalacinae. Myospalacinae, which has provided molecular evidences for Myospalacinae was classified into the family Muridae, classification and evolutionary relationships of Myospalaci- Cricetidae, Spalacidae, and the single family Myospalacidae nae. However, molecular evidences still showed that zokor in previous studies. In this study, the taxonomic status of were closer to bamboo rat rather than mole rat. By obtaining Myospalacinae and phylogenetic relationships between transcriptome data of three subfamilies, Lin et al. (2014) 38 Page 8 of 10 Yao Zou et al. suggested that zokor was more closely related to bamboo rat. 3.46 Ma and 3.82 Ma, respectively, which were consistent To further clarify the phylogenetic relationships among the with the results of this study. According to the record of three subfamilies, phylogenetic analysis could be performed literature, the rapid and intensive uplifting events of Qing- by combining multiple genes and morphological data in hai-Tibet plateau (QTP) began around 3.6 Ma (Li and Fang further study. 1998). These events had tremendous influence on the gen- eral circulation of atmosphere in Asia, and further had a huge impact on the ecological environment and biological Phylogenetic status and relationships within Myospalacinae evolution of northern China (Li and Fang 1998;Suet al. 2013). The physiognomy of the Loess plateau was formed In this study, the classification of the two genera of Eospalax by accumulation of dust from Gobi desert. As a result, it is and Myospalax and phylogenetic status within genera were speculated that the climate and ecology changes caused by affirmed based on mitochondrial Cytb gene. Extant zokors the QTP uplift in 3.6 Ma led to the divergence of two clades belong to Myospalacinae, which consists of two types, Eos- in Eospalax. palax and Myospalax. The morphological classification of the The samples of E. cansus were distinctly divided into two two types depends on whether there is convex structure in clades in phylogenetic tree. One represented the samples occipital. The occipital is distinct convex in the species of from Haiyuan and Xiji counties of Ningxia, and the other Eospalax,butflatintheMyospalax. There have been disputes represented the samples from Datong and Minhe counties of about whether these two types of zokors are at the genera level Qinghai, and Tianzhu county of Gansu provinces. These two or subgenera level. These two types were first considered as clades represented two geographical populations respec- two subgenera Eospalax and Myospalax (Allen 1940; Corbet tively. Therefore, the isolation by geographical distance and Hill 1991;Lawrence1991). However, more and more plays a key role in the intraspecific differentiation of the molecular evidences suggested the division of the two genera species. Besides, it is shown that divergent pattern of E. until the genus status for convex and flat occiput types were baileyi was characterized by allopatric divergence spatially. proposed by Zheng (1994)(Norriset al. 2004; Smith and Xie Within species of E. baileyi, the clade consists of samples 2009). Currently, more attentions have been paid to the clas- from Chengduo county of Qinghai represented the interior sification of the two genera Eospalax and Myospalax.The clade of QTP, the other clade consists of samples from phylogenetic trees constructed by ML and BI methods in this Banma county, Datong county and Guinan county of study were divided into two major clades including Myospalax Qinghai and Zhuoni county of Gansu represented the clade and Eospalax. Smaller genetic distances were found among of edge of QTP. Tang et al. (2010) reached the same con- species within Eospalax (0.1199–0.1465), larger genetic dis- clusion by investigating the phylogeography of E. baileyi tances were recorded between the species of Myospalax and based on mitochondrial Cytb gene, the population of E. Eospalax (0.1525-0.1983). The classification of the genera of baileyi were divided into the clade of the interior of QTP and Eospalax and Myospalax were reconfirmed in this study. that of the edge of QTP. The E. baileyi in Chengduo county Similarly, by analysing the mitochondrial Cytb and 12srRNA was considered as the most ancient population so far (Zhao genes of zokors, Zhou and Zhou (2008) found that the genetic et al. 2015), and some of them spread to the north and east differences between Eospalax and Myospalax were larger than afterwards, which was consistent with the results in this those within genera. The genetic differences between two study. It was speculated that the pattern of geographical genera were also found larger than those within genera (Su distribution of E. baileyi was related to Quaternary dias- et al. 2013). These findings were consistent with the results in trophisms and glaciations in QTP (Tang et al. 2010). this study. Accurate classification and phylogenetic relationships of There were a few researches on the phylogenetic rela- species are basis for understanding and studying the species. tionships of species in Eospalax due to insufficient samples. In this study, the controversial issues on taxonomy and The phylogenetic relationships of species in Eospalax have phylogeny of Myospalacinae were elucidated at molecular not been well resolved. Results in this study showed that level. The molecular evidences of taxonomy and phylogeny there were two clades within Eospalax, E. baileyi was a of Myospalacinae have been supplemented in this study, clade of early differentiation, the other four species grouped which would be significant to understanding the evolution- together as another clade. It is shown that E. baileyi was ary process and to clarifying the mechanism of differentia- relatively ancient species in this lineage of ML tree, followed tion of Myospalacinae. To make the research results more by E. smithi and E. rufescens, which were closely related. reliable, more samples should be supplemented and phylo- This was consistent with the result of phylogenetic rela- genetic analysis could be performed by combining multiple tionships in Eospalax based on Cytb sequences (Su et al. genes in further study. 2013). Phylogenetic analysis based on Bayesian method indicated that there may be two origins in Eospalax, and the Acknowledgments divergence time was estimated at 3.71 Ma during the late Pliocene. Based on D-loop and ND4, the divergence time of We sincerely thank Forest Disease and Pest Control Quarantine two clades in Eospalax were estimated by Su et al. (2013)at Station of Ningxia Hui Autonomous Region for collecting samples Taxonomy and phylogenetic relationship of zokors Page 9 of 10 38 of Gansu zokor. This work was supported by the National Key Li X. C. and Wang T. Z. 1996 The classification and phylogeny of Programme of Research and Development (2017YFD0600103-4-1) the genus Eospalax. J. Shaanxi Normal Uni. (Nat. Sci. Edi.) 3, and National Promoted Programme of scientific and technological 75–78. achievements in Forestry and Grassland (201929), and the Key Lin G. H., Wang K., Deng X. G., Nevo E., Zhao F., Su J. P. et al. 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