Phylogeographic Structure in the Chromosomally Polymorphic Rodent Cricetulus Barabensis Sensu Lato (Mammalia, Cricetidae)

Received: 8 March 2018 | Revised: 23 August 2018 | Accepted: 26 August 2018 DOI: 10.1111/jzs.12251

ORIGINAL ARTICLE

Phylogeographic structure in the chromosomally polymorphic rodent Cricetulus barabensis sensu lato (Mammalia, Cricetidae)

1A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Abstract Moscow, Russia Striped hamsters (Cricetulus barabensis sensu lato) represent a complex of chromoso- 2Department of Vertebrate Zoology, mally distinct allopatric lineages/taxa of either species or subspecies rank. They are Moscow State University, Moscow, Russia 3Institute of Pathology, City Hospital widely distributed across the steppes of eastern and central Palearctic. Phylogenetic Dessau, Dessau-Rosslau, Germany analysis of cytochrome b gene sequences based on 496 specimens from 112 locali- 4 Federal Scientific Center of the East Asia ties revealed five well‐supported lineages divergent at 2%–4%. Two of them corre- Terrestrial Biodiversity Far East Branch, Russian Academy of Sciences, Vladivostok, spond to “griseus” (2n = 22) and “pseudogriseus” (2n = 24) karyomorphs and are Russia placed as sister taxa. The “barabensis” (2n = 20) karyomorph is represented by three 5 Institute of Natural Resources, Ecology ‐ and Cryology, Siberian Branch, Russian other branches and appears non monophyletic. All mtDNA lineages are distributed Academy of Sciences, Chita, Russia allopatrically or parapatrically; no indications of gene flow between populations of 6 State Nature Biosphere Reserve different chromosomal races were found. The results of the molecular clock analysis “Daursky”, Nizhny Tsasuchey, Zabaykalsky Kray, Russia suggest that the main lineages diverged in the late Middle Pleistocene. The inferred 7Zoological Institute, Russian Academy of evolutionary scenario implies that the common ancestor of the recent lineages Sciences, Saint Petersburg, Russia belonged to the 2n = 20 karyomorph and originated in the eastern part of the con- 8Zoological Museum of Moscow State University, Moscow, Russia temporary range.

Correspondence KEYWORDS Natalia Poplavskaya, A.N. Severtsov Institute Palearctic, phylogeography, Pleistocene, Rodentia, speciation of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia. Email: [email protected]

Funding information Russian Science Foundation, Grant/Award Number: 14-50-00029; Russian Foundation for Basic Research, Grant/Award Number: 12-04-00662-a, 12-04-10047-k, 16-34- 60086 mol_а_dk, 17-44-170696

Contributing authors: Anna Bannikova (hylomys@mail.ru); Karsten Neumann ([email protected]); Marina Pavlenko ([email protected]); Irina Kartavtseva ([email protected]); Yuriy Bazhenov ([email protected]); Pavel Bogomolov ([email protected]); Alexey Abramov ([email protected]); Alexey Surov ([email protected]); Vladimir Lebedev ([email protected])

1 | INTRODUCTION Malygin, 1988). Although C. sokolovi is closely related to C. barabensis, its full species rank has now received decisive sup- In contrast to the present interglacial stage, the cold stages of Pleis- port from molecular and chromosome staining data (Poplavskaya, tocene are characterized by a radical expansion of open landscapes Romanenko, et al., 2017). across most of Palearctic Eurasia, thus causing drastic changes in dis- In this study, we evaluated the phylogeographic structure and tribution and abundance of steppe and desert species. However, our phylogenetic relations of taxa within C. b. barabensis sensu lato using current understanding of Quaternary climatic change effects on the sequences of the mitochondrial cytochrome b gene (cytb) from speci- biodiversity of Eurasian mammals is largely based on the data on for- mens covering a large portion of the range and reviewed the avail- est (for review see Hewitt, 1999, 2001; Taberlet, Fumagalli, Wust‐ able data on morphological, chromosomal and nuclear variation. To Saucy, and Cosson, 1998; Bilton et al., 1998; Arbogast & Kenagy, provide an insight into past range dynamics and historical demogra- 2001; Deffontaine et al., 2005; Oshida, Abramov, Yanagawa, & phy and to identify the historical and ecological factors that shaped Masuda, 2005; Kotlík et al., 2006; Rowe, Heske & Paige, 2006;) or the actual genetic variability of the C. barabensis species group, we tundra species (Fedorov, Goropashnaya, Jaarola, & Cook, 2003; Ban- also estimated the timing of divergence among the major geographi- nikova et al., 2010). Although present‐day steppe and desert belts cal lineages and performed ancestral area reconstructions. occupy a large part of Asia, phylogeographic studies of open‐dwelling species (Gündüz et al. 2007; Sorokin & Kholodova, 2006; Pet- 2 | MATERIALS AND METHODS rova, Zakharov, Samiya, & Abramson, 2015; Neumann et al. 2017) are still rather scarce and do not allow for the elucidation of the 2.1 | Sampling general trends in the past dynamics of arid fauna. Palearctic hamsters are an important element of small mammal The sample examined includes 496 specimens of C. barabensis s. l. communities across Eurasian steppes and deserts. Among them, collected in 112 localities representing a large part of the range striped hamsters of the Cricetulus barabensis species group are (Figure 1, Supporting Information Table S1). All animals were widespread in open landscapes of southern Siberia, Mongolia, the assigned to morphological groups (subspecies) recognized by Lebe- Russian Far East and China. The taxonomy of the group is contro- dev & Lisovskii (2008). The morphogroup corresponding to a yet versial due to the unclear status of the three allopatric kary- undescribed subspecies from East Transbaikalia is designated as C. b. omorphs: “barabensis” (2n = 20), “griseus” (2n = 22) and ssp. A. The closely related species C. sokolovi and Cricetulus longicau- “pseudogriseus” (2n = 24). They differ from each other by one or datus (Milne‐Edwards, 1867) were used as outgroups (13 individuals two Robertsonian rearrangements and X‐chromosome morphology in total). A large number of specimens were collected in the course (Kral, Radjabli, Grafodatsky, & Orlov, 1984; Romanenko et al., of fieldwork in Mongolia and other regions over the period from 2007). Some authors treat “griseus” and “pseudogriseus” as sepa- 1995 to 2013 by the joint Russian‐Mongolian biological expedition rate species (Malygin, Startsev & Zima, 1992, Gromov & Erbaeva, and were deposited in the Zoological Museum of the Lomonosov 1995), while others classify them all in the polymorphic Cricetulus Moscow State University. Cytb sequences were obtained for 486 barabensis (Pallas, 1773) sensu lato (Lebedev & Lisovskii, 2008; individuals in the recent study, and 23 sequences were added in the Lebedev, 2012). A revision of intraspecific taxonomy based on analysis from GenBank. craniometrical data suggested that striped hamsters group into Detailed information on origin of all individuals is given in the seven lineages. The “griseus” and “pseudogriseus” morphs corre- Supporting Information Table S2. spond to subspecies Cricetulus barabensis griseus (Milne‐Edwards, 1867) (Great Plain of China) and Cricetulus barabensis pseudogriseus 2.2 | DNA extraction, amplification and sequencing Orlov and Iskhakova, 1975 (Central and Eastern Mongolia, Trans- baikalia), whereas the “barabensis” karyotype is shared by five mor- Total DNA was extracted from ethanol‐preserved tissues, using a phologically distinct subspecies: Cricetulus barabensis barabensis standard protocol with proteinase K digestion, phenol‐chloroform (Altai), Cricetulus barabensis tuvinicus Iskhakova, 1974 (Tuva, Burya- deproteinization and isopropanol precipitation (Sambrook, Fritsch & tia, Western and Central Mongolia), Cricetulus barabensis xinganensis Maniatis, 1989). Wang, 1980 (Amur region), Cricetulus barabensis ferrugineus Argy- The entire cytb (1,140 bp) was amplified by polymerase chain ropulo, 1941 (Far East) and an undescribed subspecies from Trans- reaction (PCR) with the forward/reverse primer combination baikalia (Lebedev & Lisovskii, 2008). It should be noted that L14728/H15985 (Lebedev, Bannikova, Tesakov, & Abramson, 2007; C. b. griseus, the Chinese hamster, is a common laboratory animal. Ohdachi, Dokuchaev, Hasegawa, & Masuda, 2001) for 464 individu- However, the genetic variation in its natural populations is not well als. Typical conditions for cytb amplification included an initial understood. denaturation at 94°C for 3 min, 30–35 cycles of 94°C for 30 s, In addition to C. barabensis s. l., the species group also annealing at 60°C for 1 min and extension at 72°C for 1 min, fol- includes the Gobi hamster Cricetulus sokolovi, Orlov and Malygin, lowed by a final extension at 72°C for 6 min. Sequencing of the 1988, which was originally described as a full species based on amplified products was conducted using the internal primers L524 distinct karyotype morphology (Orlov et al., 1978; Orlov & and H774 (Poplavskaya, Lebedev, Bannikova, Meshcherskii, & POPLAVSKAYA ET AL. | 681

3 2 4 41 49 5 51 54 1 18 39 42 43 47 48 53 52 50 56 6 17 85 55 19 83 84 45 87 104 16 82 37 44 86 103 9 15 14 21 22 35 102 7 8 34 38 46 101 57 12 13 36 81 88 93 98 58 20 80 40 89 99 7877 91 94 59 10 11 79 90 96 97 100 23 24 26 65 66 76 106 92 95 75 25 28 107 60 61 67 70 73 108 27 29 30 74 33 72 105 32 69 31 68 71 63 109 62 64

110 111

C. b. barabensis C. b. tuvinicus C. b.ssp . A C. b. xinganensis

112 C. b. ferrugineusC. b. pseudogriseus C. b. griseus

FIGURE 1 Geographical origin of the specimens of Cricetulus barabensis sensu lato used in molecular genetic analysis. Numbers correspond to those in Table 1 and Supporting Information Table S1

Surov, 2012a). The automatic sequencing was conducted using ABI prior for tree and branch lengths was used with default parameters. PRISM BigDye Terminator v. 3.1 on an ABI 3100 Avant Sequencer The analysis included two independent runs of four chains (one cold or an AB 3500 Sequencer. For 22 individuals, a large fragment of plus three heated following the default settings). The chain length cytb (924 bp) was amplified and sequenced as described in Neu- was set at five million generations with sampling every 2,000 gener- mann et al. (2005). The final alignment was constructed manually in ations. With these settings, the effective sample size exceeded 200 Bioedit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The infor- for all estimated parameters. Tracer 1.6 software (Rambaut, Suchard, mation on primers used for amplification and sequencing is given Xie, & Drummond, 2014) was used to check for convergence and in the Table S2. determine the necessary burn‐in fraction, which was 10% of the All new haplotypes of Cricetulus obtained in this study were chain length. deposited in the GenBank with the Accession Numbers MH580901– The ultrametric Bayesian tree was reconstructed in BEAST ver. MH581079 (Table S2). The complete alignment is available from cor- 1.8.2 (Drummond, Suchard, Xie, & Rambaut, 2012) from the haplo- responding author from request. type alignment under the strict clock model. The choice of the model is validated by the results of the likelihood ratio test (de- parture from H0 of rate constancy is non-significant: p > 0.25), 2.3 | Data analysis which was conducted in PAML version 4.7 (Yang, 2007) based on the ML topology. A piecewise constant coalescent skyline prior 2.3.1 | Phylogeography and phylogeny was used with ten groups. The partitioning scheme and models A maximum likelihood (ML) tree was reconstructed from the matrix corresponded to those in the ML analysis. Two runs of 50 million of cytb haplotypes (Supporting Information Figure S1) in Treefinder, generations were conducted. Parameter convergence was assessed version October 2008 (Jobb, 2008). Appropriate models of sequence in Tracer 1.6 with burn‐in set to five million steps. The maximum evolution were selected for each of the codon positions under the clade credibility tree was generated by TreeAnnotator version Bayesian information criterion (BIC) employing the routine imple- 1.8.2 (part of the BEAST package). The dates of divergence mented in Treefinder. The bootstrap analysis included 1,000 repli- between the main lineages were estimated based on the node cates. depths of the ultrametric Bayesian tree. The tree was calibrated A Bayesian tree reconstruction was conducted in MrBayes 3.2 with the use of a set of secondary calibration points from a multi- (Ronquist, & Sanmartín, 2011). Models with either two‐ or six‐rate locus study of phylogenetic relationships in Cricetinae (Lebedev et matrix parameters were selected for each partition based on the al., 2018a). This approach was used because of the lack of reliable results of the model selection for the ML analysis. Gamma Dirichlet fossil calibrations for Cricetulus. The details of the calibration 682 | POPLAVSKAYA ET AL. method are described in the Supporting Information Appendix S1. representatively sampled local populations using Arlequin, version All the trees were visualized using FigTree 1.4.2 (http://tree.bio.e 3.5.1.2 (Schneider, Roessli & Excoffier, 2000; Excoffier & Lischer, d.ac.uk/software/figtree/). 2010), significance was tested by 1,000 permutations. The number p‐distances and K2P‐distances between the main genetic lineages of synonymous and non‐synonymous substitutions was estimated were estimated using Mega 6 (Tamura, Stecher, Peterson, Filipski, & using DnaSP (Librado and Rozas, 2009). Kumar, 2013; Kimura, 1980). To partition the total genetic variance into its hierarchical com- Nine sequences with lengths <920 bp were used only for hap- ponents (among groups, among populations within groups and logroup identification and excluded from all other analyses. within populations), we performed AMOVA (Excoffier, Smouse & Quattro, 1992) using Arlequin 3.5.1.2 (Excoffier & Lischer, 2010). Groups of populations corresponded to (a) karyomorphs “baraben- 2.3.2 | Species delimitation and spatial analysis sis,”“griseus,” and “pseudogriseus”; (b) morphogroups/subspecies To define natural groups based on the mtDNA data, we employed C. b. barabensis, C. b. tuvinicus, C. b. ssp. A, C. b. xinganensis, two methods: the Automatic Barcode Gap Discovery (ABGD) C. b. ferrugineus, C. b. griseus and C. b. pseudogriseus; and 3, 4) geo- method (Puillandre, Lambert, Brouillet, & Achaz, 2012) and the Gen- graphical regions of C. b. tuvinicus and C. b. pseudogriseus (see eral Mixed Yule‐coalescent model (GMYC) (Pons et al., 2006; Fuji- Table 1), respectively. To test the hypothesis that the observed sawa & Barraclough, 2013). pattern of geographic variation can be explained by isolation‐by The automatic identification of the “barcode gap” was performed distance, simple Mantel tests (Mantel, 1967) with 1,000 permuta- using the ABGD application available at http://wwwabi.snv.jussieu.f tions were conducted in Arlequin, version 3.5, separately for r/public/abgd/abgdweb.html under the following parameters: Pmin C. b. pseudogriseus and C. b. tuvinicus, using only population sam-

(prior minimal distance) = 0.01, Pmax (prior maximal distance) = 0.1, X ples with five or more specimens. (relative gap width) = 1.0. A matrix of either Jukes–Cantor or uncorrected p‐distances was taken as input. Only initial partitions were 2.4 | Demographic analyses considered. Single‐threshold GMYC analysis was performed employing the The signatures of population demographic changes were examined GMYC web server (http://species.h-its.org/gmyc/; Fujisawa & Barr- with Tajima's D (Tajima, 1989) and Fu's FS (Fu, 1997) neutrality aclough, 2013) with the maximum clade credibility tree produced statistics using Arlequin 3.5.1.2 (Excoffier & Lischer, 2010) and by by BEAST. Delimitation methods often tend to over‐split taxa R2 statistics estimated in DnaSP (Librado and Rozas, 2009). (Carstens, Pelletier, Reid, & Satler, 2013). This may be explained by the insufficient complexity of the GMYC model, which treats TABLE 1 Geographical origin of the tissue samples of Cricetulus all variation within a species as a coalescent within a single popu- barabensis sensu lato used in the molecular genetic analysis lation. In a structured population, a coalescent can be divided into Sample scattering and collecting phases (Wakeley, 1999), in which branch Taxa Regions Loc NN size lengths are scaled differently, thus violating the assumptions of Cricetulus barabensis s.l. GMYC. To avoid over‐splitting, we tried to reduce the contribu- C. b. griseus (2n = 22) – 11 tion of intra‐deme variation relative of interdeme variation by C. b. barabensis (2n = 20) Cb_Altai 1–619 using scattered sampling as it was done by Lebedev et al. (2018b). C. b. tuvinicus (2n = 20) 200 The total sample was reassembled so that each locality was repre- Cb_Tuva 7–19 74 sented by a single sequence selected at random. The GMYC anal- Cb_Hubsugul 20–22 6 ysis was repeated 50 times; trees with a reduced number of tips Cb_Hangai 23–33 58 were generated in Ape ver. 3.4 (Paradis, Claude & Strimmer, Cb_Buryatia 34–40 61 2004). Cb_Baikal 41 1 To examine the geographical variation at a finer scale, the Spatial C. b. ssp. A. (2n = 20) Cb_Chita 42–55 26 Analysis of Molecular Variance (SAMOVA2, Dupanloup, Schneider & C. b. xinganensis Cb_Amur 56–61 17 Excoffier, 2002) was performed with the number of a priori defined (2n = 20) groups (K) varying from 2 to 10. C. b. ferrugineus Cbf_Primorie 62–64 4 (2n = 20) 2.3.3 | Genetic structure and diversity C. b. pseudogriseus 219 (2n = 24) Cbp_CentrMongolia 65–76 51 Relationships among haplotypes and their number were inferred in Cbp_Buryatia 77–85 103 NETWORK, version 4.5.0.0 (Bandelt, Forster & Röhl, 1999) using the Cbp_Transbaikalia 86–105 49 default options. Cbp_Hentey 106–108 13 Measures of diversity (number of nucleotide substitutions, haplo- Cbp_InnerMongolia 109 3 type diversity, nucleotide diversity, etc.) were calculated for POPLAVSKAYA ET AL. | 683

To test whether the observed mismatch distribution deviates sig- 3.2 | Phylogenetic analysis and clock analysis nificantly from the predictions of the sudden expansion model (Rogers & Harpending, 1992), the Harpending's raggedness index All phylogenetic trees contain five highly supported haplotype clus- (Harpending, 1994) was calculated for each of the populations with ters of C. barabensis s. l. (Figure 2, Supporting Information Figure S1). ten or more specimens as well as for joined samples of regions and These five lineages are distributed allopatrically, and p‐distances subspecies. Calculations were performed in Arlequin 3.5.1.2 (Excof- between them vary from 2.3% to 4.2% (Table 2). The details of the fier & Lischer, 2010) using 1,000 permutations. lineage content and distribution are as follows. BEAST ver. 1.8.4 software (Drummond et al., 2012) was Lineage “C. b. barabensis + tuvinicus” (“CBBT”) includes hamsters employed to explore the demographic history using skyline plots (10 of two morphological subspecies with the “barabensis” (2n = 20) groups), which were visualized in Tracer v.1.6 (Rambaut et al., 2014). karyotype: C. barabensis s. str. (6 haplotypes in 19 individuals) from These analyses were performed based on the alignments containing the Altai at the west edge of C. barabensis s. l. range and C. b. tuvini- all individual sequences belonging to a particular lineage or originat- cus (64 haplotypes in 200 individuals), which is widely distributed in ing from a particular geographic region. Tuva, Mongolia, and Buryatia. Lineage “C. b. xinganensis + ssp. A” (“CBXS”) corresponds to two 2.5 | Phylogeographic analysis (Discreet model) other morphological groups within “barabensis”: C. b. ssp. A (16 haplotypes in 26 individuals) from the Transbaikalia region and C. b. xin- ‐ To infer the phylogeographic history, we used the discrete state con- ganensis (eight haplotypes in 17 individuals) from the Amur region. tinuous‐time Markov chain (CTMC) model (Lemey, Rambaut, Drum- The CBBT and CBXS lineages are consistently recovered as sister mond, & Suchard, 2009) of phylogeographic analysis as implemented clades. in BEAST ver.1.8.4 (Drummond et al., 2012). The CTMC phyloge- Lineage “CBF” is distant from the other two “barabensis” lin- netic–biogeographic model enables the estimation of ancestral eages, includes two haplotypes (four individuals) from the Russian ranges and migration rates between predefined geographic areas Far East (Primorie region), and thus corresponds to the subspecies using the Bayesian MCMC inference. Fourteen areas were defined C. b. ferrugineus. The position of this lineage was poorly resolved; in based on the pattern of distribution of sampling localities and geo- ML and Bayesian trees, it is placed as the sister group to the clade graphical characteristics of the range (barriers, etc.). A symmetric comprising C. b. pseudogriseus and C. b. griseus. model of diffusion without BSSVS extension was implemented. Only Lineage “CBP” includes all examined individuals of C. b. pseudo- transitions between adjacent areas were allowed (fixed loc. indica- griseus (“pseudogriseus” karyotype, 2n = 24; 86 haplotypes in 219 tors parameter). individuals) and is widely distributed in central and eastern The models and priors for the tree search used corresponded to Mongolia, southern Transbaikalia and Nei Mongol Province of those implemented in the haplotype tree reconstruction, with the China. exception of skyline parameters (given the slow convergence of the Lineage “CBG” corresponds to C. b. griseus and the “griseus” geographic analysis, we reduced the number of skyline dimensions karyomorph (2n = 22). Our sample included seven haplotypes (11 to three and fixed group and population sizes to values equal to pos- individuals) representing three natural populations from China and terior means obtained in the preliminary runs without the biogeo- the laboratory strain. This lineage is placed as a sister group to the graphic model). No outgroups were used. previous one with high support. Two independent runs were performed for a chain length of 110 The time of basal split in Cricetulus (i.e., between C. barabensis million generations, and parameters were sampled every 50,000 gen- group and C. longicaudatus) was estimated at 1.986 Myr (95% erations (10% burn‐in). For each independent run, adequate sampling highest posterior density interval – HPD: 1.214–3.573) and separa- and convergence of the chains to stationarity or distribution were tion of C. sokolovi occurred at 737 kyr (95% HPD: 443–1,634). confirmed by inspection of the MCMC samples using Tracer v.1.6 The time of the basal radiation for C. barabensis s. l. was esti- (Rambaut et al., 2014). The effective sample size (ESS) values of all mated at 262 kyr (95% HPD: 159–610). The split between the parameters were above 200. CBP and CBG lineages occurred near 126 kyr (95% HPD: 70– 334), while the CBBT lineage diverged from CBXS near 140 kyr 3 | RESULTS (95% HPD: 79–343). The times of the most recent common ancestors for CBBT, CBXS and CBP were all estimated at ~43 kyr 3.1 | Characteristics of cytb sequences (95% HPDs: 14–23 to 76–116).

The number of unique haplotypes for the samples of C. barabensis s.l. was 189 within 496 sequences. The length of sequences was 3.3 | Species delimitation complete or near complete for most specimens (Supporting Informa- The unmodified GMYC procedure suggests that the optimum num- tion Table S2). The observed number of nucleotide substitutions was ber of clusters is 24, which appears to be a gross overestimate. The 251, including 212 transitions and 39 transversions. The total num- scattered sampling modification resulted in estimates ranging from 5 ‐ ber of non synonymous changes as estimated by DnaSP was 20. to 30 with most runs inferring five groups (14 of 50 runs), which 684 | POPLAVSKAYA ET AL.

1 C. longicaudatus 1 C. sokolovi

1 C. b. barabensis CBBT + 1 C. b. tuvinicus

1 C. b. ssp. A. CBXS + 1 C. b. xinganensis 1 CBF C. b. ferrugineus C. barabensis sensu lato

0.76

1 CBP C. b. pseudogriseus FIGURE 2 Phylogenetic relationships among the lineages of Cricetulus barabensis Pleistocene 1 Gelasian sensu lato based on Bayesian analysis of Calabrian mtDNA cytb haplotypes (BEAST). Posterior Ionian probabilities (above the branches) and Tarantian 1 divergence dates of the most recent Holocene CBG C. b. griseus common ancestor in million years (below

s the time bar) are shown. Bars on the branches represent the 95% highest

l lineage

ies) My 1.99 0.4 7 0.26 0.04 al groups posterior density intervals (HPDs) for the 0.14 0.03 0.13 tMRCAs of the lineages. Correspondence

chondria

(subspec between mitochondrial lineages and

rphologic

Mo Mito morphogroups (subspecies) is shown

TABLE 2 Distances between main genetic lineages of Cricetulus barabensis sensu lato and outgroup species. Values above and below diagonal correspond to K2p distances and uncorrected p-distances, respectively

CL CS CBG CBBT CBXS CBF CBP Cricetulus longicaudatus (CL) 0.144 0.155 0.152 0.148 0.151 0.149 Cricetulus sokolovi (CS) 0.128 0.093 0.090 0.084 0.086 0.082 Cricetulus barabensis griseus (CBG) 0.138 0.086 0.043 0.039 0.040 0.024 Cricetulus barabensis barabensis + 0.136 0.083 0.042 0.024 0.041 0.040 tuvinicus (CBBT) Cricetulus barabensis xinganensis + 0.132 0.079 0.038 0.024 0.042 0.039 ssp. A (CBXS) Cricetulus barabensis ferrugineus (CBF) 0.134 0.080 0.038 0.040 0.040 0.038 Cricetulus barabensis pseudogriseus (CBP) 0.133 0.077 0.023 0.039 0.037 0.037

correspond completely to the main genetic lineages described above. the optimum solution implies partition into four subsets with CBBT Subdivision into six groups (12 of 50 runs) demonstrated uncertain and CBXS lineages lumped together. results due to among‐run variation in cluster content (Supporting Based on the value of the among group variance (Va), Information Figure S2a). SAMOVA defines four to six maximally differentiated population The results of the ABGD analysis based on the Jukes–Cantor groups (Supporting Information Figure S2c). In the case of five metric are consistent with subdivision into five major lineages (Sup- groups, the result is concordant with those from the ABGD and porting Information Figure S2b). However, with simple (p‐) distances, GMYC analyses. POPLAVSKAYA ET AL. | 685

C. b. ssp. A Cbx2 Cbt44 C. b. xinganensis Cbt6 Cbt14 Cbt40 Cbt8 Cbt9 Cbs16 Cbt20 Cbs14 Cbx3 Cbt32 Cbt21 Cbt10 Cbt39 Cbt11 Cbs12 Cbt4 Cbs13 Cbx1 Cbt37 Cbt43 Cbt12 Cbx4 Cbx7 Cbx8 Cbt29 Cbt15 Cbs11 Cbs15 Cbt16 Cbt47 Cbt36 Cbt33Cbt49 Cbt50 Cbt41 Cbt38 Cbt55 Cbs4 Cbt53 Cbt30 Cbt46 Cbs7 Cbt64 Cbt56 Cbs1 Cbt3 Cbt2 Cbt61 Cbs6 Cbt58 Cbt13 Cbt51 Cbt62 Cbt23 Cbt5 Cbs3 Cbs2 Cbt1 Cbt24 Cbt7 Cbt17 Cbt45 Cbt57 Cbt22 Cbt54 Cbb6 Cbs9 Cbt52 C. b. barabensis Cbb2 C. b. tuvinicus - Tuva Cbb3 Cbt59 C. b. tuvinicus - Hangai Cbb4 C. b. tuvinicus - Buryaa Cbt18 Cbt60 Cbb5

Cbp74 Cbp23 Cbp77 Cbp44 Cbp24 Cbp50 Cbp3 Cbp72 Cbp13 Cbp15 Cbp20 Cbp38 Cbp63 Cbp7 Cbp22 Cbp34 Cbp33 Cbp64 Cbp36 Cbp53 Cbp59 Cbp31 Cbp43 Cbp69 Cbp28 Cbp62 Cbp26 Cbp14 Cbp12 Cbp56 Cbp45 Cbp11 Cbp35 Cbp81 Cbp55 Cbp65 Cbp73 Cbp6 Cbp49 Cbp40 Cbp42 Cbp9 Cbp5 Cbp68 Cbp57 C. b. pseudogriseus Cbp41 Cbp54 Cbp39 Central Mongolia Cbp37 Cbp2 Buryaa Cbp78 Cbp1 Cbp8 Cbp52 Cbp83 Cbp29 Transbaikalia Cbp60 Cbp67 Hentey Cbp48 Cbp10 Inner Mongolia Cbp79 Cbp71 Cbp30 Cbp51 Cbp18 Cbp32 Cbp84 Cbp19 Cbp25 Cbp46 Cbp17 Cbp21 Number of individuals 1 2 3 4 8 12 16 25

FIGURE 3 Median‐joining network of haplotypes Cricetulus barabensis tuvinicus (“Cbt”‐haplotypes) + Cricetulus barabensis barabensis (“Cbb”‐ haplotypes), Cricetulus barabensis xinganensis (“Cbx”‐haplotypes) + Cricetulus barabensis ssp. A (“Cbs”‐haplotypes) and Cricetulus barabensis pseudogriseus. Number of mutations are shown by black dots on the branches 686 | POPLAVSKAYA ET AL.

Supporting Information Figure S1). In the CBXS clade, neither C. b. xinganensis nor C. b. ssp. A correspond to monophyletic groups (Figure S1); however, these two geographically isolated groups of populations share no common haplotypes.

3.4.2 | Genetic structure and within‐population variation

The results of the AMOVA analyses confirm significant genetic structuring within C. barabensis s. l. (Figure 4, Supporting Information Table S3). In designs with groups corresponding to karyomorphs and morphological subspecies, the largest fraction of variance is FIGURE 4 Results of analysis of variance components in explained by the among‐group component. Variation among popula- Cricetulus barabensis sensu lato based on cytb data (AMOVA). tions within groups is rather low, in most cases being comparable to Sectors of the diagrams represent variance components that within populations. corresponding to variation between haplotypes within local For datasets with groups corresponding to geographic (regional) populations, between local populations within groups and between populations, the among‐group and among‐population variance com- groups of populations. Numbers at the bars correspond to variance component percentages ponents are higher for C. b. tuvinicus than for C. b. pseudogriseus. The results of Mantel tests demonstrate that correlation between 3.4 | Genetic diversity and phylogeographical geographical and genetic distances is more pronounced in C. b. tu- structure vinicus (r = 0.789; p = 0.002) than in C. b. pseudogriseus (r = 0.279; p = 0.075). 3.4.1 | Haplotype networks The mean level of intrapopulation nucleotide diversity is signifi- cantly lower in C. b. tuvinicus than in C. b. pseudogriseus (Mann– To examine the relationships among haplotypes, the median‐joining Whitney test, p < 0.01) (Figure 5, Supporting Information Table S4). networks were reconstructed independently for each of the three The same result is found when comparing both haplotype and major clades (CBBT, CBXS, CBP) (Figure 3). nucleotide diversity between populations of the “barabensis” and The phylogeographic structure was revealed to be moderate. In “pseudogriseus” karyomorphs (h − p < 0.05; π − p < 0.01). most cases, the subsets of haplotypes occurring in a particular area do not form monophyletic groups; however, most of them are restricted to a certain geographic region (98.4% and 95.3% of private 3.5 | Demographic history haplotypes in C. b. tuvinicus and C. b. pseudogriseus, respectively). All haplotypes of C. b. barabensis are joined in a compact monophyletic According to the results of Tajima'sD,Fu's Fu and R2 tests (Sup- cluster within the paraphyletic C. b. tuvinicus (see also the ML‐tree in porting Information Table S5), significant departures from neutral

Haplotype diversity 12. 1 08. 06. 04. 02. 0 4 9 11 12 13 14 16 33 36 37 38 50 55 60 64 66 68 70 73 74 76 77 78 79 80 81 83 87 89 105

Nucleotide diversity 0. 008 0. 007 0. 006 0. 005 0. 004 0. 003 0. 002 0. 001 0 4 9 11 12 13 14 16 33 36 37 38 50 55 60 64 66 68 70 73 74 76 77 78 79 80 81 83 87 89 105 FIGURE 5 Genetic diversity in the C. b. barabensis C. b. tuvinicus C. b.ssp . A C. b. pseudogriseus populations of Cricetulus barabensis sensu Tuva Central Mongolia Transbaikalia lato. The numbers below columns Hangai C. b. xinganensis Buryaa Hentey correspond to numbers of localities in Buryaa Figure 1, Table 1, and Table S1 POPLAVSKAYA ET AL. | 687

C. b. barabensis C. b. tuvinicus

160 0 0 3000 120 1.E–2 1.E–2 80 2000 1.E–3 1.E–3 40 1000

–4 930 1860 2790 –4 9300 18600 0 1.E 0 1.E –4 –4 –4 02468101214 02 4 6 8 10 0 1E 2E 3E 0 5E–4 1E–3 1.5E–3 2E–3

C. b. tuvinicus - Tuva C. b. tuvinicus - Hangai

0 0 800 300

600 –2 –2 1.E 200 1.E 400 1.E–3 100 1.E–3 200 4650 9300 4650 9300 14000 0 1.E–4 0 1.E–4 0 5 10 15 –4 051015 –4 –3 –3 0 5E 1E–3 0 5E 1E 1.5E

C. b. tuvinicus - Buryatia C. b. xinganensis+ ssp .

0 1.E0 300 120 1.E–2 0 200 80 1.E–2 –3 100 1.E 40 1.E–3

0 –4 4650 9300 4650 9300 14000 1.E 0 1.E–4 0 5 10 15 –4 –3 02468101214 –4 –3 -3 0 5E 1E 0 5E 1E 1.5E

C. b. pseudogriseus C. b. pseudogriseus - Central Mongolia

0 4000 1.E 0 1.E 160 3000 0 0 120 –2 2000 1.E 1.E–2 80 1000 –3 –3 1.E 40 1.E –4 9300 18600 –4 9300 18600 0 1.E 0 1.E 0 2 4 6 8 10 12 14 16 18 –4 –3 –3 –3 –3 –3 0 5E 1E 1.5E 2E 051015 0 1E 2E

C. b. pseudogriseus - Buryatia C. b. pseudogriseus - Transbaikalia

0 0 600 –2 –2 200 1.E 1.E 400 1.E–3 100 1.E–3 200

–4 9300 18600 –4 4650 9300 0 1.E 0 1.E –3 0 5 10 15 20 –3 –4 –3 0 1E 2E 0510150 5E 1E

Mismatch distribution graphs Bayesian skyline plots X-axis: Number of pairwise differences X-axis: Time Y-axis: Frequency - In years before present - above the axis Observed Simulated -In nucleotide subtitutions - under the axis Y-axis: Effective population size Confidence interval

FIGURE 6 Mismatch distribution graphs and Bayesian skyline plots for some subspecies and regional samples of Cricetulus barabensis sensu lato 688 | POPLAVSKAYA ET AL. equilibrium expectations are evident in several samples. The from Tuva; however, other statistics do not support sudden graphs of mismatch distribution and skyline plots also demonstrate expansion. signatures of demographic expansion in some cases (Figure 6). In particular, the C. b. tuvinicus, C. b pseudogriseus and C. b. xinga- 3.6 | Spatial history nensis + ssp. A lineages show significant values of Tajima'sD,Fu's Fu and R2 statistics. Mismatch distribution plots for these datasets To reconstruct the spatial history of the striped hamsters, the poste- fit well with the predictions of the sudden expansion model, and the rior probabilities of ancestral territories were defined for six clades: skyline plots are consistent with population growth as well. (a) C. barabensis sensu lato (CBSL); (b) C. b. tuvinicus + baraben- The C. b. barabensis sample from Altai demonstrates a significant sis + xinganensis + ssp. A (CBBT + CBXS); (c) C. b. barabensis + tu- value of the R2 parameter, suggesting a recent expansion in this vinicus (CBBT); (d) C. b. xinganensis + ssp. A (CBXS); (e) region may have occurred. However, the sample size is too small to C. b. pseudogriseus + griseus (CBP + CBG); and (f) C. b. pseudogriseus allow any significant conclusions. (CBP) (see Figure 7, Supporting Information Table S6). Datasets on C. b. ssp. A (Chita) and the regional population of Taking into account regions with posterior probabilities of more C. b. pseudogriseus from eastern Transbaikalia both produce significant than 10%, the results are as follows. The ancestral territory for the statistics in all tests and show good correspondence to the sudden CBSL clade is located in the eastern part of the contemporary range— expansion model in mismatch distribution analysis. Recent growth in from Hentey to Amur and Ussuri—with the summary probability the second population is also supported by the skyline plot. of 77%. The most recent ancestor of the CBBT + CBXS clade The results of the mismatch analyses, neutrality tests and skyline occurred in the territory from Buryatia to Amur with 83% proba- plots are not always mutually concordant. Thus, the skyline shape bility. Ancestral ranges for its main subclades (CBBT and CBXS) and significant Fu's value suggest that recent population growth were restricted to Buryatia–Central Mongolia (97%) and Dauria– occurred in the regional population of C. b. tuvinicus C. b. tuvinicus Amur (93%), respectively. The result for the CBP + CBG clade

(b) (d)

NH B B D C. b. barabensis H X A + 19% C. b. tuvinicus 22% CM 11% 13% 18% CBBT 36% 38% 23% (e) C. b. xinganensis D (a) C. barabensis sensu lato + A C. b. ssp. A West Baikal Dauria Amur CBXS Tuva Byur aa 76% Altai 17%

North Hangai Hentey Xingan Central Mongolia Ussuri Inner Mongolia C. b. ferrugineus 16% 17% Central China CBF 14%

16% 10% (f) 13% South C hina D B

C. b. pseudogriseus H Regions Byur aa ()B 59% Central Mongolia (C ) CBP 19% 14% H()entey H D()auria D (c) X()ingan X Inner Mongol ia (IM ) D C. b. griseus North Hangai (NH ) U()ssuri U H X CBG A)mur (A 15% 24% IM

22% 17%

FIGURE 7 Regions defined as the most probable ancestral areas for lineages of Cricetulus barabensis sensu lato. Only regions with probabilities of 10% or higher are shown. (a) most recent common ancestor (MRCA) of C. barabensis s.l.; (b) MRCA of CBBT + CBXS lineage; (c) MRCA of CBP + CBG lineage; (d) MRCA of CBBT lineage; (e) MRCA of CBXS lineage; (f) MRCA of CBP POPLAVSKAYA ET AL. | 689 suggests a more southeasterly range including Dauria, Hentey, Xin- sungorus and Ph. campbelli, 4.5% (Neumann et al., 2006). It should be gan or Inner Mongolia (79% probability), whereas the ancestral noted that the species rank of Ph. campbelli within the last pair had area of CBP is reconstructed as Buryatia, Hentey or Dauria with been considered disputable until evidence of reproductive isolation 92% probability. was obtained (Sokolov & Vasilieva, 1993). The distance between morphologically distinct subspecies M. raddei avaricus and M. r. ni- griculus was estimated at 2.3% (Neumann et al., 2006), while that 4 | DISCUSSION between the two most divergent lineages (subspecies) of Cricetus cricetus was close to 1.5% (Neumann et al., 2005; Feoktistova et al., 4.1 | Main mitochondrial lineages of C. barabensis 2017). Important exceptions to the former cases are presented by s. l.-Taxonomic implications Allocricetulus, where the distance between chromosomally distinct The three chromosomal races of C. barabensis s. l. are found to have species A. eversmanni and A. curtatus is just 2% (GenBank: contrasting levels of genetic differentiation. While “pseudogriseus” AJ973378, KP231506, KY993923), and by Anatolian populations of and “griseus” show perfect correspondence to one morphological M. brandti, in which haplotypes differing at up to 9% were found to subspecies and one well‐supported mtDNA lineage each, populations co‐occur (Neumann et al., 2017); however, both these situations of the “barabensis” karyomorph demonstrate a more complex struc- require additional examination. Summing up the above comparative ture. The latter karyotype is shared by five morphological subspecies data, we have to conclude that the level of divergence between (Lebedev & Lisovskii, 2008), for which three well‐differentiated mito- clades within C. barabensis s. l. (2.3%–4.2%) is compatible to both chondrial lineages are now revealed. In the mtDNA trees, these species and subspecies rank. “barabensis” lineages appear rather as a paraphyletic assemblage due The data on reproductive isolation between lineages is contradic- to the separate position of the Far East C. b. ferrugineus. The latter tory as well. The differentiation among “barabensis”, “pseudogriseus” result should not be accepted at face value because it can also be and “griseus” karyotypes is explained by two Robertsonian rear- explained by introgression of mtDNA from some extinct or unstud- rangements and an inversion in the X‐chromosome (Kral et al., 1984; ied taxon. To test this hypothesis, additional multilocus analysis is Romanenko et al., 2007; Vakurin, Kartavtseva, Korablev, & Pavlenko, required. It should be noted that craniometric data support C. b. fer- 2014). Based on these changes, one cannot postulate the existence rugineus as a distinct subspecies, which is, however, relatively close to of effective reproductive barriers. Hybridization experiments showed other far eastern “barabensis”: C. b. xinganensis and C. b. ssp. A from that the three karyomorphs can produce viable and fertile hybrids Transbaikalia (Lebedev & Lisovskii, 2008). According to the mtDNA (Poplavskaya, Lebedev, Malygin, & Surov, 2012b), thus suggesting data, the latter two belong to the same major lineage (CBXS). Another the lack of post‐zygotic isolation; however, some indications to its point of disagreement between morphological and mitochondrial initial stages were revealed by a cytogenetic analysis of hybrids results concerns the relationship of C. b. tuvinicus and C. b. barabensis. (Matveevsky et al., 2014). The nominal subspecies is morphologically distinct but appears as a The occurrence of hybridization in nature was studied in zones recent offshoot from C. b. tuvinicus in the mitochondrial tree. Further of contact between “pseudogriseus” and “barabensis” located in studies are needed to determine whether this pattern can be explained Central Mongolia and Buryatia (involving C. b. tuvinicus; Poplavskaya by rapid morphological evolution or mtDNA capture. et al., 2012b) and in eastern Transbaikalia (involving C. b. ssp. A; It should be noted that although the existence of five divergent Korablev, Pavlenko, Bazhenov, & Kirilyuk, 2013). In all cases, neither mitochondrial lineages can hardly be questioned, all methods of spe- sympatry of karyomorphs nor F1 hybrids were found (Poplavskaya et cies delimitation used in this study (ABGD, GMYC, SAMOVA) under al., 2012b; Korablev et al., 2013). certain conditions could partition the data into fewer or more than However, in a single population of C. b. tuvinicus in Central Mon- five groups, gross over‐splitting by GMYC (original variant) being the golia (vicinities of Kharkhorin) the karyotype with 2n = 21 was most extreme example. This indicates the unreliability of the existing observed in two hamsters among 25 examined (Poplavskaya et al., methods of species delimitation, which stems from oversimplified 2012a), which can be attributed to hybridization with C. b. pseudo- modeling assumptions. griseus. This supposition was subsequently confirmed by the admix- Applying the genetic species concept (Bradley & Baker, 2001), ture analysis based on multilocus nuclear data, which demonstrated the status of the main lineages of C. barabensis s. l. is ambiguous. a signature of weak gene flow in population of Central Mongolia According to Bradley and Baker (2001), the level of cytb divergence (Poplavskaya, Lebedev, et al., 2017). between 2% and 11% can be indicative of both intraspecific and In contrast to that, both genetic and chromosomal data support interspecific variations. This prediction is primarily consistent with the absence of gene exchange between C. b. tuvinicus and C. b. pseu- the range of values observed between pairs of sibling species of dogriseus in southern Buryatia where populations of the two morphs Palearctic hamsters. The distance between C. barabensis and C. soko- are separated by the Selenga and the Chikoi rivers (Poplavskaya lovi (~8%) suggests species‐level differentiation (Poplavskaya, Roma- et al., 2012a; Poplavskaya, Lebedev, et al., 2017). It should be men- nenko, et al., 2017). Considering other genera, the level of tioned that the latter river can hardly be regarded as an effective divergence between Mesocricetus newtoni and M. brandti is 9%; barrier for dispersal of hamsters. The deficit of evidence for gene between M. auratus and M. raddei, 6%; and between Phodopus flow cannot be explained by the recent age of the contact zone as 690 | POPLAVSKAYA ET AL. both populations demonstrate high levels of genetic variation consis- 4.4 | Steppe mammal comparative phylogeography tent with long‐term stability. Therefore, it is unlikely that geographic isolation is responsible for the lack of natural hybridization in this Both morphological and genetic data indicate that the East Central case suggesting an important role of some other, presumably prezy- Asian steppe zone is populated by two widespread lineages of gotic barrier. striped hamsters: western (C. b. tuvinicus) and eastern (C. b. pseudo- Thus, despite the relatively low level of morphological, genetic, griseus), with the putative contact zone being located in central Mon- and karyological divergence between the major lineages, at least two golia between the Hangai and Hentey mountain areas. A similar of them appear to be genetically isolated in nature and, hence, one phylogeographic pattern is observed in the long‐tailed ground squir- may regard them as incipient species. However, any decision on the rel Urocitellus undulatus (Kapustina, Brandler, & Adiya, 2014; McLean, formal taxonomic rank of the lineages in C. barabensis s. l is largely Nyamsuren, Tchabovsky, & Cook, 2018). The species complex of the arbitrary and may remain so even when ample genetic data is avail- narrow‐headed voles (Microtus (Stenocranius) gregalis sensu lato) was able. shown to include highly differentiated lineages occurring in Hangai– West Transbaikalia and East Transbaikalia–Dauria, respectively (Pet- rova et al., 2015), which demonstrates certain resemblance to the 4.2 | Molecular variation in C. b. tuvinicus and structure in striped hamsters. Notably, some characteristic steppe C. b. pseudogriseus species such as Daurian hedgehog (Hemiechinus (Mesechinus) dauuri- The levels of genetic diversity and structuring are found to differ cus), steppe zokor (Myospalax aspalax) and Daurian ground squirrel between the two wide‐range subspecies, C. b. tuvinicus and (Spermophilus dauricus) occur only in the eastern sector of the Mon- C. b. pseudogriseus, with both nucleotide and haplotype intrapopula- golian steppe belt and reach the western limit of their distribution in tion diversity being higher in the latter. Conversely, the among‐group the area between Hangai and Hentey. Taken together, these data variation is stronger in C. b. tuvinicus. This pattern can be explained indicate eastwest subdivision and suggest the existence of two major by a higher level of migration among demes in C. b. pseudogriseus refugia in Mongolian steppe in the past. To test this hypothesis fur- and, accordingly, by higher subdivision in C. b. tuvinicus due to the ther, studies on phylogeography of other widespread steppe species effect of geographic barriers (mountain ridges, extra‐arid regions, such as Brandt's vole (Microtus (Lasiopodomys) brandtii) or Daurian forested areas), which are more pronounced in the western part of pika (Ochotona dauurica) are needed. the range. Previously, a similar contrast between the levels of intrapopulation variation in these two subspecies was revealed in a 4.5 | Evolutionary scenario study based on six nuclear loci (Poplavskaya, Lebedev, et al., 2017). According to the results of our molecular clock analysis, the basal split in Cricetulus (i.e., between C. barabensis group and C. longicauda- 4.3 | Chromosomal evolution tus) occurred in the Early Pleistocene (ca. 2.0 Myr, end of Gelasian). Karyotypes of “barabensis,”“griseus,” and “pseudogriseus” differ This estimate is younger than the earliest fossils identified as from each other by the presence or absence of the medium‐sized C. barabensis or C. cf. barabensis. For example, the earliest fossils of metacentrics, which correspond to the M. auratus elements MAU1/ C. barabensis from northern China were found in the Haiyan Forma- 10 and MAU7/2 (Romanenko et al., 2007). According to the com- tion (Yushe Basin, Shanxi province), and the age is estimated at monly accepted hypothesis, the 2n =24(“pseudogriseus”) karyotype, >2.2 Myr (Wu & Flynn, 2017). In the Transbaikalia region, in which the synteny blocks MAU1, MAU10, MAU7 and MAU2 are C. cf. barabensis was also found from the Pliocene–Pleistocene for- represented by acrocentrics, is ancestral in C. barabensis s. l. (Kral mations and included in the Itantsinian fauna complex (ca. 2.6 Myr) et al., 1984; Romanenko et al., 2007). This implies that the “griseus” and the Udunga fauna complex (ca. 3.4 Myr) (Alexeeva, 2005; Alex- and “barabensis” karyotypes are derived from 2n = 24 by two cen- eeva and Erbajeva, 2008). The apparent discrepancy between fossil tric fusions. However, FISH‐based ancestral reconstructions (Poplavs- and molecular dates may be explained by identification problems kaya, Romanenko, et al., 2017) suggested that the MAU2/7 due to a low diagnostic power of dental traits for discriminating association was present in ancestral karyotypes of the genus Cricetu- among Cricetulus. We believe that Late Pliocene “C. barabensis” fos- lus and C. barabensis sensu lato + C. sokolovi clade. The topology of sils may, in fact, belong to the common ancestor of the recent spe- the mitochondrial tree is most consistent with the scenario implying cies. At the same time, we need to note that the confidence the 2n =20(“barabensis”) ancestral karyotype. This follows from the intervals of our age estimates are relatively large and, therefore, the fact that the most parsimonious reconstruction of the ancestral con- suggested scenario should be viewed as a preliminary hypothesis. dition for MAU1/10 is metacentric, as it is shared by “griseus,” The next split, which involves C. sokolovi, appears to correspond “barabensis,” and the majority of the outgroups. Therefore, we to the Mid‐Pleistocene transition (ca. 0.8 Myr), which led to the glo- believe that chromosomal evolution in C. barabesnsis s. l. involved bal aridization of Eurasia (Head & Gibbard, 2005). In contrast to two consecutive fissions (in MAU7/2 and MAU1/10), which occurred steppe‐dwelling C. barabensis s. l., the Gobi hamster is found in the in the common ancestor of C. b. griseus – C. b. pseudogriseus (MAU7/ arid semi‐desert zone of Mongolia, where it is primarily restricted to 2) and in the C. b. pseudogriseus lineage, respectively. oases. One can hypothesize that this species originated in some POPLAVSKAYA ET AL. | 691 southerly refugium in Gobi, where it was locked during one of the The times of the most recent common ancestors of the three arid phases of Pleistocene climatic oscillations. A refugial bottleneck widespread lineages (CBBT, CBXS and CBP) are all estimated as ca. may explain the fact that the karyotype of C. sokolovi is highly rear- 40 kyr. This date corresponds to the relatively warm and dry major ranged relative to other Cricetulus (Poplavskaya, Romanenko, et al., interstadial of the Last Glacial, which was favorable for the dry 2017). steppe fauna in southern Siberia (Alexeeva, 2005). It can be sug- The basal radiation of C. barabensis s. l. into three lineages gested that at that time, the CBBT expanded westward and colo- (C. b. pseudogriseus + griseus, C. b. ferrugineus and C. b. tuvini- nized Tuva and Northwest Mongolia. cus + barabensis + xinganensis + ssp. A) is thought to have occurred Demographic reconstructions and neutrality tests indicate that in the late Middle Pleistocene (ca. 260 kyr). The discreet phylogeog- pre‐LGM expansion occurred in C. b. tuvinicus and C. b. pseudogriseus raphy analysis suggests that the ancestral area was located in the but do not reject constant population size in most of regional popu- northeastern part of the recent range eastward from Buryatia (Fig- lations. However, the neutrality tests and mismatch analysis show a ure 7). Paleoclimatic reconstructions indicate that at that time, the clear signature of expansion in Transbaikalian populations of both arid and warm climate of the early Middle Pleistocene was replaced C. b. pseudogriseus and C. b. ssp. A, which most likely occurred after by progressively colder conditions, which promoted the expansion of LGM time. The Bayesian skyline plots demonstrate growth in some forest steppes in Transbaikalia (Alexeeva, 2005) and the advance of populations but only in Holocene. the deserts in northern China (Ding, Derbyshire, Yang, Sun, & Liu, The data suggest a rather recent colonization (around LGM) of 2005). the westernmost part of the range (plains northwest of the Altai According to the phylogeographic reconstructions, the set of mountains), which is populated by the nominal subspecies. The potential ancestral areas for the 20‐chromosome clade is similar to young age of C. b. barabensis is indirectly supported by low levels of that for C. barabensis s. l., covering the south Siberian range from genetic variation observed within and among its populations. The lat- west Transbaikalia to the Amur region. Compared to this, the ter are now separated from the main part of the range by the forest ancestral area of C. b. pseudogriseus + C. b. griseus is moderately belt of the northwestern Altai, which was, however, strongly frag- shifted southward, with higher posterior probabilities for SE Mongo- mented during the cold stages of the Last Glacial (Agadjanian & Ser- lia‐Xingan. dyuk, 2005). Although the paleontological data indicate the The basal splits in these two major clades, that is, separating occurrence of striped hamsters in this region through most of Late C. b. tuvinicus + barabenis from C. b. xinganensis + ssp. A and Pleistocene (Agadjanian & Serdyuk, 2005), the disagreement with C. b. pseudogriseus from C. b. griseus, occurred at approximately the the mtDNA data can be explained by repeated cycles of coloniza- same time (ca. 120–140 kyr) and correspond to the Middle/Late tion/extinction. At the same time, it is evident that, due to potential Pleistocene boundary. This time interval includes periods of contrast- introgression, the mtDNA genealogy may not fully reflect the history ing climates ranging from the cold and dry penultimate Glacial maxi- of C. barabensis lineages. Although all hypotheses proposed here mum (MIS6) to the warm and humid Last Interglacial (MIS5e). Thus, should be verified by nuclear data, it is evident that the eventful it is unclear which conditions correlate with spatial expansion and, phylogeographic history of (striped) hamsters can provide important hence, promote diversification in hamsters. Based on the ecological clues to the past dynamics of Palearctic steppe fauna. affinities of recent Cricetulus barabensis, one may speculate that neither cold glacial phases nor humid interglacial phases are favorable ACKNOWLEDGEMENTS for population increases. It is more reasonable to expect that range expansions occurred during glacial–interglacial transitions or intersta- The authors are indebted to our late colleague Dr Vladimir Korablev dials when the climate is already warm but still dry enough for per- for invaluable contribution in the study. The authors are also grateful sistence of steppes. to A.K. Agadjanyan, O.V. Brandler, N.Yu. Feoktistova, I.G. Mescher- While the number and position of refugia during cold phases of sky, A.A. Lisovskii, N.V. Lopatina, V.V. Rosina, B.I. Sheftel and L.L. penultimate glacial cycle are uncertain, one may hypothesize that Voyta, for providing the tissue specimens of hamsters. The reported during the Kazantsevo (Eemian) interglacial (130–110 Kyr), the two study was funded by RFBR, according to the research project No. main 20‐chromosome sublineages (CBBT and CBXS) were restricted 16‐34‐60086 mol_a_dk (phylogenetic analysis and the processing of to disjoint refugial areas; following the discreet phylogeography anal- the manuscript) and Russian Science Foundation, project 14‐50‐ ysis, these areas could be located in central Mongolia/west Trans- 00029 (collection of material and genetic studies). Specimens of baikalia and the Amur region, respectively. Range fragmentation can hamsters from the Tuva, Transbaikalia, Amur region and Russian Far be linked to the interglacial expansion of forests in the Hentey–Han- East were collected with the foundation of RFBR projects No 17‐44‐ gai area (Golubeva, 1978). 170696, 12‐04‐10047‐k and No 12‐04‐00662‐a. Accordingly, it can be supposed that during the Middle/Late Pleistocene transition, the C. b. griseus lineage colonized (or recolo- nized) the southern part of the range. However, this hypothesis CONFLICT OF INTEREST should be verified with additional sampling from China. The authors declare that they have no competing interests. 692 | POPLAVSKAYA ET AL.

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