Genetic analysis of type material of the Amur lemming resolves nomenclature issues and creates challenges for the taxonomy of true lemmings (Lemmus, Rodentia: Cricetidae) in the eastern Palearctic

Genetic analysis of type material of the Amur lemming resolves nomenclature issues and creates... Abstract The proper use of species names depends entirely on verifying whether newly analysed specimens are conspecific with the type material. True lemmings (Lemmus) are the most common rodents of the Arctic tundra in the Old and New World and play an important role in the Arctic ecosystem; however, their taxonomy is far from resolved. The Amur lemming (L. amurensis) is the least studied and most enigmatic species of the genus. Its taxonomic position, distribution and nomenclature are uncertain due to a lack of cytogenetic, molecular phylogenetic and hybridization studies. Assignment of all true lemmings from the vast territory of western Beringia to this species has never properly been confirmed. Moreover, the type locality for this species was flooded by a newly created reservoir in 1974, making additional topotypes unavailable. In this context, genetic analysis of museum specimens, especially type material, has great potential. Here we report partial cytochrome b sequences extracted from all specimens identified as L. amurensis stored in the two largest mammal collections in Russian museums, including the holotype of L. amurensis and the type material of all forms currently considered as synonyms of L. amurensis. Phylogenetic analyses suggest that the range of the Amur lemming is dramatically smaller than previously assumed and is limited to the territories of Transbaikalia, South Yakutia and Amur in the eastern Palaearctic. Lemmus taxa from other territories, including L. amurensis ognevi and L. amurensis flavescens, refer to other lemming species. Our results impinge on nomenclature issues, taxonomy, divergence times and the evolutionary history of lemmings in the eastern part of the Palearctic and on the species concept as applied to the genus Lemmus. INTRODUCTION True lemmings of the genus Lemmus Link, 1795 are the most common rodents of the Arctic tundra in the Old and New World and play an important role in the Arctic ecosystem. The taxonomic subdivisions of Lemmus remain rather controversial. It is, however, commonly accepted that the genus splits into two branches with a geographical border along the Kolyma River. The ‘Palaearctic branch’ extends from Scandinavia to the western shore of the Kolyma River and the ‘Nearctic branch’ inhabits the tundra on the eastern shore of the Kolyma River, the Anadyr lowlands, the Chukotka Peninsula and on the other side of the Bering Straits. These lemming categories differ in karyotype: Palearctic branch lemmings have only acrocentric chromosomes (2n = NF [fundamental number] = 50), whereas in Nearctic branch lemmings one chromosome is subtelocentric (2n = 50, NF = 52) (Rausch & Rausch, 1975; Gileva, Kuznetsova & Cheprakov, 1984; Chernyavskiy et al., 1993; Chernyavskiy & Kartavtseva, 1999). Experimental hybridization between Palearctic and Nearctic branch lemmings produces fertile females but sterile males (Rausch & Rausch, 1975; Pokrovskiy, Kuznetsova & Cheprakov, 1984). Mitochondrial DNA data (Fedorov et al., 1999; Abramson, Kostygov & Rodchenkova, 2008) also suggest differentiation of Palearctic and Nearctic branch lemmings. The Nearctic branch of true lemmings consists of a single species – L. trimucronatus Richardson, 1825, whereas the taxonomic structure of the Palearctic branch is problematic. The number of species and criteria for their delimitation within this branch remain unclear. Conventionally, the Palearctic branch consists of three allopatric species, differing in size and colour: the Norway lemming, L. lemmus L., 1758, the polytypic Siberian lemming, L. sibiricus Kerr, 1792 and the polytypic Amur lemming, L. amurensis Vinogr., 1924. However, both coat colour and size are features that are subject to geographic, seasonal and age variation and there is a lack of characteristics in skull and dental morphology that can reliably discriminate between the three putative species. Among these, the Amur lemming is the least studied and most enigmatic species of the genus. Vinogradov (1924) described it based on a single specimen trapped in 1914, close to where the town of Zeya is now (Fig. 1: Locality 1). This is in the Amur Region and the specimen was deposited in the collection of the Zoological Institute (ZIN) in St. Petersburg (N 13722). The discovery of this lemming from 53.7°N farther south than the previous Lemmus range makes it especially interesting. Earlier, all representatives of the genus were only known from the tundra zone. Vinogradov (1925) suggested that the Amur lemming is a relict form isolated from other species of the genus. Decades later, the same author used a single specimen trapped at the Nelgese River in Yakutia (Fig. 1: Locality 6) to describe a subspecies of the Amur lemming – L. a. ognevi (Vinogradov, 1933). The site of the new specimen is ~1500 km north of the first finding. Interestingly, these two specimens are similar in size, but markedly different in colour. Later on in 1938, small numbers of Amur lemmings were trapped in the Transbaikal area (Chita Region). The next collections came 30 years later; around ten specimens were caught in the type locality in 1970. All these specimens were deposited in the Zoological Museum of Moscow University (ZMMU). However, the type locality was shortly afterwards (1974) flooded by the newly created Zeya reservoir and subsequently no one has successfully trapped lemmings in this region despite multiple attempts. Towards the end of the 20th century, a sustainable population of lemmings, referred to as L. amurensis, was discovered near the village of Chulman in South Yakutia (Chernyavskiy et al., 1980; Revin, 1983; Chernyavskiy, 1984). This site is ~500 km north of the type locality (Fig. 1: Locality 3). Only this population has been used for cytogenetic analysis and hybridization studies (Gileva et al., 1984; Pokrovskiy et al., 1984). Since then, any Lemmus trapped in the upper reaches of the Kolyma River, in the Magadan Region, 2000 km from the type locality were attributed to L. amurensis (Chernyavskiy, 1984). Moreover, lemmings from the Kamchatka Peninsula (Vinogradov, 1925) that were earlier referred to as a subspecies of L. sibiricus (Pavlinov & Rossolimo, 1987), L. s. flavescens, were redesignated as a subspecies of L. amurensis based primarily on small body size and molar features (Chernyavskiy et al., 1993). Consequently, L. flavescensVinogradov, 1925 has become a junior synonym of the Amur lemming (Carleton & Musser, 2005; Tsytsulina, 2008). Thus, in a piecemeal way, a rather wide distribution has become apparent for the Amur lemming in the northeast Palearctic. It is very important to underline that no cytogenetic, molecular phylogenetic or hybridization studies have made use of animals from the terra typica of L. amurensis. For cytogenetic and hybridization studies of L. amurensis (Gileva et al., 1984; Pokrovskiy et al., 1984), researchers have used animals from Chulman and, until now, only lemmings from one site on the east coast of the Kamchatka Peninsula have been used for the analysis of cytochrome b (cytb) sequences (Fedorov et al., 1999; Abramson et al., 2008). It should be noted that according to the holotype description, the Amur lemming differs from other species of lemmings by two main features: a significantly smaller body size and a distinctive coloration (Vinogradov, 1924). Of these features, size is particularly unreliable. For instance, a very pronounced clinal variation in size is typical for lemmings: animals from arctic tundra and islands are considerably larger than lemmings from southern populations inhabiting subarctic tundra and forest-tundra (Krivosheev & Rossolimo, 1966) and adult animals born in winter are smaller than animals of the summer cohort. The most valuable defining feature of Amur lemmings is coloration. Unlike all other lemmings, they have a practically uniform dark brown dorsal surface, sometimes with a more or less pronounced black stripe on the neck, strong rufous-brick cheeks and sides and a uniformly fulvous belly, paler than the sides (Vinogradov, 1924; Hinton, 1926). The attribution of lemmings from Chulman, eastern Kamchatka, the Kolyma uplands and the Magadan area to the Amur lemming was made exclusively based on small size and is therefore unreliable. Figure 1. View largeDownload slide A, Lemmus species ranges. B, collection sites of museum specimens originally classified as the Amur lemming. Locations are colour-coded according to cytb lineages, as shown in Figure 2. Yellow and blue stars are the lectotype and holotype accordingly. Small black dots represent collection sites of museum specimens for which cytb did not amplify (with numbers) or collection sites of specimens recorded in the literature (without numbers). Locality numbers correspond to Tables 1 and 2. *Species ranges are given according to Henttonen (2008), Linzey (2008), Tsytsulina (2008) and Tsytsulina, Formozov & Sheftel (2008) with the mitochondrial lineage distributions following Fedorov et al. (1999) and Abramson et al. (2008). Figure 1. View largeDownload slide A, Lemmus species ranges. B, collection sites of museum specimens originally classified as the Amur lemming. Locations are colour-coded according to cytb lineages, as shown in Figure 2. Yellow and blue stars are the lectotype and holotype accordingly. Small black dots represent collection sites of museum specimens for which cytb did not amplify (with numbers) or collection sites of specimens recorded in the literature (without numbers). Locality numbers correspond to Tables 1 and 2. *Species ranges are given according to Henttonen (2008), Linzey (2008), Tsytsulina (2008) and Tsytsulina, Formozov & Sheftel (2008) with the mitochondrial lineage distributions following Fedorov et al. (1999) and Abramson et al. (2008). For the present study, the goal is to obtain molecular data to clarify the taxonomy, phylogenetic relationships and nomenclature of representatives of the genus Lemmus in the eastern Palearctic using material from museum collections, including type specimens. This is especially important for the Amur lemming as it is impossible to obtain new topotypes and for the proper use of species names, it is essential to verify whether newly analysed specimens are conspecific with the type specimen (Santos et al., 2016). Thus, we first analysed cytb sequences obtained from the type material of all forms currently referred to L. amurensis and then from all available museum samples identified as L. amurensis, deposited in the two largest mammal collections in Russian museums. We compared new sequences with existing published data for Lemmus from all parts of the range of the genus in the Palearctic. MATERIAL AND METHODS Material Information on the geographic origin and year of collection of the museum specimens studied and attributed to L. amurensis is given in Table 1 and Figure 1. A total of 53 cytb sequences of other Lemmus species were retrieved from GenBank and used in the phylogenetic analysis (Table 2). Table 1. Information on museum specimens of lemmings currently attributed to Lemmus amurensis and used in this study Specimen number  Type material  Tissue number  Locality  Year of collection  GenBank accession number  Taxonomy*  ZISP 13722  L. amurensis holotype  4463  Russia, Amur Region, Zeysky District, Pikan, 53.68°N, 127.46°E (1)  1914  KX455628  L. amurensis  ZMMU s91379    4564  1970  KX455629  ZMMU s91377    4565  1970  KX455630  ZMMU s91380    4566  1970  KX455631  ZMMU s91381    4567  1970  KX455632  ZMMU s91378    4568  1970  KX455633  ZMMU s150535    4576  Russia, Yakutia, Neryungri District, Nagorny, 55.95°N, 124.91°E (2)  1978  KX455636  L. amurensis  ZISP 71779    4245  Russia, Yakutia, Neryungri District, Chulman, 56.71°N, 124.87°E (3)  1980  KX455634  L. amurensis  ZMMU s90417    4575  Russia, Transbaikal Region, Aleksandrovsky Zavod, 50.9°N, 117.9°E (4)  1938  PCR failed  –  ZMMU s34813    4574  Russia, Transbaikal Region, Chita riverhead, 52.89°N, 114.39°E (5)  1938  KX455635  L. amurensis  ZISP 16754  L. a. ognevi holotype  4464  Russia, Yakutia, Verhoyansky District, Nel’gekhe River, 64.4°N, 134.04°E (6)  1927  KX455622  L. sibiricus (East clade)  ZMMU s124119    4569  Russia, Magadan Region, Khinikanumsa River, 61.71°N, 145.79°E (7)  1980  KX455623  L. sibiricus (East clade)  ZMMU s124120    4570  1981  KX455624  ZMMU s124121    4571  1981  KX455625  ZMMU s150533    4572  Russia, Magadan Region, Yama River, 60.01°N, 152.99°E (8)  1969  KX455626  L. sibiricus (East clade)  ZMMU s88365    4573  1969  KX455627  ZISP 13896  L. flavescens paralectotype  4563  Russia, Kamchatka Peninsula, Ust’- Kamchatsk, 56.53°N, 162.11°E (9)  1908  KX455621  L. sibiricus (East clade)  ZISP 90  L. flavescens lectotype  4244  Russia, Kamchatka Peninsula, south-western coast, supposedly Ust’-Bolsheretsk (11)  1826  KX455619  L. trimucronatus  ZISP 91  L. flavescens paralectotype  4562  KX455620  ZMMU s193482    4577  Russia, Chukotka, Khatyrka riverhear, 62.61°N, 173.23°E (12)  1981  KX455618  L. trimucronatus  Specimen number  Type material  Tissue number  Locality  Year of collection  GenBank accession number  Taxonomy*  ZISP 13722  L. amurensis holotype  4463  Russia, Amur Region, Zeysky District, Pikan, 53.68°N, 127.46°E (1)  1914  KX455628  L. amurensis  ZMMU s91379    4564  1970  KX455629  ZMMU s91377    4565  1970  KX455630  ZMMU s91380    4566  1970  KX455631  ZMMU s91381    4567  1970  KX455632  ZMMU s91378    4568  1970  KX455633  ZMMU s150535    4576  Russia, Yakutia, Neryungri District, Nagorny, 55.95°N, 124.91°E (2)  1978  KX455636  L. amurensis  ZISP 71779    4245  Russia, Yakutia, Neryungri District, Chulman, 56.71°N, 124.87°E (3)  1980  KX455634  L. amurensis  ZMMU s90417    4575  Russia, Transbaikal Region, Aleksandrovsky Zavod, 50.9°N, 117.9°E (4)  1938  PCR failed  –  ZMMU s34813    4574  Russia, Transbaikal Region, Chita riverhead, 52.89°N, 114.39°E (5)  1938  KX455635  L. amurensis  ZISP 16754  L. a. ognevi holotype  4464  Russia, Yakutia, Verhoyansky District, Nel’gekhe River, 64.4°N, 134.04°E (6)  1927  KX455622  L. sibiricus (East clade)  ZMMU s124119    4569  Russia, Magadan Region, Khinikanumsa River, 61.71°N, 145.79°E (7)  1980  KX455623  L. sibiricus (East clade)  ZMMU s124120    4570  1981  KX455624  ZMMU s124121    4571  1981  KX455625  ZMMU s150533    4572  Russia, Magadan Region, Yama River, 60.01°N, 152.99°E (8)  1969  KX455626  L. sibiricus (East clade)  ZMMU s88365    4573  1969  KX455627  ZISP 13896  L. flavescens paralectotype  4563  Russia, Kamchatka Peninsula, Ust’- Kamchatsk, 56.53°N, 162.11°E (9)  1908  KX455621  L. sibiricus (East clade)  ZISP 90  L. flavescens lectotype  4244  Russia, Kamchatka Peninsula, south-western coast, supposedly Ust’-Bolsheretsk (11)  1826  KX455619  L. trimucronatus  ZISP 91  L. flavescens paralectotype  4562  KX455620  ZMMU s193482    4577  Russia, Chukotka, Khatyrka riverhear, 62.61°N, 173.23°E (12)  1981  KX455618  L. trimucronatus  Museum names are abbreviated as follows: ZISP, Zoological Institute (St. Petersburg); ZMMU, Zoological Museum of Moscow University. Locality numbers (in brackets) correspond to Figures 1 and 2. *Taxonomic attribution after cytb analysis (see Fig. 2), with sequences listed in GenBank, as indicated. View Large Table 2. List of Lemmus cytb sequences downloaded from GenBank and used in the analysis (see Fig. 2) GenBank accession  Taxonomy  Locality  Reference  FJ025977–FJ025979  L. sibiricus (East clade)  Russia, Kamchatka Peninsula (10)  Abramson et al. (2008)  FJ025980, J025981  Russia, Yakutia, Lena Delta  FJ025982, J025983  L. trimucronatus  Russia, Chukotka Peninsula  FJ025984  L. lemmus  Sweden, Vasterbotten  FJ025985  Russia, Kola Peninsula  FJ025986  L. sibiricus (West clade)  Russia, Yamal Peninsula  AJ012672  L. sibiricus (West clade)  Russia, Olenekskiy Bay  Fedorov et al. (1999)  AJ012673  L. sibiricus (East clade)  Russia, Indigirka Delta  AJ012674  Russia, West of Kolyma Delta  AJ012677  Russia, Wrangel Island  AJ012678  Russia, Kamchatka Peninsula  AY219140  L. sibiricus (West clade)  Russia, Taymyr Peninsula  Fedorov et al. (2003)  AY219143  AY219141, AY219142  L. sibiricus (East clade)  Russia, Kotelny Island  AY219144  Russia, West of Kolyma Delta  AY219145  L. lemmus  Russia, Kola Peninsula  AF348389, F348390  L. sibiricus (West clade)  Russia, West Yamal Peninsula  Fedorov & Stenseth (2001)  AF348391  L. lemmus  Norway  AF348392  Finland  JX483882–JX483888  L. sibiricus (West clade)  Russia, Amderma  Lagerholm et al. (2014)  JX483889, JX483890  Russia, Derevnya  JX483891  Russia, Pechora  JX483892  L. lemmus  Sweden, Jamtland  JX483901, JX483902  JX483893, JX483894  Sweden, Vasterbotten  JX483900  JX483896, JX483897  Sweden, Padjelanta, Norrbotten  JX483898, JX483899  Sweden, Jamtland/Vasterbotten  JX483895  Sweden, Abisko, Norrbotten  JX483903, JX483904  JX483905, JX483906  Sweden, Kebnekaise, Norrbotten  JX483907, JX483908  Sweden, Sarek, Norrbotten  GenBank accession  Taxonomy  Locality  Reference  FJ025977–FJ025979  L. sibiricus (East clade)  Russia, Kamchatka Peninsula (10)  Abramson et al. (2008)  FJ025980, J025981  Russia, Yakutia, Lena Delta  FJ025982, J025983  L. trimucronatus  Russia, Chukotka Peninsula  FJ025984  L. lemmus  Sweden, Vasterbotten  FJ025985  Russia, Kola Peninsula  FJ025986  L. sibiricus (West clade)  Russia, Yamal Peninsula  AJ012672  L. sibiricus (West clade)  Russia, Olenekskiy Bay  Fedorov et al. (1999)  AJ012673  L. sibiricus (East clade)  Russia, Indigirka Delta  AJ012674  Russia, West of Kolyma Delta  AJ012677  Russia, Wrangel Island  AJ012678  Russia, Kamchatka Peninsula  AY219140  L. sibiricus (West clade)  Russia, Taymyr Peninsula  Fedorov et al. (2003)  AY219143  AY219141, AY219142  L. sibiricus (East clade)  Russia, Kotelny Island  AY219144  Russia, West of Kolyma Delta  AY219145  L. lemmus  Russia, Kola Peninsula  AF348389, F348390  L. sibiricus (West clade)  Russia, West Yamal Peninsula  Fedorov & Stenseth (2001)  AF348391  L. lemmus  Norway  AF348392  Finland  JX483882–JX483888  L. sibiricus (West clade)  Russia, Amderma  Lagerholm et al. (2014)  JX483889, JX483890  Russia, Derevnya  JX483891  Russia, Pechora  JX483892  L. lemmus  Sweden, Jamtland  JX483901, JX483902  JX483893, JX483894  Sweden, Vasterbotten  JX483900  JX483896, JX483897  Sweden, Padjelanta, Norrbotten  JX483898, JX483899  Sweden, Jamtland/Vasterbotten  JX483895  Sweden, Abisko, Norrbotten  JX483903, JX483904  JX483905, JX483906  Sweden, Kebnekaise, Norrbotten  JX483907, JX483908  Sweden, Sarek, Norrbotten  Locality number (in brackets) corresponds to Figures 1 and 2. View Large Methods DNA extraction, amplification and sequencing DNA from museum skin samples was isolated using Qiagen’s QIAamp Tissue Kit. A 337-bp fragment of the cytb gene was amplified in three overlapping fragments using primers and PCR conditions published by Lagerholm et al. (2014). Both DNA isolation and PCR with museum samples were conducted in a room isolated from post-PCR facilities using a PCR Workstation (LAMSYSTEMS CC) and the working surface, all instruments and plastics were sterilized with UV light and chloramine-T to avoid contamination. The PCR products were purified with Omnix kit columns (Omnix, St. Petersburg) and sequenced in both directions using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit on an ABI PRISM 3130 (Applied Biosystems Inc.). Sequences were edited, assembled and aligned with BioEdit (Hall, 1999). Phylogenetic analysis Phylogenetic analysis of new and published sequences was performed using a 924-bp alignment (the missing 587 bases for 337-bp sequences from museum samples were coded with Ns). Sequences of Dicrostonyx torquatus (AF119275), Synaptomys cooperi (DQ323957) and Myopus schisticolor (EU165208) were used as outgroups. Phylogenies were reconstructed using Bayesian inference (BI) analysis and maximum likelihood (ML) approaches. To choose the best model of molecular evolution (TVM+G), we used Akaike’s information criterion (AIC) in jModelTest 2.1.4 (Darriba et al., 2012). Bayesian analysis was performed in BEAST v2.1.0 (Drummond et al., 2012) with parameters given below in the ‘Estimation of divergence dates’ section. ML analysis was performed using TreeFinder (Jobb, 2008) under the TVM+G substitution model. Bootstrap analysis employed 1000 replicates. Final trees were obtained using FigTree v1.4. (http://tree.bio.ed.ac.uk/software/figtree/). Estimation of divergence dates Divergence dates were estimated in BEAST v2.1.0 (Drummond et al., 2012). We used a relaxed lognormal clock model (Drummond et al., 2006) and the following time constraints. We set a prior on the basal node of the Lemmini clade to 3 Mya, calibrated with a lognormal distribution with the offset = 3.0 Mya. This calibration point is substantiated by the first records of ancestral forms of lemmings both in North America (Repenning, 2001) and Eurasia (Sukhov, 1976; Kowalski, 1977), from sediments of ~2.6 Mya and already subsequent to the split within the Lemmini, thus moving back the time to most recent common ancestor (MRCA) of the tribe Lemmini. Since the use of a more complicated model does not affect the result (Huelsenbeck & Ronquist, 2005), we ran the BI analysis under the GTR+I+G instead of TVM+G selected in jModelTest, and all remaining priors were set to the defaults. The analysis was performed under a birth–death model that assumes that at any time point, every lineage can undergo speciation at rate λ or go extinct at rate µ. Two replicate runs of 100 million Markov chain Monte Carlo generations each were performed, sampling trees and parameter estimators every 10000 generations. The convergence of run parameters was examined in TRACER v1.6 (Rambaut & Drummond, 2007) and the first 25 million generations were discarded as burn-in. The final time-tree was summarized using TreeAnnotator v2.0.3 (Drummond & Rambaut, 2007) based on the trees sampled after the 25% burn-in from both independent runs, using the maximum clade credibility tree option and fixing node heights as mean heights. Divergence time bars were obtained automatically in FigTree v1.4 (http://tree.bio.ed.ac.uk/software/figtree/) from the output using the 95% highest posterior density (HPD) of the ages for each node. RESULTS Data set We succeeded in amplifying cytb from 19 museum samples with one failure (Table 1). The final cytb alignment contained 72 Lemmus sequences. The 924 bp alignment reduced to the shortest sequence (337 bp museum samples) contained 61 variable sites, of which 49 were parsimony informative. Newly determined sequences were deposited in GenBank under accession numbers KX455618–KX455636 (Table 1). Phylogenetic relationships and genetic diversity The Bayesian phylogenetic tree of true lemmings shows the expected principal division, referred to here as the Palearctic and Nearctic branches (Fig. 2), with a geographic border along the Kolyma River (Fig. 1A). The average pairwise distance between these branches is 10%, in good agreement with earlier findings (Fedorov et al., 1999). The Palearctic branch is further divided into two well-defined clades. The Western Palaearctic clade (Fig. 2) includes representatives of lemmings inhabiting the arctic and subarctic tundra from Scandinavia to the Lena River. Lemmings of the Eastern Palaearctic clade (Fig. 2) inhabit tundra from the Lena River to the western bank of the Kolyma River including various Arctic islands (including Wrangel Island), South Yakutia (Fig. 1B: Localities 2, 3), the Amur Region (Fig. 1B: Locality 1), Transbaikalia (Fig. 1B: Localities 4, 5), the Kolyma uplands and the eastern shore of the Kamchatka Peninsula (Fig. 1B: Localities 7–10). The major split within the Eastern Palearctic clade separates lemmings from northeastern Siberia from the southern phylogroup that includes only lemmings from the Transbaikal area, the Amur Region and South Yakutia (Fig. 1B: Localities 1–3, 5). The latter phylogroup consists exclusively of museum samples and is of special importance as it includes the holotype of the Amur lemming together with all topotypes (Fig. 2). The geographic border on the mainland between these subdivisions within the Eastern Palaearctic clade occurs at 60°N. Figure 2. View largeDownload slide Phylogenetic tree including timings of major divergence events within the genus Lemmus species, based on cytb. The outgroups (Dicrostonyx torquatus, Myopus schisticolor and Synaptomys cooperi) are not shown. The divergence times below the nodes correspond to the mean posterior estimate of their age in kyr. The grey bars and numbers in square brackets represent the 95% HPD interval of node heights. Posterior probability values >0.90 (BI) and bootstrap support over 60% (ML) are indicated above the nodes. Numbers in brackets correspond to the collection sites in Figure 1 and Tables 1 and 2. Along the X-axis – time scale in kyr. BI, Bayesian inference; HPD, highest posterior density; ML, maximum likelihood. Figure 2. View largeDownload slide Phylogenetic tree including timings of major divergence events within the genus Lemmus species, based on cytb. The outgroups (Dicrostonyx torquatus, Myopus schisticolor and Synaptomys cooperi) are not shown. The divergence times below the nodes correspond to the mean posterior estimate of their age in kyr. The grey bars and numbers in square brackets represent the 95% HPD interval of node heights. Posterior probability values >0.90 (BI) and bootstrap support over 60% (ML) are indicated above the nodes. Numbers in brackets correspond to the collection sites in Figure 1 and Tables 1 and 2. Along the X-axis – time scale in kyr. BI, Bayesian inference; HPD, highest posterior density; ML, maximum likelihood. All of the museum samples originating both from the upper reaches of the Kolyma River and from the eastern coast of the Kamchatka Peninsula have most recently been attributed to L. amurensis (Fig. 1). They include one paralectotype of L. flavescens N 13896 at Ust’-Kamchatsk (Fig. 1B: Locality 9) and the holotype of L. a. ognevi from the Verkhoyanskiy Region (Fig. 1: Locality 6), which are phylogenetically placed within the northern phylogroup of the Eastern Palaearctic clade (Fig. 2). In conventional taxonomy, this phylogroup corresponds to L. sibiricus. Since this phylogroup was reported earlier (Fedorov et al., 1999; Abramson et al., 2008), the split within the Eastern Palearctic clade into northern and southern groups at 60°N is new to the Lemmus tree and therefore only the southern subdivision that contains the holotype can taxonomically correspond to L. amurensis. We find that the lectotype and the second paralectotype of L. flavescens (Table 1; Fig. 2: NN 4244, 4562; Fig. 1: Locality 11), trapped by Baron von Kittlitz in 1826, belong to the Nearctic branch, i.e. to L. trimucronatus. This is, therefore, the first record of this species on the Kamchatka Peninsula determined with the aid of molecular tools. Again, the other paralectotype (Table 1; Fig. 2: N 4563; Fig. 1: Locality 9) trapped almost a century later and on the other coast of the Kamchatka Peninsula is found here in the northern phylogroup of the Eastern Palearctic clade. The museum specimen N 4577 (Fig. 2) from the Koryak Plateau (Fig. 1: Locality 12) clusters (as expected) with specimens of L. trimucronatus, as this site is within the range of the latter species. Its initial attribution to L. amurensis was an evident misidentification. Divergence time estimates Molecular and fossil data were used to derive the posterior distribution of divergence dates for the crown Lemmus in the cytb tree. According to our cytb tree (Fig. 2), the divergence between the Palearctic and Nearctic branches would have occurred at ~2.1 Mya (95% HPD 1.1–2.6). Our phylogenetic reconstruction indicates that two main phases can be recognized during the diversification of Palearctic Lemmus. The first evolutionary event was the divergence of the Western Palearctic and Eastern Palearctic clades from a common ancestor, which occurred ~1 Mya (Fig. 2), while the second involved the separation of two subclades within the Eastern Palearctic clade (i.e. the L. sibiricus East clade with a northeastern distribution and the lemmings south of 60°N, corresponding to L. amurensis) around 0.68 Mya (95% HPD 0.2–0.74). The separation into L. lemmus and the L. sibiricus West clade within the Western Palearctic clade took place later, around 0.31 Mya (95% HPD 0.1–0.35). DISCUSSION Timing of divergence, biogeography and evolutionary patterns Early stages of Lemmus evolution The first remains of lemming ancestral forms appear in the fossil record almost simultaneously in Eurasia and North America (Sukhov, 1976; Kowalski, 1977; Tomida, 1987; Repenning & Grady, 1988) around 2.7 Mya. These ancestral forms in the South Urals, Russia (Sukhov, 1976) and Poland (Kowalski, 1977) dated slightly earlier than records in North America (Repenning, 2001) where their appearance coincides with the first surge of the known Northern Hemisphere continental glaciation around 2.65–2.2 Mya (Shackleton & Opdyke, 1977) and mark one of the waves of arvicoline Eurasian immigration to North America. Initially, all these findings were made with reference to the genus Synaptomys; however, later studies showed that all of these records represent the Lemmus lineage (Carls & Rabeder, 1988; Repenning & Grady, 1988; Abramson & Nadachowski, 2001). Thereafter, Lemmus fossils occur continuously in Pleistocene sites throughout northern Eurasia. It is remarkable that, contrary to other members of subfamily Arvicolinae, these ancient lemmings are very similar to modern species in molar patterns. Based on the molar evolution that can be described, a sequence of several chronospecies has been described: from L. kowalskii (Carls & Rabeder, 1988) and L. sheri (Abramson, 1992), to recent L. amurensis (Abramson, 1993). In contrast, since the first appearance of the lemming ancestral forms (Repenning & Grady, 1988) in North America around 2.7 Mya there are no records that can unequivocally be assigned to the genus Lemmus earlier than 1 Mya, when teeth indistinguishable from modern Lemmus were reported from the Cape Deceit fauna of eastern Beringia (Guthrie & Matthews, 1971). Thus, there is a time gap of almost 1 Myr between the first appearance of Lemmus ancestral forms and forms that can be attributed to the genus Lemmus proper. The other peculiarity of Lemmus evolution in North America, as compared to Eurasia, is that there are no significant southward range shifts within the Pleistocene period. In North America, fossil records of Lemmus more or less coincide with the recent distribution of the genus, whereas in Eurasia its range in the early Pleistocene reached the Pyrenees in the west and Slovakia to the south (Kowalski, 2001). Notably, lemming remains of the form that most likely may be assigned to the MRCA of L. trimucronatus and L. sibiricus + amurensis + lemmus are found on both sides of the Bering Strait (Sher et al., 1979; Repenning et al., 1987) in sediments of ~2.5–2.4 Myr. Palearctic and Nearctic branch split In this study, the main divergence within Lemmus has been dated to the early Pleistocene, at 2.1 Mya (HPD 1.1–2.6), in fairly good agreement with fossil records listed above. The date obtained is also in line with paleoclimate reconstructions. The almost simultaneous appearance of Lemmus ancestral forms on both sides of the Bering Strait coincides with the start of the first continental glaciation, 2.65 Mya (Andersen & Borns, 1997; Repenning, 2001; Cohen & Gibbard, 2012). It was a period of progressive cooling with significant drop in sea level, leading to exposure of a large portion of the continental shelf regions of the Chukchi and Bering Seas between Siberia and Alaska (Elias & Brigham-Grette, 2013). This cooling persisted until 2.0 Mya, the beginning of a Northern Hemisphere warm period (Tiglian Interglacial), which lasted for ~300000 years. This was a period of resubmergence of the Bering Strait, thus leading to the independent evolution of lemmings in Siberia and eastern Beringia. The modern distribution of L. trimucronatus in western Beringia, stretching to the eastern shore of the Kolyma River, presents a more difficult puzzle. Previous molecular studies (Fedorov et al., 2003) showed that L. trimucronatus probably moved to the Kolyma River in the Late Pleistocene period during the westward expansion from Alaska. Recent samples from both sides of the Bering Straits are genetically similar and constitute a single phylogroup. The presence of L. trimucronatus on the western coast of the Kamchatka Peninsula reported here based on the genetic study of a previously collected specimen is novel and unexpected. This finding significantly expands our knowledge of the species range. It supports the hypothesis that as part of the Late Pleistocene expansion, L. trimucronatus most likely arrived at the tundra on the western coast of the Kamchatka Peninsula from the north along one of the corridors on the sides of the glaciers on the Kamchatkan Middle Ridge (Elias & Brigham-Grette, 2013). However, the intriguing aspect of this scenario lies in the occurrence of highly fragmented populations of the L. sibiricus East clade to the east of the Middle Ridge. The presence of L. sibiricus of this clade on Wrangel Island (Fedorov et al., 1999, 2003) and eastern Kamchatka supports its probable occurrence in the Late Pleistocene over a vast territory of northeastern Russia that currently is partly occupied by L. trimucronatus. The continuous presence of lemmings in this territory is confirmed by the fossil record (Agadjanyan, 1972). One plausible explanation of the current distribution pattern is that L. trimucronatus outcompeted L. sibiricus in areas with mutual habitat. The other hypothesis implies that L. sibiricus became extinct near the Pleistocene–Holocene transition in the major part of the Chukotka Peninsula and the northern coast of the Sea of Okhotsk; edge populations of this species remained only on Wrangel Island and the eastern coast of Kamchatka before the expansion into these areas by L. trimucronatus (Fedorov et al., 2003). We suggest that the range changes associated with asynchrony in population peak numbers of L. trimucronatus and the L. sibiricus East clade during past population cycling in western Beringia may somehow explain the distributions of these species at present. It is possible that peak numbers of L. trimucronatus on the east coast of the Bering Strait in the Late Pleistocene coincided with a population crash of L. sibiricus in the Chukotka Peninsula and this discrepancy in numbers allowed L. trimucronatus to migrate and occupy the habitats of L. sibiricus. Divergence within Palearctic lemmings The main split within the Palearctic Lemmus, into the Eastern Palearctic clade (including L. sibiricus East clade and L. amurensis) and Western Palearctic clade (including L. sibiricus West clade and L. lemmus) (Fig. 2), took place around 1 Mya (0.37–1.1), according to our molecular estimations. That is synchronous with the Jaramillo episode and marks the first cryogenic epoch at the beginning of the Pleistocene in West Siberia (Arkhipov, 1987), or the Neopleistocene according to the modern scheme (Fotiev, 2009; Cohen & Gibbard, 2012). This estimate suggests that separation of these main lineages within the Palearctic Lemmus occurred much earlier than the time preceding the last glaciation, as proposed by Fedorov et al. (1999, 2003) using a smaller data set without Amur lemming samples. Lemming remains from about 1 Mya are known throughout the Palearctic from East Anglia (Harrison, Bates & Clayden, 1989) to northeastern Siberia (Sher, 1984; Abramson, 1992) and eastern Transbaikalia (Erbajeva, Alexeeva & Khenzykhenova, 2001). Although it is well known that, unlike in Europe, ice sheets had very limited distributions in East Siberia; the separation into the Eastern Palearctic and Western Palearctic clades may have resulted from isolation by mountain uplift associated with alpine glaciers and landscape remodelling due to dramatic redistribution of river systems (Korzhuev, 1974). Thus, we agree with Fedorov et al. (1999, 2003) that the ice sheet on the Verkhoyanskiy Mountain Ridge in northern Yakutia along the eastern bank of Lena River (Frenzel, Pecsi & Velichko, 1992) may have caused the isolation of lemming populations in the early Neopleistocene. The lemming remains of the early Neopleistocene in western Europe are referred to as L. kowalskii (Carls & Rabeder, 1988); in Siberia a slightly modified form from more recent sediments was described as L. sheri (Abramson, 1992). Lemmings in the eastern part of the Palearctic Within the Eastern Palearctic clade, the mean time estimation (0.68 Mya) for the split between the L. sibiricus East clade and L. amurensis (Fig. 2) roughly coincides with the glacial b stage of the Cromerian in western Europe and the early Shaitan glaciation in Siberia. The active orogeny in Transbaikalia and Yakutia (Imaev, Imaeva & Kozmin, 2005; Ovsyuchenko et al., 2010) may have generated a physical barrier for vicariant separation of the Amur lemming in southeastern Siberia and East Asia from northern populations. Late Pleistocene fossil remains of this species are found as far south as the Primorskiy Region (Tiunov & Panasenko, 2010). Nowadays, the Amur lemming is extremely rare and is declining. The only sustainable population is in South Yakutia near Chulman. Despite numerous expeditions in the Transbaikal, no one has succeeded in trapping this species since the late 1930s. Lemmings in the western part of the Palearctic Our molecular data for the split within the Western Palearctic clade into L. lemmus and the L. sibiricus West clade (from east of White Sea to the Lena River) (Figs 1, 2) gave the mean estimate for this divergence at 0.31 Mya with upper confidence limit 0.1 Mya (Fig. 2), which fully confirms to the result obtained earlier from ancient DNA (Lagerholm et al., 2014). This dating points to divergence prior to the Last Glacial Maximum (LGM). The Holocene warming and genesis of the White Sea caused the final isolation of these forms. Taxonomical considerations and species delimitation within the genus Lemmus Initially, we designed this study with DNA-barcoding ideology in mind with the goal of simple identification and naming of lemming samples using the type specimens and reference sequences. However, the results are complex and depend on the species concept used. The question of how many species of Lemmus can be distinguished is ultimately linked to species concepts. In the midst of last century, the biological species concept (BSC) dominated, defining species with an emphasis on reproductive isolation. This concept does not help much in distinguishing between conventional Palearctic species of the genus, as all hybrid combinations of L. lemmus, L. sibiricus and L. amurensis (from Chulman) give fertile offspring (Gileva et al., 1984; Pokrovskiy et al., 1984). However, many researchers have argued that reproductive isolation is a poor criterion in the case of allopatric species and thus prefer to decide species status on the basis of distinct diagnosable features, i.e. coloration and size, in agreement with the phylogenetic species concept (PSC). The mitochondrial cytb lineages, however, disagree with conventional taxonomy. According to results presented here, L. sibiricus appears to be paraphyletic in relation to both L. lemmus and L. amurensis. Thus, interpreting the mitochondrial tree in accordance with the PSC, we can either consider all the Palearctic forms as one polytypic L. lemmus species with four subspecies, or recognize four monophyletic species. Both decisions will require a new scientific name for L. sibiricus within the Eastern Palearctic clade. However, recalling that a gene tree is not equal to a species tree, and the latter is impossible to infer from studying only one locus, it is highly unreasonable to give taxonomic names to mitochondrial lineages. The other species concept that is relevant here is the evolutionary species concept (ESC). Although our data include only a fragment of a mitochondrial gene, they reflect past fragmentation events and times of isolation. The core point of the ESC is that ‘species are historical, temporal, and spatial entities’ (Wiley, 1978). Considering the true lemmings in this context, there is no support to qualify the Eastern and Western Palearctic Lemmus mitochondrial lineages as independent species, as at present there is no barrier in the tundra along the Lena River that could prevent mixing of mitochondrial lineages. From our viewpoint, the only reason why there are no records of populations with mixed Eastern and Western Palearctic clade haplotypes is insufficient sampling in these remote regions of the Arctic. The Verkhoyanskiy Ridge evidently served as a barrier isolating these phylogroups in past glacial epochs, when lemmings dominated over vast territories of the Palearctic and the southern border of their distribution was shifted significantly southward compared to the present. However, proceeding from ESC principles, there is a strong temptation to give the allopatric Amur lemming a species status, relying on data of its long independent evolutionary history, sharp differences in both coloration and size and very restricted and isolated current distribution. Notably, among recent lemmings, specimens with molar characteristics typical for Eopleistocene–Early Neopleistocene forms occur sporadically only among L. amurensis. The listed features comply with the ESC and in part with the PSC and the BSC. The species status of the Amur lemming, in the light of current data, may be supported by a conservation perspective. Contrary to the opinions given in the IUCN (International Union for Conservation of Nature) Red List of Threatened Species (Tsytsulina, 2008), and by Shenbrot & Krasnov (2005) and Carleton & Musser (2005), our data show that L. amurensis is not widespread and its status as Least Concern (LC) reflects incorrect sample identification leading to incorrect ideas of its distribution. This species is not only rare, but it has disappeared from most places where it was reported in the first half of the 20th century, including the terra typical, which was flooded by the construction of the Zeya reservoir. Ever since Fetisov trapped several specimens (deposited in ZMMU) from various sites in the southeast Transbaikal region in the first half of the 1900s, no one has been able to trap these animals despite numerous field attempts. The only sustainable population is in the Chulman River, South Yakutia area and, in our opinion, the conservation status of the Amur lemming needs to be reclassified as Vulnerable/Near Threatened (VU/NT). Nomenclature notes The results obtained in the current study, irrespective from the issue on the number of species in the genus, highlight a nomenclatural challenge. It is impossible to retain the species L. lemmus and L. sibiricus within the Palearctic branch as currently used, since L. sibiricus is clearly a paraphyletic relative of L. lemmus. Therefore, L. sibiricus should be considered a junior synonym of L. lemmus, or the L. sibiricus West clade and the L. sibiricus East clade (Fig. 2) should be assigned species or subspecies names. Thus, sibiricus could be used as a species or subspecies name for the L. sibiricus West clade and ognevi could be used for the L. sibiricus East clade. The name L. flavescens is a ‘nomen nudum’ and existed only in the form of Brandt’s handwritten labels of specimens collected by von Kittlitz during his expedition to Kamchatka in 1826–1828 until Vinogradov (1925) temporally fixed this name and designated the type series specimens. It is interesting that Vinogradov mentioned differences in coloration between the von Kittlitz specimens and N 13896 from Ust’-Kamchatsk collected by Ryabushinskiy almost a century later in 1908. The von Kittlitz specimens do not have exact coordinates with the geographic origin of the specimens; the labels indicate ‘Kamchatka’. However, the detailed description of expedition routes provided by numerous watercolour paintings (Strecker, 2011) allows us to consider with high probability that von Kittlitz collected these specimens from the southwest coast of Kamchatka in the area of modern Ust’-Bolsheretsk. According to our results the lectotype of L. flavescens (N 90) belongs to L. trimucronatus, therefore the name L. flavescens should be considered a junior synonym of L. trimucronatus. The specimen from Ust’-Kamchatsk, as shown above, explicitly clusters within the L. sibiricus East clade and should be withdrawn from the type seria. ACKNOWLEDGEMENTS We are grateful to V. S. Lebedev, curator of mammal collection at ZMMU, for giving us access to samples from the core collection of the museum; we thank F. N. Golenishchev, curator of mammal collection at ZISP. We thank the two anonymous referees whose critical comments helped to improve the manuscript. We are particularly grateful to prof. Jeremy Searle, Department of Ecology and Evolutionary Biology, Cornell University, who has done enormous work on the manuscript to improve the English and gave many valuable comments. This study was conducted in Zoological Institute RAS under the research theme N 01201376804 and financially supported from the grant of RFBR N 15-04-04602. REFERENCES Abramson NI. 1992. A new species of lemming from Eopleistocene of North east Siberia (Mammalia: Cricetidae). Zoosystematica Rossica  1: 156– 160. Abramson NI. 1993. The genus Lemmus in Eurasia in the Late Cenozoic. Trudy Zoologicheskogo Instituta AN SSSR  249: 146– 157 (in Russian). Abramson NI, Kostygov AYu, Rodchenkova EN. 2008. The taxonomy and phylogeography of Palaearctic true lemmings (Lemmus, Cricetidae, Rodentia): new insights from cyt b data. Russian Journal Theriology  7: 17– 23. Google Scholar CrossRef Search ADS   Abramson NI, Nadachowski A. 2001. 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Genetic analysis of type material of the Amur lemming resolves nomenclature issues and creates challenges for the taxonomy of true lemmings (Lemmus, Rodentia: Cricetidae) in the eastern Palearctic

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© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society
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Abstract

Abstract The proper use of species names depends entirely on verifying whether newly analysed specimens are conspecific with the type material. True lemmings (Lemmus) are the most common rodents of the Arctic tundra in the Old and New World and play an important role in the Arctic ecosystem; however, their taxonomy is far from resolved. The Amur lemming (L. amurensis) is the least studied and most enigmatic species of the genus. Its taxonomic position, distribution and nomenclature are uncertain due to a lack of cytogenetic, molecular phylogenetic and hybridization studies. Assignment of all true lemmings from the vast territory of western Beringia to this species has never properly been confirmed. Moreover, the type locality for this species was flooded by a newly created reservoir in 1974, making additional topotypes unavailable. In this context, genetic analysis of museum specimens, especially type material, has great potential. Here we report partial cytochrome b sequences extracted from all specimens identified as L. amurensis stored in the two largest mammal collections in Russian museums, including the holotype of L. amurensis and the type material of all forms currently considered as synonyms of L. amurensis. Phylogenetic analyses suggest that the range of the Amur lemming is dramatically smaller than previously assumed and is limited to the territories of Transbaikalia, South Yakutia and Amur in the eastern Palaearctic. Lemmus taxa from other territories, including L. amurensis ognevi and L. amurensis flavescens, refer to other lemming species. Our results impinge on nomenclature issues, taxonomy, divergence times and the evolutionary history of lemmings in the eastern part of the Palearctic and on the species concept as applied to the genus Lemmus. INTRODUCTION True lemmings of the genus Lemmus Link, 1795 are the most common rodents of the Arctic tundra in the Old and New World and play an important role in the Arctic ecosystem. The taxonomic subdivisions of Lemmus remain rather controversial. It is, however, commonly accepted that the genus splits into two branches with a geographical border along the Kolyma River. The ‘Palaearctic branch’ extends from Scandinavia to the western shore of the Kolyma River and the ‘Nearctic branch’ inhabits the tundra on the eastern shore of the Kolyma River, the Anadyr lowlands, the Chukotka Peninsula and on the other side of the Bering Straits. These lemming categories differ in karyotype: Palearctic branch lemmings have only acrocentric chromosomes (2n = NF [fundamental number] = 50), whereas in Nearctic branch lemmings one chromosome is subtelocentric (2n = 50, NF = 52) (Rausch & Rausch, 1975; Gileva, Kuznetsova & Cheprakov, 1984; Chernyavskiy et al., 1993; Chernyavskiy & Kartavtseva, 1999). Experimental hybridization between Palearctic and Nearctic branch lemmings produces fertile females but sterile males (Rausch & Rausch, 1975; Pokrovskiy, Kuznetsova & Cheprakov, 1984). Mitochondrial DNA data (Fedorov et al., 1999; Abramson, Kostygov & Rodchenkova, 2008) also suggest differentiation of Palearctic and Nearctic branch lemmings. The Nearctic branch of true lemmings consists of a single species – L. trimucronatus Richardson, 1825, whereas the taxonomic structure of the Palearctic branch is problematic. The number of species and criteria for their delimitation within this branch remain unclear. Conventionally, the Palearctic branch consists of three allopatric species, differing in size and colour: the Norway lemming, L. lemmus L., 1758, the polytypic Siberian lemming, L. sibiricus Kerr, 1792 and the polytypic Amur lemming, L. amurensis Vinogr., 1924. However, both coat colour and size are features that are subject to geographic, seasonal and age variation and there is a lack of characteristics in skull and dental morphology that can reliably discriminate between the three putative species. Among these, the Amur lemming is the least studied and most enigmatic species of the genus. Vinogradov (1924) described it based on a single specimen trapped in 1914, close to where the town of Zeya is now (Fig. 1: Locality 1). This is in the Amur Region and the specimen was deposited in the collection of the Zoological Institute (ZIN) in St. Petersburg (N 13722). The discovery of this lemming from 53.7°N farther south than the previous Lemmus range makes it especially interesting. Earlier, all representatives of the genus were only known from the tundra zone. Vinogradov (1925) suggested that the Amur lemming is a relict form isolated from other species of the genus. Decades later, the same author used a single specimen trapped at the Nelgese River in Yakutia (Fig. 1: Locality 6) to describe a subspecies of the Amur lemming – L. a. ognevi (Vinogradov, 1933). The site of the new specimen is ~1500 km north of the first finding. Interestingly, these two specimens are similar in size, but markedly different in colour. Later on in 1938, small numbers of Amur lemmings were trapped in the Transbaikal area (Chita Region). The next collections came 30 years later; around ten specimens were caught in the type locality in 1970. All these specimens were deposited in the Zoological Museum of Moscow University (ZMMU). However, the type locality was shortly afterwards (1974) flooded by the newly created Zeya reservoir and subsequently no one has successfully trapped lemmings in this region despite multiple attempts. Towards the end of the 20th century, a sustainable population of lemmings, referred to as L. amurensis, was discovered near the village of Chulman in South Yakutia (Chernyavskiy et al., 1980; Revin, 1983; Chernyavskiy, 1984). This site is ~500 km north of the type locality (Fig. 1: Locality 3). Only this population has been used for cytogenetic analysis and hybridization studies (Gileva et al., 1984; Pokrovskiy et al., 1984). Since then, any Lemmus trapped in the upper reaches of the Kolyma River, in the Magadan Region, 2000 km from the type locality were attributed to L. amurensis (Chernyavskiy, 1984). Moreover, lemmings from the Kamchatka Peninsula (Vinogradov, 1925) that were earlier referred to as a subspecies of L. sibiricus (Pavlinov & Rossolimo, 1987), L. s. flavescens, were redesignated as a subspecies of L. amurensis based primarily on small body size and molar features (Chernyavskiy et al., 1993). Consequently, L. flavescensVinogradov, 1925 has become a junior synonym of the Amur lemming (Carleton & Musser, 2005; Tsytsulina, 2008). Thus, in a piecemeal way, a rather wide distribution has become apparent for the Amur lemming in the northeast Palearctic. It is very important to underline that no cytogenetic, molecular phylogenetic or hybridization studies have made use of animals from the terra typica of L. amurensis. For cytogenetic and hybridization studies of L. amurensis (Gileva et al., 1984; Pokrovskiy et al., 1984), researchers have used animals from Chulman and, until now, only lemmings from one site on the east coast of the Kamchatka Peninsula have been used for the analysis of cytochrome b (cytb) sequences (Fedorov et al., 1999; Abramson et al., 2008). It should be noted that according to the holotype description, the Amur lemming differs from other species of lemmings by two main features: a significantly smaller body size and a distinctive coloration (Vinogradov, 1924). Of these features, size is particularly unreliable. For instance, a very pronounced clinal variation in size is typical for lemmings: animals from arctic tundra and islands are considerably larger than lemmings from southern populations inhabiting subarctic tundra and forest-tundra (Krivosheev & Rossolimo, 1966) and adult animals born in winter are smaller than animals of the summer cohort. The most valuable defining feature of Amur lemmings is coloration. Unlike all other lemmings, they have a practically uniform dark brown dorsal surface, sometimes with a more or less pronounced black stripe on the neck, strong rufous-brick cheeks and sides and a uniformly fulvous belly, paler than the sides (Vinogradov, 1924; Hinton, 1926). The attribution of lemmings from Chulman, eastern Kamchatka, the Kolyma uplands and the Magadan area to the Amur lemming was made exclusively based on small size and is therefore unreliable. Figure 1. View largeDownload slide A, Lemmus species ranges. B, collection sites of museum specimens originally classified as the Amur lemming. Locations are colour-coded according to cytb lineages, as shown in Figure 2. Yellow and blue stars are the lectotype and holotype accordingly. Small black dots represent collection sites of museum specimens for which cytb did not amplify (with numbers) or collection sites of specimens recorded in the literature (without numbers). Locality numbers correspond to Tables 1 and 2. *Species ranges are given according to Henttonen (2008), Linzey (2008), Tsytsulina (2008) and Tsytsulina, Formozov & Sheftel (2008) with the mitochondrial lineage distributions following Fedorov et al. (1999) and Abramson et al. (2008). Figure 1. View largeDownload slide A, Lemmus species ranges. B, collection sites of museum specimens originally classified as the Amur lemming. Locations are colour-coded according to cytb lineages, as shown in Figure 2. Yellow and blue stars are the lectotype and holotype accordingly. Small black dots represent collection sites of museum specimens for which cytb did not amplify (with numbers) or collection sites of specimens recorded in the literature (without numbers). Locality numbers correspond to Tables 1 and 2. *Species ranges are given according to Henttonen (2008), Linzey (2008), Tsytsulina (2008) and Tsytsulina, Formozov & Sheftel (2008) with the mitochondrial lineage distributions following Fedorov et al. (1999) and Abramson et al. (2008). For the present study, the goal is to obtain molecular data to clarify the taxonomy, phylogenetic relationships and nomenclature of representatives of the genus Lemmus in the eastern Palearctic using material from museum collections, including type specimens. This is especially important for the Amur lemming as it is impossible to obtain new topotypes and for the proper use of species names, it is essential to verify whether newly analysed specimens are conspecific with the type specimen (Santos et al., 2016). Thus, we first analysed cytb sequences obtained from the type material of all forms currently referred to L. amurensis and then from all available museum samples identified as L. amurensis, deposited in the two largest mammal collections in Russian museums. We compared new sequences with existing published data for Lemmus from all parts of the range of the genus in the Palearctic. MATERIAL AND METHODS Material Information on the geographic origin and year of collection of the museum specimens studied and attributed to L. amurensis is given in Table 1 and Figure 1. A total of 53 cytb sequences of other Lemmus species were retrieved from GenBank and used in the phylogenetic analysis (Table 2). Table 1. Information on museum specimens of lemmings currently attributed to Lemmus amurensis and used in this study Specimen number  Type material  Tissue number  Locality  Year of collection  GenBank accession number  Taxonomy*  ZISP 13722  L. amurensis holotype  4463  Russia, Amur Region, Zeysky District, Pikan, 53.68°N, 127.46°E (1)  1914  KX455628  L. amurensis  ZMMU s91379    4564  1970  KX455629  ZMMU s91377    4565  1970  KX455630  ZMMU s91380    4566  1970  KX455631  ZMMU s91381    4567  1970  KX455632  ZMMU s91378    4568  1970  KX455633  ZMMU s150535    4576  Russia, Yakutia, Neryungri District, Nagorny, 55.95°N, 124.91°E (2)  1978  KX455636  L. amurensis  ZISP 71779    4245  Russia, Yakutia, Neryungri District, Chulman, 56.71°N, 124.87°E (3)  1980  KX455634  L. amurensis  ZMMU s90417    4575  Russia, Transbaikal Region, Aleksandrovsky Zavod, 50.9°N, 117.9°E (4)  1938  PCR failed  –  ZMMU s34813    4574  Russia, Transbaikal Region, Chita riverhead, 52.89°N, 114.39°E (5)  1938  KX455635  L. amurensis  ZISP 16754  L. a. ognevi holotype  4464  Russia, Yakutia, Verhoyansky District, Nel’gekhe River, 64.4°N, 134.04°E (6)  1927  KX455622  L. sibiricus (East clade)  ZMMU s124119    4569  Russia, Magadan Region, Khinikanumsa River, 61.71°N, 145.79°E (7)  1980  KX455623  L. sibiricus (East clade)  ZMMU s124120    4570  1981  KX455624  ZMMU s124121    4571  1981  KX455625  ZMMU s150533    4572  Russia, Magadan Region, Yama River, 60.01°N, 152.99°E (8)  1969  KX455626  L. sibiricus (East clade)  ZMMU s88365    4573  1969  KX455627  ZISP 13896  L. flavescens paralectotype  4563  Russia, Kamchatka Peninsula, Ust’- Kamchatsk, 56.53°N, 162.11°E (9)  1908  KX455621  L. sibiricus (East clade)  ZISP 90  L. flavescens lectotype  4244  Russia, Kamchatka Peninsula, south-western coast, supposedly Ust’-Bolsheretsk (11)  1826  KX455619  L. trimucronatus  ZISP 91  L. flavescens paralectotype  4562  KX455620  ZMMU s193482    4577  Russia, Chukotka, Khatyrka riverhear, 62.61°N, 173.23°E (12)  1981  KX455618  L. trimucronatus  Specimen number  Type material  Tissue number  Locality  Year of collection  GenBank accession number  Taxonomy*  ZISP 13722  L. amurensis holotype  4463  Russia, Amur Region, Zeysky District, Pikan, 53.68°N, 127.46°E (1)  1914  KX455628  L. amurensis  ZMMU s91379    4564  1970  KX455629  ZMMU s91377    4565  1970  KX455630  ZMMU s91380    4566  1970  KX455631  ZMMU s91381    4567  1970  KX455632  ZMMU s91378    4568  1970  KX455633  ZMMU s150535    4576  Russia, Yakutia, Neryungri District, Nagorny, 55.95°N, 124.91°E (2)  1978  KX455636  L. amurensis  ZISP 71779    4245  Russia, Yakutia, Neryungri District, Chulman, 56.71°N, 124.87°E (3)  1980  KX455634  L. amurensis  ZMMU s90417    4575  Russia, Transbaikal Region, Aleksandrovsky Zavod, 50.9°N, 117.9°E (4)  1938  PCR failed  –  ZMMU s34813    4574  Russia, Transbaikal Region, Chita riverhead, 52.89°N, 114.39°E (5)  1938  KX455635  L. amurensis  ZISP 16754  L. a. ognevi holotype  4464  Russia, Yakutia, Verhoyansky District, Nel’gekhe River, 64.4°N, 134.04°E (6)  1927  KX455622  L. sibiricus (East clade)  ZMMU s124119    4569  Russia, Magadan Region, Khinikanumsa River, 61.71°N, 145.79°E (7)  1980  KX455623  L. sibiricus (East clade)  ZMMU s124120    4570  1981  KX455624  ZMMU s124121    4571  1981  KX455625  ZMMU s150533    4572  Russia, Magadan Region, Yama River, 60.01°N, 152.99°E (8)  1969  KX455626  L. sibiricus (East clade)  ZMMU s88365    4573  1969  KX455627  ZISP 13896  L. flavescens paralectotype  4563  Russia, Kamchatka Peninsula, Ust’- Kamchatsk, 56.53°N, 162.11°E (9)  1908  KX455621  L. sibiricus (East clade)  ZISP 90  L. flavescens lectotype  4244  Russia, Kamchatka Peninsula, south-western coast, supposedly Ust’-Bolsheretsk (11)  1826  KX455619  L. trimucronatus  ZISP 91  L. flavescens paralectotype  4562  KX455620  ZMMU s193482    4577  Russia, Chukotka, Khatyrka riverhear, 62.61°N, 173.23°E (12)  1981  KX455618  L. trimucronatus  Museum names are abbreviated as follows: ZISP, Zoological Institute (St. Petersburg); ZMMU, Zoological Museum of Moscow University. Locality numbers (in brackets) correspond to Figures 1 and 2. *Taxonomic attribution after cytb analysis (see Fig. 2), with sequences listed in GenBank, as indicated. View Large Table 2. List of Lemmus cytb sequences downloaded from GenBank and used in the analysis (see Fig. 2) GenBank accession  Taxonomy  Locality  Reference  FJ025977–FJ025979  L. sibiricus (East clade)  Russia, Kamchatka Peninsula (10)  Abramson et al. (2008)  FJ025980, J025981  Russia, Yakutia, Lena Delta  FJ025982, J025983  L. trimucronatus  Russia, Chukotka Peninsula  FJ025984  L. lemmus  Sweden, Vasterbotten  FJ025985  Russia, Kola Peninsula  FJ025986  L. sibiricus (West clade)  Russia, Yamal Peninsula  AJ012672  L. sibiricus (West clade)  Russia, Olenekskiy Bay  Fedorov et al. (1999)  AJ012673  L. sibiricus (East clade)  Russia, Indigirka Delta  AJ012674  Russia, West of Kolyma Delta  AJ012677  Russia, Wrangel Island  AJ012678  Russia, Kamchatka Peninsula  AY219140  L. sibiricus (West clade)  Russia, Taymyr Peninsula  Fedorov et al. (2003)  AY219143  AY219141, AY219142  L. sibiricus (East clade)  Russia, Kotelny Island  AY219144  Russia, West of Kolyma Delta  AY219145  L. lemmus  Russia, Kola Peninsula  AF348389, F348390  L. sibiricus (West clade)  Russia, West Yamal Peninsula  Fedorov & Stenseth (2001)  AF348391  L. lemmus  Norway  AF348392  Finland  JX483882–JX483888  L. sibiricus (West clade)  Russia, Amderma  Lagerholm et al. (2014)  JX483889, JX483890  Russia, Derevnya  JX483891  Russia, Pechora  JX483892  L. lemmus  Sweden, Jamtland  JX483901, JX483902  JX483893, JX483894  Sweden, Vasterbotten  JX483900  JX483896, JX483897  Sweden, Padjelanta, Norrbotten  JX483898, JX483899  Sweden, Jamtland/Vasterbotten  JX483895  Sweden, Abisko, Norrbotten  JX483903, JX483904  JX483905, JX483906  Sweden, Kebnekaise, Norrbotten  JX483907, JX483908  Sweden, Sarek, Norrbotten  GenBank accession  Taxonomy  Locality  Reference  FJ025977–FJ025979  L. sibiricus (East clade)  Russia, Kamchatka Peninsula (10)  Abramson et al. (2008)  FJ025980, J025981  Russia, Yakutia, Lena Delta  FJ025982, J025983  L. trimucronatus  Russia, Chukotka Peninsula  FJ025984  L. lemmus  Sweden, Vasterbotten  FJ025985  Russia, Kola Peninsula  FJ025986  L. sibiricus (West clade)  Russia, Yamal Peninsula  AJ012672  L. sibiricus (West clade)  Russia, Olenekskiy Bay  Fedorov et al. (1999)  AJ012673  L. sibiricus (East clade)  Russia, Indigirka Delta  AJ012674  Russia, West of Kolyma Delta  AJ012677  Russia, Wrangel Island  AJ012678  Russia, Kamchatka Peninsula  AY219140  L. sibiricus (West clade)  Russia, Taymyr Peninsula  Fedorov et al. (2003)  AY219143  AY219141, AY219142  L. sibiricus (East clade)  Russia, Kotelny Island  AY219144  Russia, West of Kolyma Delta  AY219145  L. lemmus  Russia, Kola Peninsula  AF348389, F348390  L. sibiricus (West clade)  Russia, West Yamal Peninsula  Fedorov & Stenseth (2001)  AF348391  L. lemmus  Norway  AF348392  Finland  JX483882–JX483888  L. sibiricus (West clade)  Russia, Amderma  Lagerholm et al. (2014)  JX483889, JX483890  Russia, Derevnya  JX483891  Russia, Pechora  JX483892  L. lemmus  Sweden, Jamtland  JX483901, JX483902  JX483893, JX483894  Sweden, Vasterbotten  JX483900  JX483896, JX483897  Sweden, Padjelanta, Norrbotten  JX483898, JX483899  Sweden, Jamtland/Vasterbotten  JX483895  Sweden, Abisko, Norrbotten  JX483903, JX483904  JX483905, JX483906  Sweden, Kebnekaise, Norrbotten  JX483907, JX483908  Sweden, Sarek, Norrbotten  Locality number (in brackets) corresponds to Figures 1 and 2. View Large Methods DNA extraction, amplification and sequencing DNA from museum skin samples was isolated using Qiagen’s QIAamp Tissue Kit. A 337-bp fragment of the cytb gene was amplified in three overlapping fragments using primers and PCR conditions published by Lagerholm et al. (2014). Both DNA isolation and PCR with museum samples were conducted in a room isolated from post-PCR facilities using a PCR Workstation (LAMSYSTEMS CC) and the working surface, all instruments and plastics were sterilized with UV light and chloramine-T to avoid contamination. The PCR products were purified with Omnix kit columns (Omnix, St. Petersburg) and sequenced in both directions using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit on an ABI PRISM 3130 (Applied Biosystems Inc.). Sequences were edited, assembled and aligned with BioEdit (Hall, 1999). Phylogenetic analysis Phylogenetic analysis of new and published sequences was performed using a 924-bp alignment (the missing 587 bases for 337-bp sequences from museum samples were coded with Ns). Sequences of Dicrostonyx torquatus (AF119275), Synaptomys cooperi (DQ323957) and Myopus schisticolor (EU165208) were used as outgroups. Phylogenies were reconstructed using Bayesian inference (BI) analysis and maximum likelihood (ML) approaches. To choose the best model of molecular evolution (TVM+G), we used Akaike’s information criterion (AIC) in jModelTest 2.1.4 (Darriba et al., 2012). Bayesian analysis was performed in BEAST v2.1.0 (Drummond et al., 2012) with parameters given below in the ‘Estimation of divergence dates’ section. ML analysis was performed using TreeFinder (Jobb, 2008) under the TVM+G substitution model. Bootstrap analysis employed 1000 replicates. Final trees were obtained using FigTree v1.4. (http://tree.bio.ed.ac.uk/software/figtree/). Estimation of divergence dates Divergence dates were estimated in BEAST v2.1.0 (Drummond et al., 2012). We used a relaxed lognormal clock model (Drummond et al., 2006) and the following time constraints. We set a prior on the basal node of the Lemmini clade to 3 Mya, calibrated with a lognormal distribution with the offset = 3.0 Mya. This calibration point is substantiated by the first records of ancestral forms of lemmings both in North America (Repenning, 2001) and Eurasia (Sukhov, 1976; Kowalski, 1977), from sediments of ~2.6 Mya and already subsequent to the split within the Lemmini, thus moving back the time to most recent common ancestor (MRCA) of the tribe Lemmini. Since the use of a more complicated model does not affect the result (Huelsenbeck & Ronquist, 2005), we ran the BI analysis under the GTR+I+G instead of TVM+G selected in jModelTest, and all remaining priors were set to the defaults. The analysis was performed under a birth–death model that assumes that at any time point, every lineage can undergo speciation at rate λ or go extinct at rate µ. Two replicate runs of 100 million Markov chain Monte Carlo generations each were performed, sampling trees and parameter estimators every 10000 generations. The convergence of run parameters was examined in TRACER v1.6 (Rambaut & Drummond, 2007) and the first 25 million generations were discarded as burn-in. The final time-tree was summarized using TreeAnnotator v2.0.3 (Drummond & Rambaut, 2007) based on the trees sampled after the 25% burn-in from both independent runs, using the maximum clade credibility tree option and fixing node heights as mean heights. Divergence time bars were obtained automatically in FigTree v1.4 (http://tree.bio.ed.ac.uk/software/figtree/) from the output using the 95% highest posterior density (HPD) of the ages for each node. RESULTS Data set We succeeded in amplifying cytb from 19 museum samples with one failure (Table 1). The final cytb alignment contained 72 Lemmus sequences. The 924 bp alignment reduced to the shortest sequence (337 bp museum samples) contained 61 variable sites, of which 49 were parsimony informative. Newly determined sequences were deposited in GenBank under accession numbers KX455618–KX455636 (Table 1). Phylogenetic relationships and genetic diversity The Bayesian phylogenetic tree of true lemmings shows the expected principal division, referred to here as the Palearctic and Nearctic branches (Fig. 2), with a geographic border along the Kolyma River (Fig. 1A). The average pairwise distance between these branches is 10%, in good agreement with earlier findings (Fedorov et al., 1999). The Palearctic branch is further divided into two well-defined clades. The Western Palaearctic clade (Fig. 2) includes representatives of lemmings inhabiting the arctic and subarctic tundra from Scandinavia to the Lena River. Lemmings of the Eastern Palaearctic clade (Fig. 2) inhabit tundra from the Lena River to the western bank of the Kolyma River including various Arctic islands (including Wrangel Island), South Yakutia (Fig. 1B: Localities 2, 3), the Amur Region (Fig. 1B: Locality 1), Transbaikalia (Fig. 1B: Localities 4, 5), the Kolyma uplands and the eastern shore of the Kamchatka Peninsula (Fig. 1B: Localities 7–10). The major split within the Eastern Palearctic clade separates lemmings from northeastern Siberia from the southern phylogroup that includes only lemmings from the Transbaikal area, the Amur Region and South Yakutia (Fig. 1B: Localities 1–3, 5). The latter phylogroup consists exclusively of museum samples and is of special importance as it includes the holotype of the Amur lemming together with all topotypes (Fig. 2). The geographic border on the mainland between these subdivisions within the Eastern Palaearctic clade occurs at 60°N. Figure 2. View largeDownload slide Phylogenetic tree including timings of major divergence events within the genus Lemmus species, based on cytb. The outgroups (Dicrostonyx torquatus, Myopus schisticolor and Synaptomys cooperi) are not shown. The divergence times below the nodes correspond to the mean posterior estimate of their age in kyr. The grey bars and numbers in square brackets represent the 95% HPD interval of node heights. Posterior probability values >0.90 (BI) and bootstrap support over 60% (ML) are indicated above the nodes. Numbers in brackets correspond to the collection sites in Figure 1 and Tables 1 and 2. Along the X-axis – time scale in kyr. BI, Bayesian inference; HPD, highest posterior density; ML, maximum likelihood. Figure 2. View largeDownload slide Phylogenetic tree including timings of major divergence events within the genus Lemmus species, based on cytb. The outgroups (Dicrostonyx torquatus, Myopus schisticolor and Synaptomys cooperi) are not shown. The divergence times below the nodes correspond to the mean posterior estimate of their age in kyr. The grey bars and numbers in square brackets represent the 95% HPD interval of node heights. Posterior probability values >0.90 (BI) and bootstrap support over 60% (ML) are indicated above the nodes. Numbers in brackets correspond to the collection sites in Figure 1 and Tables 1 and 2. Along the X-axis – time scale in kyr. BI, Bayesian inference; HPD, highest posterior density; ML, maximum likelihood. All of the museum samples originating both from the upper reaches of the Kolyma River and from the eastern coast of the Kamchatka Peninsula have most recently been attributed to L. amurensis (Fig. 1). They include one paralectotype of L. flavescens N 13896 at Ust’-Kamchatsk (Fig. 1B: Locality 9) and the holotype of L. a. ognevi from the Verkhoyanskiy Region (Fig. 1: Locality 6), which are phylogenetically placed within the northern phylogroup of the Eastern Palaearctic clade (Fig. 2). In conventional taxonomy, this phylogroup corresponds to L. sibiricus. Since this phylogroup was reported earlier (Fedorov et al., 1999; Abramson et al., 2008), the split within the Eastern Palearctic clade into northern and southern groups at 60°N is new to the Lemmus tree and therefore only the southern subdivision that contains the holotype can taxonomically correspond to L. amurensis. We find that the lectotype and the second paralectotype of L. flavescens (Table 1; Fig. 2: NN 4244, 4562; Fig. 1: Locality 11), trapped by Baron von Kittlitz in 1826, belong to the Nearctic branch, i.e. to L. trimucronatus. This is, therefore, the first record of this species on the Kamchatka Peninsula determined with the aid of molecular tools. Again, the other paralectotype (Table 1; Fig. 2: N 4563; Fig. 1: Locality 9) trapped almost a century later and on the other coast of the Kamchatka Peninsula is found here in the northern phylogroup of the Eastern Palearctic clade. The museum specimen N 4577 (Fig. 2) from the Koryak Plateau (Fig. 1: Locality 12) clusters (as expected) with specimens of L. trimucronatus, as this site is within the range of the latter species. Its initial attribution to L. amurensis was an evident misidentification. Divergence time estimates Molecular and fossil data were used to derive the posterior distribution of divergence dates for the crown Lemmus in the cytb tree. According to our cytb tree (Fig. 2), the divergence between the Palearctic and Nearctic branches would have occurred at ~2.1 Mya (95% HPD 1.1–2.6). Our phylogenetic reconstruction indicates that two main phases can be recognized during the diversification of Palearctic Lemmus. The first evolutionary event was the divergence of the Western Palearctic and Eastern Palearctic clades from a common ancestor, which occurred ~1 Mya (Fig. 2), while the second involved the separation of two subclades within the Eastern Palearctic clade (i.e. the L. sibiricus East clade with a northeastern distribution and the lemmings south of 60°N, corresponding to L. amurensis) around 0.68 Mya (95% HPD 0.2–0.74). The separation into L. lemmus and the L. sibiricus West clade within the Western Palearctic clade took place later, around 0.31 Mya (95% HPD 0.1–0.35). DISCUSSION Timing of divergence, biogeography and evolutionary patterns Early stages of Lemmus evolution The first remains of lemming ancestral forms appear in the fossil record almost simultaneously in Eurasia and North America (Sukhov, 1976; Kowalski, 1977; Tomida, 1987; Repenning & Grady, 1988) around 2.7 Mya. These ancestral forms in the South Urals, Russia (Sukhov, 1976) and Poland (Kowalski, 1977) dated slightly earlier than records in North America (Repenning, 2001) where their appearance coincides with the first surge of the known Northern Hemisphere continental glaciation around 2.65–2.2 Mya (Shackleton & Opdyke, 1977) and mark one of the waves of arvicoline Eurasian immigration to North America. Initially, all these findings were made with reference to the genus Synaptomys; however, later studies showed that all of these records represent the Lemmus lineage (Carls & Rabeder, 1988; Repenning & Grady, 1988; Abramson & Nadachowski, 2001). Thereafter, Lemmus fossils occur continuously in Pleistocene sites throughout northern Eurasia. It is remarkable that, contrary to other members of subfamily Arvicolinae, these ancient lemmings are very similar to modern species in molar patterns. Based on the molar evolution that can be described, a sequence of several chronospecies has been described: from L. kowalskii (Carls & Rabeder, 1988) and L. sheri (Abramson, 1992), to recent L. amurensis (Abramson, 1993). In contrast, since the first appearance of the lemming ancestral forms (Repenning & Grady, 1988) in North America around 2.7 Mya there are no records that can unequivocally be assigned to the genus Lemmus earlier than 1 Mya, when teeth indistinguishable from modern Lemmus were reported from the Cape Deceit fauna of eastern Beringia (Guthrie & Matthews, 1971). Thus, there is a time gap of almost 1 Myr between the first appearance of Lemmus ancestral forms and forms that can be attributed to the genus Lemmus proper. The other peculiarity of Lemmus evolution in North America, as compared to Eurasia, is that there are no significant southward range shifts within the Pleistocene period. In North America, fossil records of Lemmus more or less coincide with the recent distribution of the genus, whereas in Eurasia its range in the early Pleistocene reached the Pyrenees in the west and Slovakia to the south (Kowalski, 2001). Notably, lemming remains of the form that most likely may be assigned to the MRCA of L. trimucronatus and L. sibiricus + amurensis + lemmus are found on both sides of the Bering Strait (Sher et al., 1979; Repenning et al., 1987) in sediments of ~2.5–2.4 Myr. Palearctic and Nearctic branch split In this study, the main divergence within Lemmus has been dated to the early Pleistocene, at 2.1 Mya (HPD 1.1–2.6), in fairly good agreement with fossil records listed above. The date obtained is also in line with paleoclimate reconstructions. The almost simultaneous appearance of Lemmus ancestral forms on both sides of the Bering Strait coincides with the start of the first continental glaciation, 2.65 Mya (Andersen & Borns, 1997; Repenning, 2001; Cohen & Gibbard, 2012). It was a period of progressive cooling with significant drop in sea level, leading to exposure of a large portion of the continental shelf regions of the Chukchi and Bering Seas between Siberia and Alaska (Elias & Brigham-Grette, 2013). This cooling persisted until 2.0 Mya, the beginning of a Northern Hemisphere warm period (Tiglian Interglacial), which lasted for ~300000 years. This was a period of resubmergence of the Bering Strait, thus leading to the independent evolution of lemmings in Siberia and eastern Beringia. The modern distribution of L. trimucronatus in western Beringia, stretching to the eastern shore of the Kolyma River, presents a more difficult puzzle. Previous molecular studies (Fedorov et al., 2003) showed that L. trimucronatus probably moved to the Kolyma River in the Late Pleistocene period during the westward expansion from Alaska. Recent samples from both sides of the Bering Straits are genetically similar and constitute a single phylogroup. The presence of L. trimucronatus on the western coast of the Kamchatka Peninsula reported here based on the genetic study of a previously collected specimen is novel and unexpected. This finding significantly expands our knowledge of the species range. It supports the hypothesis that as part of the Late Pleistocene expansion, L. trimucronatus most likely arrived at the tundra on the western coast of the Kamchatka Peninsula from the north along one of the corridors on the sides of the glaciers on the Kamchatkan Middle Ridge (Elias & Brigham-Grette, 2013). However, the intriguing aspect of this scenario lies in the occurrence of highly fragmented populations of the L. sibiricus East clade to the east of the Middle Ridge. The presence of L. sibiricus of this clade on Wrangel Island (Fedorov et al., 1999, 2003) and eastern Kamchatka supports its probable occurrence in the Late Pleistocene over a vast territory of northeastern Russia that currently is partly occupied by L. trimucronatus. The continuous presence of lemmings in this territory is confirmed by the fossil record (Agadjanyan, 1972). One plausible explanation of the current distribution pattern is that L. trimucronatus outcompeted L. sibiricus in areas with mutual habitat. The other hypothesis implies that L. sibiricus became extinct near the Pleistocene–Holocene transition in the major part of the Chukotka Peninsula and the northern coast of the Sea of Okhotsk; edge populations of this species remained only on Wrangel Island and the eastern coast of Kamchatka before the expansion into these areas by L. trimucronatus (Fedorov et al., 2003). We suggest that the range changes associated with asynchrony in population peak numbers of L. trimucronatus and the L. sibiricus East clade during past population cycling in western Beringia may somehow explain the distributions of these species at present. It is possible that peak numbers of L. trimucronatus on the east coast of the Bering Strait in the Late Pleistocene coincided with a population crash of L. sibiricus in the Chukotka Peninsula and this discrepancy in numbers allowed L. trimucronatus to migrate and occupy the habitats of L. sibiricus. Divergence within Palearctic lemmings The main split within the Palearctic Lemmus, into the Eastern Palearctic clade (including L. sibiricus East clade and L. amurensis) and Western Palearctic clade (including L. sibiricus West clade and L. lemmus) (Fig. 2), took place around 1 Mya (0.37–1.1), according to our molecular estimations. That is synchronous with the Jaramillo episode and marks the first cryogenic epoch at the beginning of the Pleistocene in West Siberia (Arkhipov, 1987), or the Neopleistocene according to the modern scheme (Fotiev, 2009; Cohen & Gibbard, 2012). This estimate suggests that separation of these main lineages within the Palearctic Lemmus occurred much earlier than the time preceding the last glaciation, as proposed by Fedorov et al. (1999, 2003) using a smaller data set without Amur lemming samples. Lemming remains from about 1 Mya are known throughout the Palearctic from East Anglia (Harrison, Bates & Clayden, 1989) to northeastern Siberia (Sher, 1984; Abramson, 1992) and eastern Transbaikalia (Erbajeva, Alexeeva & Khenzykhenova, 2001). Although it is well known that, unlike in Europe, ice sheets had very limited distributions in East Siberia; the separation into the Eastern Palearctic and Western Palearctic clades may have resulted from isolation by mountain uplift associated with alpine glaciers and landscape remodelling due to dramatic redistribution of river systems (Korzhuev, 1974). Thus, we agree with Fedorov et al. (1999, 2003) that the ice sheet on the Verkhoyanskiy Mountain Ridge in northern Yakutia along the eastern bank of Lena River (Frenzel, Pecsi & Velichko, 1992) may have caused the isolation of lemming populations in the early Neopleistocene. The lemming remains of the early Neopleistocene in western Europe are referred to as L. kowalskii (Carls & Rabeder, 1988); in Siberia a slightly modified form from more recent sediments was described as L. sheri (Abramson, 1992). Lemmings in the eastern part of the Palearctic Within the Eastern Palearctic clade, the mean time estimation (0.68 Mya) for the split between the L. sibiricus East clade and L. amurensis (Fig. 2) roughly coincides with the glacial b stage of the Cromerian in western Europe and the early Shaitan glaciation in Siberia. The active orogeny in Transbaikalia and Yakutia (Imaev, Imaeva & Kozmin, 2005; Ovsyuchenko et al., 2010) may have generated a physical barrier for vicariant separation of the Amur lemming in southeastern Siberia and East Asia from northern populations. Late Pleistocene fossil remains of this species are found as far south as the Primorskiy Region (Tiunov & Panasenko, 2010). Nowadays, the Amur lemming is extremely rare and is declining. The only sustainable population is in South Yakutia near Chulman. Despite numerous expeditions in the Transbaikal, no one has succeeded in trapping this species since the late 1930s. Lemmings in the western part of the Palearctic Our molecular data for the split within the Western Palearctic clade into L. lemmus and the L. sibiricus West clade (from east of White Sea to the Lena River) (Figs 1, 2) gave the mean estimate for this divergence at 0.31 Mya with upper confidence limit 0.1 Mya (Fig. 2), which fully confirms to the result obtained earlier from ancient DNA (Lagerholm et al., 2014). This dating points to divergence prior to the Last Glacial Maximum (LGM). The Holocene warming and genesis of the White Sea caused the final isolation of these forms. Taxonomical considerations and species delimitation within the genus Lemmus Initially, we designed this study with DNA-barcoding ideology in mind with the goal of simple identification and naming of lemming samples using the type specimens and reference sequences. However, the results are complex and depend on the species concept used. The question of how many species of Lemmus can be distinguished is ultimately linked to species concepts. In the midst of last century, the biological species concept (BSC) dominated, defining species with an emphasis on reproductive isolation. This concept does not help much in distinguishing between conventional Palearctic species of the genus, as all hybrid combinations of L. lemmus, L. sibiricus and L. amurensis (from Chulman) give fertile offspring (Gileva et al., 1984; Pokrovskiy et al., 1984). However, many researchers have argued that reproductive isolation is a poor criterion in the case of allopatric species and thus prefer to decide species status on the basis of distinct diagnosable features, i.e. coloration and size, in agreement with the phylogenetic species concept (PSC). The mitochondrial cytb lineages, however, disagree with conventional taxonomy. According to results presented here, L. sibiricus appears to be paraphyletic in relation to both L. lemmus and L. amurensis. Thus, interpreting the mitochondrial tree in accordance with the PSC, we can either consider all the Palearctic forms as one polytypic L. lemmus species with four subspecies, or recognize four monophyletic species. Both decisions will require a new scientific name for L. sibiricus within the Eastern Palearctic clade. However, recalling that a gene tree is not equal to a species tree, and the latter is impossible to infer from studying only one locus, it is highly unreasonable to give taxonomic names to mitochondrial lineages. The other species concept that is relevant here is the evolutionary species concept (ESC). Although our data include only a fragment of a mitochondrial gene, they reflect past fragmentation events and times of isolation. The core point of the ESC is that ‘species are historical, temporal, and spatial entities’ (Wiley, 1978). Considering the true lemmings in this context, there is no support to qualify the Eastern and Western Palearctic Lemmus mitochondrial lineages as independent species, as at present there is no barrier in the tundra along the Lena River that could prevent mixing of mitochondrial lineages. From our viewpoint, the only reason why there are no records of populations with mixed Eastern and Western Palearctic clade haplotypes is insufficient sampling in these remote regions of the Arctic. The Verkhoyanskiy Ridge evidently served as a barrier isolating these phylogroups in past glacial epochs, when lemmings dominated over vast territories of the Palearctic and the southern border of their distribution was shifted significantly southward compared to the present. However, proceeding from ESC principles, there is a strong temptation to give the allopatric Amur lemming a species status, relying on data of its long independent evolutionary history, sharp differences in both coloration and size and very restricted and isolated current distribution. Notably, among recent lemmings, specimens with molar characteristics typical for Eopleistocene–Early Neopleistocene forms occur sporadically only among L. amurensis. The listed features comply with the ESC and in part with the PSC and the BSC. The species status of the Amur lemming, in the light of current data, may be supported by a conservation perspective. Contrary to the opinions given in the IUCN (International Union for Conservation of Nature) Red List of Threatened Species (Tsytsulina, 2008), and by Shenbrot & Krasnov (2005) and Carleton & Musser (2005), our data show that L. amurensis is not widespread and its status as Least Concern (LC) reflects incorrect sample identification leading to incorrect ideas of its distribution. This species is not only rare, but it has disappeared from most places where it was reported in the first half of the 20th century, including the terra typical, which was flooded by the construction of the Zeya reservoir. Ever since Fetisov trapped several specimens (deposited in ZMMU) from various sites in the southeast Transbaikal region in the first half of the 1900s, no one has been able to trap these animals despite numerous field attempts. The only sustainable population is in the Chulman River, South Yakutia area and, in our opinion, the conservation status of the Amur lemming needs to be reclassified as Vulnerable/Near Threatened (VU/NT). Nomenclature notes The results obtained in the current study, irrespective from the issue on the number of species in the genus, highlight a nomenclatural challenge. It is impossible to retain the species L. lemmus and L. sibiricus within the Palearctic branch as currently used, since L. sibiricus is clearly a paraphyletic relative of L. lemmus. Therefore, L. sibiricus should be considered a junior synonym of L. lemmus, or the L. sibiricus West clade and the L. sibiricus East clade (Fig. 2) should be assigned species or subspecies names. Thus, sibiricus could be used as a species or subspecies name for the L. sibiricus West clade and ognevi could be used for the L. sibiricus East clade. The name L. flavescens is a ‘nomen nudum’ and existed only in the form of Brandt’s handwritten labels of specimens collected by von Kittlitz during his expedition to Kamchatka in 1826–1828 until Vinogradov (1925) temporally fixed this name and designated the type series specimens. It is interesting that Vinogradov mentioned differences in coloration between the von Kittlitz specimens and N 13896 from Ust’-Kamchatsk collected by Ryabushinskiy almost a century later in 1908. The von Kittlitz specimens do not have exact coordinates with the geographic origin of the specimens; the labels indicate ‘Kamchatka’. However, the detailed description of expedition routes provided by numerous watercolour paintings (Strecker, 2011) allows us to consider with high probability that von Kittlitz collected these specimens from the southwest coast of Kamchatka in the area of modern Ust’-Bolsheretsk. According to our results the lectotype of L. flavescens (N 90) belongs to L. trimucronatus, therefore the name L. flavescens should be considered a junior synonym of L. trimucronatus. The specimen from Ust’-Kamchatsk, as shown above, explicitly clusters within the L. sibiricus East clade and should be withdrawn from the type seria. ACKNOWLEDGEMENTS We are grateful to V. S. Lebedev, curator of mammal collection at ZMMU, for giving us access to samples from the core collection of the museum; we thank F. N. Golenishchev, curator of mammal collection at ZISP. We thank the two anonymous referees whose critical comments helped to improve the manuscript. We are particularly grateful to prof. Jeremy Searle, Department of Ecology and Evolutionary Biology, Cornell University, who has done enormous work on the manuscript to improve the English and gave many valuable comments. This study was conducted in Zoological Institute RAS under the research theme N 01201376804 and financially supported from the grant of RFBR N 15-04-04602. REFERENCES Abramson NI. 1992. A new species of lemming from Eopleistocene of North east Siberia (Mammalia: Cricetidae). Zoosystematica Rossica  1: 156– 160. Abramson NI. 1993. The genus Lemmus in Eurasia in the Late Cenozoic. Trudy Zoologicheskogo Instituta AN SSSR  249: 146– 157 (in Russian). Abramson NI, Kostygov AYu, Rodchenkova EN. 2008. The taxonomy and phylogeography of Palaearctic true lemmings (Lemmus, Cricetidae, Rodentia): new insights from cyt b data. Russian Journal Theriology  7: 17– 23. Google Scholar CrossRef Search ADS   Abramson NI, Nadachowski A. 2001. 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