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New insights into the taxonomy and phylogeny of social voles inferred from mitochondrial cytochrome b sequences
Mammalian Biology 77 (2012) 178–182
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Mammalian Biology journal homepage: www.elsevier.de/mambio
Original Investigation
New insights into the taxonomy and phylogeny of social voles inferred from mitochondrial cytochrome b sequences Boris Kryˇstufek a , Tanya Zorenko b , Elena V. Buzan a,∗ a b
Science and Research Centre, University of Primorska, SI-6000 Koper, Slovenia Department of Biology, University of Latvia, LV-1586, Riga, Latvia
a r t i c l e
i n f o
Article history: Received 17 August 2011 Accepted 23 November 2011 Keywords: Microtus socialis Microtus paradoxus Arvicolinae Cryptic species Species delimitation Molecular systematics
a b s t r a c t We sequenced the entire cytochrome b gene in Microtus paradoxus from Turkmenistan and Microtus socialis from Crimea and Kalmykia. Phylogenetic relationships among social voles were reconstructed by the inclusion into analyses of a further 23 published haplotypes belonging to six species. The two probabilistic methods which were used in phylogenetic analyses, the Bayesian inference and Maximum Likelihood, yielded very similar results. Both trees showed two highly divergent lineages which were further subdivided into seven species. The socialis lineage encompassed four species (M. socialis, M. irani, M. anatolicus, and M. paradoxus), and the remaining three species clustered into the guentheri lineage (M. guentheri, M. hartingi, M. dogramacii). The ranges for nucleotide divergences between seven species of social voles (4.95–9.28% and 4.18–8.81% for mean and net divergences, respectively) mainly exceeded 4.3%, which is frequently regarded as the conservative cut-off between sibling species in the specious genus Microtus. © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved.
Introduction The arvicoline genus Microtus gained its species richness over the course of a fairly recent radiation pulse and is still in an ongoing speciation process (Jaarola et al., 2004). The entire group abounds with cryptic species which have complicated taxonomic settings throughout the 20th century. Chromosomal and molecular studies have demonstrated that two species groups underwent the most dramatic radiation in the western Palaearctics, namely the pine voles (subgenus Terricola) and the social voles, which are either classified in the subgenus Sumeriomys (Argyropulo, 1933; Gromov and Erbajeva, 1995; Shenbrot and Krasnov, 2005) or as a socialis species group within the subgenus Microtus (Jaarola et al., 2004; Musser and Carleton, 2005). The last name steams from M. socialis which is the oldest name in the species group and the type species for Sumeryoms. Chromosomal evidence had already begun to satisfactorily resolve species limits of Terricola by the 1970s when the taxonomic richness of social voles was still heavily underestimated with up to three species recognized (Corbet, 1978). Subsequent studies increased this number to eight species (Musser and Carleton, 2005) and there is little doubt that southwestern Asia was the center of speciation and diversification for social voles (Kryˇstufek et al., 2009; Zorenko, 2010).
∗ Corresponding author. Tel.: +386 5 66 37700; fax: +386 66 37710. E-mail address: [email protected] (E.V. Buzan).
Social voles are a monophyletic division of the genus Microtus (Musser and Carleton, 2005). These are small to medium arvicolines with dense and soft pelage, relatively short ears and tail, five plantar pads, shallow skull with broad and rounded brain-case, flat interorbital region with no crest, enlarged os petromastoideum and bullae, and with molars closely resembling the pattern seen in Microtus arvalis (Kryˇstufek and Vohralík, 2005). Musser and Carleton (2005) discerned eight species of social voles which differ in cytochrome b (cyt b) sequences (Kryˇstufek et al., 2009; Thanou et al., 2011), diploid numbers of chromosomes (Zima and Král, 1984; Kefelio˘glu and Kryˇstufek, 1999), cranial morphology, dental patterns and the shape of the baculum (Zorenko, 2000; Golenishchev et al., 2002; Kryˇstufek and Vohralík, 2005; Yi˘git et al., 2006; Kryˇstufek et al., 2010). These species developed at least partly effective mechanisms of prezygotic and postzygotic reproductive isolation, such as an impaired synchronicity of reproductive behavior, aggression and suppression of the female sexual cycle in interspecific trials and partial or complete sterility of offspring (Zorenko and Aksenova, 1989; Zorenko et al., 1997; Zorenko, 2000). Hybrids were so far not reported in nature, possibly also due to prevailing allopatry in this arvicoline group. The range encompass dry steppes and semi-deserts of eastern Europe, western and central Asia (from the River Dnieper and Crimea to Dzungaria in the east and to Iran and Israel in the south), south-eastern Europe (the Balkans) and, very marginally, of northern Africa (Cyrenaica in Lybia), where they are the only representatives of Arvicolinae. Microtus socialis occupies
1616-5047/$ – see front matter © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.mambio.2011.11.007
B. Kryˇstufek et al. / Mammalian Biology 77 (2012) 178–182 Table 1 Species, sample location, haplotype acronym and accession number of cyt. b sequences for Microtus paradoxus and M. socialis. Sequences AY513829-31 were published by Jaarola et al. (2004). Species
Sample location
Haplotype
M. paradoxus
Central Kopet Dag, Turkmenistan
M. socialis
Iora River, Georgia
P1 P2 P3 S1 S2 S3 S4 S5 S6
Reine, Iran Crimea, Ukraine Kalmykia, Russia
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primer annealing (1 min at 52 ◦ C), and extension (2 min at 72 ◦ C). Forward and reverse sequencing was performed on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) using BigDye chemistry (Applied Biosystems).
GeneBank Accession No.
Sequence analyses AY513829 AY513830 AY513831
the largest area, extending from eastern Europe and Anatolia to Dzungaria. The remaining species are restricted to relatively small areas along the south-western periphery of the range of M. socialis. Ranges are imperfectly known due to unresolved taxonomy of the populations in the Near East (cf. Shenbrot and Krasnov, 2005). Several studies have demonstrated that molecular data was able to provide vital insights into current species limits and past cladogenetic events in Microtus (Jaarola et al., 2004). The taxonomic status has so far been assessed by molecular markers for six species of social voles (Kryˇstufek et al., 2009) and in this study we provide new sequences of the mitochondrial cyt b gene for the paradox vole Microtus paradoxus, which has not been sequenced yet. We further demonstrate the usefulness of a cyt b gene for the reconstruction of the phylogenetic history of social voles and for setting species boundaries in the group. We set out to address two main questions: (i) are genetic distances between the paradox vole and the remaining social voles indicative of interspecific divergence and (ii) what is the basal dichotomy in social voles? Material and methods Samples This study utilized six specimens of social voles (Table 1) and a further 23 haplotypes belonging to six species were downloaded from GenBank (Jaarola et al., 2004, Kryˇstufek et al., 2009): M. hartingi from Macedonia (Accession No. FJ767744), Greece (AY513804), and Turkey (FJ767745-7, FJ767751-2); M. guentheri from Syria (FJ767743, AY513805) and Israel (AY513806); M. dogramacii from Turkey (AY513793-5); M. anatolicus from Turkey (FJ767740-2); M. irani from Turkey (FJ767748-50) and Iran (FJ767739); and M. socialis from Georgia and Iran (Table 1). DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing A 2 × 2 mm sample of tissue was dissected from ethanolpreserved tissue and air-dried under sterile conditions. DNA was extracted using a QIAamp DNA Mini kit (Qiagen). Three overlapping cyt b fragments of 613 bp, 320 bp and 470 bp were amplified using the primers L14727-SP, H15348ASP, H15915-SP (Jaarola & Searle 2002), L15162Marv, L15408Marv (Haynes et al., 2003) and H15497-SP (Jaarola et al., 2004). The alignment of these fragments yielded high quality sequence data for the entire cyt b gene (1140 bp). A polymerase chain reaction (PCR) was performed in a total volume of 25 l containing: 3 mM MgCl2 , 0.3 mM of forward and reverse primers, 0.2 mM dNTPs and 1 unit Taq polymerase (Fermentas) was supplied in buffer containing (NH4 )2 SO4 . Cycling conditions included an initial denaturation step at 94 ◦ C for 2 min, followed by 40 cycles of the following: denaturation (30 s at 96 ◦ C),
The program CodonCode Aligner 1.63 (Ewing et al., 1998) was used to align forward and reverse sequences. The resulting consensus sequences for each individual were aligned using ClustalW 4.0, implemented in the MEGA package 4.0 (Tamura et al., 2007) in combination with Bioedit 7.09 (Hall, 2004). Considering the data regarding the presence of pseudogenes in mitochondrial phylogenies (Dubey et al., 2009), the sequences were checked for the absence of stop-codons and chimeric sequences. Nucleotide, amino acid composition and genetic distances were analysed assuming a Kimura 2 parameter (K2P) sequence evolution with 10,000 bootstraps in the MEGA program (Tamura et al., 2007). The total number of polymorphic sites at each position was estimated using the DAMBE 4.2.13 package (Xia, 2000; Xia and Xie, 2001). Phylogenetic methods The most appropriate models of DNA substitution for the data were identified using MRMODELTEST 2.3 (Nylander, 2004). Both the Akaike Information Criterion (AIC) and the hierarchical Likelihood Ratio Test (hLRT) were used. Phylogenetic analysis was conducted with the Bayesian inference (BI), using the program MRBAYES 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003), and Maximum Likelihood (ML) as implemented in the program PhyML 2.4.5 (Guindon and Gascuel, 2003; Anisimova and Gascuel, 2006). Branch support, which estimates a reliability of evolutionary trees is assessed differently by the two algorithms. The BI inference of phylogeny express branch support as posterior probabilities (PP) while ML algorithm reveals nonparametric bootstrap supports (BP). Both supports are of interest to phylogenetic reconstruction as potential upper and lower bounds of node reliability. They are not interchangeable and cannot be directly compared (Douady et al., 2003). The phylogenetic inferences were performed with a general time-reversible model (GTR) + gamma distribution (G) + proportion of invariable sites (I) (G = 0.3988 and I = 0.4570). The four Monte Carlo Markov chains were run simultaneously for 106 generations, with the resulting trees sampled every 10 generations. Convergence for PP was checked by examining the generation plot visualized with TRACER v1.4 (Rambaud and Drummond, 2007). The GTRGAMMA model was used for ML analysis. Branch support (BP) in the ML tree was estimated by 1000 bootstrap replicates. The topologies resulting from these two methods were compared using a Shimodaira–Hasegawa test (Shimodaira and Hasegawa, 1999) implemented in PAUP* 4.010b (Swofford, 2002) with 1000 bootstrap replicates. Genetic diversity To assess whether genetic diversity differed between species, the nucleotide () diversity within each clade was estimated using DnaSP 4.90.1 (Rozas et al., 2003). Results Sequence data Altogether, six new haplotypes were found in our material generating a total dataset of 29 different social vole cyt b haplotypes.
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Fig. 1. Bayesian inference tree reconstructed from cytochrome b sequences of social voles and rooted with Microtus agrestis, M. subterraneus, M. kirgisorum, and M. arvalis. The branching pattern and branch lengths follow the Bayesian analysis, whereas the first and second numbers on the branches correspond to posterior probability values and bootstrap support in the maximum likelihood tree analyses, respectively. The triangles represent species of social voles which are based entirely on published haplotypes.
Within the 1140-bp long sequences considered here, 209 polymorphic sites were found with a total of 231 mutations, 184 of which were parsimony-informative. No stop-codons, insertions or deletions were observed in the alignment. As expected under neutral evolution (Martin and Palumbi, 1993), the majority of polymorphic sites were at third positions (134 variable sites, 64% of all variable sites), followed by first positions (50 variable sites, 24% of all variable sites) and second positions (25 variable sites, 12% of all variable sites). The average ratio of transitions/transversions was 4.5. Nucleotide composition was characterized by a deficit of guanines (13.08%), similar to previous findings in arvicolins (Buzan et al., 2008) and mammals (Irwin et al., 1991).
M. socialis lacked statistically supported nodes and failed to show geographical associations. The three haplotypes from Turkmenistan, which were closely clustered together, were interpreted as representing M. paradoxus. These haplotypes were the most closely related to M. socialis (mean and net divergence of 6.34% ± 0.78 and 5.17% ± 0.69, respectively; Table 2). In our results, the ranges for nucleotide divergences between seven species of social voles were 4.95–9.28% and 4.18–8.81% for mean and net divergences, respectively. The lowest divergences were between M. hartingi and M. dogramacii and the highest values of K2P estimator separated M. hartingi from M. anatolicus (mean divergences) and M. paradoxus (net divergences; Table 2).
Phylogenetic analysis
Discussion
Phylogenetic relationships among the cyt b haplotypes of social voles obtained with the two probabilistic methods (ML and BI) yielded very similar results. The Shimodaira–Hasegawa test did not reveal significant differences between these trees (P = 0.283); consequently, only the BI tree is shown (Fig. 1). Both trees showed two highly divergent lineages, which were strongly supported by bootstrap replicates (BP = 100%) and Bayesian posterior probabilities (PP = 0.97). These two lineages, the socialis lineage and the guentheri lineage, were further subdivided into seven species. All nodes within the guentheri lineage benefited from a significant albeit moderate support (PP > 0.80, BP > 95%). Microtus guentheri hold a basal position against a clade of two sister species, M. hartingi and M. dogramacii. The basal polytomy among the four species (M. anatolicus, M. irani, M. socialis, and M. paradoxus) of the socialis lineage was not resolved. New haplotypes of M. socialis from Crimea and Kalmykia clustered with the reference conspecific haplotypes from Iran and Georgia. Further substructuring within
Our study fully confirms the taxonomic status of M. paradoxus as an independent species. The paradox vole was described on the basis of material from the vicinity of Ashkhabad, Turkmenistan, as a species in its own right Chilotus paradoxus Ognev and Heptner, 1928. Afterwards it was synonymized with M. socialis (Argyropulo, 1933; Corbet, 1978; Golenishchev and Abramson, 2011) or with M. irani (Pavlinov et al., 1998; Marinina, 2005). Zykov and Zagorodnjuk (1988) argued for a specific status of the paradox vole, which was accepted by several subsequent authors (Zorenko and Golenischev, 2002a,b; Musser and Carleton, 2005; Shenbrot and Krasnov, 2005). This vole hybridized in captivity with M. schidlovskii and M. socialis (socialis lineage), and even with M. hartingi (guentheri lineage; Zorenko et al., 1997). Within its current taxonomic scope (Gromov and Erbajeva, 1995; Musser and Carleton, 2005), M. paradoxus is allopatric with respect to all other social voles and occupies a small range of about 5 × 104 km2 in the Kopet Dagh Mts. of southern Turkmenistan (Marinina, 2005) and adjacent parts of Khorassan in northern Iran (Shenbrot and Krasnov, 2005). It is common and
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Table 2 K2P divergences (mean ± SD) between seven species of social voles. Mean distances are above the diagonal and net distances are below the diagonal. 1 1. M. paradoxus 2. M. hartingi 3. M. guentheri 4. M. dogramacii 5. M. socialis 6. M. irani 7. M. anatolicus
8.81 7.53 6.50 5.17 5.74 7.08
± ± ± ± ± ±
1.03 0.89 0.81 0.69 0.82 0.91
2
3
4
5
9.22 ± 1.05
7.81 ± 0.93 6.75 ± 0.82
6.72 ± 0.78 4.95 ± 0.72 5.76 ± 0.79
6.34 8.24 7.41 5.73
6.06 4.32 6.66 6.28 8.78
± ± ± ± ±
0.79 0.70 0.86 0.84 1.07
5.26 5.96 6.70 7.52
± ± ± ±
0.75 0.80 0.88 0.89
widespread in the river valleys (e.g. Sumbar, Chandyr, Chuli, Firjuza; unpublished observations by T.Z.). Divergences between M. paradoxus and the remaining six species of social voles in our interpretations (6.34–9.22% for mean divergence and 5.14–8.81% for net divergence) exceed the intraspecific variation in Microtus in general (≤4.4%; Jaarola et al., 2004) and in social voles in particular (up to 3.19% ± 0.50 in M. irani; Kryˇstufek et al., 2009). Furthermore, the interspecific variation of social voles in our analysis (>4.95% and >4.10% for mean and net divergences, respectively; Table 2) mainly exceeds 4.3%, which was suggested as a conservative cut-off between sibling species in the specious genus Microtus (Jaarola et al., 2004). Our study retrieved a basal cladogenesis of social voles into two lineages, the guentheri lineage and the socialis lineage. An early split into two lineages is concordant with fossil evidence. Two distinct social vole morphotypes were already present in southwestern Palaearctics during the Middle Pleistocene: M. socialis in Transcaucasia (Gromov and Polyakov, 1992) and M. guentheri in western Anatolia (Storch, 1975) and south eastern Europe (Santel and Koenigswald, 1998; Kowalski, 2001). Admittedly, the fossil history of social voles is poorly documented because isolated molars contain only limited taxonomic information (Gromov and Polyakov, 1992). The two lineages differ in the diploid numbers of chromosomes which are low in the guentheri lineage (between 48 and 54), but high in the socialis lineage (between 60 and 64; Zima and Král, 1984; Kefelio˘glu and Kryˇstufek, 1999). Molecular evidence nested four species within the guentheri lineage (M. hartingi, M. guentheri, M. dogramacii, and M. mustersi; cf. Thanou et al., 2011, and the above results). The phylogenetic position of Microtus qazvinensis from northwestern Iran is still not known. This vole displays the same diploid number as M. guentheri and M. hartingi (2n = 54), but did not produce fertile hybrids in cross-breeding trials with either of these two species (Golenishchev et al., 2002). We recommend a taxonomic revision of M. quasvinensis based on molecular data. A further four species of social voles (M. socialis, M. irani, M. anatolicus, and M. paradoxus) form the socialis lineage. Musser and Carleton (2005) suggested that M. paradoxus stems from the M. guentheri lineage; however, such a scenario is not concordant with our molecular tree (Fig. 1). Species richness of social voles possibly originates from past fragmentation with subsequent allopatric speciation in refugial areas in southwestern Asia (Kryˇstufek et al., 2009). Vicariances were possibly triggered by the dynamics of the interconnected lakes in Anatolia (Kosswig, 1955), and by aridization, the tectonics of the Kopet Dagh Mts., and transgressions of the Caspian Sea further east (Zubakov and Borzenkova, 1990). Rapidly evolving social voles provide an excellent model system to detail the glacial and postglacial history of grassland biota in the southwestern Asia. Acknowledgements We would like to thank Ms. Karolyn Close for English editing. An anonymous reviewer provided valuable comments on an earlier draft.
4.34 ± 0.65 5.08 ± 0.74 7.16 ± 0.93
6 ± ± ± ±
0.78 0.92 0.85 0.72
4.18 ± 0.60 5.31 ± 0.69
6.69 7.65 7.90 6.26 6.30
7 ± ± ± ± ±
0.87 0.91 0.92 0.0.80 0.73
7.18 9.28 7.90 7.47 6.57 6.09
± ± ± ± ± ±
0.90 1.09 0.91 0.94 0.75 0.79
5.04 ± 0.73
References Anisimova, M., Gascuel, O., 2006. Approximate likelihood ratio test for branches: a fast, accurate and powerful alternative. Syst. Biol. 55, 539–552. Argyropulo, A.J., 1933. Über zwei neue paläarktische Wühlmäuse. Z. Säugetierkunde 8, 180–183. Buzan, E., Krystufek, B., Hanfling, B., Hutchinson, W.F., 2008. Mitochondrial phylogeny of Arvicolinae based on comprehensive taxonomic sampling yields new insights. Biol. J. Linn. Soc. 94, 825–835. Corbet, G.B., 1978. The Mammals of the Palaearctic Region: A Taxonomic Review. British Museum (Natural History), London. Douady, C.J., Delsuc, F., Boucher, Y., Doolittle, W.F., Douzery, E.J.P., 2003. Comparison of Bayesian and maximum likelihood bootstrap measures of phylogenetic reliability. Mol. Biol. Evol. 20, 248–254. Dubey, S., Michaux, J., Brünner, H., Hutterer, R., Vogel, P., 2009. False phylogenies on wood mice due to cryptic cytochrome-b pseudogene. Mol. Phyl. Evol. 50, 633–641. Ewing, B., Hillier, L., Wendl, M.C., Green, P., 1998. Base-calling of automated sequencer traces using Phred. I. Accuracy assessment. Gen. Res. 8, 175–185. Golenishchev, F.N., Abramson, N.I., 2011. New data on phylogeography of voles’ subgenus Sumeriomys. In: Mammals of Russia and Adjacent Territories. Moscow, p. 117. Golenishchev, F.V., Malikov, V.G., Nazari, F., Vaziri, A.Sh., Sablina, O.V., Polyakov, A.V., 2002. New species of vole of “guentheri” group (Rodentia, Arvicolinae, Microtus) from Iran. Russian J. Theriol. 1, 117–123. Gromov, I.M., Erbajeva, M.A., 1995. The Mammals of Russia and Adjacent Territories: Lagomorphs and Rodents. Russian Academy of Sciences, Zoological Institute, St. Petersburg. Gromov, I.M., Polyakov, I.Ya., 1992. Fauna of the USSR. Mammals. Vol. III, No. 8. Voles (Microtinae). Smithsonian Institution Libraries, Washington, D.C. Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate phylogenies by maximum likelihood. Syst. Biol. 52, 696–704. Hall, T., 2004. Bioedit 7.0.0. North Carolina State University, Raleigh, NC. Haynes, S., Jaarola, M., Searle, J.B., 2003. Phylogeography of the common vole (Microtus arvalis) with particular emphasis on the colonization of the Orkney archipelago. Mol. Ecol. 12, 951–956. Huelsenbeck, J.P., Ronquist, F., 2001. MrBayes: Bayesian inference of phylogeny. Bioinformatics 17, 754–755. Irwin, D.M., Kocher, T.D., Wilson, A.C., 1991. Evolution of the cytochrome b gene of mammals. J. Mol. Evol. 32, 128–144. Jaarola, M., Searle, J.B., 2002. Phylogeography of field voles (Microtus agrestis) in Eurasia inferred from mitochondrial DNA sequences. Mol. Ecol. 11, 2613–2621. Jaarola, M., Martínková, N., Gündüz, I˙ ., Brunhoff, C., Zima, J., Nadachowski, A., Amori, G., Bulatova, N.S., Chondropoulos, B., Fraguedakis-Tsolis, S., González-Esteban, J., López-Fuster, M.J., Kandaurov, A.S., Kefelio˘glu, H., Mathias, M.L., Villatem, I., Searle, J.B., 2004. Molecular phylogeny of the speciose vole genus Microtus (Arvicolinae, Rodentia) inferred from mitochondrial DNA sequences. Mol. Phyl. Evol. 33, 647–663. Kefelio˘glu, H., Kryˇstufek, B., 1999. The taxonomy of Microtus socialis group (Rodentia: Microtinae) in Turkey, with the description of a new species. J. Nat. Hist. 33, 289–303. Kosswig, C., 1955. Zoogeography of the Near East. Syst. Zool. 4, 49–73. Kowalski, K., 2001. Pleistocene rodents of Europe. Folia Quarternaria 72, 1–389. Kryˇstufek, B., Vohralík, V., 2005. Mammals of Turkey and Cyprus. Rodentia I: Sciuridae, Dipodidae, Gliridae, Arvicolinae. Annales Majora, Koper, Slovenia. Kryˇstufek, B., Buˇzan, E.V., Vohralík, V., Zareie, R., Özkan, B., 2009. Mitochondrial cytochrome b sequence yields new insight into the speciation of social voles in south-west Asia. Biol. J. Linn. Soc. 98, 121–128. Kryˇstufek, B., Vohralík, V., Zima, J., Koubinova, D., Buˇzan, E.V., 2010. A new subspecies of the Iranian vole, Microtus irani Thomas, 1921, from Iran. Zool. Middle East 50, 11–20. Marinina, L.S., 2005. Iranskaya polevka Microtus irani (Thomas, 1921). In: Kucheruk, V.V., Khlyap, L.A. (Eds.), Lagomorpha and Rodents of the Middle Asia’s deserts. Geos, Moskva, pp. 191–197. Martin, A.P., Palumbi, S.R., 1993. Protein evolution in different cellular environments: cytochrome b in sharks and mammals. Mol. Biol. Evol. 10, 873–891. Musser, G.G., Carleton, M.D., 2005. Superfamily Muroidae. In: Wilson, D.E., Reeder, D.A.M. (Eds.), Mammal species of the World. A taxonomic and geographic reference, vol. 2, 3rd ed. John Hopkins Univ. Press, Baltimore, pp. 894–1531. Nylander, J.A.A., 2004. MrModeltest v 2.2. Uppsala University, Department of Systematic Zoology, Uppsala.
182
B. Kryˇstufek et al. / Mammalian Biology 77 (2012) 178–182
Pavlinov, I.Ya., Rossolimo, O.L., 1998. Systematics of the Mammals of the USSR. Moscow University Press, Moscow. Rambaud, A., Drummond, A.J., 2007. Tracer, version 1.4, http://beast.bio.ed.ac.uk/Tracer. Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Rozas, J., Sanchez-DelBarrio, J.C., Messeguer, X., Rozas, R., 2003. DNASP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19, 2496–2497. Santel, W., Koenigswald von, W., 1998. Preliminary report on the middle Pleistocene small mammal fauna from Yarimburgaz Cave in Turkish Thrace. Eiszeitalter u. Gegenwart 48, 162–169. Shimodaira, H., Hasegawa, M., 1999. Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol. Biol. Evol. 16, 1114–1116. Shenbrot, G.I., Krasnov, B.R., 2005. An Atlas of the Geographic Distribution of the Arvicoline Rodents of the World (Rodentia, Muridae: Arvicolinae). Pensoft, Sofia. Storch, G., 1975. Eine mittelpleistozane Nager-Fauna von der Insel Chios, Agais. Senckenbergiana Biol. 56, 165–189. Swofford, D.L., 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods), Version 4. 0b10. Sinauer Associates, Sunderland, MA. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Thanou, E., Fraguedakis-Tsolis, St., Chondropoulos, B., Paragamian, K., Lymberakis, P., 2011. Ancient DNA clarifies the systematic and phylogenetic status of the only vole species living in Africa. BioSystematics Berlin 2011. Programme and Abstracts. Freie Universität Berlin, Berlin, pp. 355–356, http://www.biosystberlin-2011.de/Biosystematics Abstracts.pdf. Xia, X., 2000. Data Analysis in Molecular Biology and Evolution. Kluwer Acad. Publ., Boston, MA.
Xia, X., Xie, Z., 2001. DAMBE: data analysis in molecular biology and evolution. J. Heredity 92, 371–373. Yi˘git, N., C¸olak, E., Sözen, M., Karatas¸, A., 2006. Rodents of Türkiye. Meteksan Co., Ankara. Zima, J., Král, B., 1984. Karyotypes of European mammals II. Acta Sc. Nat. Brno 18 (8), 1–62. Zorenko, T.A., 2000. Morphology of genitals and copulatory behavior in social voles of the subgenus Sumeriomys (Rodentia, Arvicolinae). Zool. Zh. 79, 990–999. Zorenko, T., 2010. Formation of a modern area of steppe voles’ subgenus Sumeriomys. In: 12th Rodent et Spatium Conf., Zonguldak, Turkiye, pp. 32–33. Zorenko, T., Aksenova, T., 1989. The morphology of genitals, copulatory behavior and ‘ the problem of reproductive isolation in voles of tribe Microtini. In: Zoologijas ¯ as ¯ problemas, ¯ aktual Latv. Univ., Riga, pp. 111–132. Zorenko, T., Golenischev, F., 2002a. Copet Dag’s social vole Microtus paradoxus. In: Formozov, N. (Ed.), Rodents of the Former USSR. An Estimation of the Status and the Plan of Nature Protection Actions. , http://bcc.seu.ru/programs/rodent/species/microtus paradoxus.html. Zorenko, T., Golenischev, F., 2002b. Social vole Microtus socialis. In: Formozov, N. (Ed.), Rodents of the Former USSR. An Estimation of the Status and the Plan of Nature Protection Actions. , Red. N. Formozov: http://bcc.seu.ru/programs/rodent/species/microtus socialis.html. Zorenko, T.A., Golenishchev, F.N., Skinderskaya, I.A., 1997. Behavior in social voles of subgenus Sumeriomys (Rodenria, Arvicolinae) in hybridization. Baltic J. Lab. Anim. Sci. 7, 77–102. Zubakov, V.A., Borzenkova, I.I., 1990. Global Palaeocliate of the Late Cenozoic. Elsevier Sci. Publ., Amsterdam, the Netherlands. Zykov, A.E., Zagorodnjuk, I.V., 1988. O sistematicheskom polozhenii obshchestvennoj polevki (Mammalia, Rodentia) iz Kopetdaga. Vest. Zool., Kiev. 1988 (5), 46–52.
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Mammalian Biology journal homepage: www.elsevier.de/mambio
Original Investigation
New insights into the taxonomy and phylogeny of social voles inferred from mitochondrial cytochrome b sequences Boris Kryˇstufek a , Tanya Zorenko b , Elena V. Buzan a,∗ a b
Science and Research Centre, University of Primorska, SI-6000 Koper, Slovenia Department of Biology, University of Latvia, LV-1586, Riga, Latvia
a r t i c l e
i n f o
Article history: Received 17 August 2011 Accepted 23 November 2011 Keywords: Microtus socialis Microtus paradoxus Arvicolinae Cryptic species Species delimitation Molecular systematics
a b s t r a c t We sequenced the entire cytochrome b gene in Microtus paradoxus from Turkmenistan and Microtus socialis from Crimea and Kalmykia. Phylogenetic relationships among social voles were reconstructed by the inclusion into analyses of a further 23 published haplotypes belonging to six species. The two probabilistic methods which were used in phylogenetic analyses, the Bayesian inference and Maximum Likelihood, yielded very similar results. Both trees showed two highly divergent lineages which were further subdivided into seven species. The socialis lineage encompassed four species (M. socialis, M. irani, M. anatolicus, and M. paradoxus), and the remaining three species clustered into the guentheri lineage (M. guentheri, M. hartingi, M. dogramacii). The ranges for nucleotide divergences between seven species of social voles (4.95–9.28% and 4.18–8.81% for mean and net divergences, respectively) mainly exceeded 4.3%, which is frequently regarded as the conservative cut-off between sibling species in the specious genus Microtus. © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved.
Introduction The arvicoline genus Microtus gained its species richness over the course of a fairly recent radiation pulse and is still in an ongoing speciation process (Jaarola et al., 2004). The entire group abounds with cryptic species which have complicated taxonomic settings throughout the 20th century. Chromosomal and molecular studies have demonstrated that two species groups underwent the most dramatic radiation in the western Palaearctics, namely the pine voles (subgenus Terricola) and the social voles, which are either classified in the subgenus Sumeriomys (Argyropulo, 1933; Gromov and Erbajeva, 1995; Shenbrot and Krasnov, 2005) or as a socialis species group within the subgenus Microtus (Jaarola et al., 2004; Musser and Carleton, 2005). The last name steams from M. socialis which is the oldest name in the species group and the type species for Sumeryoms. Chromosomal evidence had already begun to satisfactorily resolve species limits of Terricola by the 1970s when the taxonomic richness of social voles was still heavily underestimated with up to three species recognized (Corbet, 1978). Subsequent studies increased this number to eight species (Musser and Carleton, 2005) and there is little doubt that southwestern Asia was the center of speciation and diversification for social voles (Kryˇstufek et al., 2009; Zorenko, 2010).
∗ Corresponding author. Tel.: +386 5 66 37700; fax: +386 66 37710. E-mail address: [email protected] (E.V. Buzan).
Social voles are a monophyletic division of the genus Microtus (Musser and Carleton, 2005). These are small to medium arvicolines with dense and soft pelage, relatively short ears and tail, five plantar pads, shallow skull with broad and rounded brain-case, flat interorbital region with no crest, enlarged os petromastoideum and bullae, and with molars closely resembling the pattern seen in Microtus arvalis (Kryˇstufek and Vohralík, 2005). Musser and Carleton (2005) discerned eight species of social voles which differ in cytochrome b (cyt b) sequences (Kryˇstufek et al., 2009; Thanou et al., 2011), diploid numbers of chromosomes (Zima and Král, 1984; Kefelio˘glu and Kryˇstufek, 1999), cranial morphology, dental patterns and the shape of the baculum (Zorenko, 2000; Golenishchev et al., 2002; Kryˇstufek and Vohralík, 2005; Yi˘git et al., 2006; Kryˇstufek et al., 2010). These species developed at least partly effective mechanisms of prezygotic and postzygotic reproductive isolation, such as an impaired synchronicity of reproductive behavior, aggression and suppression of the female sexual cycle in interspecific trials and partial or complete sterility of offspring (Zorenko and Aksenova, 1989; Zorenko et al., 1997; Zorenko, 2000). Hybrids were so far not reported in nature, possibly also due to prevailing allopatry in this arvicoline group. The range encompass dry steppes and semi-deserts of eastern Europe, western and central Asia (from the River Dnieper and Crimea to Dzungaria in the east and to Iran and Israel in the south), south-eastern Europe (the Balkans) and, very marginally, of northern Africa (Cyrenaica in Lybia), where they are the only representatives of Arvicolinae. Microtus socialis occupies
1616-5047/$ – see front matter © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.mambio.2011.11.007
B. Kryˇstufek et al. / Mammalian Biology 77 (2012) 178–182 Table 1 Species, sample location, haplotype acronym and accession number of cyt. b sequences for Microtus paradoxus and M. socialis. Sequences AY513829-31 were published by Jaarola et al. (2004). Species
Sample location
Haplotype
M. paradoxus
Central Kopet Dag, Turkmenistan
M. socialis
Iora River, Georgia
P1 P2 P3 S1 S2 S3 S4 S5 S6
Reine, Iran Crimea, Ukraine Kalmykia, Russia
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primer annealing (1 min at 52 ◦ C), and extension (2 min at 72 ◦ C). Forward and reverse sequencing was performed on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) using BigDye chemistry (Applied Biosystems).
GeneBank Accession No.
Sequence analyses AY513829 AY513830 AY513831
the largest area, extending from eastern Europe and Anatolia to Dzungaria. The remaining species are restricted to relatively small areas along the south-western periphery of the range of M. socialis. Ranges are imperfectly known due to unresolved taxonomy of the populations in the Near East (cf. Shenbrot and Krasnov, 2005). Several studies have demonstrated that molecular data was able to provide vital insights into current species limits and past cladogenetic events in Microtus (Jaarola et al., 2004). The taxonomic status has so far been assessed by molecular markers for six species of social voles (Kryˇstufek et al., 2009) and in this study we provide new sequences of the mitochondrial cyt b gene for the paradox vole Microtus paradoxus, which has not been sequenced yet. We further demonstrate the usefulness of a cyt b gene for the reconstruction of the phylogenetic history of social voles and for setting species boundaries in the group. We set out to address two main questions: (i) are genetic distances between the paradox vole and the remaining social voles indicative of interspecific divergence and (ii) what is the basal dichotomy in social voles? Material and methods Samples This study utilized six specimens of social voles (Table 1) and a further 23 haplotypes belonging to six species were downloaded from GenBank (Jaarola et al., 2004, Kryˇstufek et al., 2009): M. hartingi from Macedonia (Accession No. FJ767744), Greece (AY513804), and Turkey (FJ767745-7, FJ767751-2); M. guentheri from Syria (FJ767743, AY513805) and Israel (AY513806); M. dogramacii from Turkey (AY513793-5); M. anatolicus from Turkey (FJ767740-2); M. irani from Turkey (FJ767748-50) and Iran (FJ767739); and M. socialis from Georgia and Iran (Table 1). DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing A 2 × 2 mm sample of tissue was dissected from ethanolpreserved tissue and air-dried under sterile conditions. DNA was extracted using a QIAamp DNA Mini kit (Qiagen). Three overlapping cyt b fragments of 613 bp, 320 bp and 470 bp were amplified using the primers L14727-SP, H15348ASP, H15915-SP (Jaarola & Searle 2002), L15162Marv, L15408Marv (Haynes et al., 2003) and H15497-SP (Jaarola et al., 2004). The alignment of these fragments yielded high quality sequence data for the entire cyt b gene (1140 bp). A polymerase chain reaction (PCR) was performed in a total volume of 25 l containing: 3 mM MgCl2 , 0.3 mM of forward and reverse primers, 0.2 mM dNTPs and 1 unit Taq polymerase (Fermentas) was supplied in buffer containing (NH4 )2 SO4 . Cycling conditions included an initial denaturation step at 94 ◦ C for 2 min, followed by 40 cycles of the following: denaturation (30 s at 96 ◦ C),
The program CodonCode Aligner 1.63 (Ewing et al., 1998) was used to align forward and reverse sequences. The resulting consensus sequences for each individual were aligned using ClustalW 4.0, implemented in the MEGA package 4.0 (Tamura et al., 2007) in combination with Bioedit 7.09 (Hall, 2004). Considering the data regarding the presence of pseudogenes in mitochondrial phylogenies (Dubey et al., 2009), the sequences were checked for the absence of stop-codons and chimeric sequences. Nucleotide, amino acid composition and genetic distances were analysed assuming a Kimura 2 parameter (K2P) sequence evolution with 10,000 bootstraps in the MEGA program (Tamura et al., 2007). The total number of polymorphic sites at each position was estimated using the DAMBE 4.2.13 package (Xia, 2000; Xia and Xie, 2001). Phylogenetic methods The most appropriate models of DNA substitution for the data were identified using MRMODELTEST 2.3 (Nylander, 2004). Both the Akaike Information Criterion (AIC) and the hierarchical Likelihood Ratio Test (hLRT) were used. Phylogenetic analysis was conducted with the Bayesian inference (BI), using the program MRBAYES 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003), and Maximum Likelihood (ML) as implemented in the program PhyML 2.4.5 (Guindon and Gascuel, 2003; Anisimova and Gascuel, 2006). Branch support, which estimates a reliability of evolutionary trees is assessed differently by the two algorithms. The BI inference of phylogeny express branch support as posterior probabilities (PP) while ML algorithm reveals nonparametric bootstrap supports (BP). Both supports are of interest to phylogenetic reconstruction as potential upper and lower bounds of node reliability. They are not interchangeable and cannot be directly compared (Douady et al., 2003). The phylogenetic inferences were performed with a general time-reversible model (GTR) + gamma distribution (G) + proportion of invariable sites (I) (G = 0.3988 and I = 0.4570). The four Monte Carlo Markov chains were run simultaneously for 106 generations, with the resulting trees sampled every 10 generations. Convergence for PP was checked by examining the generation plot visualized with TRACER v1.4 (Rambaud and Drummond, 2007). The GTRGAMMA model was used for ML analysis. Branch support (BP) in the ML tree was estimated by 1000 bootstrap replicates. The topologies resulting from these two methods were compared using a Shimodaira–Hasegawa test (Shimodaira and Hasegawa, 1999) implemented in PAUP* 4.010b (Swofford, 2002) with 1000 bootstrap replicates. Genetic diversity To assess whether genetic diversity differed between species, the nucleotide () diversity within each clade was estimated using DnaSP 4.90.1 (Rozas et al., 2003). Results Sequence data Altogether, six new haplotypes were found in our material generating a total dataset of 29 different social vole cyt b haplotypes.
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Fig. 1. Bayesian inference tree reconstructed from cytochrome b sequences of social voles and rooted with Microtus agrestis, M. subterraneus, M. kirgisorum, and M. arvalis. The branching pattern and branch lengths follow the Bayesian analysis, whereas the first and second numbers on the branches correspond to posterior probability values and bootstrap support in the maximum likelihood tree analyses, respectively. The triangles represent species of social voles which are based entirely on published haplotypes.
Within the 1140-bp long sequences considered here, 209 polymorphic sites were found with a total of 231 mutations, 184 of which were parsimony-informative. No stop-codons, insertions or deletions were observed in the alignment. As expected under neutral evolution (Martin and Palumbi, 1993), the majority of polymorphic sites were at third positions (134 variable sites, 64% of all variable sites), followed by first positions (50 variable sites, 24% of all variable sites) and second positions (25 variable sites, 12% of all variable sites). The average ratio of transitions/transversions was 4.5. Nucleotide composition was characterized by a deficit of guanines (13.08%), similar to previous findings in arvicolins (Buzan et al., 2008) and mammals (Irwin et al., 1991).
M. socialis lacked statistically supported nodes and failed to show geographical associations. The three haplotypes from Turkmenistan, which were closely clustered together, were interpreted as representing M. paradoxus. These haplotypes were the most closely related to M. socialis (mean and net divergence of 6.34% ± 0.78 and 5.17% ± 0.69, respectively; Table 2). In our results, the ranges for nucleotide divergences between seven species of social voles were 4.95–9.28% and 4.18–8.81% for mean and net divergences, respectively. The lowest divergences were between M. hartingi and M. dogramacii and the highest values of K2P estimator separated M. hartingi from M. anatolicus (mean divergences) and M. paradoxus (net divergences; Table 2).
Phylogenetic analysis
Discussion
Phylogenetic relationships among the cyt b haplotypes of social voles obtained with the two probabilistic methods (ML and BI) yielded very similar results. The Shimodaira–Hasegawa test did not reveal significant differences between these trees (P = 0.283); consequently, only the BI tree is shown (Fig. 1). Both trees showed two highly divergent lineages, which were strongly supported by bootstrap replicates (BP = 100%) and Bayesian posterior probabilities (PP = 0.97). These two lineages, the socialis lineage and the guentheri lineage, were further subdivided into seven species. All nodes within the guentheri lineage benefited from a significant albeit moderate support (PP > 0.80, BP > 95%). Microtus guentheri hold a basal position against a clade of two sister species, M. hartingi and M. dogramacii. The basal polytomy among the four species (M. anatolicus, M. irani, M. socialis, and M. paradoxus) of the socialis lineage was not resolved. New haplotypes of M. socialis from Crimea and Kalmykia clustered with the reference conspecific haplotypes from Iran and Georgia. Further substructuring within
Our study fully confirms the taxonomic status of M. paradoxus as an independent species. The paradox vole was described on the basis of material from the vicinity of Ashkhabad, Turkmenistan, as a species in its own right Chilotus paradoxus Ognev and Heptner, 1928. Afterwards it was synonymized with M. socialis (Argyropulo, 1933; Corbet, 1978; Golenishchev and Abramson, 2011) or with M. irani (Pavlinov et al., 1998; Marinina, 2005). Zykov and Zagorodnjuk (1988) argued for a specific status of the paradox vole, which was accepted by several subsequent authors (Zorenko and Golenischev, 2002a,b; Musser and Carleton, 2005; Shenbrot and Krasnov, 2005). This vole hybridized in captivity with M. schidlovskii and M. socialis (socialis lineage), and even with M. hartingi (guentheri lineage; Zorenko et al., 1997). Within its current taxonomic scope (Gromov and Erbajeva, 1995; Musser and Carleton, 2005), M. paradoxus is allopatric with respect to all other social voles and occupies a small range of about 5 × 104 km2 in the Kopet Dagh Mts. of southern Turkmenistan (Marinina, 2005) and adjacent parts of Khorassan in northern Iran (Shenbrot and Krasnov, 2005). It is common and
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Table 2 K2P divergences (mean ± SD) between seven species of social voles. Mean distances are above the diagonal and net distances are below the diagonal. 1 1. M. paradoxus 2. M. hartingi 3. M. guentheri 4. M. dogramacii 5. M. socialis 6. M. irani 7. M. anatolicus
8.81 7.53 6.50 5.17 5.74 7.08
± ± ± ± ± ±
1.03 0.89 0.81 0.69 0.82 0.91
2
3
4
5
9.22 ± 1.05
7.81 ± 0.93 6.75 ± 0.82
6.72 ± 0.78 4.95 ± 0.72 5.76 ± 0.79
6.34 8.24 7.41 5.73
6.06 4.32 6.66 6.28 8.78
± ± ± ± ±
0.79 0.70 0.86 0.84 1.07
5.26 5.96 6.70 7.52
± ± ± ±
0.75 0.80 0.88 0.89
widespread in the river valleys (e.g. Sumbar, Chandyr, Chuli, Firjuza; unpublished observations by T.Z.). Divergences between M. paradoxus and the remaining six species of social voles in our interpretations (6.34–9.22% for mean divergence and 5.14–8.81% for net divergence) exceed the intraspecific variation in Microtus in general (≤4.4%; Jaarola et al., 2004) and in social voles in particular (up to 3.19% ± 0.50 in M. irani; Kryˇstufek et al., 2009). Furthermore, the interspecific variation of social voles in our analysis (>4.95% and >4.10% for mean and net divergences, respectively; Table 2) mainly exceeds 4.3%, which was suggested as a conservative cut-off between sibling species in the specious genus Microtus (Jaarola et al., 2004). Our study retrieved a basal cladogenesis of social voles into two lineages, the guentheri lineage and the socialis lineage. An early split into two lineages is concordant with fossil evidence. Two distinct social vole morphotypes were already present in southwestern Palaearctics during the Middle Pleistocene: M. socialis in Transcaucasia (Gromov and Polyakov, 1992) and M. guentheri in western Anatolia (Storch, 1975) and south eastern Europe (Santel and Koenigswald, 1998; Kowalski, 2001). Admittedly, the fossil history of social voles is poorly documented because isolated molars contain only limited taxonomic information (Gromov and Polyakov, 1992). The two lineages differ in the diploid numbers of chromosomes which are low in the guentheri lineage (between 48 and 54), but high in the socialis lineage (between 60 and 64; Zima and Král, 1984; Kefelio˘glu and Kryˇstufek, 1999). Molecular evidence nested four species within the guentheri lineage (M. hartingi, M. guentheri, M. dogramacii, and M. mustersi; cf. Thanou et al., 2011, and the above results). The phylogenetic position of Microtus qazvinensis from northwestern Iran is still not known. This vole displays the same diploid number as M. guentheri and M. hartingi (2n = 54), but did not produce fertile hybrids in cross-breeding trials with either of these two species (Golenishchev et al., 2002). We recommend a taxonomic revision of M. quasvinensis based on molecular data. A further four species of social voles (M. socialis, M. irani, M. anatolicus, and M. paradoxus) form the socialis lineage. Musser and Carleton (2005) suggested that M. paradoxus stems from the M. guentheri lineage; however, such a scenario is not concordant with our molecular tree (Fig. 1). Species richness of social voles possibly originates from past fragmentation with subsequent allopatric speciation in refugial areas in southwestern Asia (Kryˇstufek et al., 2009). Vicariances were possibly triggered by the dynamics of the interconnected lakes in Anatolia (Kosswig, 1955), and by aridization, the tectonics of the Kopet Dagh Mts., and transgressions of the Caspian Sea further east (Zubakov and Borzenkova, 1990). Rapidly evolving social voles provide an excellent model system to detail the glacial and postglacial history of grassland biota in the southwestern Asia. Acknowledgements We would like to thank Ms. Karolyn Close for English editing. An anonymous reviewer provided valuable comments on an earlier draft.
4.34 ± 0.65 5.08 ± 0.74 7.16 ± 0.93
6 ± ± ± ±
0.78 0.92 0.85 0.72
4.18 ± 0.60 5.31 ± 0.69
6.69 7.65 7.90 6.26 6.30
7 ± ± ± ± ±
0.87 0.91 0.92 0.0.80 0.73
7.18 9.28 7.90 7.47 6.57 6.09
± ± ± ± ± ±
0.90 1.09 0.91 0.94 0.75 0.79
5.04 ± 0.73
References Anisimova, M., Gascuel, O., 2006. Approximate likelihood ratio test for branches: a fast, accurate and powerful alternative. Syst. Biol. 55, 539–552. Argyropulo, A.J., 1933. Über zwei neue paläarktische Wühlmäuse. Z. Säugetierkunde 8, 180–183. Buzan, E., Krystufek, B., Hanfling, B., Hutchinson, W.F., 2008. Mitochondrial phylogeny of Arvicolinae based on comprehensive taxonomic sampling yields new insights. Biol. J. Linn. Soc. 94, 825–835. Corbet, G.B., 1978. The Mammals of the Palaearctic Region: A Taxonomic Review. British Museum (Natural History), London. Douady, C.J., Delsuc, F., Boucher, Y., Doolittle, W.F., Douzery, E.J.P., 2003. Comparison of Bayesian and maximum likelihood bootstrap measures of phylogenetic reliability. Mol. Biol. Evol. 20, 248–254. Dubey, S., Michaux, J., Brünner, H., Hutterer, R., Vogel, P., 2009. False phylogenies on wood mice due to cryptic cytochrome-b pseudogene. Mol. Phyl. Evol. 50, 633–641. Ewing, B., Hillier, L., Wendl, M.C., Green, P., 1998. Base-calling of automated sequencer traces using Phred. I. Accuracy assessment. Gen. Res. 8, 175–185. Golenishchev, F.N., Abramson, N.I., 2011. New data on phylogeography of voles’ subgenus Sumeriomys. In: Mammals of Russia and Adjacent Territories. Moscow, p. 117. Golenishchev, F.V., Malikov, V.G., Nazari, F., Vaziri, A.Sh., Sablina, O.V., Polyakov, A.V., 2002. New species of vole of “guentheri” group (Rodentia, Arvicolinae, Microtus) from Iran. Russian J. Theriol. 1, 117–123. Gromov, I.M., Erbajeva, M.A., 1995. The Mammals of Russia and Adjacent Territories: Lagomorphs and Rodents. Russian Academy of Sciences, Zoological Institute, St. Petersburg. Gromov, I.M., Polyakov, I.Ya., 1992. Fauna of the USSR. Mammals. Vol. III, No. 8. Voles (Microtinae). Smithsonian Institution Libraries, Washington, D.C. Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate phylogenies by maximum likelihood. Syst. Biol. 52, 696–704. Hall, T., 2004. Bioedit 7.0.0. North Carolina State University, Raleigh, NC. Haynes, S., Jaarola, M., Searle, J.B., 2003. Phylogeography of the common vole (Microtus arvalis) with particular emphasis on the colonization of the Orkney archipelago. Mol. Ecol. 12, 951–956. Huelsenbeck, J.P., Ronquist, F., 2001. MrBayes: Bayesian inference of phylogeny. Bioinformatics 17, 754–755. Irwin, D.M., Kocher, T.D., Wilson, A.C., 1991. Evolution of the cytochrome b gene of mammals. J. Mol. Evol. 32, 128–144. Jaarola, M., Searle, J.B., 2002. Phylogeography of field voles (Microtus agrestis) in Eurasia inferred from mitochondrial DNA sequences. Mol. Ecol. 11, 2613–2621. Jaarola, M., Martínková, N., Gündüz, I˙ ., Brunhoff, C., Zima, J., Nadachowski, A., Amori, G., Bulatova, N.S., Chondropoulos, B., Fraguedakis-Tsolis, S., González-Esteban, J., López-Fuster, M.J., Kandaurov, A.S., Kefelio˘glu, H., Mathias, M.L., Villatem, I., Searle, J.B., 2004. Molecular phylogeny of the speciose vole genus Microtus (Arvicolinae, Rodentia) inferred from mitochondrial DNA sequences. Mol. Phyl. Evol. 33, 647–663. Kefelio˘glu, H., Kryˇstufek, B., 1999. The taxonomy of Microtus socialis group (Rodentia: Microtinae) in Turkey, with the description of a new species. J. Nat. Hist. 33, 289–303. Kosswig, C., 1955. Zoogeography of the Near East. Syst. Zool. 4, 49–73. Kowalski, K., 2001. Pleistocene rodents of Europe. Folia Quarternaria 72, 1–389. Kryˇstufek, B., Vohralík, V., 2005. Mammals of Turkey and Cyprus. Rodentia I: Sciuridae, Dipodidae, Gliridae, Arvicolinae. Annales Majora, Koper, Slovenia. Kryˇstufek, B., Buˇzan, E.V., Vohralík, V., Zareie, R., Özkan, B., 2009. Mitochondrial cytochrome b sequence yields new insight into the speciation of social voles in south-west Asia. Biol. J. Linn. Soc. 98, 121–128. Kryˇstufek, B., Vohralík, V., Zima, J., Koubinova, D., Buˇzan, E.V., 2010. A new subspecies of the Iranian vole, Microtus irani Thomas, 1921, from Iran. Zool. Middle East 50, 11–20. Marinina, L.S., 2005. Iranskaya polevka Microtus irani (Thomas, 1921). In: Kucheruk, V.V., Khlyap, L.A. (Eds.), Lagomorpha and Rodents of the Middle Asia’s deserts. Geos, Moskva, pp. 191–197. Martin, A.P., Palumbi, S.R., 1993. Protein evolution in different cellular environments: cytochrome b in sharks and mammals. Mol. Biol. Evol. 10, 873–891. Musser, G.G., Carleton, M.D., 2005. Superfamily Muroidae. In: Wilson, D.E., Reeder, D.A.M. (Eds.), Mammal species of the World. A taxonomic and geographic reference, vol. 2, 3rd ed. John Hopkins Univ. Press, Baltimore, pp. 894–1531. Nylander, J.A.A., 2004. MrModeltest v 2.2. Uppsala University, Department of Systematic Zoology, Uppsala.
182
B. Kryˇstufek et al. / Mammalian Biology 77 (2012) 178–182
Pavlinov, I.Ya., Rossolimo, O.L., 1998. Systematics of the Mammals of the USSR. Moscow University Press, Moscow. Rambaud, A., Drummond, A.J., 2007. Tracer, version 1.4, http://beast.bio.ed.ac.uk/Tracer. Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Rozas, J., Sanchez-DelBarrio, J.C., Messeguer, X., Rozas, R., 2003. DNASP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19, 2496–2497. Santel, W., Koenigswald von, W., 1998. Preliminary report on the middle Pleistocene small mammal fauna from Yarimburgaz Cave in Turkish Thrace. Eiszeitalter u. Gegenwart 48, 162–169. Shimodaira, H., Hasegawa, M., 1999. Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol. Biol. Evol. 16, 1114–1116. Shenbrot, G.I., Krasnov, B.R., 2005. An Atlas of the Geographic Distribution of the Arvicoline Rodents of the World (Rodentia, Muridae: Arvicolinae). Pensoft, Sofia. Storch, G., 1975. Eine mittelpleistozane Nager-Fauna von der Insel Chios, Agais. Senckenbergiana Biol. 56, 165–189. Swofford, D.L., 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods), Version 4. 0b10. Sinauer Associates, Sunderland, MA. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Thanou, E., Fraguedakis-Tsolis, St., Chondropoulos, B., Paragamian, K., Lymberakis, P., 2011. Ancient DNA clarifies the systematic and phylogenetic status of the only vole species living in Africa. BioSystematics Berlin 2011. Programme and Abstracts. Freie Universität Berlin, Berlin, pp. 355–356, http://www.biosystberlin-2011.de/Biosystematics Abstracts.pdf. Xia, X., 2000. Data Analysis in Molecular Biology and Evolution. Kluwer Acad. Publ., Boston, MA.
Xia, X., Xie, Z., 2001. DAMBE: data analysis in molecular biology and evolution. J. Heredity 92, 371–373. Yi˘git, N., C¸olak, E., Sözen, M., Karatas¸, A., 2006. Rodents of Türkiye. Meteksan Co., Ankara. Zima, J., Král, B., 1984. Karyotypes of European mammals II. Acta Sc. Nat. Brno 18 (8), 1–62. Zorenko, T.A., 2000. Morphology of genitals and copulatory behavior in social voles of the subgenus Sumeriomys (Rodentia, Arvicolinae). Zool. Zh. 79, 990–999. Zorenko, T., 2010. Formation of a modern area of steppe voles’ subgenus Sumeriomys. In: 12th Rodent et Spatium Conf., Zonguldak, Turkiye, pp. 32–33. Zorenko, T., Aksenova, T., 1989. The morphology of genitals, copulatory behavior and ‘ the problem of reproductive isolation in voles of tribe Microtini. In: Zoologijas ¯ as ¯ problemas, ¯ aktual Latv. Univ., Riga, pp. 111–132. Zorenko, T., Golenischev, F., 2002a. Copet Dag’s social vole Microtus paradoxus. In: Formozov, N. (Ed.), Rodents of the Former USSR. An Estimation of the Status and the Plan of Nature Protection Actions. , http://bcc.seu.ru/programs/rodent/species/microtus paradoxus.html. Zorenko, T., Golenischev, F., 2002b. Social vole Microtus socialis. In: Formozov, N. (Ed.), Rodents of the Former USSR. An Estimation of the Status and the Plan of Nature Protection Actions. , Red. N. Formozov: http://bcc.seu.ru/programs/rodent/species/microtus socialis.html. Zorenko, T.A., Golenishchev, F.N., Skinderskaya, I.A., 1997. Behavior in social voles of subgenus Sumeriomys (Rodenria, Arvicolinae) in hybridization. Baltic J. Lab. Anim. Sci. 7, 77–102. Zubakov, V.A., Borzenkova, I.I., 1990. Global Palaeocliate of the Late Cenozoic. Elsevier Sci. Publ., Amsterdam, the Netherlands. Zykov, A.E., Zagorodnjuk, I.V., 1988. O sistematicheskom polozhenii obshchestvennoj polevki (Mammalia, Rodentia) iz Kopetdaga. Vest. Zool., Kiev. 1988 (5), 46–52.