Molecular Phylogenetics and Evolution 76 (2014) 18–29

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Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Multilocus systematics and non-punctuated evolution of Holarctic Myodini (Rodentia: Arvicolinae) Brooks A. Kohli a,⇑, Kelly A. Speer a,1, C. William Kilpatrick b, Nyamsuren Batsaikhan c, Darmaa Damdinbaza c, Joseph A. Cook a a b c

Department of Biology and Museum of Southwestern Biology, University of New Mexico, Albuquerque, NM 87131-1051, USA Department of Biology, University of Vermont, Burlington, VT 05405, USA Department of Zoology, Faculty of Biology, National University of Mongolia, Ulaan Bataar, Mongolia

a r t i c l e

i n f o

Article history: Received 7 September 2013 Revised 6 February 2014 Accepted 18 February 2014 Available online 1 March 2014 Keywords: Holarctic Lineage through time Mitochondrial introgression Paraphyly Punctuated diversification Species tree

a b s t r a c t The tribe Myodini consists of five genera of forest and alpine voles (Alticola, Caryomys, Eothenomys, Hyperacrius and Myodes) distributed throughout the Holarctic. Because mitochondrial evidence has revealed paraphyly and polyphyly among genera, we apply the first multilocus tests to clarify taxonomy and phylogenetic relationships. Our analyses of 28 of 36 species within Myodini, including three not previously sequenced (A. montosa, A. albicaudus, and H. fertilis), identify four distinct clades and provide the first molecular evidence that Hyperacrius may not belong in Myodini. Myodes is paraphyletic, while polyphyly of Alticola reflects apparent ancient mitochondrial introgression. Diversification in this tribe was hypothesized to be tightly linked to Late Cenozoic climatic events, however, lineage through time analysis indicates diversification over the last 4 My was gradual and not strongly punctuated. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The subfamily Arvicolinae (voles and lemmings) includes geographically widespread rodents that inhabit grassland, forest, alpine, and tundra habitats throughout the Northern Hemisphere (Gromov and Polyakov, 1977). With over 150 extant species (Carleton and Musser, 2005), Arvicolinae represents one of the largest radiations of mammals within the last 5 million years (My) and a relatively extensive fossil record has allowed detailed examination of evolution and dispersal across high latitudes (Chaline et al., 1999; Repenning et al., 1990). Paleontological (Repenning et al., 1990) and molecular (Conroy and Cook, 1999) studies suggest this group underwent punctuated bursts of dispersal and diversification in correlation with glacial-interglacial cycles. The punctuated diversification hypothesis is consistent with clusters of closely related genera and species, which have stymied multiple efforts to resolve evolutionary relationships through morphologic and molecular methods (Abramson et al., 2009b; Chaline and Graf, 1988; Galewski et al., 2006; Robovsky et al., 2008). ⇑ Corresponding author. E-mail address: [email protected] (B.A. Kohli). Present address: Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA. 1

http://dx.doi.org/10.1016/j.ympev.2014.02.019 1055-7903/Ó 2014 Elsevier Inc. All rights reserved.

Punctuated speciation within Arvicolinae has been examined in a few clades (e.g. genus Microtus), but the Tribe Myodini was not thoroughly explored (Buzan et al., 2008; Conroy and Cook, 1999). Although Myodini is monophyletic (Buzan et al., 2008; Robovsky et al., 2008), numerous relationships remain unresolved within the tribe, both among and within genera (see Section 1.1). Fossil evidence indicates an Asian origin for the tribe and its five genera (Chaline and Graf, 1988; Repenning et al., 1990; Zheng and Li, 1990). Highest extant diversity is in central Asia, but species range from southern China and the Tibetan Plateau to the Arctic Circle and across subarctic and boreal habitats of Eurasia and North America (Fig. 1) (Carleton and Musser, 2005; Repenning et al., 1990). Diversification within Myodini was linked to glacial cycles (Cook et al., 2004; Repenning et al., 1990) and geomorphic events such as Tibetan Plateau uplift (Liu et al., 2012; Luo et al., 2004) over the past 3.5 My. Glaciations occurred in repeated, punctuated bouts throughout the Quaternary and altered species distributions and population connectivity. Over the last 1.2 My, glacial periodicity increased to roughly every 100 Ky (Abe-Ouchi et al., 2013), affecting ice coverage, sea levels, and climate regimes on a continental scale and influencing the role of geographic barriers in biotic diversification (Ehlers and Gibbard, 2007).

B.A. Kohli et al. / Molecular Phylogenetics and Evolution 76 (2014) 18–29

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Fig. 1. Distribution of the 5 nominal genera of Myodini (Source: IUCN): Alticola (diagonal cross-hatching), central to northeast Asia; Myodes (stippling), Europe to North America; Eothenomys (vertical lines), south-central China; Caryomys (horizontal lines), central China; and Hyperacrius (black shading), northern India and Pakistan. Distributions reflect the combined range of all species within each genus and is not limited to those sampled in this study.

Systematic uncertainty regarding the five Myodini genera (Carleton and Musser, 2005), Alticola Blanford, 1881, Myodes Pallas, 1811, Caryomys Thomas, 1911, Eothenomys Miller, 1896, and Hyperacrius Miller, 1896, is a consequence of sparse fossils, convergent or parallel morphological evolution, relatively recent diversification, limited molecular sampling, and difficulty in obtaining samples (Chaline and Graf, 1988; Gromov and Polyakov, 1977; Lebedev et al., 2007). Phylogenetic studies of Myodini have primarily utilized mitochondrial DNA (mtDNA) (Cook et al., 2004; Lebedev et al., 2007; Liu et al., 2012; Luo et al., 2004) or very limited nuclear DNA (nucDNA) (Boratynski et al., 2011; Runck et al., 2009), and have revealed cases of hybridization among nominal species, and paraphyly or polyphyly among genera. Despite efforts to increase taxonomic sampling (Lebedev et al., 2007; Liu et al., 2012; Luo et al., 2004), this is the first study to include samples of all five genera and a nucDNA perspective. Multilocus phylogenetics is essential for characterizing recent radiations, such as Myodini, due to possible hybridization, horizontal gene transfer, or incomplete lineage sorting (Maddison, 1997; Rosenberg, 2002). Examining this widely distributed tribe provides a broader understanding of how Quaternary climatic and geological events affected evolution of subarctic organisms. 1.1. Description of Myodini genera Following the taxonomy of Carleton and Musser (2005), we briefly describe systematics of Myodini genera from reviews of Caryomys and Eothenomys (Liu et al., 2012; Luo et al., 2004), Alticola and Myodes (Abramson and Lissovsky, 2012; Lebedev et al., 2007), and Hyperacrius (Phillips, 1969). Myodes (red-backed voles) is the sole Holarctic genus, occurring throughout northern Eurasia and North America (Fig. 1) (Carleton and Musser, 2005). In these predominantly forest-associated voles, three species are known to hybridize and/or exhibit mitochondrial introgression. Myodes rutilus interbreeds with parapatric M. gapperi and sympatric M. glareolus where their ranges meet in North America and Europe, respectively (Abramson et al., 2009a; Deffontaine et al., 2005;

Runck et al., 2009; Tegelström, 1987). Elsewhere, highly structured intraspecific variation within a few nominal species (e.g. M. gapperi) actually may represent multiple cryptic species (Cook et al., 2004). Other studies based on mtDNA and chromosomal evidence indicated Myodes is paraphyletic, with the subgenus Craseomys sister to a clade containing all other Myodes and the genus Alticola (Gromov and Polyakov, 1977; Lebedev et al., 2007). Craseomys includes M. rufocanus, Korean (M. regulus) and Japanese (M. smithii, M. andersoni, M. imaizumii, M. rex) red-backed voles. A recent monograph on Russian mammals listed Craseomys Miller, 1900, as a separate genus (Abramson and Lissovsky, 2012), but we choose to retain the nomenclature of Carleton and Musser (2005) until a comprehensive review of the taxonomy is completed. To reflect the ambiguity of the name Craseomys we use quotation marks herein when directly referring to it as a genus. Most species of Alticola (montane voles) are found in central Asia and typically inhabit steppe, rocky montane, and alpine habitats (Gromov and Polyakov, 1977). The exception is A. lemminus, found in tundra of northeastern Siberia (Gromov and Polyakov, 1977). Based on mtDNA, A. lemminus and A. macrotis are more closely related to Myodes than to other Alticola, suggesting the need to revise higher taxonomy (Cook et al., 2004; Lebedev et al., 2007). Hyperacrius is hypothesized to be derived from Alticola (Phillips, 1969). The two species of Hyperacrius are semi-fossorial and found in high altitude forests and alpine meadows in northern India and northern Pakistan (Phillips, 1969). Recent morphological analysis suggests a close relationship to Prometheomys and Ellobius (fossorial arvicolines) rather than Myodini (Robovsky et al., 2008), but molecular assessments were lacking. Diversity of forest-dwelling Eothenomys is centered on the southeastern Tibetan Plateau. All species have relatively limited distributions except E. melanogaster, which extends across central China to Taiwan. Despite morphologic and molecular investigations, taxonomy of Eothenomys remains unresolved (Kaneko, 1996; Liu et al., 2012; Luo et al., 2004); however, a mtDNA-based study concluded that Eothenomys should be split into multiple genera (Liu et al., 2012).

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Detailed morphometric investigation of Caryomys identified two species endemic to China (Kaneko, 1992). Although Caryomys was considered a subgenus within Eothenomys (Carleton and Musser, 2005), recent analysis combining mtDNA and morphologic data supports monophyly of Caryomys as sister to Eothenomys (Liu et al., 2012).

primers were used to amplify 400 bp segments (Kohli, 2013). Primer pairs IRBPA/B (Stanhope et al., 1996), G6pd-int1L/H, and ETS2F/R (Lyons et al., 1997) were used for IRBP, G6pd and ETS2 genes, respectively. For amplification, PCR reagents and conditions followed established protocols and annealing temperatures were as follows: cyt b, 50 °C; G6pd and IRBP, 56 °C; ETS2, 63 °C. PCR cleanup was performed with ExoSAP-IT (Affymetrix Inc., Santa Clara, California). Cycle sequencing used the ABI BigDye version 3.1 Sequencing Kit (Applied Biosystems, Foster City, California) following Platt et al. (2007), and reactions were cleaned using ethanol/EDTA/sodium acetate precipitation (Applied Biosystems).Sequences were edited with SEQUENCHER v4.9 (GeneCodesCorporation) and checked by eye. Complementary strands were aligned to create composite sequences and deposited in GenBank (Accession number KJ556566-KJ556825; Appendix B). Gaps due to indels were coded as missing data, and did not create alignment problems. To ensure genuine mtDNA was sequenced, cyt b sequences were translated to amino acids and inspected for anomalies. Four iterations of the program PHASE v2.1 (Stephens et al., 2001) was used to infer nuclear alleles. Only likelihood values over 0.9 were assigned base values.

1.2. Objectives We aim to resolve taxonomic uncertainty within Myodini and explore the mode and tempo of evolution within the tribe including mtDNA analysis of 28 (of 36) species and multilocus analysis of 19 species to address these questions: (1) Does taxonomy correctly reflect higher-level evolutionary relationships or do multilocus analyses indicate paraphyly or polyphyly of genera? (2) Are molecular analyses consistent with current species delimitations? (3) Is diversification within the tribe punctuated as a consequence of episodic environmental events? 2. Methods 2.1. Sampling and laboratory techniques

2.2. Gene tree reconstruction

We sequenced 19 Myodini species (Appendix A) and added sequences of nine others from GenBank, totaling 28 of 36 species in the tribe (Appendix C). Remaining species are rare in museum archives. Representatives of two other Arvicoline genera (Microtus and Dicrostonyx) were used as outgroups. Geographic sampling extends across the northern hemisphere (Fig. 1; Appendix A) including specimens of Alticola, Myodes, and Hyperacrius obtained from fieldwork in Mongolia and Pakistan over the last decade or the Museum of Southwestern Biology, University of Alaska Museum of the North, University of Washington Burke Museum, and Royal Ontario Museum. Genomic DNA was extracted from frozen ( 80 °C), ethanol-preserved, or dried samples using commercial kits (Qiagen Inc., Valencia, California). Ethanol-preserved tissues and skin samples were washed in STE or PBS buffer prior to extraction; otherwise, all procedures were the same. We used polymerase chain reaction (PCR) to amplify four independent loci: 671–1143 bp of mtDNA cytochrome-b gene (cyt b), and three nucDNA loci including 1059 bp of the first exon of interphotoreceptor retinoid binding protein gene (IRBP) (Stanhope et al., 1996), 569 bp of intron 1 of glucose 6-phosphate dehydrogenase (G6pd) (Iwasa and Suzuki, 2002), and 926 bp of V-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) (Lyons et al., 1997) genes. IRBP and G6pd were selected because both have been successfully sequenced for members of Myodini in previous studies; ETS2 was chosen after screening a subset of CATS primers for effectiveness and variability (Lyons et al., 1997). MSB05 and MSB14 (Hope et al., 2010) or VOLE14 (Hadly et al., 2004) primers were used for double-stranded amplifications and sequencing of cyt b. For degraded DNA, internal

For nucDNA genealogies, a subset of individuals was selected from mtDNA sampling (Table 1; Appendix B). GenBank yielded sequences for all genes: ETS2 (N = 1), IRBP (N = 4), G6pd (N = 24), cyt b (N = 77) (Appendix C). MrModeltest v2.3 (Nylander, 2004) was used to find the best model of evolution for each gene (and cyt b codon position) based on the Akaike Information Criterion. Gene trees were reconstructed using Bayesian methods in MrBayes 3.2.1 (Huelsenbeck and Ronquist, 2001). For phylogenetic analysis, missing data due to short sequence length was coded as such, whereas ambiguous base assignments were coded separately as N. Phylogenies were reconstructed using Markov chain Monte Carlo (MCMC) procedures run for 10 million generations for nucDNA and 15 million generations for mtDNA, sampling every 1000 generations, with four independent chains and a burnin of 25%. Sufficient mixing was assessed by examining the split of the standard deviations in the MrBayes output file, ensuring it was less than 0.01 after burnin. 2.3. Species tree estimation and divergence dates BEAUti v7.0.1 was used to set up species tree runs in the Bayesian reconstruction program BEAST (Drummond and Rambaut, 2007), utilizing the *BEAST algorithm (Heled and Drummond, 2010). A subset of 1–4 individuals per species sequenced over all loci was used. We initially allowed all molecular clocks to vary (lognormal relaxed clock prior), but when preliminary runs supported a clock-like rate of evolution (i.e. the ucld.stdev parameter estimate was close to 0) a strict clock prior was applied. Mutation

Table 1 Genetic locus information and sampling. Sample size (Myodini only, N), number of segregating sites (S), number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (p), model of evolution as determined by ModelTest and used in tree construction, and ⁄BEAST estimated mutation rate and 95% CI are included for each gene for all species. Sites with gaps or missing data were excluded for calculation of summary statistics. Locus

N (gene trees)

N (species trees)

Length (bp)

Genomic location

S

H

Hd

p

Model of evolution

Estimated mutation rate (%/My)

G6pd ETS2 IRBP Cyt b Pos1 Pos2 Pos3

69 45 53 189

58 44 50 65

587 893 1059 671–1143

X chromosome Nuclear intron Nuclear exon MtDNA

39 102 58 229

26 46 42 124

0.953 0.983 0.978 0.992

0.0176 0.0263 0.0228 0.0838

K80 + C HKY + C HKY + I + C GTR + I + C SYM + I + C HKY + I + C GTR + I + C

0.34 0.53 0.44 3.84

(0.25–0.43) (0.38–0.69) (0.32–0.57) (2.90–4.87)

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rates were estimated for each gene. Models of evolution were input as priors as informed by MrModelTest. Using UPGMA starting trees, 500 million MCMC generations were run for each species tree. To explore the possibility of mtDNA capture between species, nucDNA data were run separately and compared to the species tree that included mtDNA. We assessed convergence by examining run parameters in TRACER, making sure ESSs were >200 and then performing at least two identical runs to ensure that runs starting from unique random points were converging (Drummond and Rambaut, 2007). BEAST simultaneously estimates divergence dates during phylogeny reconstruction (Drummond and Rambaut, 2007) based on the multilocus species tree (Sanchez-Gracia and Castresana, 2012). To calibrate date estimates, information from three Myodini fossils were input as priors. The earliest known fossil assigned to each of the following genera were used to set a hard minimum bound on the age of each group: Alticola, 1.5 My (Serdyuk and Tesakov, 2006); Myodes, 2.6 My (Repenning et al., 1990); Eothenomys, 2 My (Liu et al., 2012). Exponential priors were then adjusted for

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each time to most recent common ancestor (tMRCA) fossil calibration so that 97.5% of the prior distribution fell between the hard minimum and 5.5 My (Ho and Phillips, 2009), the most recent possible origin of Arvicolinae according to fossil evidence (Chaline et al., 1999). 2.4. Genetic diversity and sequence divergence Summary statistics were calculated using DnaSP v5.10 (Librado and Rozas, 2009) to assess intraspecific genetic diversity and compare variability at each locus. Uncorrected sequence divergence between species was calculated by average pairwise distance for each gene in MEGA v5 (Tamura et al., 2011). Sites with gaps or missing data were excluded for calculation of summary statistics and genetic distance. 2.5. Patterns and processes of diversification To begin characterizing diversification of Myodini and assess putative links to episodic environmental changes, we investigated

Fig. 2. Bayesian gene trees for four independent loci from up to 28 species of Myodini. Cyt b was partitioned by codon position. Posterior probabilities P0.90 are indicated at nodes by an asterisk. (A) Cytochrome b, (B) ETS2, (C) IRBP, (D) G6pd. A subset of the individuals in the Cyt b tree were analyzed for all other gene trees. Species names follow Carleton and Musser (2005).

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the rate of lineage accumulation with a lineage-through-time (LTT) plot (Silberfeld et al., 2010; Tolley et al., 2008). We used the gamma statistic to test the assumption of a constant diversification rate, applying the Monte Carlo constant rates (MCCR) test to correct for incomplete taxon sampling (Pybus and Harvey, 2000). An LTT plot was created in GENIE (Pybus and Rambaut, 2002), using a four-gene, 17-taxon BEAST species tree chronogram (containing no outgroups) as the input tree. Despite achieving greater taxon sampling by using only mtDNA, LTT plots based on unresolved or spurious relationships (e.g. due to hybridization) can misrepresent diversification patterns, so integrating data from the greatest number of genes is preferable (Barraclough and Nee, 2001; Pybus and Harvey, 2000). To determine whether rates of diversification varied through time, an expected or ‘‘constant-rate’’ lineage accumulation curve and 95% confidence intervals (CI) were generated by simulating 1000 random trees in Mesquite (Maddison and Maddison, 2011) and utilizing the ‘‘Uniform speciation with sampling’’ option to create trees with the number of taxa equal to complete taxon sampling before randomly pruning branches to match the number in the original data set (input species tree) (Maddison and Maddison, 2011). This method reduces bias from incomplete taxon sampling by accounting for a wider array of potential branching patterns that missing taxa may represent (Barraclough and Nee, 2001; Pybus and Harvey, 2000). Gamma estimates for the 1000 simulated trees were then generated in GENIE to create the gamma distribution and determine the critical value for the test (one-tailed,

p < 0.05). GENIE was also used to construct LTT plots for the simulated trees, which were then summarized to determine the expected curve and 95% CI. 3. Results Sequences of nucDNA and cyt b were obtained for 19 species in Myodini (Appendix B). We were unable to sequence all four genes for A. montosa and H. fertilis, but these species are in cyt b and G6pd trees. Seventeen species were included in species tree analyses. With GenBank sequences, 28 of 36 Myodini species were included in mtDNA analyses, the first to include all five genera. 3.1. Gene tree reconstruction All Bayesian genealogies were moderately to well resolved (Fig. 2). Each genealogy supports two primary clades: one with M. rufocanus and associated species (subgenus Craseomys), and one containing all other Myodes and Alticola. Thus, Myodes is rendered paraphyletic due to the sister relationship of Craseomys subgenus to the Alticola-Myodes clade. A third basal group includes Eothenomys and Caryomys, but is only monophyletic in the cyt b and IRBP genealogies. MtDNA generally supports current taxonomy except for the two cases of apparent polyphyly in Alticola (A. macrotis and A. lemminus grouping with Myodes). These exceptions did not change with increased intra- and inter-specific sampling (Bayesian posterior

A. barakshin

A. strelzowi A. semicanus A. albicaudus A. argentatus A. lemminus

M. rutilus M. rutilus M. gapperi M. californicus M. glareolus M. centralis M. rufocanus M. andersoni M. smithii E. miletus E. proditor C. eva Dicrostonyx Microtus

Fig. 2 (continued)

B.A. Kohli et al. / Molecular Phylogenetics and Evolution 76 (2014) 18–29

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A. barakshin

A. semicanus A. strelzowi A. argentatus A. albicaudus A. argentatus A. lemminus M. glareolus M. californicus M. rutilus M. centralis M. gapperi M. rufocanus M. andersoni M. smithii E. proditor E. melanogaster E. miletus C. eva Microtus Dicrostonyx Fig. 2 (continued)

probability (PP) of 1.0; Fig. 2A). In contrast, nucDNA genealogies place A. lemminus with other Alticola (0.84–0.91 PP) and thus nominal genera are supported. We were unable to obtain samples of A. macrotis to sequence nucDNA. For the first time, a representative of Hyperacrius was sequenced, as well as two species of Alticola. Alticola albicaudus was sequenced at all four loci while H. fertilis and A. montosa were included in cyt b and G6pd trees. Both Alticola consistently grouped within the genus (Fig. 2A–D). Hyperacrius fertilis is phylogenetically distant from Alticola, the presumed sister lineage to Hyperacrius, and instead appears basal to Myodini or is nested within outgroups (Fig. 2A and D). Although the sequence length of H. fertilis cyt b was 888 bp, several other sequences included in the analysis (especially Alticola sequences obtained from GenBank) were even shorter, and this does not appear to have influenced the results. Similarly, we were only able to obtain sequence for roughly half of G6pd for H. fertilis, but the successful section included several indels the species shared only with outgroups.

nomys and Caryomys and all major clades from individual genealogies. Species level relationships of Myodes (excluding the Craseomys subgenus) remain unresolved; a polytomy was also recovered in previous molecular analyses (Cook et al., 2004; Lebedev et al., 2007). Divergence date estimates from opposing species trees have overlapping 95% confidence intervals, although the tree based only on nucDNA has wider CIs. The estimated tMRCA of Myodini is about 4 million years ago (Ma) (Fig. 3). Divergence of Myodini from Microtus and Dicrostonyx is estimated at 5–7 Ma. The tMRCA of Caryomys and Eothenomys is roughly 3 Ma, similar to the split between the ‘‘Craseomys’’ and Alticola-Myodes clades, while tMRCA for Alticola and Myodes is estimated at 2 Ma. ‘‘Craseomys’’ has the most recent tMRCA of the sampled genera (1.4 Ma) followed by Alticola and Myodes (both near 1.7 Ma), and Eothenomys (2.5 Ma). Estimated mutation rates for nucDNA loci were about an order of magnitude lower than the cyt b rate of 3.8% per My (Table 1).

3.2. Species tree estimation and divergence dates

3.3. Genetic diversity and sequence divergence

Species trees based on all loci were not significantly different from trees based on only nucDNA, except regarding monophyly of Alticola (Fig. 3). When only nucDNA is analyzed, A. lemminus is placed with other Alticola with strong support (PP = 0.97), but when mtDNA and nucDNA are used, monophyly is equivocal (PP < 0.74). Species trees also recovered the basal position of Eothe-

Each gene showed high haplotype number and haplotype diversity (Table 1). Nucleotide diversity is higher in cyt b than nuclear genes, as expected due to more rapid accumulation of mutations in mtDNA. Sequence divergence within and among genera is roughly an order of magnitude greater for cyt b than for nuclear genes (Table 2) and generally overlap with previously reported val-

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A. albicaudus A. montosa A. barakshin A. argentatus A. lemminus A. strelzowi A. semicanus M. rutilus M. gapperi M. californicus M. centralis M. glareolus

M. rufocanus M. regulus M. rufocanus M. rex M. smithii M. andersoni C. eva

M. smithii

E. miletus E. proditor H. fertilis Microtus gregalis

Microtus pennsylvanicus Dicrostonyx

Fig. 2 (continued)

ues for Myodini and other arvicoline rodents (Buzan et al., 2008; Lebedev et al., 2007; Liu et al., 2012). Hyperacrius consistently exhibits highest average divergence from all other genera for both cyt b and G6pd. Divergence values among species of Alticola and Myodes cluster according to generic or subgeneric (Craseomys) boundaries (Table 3).

Variation in rates of diversification through time are not statistically significant (gamma statistic = 3.8; critical gamma value = 4.24, p > 0.05). There is no evidence for departure from a constant diversification rate through time for Myodini.

tered regional climate patterns and created dramatic topographic variation (Li and Fang, 1999; Zhisheng et al., 2001). These dominant factors have been linked to pulses of diversification in northern taxa, including arvicoline rodents, through fossil and genetic evidence (Conroy and Cook, 1999; Liu et al., 2012; Repenning et al., 1990). However, our expanded sampling, multilocus approach, and tests of lineage accumulation rates indicate the hypothesized tight relationship between environment and diversification may be equivocal for some arvicoline groups, such as Myodini. This is the most complete characterization of molecular evolution for Myodini and more generally sheds light on the role of Late Cenozoic environmental events in the evolution and radiation of Holarctic taxa.

4. Discussion

4.1. Multilocus insights and systematics

The Late Cenozoic was a period of dynamic environmental change; for example, repeated climate fluctuations increased in intensity and frequency during the Quaternary, creating conditions hypothesized as paramount to evolution of northern taxa (Ehlers and Gibbard, 2007; Miller et al., 2010; Velichko et al., 2005; Webb and Bartlein, 1992). Changes in global heat and moisture transport strongly affected widespread glaciation (e.g. northern North America and Europe) and changes in relatively ice-free regions such as western China and much of central and northern Asia (Ni et al., 2010; Tarasov et al., 2000). Climate change in Asia was compounded by Tibetan Plateau uplift (1.5–3.5 Ma), that further al-

4.1.1. Newly sequenced species We provide the first molecular sequences for three species to clarify their phylogenetic positions, but we still lack specimens for eight species. Inclusion of H. fertilis as a member of Myodini should be comprehensively reviewed, as our preliminary genetic results reveal significant uncertainty; a finding concordant with morphological analyses aligning it with other fossorial arvicolids, Prometheomys and Ellobius (Robovsky et al., 2008) rather than with Alticola (Phillips, 1969). Monophyly of A. albicaudus and A. argentatus is supported by low pairwise divergence values at all loci (Table 2; Figs. 2 and 3). A strongly supported mtDNA clade includes A.

3.4. Patterns and processes of diversification

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A Alticola

Tribe Myodini

Myodes

“Craseomys” Eothenomys Caryomys

B Alticola

Tribe Myodini

Myodes

“Craseomys” Eothenomys Caryomys

Fig. 3. Species tree for 17 species of Myodini based on (A) 3 nuclear genes and cyt b or B) 3 nuclear genes, constructed using ⁄BEAST to estimate divergence dates. Microtus and Dicrostonyx are included as outgroups. Analyses were run for 500 million MCMC generations and dated fossils of Alticola, Myodes, and Eothenomys were used to calibrate date estimates. A time scale is included showing past to present (left to right) in millions of years. Posterior probability is reported above nodes. Blue bars represent the 95% confidence intervals for date estimates. Species names follow Carleton and Musser (2005). The uncertain rank of monophyletic Craseomys as either a subgenus or genus is reflected by the quotation marks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

argentatus, A. montosa, and A. albicaudus (Fig. 2A and D), which all occur on the Tibetan Plateau and is consistent with their current assignment to the nominative subgenus, Alticola. Nesting of A. albicaudus individuals within A. argentatus in IRBP and cyt b trees, however, indicates that species boundaries require further study. 4.1.2. Cases of paraphyly and polyphyly Morphological, karyotypic, and genetic studies of Myodini have revealed that some genera formed paraphyletic or polyphyletic associations (Cook et al., 2004; Iwasa and Suzuki, 2002; Lebedev et al., 2007; Luo et al., 2004). Phylogenetic relationships have remained problematic due to the tribe’s recent radiation and episodes of mtDNA introgression and hybridization (Deffontaine et al., 2005; Runck et al., 2009). Multilocus molecular analysis supports monophyly of Alticola (montane voles), contrary to mtDNA evidence excluding A. lemminus from the genus (Cook et al., 2004; Lebedev et al., 2007). Alticola lemminus is unusual among montane voles with their Arctic distribution and associated

morphological features (e.g. white winter pelage). This species also may have captured mtDNA from M. rutilus, as evidenced by contrasting nucDNA and mtDNA perspectives (Figs. 2 and 3) and genetic distance information (Table 2). These species’ ranges overlap throughout much of northeastern Siberia (Fig. 1) but they inhabit different habitats. The magnitude of cyt b sequence divergence (6%), while less than comparisons between M. rutilus and all other species of Myodes, indicates relatively ancient introgression. Alternatively, incomplete lineage sorting may be producing this relationship. At least two other species of Myodes have acquired mtDNA from the northerly-distributed M. rutilus (Deffontaine et al., 2005; Runck et al., 2009). Introgressed individuals of M. gapperi and M. glareolus are predominantly found at the northern edge of their respective ranges, where they are parapatric or sympatric with M. rutilus. We identified one hybrid individual with the mtDNA signature of M. rutilus, but IRBP and G6pd alleles characteristic of M. gapperi (Appendix B). Myodes rutilus is more closely associated with tundra

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Table 2 Range of average uncorrected pairwise divergence among species, as compared within and between genera of Myodini. Gene tree sampling sets for each of the four genes studied were used in the analysis. A dash indicates that less than two species of the genus were successfully sequenced. Sites with gaps or missing data were excluded from the analysis. Myodes excludes the monophyletic group Craseomys, considered either a subgenus of Myodes or a separate genus altogether; it is shown separately. Hyperacrius is excluded from within genera comparisons because only one species was included in our analysis. All standard errors are 80%) taxon sampling should refine these observations (Cusimano and Renner, 2010). Simultaneous diversification, as indicated by polytomies in the species tree, is intriguing from an evolutionary standpoint despite lack of overall agreement with a punctuated model of diversification. For example, M. rutilus exemplifies inter- and intra-specific

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B.A. Kohli et al. / Molecular Phylogenetics and Evolution 76 (2014) 18–29

Table 3 Uncorrected pairwise sequence divergence of four genes (A. cyt b, B. G6pd, C. ETS2, D. IRBP) between species of Alticola and Myodes. Sites with gaps or missing data were excluded from the analysis. Solid lines demarcate current genus boundaries; dotted lines demarcate proposed genus boundaries (‘‘Craseomys’’). Species are named with the first three letters of their specific epithet, as identified in the Appendix. Cyt b

A alb.

A arg.

A bar.

A lem.

A mac.

A mon.

Panel A A alb. A arg. A bar. A lem. A mac. A mon. A sem. A str.

.038 .069 .088 .098 .060 .082 .080

.078 .090 .103 .058 .086 .089

.088 .102 .078 .086 .088

.094 .081 .091 .095

.091 .097 .112

.078 .096

.052

M M M M M M M M M

.100 .098 .082 .092 .092 .088 .092 .091 .089

.097 .096 .087 .095 .092 .081 .091 .096 .093

.097 .087 .083 .091 .088 .074 .090 .087 .081

.074 .078 .067 .075 .060 .089 .081 .092 .091

.090 .051 .083 .062 .093 .098 .108 .106 .110

.090 .096 .078 .086 .089 .076 .083 .096 .095

G6pd

A alb.

A arg.

A bar.

A lem.

A mon.

A sem.

Panel B A alb. A arg. A bar. A lem. A mon. A sem. A str.

.004 .004 .008 .004 .012 .004

.008 .012 .008 .017 .008

.012 .008 .017 .008

.012 .012 .012

.017 .008

.008

M M M M M M M M M M

.008 .008 .008 .010 .014 .008 .008 .010 .015 .008

.012 .012 .012 .014 .018 .012 .012 .014 .020 .012

.012 .012 .012 .014 .018 .012 .012 .013 .020 .012

.017 .017 .017 .018 .022 .017 .017 .018 .024 .017

.012 .012 .012 .014 .018 .012 .012 .014 .020 .012

.021 .021 .021 .022 .027 .021 .021 .022 .028 .021

cal. cen. gap. gla. rut. rex ruf. and. smi.

cal. cen. gap. gla. rut. reg. rex ruf. and. smi.

ETS2

A alb.

A arg.

A bar.

A lem.

A sem.

Panel C A alb. A arg. A bar. A lem. A sem. A str.

0.008 0.009 0.011 0.012 0.011

0.011 0.013 0.014 0.013

0.014 0.011 0.010

0.017 0.016

0.014

M M M M M M M M

0.012 0.015 0.016 0.014 0.013 0.030 0.031 0.031

0.014 0.018 0.018 0.016 0.015 0.032 0.033 0.033

0.015 0.019 0.019 0.017 0.015 0.033 0.030 0.030

0.014 0.017 0.017 0.015 0.013 0.032 0.032 0.032

IRBP

A alb.

A arg.

A bar.

Panel D A alb. A arg. A bar. A lem. A sem. A str.

0.001 0.015 0.015 0.013 0.013

0.015 0.015 0.013 0.013

M M M M M M M M

0.027 0.022 0.022 0.016 0.022 0.033 0.030 0.026

0.027 0.023 0.023 0.016 0.022 0.033 0.030 0.026

cal. cen. gap. gla. rut. ruf. and. smi.

cal. cen. gap. gla. rut. ruf. and. smi.

A sem.

A str.

M cal.

M cen.

M gap.

M gla.

M rut.

M rex

M ruf.

M and.

.096 .088 .084 .090 .096 .076 .092 .101 .099

.102 .104 .093 .103 .098 .087 .102 .105 .098

.064 .069 .078 .068 .076 .095 .093 .090

.068 .049 .068 .085 .090 .100 .097

.064 .064 .077 .084 .092 .092

.074 .088 .093 .095 .099

.087 .088 .093 .092

.059 .061 .057

.063 .064

.026

A str.

M cal.

M cen.

M gap.

M gla.

M rut.

M reg.

M rex

M ruf.

M and.

.012 .012 .012 .014 .018 .012 .012 .014 .020 .012

.017 .000 .018 .014 .017 .017 .018 .024 .017

.017 .002 .022 .017 .017 .018 .024 .017

.018 .014 .017 .017 .018 .024 .017

.024 .018 .018 .019 .025 .018

.022 .022 .024 .030 .022

.000 .001 .015 .008

.001 .015 .008

.017 .010

M smi.

M smi.

.007

A str.

M cal.

M cen.

M gap.

M gla.

M rut.

M ruf.

M and.

0.019 0.022 0.020 0.017 0.019 0.037 0.033 0.033

0.017 0.021 0.021 0.019 0.018 0.035 0.032 0.032

0.019 0.008 0.017 0.011 0.033 0.033 0.033

0.022 0.017 0.016 0.037 0.031 0.033

0.018 0.015 0.037 0.037 0.037

0.017 0.035 0.033 0.035

0.033 0.034 0.033

0.026 0.028

0.008

A lem.

A sem.

A str.

M cal.

M cen.

M gap.

M gla.

M rut.

M ruf.

M and.

0.015 0.008 0.006

0.013 0.010

0.006

0.027 0.027 0.027 0.016 0.022 0.033 0.030 0.026

0.022 0.022 0.022 0.014 0.017 0.028 0.027 0.023

0.026 0.026 0.026 0.014 0.021 0.032 0.029 0.025

0.025 0.025 0.025 0.014 0.020 0.031 0.028 0.024

0.025 0.025 0.021 0.013 0.036 0.033 0.030

0.005 0.016 0.013 0.026 0.025 0.021

0.021 0.013 0.031 0.030 0.026

0.016 0.023 0.020 0.016

0.027 0.025 0.021

0.011 0.007

0.005

M smi.

M smi.

28

B.A. Kohli et al. / Molecular Phylogenetics and Evolution 76 (2014) 18–29

episodic diversification. Unresolved relationships among M. rutilus, M. gapperi, and M. glareolus (Figs. 2 and 3) (Cook et al., 2004; Lebedev et al., 2007) are analogous to isolation of central, eastern and western lineages of the Holarctic M. rutilus, respectively (Kohli, 2013). During glacial phases M. gapperi was isolated south of continental ice sheets in North America (Runck and Cook, 2005), M. glareolus persisted and diversified in several western Eurasian refugia (Deffontaine et al., 2005; Kotlik et al., 2006), and M. rutilus was limited to regions between the Ural Mountains and Beringia. Relationships at both intra- and inter-specific scales collapse to polytomies (Figs. 2 and 3) and suggest synchronous isolation of lineages. Given the cyclic nature of environmental change in high-latitude ecosystems over the last 2.5 million years, repeated evolutionary outcomes at multiple phylogenetic levels is plausible. Following the origin of modern Myodini genera, diversification of clades was more gradual than previously suspected. Idiosyncratic biotic responses to glacial cycles (due to ecological affinities, demography, population connectivity, etc.) may have contributed to the lack of punctuated diversification of these predominantly Asian voles. Although most of Asia was relatively ice-free during glacial periods, climate cycles also dramatically altered biotic communities in unglaciated central and northern Asia (Harrison et al., 2001; Tarasov et al., 2000; Velichko et al., 2005), where species of Alticola and Myodes occurred. In contrast, diversification of Eothenomys has been linked to three major episodes of Tibetan Plateau uplift (Liu et al., 2012; Luo et al., 2004) that occurred at 3.6, 2.5, and 1.7 Ma (Li and Fang, 1999). Uplift is responsible for diversification of other regional species (Zhang and Ge, 2007), but our limited multilocus data for Eothenomys do not support a correlation between uplift and increased diversification. The broad geographic range of Myodes likely facilitated diversification as a consequence of glacial cycling and isolation in multiple refugia across the Holarctic, a process that was accentuated by repeated connection of North America to Eurasia via the Bering Land Bridge. Species of Myodes dispersed eastward through Beringia at least twice (Cook et al., 2004; Repenning, 2001; Runck et al., 2009). Close affinity of M. rufocanus with Korean and Japanese redbacked voles (‘‘Craseomys’’) supports their independent diversification in east Asia, which agrees with studies suggesting isolation of M. rufocanus in the Asian Far East by the mid-Pleistocene (0.7 Ma) (Chaline and Graf, 1988). The tMRCA estimate of 1.4 Ma for ‘‘Craseomys’’ significantly predates that minimum fossil-based estimate. Alticola’s association with arid, rocky environments is unique among Myodini and may be central to the diversification of Alticola in central Asia. Discovery of mtDNA polyphyly of two Alticola species led to the hypothesis that the ‘‘Alticola ecotype’’ had evolved multiple times from different species of Myodes, presumably because of an adaptive advantage in rocky alpine habitats (Lebedev et al., 2007). Multilocus data, however, support a single evolutionary transition from a forest-associated ancestor to one that inhabited rocky habitats and subsequently diversified into 12 species. The basal position of A. lemminus among Alticola is consistent with a morphological assessment that characterized it and A. macrotis (formerly considered a single species) as ‘‘transitional’’ between Alticola and Myodes (Gromov and Polyakov, 1977). 4.3. Conclusions By using a multilocus approach to sample all five genera of Myodini, long-standing taxonomic issues were resolved. Monophyly of Alticola is supported by nucDNA, with cases of ancient mitochondrial introgression now detected that falsify previous hypotheses of polyphyly. In contrast, Myodes as defined by Carleton and Musser (2005), is paraphyletic, so we support elevating Craseomys to genus. Phylogenetic analysis of Hyperacrius raises

questions about its inclusion in the tribe. With an improved taxonomic framework, long held assumptions about the mode and tempo of evolution of Myodini were explicitly tested for the first time. Contrary to previous generalizations tying the tribe’s radiation to distinct environmental events, evidence for pulses of diversification is equivocal within the tribe over the last 4 My. However, this preliminary assessment of Myodini, which also identified cryptic diversity within several species, should be expanded through additional geographic, taxonomic and genetic sampling. By fully elucidating the recent radiation of this broadly distributed and ecologically diverse tribe, we will uncover the processes shaping biodiversity in northern high-latitudes and improve forecasts of the effects of future climate change on high latitude systems. Acknowledgments Some phylogenetic analyses were facilitated by the University of Alaska Fairbanks, Life Science Informatics Portal, accessed online at http://biotech.inbre.alaska.edu/. Funding was received through the National Science Foundation under Grants NSF-DEB 0731350 (to S. Gardner, A.T. Peterson, and J. Cook) and NSF 1258010 (to J. Cook, E. Hoberg, K. Galbreath and E. DeChaine). The University of New Mexico Department of Biology Caughran Scholarship, the Graduate and Professional Students Association, and the Biology Graduate Students Association also financially supported this work. Members of the Cook Lab at UNM assisted with work and reviewed earlier drafts of this manuscript, especially K. Bell, J. Malaney, and Y. Sawyer. Special thanks to A. Hope for collecting preliminary data and providing insightful comments. Vadim Fedorov generously provided specimens. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev. 2014.02.019. References Abe-Ouchi, A., Saito, F., Kawamura, K., Raymo, M.E., Okuno, J.i., Takahashi, K., Blatter, H., 2013. Insolation-driven 100,000-year glacial cycles and hysteresis of icesheet volume. Nature 500, 190–193. Abramson, N.I., Lissovsky, A.A., 2012. Subfamily arvicolinae. In: Pavlinov, I.Y., Lissovsky, A.A. (Eds.), The Mammals of Russia: A Taxonomic and Geographic Reference. KMK Scientific Press, Moscow, pp. 220–276. Abramson, N., Rodchenkova, E., Kostygov, A.Y., 2009a. Genetic variation and phylogeography of the bank vole (Clethrionomys glareolus, Arvicolinae, Rodentia) in Russia with special reference to the introgression of the mtDNA of a closely related species, red-backed vole (Cl. rutilus). Russ. J. Genet. 45, 533– 545. Abramson, N.I., Lebedev, V.S., Tesakov, A.S., Bannikova, A.A., 2009b. Supraspecies relationships in the subfamily arvicolinae (Rodentia, Cricetidae): an unexpected result of nuclear gene analysis. Mol. Biol. 43, 834–846. Alves, P.C., Melo-Ferreira, J., Freitas, H., Boursot, P., 2008. The ubiquitous mountain hare mitochondria: multiple introgressive hybridization in hares, genus Lepus. Philos. Trans. Roy. Soc. B: Biol. Sci. 363, 2831–2839. Barraclough, T.G., Nee, S., 2001. Phylogenetics and speciation. Trends Ecol. Evol. 16, 391–399. Boratynski, Z., Alves, P.C., Berto, S., Koskela, E., Mappes, T., Melo-Ferreira, J., 2011. Introgression of mitochondrial DNA among Myodes voles: consequences for energetics? BMC Evol. Biol. 11, 355. Buzan, E.V., Krystufek, B., Hanfling, B., Hutchinson, W.F., 2008. Mitochondrial phylogeny of Arvicolinae using comprehensive taxonomic sampling yields new insights. Biol. J. Linn. Soc. 94, 825–835. Carleton, M.D., Musser, G.G., 2005. Order Rodentia. In: Wilson, D.E., Reeder, D.M. (Eds.), Mammal Species of the World. The Johns Hopkins University Press, Baltimore, Maryland. Chaline, J., Graf, J.D., 1988. Phylogeny of the Arvicolidae (Rodentia): biochemical and paleontological evidence. J. Mammal., 22–33. Chaline, J., Brunet-Lecomte, P., Montuire, S., Viriot, L., Courant, F., 1999. Anatomy of the arvicoline radiation (Rodentia): palaeogeographical, palaeoecological history and evolutionary data. Annales Zoologici Fennici. Suomen Biologian Seura Vanamo, Helsinki, 1964-, pp. 239–267.

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