Phylogeny and Genetic History of the Siberian Salamander

Molecular Phylogenetics and Evolution 67 (2013) 348–357

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Molecular Phylogenetics and Evolution

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Phylogeny and genetic history of the Siberian salamander (Salamandrella keyserlingii, Dybowski, 1870) inferred from complete mitochondrial genomes ⇑ Boris Malyarchuk , Miroslava Derenko, Galina Denisova

Institute of Biological Problems of the North, Magadan 685000, Russia article info abstract

Article history: We assessed phylogeny of the Siberian salamander (Salamandrella keyserlingii, Dybowski, 1870), the most Received 31 October 2012 northern ectothermic, terrestrial vertebrate in Eurasia, by sequence analysis of complete mitochondrial Revised 28 January 2013 genomes in 26 specimens from different localities (China, Khabarovsk region, Sakhalin, Yakutia, Magadan Accepted 3 February 2013 region, Chukotka, Kamchatka, Ural, European part of Russia). In addition, a complete mitochondrial gen- Available online 12 February 2013 ome of the Schrenck salamander, Salamandrella schrenckii, was determined for the ﬁrst time. Bayesian phylogenetic analysis of the entire mtDNA genomes of S. keyserlingii demonstrates that two haplotype Keywords: clades, AB and C, radiated about 1.4 million years ago (Mya). Bayesian skyline plots of population size Mitochondrial DNA change through time show an expansion around 250 thousand years ago (kya) and then a decline around Salamandrella keyserlingii Phylogeny the Last Glacial Maximum (25 kya) with subsequent restoration of population size. Climatic changes dur- Molecular phylogeography ing the Quaternary period have dramatically affected the population genetic structure of the Siberian sal- Adaptation amanders. In addition, complete mtDNA sequence analysis allowed us to recognize that the vast area of Northern Eurasia was colonized only by the Siberian salamander clade C1b during the last 150 kya. Meanwhile, we were unable to ﬁnd evidence of molecular adaptation in this clade by analyzing the whole mitochondrial genomes of the Siberian salamanders. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction River Basin and the northern Sikhote-Alin Mountains region can be considered the center of genetic diversiﬁcation and, possibly, the The Siberian salamander (Salamandrella keyserlingii, Dybowski, site of origin of S. keyserlingii (Malyarchuk et al., 2010). This conclu- 1870) occupies the widest range compared to other Palearctic sal- sion is based on the fact that only this part of the range shows max- amanders, extending from Hokkaido in the east to the North-East imum diversity of the mtDNA clades. All mtDNA monophyletic of Europe in the west, and from Chukotkan peninsula in the north haplogroups – clades A, B, C1, C2, and C3 – were detected there, to northern China in the south (Borkin et al., 1984). Apparently, while the whole range westward and northward from the Kha- this wide distribution of the Siberian salamander was promoted barovsk region is populated by the Siberian salamanders belonging by the fact that this species can tolerate extremely low winter tem- only to clade C1 (Malyarchuk et al., 2010). These data suggest that peratures (up to À40 °C) (Berman et al., 1984). Morphologists re- such selective dispersal pattern of the Siberian salamander could ported homogeneity of the Siberian salamander populations be mediated by selection of the animals most adapted to habitation across most of the species range, albeit they suggested the pres- in low temperature conditions. Interestingly, a close relative of the ence of some variety at the southeast of the range (Borkin et al., Siberian Salamander, the Schrenck salamander, did not extend its 1984). Molecular genetic studies, based on both mitochondrial range as far as the Siberian salamander, even though it is thought and nuclear genomic data, revealed a cryptic species, the Schrenck to be a more ancient species based on the ages of the cytochrome b salamander (S. schrenckii), in the southeast of Russia (Primorye and gene haplotype divergence (Berman et al., 2005; Matsui et al., Khabarovsk regions) (Berman et al., 2005; Matsui et al., 2008; 2008; Malyarchuk et al., 2009). Thus, as only the Siberian salaman- Poyarkov and Kuzmin, 2008; Malyarchuk et al., 2009). der occupies the most northern areas, it is possible that this species More detailed phylogeographic investigation of mitochondrial had an earlier opportunity to expand into formerly inaccessible DNA (mtDNA) cytochrome b gene variation in populations of the territory and subsequently blocked the Schrenck salamander from Siberian salamander allowed us to establish that the middle Amur entering the same territory. To obtain more information concerning the phylogeny and genetic history of the Siberian salamander, we analyzed here the ⇑ Corresponding author. Address: Institute of Biological Problems of the North, whole mitochondrial genome sequences in the Siberian salaman- Far East Branch of the Russian Academy of Sciences, Portovaya Str., 18, Magadan der, as well as in the related Schrenck salamander, reported in this 685000, Russia. Fax: +7 4132 63 44 63. E-mail address: [email protected] (B. Malyarchuk). study for the ﬁrst time.

1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2013.02.004 B. Malyarchuk et al. / Molecular Phylogenetics and Evolution 67 (2013) 348–357 349

2. Materials and methods The confidence of branches in NJ and ML trees was assessed using non-parametric bootstrapping searches of 1000 replicates. Topolo- 2.1. Samples collection, DNA extraction, PCR amplification and gies of NJ and ML trees with bootstrap values 70% or greater were sequencing regarded as sufficiently resolved (Huelsenbeck and Hillis, 1993). Maximum parsimony (MP) networks of complete mtDNA genomes We examined a total of 26 Siberian salamanders from different were constructed by the median-joining (MJ) algorithm (Bandelt regions of Eurasia (Table 1, Fig. 1) and one Schrenck salamander (S. et al., 1999), using the Network 4.6 and DNA Alignment 1.3 pack- schrenckii) from the Primorye region (Russia). Total DNA was puri- ages (www.fluxus-engineering.com). Homoplasic sites were re- fied from various tissues of adults, larvae, and embryos, which vealed by use of the phylogenetic network analysis as were frozen or fixed with 70% ethanol. DNA was extracted by the implemented in the Network 4.6 program. standard method involving tissue lysis in a solution containing Bayesian inference (BI) of phylogeny using the Markov Chain 100 mM Tris–HCl (pH 8.0), 10 mM EDTA, 100 mM NaCl, 1% sodium Monte Carlo technique (MCMC) was produced from whole mtDNA dodecylsulfate, and 0.2 mg/ml proteinase K (Sigma, USA) at 56 °C sequences and the 16S rRNA-CO1 gene haplotypes by means of the for 12–16 h, with subsequent phenol–chloroform deproteinization. program BEAST v.1.6.1 (Drummond and Rambaut, 2007) with the The sequencing strategy proposed by Zhang and Wake (2009) HKY+G+I model of DNA substitution. We initiated three indepen- was used for the Siberian salamander mtDNA sequencing. A suite dent analyses with a random starting tree that ran for 30 million of 22 PCR primers was used to amplify overlapping fragments that generations sampled every 3000 steps, with the first 3 million gen- covered the entire mitochondrial genome of the Siberian salaman- erations regarded as burn-in. Posterior probabilities 95% or greater der (Table S1). Initially, primers were designed on the basis of were considered significant support (Leaché and Reeder, 2002). We complete mtDNA sequence of S. keyserlingii presented in GenBank used the mtDNA sequence of the Schrenck salamander as an out- under accession number DQ333814 (Zhang et al., 2006). For PCR group. Changes in effective population size across time were in- fragments of the Schrenck salamander mtDNA, specific primers ferred using Bayesian skyline analyses, which enable estimation were designed according to newly obtained sequences to facilitate of past population dynamics through time from a sample of mod- primer walking. ern DNA sequences (Drummond et al., 2005). The Bayesian skyline Sequencing was performed directly with the corresponding PCR plots (BSPs) produced from the whole mtDNA sequences were ob- primers using the BigDye Terminator version 3.1 cycle sequencing tained with a piecewise linear model using BEAST v.1.6.1. kit (Applied Biosystems) in an ABI 3130 or ABI 3500xL genetic ana- In Bayesian analysis, to test the hypothesis of clock-like evolu- lyzers following the manufacturer’s instructions. Multiple align- tion, we have used the uncorrelated relaxed lognormal molecular ments of nucleotide sequences were prepared for complete clock and measured the ucld.stdev parameter (the standard devia- mitochondrial genomes by using ClustalW at default settings as tion of the uncorrelated lognormal relaxed clock). Parameter esti- implemented in MEGA 5.05 (Tamura et al., 2011). mate close to 0.0 (ucld.stdev = 0.129–0.135) indicates that genomic evolution is nearly clock-like, with no variation among lineages in 2.2. Data analysis a tree. To estimate times to the most recent common ancestors (TMRCA) of the Siberian salamander mtDNA clades, a relaxed molec- To reconstruct the tree topology for complete mtDNAs and the ular clock with the evolutionary rate for salamanders (0.64% per mil- 16S rRNA-CO1 gene haplotypes, neighbor-joining (NJ) and maxi- lion years (My) per lineage), as proposed by Weisrock et al. (2001), mum likelihood (ML) analyses were used as implemented in MEGA was used. This rate was calculated based on variation of the segment 5.05 (Tamura et al., 2011). NJ-analysis was based on Kimura 2- of the mitochondrial genome encompassing three protein genes parameter model (Kimura, 1980). For ML analysis, we chose the (ND1, ND2, and CO1) and eight tRNA genes, and derived from four HKY (Hasegawa–Kishino–Yano) model of evolution with the gam- geologic events in salamandrid salamanders (Weisrock et al., 2001). ma shape parameter and proportion of invariable sites (HKY+G+I) Haplotype and nucleotide diversity indices and their variances as the best model of evolution that fits our data, by MEGA 5.05. as well as neutrality tests (Tajima’s D, Fu and Li’s DÃ, Fu and Li’s

Table 1 Distribution of sequenced S. keyserlingii samples in Eurasian populations.

Population mtDNA clades A B C1a C1b1 C1b2 C2 Khabarovsk SK8321 (JX508761), SK8391 (JX508762) SK8191 (JX508758), SK8250 (JX508759) SK8070 (JX508757) SK8301 (JX508760), SK8440 (JX508763) Jewish autonomy SK8021 (JX508756) SK921 (JX508754) SK1001 (JX508755) Sakhalin SK363 (JX508742) SK364 (JX508743), SK430 (JX508744) Buryatia SK721 (JX508750) China SK471 (JX508746), SK472 (JX508747) Tomsk SK314 (JX508741) Sverdlovsk SK224 (JX508740) Arkhangelsk SK741 (JX508751) Yakutia SK441A (JX508745) SK711 (JX508749), SKN9 (JX508764) Magadan SK701 (JX508748), SK745 (JX508752) Chukotka SK181 (JX508739) Kamchatka SK751 (JX508753)

Note: GenBank accession numbers are shown in parentheses. 350 B. Malyarchuk et al. / Molecular Phylogenetics and Evolution 67 (2013) 348–357

Kola Peninsula

SK181 SK741 SK441A

SKN9 SK701 SK224 SK711 SK745 Kamchatka

SK751 SK314 SK721

Lake Baikal Sakhalin

SK8070 SK430 A

SK8191 SK8391 B SK921 SK8301 C1a SK8321 SK1001 SK8250 C1b1 SK8021 SK8440 SK471 C1b2 SK472 C2

SK364

SK363

Fig. 1. Map showing sampled localities of S. keyserlingii and geographical distribution of the main mtDNA clades.

FÃ) were calculated using DnaSP version 5.0 software package (Lib- gram. Calculations of different estimators of the population param- rado and Rozas, 2009). Gene conversion was searched using an eter h – h(S) and h(Pi) – were performed using Arlequin 3.5 algorithm (Betrán et al., 1997) as implemented in the DnaSP pro- software package (Excofﬁer and Lischer, 2010). B. Malyarchuk et al. / Molecular Phylogenetics and Evolution 67 (2013) 348–357 351

Sliding-window analysis was performed on the aligned com- interspecies divergence equal to 7.8% (Table S2). The length of plete mitochondrial genome nucleotide sequences of Salamandrella the Schrenck salamander mitochondrial genome is 16,342 np, species, as well as some representatives of Hynobiidae family, such and the size of the Siberian salamander mtDNA varied from as Onychodactylus fischeri (NC_008089; Zhang et al., 2006), Hynobi- 16,334 to 16,340 nps. In the Siberian salamander mtDNAs, 670 us amjiensis (NC_008076; Zhang et al., 2006), Ranodon sibiricus positions were variable and 369 were parsimony-informative. (NC_004021; Zhang et al., 2003) and Batrachuperus tibetanus Complete mitochondrial genomes of Salamandrella species were (NC_008085; Zhang et al., 2006). Pairwise comparisons were per- used to estimate interspecific nucleotide divergence (Dxy) and formed to estimate interspecific divergence (Dxy; average number intraspecific nucleotide diversity (Pi) across the genome as re- of nucleotide substitutions per site between species) and intraspe- vealed by sliding-window analysis using DnaSP (Librado and Ro- cific nucleotide diversity (Pi) using sliding windows of 300 nucleo- zas, 2009). Lowest divergence and diversity values were observed tide positions (np) and steps of 10 np as implemented in the DnaSP in the comparison between D-loop regions: Dxy values varied from program. 0.2% to 1%, and Pi values varied from 0% to 0.1%. Gene by gene Dxy The ratio of the number of nonsynonymous substitutions per and Pi values were highly variable (Fig. S1). The highest interspe- nonsynonymous sites (KA) to the number of synonymous substitu- cific differences were observed in ND2, ND4L and ND4 genes (up tions per synonymous sites (KS) indicates the level of selection to 15%), and in ND5 and CYTB (up to 12%). Nucleotide diversity in against nonsynonymous substitutions relative to synonymous S. keyserlingii varies almost similarly with Dxy, with highest values ones. KA/KS distributions in pairwise comparisons between DNA se- of Pi (about 1.5%) detected in ND2, CO1, CO3, ND4L, ND4 and CYTB. quences were calculated (Nei and Gojobori, 1986) using DnaSP. Ka/ Interspecific divergence and intraspecific diversity values are not Ks > 1 indicates positive selection, Ka/Ks < 1 negative (purifying) high in 12S and 16S rRNA genes, being estimated at interval of selection, and Ka/Ks = 1 neutrality. A codon-based Z-test was used 1.5–6% for Dxy and 0.08–0.6% for Pi. to test for a significant difference between two estimates, with the Calculations of the nucleotide diversity in the D-loop region, variance of the difference computed using the bootstrap method rRNA genes and in the structural genes suggested that the variation (1000 replicates) in MEGA 5.05. among the 12S and 16S rRNA genes is quite low in comparison Using the DnaSP program, the McDonald–Kreitman test was with the protein-coding genes (Table S2). The variation in the D- performed for comparison of between-species divergence (D) and loop region was also lower than that in structural genes (with within-species polymorphism (P) at nonsynonymous (DA, PA) and the exception of the CO2 gene), being 0.6%. synonymous (DS, PS) sites to infer adaptive protein evolution Nucleotide sequences of 16S rRNA and CO1 genes appear to be (McDonald and Kreitman, 1991). For between-species divergence the commonly used mitochondrial ‘‘barcodes’’ for amphibians estimates, the mtDNA sequence of S. schrenckii was used. NI is a (Vences et al., 2005; Maya-Soriano et al., 2012; Xia et al., 2012). Re- neutrality index calculated as a ratio of PA/PS to DA/DS, and its value sults of hynobiid barcoding have shown that the standard barcod- should be 1.0 under selective neutrality. In the presence of nega- ing marker CO1 (with its divergence threshold value equal in tive selection NI should be >1, while in the presence of positive amphibians to 10%), in contrast to 16S rRNA, can identify species selection NI should be <1. of Asiatic salamanders (Xia et al., 2012). However, interspecific Significant physicochemical amino acid changes among resi- comparisons at the complete mtDNA-based level indicate that dues in mitochondrial protein-coding genes were identified by highest values of Dxy (>20%) are characteristic for ND2 and CYTB the algorithm implemented in TreeSAAP 3.2, which compares the genes, so in hynobiids, both CO1 and 16S rRNA regions used in bar- observed distribution of physicochemical changes inferred from a coding studies are of relatively low divergence. To reach this con- phylogenetic tree with an expected distribution based on the clusion, we compared the mitochondrial genomes of S. keyserlingii assumption of completely random amino acid replacement ex- with S. schrenckii, as well as other hinobiid species in genera Hyno- pected under the condition of selective neutrality (Woolley et al., bius, Batrachuperus, Ranodon and Onychodactylus (data not shown). 2003). Positive selection is detected when the number of inferred amino acid replacements significantly exceeds the number ex- 3.2. The phylogeny of mtDNA clades in the Siberian salamander pected by chance alone, yielding positive Z-scores. Z-scores from 1 to 3 indicate small variation in the amino acid characteristics, Mitochondrial genomes were employed to build a detailed phy- while scores from 6 to 8 represent the most radical substitutions, logeny (Fig. 2). Bayesian phylogenetic tree revealed two nonover- those that cause local directional shifts in biochemical function, lapping clades, AB and C, with maximal nodal support (100% structure, or both (McClellan et al., 2005). Therefore, in the present posterior probabilities). In turn, clade AB consists of clades A and study, to detect strong directional selective pressures as a conse- B. This result was further supported by the ML and NJ trees (data quence of adaptive evolution, only changes corresponding to cate- not shown). Median-network analysis designed for the reconstruc- gories 6–8 at the P 6 0.001 level were considered, following tion of all possible MP trees from a given data set also allowed the McClellan et al. (2005). identification of clade AB (with interior clades A and B) and clade C, each characterized by substantial substructure (Fig. S2). Among the 26 complete sequences, 5 belonged to clades A and B 3. Results (Fig. 1 and Fig. S2). Thus, most mtDNAs formed a separate large clade C, differing by 156 mutations from the AB branch. Mitochon- 3.1. Sequence data analysis drial genomes belonging to clade C clustered within two clades, C1 and C2. Clade C2 includes four haplotypes, two of which belong to We sequenced entire mitochondrial genomes of 26 Siberian sal- clade C2a1, which is specific for the Sakhalin population of the amander samples (GenBank accession numbers JX508739- Siberian salamander. Continental C2aÃ-sequences found in the JX508764) and one Schrenck salamander (JX508765) (Table 1). Khabarovsk region (SK8250) formed a separate branch, differing The final alignment used for phylogenetic analysis consisted of by 41 mutations from the Sakhalin C2a1-clade (Fig. S2). 16356 bp, of which 1692 were variable and 438 were parsimony- Clade C1 is the most represented in our phylogeny with 17 dis- informative. Each of the sequences represents a unique haplotype. tinct haplotypes. This clade harbors both the Far Eastern and North Mitochondrial genomes of two Salamandrella species differ in Eurasian samples. Clade C1a comprises three samples from Far East length by 3 point deletions and 6 point insertions of nucleotides (Khabarovsk region), whereas C1b connects mtDNA sequences and are characterized by 1050 fixed nucleotide differences, with from a wide range of geographical areas, including North-East 352 B. Malyarchuk et al. / Molecular Phylogenetics and Evolution 67 (2013) 348–357

100/100/100 SK8440

66/92/100 SK8301 C1a SK8191

74/94/100 SK472 C1 SK701

SK181 99/99/100 100/100/100 SK751

90/95/100 SK441a

100/99/100 SK745

SK471 C1b SK921 29/-/100 C 100/99/100 SK741 100/99/100 SK721

100/99/100 SK711

100/100/100 SK224

57/61/97 SKN9

SK314 S. keyserlingii 100/100/100 SK430 100/100/100 100/100/100 SK364

SK8250 C2 -/68/100 SK1001 B SK363 AB 100/100/100 SK8391 100/99/100 A SK8070

100/100/100 SK8321

100/100/100 SK8021 S. schrenckii SS77

Fig. 2. Complete mtDNA based Bayesian inference (BI) of the intraspeciﬁc phylogeny of the Siberian salamanders (S. keyserlingii) under the HKY+I+G model of nucleotide substitutions. The Schrenck salamander (S. schrenckii) was used as an outgroup. The analysis was run for 30 million iterations, with the ﬁrst 10% discarded as burn-in. The statistical supports (in %) are listed in the order NJ/ML/BI.

China, the whole of Siberia and North-East Europe. C1b is the most and C2 (0.47%), C1a and C1b (0.042%), and C1b1 and C1b2 structured clade, with two major clades, C1b1 and C1b2. Five se- (0.028%). Analysis of the median network (Fig. S2) indicates that quences from North-East Asia (Magadan region, Chukotka and among 670 variable sites, 79 sites had recurrent nucleotide substi- Kamchatka) formed clade C1b1. C1b2 comprises three further tutions. Homoplasic events [i.e. independent (parallel) mutations clades: Chinese clade C1b2b (from Heilongjiang Province) marked occurring at particular sites during the evolution DNA lineages] by only two mutations, separate branch C1b2Ã found in the Far East were identified in 52 sites, and reversions (or back mutations) (Jewish autonomous region) and clade C1b2a represented by sala- were observed in 32 sites. manders from several geographical areas: South, Central and West Simultaneous occurrence of several homoplasies in relatively Siberia, the Urals, and North-East Europe (Fig. S2). short nucleotide tracts provides a means for evaluating the possi- Sample DQ333814 sequenced by Zhang et al. (2006) originates bility of recombination between maternal and paternal mtDNA, from China (Heilongjiang Province) and according to our results which has been suggested to take place in animals (Tsaousis can be classified as belonging to clade C1b2a. However, we do et al., 2005; Sun et al., 2011b; Malyarchuk, 2012). Application of not use this sample in our further analyses because some sequence the gene conversion algorithm (as implemented in DnaSP pro- ambiguities were revealed in the DQ333814 sample [i.e., unidenti- gram) allowed us to identify two tracts characterized by homo- fied nucleotides and multiple amino acid substitutions in ND2 plasy at two and three sites in nucleotide sequences of 80 and (Y59H, H92L, L93F, S183F, V193G) and CYTB (K312N, Q313K) genes 323 np in length, respectively (Fig. S2). Both nucleotide tracts de- in comparison with our data set]. tected in clade B individuals (SK363 and SK8391) were, in fact, The nucleotide divergence (Dxy) between clades AB and C is clade C-specific. Therefore, this may indicate that these homoplasic 1.53%, much higher than divergence values between clades C1 events had arisen as either parallel mutations or a consequence of B. Malyarchuk et al. / Molecular Phylogenetics and Evolution 67 (2013) 348–357 353

Table 2 parameters as the average number of pairwise nucleotide differ- Time estimates of S. keyserlingii mtDNA haplogroups obtained using Bayesian ences and nucleotide diversity (Table 3). Only clade C displayed analysis. significantly negative values of sequence-based neutrality tests Species/clades Sample size Time (and 95% HPD of the posterior (Tajima’s D and Fu and Li’s FÃ), suggesting recent demographic probability distribution), in Mya expansion or the influence of positive or weak negative (purifying) S. keyserlingii 26 1.389 (1.153–1.636) selection (Tajima, 1989; Rogers and Harpending, 1992; Aris-Brosou AB 5 0.776 (0.618–0.944) and Excoffier, 1996). Results of the MacDonald–Kreitman test C 21 0.424 (0.362–0.495) (with the use of S. schrenckii as an outgroup for interspecies anal- C1 17 0.372 (0.315–0.431) C1a 3 0.34 (0.281–0.4) yses) also demonstrated that all but one clade (AB) underwent sta- C1b 14 0.307 (0.253–0.361) tistically significant purifying selection (NI > 1) (Table 4). To C1b1 5 0.079 (0.047–0.114) identify which of the mitochondrial proteins were most influenced C1b2a 6 0.147 (0.106–0.192) by purifying selection, we applied the MacDonald–Kreitman test to C2 4 0.404 (0.333–0.481) each of the 13 mitochondrial protein-coding genes (Table S3). Our results suggest that six genes (ND1, ND5, CO1, CO2, CO3 and ATP6), the recombination. Future studies may help resolve above the mainly in clade C, have undergone stronger selective constraints problem (Chen et al., 2009; Sun et al., 2011b). than others. We estimated the ratio of the number of nonsynonymous sub-

3.3. Age estimates of the Siberian salamander mtDNA clades stitutions per nonsynonymous sites (KA) to the number of synonymous substitutions per synonymous sites (KS) and found low KA/KS The Bayesian estimate of the age of mtDNA diversity in the values in both clades (KA/KS = 0.04 and 0.1 in clades AB and C, Siberian salamanders based on the 26 complete mtDNA sequences respectively), indicating the influence of negative selection (Ta- is about 1.36 My according to the mutation rate proposed by Weis- ble 3). Meanwhile, in the Siberian salamanders, relatively high rock et al. (2001) (Table 2). Evolutionary ages of clades C, C1, C1a, KA/KS value (0.258) has been detected in North-East Asian clade C1b and C2 are very close to each other, with values ranging from C1b1. This is due to very low synonymous substitution rate 310 to 420 ky, while the age of clade AB is higher, corresponding to (KS = 0.003), on the order less than in other clades (Table 3). Addi- 780 ky. Much lower age estimates were obtained for clades C1b1 tional analysis of purifying selection using Z-test has shown that (80 ky) and C1b2a (150 ky), specific for North Eurasian regions. the possibility of relaxation of purifying selection for clade C1b1 We utilized our samples of complete mtDNA genome sequences cannot be excluded, because values of P were of two kinds: to estimate population size changes over time, using Bayesian sky- P = 0.027 using the Li-Wu-Luo method (Li et al., 1985), but line analysis. The BSPs for species and major clades of the Siberian P = 0.113 using the Kumar method (Nei and Kumar, 2000). Conse- salamander mtDNAs are generally similar, showing a decline of quently, Z-test did not unambiguously support positive selection, population size around 400–250 kya, followed by expansion, and although it is possible that the branch leading to the common then another decline about 25 kya with subsequent restoration of ancestor of C1b1-Siberian salamanders has undergone relaxation population size (Fig. 3 and Fig. S3). Thus, the BSPs suggest that of purifying selective pressure, connected probably with adapta- the Siberian salamanders of all clades (the Far Eastern A, B, C1a tion to extreme environments of the North-East Asia. However, and C2, as well as North Eurasian C1b) went through a bottleneck additional studies are required to confirm this suggestion due to in the cold period around the Last Glacial Maximum about 25 kya. the small sample size of C1b1-Siberian salamanders analyzed. The program TreeSAAP indicated that 20 amino acid sites in dif- 3.4. Testing for the signatures of selection in the Siberian salamander ferent genes of the Siberian salamanders have been affected by mitochondrial genome positive selection during mtDNA evolution (Table S4, Fig. S2). The Z-scores for these substitutions were positive and significant Haplotypes in clade AB are much more divergent from each (z > 3.09, P < 0.001), indicating radical changes in the physicochem- other than are those in clade C taking into consideration such ical amino acid properties. Singular and clade-specific amino acid

1,0E2

1,0E1

P 1,0E0

1,0E-1 0,0 0,25 0,5 0,75 1,0 Time (million years ago)

Fig. 3. Bayesian skyline plot derived from 26 complete mitochondrial genomes of the Siberian salamanders. The x-axis is the time from present in units of million years, and the y-axis is equal to Nel (the product of the effective population size and mutation rate). The thick solid line is the median estimate and the thin lines show the 95% highest posterior density limits estimated with 30 million chains with the ﬁrst 3 million generations regarded as burn-in. 354 B. Malyarchuk et al. / Molecular Phylogenetics and Evolution 67 (2013) 348–357

Table 3

Parameters estimated using the neutrality tests and the KA/KS approach along the complete mtDNA sequence of S. keyserlingii.

Species/clades nk Pi Neutrality tests KA KS KA/KS Tajima’s D Fu and Li’s DÃ Fu and Li’s FÃ

S. keyserlingii 26 121.7 0.008 À1.267 À1.257 À1.487 0.002 0.032 0.056 AB 5 105.2 0.006 0.333 0.348 0.376 0.002 0.059 0.039 C 21 58.7 0.004 À1.825 À2.462 À2.652 0.009 0.086 0.101 C1 17 49.3 0.003 À1.599 À2.03 À2.209 0.006 0.059 0.098 C1b 14 39.5 0.002 À1.252 À1.83 À1.921 0.004 0.038 0.105 C1b1 5 10.3 0.001 À0.183 À0.112 À0.136 0.001 0.003 0.258 C1b2 9 38.9 0.002 À1.264 À1.494 À1.616 0.003 0.032 0.084 C2 4 61.8 0.004 À0.579 À0.579 À0.622 0.003 0.029 0.1

Note: n, number of sequences used; k, average number of nucleotide differences; Pi, nucleotide diversity; KA/KS, ratio of nonsynonymous and synonymous substitutions at nonsynonymous and synonymous sites in mtDNA protein-coding genes (except the ND6). Statistically signiﬁcant values (P < 0.05) are shown in bold.

Table 4 were found in Heilongjiang, this indicates that ﬁrst split could oc- Negative selection acting on mtDNA clades of S. keyserlingii (except the clade AB) cur near the Manchurian region. revealed by the McDonald–Kreitman test.

Species/clades DA DS PA PS NI P 4. Discussion S. keyserlingii 84 742 98 441 1.963 0.00003 AB 100 841 19 151 1.058 0.89 C 93 835 72 220 2.938 0 The present study demonstrates that the highest diversity of the C1 99 858 48 150 2.773 0.00001 Siberian salamander mtDNA clades is observed in the Russian Far C1b 104 872 33 96 2.882 0.00001 East (the northern Sikhote-Alin Mountains and the middle Amur C1b1 111 900 7 8 7.095 0.00066 River Basin) and Manchuria. This suggests that these territories C1b2 105 879 22 83 2.219 0.0036 C2 103 878 24 74 2.765 0.0002 can be considered the center of genetic diversification of S. keyserlingii. This is generally consistent with the results of previous stud- Note: Protein-coding genes (except the ND6) were analyzed. Mitochondrial genome ies of cytochrome b gene variation in the Siberian salamander of S. schrenckii was used for interspecific comparison. Number of nonsynonymous populations (Poyarkov and Kuzmin, 2008; Malyarchuk et al., and synonymous substitutions that were fixed between species (DA, DS) and that were polymorphic within one of those species (PA, PS) is shown. NI is neutrality 2010). Salamandrella is one of multiple taxa for which the Russian index calculated as described in Section 2. P-values were determined with Fisher’s Far East, the northern part of the Korean Peninsula and North-East exact test. Significant values are shown in bold. China demonstrate distinct genetic subdivisions explained by eco- logical factors accompanying the changing climatic conditions during the Pleistocene (Serizawa et al., 2002; Oshida et al., 2005; replacements were distributed equally (Table S4). Radical amino Haring et al., 2007; Fedorov et al., 2008; Li et al., in press). For in- acid changes defining the whole clades appear to be most impor- stance, the Primorye region is considered a refugium, well-known tant. For instance, five changes at once – in genes ND1 (I270V), for its highly stable natural conditions, at least, in the Late Quater- ND2 (A321T), CO3 (V122I and V160I) and ND6 (I48V) – were de- nary; unlike other areas of the boreal Palearctic, this region did not tected in the trunk of clade C, implying that positive selection on suffer widespread extinction of populations during glaciations mtDNA genes could be caused by adaptive divergence of the Sibe- (Nazarenko, 1990). rian salamanders. At the same time, all clade C branches are influ- Our study suggests that genetic history of the Siberian salaman- enced by strong negative selection (Table 4). Meanwhile, some der was affected by Pleistocene glacial cycles. Using Bayesian sky- relaxation of negative selection observed for clade C1b1 (in the line plot analysis, an expansion of population size of S. keyserlingii form of K /K ) is not accompanied by the accumulation of radical A S is placed in the Middle Pleistocene during the Mindel-Riss intergla- amino acid replacements, since there is only one such change cial (= Tobolsk, 200–375 kya). The Siberian salamander later expe- (Y206H in ND5 gene) in the trunk of the clade C1b1. rienced a severe bottleneck around 25 kya, coincident with the coldest period around the Last Glacial Maximum. During this per- 3.5. Comparison with mtDNA variability data (16S rRNA and CO1 iod, Siberia was covered by many local, especially highland, glaci- genes) in Chinese populations of the Siberian salamander reveals a ations (Velichko, 1984; Schirrmeister et al., 2002), which deep split in Manchuria prevented amphibian migrations. Results of molecular dating suggest that the Mindel-Riss interglacial was the most probable time Recently, Xia et al. (2012) analyzed variability of two mitochon- of formation of major Siberian salamander clades (such as C, C1 drial genes (16S rRNA and CO1) in different species of hynobiid sal- and C2) in the Far East and neighboring regions, while some clades amanders, including Salamandrella species from Heilongjiang and (C1b2a) may have started to colonize the western part of North Jilin Provinces of China. They found high levels of mtDNA diver- Eurasia during the warm Kazantsevo interglacial (130–100 kya), gence in Salamandrella (>5%), suggesting that two species can be and another clade (C1b1) may have reached North-East Asia (up recognized in North-East China. We have performed a comparative to Chukotka) in the Early Zyryan stage (100–50 kya). analysis of combined 16S rRNA-CO1 mtDNA fragment (1049 np in However, results of molecular dating should be considered ten- length) in different populations of North Eurasia and found that tative because of uncertainties concerning the mutation rates. four Siberian salamanders from Xia et al. (2012) are clustered with Unfortunately, hynobiid salamanders lack calibration points that our S. schrenckii sample, while the remaining six samples belong to could be used in the BEAST analyses of complete mtDNA genomes. S. keyserlingii species (Fig. 4). Most importantly, the mtDNA haplo- Another problem is that the Siberian salamander data analyzed types from Heilongjiang clustered within clade C, while two Sibe- here do not represent all branches of mtDNA phylogeny revealed rian salamander specimens (JN165809 and JN165810) form an in previous studies of mtDNA segments (Matsui et al., 2008; Xia independent branch (clade D), which constitutes the earliest split et al., 2012). For instance, there is a divergent Hokkaido-specific in the mtDNA phylogeny of S. keyserlingii. As clade D sequences haplotype belonging to clade AB, but its position within this clade B. Malyarchuk et al. / Molecular Phylogenetics and Evolution 67 (2013) 348–357 355

SKN9 SK741 721 SK751 SK701 181 441a 745 SK921 SK224 XM2057 XM2059 SK314 711 SK472 SK8301 86/85/100 SK8440 SK1001 SK471 XM2055 79/80/100 XM2056 95/71/100 SK8250 C SK430 82/76/100 SK364 98/88/100 SK8191 ABC B SK8391 AB 91/81/100 SK363 100/100/100 97/91/100 A SK8070 ABCD 99/98/100 SK8021 SK8321 S. keyserlingii D XM1879 99/90/100 XM1880 S. schrenckii SS77 99/77/100 XM2107 99/99/100 XM1882 95/74/100 XM1802 88/94/100 XM1804

Fig. 4. Neighbour-joining tree illustrating the phylogenetic relationships among the 16S rRNA-CO1 gene haplotypes in Salamandrella genus. The statistical supports (>70% obtained in ML and NJ analysis and >95% in Bayesian inference) are listed in the order NJ/ML/BI. Our samples correspond to those shown in the Table 1, while the others (designated by the use of ‘‘XM’’) have been taken from Xia et al. (2012). SS77 is S. schrenckii specimen from the Primorye region (Russia). is still unclear (Matsui et al., 2008; Malyarchuk et al., 2010). In Castellana et al., 2011; Sun et al., 2011a). Meanwhile, relaxation addition, Manchurian clade D requires further analysis with com- of purifying selection has been detected in the North-East Asian plete mtDNA genomes. Thus, further sequencing of the entire clade C1b1. However, this was an artifact of decreasing the synon- mitochondrial genomes of the Siberian salamanders from these ymous substitution rate (Table 3). In addition, clade C1b1 is char- clades may clarify the estimation of divergence time between acterized by the lowest values of molecular diversity indexes, mtDNA branches and the origin of S. keyserlingii. associated with the effective population size and the mutation rate

Unique cold tolerance of the Siberian salamander, as well as the (Ewens, 1972)(Table S5). As KA/KS values should negatively scale ability of larvae to survive at very low concentrations of oxygen with effective population size, according to the nearly neutral the-

(Ishchenko et al., 1995), allowed the Siberian salamander to colo- ory (Ohta, 1992), it is possible that the higher ratio of KA/KS found nize various water pools and wetlands formed during the Early in clade C1b1-Siberian salamanders is due to their low effective Holocene warming and the Pleistocene glaciers melting (Malyar- population size. In this case, however, it is unclear whether this chuk et al., 2010; Malyarchuk et al., 2011). In the present study, was caused by extreme environments in the North. we have found that the vast area of Northern Eurasia was colo- It is probable that adaptation to the North environments might nized, probably during the last 150 ky, by the Siberian salamander have led to appearance of amino acid changes in mtDNA-encoded haplotype clade C1b alone. We have tested for selection in the proteins that were advantageous in those climatic conditions and Siberian salamander mitochondrial genome and found that the have allowed the Siberian salamanders to expand their range. Previ- mitochondrial proteins were most influenced by purifying selec- ously, we speculated that radical amino acid change at site 160 of tion. This pattern appears to be characteristic of mitochondrial cytochrome b, which was found in the Siberian salamanders belong- genomes of many species (Popadin et al., 2007; Shen et al., 2009; ing to clade C, may have some influence on bioenergetic functions, 356 B. Malyarchuk et al. / Molecular Phylogenetics and Evolution 67 (2013) 348–357 such as production of ATP and cellular respiration (Malyarchuk et al., Ishchenko, V.G., Kuzmin, S.L., Kuranova, V.N., Tagirova, V.T., Berman, D.I., Basarukin, 2010; see also McClellan and McCracken, 2001; Fink et al., 2004; A.M., 1995. 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A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative on most sites in a gene sequence (Ruiz-Pesini et al., 2004; Zhang likelihood of nucleotide and codon changes. Mol. Biol. Evol. 2, 150–174. et al., 2005; da Fonseca et al., 2008; Shen et al., 2009). Further studies Li, B., Malyarchuk, B., Ma, Z., Derenko, M., Zhao, J., Zhou, X., in press. Phylogeography of nuclear genes are needed to identify the signals of adaptive evolu- of sable (Martes zibellina L. 1758) in the southeast portion of its range based on mitochondrial DNA variation: highlighting the evolutionary history of the sable. tion of the Siberian salamander. Acta Theriol. http://dx.doi.org/10.1007/s13364-012-0100-2. (on-line published October 25, 2012). Acknowledgments Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452. Malyarchuk, B.A., 2012. 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