Different Post-Pleistocene Histories of Eurasian Parids

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1 Journal of Heredity 26:97(4): doi:1.193/jhered/esl11 Advance Access publication July 14, 26 ª The American Genetic Association. 26. All rights reserved. For permissions, please Different Post-Pleistocene Histories of Eurasian Parids ALEXANDRA PAVLOVA, SIEVERT ROHWER, SERGEI V. DROVETSKI, AND ROBERT M. ZINK From the Bell Museum of Natural History, University of Minnesota, St Paul, MN 5518 (Pavlova and Zink); Burke Museum and Department of Zoology, University of Washington, Seattle, WA (Rohwer); and Department of Biological Sciences, University of Alaska, Anchorage, AL 9958 (Drovetski). Alexandra Pavlova is now at the School of Biological Sciences, Monash University, Clayton, Victoria 38, Australia. Address correspondence to R. M. Zink, 1 Ecology Building, 1987 Upper Buford Circle, St Paul, MN 5518, or zinkx3@umn.edu. Abstract Previous phylogeographic studies of the great tit (Parus major) and the willow tit (Parus montanus) found a general absence of phylogeographic structure for both species and suggested that each species underwent range contraction during the last Ice Age and survived in relatively low numbers, P. major in southern Europe and P. montanus in southeastern Asia. However, prior studies did not sample the entire range of either species. We analyzed sequence data for the complete mitochondrial ND2 gene from 87 P. major and 139 P. montanus from 15 new Eurasian localities, both to test prior conclusions and to provide better coverage of each species range. Our analyses confirmed the absence of phylogeographic structure in P. major and P. montanus and supported the prior refuge hypothesis for P. major. For P. montanus, we concluded that besides surviving the Ice Age in southeastern Asia, as previously hypothesized, it apparently sustained a relatively large population in northern Eurasian riverine thickets and then expanded eastward. Genetic diversity was low in P. major (p 5.12, h 5.64) and moderate in P. montanus (p 5.21, h 5.88), suggesting higher long-term effective population sizes and the older ages of populations in P. montanus. If molecular substitution rates are similar, P. montanus colonized its current Eurasian range earlier than P. major. Differences between prior studies and ours likely result from sampling gaps in earlier studies. Phylogeographic studies have revealed that many North American and European species were strongly affected by Pleistocene glaciations (Hewitt 1996, 1999, 24; Taberlet and others 1998). Relatively few studies, however, examine species with wide Eurasian distributions (Irwin and others 21; Kvist and others 21, 23; Liebers and others 21; Zink, Drovetski, and Rohwer 22; Zink, Rohwer, and others 22; Pavlova and others 23; Zink and others 23; Drovetski, Zink, Rohwer, and others 24; Godoy and others 24; Pavlova 24; Pavlova, Zink, and Rohwer 25; Pavlova, Zink, Rohwer, and others 25) because of the difficulties in obtaining samples. As a result, many studies of Palearctic species sample the European part of the species ranges but only include a few Asian samples. Such sampling gaps can affect the conclusions of phylogeographic studies (Zink and others 23). In this paper, we analyze new and published mitochondrial DNA (mtdna) sequences representing most of the range of 2 widespread Eurasian passerine birds, the great tit (Parus major) and the willow tit (Parus montanus). Our goals are to test prior conclusions about the recent histories of these species and their taxonomic status. Parus major In a review of morphological variation, Harrap and Quinn (1995) distinguished 3 major groups in the P. major species complex: major (yellow-bellied with green upperparts; 11 subspecies inhabiting Europe, northwest Africa, and northern Asia; Figure 1A), minor (whitish belly and green upperparts; 9 subspecies inhabiting southeast Russia, Japan, and northern Southeast Asia), and cinereus (whitish belly and blue-gray upperparts; 13 subspecies inhabiting northeast Iran, south Afghanistan, India, and Indonesia). A fourth group, the Turkestan tit (Parus bokharensis), is often included in the major group, but it is considered a species by Harrap and Quinn (1995). Parus bokharensis resembles cinereus but is slightly smaller and longer tailed and occurs in the lowlands of central Asia from Turkmenia to Mongolia (Cramp and Perrins 1993). Songs of the major group differ substantially from songs of the other groups (Ivankina and others 1997). Formozov and others (1993) suggested that major and bokharensis are the most distant from each other among the 4 groups based on morphological characters; however, their view was not supported by the mtdna study of Kvist and others (23), which 389

2 Journal of Heredity 26:97(4) Figure 1. (A) Distribution of collecting localities for the great tit Parus major, Turkestan tit Parus bokharensis (Kirghizia and Almaty), and Japanese tit Parus minor (Amur, Primor e, Japan, North Korea, and China): filled circles indicate new samples and open circles samples of Kvist and others (23) (some localities are pooled to simplify analysis). (B) Collecting localities for the willow tit Parus montanus samples: filled circles indicate new samples and open circles samples of Kvist and others (21). Shaded areas represent the distributions of (A) P. major and (B) P. montanus (following Harrap and Quinn 1996). found a clade consisting of 2 pairs of sister taxa, major and bokharensis and cinereus and minor. Thus, although some hybridization does occur where group ranges meet (Kerimov and Formozov 1986; Formozov and others 1993; Nazarenko and others 1999; Fedorov and others forthcoming), the 4 groups have had independent evolutionary histories and, therefore, should be considered phylogenetic species (Kvist and others 23). In this paper, we consider the great tit (P. major), the Turkestan tit (P. bokharensis), and the Japanese tit (Parus minor) separate species (we lack Parus cinereus), as do some other authors (Stepanyan 23). Kvist and others (1999) studied genetic variation in 6 European populations of P. major using sequence data from the complete mitochondrial (mtdna) control region (CR) for 68 birds (Figure 1A, Table 1). They found an absence of phylogeographic structure and extensive gene flow among populations, for which they inferred recent demographic expansions. Kvist and others (1999) hypothesized that the Balkans was a refugium during the last glacial maximum (2 years ago) and that P. major subsequently expanded rapidly to northern Europe. More recently, Kvist and others (23) examined partial CR sequences of 125 P. major expanding the initial sampling to Portugal and the United Kingdom in Europe and to 3 Asian and 1 African localities (open circles in Figure 1A, Table 1); they also included sequences of 3 individuals of P. bokharensis from Kirghizia and 44 individuals of P. minor from 4 southeastern Asian populations (Figure 1A, Table 1). Kvist and others (23) found 39

3 Pavlova et al. Phylogeography of P. major and P. montanus Table 1. Genetic characteristics of the great tit (Parus major), Japanese tit (Parus minor), and Turkestan tit (Parus bokharensis) populations (Figure 1A); sites with missing data are excluded. Source ND2: this study, partial CR: Kvist and others (23), and complete CR: Kvist and others (1999) Species Source/sequence length/polymorphic sites Collecting locality Sample size Number of haplotypes Haplotype diversity Nucleotide diversity P. major, great tit ND2/139/34 Norway Kursk þ Oryol 6 þ Smolensk Crimea Krasnodar Moscow Medvedevo 2 1 Astrakhan Yekaterinburg 1 1 Krasnoyarsk 1 1 Irkutsk Buryatiya 4 1 Mongolia 3 1 Magadan 7 1 Pooled major Partial CR/578/52 United Kingdom Portugal Spain* Germany* The Netherlands* Estonia* Sweden* Finland Urals Amur Kirghizia Corsica 2 1 Morocco 1 Pooled major P. bokharensis, Turkestan tit P. minor, Japanese tit Complete CR/1189/52 Spain* a.245 Germany* a.142 The Netherlands* 6.6 a.113 Estonia* a.132 Sweden* a.185 Finland, Kilpisjarvi a.127 Finland, Oulu a.149 Finland, Harjavalta 1 1. a.33 Pooled major a.187 ND2/141/2 Almaty Partial CR/578/11 Kirghizia ND2/14/13 Primor e Partial CR 575/24 Amur Japan North Korea China Pooled minor The populations for which most pairwise U ST values were significant are shown in bold. Asterisks indicate populations for which 2 subsets of data are available. ÿ a The values of haplotype diversity, originally reported in Table 2 of Kvist and others (1999) were calculated as 1 ÿ P fi 2 and were as follows Spain:.815, Germany:.75, The Netherlands:.5, Estonia:.79, Sweden:.781, Finland (Kilpisjarvi):.781, ÿ Finland (Oulu):.78, Finland (Harjavalta):.9, Pooled:.863. All values of haplotype diversity reported in Tables 1 and 2 of this paper are calculated as 1 ÿ P fi 2 n=ðn ÿ 1Þ (Nei 1987, p. 18). low genetic diversity within P. major and concluded that the genetic variation within populations of P. major was reduced drastically because of population bottlenecks during Ice Age range contractions. Although the starlike haplotype network of Kvist and others (23; their Figure 2) suggested no overall phylogeographic structure, their analysis of molecular variance (AMOVA) showed some overall population structure (U ST 5.12) and pairwise population U ST values for the 391

4 Journal of Heredity 26:97(4) Figure 2. Median-joining ND2 haplotype networks for Parus major (white circles on large network) and Parus bokharensis (gray circles on large network) and Parus minor from Primor e (small network). Each circle represents a unique haplotype, and the size of the circle is proportional to the frequency of the haplotype in the sample. Small black dots represent unsampled haplotypes, and lines connecting circles represent single mutations. Numbers within circles indicate the total number of individuals sharing the same haplotype; numbers in parentheses indicate the number of individuals from a particular locality sharing the same haplotype (if different from 1). Locality abbreviations are as follows: NOR, Norway; KUR, Kursk; ORY, Oryol; SMO, Smolensk; CRI, Crimea; KRD, Krasnodar; MOS, Moscow; MED, Medvedevo; AST, Astrakhan ; YEK, Yekaterinburg; KRY, Krasnoyarsk; IRK, Irkutsk; BUR, Buryatiya; MON, Mongolia; MAG, Magadan; ALM, Almaty; PRI, Primor e. geographically isolated populations from Kirghizia and the United Kingdom were large and significant. Based on higher genetic diversity in the 3 individuals of P. bokharensis, it was concluded that P. bokharensis was historically stable, whereas both P. major and P. minor experienced Late Pleistocene or Holocene population reductions and subsequent expansions. Kvist and others (23) also suggested that P. major had a larger and more recent population expansion than P. minor. Parus montanus This species ranges from northeastern France through central and northern Europe and Russia to the Pacific coast (Cramp and Perrins 1993, Figure 1B). Geographic variation in morphology is clinal and connects most of the continental subspecies, which also differ in song types and coloration (Harrap and Quinn 1995). Harrap and Quinn (1995) divided P. montanus into 3 subspecies groups: salicarius (northern Eurasia, 6 subspecies), kamtschatkensis (Pacific rim in eastern Asia, 4 subspecies), and montanus (mountains of central Europe, 1 subspecies); they considered the songar tit (Parus songarus) of central Asia a separate species, as we do in this paper. Kvist and others (21) and Salzburger and others (22), using sequences of the mitochondrial CR and cytochrome b, respectively, found no mtdna structuring associated with subspecies groups within P. montanus, whereas the songar tit, considered by them as subspecies of P. montanus, was found to be polyphyletic and consisting of 3 distinct lineages: weigoldicus, affinis, and songarus. On the basis of close relationships among 6 Eurasian subspecies of P. montanus, Salzburger and others (22) suggested that this species spread over major parts of Eurasia during the Pleistocene and underwent rapid morphological and vocal divergence. Kvist and others (1998) compared complete mitochondrial CR sequences from Finland and Swedish populations and concluded that individuals from both localities belong to one panmictic population with high long-term effective population size. Later, Kvist and others (21) studied partial CR sequences of birds from 6 western European localities, 6 samples from eastern Asia, and 1 sample from the Urals (open circles in Figure 1B, Table 2) and found no phylogeographic structure; however, their AMOVA showed that populations were subdivided overall (U ST 5.15), and all pairwise population U ST values for Japan and most U ST values for Latvia were large and significant. Kvist and others (21) attributed the general lack of phylogeographic structure to homogeneous habitat throughout the distribution, absence of geographic barriers, high gene flow, and a high long-term effective population size and, based on trend of westward decrease in genetic diversity, suggested that P. montanus expanded through the Palearctic from southeastern Asia after the last Ice Ages. Summary of Previous Work on Parid Phylogeography Several studies have shown a general lack of phylogeographic structure and evidence of overall population subdivision in P. major and P. montanus (sensu strictu). However, except for the Urals, samples from eastern Europe and western Asia were lacking (open circles in Figure 1 indicate prior samples). 392

5 Pavlova et al. Phylogeography of P. major and P. montanus Table 2. Genetic characteristics of willow tit (Parus montanus) populations (Figure 1B); sites with missing data are excluded. Source ND2: this paper, partial CR: Kvist and others (21), and complete CR: Kvist and others (1998) Source/sequence length/polymorphic sites Collecting locality Sample size Number of haplotypes Haplotype diversity Nucleotide diversity ND2/139/63 Moscow þ Smolensk 12 þ Medvedevo Mezen Yekaterinburg 1 1 Noyabr sk Tyva þ Gorno-Altay 7 þ Krasnoyarsk 1 1 Irkutsk Buryatiya Mongolia Anadyr Primor e* Khabarovsk* Magadan* Kamchatka* Pooled montanus Partial CR/592/56 Alps Germany Poland Finland Sweden Latvia Urals Ussuri* Amur* Magadan* Kamchatka* Sakhalin Japan Pooled montanus a Complete CR/127/52 Finland Sweden Pooled The populations for which most pairwise U ST values were significant are shown in bold. Asterisks indicate populations for which 2 subsets of data are available. a Top row values reported in Table 1 (p. 932) of Kvist and others 21, bottom row values reported on page 934 of the text (nucleotide diversity of.671 was calculated by us in DNASP and was not reported in original paper). Species-level evolutionary conclusions should not be made with confidence until the complete geographic range is sampled. This concern motivated us to add new localities (black circles in Figure 1) from previously unstudied areas. If the conclusions of Kvist and others (21, 23) are correct, then our expanded analyses should reveal 1) a general absence of geographic structure on the haplotypes trees of both species, 2) a decrease in genetic diversity consistent with the direction of expansion: eastward and northward in P. major and westward and northward in P. montanus, 3) signatures of expansion for populations of both species, and 4) earlier expansions in southeastern populations of P. montanus relative to the northern and western populations. Because our new samples overlap to some degree with those used in the studies reviewed above, we chose to sequence the mitochondrial coding gene ND2. Thus, in addition to testing prior phylogeographic conclusions, we were able to compare genetic estimates derived from the noncoding CR with those from ND2. Materials and Methods New Sampling We analyzed 87 individuals of P. major from 15 Eurasian localities, 4 individuals of P. bokharensis from Almaty, and 29 individuals of P. minor from Primor e (black circles in Figure 1A, Table 1). For P. montanus, we analyzed 139 individuals from 17 localities (Figure 1B, Table 2). We used the marsh tit Parus palustris (GenBank number AY734248) as an out-group for the haplotype tree of P. montanus and P. minor for P. major. All birds were collected during the breeding season. From nearly all specimens, a study skin and spread wing 393

6 Journal of Heredity 26:97(4) were preserved and deposited at the Burke Museum, University of Washington, Seattle; the Moscow State University Zoological Museum, Moscow, Russia; the Bell Museum, University of Minnesota, St Paul; or the State Darwin Museum, Moscow, Russia. Tissue samples were preserved in the field either in 96% ethanol or in lysis buffer (Longmire and others 1997) or frozen in liquid nitrogen. Molecular Laboratory Methods Isolation and purification of DNA was performed using QIAamp Tissue Kit (Qiagen, Valencia, CA). Polymerase chain reaction (PCR) (Saiki and others 1988) with Perkin- Elmer PCR reagents and primers L5215 (Hackett 1996) and H164 (Drovetski, Zink, Fadeev, and others 24) was used to amplify the complete ND2 gene; PCR conditions are explained in Drovetski, Zink, Fadeev, and others (24). PCR products were cleaned with Qiaquick PCR Purification Kit (Qiagen) and directly sequenced on ABI 37 automated sequencer using BIGDYE versions 3. and 3.1 chemistry (Applied Biosystems, Foster City, CA) and primers L5215, H164, L347 (Drovetski, Zink, Fadeev, and others 24), and H5578 (Hackett 1996). Data Analysis Sequences were aligned and edited in SEQUENCHER (Gene Codes Corporation, Ann Arbor, MI). Sequence data have been deposited in GenBank (Accession numbers AY AY732729, AY AY734251, and AY AY7337). Mitochondrial origin of sequenced DNA fragments was supported by the absence of stop codons and the existence of a large number of haplotypes, which are inconsistent with nuclear copies (Zhang and Hewitt 1996). A matrix of variable sites was used as input for constructing a median-joining network (Bandelt and others 1999) using software NETWORK version 4.11 ( assigning equal weights to all sites and with default value of epsilon 5. Maximum likelihood (ML) model and parameters were determined by the hierarchical likelihood ratio test in MODELTEST 3.6 (Posada and Crandall 1998). PAUP* (Swofford 2) and PHYML (Guindon and Gascuel 23) were used to perform ML tree searches. SEQBOOT and CONSENSE were used from the PHYLIP program package (Felsenstein 1993). SEQBOOT version 3.5c was used to generate 1 bootstrapped sets of data from which ML trees were constructed in PHYML. Then, CONSENSE version 3.5c was used to compute a majority rule consensus tree. DNASP version 4. (Rozas and others 23) was used to calculate the number of haplotypes in populations, nucleotide diversity (p, Nei 1987; Equation 1.5), haplotype diversity (h; Nei 1987; Equation 8.4), and theta (H 5 2Nel, where Ne is the effective population size and l is the mutation rate) from the number of polymorphic sites per nucleotide (Tajima 1996) and to perform the R 2 test for detecting population growth (Ramos-Onsins and Rozas 22). We used ARLEQUIN version 2. (Schneider and others 2) to perform an AMOVA (Excoffier and others 1992) taking into account haplotype frequencies and the distances between haplotypes, to compute pairwise population U ST, mismatch distributions, time since population expansion (s), effective population sizes before (H ) and after (H 1 ) expansion, and Tajima s D (Tajima 1996) and Fu s Fs (Fu 1997) tests of selective neutrality for localities with sample sizes greater than 1 individuals (in some cases, samples were pooled if they were not significantly differentiated). The latter 2 tests were used as indicators of population expansion. Sites with ambiguous characters were removed. To test the empirical mismatch distribution against a model of sudden expansion, we used the generalized nonlinear least squares approach (Schneider and Excoffier 1999) implemented in ARLEQUIN. We compared our estimates of p with those of Kvist and others (21, 23) and regressed values of p against latitude and longitude for samples of 3 or more individuals expecting smaller values to be observed on the leading edge of population expansion (Hewitt 1999, 2). Results and Discussion The Great Tit P. major Genetic Variability and Gene Flow We analyzed 139 aligned nucleotides of ND2 after removing sites with missing characters. The 34 polymorphic sites (9 of them parsimony informative) resolved 24 haplotypes among the 87 individuals (Table 1). The most common haplotype (haplotype A in Figure 2) was shared by 52 individuals (6% of all sampled individuals) from 13 populations: Norway (1 individual), Kursk (2), Smolensk (2), Crimea (1), Krasnodar (4), Moscow (16), Medvedevo (2), Astrakhan (4), Krasnoyarsk (1), Irkutsk (5), Buryatiya (4), Mongolia (3), and Magadan (7). Three more haplotypes were shared among localities (Figure 2). Overall haplotype diversity was.638 and ranged from in Buryatiya, Mongolia, and Magadan, where all individuals shared the most common haplotype, to 1 in Norway and Crimea (Table 1). Overall nucleotide diversity (p) averaged.12 and ranged from. in Buryatiya, Mongolia, and Magadan to.26 in Crimea (Table 1). Northern populations had lower p, both for our data and for those of Kvist and others (23) (Figure 3A), but this trend was not significant (P..5). Our data, but not those of Kvist and others (23), showed a significant trend toward eastern birds having lower p (R , P 5.3) (Figure 3B), suggesting eastward dispersal. A general lack of population subdivision was suggested by the overall nonsignificant U ST value (P..5, Table 1). However, several pairwise population comparisons yielded significant (P,.5) U ST values: Krasnodar Magadan (U ST 5.7), Crimea Buryatiya (.11), and Astrakhan Irkutsk (.5), indicating that gene flow between eastern and western populations might be restricted. Molecular Phylogenetic Analysis and Phylogeography MODELTEST 3.6 indicated TrN þ G (Tamura and Nei 1993) with alpha 5.69 as the best-fit model of sequence 394

7 Pavlova et al. Phylogeography of P. major and P. montanus A Nucleotide diversity vs latitude P. major 9 Nucleotide diversity R 2 =.23 P =.13 3 R 15 2 =.6 P = North latitude B Nucleotide diversity Nucleotide diversity vs longitude P. major R 2 =.1 P = R 2 =.43 P = East longitude C Nucleotide diversity Nucleotide diversity vs latitude P. montanus R 2 =.2 P =.64 R 2 =.2 P = North latitude D Nucleotide diversity Nucleotide diversity vs longitude P. montanus R 2 =.4 P =.53 3 R 2 =.6 15 P = East longitude Figure 3. Nucleotide diversity (in units of p 1 4 ) for the Parus major (A, B) and Parus montanus (C, D) populations plotted against north latitude (A, C) and east longitude (B, D). Filled circles indicate our data and open circles data of Kvist and others (23) for P. major and those of Kvist and others (21) for P. montanus (Tables 1 and 2). Solid lines are linear regression lines for our data and dashed lines for data of Kvist and others (21, 23). The values of R 2 and the corresponding P values for our data are in the lower part of each graph. evolution. On the ML tree found by PHYML (not shown), all 24 haplotypes of P. minor clustered together (this clade was recovered by 1% of 1 ML bootstrapped trees), as well as the 2 haplotypes of P. bokharensis from Almaty (99% bootstrap support). However, the P. major clade received only 69% bootstrap support and was not recovered by the ML tree found by PAUP* because the P. bokharensis clade was embedded in it, making P. major paraphyletic. On the haplotype network (Figure 2), P. bokharensis was separated from P. major by 1 mutational steps. Intraspecific genealogies can be multifurcating due to ancestral and descendant haplotypes coexisting in populations, which violates the assumption of bifurcating trees employed in ML algorithm (Posada and Crandall 21). Thus, the network likely provides a better estimate of amonghaplotype relationships, and we conclude that both P. major and P. bokharensis are evolutionarily independent lineages. In general, our results (Figure 2) confirm and extend those of Kvist and others (1999, 23) that P. major is not structured geographically. This inference is strengthened by the observation that the most common haplotype was shared by more than a third of the individuals and distributed throughout Eurasia (Table 1), including our new sites. For example, most (2 of 24) individuals from our Asian localities (Yekaterinburg, Krasnoyarsk, Irkutsk, Buryatiya, Mongolia, and Magadan) shared the most common haplotype. The widespread occurrence of common haplotypes is explicable given known patterns of dispersal. Although young P. major normally disperse less than 1 km on average from their natal grounds, northern and eastern populations are eruptive migrants, moving in highly variable numbers depending on the food supply... (Harrap and Quinn 1995, p. 356), and some invading birds remain to breed in the wintering areas. This dispersal pattern can explain the general absence of differentiation in continental tits. The insignificant overall U ST for our data also suggests that P. major is not geographically subdivided, although several significant pairwise values suggested restricted gene flow between some eastern and western populations. Kvist and others (23) also found evidence of differentiation for 2 geographically isolated populations, United Kingdom and Kirghizia, for which significant pairwise U ST values were found. This local differentiation is apparently attributable to relatively low natal dispersal distances (Verhulst and others 1997). In addition, 19 of 2 unique haplotypes were sampled from European populations (Figure 2). These results support the hypothesis of recent colonization of eastern Eurasia and can account for some of the east west population differences. Therefore, although earlier studies were limited in their sampling of the range, the expanded analysis presented here corroborates the hypothesis that there is little geographic structure across the huge Palearctic range of P. major. The 395

8 Journal of Heredity 26:97(4) Table 3. Estimates of effective population size and demographic changes from ND2 data for samples of 1 or more individuals Species Locality H S s H H 1 Tajima s D Fu s Fs R 2 Parus major Moscow ÿ2.2 ÿ Pooled ÿ2.6 ÿ Parus minor Primor e.32 ne ne ne ÿ2.1 ÿ Parus montanus Moscow ÿ1.8 ÿ Irkutsk ÿ2.1 ÿ ns Buryatiya ns ns.154 ns Anadyr ns ÿ1.2.2 ns Primor e ÿ2. ÿ Magadan ÿ1.9 ÿ Pooled ÿ2.5 ÿ Pooled reduced ÿ2.5 ÿ Pooled reduced refers to calculations omitting differentiated sampling sites of Parus montanus (Medvedevo, Anadyr, and Kamchatka). H S is estimated (in DNASP) from the number of segregating sites per nucleotide; s, H, and H 1 (calculated in ARLEQUIN) are parameters of the demographic expansion model of Schneider and Excoffier (1999). All mismatch distributions except Irkutsk were unimodal, and none differed from the model of sudden expansion. Given values of Tajima s D, Fu s Fs (ARLEQUIN), and R 2 (DNASP) are significant at the P,.5 level unless denoted as ns, not significant, or ne, no estimates available. general absence of phylogeographic structure as the result of postglacial range expansion and low genetic diversity was reported for 2 northern species of North American chickadees, Poecile atricapillus and Poecile hudsonicus (Gill and others 1993). Other forest-dwelling birds also appear to lack phylogeographic structure across much of northern Eurasia (e.g., Zink, Drovetski, and Rohwer 22; Zink, Rohwer, and others 22). The mtdna data appear to conflict with 2 recent studies from the United Kingdom that revealed the existence of natural selection acting on local differences in life-history traits. Postma and van Noordwijk (25) discovered differences in clutch size between subpopulations separated by only a few kilometers. Garant and others (25) discovered that the mean mass of P. major decreased in one part of Wytham woods, whereas clutch size remained constant to the north. A significant genetic component to variation in these fitness or fitness-linked traits was documented in both studies. If these represented genome-wide patterns of differentiation, the mtdna tree should have revealed more structure. Thus, the mtdna data suggest that these local adaptations are very recently evolved. Population Expansion Kvist and others (23) suggested that P. major populations underwent a drastic reduction in size as a result of range contraction during the last glacial maximum. Survival in low numbers in a small southwestern refugium (possibly Balkans, Kvist and others 1999) was thought to have been followed by northeastward range expansion and subsequent demographic expansion. Although many of our individual samples are small, our mismatch distributions and values of Tajima s D, Fu s Fs, and R 2 are consistent with these hypotheses of population and range expansion (Table 3, Figure 4), as expected for north temperate populations (Hewitt 24). To test for the effects of natural selection, Zink (25) analyzed our data set using 2 partitions of the ND2 gene (surface and transmembrane portions and synonymous nonsynonymous sites). Both partitions yielded similar estimates of U ST, which suggested demographic expansion as the cause of the shallow tree for P. major and not directional or stabilizing natural selection (Zink 25). Notes on Evolutionary History of P. bokharensis Kvist and others (23) suggested that there was less or no historic habitat reduction for P. bokharensis because it prefers deserts and semideserts, which is the same kind of environment that existed during the Ice Ages within its present range based on the reconstruction by Adams (1997). The estimate of Kvist and others (23) of p (from 3 individuals) for P. bokharensis led them to conclude that populations have been stable for a long time because... [they]... possess more diversity than the minor and major groups. We did not find a higher p in P. bokharensis with our sample of 4 birds. We suggest more samples are needed to clarify the history of P. bokharensis. The Willow Tit P. montanus Genetic Variability and Gene Flow We analyzed 139 aligned bp of ND2 after removing sites with ambiguous characters. The 63 polymorphic sites (26 parsimony informative) resolved 58 haplotypes among the 139 individuals (Table 2). The most common haplotype (haplotype A in Figure 5) was shared by 44 individuals (32% of all individuals) from 12 populations: Moscow (N 5 2), Medvedevo (1), Mezen (2), Noyabr sk (1), Gorno-Altay (2), Tyva (1), Irkutsk (11), Buryatiya (8), Mongolia (3), Primor e (5), Khabarovsk (2), and Magadan (6) (Figure 5). Two other common haplotypes were found: haplotype B (Figure 5) had a restricted Far Eastern distribution and occurred in 13 individuals from Kamchatka, Magadan, and Anadyr and haplotype C was shared by 12 individuals from Mezen, Noyabr sk, Irkutsk, Buryatiya, Mongolia, Primor e, and Khabarovsk. Several more haplotypes were shared among populations (Figure 5). The occurrence of widespread haplotypes indicates considerable recent gene flow. 396

9 Pavlova et al. Phylogeography of P. major and P. montanus Frequency P. major pooled P. major Moscow P. minor Primor'e Frequency Frequency.5 P. montanus pooled.5 P. montanus Moscow.5 P. montanus Primor'e P. montanus Irkutsk.5 P. montanus Buryatia.5 P. montanus Magadan Number of pairwise differences Pairwise differences Pairwise differences Figure 4. Mismatch distributions for Parus major, Parus minor, and Parus montanus (ND2 data). Black dots indicate the observed frequency, solid lines the model distribution as calculated by ARLEQUIN version 2. (Schneider and others 2), and dashed lines the mean of the distribution. Overall haplotype diversity (.88) was moderate and ranged from.38 in Kamchatka to 1 in Medvedevo (Table 2). Overall nucleotide diversity was.21 and ranged from.4 in Anadyr to.32 in Mezen (Table 2). Relatively high genetic diversity within P. montanus and the relatively large number of haplotypes found in our data and those of Kvist and others (21) (Table 2) suggest a high longterm effective population size and an absence of recent bottlenecks. Regressions of p on latitude were not significant (P..5) for our data or for those of Kvist and others (21) (Figure 3C). Longitude explained 6% of the variance of p in our data with eastern birds having lower p (R 2 5.6, P,.5), but no such trend was found for the data of Kvist and others (21) (Figure 3D). Kvist and others (21) reported a westward decrease in nucleotide diversity for salicarius group (which includes most of the species distribution excluding Kamchatka, Sakhalin, and Japan), from which they concluded that during the last Ice Ages P. montanus occupied large regions of the southeastern Palearctic and colonized the European (western) part of the range from the east. However, our regression analysis of their nucleotide diversity data did not find this trend to be a statistically significant (with or without Kamchatka, Sakhalin, and Japan). Furthermore, our data show a significant trend in which the eastern birds have lower nucleotide diversity (Figure 3D), which might be a trace of past eastward range expansion. This does not mean, however, that our results contradict those of Kvist and others (21). Low genetic diversity in European samples is expected because during last glacial maximum a large part of western Europe was under glacial ice and was recolonized recently. As Figure 1 demonstrates, the western samples of P. montanus of Kvist and others (21) are from western Europe, and the eastward increase of nucleotide diversity might have resulted from their sampling design. The discrepancy in interpretation reinforces the need to have adequate geographic coverage. Molecular Phylogenetic Analysis and Phylogeography MODELTEST 3.6 suggested TrN þ G (Tamura and Nei 1993) with alpha as a most probable model of sequence evolution. This was the same model selected for the P. major group, suggesting similar sequence evolution of ND2 gene in different parids. The monophyly of P. montanus was supported by 1% of 1 ML bootstrap support replicates. The ML haplotype tree (not shown) was geographically 397

10 Journal of Heredity 26:97(4) Figure 5. Median-joining haplotype network for Parus montanus ND2 haplotypes. Each circle represents a unique haplotype, and the size of the circle is proportional to the frequency of the haplotype in the sample. Small black dots represent unsampled haplotypes, and lines connecting circles represent single mutations. Numbers within the circles indicate the total number of individuals sharing the same haplotype; numbers in parenthesis indicate number of individuals from particular locality sharing the same haplotype (if different from 1). Gray areas in the circles indicate the proportion of individuals from the Far Eastern populations (Anadyr, Primor e, Khabarovsk, Magadan, and Kamchatka) sharing the same haplotype. Locality abbreviations are as follows: SMO, Smolensk; MOS, Moscow; MED, Medvedevo; MEZ, Mezen ; YEK, Yekaterinburg; NOY, Noyabr sk; GOR, Gorno-Altay; TYV, Tyva; KRY, Krasnoyarsk; IRK, Irkutsk; BUR, Buryatiya; MON, Mongolia; ANA, Anadyr ; PRI, Primor e; KHA, Khabarovsk; MAG, Magadan; KAM, Kamchatka. unstructured, which can be seen in the network (Figure 5). Kvist and others (21) also found a geographically unstructured haplotype network and attributed it to high gene flow. However, they also suggested that gene flow might not be currently high because of geographic differences in morphology and voice. Our haplotype network (Figure 5) shows that some haplotypes are restricted to Asia, suggesting limited gene exchange for some eastern localities. Furthermore, our AMOVA showed that geographic localities explain 1.8% of the total variation (P,.5). Most pairwise U ST values involving Anadyr (range.62) and Kamchatka (range.37) were large and significant (Table 4), indicating restricted or no gene flow between these and more western populations. Zink (25) explored the nature of high U ST values for these populations and found that they occurred because of a replacement substitution at base position 452 that was shared by all Anadyr individuals and 4 of 5 birds from Kamchatka (haplotype B and 2 adjacent haplotypes from Anadyr in Figure 5). Zink (25) pointed out that although significant U ST values might be a signature of incipient divergence caused by geographic isolation, they also might indicate a selection for a new favorable haplotype. Because only a single substitution is involved, a definitive conclusion is unwarranted. Several pairwise U ST values involving Medvedevo (range.18) were also significant (Table 4), further supporting restricted gene flow between western and eastern populations. Significant pairwise U ST values that are found for 2 northeastern populations (Anadyr and Kamchatka, subspecies anadyrensis and kamtchatkensis) and the population from Japan (subspecies restrictus), Medvedevo, and Latvia (both belong to subspecies borealis), suggest that some populations of P. montanus are differentiated. Populations diverging from a common ancestor go through a series of stages represented by gene trees that are polyphyletic, then paraphyletic, and finally reciprocally monophyletic with respect to geography (Avise 2). The existence of significant U ST values and a haplotype tree that is not reciprocally monophyletic, but has some limited differentiation, suggests that P. montanus is at an intermediate stage of differentiation. The pattern of incomplete genetic differentiation coincides in part with phenotypic (subspecific) divergence. 398

11 Pavlova et al. Phylogeography of P. major and P. montanus Table 4. Significant pairwise U ST values for Parus montanus populations (ND2 data) Medvedevo Anadyr Kamchatka Moscow þ Smolensk Medvedevo Mezen.27 Noyabr sk Gorno-Altay þ Tyva Irkutsk Buryatiya Mongolia Primor e Khabarovsk.5.24 Magadan Population and Range Expansion Kvist and others (21) suggested that during Pleistocene cooling P. montanus was restricted to southeastern Asia and colonized Eurasia (westward) when the climate warmed. However, if P. montanus s range expanded from a refugium, then a large number of haplotypes from the refugial population is expected to be unique and most haplotypes from recently colonized areas are expected to be closely related to the common haplotype. Such a pattern was observed for P. major (Figure 2), consistent with the findings of Kvist and others (23). However, on haplotype networks for P. montanus (Figure 5 in this paper and Figure 2 in Kvist and others [21]), haplotypes from the western and most eastern, potentially refugial, populations were randomly distributed (although in Kvist and others 21 no eastern individuals shared the most common haplotype). Reconstructions of biomes during the last glacial maximum based on pollen and plant macrofossils (Tarasov and others 1999) suggest that much of northeastern Europe and northwestern Asia were covered by tundra. Although P. montanus typically breeds in coniferous and mixed forests, it also inhabits woody shrubs and riverine thickets extending to the tree line in northern Eurasia and in mountainous regions (Harrap and Quinn 1995). Thus, Pleistocene tundra in northern Eurasia could have provided suitable habitats for P. montanus. The existence of substantial genetic diversity in western populations (see Figure 4 for comparison with P. major) suggests that P. montanus, unlike P. major, was not subjected to drastic range contractions and during climate warming could have expanded eastward, as suggested by our data, as well as westward to Europe, as Kvist and others (21) suggested. It is also possible that historic range of P. montanus was split during the last Pleistocene cooling and some birds survived in refugial forests of eastern Asia. The mtdna data are consistent with the hypothesis of range and population expansion. Mismatch distributions (Figure 4) for P. montanus populations did not differ from the model of sudden population expansion and were unimodal for all localities except Irkutsk. Population growth was also suggested by most values of Tajima s D, Fu s Fs, and R 2 for all populations except Buryatiya. Thus, both P. major and P. montanus appear to have responded to post Ice Age habitat amelioration by range and population expansion, although as discussed below, the details differ. Different Evolutionary Histories of P. major, P. minor, and P. montanus During the last glacial maximum, some broadleaved forests of southeastern Asia were replaced by grassland vegetation. However, this part of the continent also contained several large refugial forests (Adams 1997) and, therefore, was a potential refuge for forest-dwelling birds. Although P. montanus, like P. major and P. minor, is a cavity nester and its range largely overlaps with that of P. major, the evolutionary history of P. montanus likely differed from that of its congeners, as evidenced by higher values of p, H, and h and the low percentage of individuals sharing a common haplotype (Table 5). A significant range restriction associated with Pleistocene cooling would be expected for P. major because they require forested habitats and do not live in the brushy habitats of land underlain with permafrost. Eastward decrease of genetic diversity (Figure 3B) and low genetic diversity suggest that P. major might have survived Pleistocene glaciations in small numbers in refugial forests of southern Europe and subsequently expanded into eastern Eurasia. In contrast to P. major, P. montanus, whose range extends farther north, did not undergo as severe a population bottleneck but instead survived in relatively large numbers in narrow riparian thickets in the widespread north Eurasian ice-free areas where large river Table 5. Summary of genetic characteristics for 3 species of Parus based on different sets of individuals, populations, and gene regions. Source partial CR: data of Kvist and others (21) (P. montanus) and Kvist and others (23) (P. major and P. minor), ND2: this paper Species Data set % Most com. hapl. h p H S Total U ST s Tajima s D Sample size P. major Partial CR * 1.88 ÿ2.52* 125 P. minor * 2.11 ÿ2.5* 44 P. montanus * 3.51 ÿ2.6* 94 P. major ND ns.51 ÿ2.57* 87 P. minor ÿ2.22* 29 P. montanus * 1.5 ÿ2.55* 139 P. montanus ND2 reduced ÿ2.58* 118 ND2 reduced refers to calculations omitting sampling sites that are differentiated (Medvedevo, Anadyr, and Kamchatka); % most com. hapl., proportion of individuals sharing most common haplotype. Values of h, p, H S, s, and Tajima s D were calculated in DNASP and U ST in ARLEQUIN. Asterisk indicates significance at P,.5 level; ns, not significant. 399

12 Journal of Heredity 26:97(4) valleys were free of permafrost, as well as in refugial forests of southeastern Asia. The present range of P. minor is restricted to southeastern Asia, and it is thought that the species only recently has been expanding northward (Smirenskiy 1986; Harrap and Quinn 1995). In fact, all but 5 haplotypes of P. minor in Kvist and others (23) were unique and clustered according to their geographic localities on the haplotype network (their Figure 1), whereas only 2 of the 5 common haplotypes were shared between populations. A nonrandom distribution of haplotypes and the large significant overall U ST for P. minor (.28, Table 5) indicate significant population subdivision, inconsistent with high gene flow expected from range expansion. For our only sample of P. minor (from Primor e), significantly negative Tajima s D and Fu s Fs, significant R 2 value (Table 3), and a starlike network where 46% individuals shared the most common haplotype (Figure 2) indicated population growth, consistent with recent colonization of Primor e. On the basis of similar nucleotide diversity for P. major and P. minor, but higher theta within P. major (the results consistent with our findings, Table 5), Kvist and others (23) suggested a greater degree of population expansion in P. major than in P. minor because theta is more influenced by current population size, whereas p reflects the historic size (Tajima 1989). Given that the range of P. major is roughly 4 times greater than that of P. minor, this conclusion is not surprising. Time since expansion (s, Table 5) was small in P. major, intermediate in P. minor, and large in P. montanus for all data sets, indicating that expansion in P. major was relatively recent. In fact, range expansion of P. major is known to have mirrored the expansion of human settlements (Harrap and Quinn 1995), and in some regions of the Russian Far East, the species presence is restricted to human habitations. In addition, introgression of major haplotypes into minor populations in the Amur region hybrid zone and absence of foreign haplotypes in allopatric P. minor populations provide indirect evidence toward high philopatry in P. minor (Fedorov and others forthcoming). Thus, the genetic analyses of 3 codistributed sedentary parids suggest that each species responded differently to the environmental changes of the Ice Ages. These different genetic signatures are likely a result of differences in habitat use and dispersal abilities. Comparison of Genetic Parameters Derived from CR and ND2 Regions of the mtdna genome are known to evolve at different rates, owing in part to selective constraints. The CR is often considered the most variable, although this is not always the case (Ruokonen and Kvist 22). Despite the potential for different regions of mtdna to evolve at different rates, the entire gene has a single history owing to linkage. Nonetheless, it is of interest to make direct comparisons of different gene regions as studies often compare levels of variability across gene regions (Ruokonen and Kvist 22). For the 5 European populations of P. major, estimates of nucleotide diversity were on average 1.64 times higher when computed from the partial 5#-end of the CR (Kvist and others 23) than those calculated from the complete CR sequences (Kvist and others 1999) (Table 1), indicating that within species the 5#-end of the mitochondrial CR is more variable than the 3#-end. This result was reported earlier for intraspecific comparisons in P. major, Parus caeruleus, and P. montanus (Ruokonen and Kvist 22). However, for the 2 Scandinavian populations of P. montanus studied by Kvist and others (1998, 21), the results were not consistent: p Finland was higher when estimated from partial (5#-end) CR, whereas p Sweden was higher when estimated from complete CR sequences. Such inconsistency in the relative distribution of variation in CR sequences makes it hard to compare estimates of p obtained from different mitochondrial gene regions. Although Ruokonen and Kvist (22) suggested that the distribution of variation in CR is similar within genera, nucleotide diversity estimated from the 3#-end of the CR sequences for P. major, P. minor, and P. montanus (A Pavlova, RM Zink, S Rohwer, and S Drovetski, unpublished data; GenBank sequence numbers AY AY and AY ) yielded comparative results very different than those obtained from ND2 for the same sets of individuals. For example, p from the 3#-end of CR was.7,.34, and.26 and H was.46,.114, and.125 for P. major, P. minor, and P. montanus, respectively, whereas estimates of p from ND2 or 5#-end of CR were similarly low for P. major and P. minor and H was smallest in P. minor (Table 5). This example demonstrates that different parts of mitochondrial genome, and even parts of the same gene region, evolve with different rates in different taxa (even within genera), and care must be used when interpreting results. Thus, molecular clock rates calibrated for coding genes should not be applied for CR data to avoid substantial overestimation of the ages of population/species splits. Furthermore, calibrations for different parts of the CR are not general. Values of nucleotide diversity estimated from our ND2 data were consistently lower than those calculated from the partial CR data of Kvist and others (21, 23). For the 4 eastern populations of P. montanus (Magadan, Kamchatka, Khabarovsk [Amur], and Primor e [Ussuri]), our samples and those of Kvist and others (21) overlapped (Figure 1B, Table 2). Comparisons of estimates of genetic diversity calculated from ND2 and partial CR for these populations showed that estimates of p from partial CR are on average 2.9 times higher than from ND2 and estimates of h are 1.4 times higher from partial CR than from ND2 (Table 5). Therefore, for Parus species, the 5#-end of CR yields more intraspecific variation than either complete CR or ND2, and estimates of genetic diversity obtained from different genes cannot be compared directly. Because it is always possible that CR sequences are nuclear in origin, it is important to confirm general findings with a coding gene such as ND2, which can be more easily checked for mitochondrial authenticity. Genetic parameters estimated from ND2 showed a pattern similar to the one from partial CR studies of Kvist and colleagues (Table 5). In all studies, haplotype diversity was low in P. major and 4