Different Post-Pleistocene Histories ofEurasian ParidsALEXANDRA PAVLOVA, SIEVERT ROHWER, SERGEI V. DROVETSKI, AND ROBERT M. ZINK

From the Bell Museum of Natural History, University of Minnesota, St Paul, MN 55108 (Pavlova and Zink); Burke Museumand Department of Zoology, University of Washington, Seattle, WA 98195-3010 (Rohwer); and Department ofBiological Sciences, University of Alaska, Anchorage, AL 99508 (Drovetski). Alexandra Pavlova is now at the Schoolof Biological Sciences, Monash University, Clayton, Victoria 3800, Australia.

Address correspondence to R. M. Zink, 100 Ecology Building, 1987 Upper Buford Circle, St Paul, MN 55108, ore-mail: zinkx003@umn.edu.

Abstract

Previous phylogeographic studies of the great tit (Parus major) and the willow tit (Parus montanus) found a general absence ofphylogeographic structure for both species and suggested that each species underwent range contraction during the last IceAge and survived in relatively low numbers, P. major in southern Europe and P. montanus in southeastern Asia. However, priorstudies did not sample the entire range of either species. We analyzed sequence data for the complete mitochondrialND2 genefrom 87 P. major and 139 P. montanus from 15 new Eurasian localities, both to test prior conclusions and to provide bettercoverage of each species’ range. Our analyses confirmed the absence of phylogeographic structure in P. major and P. montanusand supported the prior refuge hypothesis for P. major. For P. montanus, we concluded that besides surviving the Ice Age insoutheastern Asia, as previously hypothesized, it apparently sustained a relatively large population in northern Eurasian riv-erine thickets and then expanded eastward. Genetic diversity was low in P. major (p 5 0.0012, h 5 0.64) and moderate inP. montanus (p5 0.0021, h5 0.88), suggesting higher long-term effective population sizes and the older ages of populations inP. 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 NorthAmerican and European species were strongly affected byPleistocene glaciations (Hewitt 1996, 1999, 2004; Taberletand others 1998). Relatively few studies, however, examinespecies with wide Eurasian distributions (Irwin and others2001; Kvist and others 2001, 2003; Liebers and others2001; Zink, Drovetski, and Rohwer 2002; Zink, Rohwer,and others 2002; Pavlova and others 2003; Zink andothers 2003; Drovetski, Zink, Rohwer, and others 2004;Godoy and others 2004; Pavlova 2004; Pavlova, Zink, andRohwer 2005; Pavlova, Zink, Rohwer, and others 2005) be-cause of the difficulties in obtaining samples. As a result,many studies of Palearctic species sample the European partof the species’ ranges but only include a few Asian samples.Such sampling gaps can affect the conclusions of phylogeo-graphic studies (Zink and others 2003). In this paper, weanalyze new and published mitochondrial DNA (mtDNA)sequences representing most of the range of 2 widespreadEurasian passerine birds, the great tit (Parus major) and thewillow tit (Parus montanus). Our goals are to test prior conclu-sions about the recent histories of these species and theirtaxonomic 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 sub-

species 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 Tur-

kestan 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 mor-

phological characters; however, their view was not supported

by the mtDNA study of Kvist and others (2003), which

Journal of Heredity 2006:97(4):389–402doi:10.1093/jhered/esl011Advance Access publication July 14, 2006

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found a clade consisting of 2 pairs of sister taxa, major andbokharensis and cinereus and minor. Thus, although some hy-bridization does occur where group ranges meet (Kerimovand Formozov 1986; Formozov and others 1993; Nazarenkoand others 1999; Fedorov and others forthcoming), the4 groups have had independent evolutionary histories and,therefore, should be considered phylogenetic species (Kvistand others 2003). In this paper, we consider the great tit(P. major), the Turkestan tit (P. bokharensis), and the Japanesetit (Parus minor) separate species (we lack Parus cinereus), as dosome other authors (Stepanyan 2003).

Kvist and others (1999) studied genetic variation in 6 Eu-ropean populations of P. major using sequence data from thecomplete mitochondrial (mtDNA) control region (CR) for 68

birds (Figure 1A, Table 1). They found an absence of phy-logeographic structure and extensive gene flow among pop-ulations, for which they inferred recent demographicexpansions. Kvist and others (1999) hypothesized that theBalkans was a refugium during the last glacial maximum(20 000 years ago) and that P. major subsequently expandedrapidly to northern Europe. More recently, Kvist and others(2003) examined partial CR sequences of 125 P. major

expanding the initial sampling to Portugal and the UnitedKingdom in Europe and to 3 Asian and 1 African localities(open circles in Figure 1A, Table 1); they also includedsequences of 3 individuals of P. bokharensis from Kirghiziaand 44 individuals of P. minor from 4 southeastern Asian pop-ulations (Figure 1A, Table 1). Kvist and others (2003) found

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 (2003) (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 (2001). Shaded areas

represent the distributions of (A) P. major and (B) P. montanus (following Harrap and Quinn 1996).

Journal of Heredity 2006:97(4)

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low genetic diversity within P. major and concluded that thegenetic variation within populations of P. major was reduceddrastically because of population bottlenecks during Ice Agerange contractions. Although the starlike haplotype network

of Kvist and others (2003; their Figure 2) suggested no over-all phylogeographic structure, their analysis of molecular var-iance (AMOVA) showed some overall population structure(UST 5 0.12) and pairwise population UST values for the

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 (2003), and complete CR:Kvist and others (1999)

Species

Source/sequencelength/polymorphicsites

Collectinglocality

Samplesize

Number ofhaplotypes

Haplotypediversity

Nucleotidediversity

P. major, great tit ND2/1039/34 Norway 3 3 1.000 0.00128Kursk þ Oryol 6 þ 2 7 0.960 0.00193Smolensk 3 2 0.667 0.00064Crimea 3 3 1.000 0.00257Krasnodar 5 2 0.400 0.00077Moscow 31 12 0.727 0.00139Medvedevo 2 1 — —Astrakhan’ 8 4 0.750 0.00162Yekaterinburg 1 1 — —Krasnoyarsk 1 1 — —Irkutsk 8 3 0.607 0.00114Buryatiya 4 1 0 0Mongolia 3 1 0 0Magadan 7 1 0 0Pooled major 87 24 0.638 0.00115

Partial CR/578/52 United Kingdom 10 5 0.844 0.00300Portugal 14 10 0.945 0.00373Spain* 9 7 0.917 0.00384Germany* 8 5 0.857 0.00247The Netherlands* 6 3 0.600 0.00173Estonia* 9 6 0.833 0.00269Sweden* 8 6 0.893 0.00247Finland 28 17 0.794 0.00243Urals 9 7 0.917 0.00336Amur 15 4 0.695 0.00277Kirghizia 6 5 0.933 0.00277Corsica 2 1 — —Morocco 1 — — —Pooled major 125 57 0.885 0.00325

CompleteCR/1189/52

Spain* 9 0.917a 0.00245Germany* 8 0.857a 0.00142The Netherlands* 6 0.600a 0.00113Estonia* 9 0.889a 0.00132Sweden* 8 0.893a 0.00185Finland, Kilpisjarvi 8 0.893a 0.00127Finland, Oulu 10 0.867a 0.00149Finland, Harjavalta 10 1.000a 0.00330Pooled major 68 0.876a 0.00187

P. bokharensis,Turkestan tit

ND2/1041/2 Almaty 4 2 0.500 0.00076Partial CR/578/11 Kirghizia 3 3 1.000 0.01269

P. minor,Japanese tit

ND2/1040/13 Primor’e 29 14 0.800 0.00117Partial CR 575/24 Amur 18 15 0.948 0.00383

Japan 14 6 0.736 0.00176North Korea 4 4 1.000 0.00725China 8 6 0.893 0.00354Pooled minor 44 29 0.908 0.00368

The populations for which most pairwise UST values were significant are shown in bold. Asterisks indicate populations for which 2 subsets of data

are available.a The values of haplotype diversity, originally reported in Table 2 of Kvist and others (1999) were calculated as 1�

Pf 2i

� �and were as follows—Spain: 0.815,

Germany: 0.75, The Netherlands: 0.5, Estonia: 0.79, Sweden: 0.781, Finland (Kilpisjarvi): 0.781, Finland (Oulu): 0.78, Finland (Harjavalta): 0.9, Pooled: 0.863.

All values of haplotype diversity reported in Tables 1 and 2 of this paper are calculated as 1�P

f 2i� �

n=ðn� 1Þ (Nei 1987, p. 180).

Pavlova et al. � Phylogeography of P. major and P. montanus

391

geographically isolated populations from Kirghizia and theUnited Kingdom were large and significant. Based on highergenetic diversity in the 3 individuals of P. bokharensis, itwas concluded that P. bokharensis was historically stable,whereas both P. major and P. minor experienced Late Pleisto-cene or Holocene population reductions and subsequentexpansions. Kvist and others (2003) also suggested thatP. major had a larger and more recent population expansionthan P. minor.

Parus montanus

This species ranges from northeastern France through cen-tral and northern Europe and Russia to the Pacific coast(Cramp and Perrins 1993, Figure 1B). Geographic variationin morphology is clinal and connects most of the continentalsubspecies, which also differ in song types and coloration(Harrap and Quinn 1995). Harrap and Quinn (1995) dividedP. montanus into 3 subspecies groups: salicarius (northern Eur-asia, 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 (2001) and Salzburger and others (2002),using sequences of the mitochondrial CR and cytochrome b,respectively, found no mtDNA structuring associated withsubspecies groups within P. montanus, whereas the songartit, considered by them as subspecies of P. montanus, wasfound to be polyphyletic and consisting of 3 distinct lineages:weigoldicus, affinis, and songarus. On the basis of close relation-

ships among 6 Eurasian subspecies of P. montanus, Salzburgerand others (2002) suggested that this species spread over ma-jor parts of Eurasia during the Pleistocene and underwentrapid morphological and vocal divergence. Kvist and others(1998) compared complete mitochondrial CR sequencesfrom Finland and Swedish populations and concluded thatindividuals from both localities belong to one panmictic pop-ulation with high long-term effective population size. Later,Kvist and others (2001) studied partial CR sequences of birdsfrom 6 western European localities, 6 samples from easternAsia, 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 subdividedoverall (UST 5 0.15), and all pairwise population UST valuesfor Japan and most UST values for Latvia were large and sig-nificant. Kvist and others (2001) attributed the general lack ofphylogeographic structure to homogeneous habitat through-out the distribution, absence of geographic barriers, highgene flow, and a high long-term effective population sizeand, based on trend of westward decrease in genetic diversity,suggested that P. montanus expanded through the Palearcticfrom southeastern Asia after the last Ice Ages.

Summary of Previous Work on Parid Phylogeography

Several studies have shown a general lack of phylogeographicstructure and evidence of overall population subdivision inP. major and P. montanus (sensu strictu). However, exceptfor the Urals, samples from eastern Europe and western Asiawere lacking (open circles in Figure 1 indicate prior samples).

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.

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Journal of Heredity 2006:97(4)

Species-level evolutionary conclusions should not be madewith confidence until the complete geographic range is sam-pled. This concern motivated us to add new localities (blackcircles in Figure 1) from previously unstudied areas. If theconclusions of Kvist and others (2001, 2003) are correct, thenour expanded analyses should reveal 1) a general absence ofgeographic structure on the haplotypes trees of both species,2) a decrease in genetic diversity consistent with the directionof expansion: eastward and northward in P. major and west-ward and northward in P. montanus, 3) signatures of expansionfor populations of both species, and 4) earlier expansions insoutheastern populations of P. montanus relative to the north-ern and western populations. Because our new samples over-lap to some degree with those used in the studies reviewedabove, we chose to sequence the mitochondrial coding geneND2. Thus, in addition to testing prior phylogeographic con-

clusions, we were able to compare genetic estimates derivedfrom the noncoding CR with those from ND2.

Materials and MethodsNew Sampling

We analyzed 87 individuals of P. major from 15 Eurasianlocalities, 4 individuals of P. bokharensis from Almaty, and29 individuals of P. minor from Primor’e (black circles inFigure 1A, Table 1). For P. montanus, we analyzed 139 indi-viduals from 17 localities (Figure 1B, Table 2). We used themarsh tit Parus palustris (GenBank number AY734248) as anout-group for the haplotype tree of P. montanus and P. minor

for P. major. All birds were collected during the breeding sea-son. From nearly all specimens, a study skin and spread wing

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 (2001), and complete CR: Kvist and others (1998)

Source/sequencelength/polymorphicsites

Collectinglocality

Samplesize

Number ofhaplotypes

Haplotypediversity

Nucleotidediversity

ND2/1039/63 Moscow þ Smolensk 12 þ 2 12 0.978 0.00253Medvedevo 6 6 1.000 0.00269Mezen’ 8 7 0.964 0.00323Yekaterinburg 1 1 — —Noyabr’sk 5 4 0.900 0.00212Tyva þ Gorno-Altay 7 þ 2 7 0.917 0.00192Krasnoyarsk 1 1 — —Irkutsk 23 10 0.767 0.00189Buryatiya 12 4 0.561 0.00077Mongolia 8 5 0.857 0.00148Anadyr’ 10 3 0.378 0.00038Primor’e* 13 9 0.872 0.00190Khabarovsk* 7 5 0.905 0.00238Magadan* 17 11 0.880 0.00198Kamchatka* 5 2 0.400 0.00231Pooled montanus 139 58 0.884 0.00210

Partial CR/592/56 Alps 8 6 0.893 0.00767Germany 4 3 0.833 0.00508Poland 2 2 1.000 0.00677Finland 13 11 0.962 0.00620Sweden 12 8 0.892 0.00397Latvia 8 6 0.929 0.00387Urals 13 12 0.987 0.00677Ussuri* 5 4 0.900 0.00575Amur* 4 4 1.000 0.00592Magadan* 7 7 1.000 0.00806Kamchatka* 7 5 0.857 0.00419Sakhalin 4 4 1.000 0.00733Japan 7 5 0.905 0.00612Pooled montanusa 94 71 0.981 0.01114

62 0.976 0.00671Complete CR/1207/52 Finland 13 13 1.000 0.00550

Sweden 12 12 1.000 0.00500Pooled 25 25 0.00530

The populations for which most pairwise UST 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 2001, bottom row values reported on page 934 of the text (nucleotide diversity of 0.00671 was

calculated by us in DNASP and was not reported in original paper).

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Pavlova et al. � Phylogeography of P. major and P. montanus

were preserved and deposited at the Burke Museum, Univer-sity of Washington, Seattle; the Moscow State UniversityZoological Museum, Moscow, Russia; the Bell Museum, Uni-versity of Minnesota, St Paul; or the State Darwin Museum,Moscow, Russia. Tissue samples were preserved in the fieldeither in 96% ethanol or in lysis buffer (Longmire and others1997) or frozen in liquid nitrogen.

Molecular Laboratory Methods

Isolation and purification of DNA was performed usingQIAamp Tissue Kit (Qiagen, Valencia, CA). Polymerasechain reaction (PCR) (Saiki and others 1988) with Perkin-Elmer PCR reagents and primers L5215 (Hackett 1996)and H1064 (Drovetski, Zink, Fadeev, and others 2004)was used to amplify the completeND2 gene; PCR conditionsare explained in Drovetski, Zink, Fadeev, and others (2004).PCR products were cleaned with Qiaquick PCR PurificationKit (Qiagen) and directly sequenced on ABI 3700 automatedsequencer using BIGDYE versions 3.0 and 3.1 chemistry(Applied Biosystems, Foster City, CA) and primers L5215,H1064, L347 (Drovetski, Zink, Fadeev, and others 2004),and H5578 (Hackett 1996).

Data Analysis

Sequences were aligned and edited in SEQUENCHER 3.1.1(Gene Codes Corporation, Ann Arbor, MI). Sequencedata have been deposited in GenBank (Accession num-bers AY732613–AY732729, AY734249–AY734251, andAY732869–AY733007). Mitochondrial origin of sequencedDNA fragments was supported by the absence of stopcodons and the existence of a large number of haplotypes,which are inconsistent with nuclear copies (Zhang andHewitt 1996). A matrix of variable sites was used as inputfor constructing a median-joining network (Bandelt andothers 1999) using software NETWORK version 4.110(http://www.fluxus-engineering.com) assigning equal weightsto all sites and with default value of epsilon 5 0.

Maximum likelihood (ML) model and parameters weredetermined by the hierarchical likelihood ratio test inMODELTEST 3.06 (Posada and Crandall 1998). PAUP*(Swofford 2000) and PHYML (Guindon and Gascuel2003) were used to perform ML tree searches. SEQBOOTandCONSENSEwere used from the PHYLIPprogram pack-age (Felsenstein 1993). SEQBOOT version 3.5c was used togenerate 100 bootstrapped sets of data from which ML treeswere constructed in PHYML. Then, CONSENSE version3.5c was used to compute a majority rule consensus tree.

DNASP version 4.00 (Rozas and others 2003) was usedto calculate the number of haplotypes in populations, nucle-otide diversity (p, Nei 1987; Equation 10.5), haplotype diver-sity (h; Nei 1987; Equation 8.4), and theta (H5 2Nel, whereNe is the effective population size and l is the mutationrate) from the number of polymorphic sites per nucleotide(Tajima 1996) and to perform the R2 test for detecting pop-ulation growth (Ramos-Onsins and Rozas 2002). We usedARLEQUIN version 2.000 (Schneider and others 2000) toperform an AMOVA (Excoffier and others 1992) taking into

account haplotype frequencies and the distances betweenhaplotypes, to compute pairwise population UST, mismatchdistributions, time since population expansion (s), effectivepopulation sizes before (H0) and after (H1) expansion, andTajima’s D (Tajima 1996) and Fu’s Fs (Fu 1997) tests of se-lective neutrality for localities with sample sizes greater than10 individuals (in some cases, samples were pooled if theywere not significantly differentiated). The latter 2 tests wereused as indicators of population expansion. Sites with ambig-uous characters were removed. To test the empirical mis-match 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 andothers (2001, 2003) and regressed values of p against latitudeand longitude for samples of 3 or more individuals expectingsmaller values to be observed on the leading edge of popu-lation expansion (Hewitt 1999, 2000).

Results and DiscussionThe Great Tit P. major

Genetic Variability and Gene Flow

We analyzed 1039 aligned nucleotides of ND2 after remov-ing sites with missing characters. The 34 polymorphic sites(9 of them parsimony informative) resolved 24 haplotypesamong the 87 individuals (Table 1). The most common hap-lotype (haplotype A in Figure 2) was shared by 52 individuals(60% 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), andMagadan (7). Three more haplotypes were shared amonglocalities (Figure 2). Overall haplotype diversity was 0.638and ranged from 0 in Buryatiya, Mongolia, and Magadan,where all individuals shared the most common haplotype,to 1 in Norway and Crimea (Table 1). Overall nucleotide di-versity (p) averaged 0.0012 and ranged from 0.0 in Buryatiya,Mongolia, and Magadan to 0.0026 in Crimea (Table 1).Northern populations had lower p, both for our data andfor those of Kvist and others (2003) (Figure 3A), but thistrend was not significant (P. 0.05). Our data, but not thoseof Kvist and others (2003), showed a significant trend to-ward eastern birds having lower p (R2 5 0.43, P 5 0.03)(Figure 3B), suggesting eastward dispersal. A general lackof population subdivision was suggested by the overallnonsignificant UST value (P . 0.05, Table 1). However,several pairwise population comparisons yielded significant(P , 0.05) UST values: Krasnodar–Magadan (UST 5 0.07),Crimea–Buryatiya (0.11), and Astrakhan’–Irkutsk (0.05),indicating that gene flow between eastern and westernpopulations might be restricted.

Molecular Phylogenetic Analysis and Phylogeography

MODELTEST 3.06 indicated TrN þ G (Tamura and Nei1993) with alpha 5 0.0069 as the best-fit model of sequence

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evolution. On the ML tree found by PHYML (not shown), all24 haplotypes of P. minor clustered together (this clade wasrecovered by 100% of 100 ML bootstrapped trees), as well asthe 2 haplotypes of P. bokharensis from Almaty (99% boot-strap support). However, the P. major clade received only 69%bootstrap support and was not recovered by the ML treefound by PAUP* because the P. bokharensis clade was embed-ded in it, making P. major paraphyletic. On the haplotype net-work (Figure 2), P. bokharensis was separated from P. major by10 mutational steps. Intraspecific genealogies can be multi-furcating due to ancestral and descendant haplotypes coexist-ing in populations, which violates the assumption of bifurcatingtrees employed in ML algorithm (Posada and Crandall 2001).Thus, the network likely provides a better estimate of among-haplotype 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, 2003) that P. major is notstructured geographically. This inference is strengthenedby the observation that the most common haplotype wasshared by more than a third of the individuals and distributedthroughout Eurasia (Table 1), including our new sites. Forexample, most (20 of 24) individuals from our Asian localities(Yekaterinburg, Krasnoyarsk, Irkutsk, Buryatiya, Mongolia,and Magadan) shared the most common haplotype. Thewidespread occurrence of common haplotypes is explicable

given known patterns of dispersal. Although young P. majornormally disperse less than 10 km on average from their natalgrounds, ‘‘northern and eastern populations are eruptivemigrants, moving in highly variable numbers dependingon the food supply . . . ’’ (Harrap and Quinn 1995,p. 356), and some invading birds remain to breed in the win-tering areas. This dispersal pattern can explain the generalabsence of differentiation in continental tits.

The insignificant overall UST for our data also suggeststhat P. major is not geographically subdivided, although sev-eral significant pairwise values suggested restricted gene flowbetween some eastern and western populations. Kvist andothers (2003) also found evidence of differentiation for2 geographically isolated populations, United Kingdomand Kirghizia, for which significant pairwise UST values werefound. This local differentiation is apparently attributable torelatively low natal dispersal distances (Verhulst and others1997). In addition, 19 of 20 unique haplotypes were sampledfrom European populations (Figure 2). These results sup-port the hypothesis of recent colonization of eastern Eurasiaand can account for some of the east–west populationdifferences.

Therefore, although earlier studies were limited in theirsampling of the range, the expanded analysis presented herecorroborates the hypothesis that there is little geographicstructure across the huge Palearctic range of P. major. The

A Nucleotide diversity vs latitudeP. major

Nucleotide diversity vs latitudeP. montanus

Nucleotide diversity vs longitudeP. montanus

0

15

30

45

60

75

90

30 35 40 45 50 55 60 65 70North latitude

Nuc

leot

ide

dive

rsity

Nuc

leot

ide

dive

rsity

Nuc

leot

ide

dive

rsity

Nuc

leot

ide

dive

rsity

C

0

15

30

45

60

75

90

30 35 40 45 50 55 60 65 70North latitude East longitude

B Nucleotide diversity vs longitudeP. major

0

15

30

45

60

75

90

East longitude

D

0

15

30

45

60

75

90

-20 0 20 40 60 80 100 120 140 160

0 20 40 60 80 100 120 140 160 180

R2 = 0.23P = 0.13

R2 = 0.06P = 0.47

R2 = 0.01P = 0.8

R2 = 0.43P = 0.03

R2 = 0.04P = 0.53

R2 = 0.6P = 0.03

R2 = 0.02P = 0.64

R2 = 0.02P = 0.62

Figure 3. Nucleotide diversity (in units of p � 104) 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 (2003) for P. major and those of Kvist and others (2001) 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 (2001, 2003). The values of R2 and the corresponding P values

for our data are in the lower part of each graph.

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general absence of phylogeographic structure as the result ofpostglacial range expansion and low genetic diversity wasreported for 2 northern species of North American chicka-dees, Poecile atricapillus and Poecile hudsonicus (Gill and others1993). Other forest-dwelling birds also appear to lack phylo-geographic structure across much of northern Eurasia (e.g.,Zink, Drovetski, and Rohwer 2002; Zink, Rohwer, andothers 2002).

The mtDNA data appear to conflict with 2 recent studiesfrom the United Kingdom that revealed the existence of nat-ural selection acting on local differences in life-history traits.Postma and van Noordwijk (2005) discovered differences inclutch size between subpopulations separated by only a fewkilometers. Garant and others (2005) discovered that themean mass of P. major decreased in one part of Wythamwoods, whereas clutch size remained constant to the north.A significant genetic component to variation in these fitnessor fitness-linked traits was documented in both studies. Ifthese represented genome-wide patterns of differentiation,the mtDNA tree should have revealed more structure. Thus,the mtDNA data suggest that these local adaptations are veryrecently evolved.

Population Expansion

Kvist and others (2003) suggested that P. major populationsunderwent a drastic reduction in size as a result of range con-traction during the last glacial maximum. Survival in lownumbers in a small southwestern refugium (possibly Balkans,Kvist and others 1999) was thought to have been followed bynortheastward range expansion and subsequent demographicexpansion. Although many of our individual samples aresmall, our mismatch distributions and values of Tajima’sD, Fu’s Fs, and R2 are consistent with these hypotheses ofpopulation and range expansion (Table 3, Figure 4), asexpected for north temperate populations (Hewitt 2004).

To test for the effects of natural selection, Zink (2005)analyzed our data set using 2 partitions of the ND2 gene(surface and transmembrane portions and synonymous–

nonsynonymous sites). Both partitions yielded similar esti-mates of UST, which suggested demographic expansion asthe cause of the shallow tree for P. major and not directionalor stabilizing natural selection (Zink 2005).

Notes on Evolutionary History of P. bokharensis

Kvist and others (2003) suggested that there was less or nohistoric habitat reduction for P. bokharensis because it prefersdeserts and semideserts, which is ‘‘the same kind of environ-ment that existed during the Ice Ages within its presentrange’’ based on the reconstruction by Adams (1997). Theestimate of Kvist and others (2003) of p (from 3 individuals)for P. bokharensis led them to conclude that ‘‘populations havebeen stable for a long time because . . . [they] . . . possessmore diversity than the minor and major groups.’’ We did notfind a higher p in P. bokharensis with our sample of 4 birds.We suggest more samples are needed to clarify the history ofP. bokharensis.

The Willow Tit P. montanus

Genetic Variability and Gene Flow

We analyzed 1039 aligned bp of ND2 after removing siteswith ambiguous characters. The 63 polymorphic sites (26parsimony informative) resolved 58 haplotypes among the139 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 othercommon haplotypes were found: haplotype B (Figure 5)had a restricted Far Eastern distribution and occurred in13 individuals from Kamchatka, Magadan, and Anadyr’and haplotype C was shared by 12 individuals from Mezen’,Noyabr’sk, Irkutsk, Buryatiya, Mongolia, Primor’e, andKhabarovsk. Several more haplotypes were shared amongpopulations (Figure 5). The occurrence of widespread hap-lotypes indicates considerable recent gene flow.

Table 3. Estimates of effective population size and demographic changes from ND2 data for samples of 10 or more individuals

Species Locality HS s H0 H1 Tajima’s D Fu’s Fs R2

Parus major Moscow 0.0041 1.2 0 283 �2.2 �7.6 0.052Pooled 0.0065 1.3 0 3 �2.6 �26.1 0.022

Parus minor Primor’e 0.0032 ne ne ne �2.1 �13.1 0.046Parus montanus Moscow 0.0041 2.7 0 6930 �1.8 �9.4 0.073

Irkutsk 0.0044 1.4 0 121 �2.1 �4.1 0.092 nsBuryatiya 0.0013 0.8 0 31 ns ns 0.154 nsAnadyr’ 0.0007 0.5 0 725 ns �1.2 0.2 nsPrimor’e 0.0037 2 0 322 �2.0 �5.3 0.086Magadan 0.0040 2.2 0 2310 �1.9 �6.9 0.059Pooled 0.0110 2 0 1397 �2.5 �27.2 0.015Pooled reduced 0.0106 2.1 0 23 �2.5 �27.3 0.017

‘‘Pooled reduced’’ refers to calculations omitting differentiated sampling sites of Parus montanus (Medvedevo, Anadyr’, and Kamchatka). HS is estimated

(in DNASP) from the number of segregating sites per nucleotide; s, H0, and H1 (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 R2 (DNASP) are significant at the P , 0.05 level unless denoted as ns, not significant, or ne, no

estimates available.

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Overall haplotype diversity (0.88) was moderate andranged from 0.38 in Kamchatka to 1 in Medvedevo (Table 2).Overall nucleotide diversity was 0.0021 and ranged from0.0004 in Anadyr’ to 0.0032 in Mezen’ (Table 2). Relativelyhigh genetic diversity within P. montanus and the relativelylarge number of haplotypes found in our data and thoseof Kvist and others (2001) (Table 2) suggest a high long-term effective population size and an absence of recentbottlenecks.

Regressions of p on latitude were not significant (P .

0.05) for our data or for those of Kvist and others (2001)(Figure 3C). Longitude explained 60% of the variance ofp in our data with eastern birds having lower p (R2 5 0.6,P, 0.05), but no such trend was found for the data of Kvistand others (2001) (Figure 3D). Kvist and others (2001)reported a westward decrease in nucleotide diversity forsalicarius group (which includes most of the species’ distribu-tion excluding Kamchatka, Sakhalin, and Japan), from whichthey concluded that during the last Ice Ages P. montanus

occupied large regions of the southeastern Palearctic andcolonized the European (western) part of the range fromthe east. However, our regression analysis of their nucleotidediversity data did not find this trend to be a statistically sig-nificant (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 (2001). 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 (2001)

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 3.06 suggested TrN þ G (Tamura and Nei

1993) with alpha 5 0.0153 as a most probable model of se-

quence evolution. This was the same model selected for the

P. major group, suggesting similar sequence evolution ofND2

gene in different parids. The monophyly of P. montanus was

supported by 100% of 100 ML bootstrap support replicates.

The ML haplotype tree (not shown) was geographically

P. major pooled

0

0.1

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0.5Fr

eque

ncy

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0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9

0 1 2 3 4 5 6 7 8 90 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9

0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9Number 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 2.000 (Schneider and others 2000), and dashed

lines the mean of the distribution.

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Pavlova et al. � Phylogeography of P. major and P. montanus

unstructured, which can be seen in the network (Figure 5).Kvist and others (2001) also found a geographically unstruc-tured haplotype network and attributed it to high gene flow.However, they also suggested that gene flow might not becurrently high because of geographic differences in morphol-ogy and voice. Our haplotype network (Figure 5) shows thatsome haplotypes are restricted to Asia, suggesting limitedgene exchange for some eastern localities. Furthermore,our AMOVA showed that geographic localities explain10.8% of the total variation (P , 0.05). Most pairwiseUST values involving Anadyr’ (range 0–0.62) and Kamchatka(range 0–0.37) were large and significant (Table 4), indicatingrestricted or no gene flow between these and more westernpopulations. Zink (2005) explored the nature of highUST val-ues for these populations and found that they occurred be-cause of a replacement substitution at base position 452 thatwas shared by all Anadyr’ individuals and 4 of 5 birds fromKamchatka (haplotype B and 2 adjacent haplotypes fromAnadyr’ in Figure 5). Zink (2005) pointed out that althoughsignificant UST values might be a signature of incipient diver-gence caused by geographic isolation, they also might indicate

a selection for a new favorable haplotype. Because only a sin-gle substitution is involved, a definitive conclusion is unwar-ranted. Several pairwise UST values involving Medvedevo(range 0–0.18) were also significant (Table 4), further sup-porting restricted gene flow between western and easternpopulations. Significant pairwise UST values that are foundfor 2 northeastern populations (Anadyr’ and Kamchatka,subspecies anadyrensis and kamtchatkensis) and the populationfrom Japan (subspecies restrictus), Medvedevo, and Latvia(both belong to subspecies borealis), suggest that some pop-ulations of P. montanus are differentiated.

Populations diverging from a common ancestor gothrough a series of stages represented by gene trees thatare polyphyletic, then paraphyletic, and finally reciprocallymonophyletic with respect to geography (Avise 2000). Theexistence of significant UST values and a haplotype tree thatis not reciprocally monophyletic, but has some limited differ-entiation, suggests that P. montanus is at an intermediate stageof differentiation. The pattern of incomplete genetic dif-ferentiation coincides in part with phenotypic (subspecific)divergence.

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.

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Population and Range Expansion

Kvist and others (2001) suggested that during Pleistocenecooling P. montanus was restricted to southeastern Asia andcolonized 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 popu-lation is expected to be unique and most haplotypes fromrecently colonized areas are expected to be closely relatedto the common haplotype. Such a pattern was observedfor P. major (Figure 2), consistent with the findings of Kvistand others (2003). However, on haplotype networks forP. montanus (Figure 5 in this paper and Figure 2 in Kvistand others [2001]), haplotypes from the western and mosteastern, potentially refugial, populations were randomlydistributed (although in Kvist and others 2001 no easternindividuals shared the most common haplotype). Recon-structions of biomes during the last glacial maximum basedon pollen and plant macrofossils (Tarasov and others 1999)suggest that much of northeastern Europe and northwesternAsia were covered by tundra. Although P. montanus typicallybreeds in coniferous and mixed forests, it also inhabits woodyshrubs and riverine thickets extending to the tree line innorthern Eurasia and in mountainous regions (Harrap andQuinn 1995). Thus, Pleistocene tundra in northern Eurasiacould have provided suitable habitats for P. montanus. Theexistence of substantial genetic diversity in western popula-tions (see Figure 4 for comparison with P. major) suggests that

P. montanus, unlike P. major, was not subjected to drastic rangecontractions and during climate warming could have ex-panded eastward, as suggested by our data, as well as west-ward to Europe, as Kvist and others (2001) suggested. It isalso possible that historic range of P. montanus was split dur-ing the last Pleistocene cooling and some birds survived inrefugial forests of eastern Asia.

The mtDNA data are consistent with the hypothesis ofrange and population expansion. Mismatch distributions(Figure 4) for P. montanus populations did not differ from themodel of sudden population expansion and were unimodalfor all localities except Irkutsk. Population growth was alsosuggested by most values of Tajima’s D, Fu’s Fs, and R2 forall populations except Buryatiya. Thus, both P. major andP. montanus appear to have responded to post–Ice Age habitatamelioration by range and population expansion, although asdiscussed below, the details differ.

Different Evolutionary Histories of P. major, P. minor, andP. montanus

During the last glacial maximum, some broadleaved forestsof southeastern Asia were replaced by grassland vegetation.However, this part of the continent also contained severallarge refugial forests (Adams 1997) and, therefore, was apotential refuge for forest-dwelling birds. AlthoughP. montanus,like P. major and P. minor, is a cavity nester and its range largelyoverlaps with that of P. major, the evolutionary history ofP. montanus likely differed from that of its congeners, as evi-denced by higher values of p, H, and h and the low percent-age of individuals sharing a common haplotype (Table 5).A significant range restriction associated with Pleistocenecooling would be expected for P. major because they requireforested habitats and do not live in the brushy habitats of landunderlain with permafrost. Eastward decrease of geneticdiversity (Figure 3B) and low genetic diversity suggest thatP. major might have survived Pleistocene glaciations in smallnumbers in refugial forests of southern Europe and subse-quently expanded into eastern Eurasia. In contrast to P. major,P. montanus, whose range extends farther north, did not un-dergo as severe a population bottleneck but instead survivedin relatively large numbers in narrow riparian thickets in thewidespread north Eurasian ice-free areas where large river

Table 4. Significant pairwise UST values for Parus montanuspopulations (ND2 data)

Medvedevo Anadyr’ Kamchatka

Moscow þ Smolensk 0.36 0.19Medvedevo — 0.51 0.25Mezen’ 0.27Noyabr’sk 0.59 0.28Gorno-Altay þ Tyva 0.10 0.46 0.22Irkutsk 0.11 0.39 0.24Buryatiya 0.18 0.62 0.37Mongolia 0.56 0.27Primor’e 0.10 0.39 0.20Khabarovsk 0.50 0.24Magadan 0.11 0.37 0.19

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 (2001) (P. montanus) and Kvist and others (2003) (P. major and P. minor), ND2: this paper

Species Data set % Most com. hapl. h p HS Total UST s Tajima’s D Sample size

P. major Partial CR 36.8 0.855 0.00325 0.01698 0.12* 1.88 �2.52* 125P. minor 15.9 0.908 0.00368 0.00921 0.28* 2.11 �2.05* 44P. montanus 11.7 0.976 0.00671 0.02558 0.15* 3.51 �2.06* 94P. major ND2 59.7 0.638 0.00115 0.00650 ns 0.51 �2.57* 87P. minor 45.8 0.800 0.00117 0.00318 — 1.21 �2.22* 29P. montanus 31.7 0.884 0.00210 0.01101 0.11* 1.50 �2.55* 139P. montanus ND2 reduced — 0.857 0.00204 0.01062 — 1.34 �2.58* 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, HS, s, and Tajima’s D were calculated in DNASP and UST in ARLEQUIN. Asterisk indicates

significance at P , 0.05 level; ns, not significant.

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Pavlova et al. � Phylogeography of P. major and P. montanus

valleys were free of permafrost, as well as in refugial forestsof southeastern Asia.

The present range of P. minor is restricted to southeasternAsia, and it is thought that the species only recently has beenexpanding northward (Smirenskiy 1986; Harrap and Quinn1995). In fact, all but 5 haplotypes of P. minor in Kvistand others (2003) were unique and clustered according totheir geographic localities on the haplotype network (theirFigure 1), whereas only 2 of the 5 common haplotypes wereshared between populations. A nonrandom distribution ofhaplotypes and the large significant overall UST for P. minor(0.28, Table 5) indicate significant population subdivision, in-consistent with high gene flow expected from range expan-sion. For our only sample of P. minor (from Primor’e),significantly negative Tajima’s D and Fu’s Fs, significantR2 value (Table 3), and a starlike network where 46% indi-viduals shared the most common haplotype (Figure 2) indi-cated population growth, consistent with recent colonizationof Primor’e.

On the basis of similar nucleotide diversity for P. majorand P. minor, but higher theta within P. major (the results con-sistent with our findings, Table 5), Kvist and others (2003)suggested a greater degree of population expansion in P. majorthan in P. minor because theta is more influenced by currentpopulation size, whereas p reflects the historic size (Tajima1989). Given that the range of P. major is roughly 4 timesgreater than that of P. minor, this conclusion is not surprising.Time since expansion (s, Table 5) was small in P. major, in-termediate in P. minor, and large in P. montanus for all data sets,indicating that expansion in P. major was relatively recent. Infact, range expansion of P. major is known to have mirroredthe expansion of human settlements (Harrap and Quinn1995), and in some regions of the Russian Far East, the spe-cies’ presence is restricted to human habitations. In addition,introgression of major haplotypes into minor populations inthe Amur region hybrid zone and absence of foreign haplo-types in allopatric P. minor populations provide indirect evi-dence toward high philopatry in P. minor (Fedorov and othersforthcoming).

Thus, the genetic analyses of 3 codistributed sedentaryparids suggest that each species responded differently tothe environmental changes of the Ice Ages. These differentgenetic signatures are likely a result of differences in habitatuse and dispersal abilities.

Comparison of Genetic Parameters Derived fromCR and ND2

Regions of the mtDNA genome are known to evolve at dif-ferent rates, owing in part to selective constraints. The CR isoften considered the most variable, although this is not al-ways the case (Ruokonen and Kvist 2002). Despite the po-tential for different regions of mtDNA to evolve at differentrates, the entire gene has a single history owing to linkage.Nonetheless, it is of interest to make direct comparisonsof different gene regions as studies often compare levelsof variability across gene regions (Ruokonen and Kvist2002). For the 5 European populations of P. major, estimates

of nucleotide diversity were on average 1.64 times higherwhen computed from the partial 5#-end of the CR (Kvistand others 2003) than those calculated from the completeCR sequences (Kvist and others 1999) (Table 1), indicatingthat within species the 5#-end of the mitochondrial CR ismore variable than the 3#-end. This result was reported ear-lier for intraspecific comparisons in P. major, Parus caeruleus,and P. montanus (Ruokonen and Kvist 2002). However, forthe 2 Scandinavian populations of P. montanus studied byKvist and others (1998, 2001), the results were not consistent:pFinland was higher when estimated from partial (5#-end) CR,whereas pSweden was higher when estimated from completeCR sequences. Such inconsistency in the relative distributionof variation in CR sequences makes it hard to compare esti-mates of p obtained from different mitochondrial generegions. Although Ruokonen and Kvist (2002) suggested thatthe distribution of variation in CR is similar within genera,nucleotide diversity estimated from the 3#-end of the CRsequences for P. major, P. minor, and P. montanus (A Pavlova,RM Zink, S Rohwer, and S Drovetski, unpublished data;GenBank sequence numbers AY732496–AY732612 andAY732730–732868) yielded comparative results very differ-ent than those obtained from ND2 for the same sets of indi-viduals. For example, p from the 3#-end of CR was 0.0007,0.0034, and 0.0026 andH was 0.0046, 0.0114, and 0.0125 forP. major, P. minor, and P. montanus, respectively, whereas esti-mates of p fromND2 or 5#-end of CR were similarly low forP. major and P. minor andH was smallest in P. minor (Table 5).This example demonstrates that different parts of mitochon-drial genome, and even parts of the same gene region, evolvewith different rates in different taxa (even within genera), andcare must be used when interpreting results. Thus, molecularclock rates calibrated for coding genes should not be appliedfor CR data to avoid substantial overestimation of the ages ofpopulation/species splits. Furthermore, calibrations for differ-ent parts of the CR are not general.

Values of nucleotide diversity estimated from our ND2

data were consistently lower than those calculated from thepartial CR data of Kvist and others (2001, 2003). For the4 eastern populations of P. montanus (Magadan, Kamchatka,Khabarovsk [Amur], and Primor’e [Ussuri]), our samples andthose of Kvist and others (2001) overlapped (Figure 1B,Table 2). Comparisons of estimates of genetic diversity cal-culated from ND2 and partial CR for these populationsshowed that estimates of p from partial CR are on average2.9 times higher than from ND2 and estimates of h are 1.4times higher from partial CR than from ND2 (Table 5).Therefore, for Parus species, the 5#-end of CR yields moreintraspecific variation than either complete CR or ND2,and estimates of genetic diversity obtained from differentgenes cannot be compared directly.

Because it is always possible that CR sequences are nu-clear in origin, it is important to confirm general findings witha coding gene such as ND2, which can be more easilychecked for mitochondrial authenticity. Genetic parametersestimated from ND2 showed a pattern similar to the onefrom partial CR studies of Kvist and colleagues (Table 5).In all studies, haplotype diversity was low in P. major and

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higher in P. minor and P. montanus, whereas nucleotide diver-sity was similar in P. major and P. minor and higher inP. montanus (Table 5). For P. montanus, we also computed ge-netic parameters for ourND2 data removing 3 sites (Anadyr’,Kamchatka, and Medvedevo) that had the majority of signif-icant pairwise UST values (Table 5) because population sub-structure, found both in Kvist and others (2003) and in ourstudy (Table 4), could confound the estimation of theseparameters. The relative values of the genetic parametersdid not change. Thus, although absolute values for parame-ters cannot be directly compared across gene regions, theoverall phylogeographic pattern is consistent.

AcknowledgmentsWe are grateful to B. Eddy, H. Furguson, and to the late G. Eddy for funding

fieldwork. Additional support came from the National Science Foundation

(DEB 9707496 and DEB 0212832) and the Dayton-Wilkie Natural History

fund. Tissue samples were provided by Burke Museum of Natural History

and Culture, University of Washington; Moscow State University Zoological

Museum, Moscow, Russia; and the Bell Museum of Natural History, Uni-

versity of Minnesota. We thank the Burke Museum for curatorial assistance

and M. Westberg for laboratory assistance. We also thank A. Andreev, V.

Andreev, Yu. Lohman, D. Banin, I. Karagodin, Ya. Red’kin, B. Schmidt,

V. Rohwer, C. S. Wood, X. Pu, V. Masterov, R. Faucett, G. Voelker,

V. Sotnikov, S. Birks, B. Barber, I. Fadeev, E. Nesterov, E. Koblik, and

A. Jones for logistical help with expeditions and collecting.

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Received March 9, 2005Accepted May 21, 2006

Corresponding Editor: Rob Fleischer

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