J Ornithol (2012) 153:405–411 DOI 10.1007/s10336-011-0756-4

ORIGINAL ARTICLE

Phylogeography and population structure of Kru¨per’s Nuthatch Sitta krueperi from Turkey based on microsatellites and mitochondrial DNA

Tamer Albayrak • Javier Gonzalez • Sergei V. Drovetski • Michael Wink

Received: 28 October 2010 / Revised: 22 August 2011 / Accepted: 1 September 2011 / Published online: 23 September 2011 Ó Dt. Ornithologen-Gesellschaft e.V. 2011

Abstract Kru¨per’s Nuthatch Sitta krueperi is endemic to contact after glacial retreat. The identified populations of Anatolia, Lesvos Island and the western Caucasus region this ‘near threatened’ species should be treated as separate where it inhabits coniferous forests. To infer the phyloge- conservation units. ographic structure of Kru¨per’s Nuthatch, we analyzed partial sequences of the mitochondrial cytochrome oxidase Keywords Population genetics Á Evolution Á Anatolia Á subunit I gene (COI; 605 bp) and allele variation at eight Cytochrome oxidase subunit 1 gene Á Sitta krueperi microsatellite loci from birds sampled across five localities in Turkey. A total of ten COI haplotypes were found Zusammenfassung among 68 individuals revealing three distinct haplotype groups; one each in northwestern, northeastern, and Phylogeographie und Populationsstruktur des Tu¨r- southern Turkey. However, there was evidence for sec- kenkleibers Sitta krueperi in der Tu¨rkei auf Grund ondary gene flow between the northwestern and the other vom Mikrosatelliten und mitochondrialer DNA two regions. Significant microsatellite differentiation was also found between northern and southern localities, but Der Tu¨rkenkleiber, Sitta krueperi, ist eine endemische not between the two northern ones. This genetic structure is Vogelart in Anatolien, Lesbos und im westlichen Kauka- consistent with the isolation of these populations in dif- sus, wo er besonders Nadelwa¨lder besiedelt. Zur Untersu- ferent glacial refugia followed by establishing secondary chung seiner molekularen Phylogeographie wurden Teilsequenzen der mitochondrialen Cytochrom-Oxidase (COI, 605 Nucleotide) und die Allelvariation von 8 poly- Communicated by T. Friedl. morphen Mikrosatellitenloci von 5 Kleiberpopulation in der Tu¨rkei untersucht. Unter den 68 untersuchten Indivi- Electronic supplementary material The online version of this article (doi:10.1007/s10336-011-0756-4) contains supplementary duen wurden 10 COI-Haplotypen ermittelt, die sich in drei material, which is available to authorized users. Hauptgruppen in der nordwestlichen, nordo¨stlichen und su¨dlichen Tu¨rkei abbilden lassen. Ein sekunda¨rer Genfluss & T. Albayrak ( ) zwischen der nordwestlichen Population zu den beiden Department of Biology, Faculty of Science and Art, Mehmet Akif Ersoy University, Ortulu Yerleskesi, 15030 Burdur, Turkey anderen wird vermutet. Die Mikrosatelliten-Analyse weist e-mail: [email protected] signifikante Unterschiede zwischen den no¨rdlichen und su¨dlichen Populationen, nicht aber zwischen den no¨rdli- J. Gonzalez Á M. Wink chen Populationen auf. Die genetischen Daten sprechen fu¨r Department of Biology, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld, eine Isolierung der Populationen in unterschiedliche Eis- 364, 69120 Heidelberg, Germany zeitrefugien und die Entwicklung sekunda¨rer Kontaktzonen nach der letzten Eiszeit. Die in dieser Arbeit ermittelten S. V. Drovetski genetischen Gruppen sollten als getrennte Conservation CIBIO, Centro de Investigac¸a˜o em Biodiversidade e Recursos ¨ Gene´ticos Campus, Agra´rio de Vaira˜o Rua Padre Armando Units erhalten werden, um das Uberleben dieser auf der Quintas - Crasto, 4485-661 Vaira˜o, Portugal Vorwarnliste stehenden Art zu sichern. 123 406 J Ornithol (2012) 153:405–411

Introduction profoundly affected by advances and retreats of glacial ice during the Pleistocene. Successive retreats and expansions Nuthatches (Sitta, Passeriformes) inhabit different types of of glacial ice may have resulted in isolation in multiple forests throughout most of the northern hemisphere. refugia, postglacial colonization, and secondary contact Kru¨per’s Nuthatch Sitta krueperi belongs to the ‘canadensis’ (Hewitt 2000; Hampe et al. 2003). The use of multiple group of relatively small nuthatches which was considered a genetic markers with different modes of inheritance and single species until Vaurie’s work (1957). Currently, this rates of evolution is often needed to infer complex phy- group consists of six species: Red-breasted Nuthatch logeographic histories (Howes et al. 2006). Due to its rapid (S. canadensis), Corsican Nuthatch (S. whiteheadi), Kabylie coalescence time, mitochondrial DNA has proven very Nuthatch (S. ledanti), Chinese Nuthatch (S. villosa), Yunnan useful for revealing the phylogeographic structure of sev- Nuthatch (S. yunnanensis) and Kru¨per’s Nuthatch (Lo¨hrl eral widespread Eurasian avian species (Drovetski et al. 1988; Harrap and Quinn 1996;Matthysen1998;Pasquet 2005; Zink et al. 2008). However, great dispersal abilities 1998). A phylogenetic analysis of a short fragment (491 bp) and large home ranges of birds often require the use of of the mitochondrial cytochrome b gene revealed that the complementary, fast-evolving markers such as microsat- Kabylie and Kru¨per’s Nuthatches represent the sister clade to ellites, to detect genetic differentiation among populations. a clade containing Red-breasted, Corsican and Chinese Nut- In this study, we use a combination of mitochondrial hatches (Pasquet 1998). These two major clades may have DNA sequences of cytochrome oxidase subunit 1 gene and diverged approximately 5 million years ago (MYA), whereas variation at eight microsatellite loci to analyze the phy- Kru¨per’s and Kabyile Nuthaches may have diverged 1–2 logeographic structure in Kru¨per’s Nuthatch among five MYA (Pasquet 1998). localities across Turkey. The distribution of Kru¨per’s Nuthatch is primarily confined to coastal areas of the Anatolian Peninsula. Out- side Anatolia, this species is found on the adjacent Lesvos Methods Island and in the western Caucasus region (Harrap and Quinn 1996; Hagemeijer and Blair 1997; Albayrak and Sampling, DNA isolation and sequencing Erdogan 2005a; Hoyo et al. 2008; Fig. 1) and is considered of the mitochondrial COI gene a ‘near threatened’ species by the IUCN (2009). Kru¨per’s Nuthatch is a sedentary bird inhabiting coniferous forests Sixty-seven Kru¨per’s Nuthatches were captured with mist- from sea level up to the timberline exhibiting limited post- nets at five localities in Turkey. Blood or feathers were breeding dispersal and seasonal altitudinal movements obtained from each individual from the following habitats (Cramps and Perrins 1993; Albayrak and Erdogan 2005b; and localities: (1) red pine forests (Pinus burutia): Research Hoyo et al. 2008; Albayrak et al. 2010). Forest, Antalya (BUK, 650 m.a.s.l, n = 19), Adrasan (ADR, The Anatolia Peninsula is a mountainous area with 200 m.a.s.l, n = 5), and Aladag˘lar Mountains (ALA, complex topography (Hughes et al. 2006). It was 930 m.a.s.l, n = 13); and (2) black pine forests (Pinus

Fig. 1 Sampling localities, mitochondrial DNA haplotypes and mismatch distributions of Kru¨per’s Nuthatch Sitta krueperi; the shaded area represents the range of the species; black line indicate political boundaries

123 J Ornithol (2012) 153:405–411 407 nigra): Kazdag˘lari Mountains (KAZ, 800 m.a.s.l, n = 15) re-run on different gels and used for relative comparisons and Kartalkaya Mountain (KAR, 1,300 m.a.s.l, n = 15; in order to confirm the identification of alleles and validate Fig. 1). Also, one Kru¨per’s Nuthach was obtained from scoring results. Lesvos Island (LES, red pine forests, 400 m.a.s.l). Total DNA was isolated using standard Proteinase K Data analyses (Merck, Darmstadt) and phenol/chloroform procedures (Sambrook et al. 1989). We sequenced 605 base pairs (bp) Sequences were aligned manually with BioEdit version of the mitochondrial cytochrome c oxidase subunit 1 (COI) 7.0.9.0 (Hall 2004). Basic statistics and average uncor- gene using the primers passerF1 and passerR1 (Lohman rected p-distances were calculated with MEGA version 4.0 et al. 2009). The PCR amplifications were performed in (Tamura et al. 2007). Median-joining networks were 50-ll reaction volumes containing 19 PCR buffer (Bioron, reconstructed for mitochondrial haplotypes using the pro- Ludwigshafen), 100 lM dNTPs, 0.2 units of Taq DNA gram Network version 4.2.0.1 (Bandelt et al. 1999). polymerase (Bioron, Ludwingshafen), 200 ng of DNA and Nucleotide diversity, haplotype diversity, mismatch distri- 5 pmol of primers. Optimal annealing temperature was butions and R2 test of demographic/population expansion determined by gradient PCR in a Tgradient thermocycler were calculated in DnaSP version 5 (Librado and Rozas (Biometra). Thermal cycling was performed under the 2009) for each population. following conditions: 5 min at 94°C, followed by 38 cycles The departure from Hardy–Weinberg equilibrium of 50 s at 94°C, 40 s at 55.6°C, 1 min at 72°C and a final (HWE) and linkage disequilibrium (exact tests) were tested extension at 72°C for 10 min. PCR products were precip- for each microsatellite locus for each population using itated with 4 M NH4Ac and ethanol (1:1:6) via centrifu- GENEPOP version 4.0 (10,000 dememorizations, 100 gation for 15 min at 13,000 rpm. batches and 5,000 iterations per batch; Raymond and Sequencing was performed using an ABI 3730 automated Rousset 1995; Rousset 2008). A sequential Bonferroni capillary sequencer (Applied Biosystems) with the ABI correction (Rice 1989) was used for these tests. Mean

Prism Big Dye Terminator Cycle Sequencing Ready Reac- number of alleles (A), observed (HO) and expected (HE) tion Kit version 3.1 by STARSEQ (Mainz, Germany). In order heterozygosity were calculated using ARLEQUIN version to confirm observed substitutions, both strands were 3.1 (Excoffier et al. 2005). FST distances were also calcu- sequenced. The sequences were deposited in GenBank under lated with ARLEQUIN and their significance was tested the accession numbers HM469464–HM469531. using 22,000 permutations. Each locus was tested for consistency with HWE in each locality with MICRO- Microsatellites analysis CHECKER (Van Oosterhout et al. 2004). We used a Bayesian clustering method implemented in We selected eight polymorphic STR loci from a broad set STRUCTURE version 2.2 (Pritchard et al. 2000; Falush of loci amplified with STR primers from different species et al. 2003) to infer the population structure. This analysis of songbirds: Ase29, Ase48 (Richardson et al. 2000), Pca8 considered the individuals without any group origin (Dawson et al. 2000), Pij23 (Saito et al. 2005), Spu4-E7, information. In order to identify the correct number of SpuL5-22, SpuL6-2 (Haas et al. 2009) and Syl14 (Segelb- independent genetic groups (K), a series of ten independent acher et al. 2008; supplementary Table). We analyzed 55 runs with K = 1–7, where K is the different number of individuals of Kru¨per’s Nuthatches from five localities: 9 subpopulations, were used with an admixture model and individuals from BUK, 5 from ADR, 15 from ALA, 13 correlated allele frequencies. Throughout the analysis, the from KAZ, and 13 from KAR. The PCR was performed burn-in period was fixed at 100,000 and the number of with 60 ng of total DNA in 25 ll of reaction volume MCMC runs was set at 50,000. We performed two repli- containing 10 pmol of each primer, 25 lM of dGTP, dCTP cates for each run. and dTTP, and 12.5 lM of dATP, 19 PCR buffer (Bioron), 0.15 units of Taq polymerase (Bioron), 1 lCi [a-33P]- dATP (Amersham Biosciences). Thermocycling was per- Results formed under the following conditions: 5 min at 94°C, followed by 38 cycles of 50 s at 94°C, 40 s at 50–58°C, Sequence variation 1.5 min at 72°C, with a final extension at 72°C for 10 min. PCR products were separated by vertical PAGE (poly- We detected 10 unique COI haplotypes among 68 Kru¨per’s acrylamide gel electrophoresis) at 65 W for 1.5 h (length Nuthatches (Figs. 1 and 2) on the basis of 10 variable sites 40 cm). After drying, the gel was exposed to an X-ray film (all 10 transitions). Three of these haplotypes had high (Hyperfilm-MP; Amersham) for 1–2 days and developed frequencies in different geographic areas. The most fre- (X-ray developer and fixer; Kodak). Some samples were quent haplotype I (n = 25) was found only in the southern 123 408 J Ornithol (2012) 153:405–411

Anatolia (BUK, ADR, and ALA). The second most fre- southern localities. The haplotype found on LES was the quent haplotype II (n = 19) dominated northwestern most common in the northwestern locality (KAZ; Fig. 1). Anatolia (KAZ; 13 of 15 birds), but a single individual Significant genetic differentiation in COI sequences was carrying that haplotype was found in BUK, ADR, and LES found among most of the sampling areas, except between and 3 more birds in KAR. The last high-frequency haplo- BUK and ALA (Table 1) and between combined Antalya type IX (n = 12) dominated in northeastern Anatolia localities (ADR ? BUK) and ALA (FST = 0.045, P = (KAR; 10 of 15 birds), but was also found in 2 birds from 0.159). KAZ (Fig. 2). The other seven haplotypes had low fre- quency and all but one (IV) differed by one mutation from Microsatellite analysis the high-frequency haplotypes (Fig. 2).

The pairwise FST values supported geographic struc- The mean number of alleles per locus varied from 4.1 to turing of the COI haplotypes. They were significantly 7.0 across populations (Table 2). Among eight microsat- greater than 0 for all comparisons involving northwestern ellite loci, we observed a total of 80 alleles with a mean of (KAZ) and northeastern (KAR) localities (P \ 0.05). 8.0 alleles per locus (Table 3). Two loci had the greatest

However, among the southern localities, only FST values number of alleles: SpuL5-22 had 19 alleles and Pca8 had involving ADR (n = 5) were significant whereas FST val- 13 alleles. Three localities had specific alleles: ALA had 6, ues for ALA (n = 13) and BUK (n = 19) were not BUK had 3, and KAR had 10 (Table 3). The mean (Table 1). Because the ADR locality is only comprised of expected and observed heterozygosities were 0.718 and five individuals, analyses involving the relative frequencies 0.610, respectively (Table 2). of this locality compared with the others should be inter- The linkage equilibrium was rejected for only 2 pairs of preted cautiously. alleles out of 140 comparisons in KAZ: between SpuL5-22 Mismatch distribution was unimodal and population and SpuL6-2 (P = 0.012) and between SpuL6-2 and Spu4- expansion was not rejected for southern populations (ADR, E7 (P = 0.014). After sequential Bonferroni correction, BUK, ALA). Mismatch distribution for KAZ and KAR however, exact tests for genotypic linkage disequilibrium were bimodal and demographic expansion was not sup- became non-significant. These test results indicate that all ported in either northern populations. our markers segregate independently. Majority of loci were The largest haplotype diversity was found in ADR consistent with HWE in all localities (Table 3). Deviation (0.900) followed by KAR and BUK (Table 2). There were from HWE was found in KAZ, KAR, and ALA (Table 2), eight COI haplotypes in southern localities (ALA, BUK indicating potential inbreeding, assortative mating or null and ADR) whereas only three haplotypes were found in alleles. Although they had heterozygote deficiency in one northern localities (KAR and KAZ). Two haplotypes were to three loci, none of the loci had heterozygote deficiency private for northern and seven haplotypes were specific for in all populations suggesting that selection did not signif- icantly affect any of our microsatellite loci (Table 3).

Pair-wise FST values based on microsatellites show significant genetic differentiation among most localities (Table 1). Non-significant values were obtained between neighboring south Anatolian localities: BUK–ADR and BUK–ALA. The Bayesian clustering analyses suggested that the individuals can be partitioned into two clusters (K = 2): the southern (BUK, ADR, ALA) and northern (KAZ, KAR). Although cluster assignment of ALA samples was split between southern and northern clusters 0.76:0.24. (Fig. 3).

Discussion

The network of COI haplotypes identified three largely geographically concordant haplogroups (Fig. 2). A few Fig. 2 Minimum spanning network of Kru¨per’s Nuthatch haplotypes. haplotypes (II, IX) shared by northeastern (KAZ) and The area of each circle is approximately proportional to the number of individuals sharing that haplotype. The number in parentheses equals northwestern (KAR) localities and between northwestern the number of individuals sharing that haplotype (KAZ) and southern (BUK, ADR) localities indicate 123 J Ornithol (2012) 153:405–411 409

Table 1 Pairwise FST values among the five Kru¨per’s Nuthatch Sitta krueperi localities ALA BUK ADR KAZ KAR

ALA 0.085 ns (441) 0.069* (463) 0.511*** (783) 0.593*** (440) BUK 0.038 ns 0.119* (81) 0.325*** (459) 0.584*** (422) ADR 0.112* 0.056 ns 0.325** (518) 0.496** (496) KAZ 0.075** 0.093*** 0.168*** 0.559*** (439) KAR 0.061** 0.068** 0.125*** 0.010 ns

FST values based on microsatellite data are presented below the diagonal and those based on COI sequence data are shown above the diagonal. Numbers in parentheses indicate the distance in km between the localities ns not significant * 0.01 \ P \ 0.05 ** 0.001 \ P \ 0.01 *** P \ 0.001

Table 2 Summary statistics (±SD) of genetic diversity in Kru¨per’s Nuthatch populations Sampling area Mitochondrial diversity Microsatellite diversity

-3 nShHd p (910 ) nA HE HO P

ALA 13 3 3 0.30 ± 0.16 0.76 ± 0.45 15 7.0 ± 3.1 0.77 ± 0.12 0.60 ± 0.27 0.000 BUK 19 3 4 0.51 ± 0.12 1.02 ± 0.29 9 6.1 ± 2.0 0.78 ± 0.19 0.71 ± 0.31 ns ADR 5 3 4 0.90 ± 0.16 2.31 ± 0.53 5 4.1 ± 1.6 0.70 ± 0.17 0.60 ± 0.36 ns KAZ 15 3 2 0.25 ± 0.13 1.23 ± 0.65 13 5.0 ± 1.8 0.62 ± 0.27 0.54 ± 0.27 0.004 KAR 15 4 3 0.53 ± 0.13 2.11 ± 0.63 13 6.5 ± 2.9 0.72 ± 0.18 0.60 ± 0.21 0.002

Haplotype diversity (Hd) and nucleotide diversity (p) expected heterozygosity (HE) and observed heterozygosity (HO) for each sampling area. P values for heterozygote deficiency n number of individuals, S polymorphic sites, h number of haplotypes, A mean number of allele per locus, ns not significant

Table 3 Microsatellite polymorphism Ase29 Ase48 Pca8 Pij23 Spu4-E7 SpuL5-22 SpuL6-2 Syl14

T 5 (2–5) 7 (4–6) 13 (5–10) 8 (3–7) 9 (3–7) 19 (6–13) 10 (4–8) 9 (2–7)

Np 3.2 ± 1.3 5.2 ± 0.8 7.6 ± 2.1 5.0 ± 2.0 4.8 ± 1.8 9.2 ± 2.9 6.2 ± 1.6 4.8 ± 1.8 BUKa –/HWE –/HWE –/HWE –/HWE 2/HWE –/HWE –/HWE 1/HD ADRa –/HWE –/HWE –/HWE –/HWE –/HWE –/HWE –/HWE –/HWE ALAa 1/HWE –/HWE 1/HWE –/HD –/HD 2/HWE 2/HWE –/HD KAZa –/HWE –/HWE –/HWE –/HWE –/HWE –/HWE –/HWE –/HD KARa –/HWE –/HWE 3/HWE –/HWE 1/HD 3/HWE 1/HWE 2/HWE

T total number of alleles observed within populations (range), Np mean number of alleles per population ±SD, HWE Hardy–Weinberg equi- librium, HD Heterozygote deficiency a Values represent the number of specific alleles in every population recently established geneflow (Figs. 1, 2). There is no results for northern populations (KAZ and KAR) were not evidence of geneflow between northeastern (KAR) and consistent with the recent demographic expansion despite southeastern (ALA) localities (Fig. 1). These results sug- the fact that only three haplotypes were found in both of gest that geneflow follows the species range and that these populations. Their bimodal mismatch distribution unsuitable habitats in central Anatolia represent a barrier to suggested that admixture of haplotypes from two previ- geneflow. ously isolated lineages might have occurred. The unimodal mismatch distribution and R2 test results The geographic distribution of the three haplogroups indicated that southern populations (ADR, BUK, and ALA) coincides with the location of the three potential Last experienced a recent demographic expansion. The R2 test Glacial Maximum (LGM) refugia within Anatolia. During

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geneflow between the three formerly isolated populations of the Kru¨per’s Nuthatch. Over the last 2,000 years, and most markedly in the last five centuries, anthropogenic impacts have resulted in a significant reduction of forested areas in Anatolia (Colak and Rotherham 2006). Turkey has 21.18 million ha of forest, covering 26% of the land surface of the country (Ministry of Environment and Forestry Turkey: http://www.ogm.gov.tr). The forest area has declined to 26% as a result of over-grazing, over-cutting, fires, spread of agricultural lands, wars, etc., and steppe has increased to 24% in the last 4,000 years (Colak and Rotherham 2006). The continuing fragmentation of breeding habitats may increase the extent of this isolation in the future. Further genetic studies of populations at the edges of the Kru¨per’s Fig. 3 Results of the Bayesian clustering analysis of microsatellite Nuthatch range in the Caucasus Mountains and on Lesvos alleles using an admixture model Island may provide further insights into the species’ phy- logeographic history and population connectivity. Our results are of considerable importance for effective LGM, Taurus Mountains in the south and north Anatolian conservation strategy of the near threatened Kru¨per’s Mountains in the north were glaciated (Hughes et al. 2006) Nuthatch. The genetic differentiation found among popu- and formed barriers for the geneflow between isolated lations points to the existence of three independent man- populations. Kru¨per’s Nuthatch might have been isolated in agement units in Turkey. Although currently there is some three different low elevation regions: southern, north- geneflow among these populations, detrimental anthropo- western, and northeastern Turkey/Georgia. At the end of genic effects on suitable habitat (e.g., logging, deforesta- the last glacial period, north Anatolian Mountain glaciers tion for agricultural purposes) might result in could have melted first due to their lower elevation com- fragmentation of the habitat and sever the geneflow among pared with the elevation of the south Anatolian Taurus populations. Such an isolation might cause inbreeding and Mountains (Hughes et al. 2006), allowing earlier secondary decline of the Kru¨per’s Nuthatch populations. geneflow between northern refugial populations. Unfortu- nately, we do not have a reliable mutation rate calibration Acknowledgments This study was partly supported by TUBITAK to use a molecular clock for testing this glacial refugium and DFG (WI 719/32-1, AOBJ: 565239) cooperation. We thank Alice Cibois and Eric Pasquet for valuable comments, Muhammad Arshad hypothesis. and Hedi Sauer-Gu¨rth for technical assistance in the laboratory and The number of microsatellite alleles per locus varied Metin Balc¸ay kindly helped in the field work. from 5 to 19 with a mean of 8 across all populations. Only northeastern (KAR) and southern populations (ALA and BUK) had private alleles (10, 6, and 3, respectively) References whereas the northwestern population (KAZ) had none. This distribution of unique alleles supports the pattern of dis- Albayrak T, Erdogan A (2005a) Breeding ecology of Krueper’s tribution of COI haplotypes. nuthatch (Sitta krueperi) near Antalya, Turkey. Isr J Zool Microsatellite pair-wise FST values indicate the presence 51:309–314 of significant genetic differences between northern (KAZ, Albayrak T, Erdogan A (2005b) Observation on some behaviours of KAR) and southern localities (ALA, BUK, ADR). Our Krueper’s nuthatch (Sitta krueperi), a little-known West Pale- arctic bird. Turk J Zool 29:177–181 Bayesian clustering analyses also partitioned individuals Albayrak T, Bairlein F, Erdog˘an A (2010) Habitat parameters and into the same two clusters: the northern (KAZ, KAR) and breeding density of Kru¨per’s Nuthatch Sitta krueperi in south southern (BUK, ADR, ALA). The lack of differentiation eastern Turkey. Pol J Ecol 58:545–552 between KAZ and KAR appears to contradict the results of Bandelt HJ, Forster P, Rohl A (1999) Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol 16:37–48 our COI analysis. We believe, however, the secondary Colak AH, Rotherham ID (2006) A review of the forest vegetation of geneflow between these two localities may be responsible Turkey: its status past and present and its future conservation. for the admixture of microsatellite alleles. Biol Environ 106:343–354 Therefore, our results support the hypothesis which Cramps S, Perrins CM (1993) The birds of western palearctic. Oxford University Press, Oxford suggests the presence of three glacial refugia in Turkey Dawson DA, Hanotte O, Greig C, Stewart IRK, Burke T (2000) during the LGM and recently established secondary Polymorphic microsatellites in the blue tit Parus caeruleus and

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