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Biochem Genet (2010) 48:125–140 DOI 10.1007/s10528-009-9305-8

Genetic Differentiation Among Peripheral Populations of Bombina bombina from Thrace and Anatolia: An Allozyme Analysis Nursen Alpagut-Keskin • Ethem I. Cevik Huseyin Arikan



Received: 18 January 2008 / Accepted: 13 August 2009 / Published online: 22 November 2009 Ó Springer Science+Business Media, LLC 2009

Abstract Genetic structures of Bombina bombina populations, located as peripheral isolates in Turkish Thrace and northwestern Anatolia, were analyzed by polyacrylamide gel electrophoresis using 20 allozyme loci, to investigate the populations’ current genetic variation and possible colonization history. Significant genetic variability was detected in most of the loci and all populations. Allozyme pairwise FST matrices and distribution of allele frequencies indicate their very close genetic relationships and relatively recent formation. Mean genetic distance values between Thracian and Anatolian populations indicate a Middle or Upper Pleistocene lineage separation before the formation of the Bosporus as an isolating geographic barrier. All the samples show substantial heterozygosity excess, and there was statistically significant evidence of recent bottlenecks. The extent and patterns of genetic divergence indicate that the Anatolian and Thracian populations have probably experienced bottlenecks, and incipient speciation may have occurred in Anatolian populations of B. bombina. Keywords Fire-bellied toad  Bombina bombina  Peripheral isolates  Allozyme variations  Founder effects  Bottleneck  Anatolia  Thrace

Introduction The small, isolated populations on the periphery of a species’ distribution range are considered to have an important role in the process of speciation and divergence (Avise 1994; Gavrilets et al. 2000). Mayr (1954, 1963, 1982) proposed that when a new population was founded outside the continuous species range by a small founder group, rapid genetic changes could be achieved in a population isolate by N. Alpagut-Keskin (&)  E. I. Cevik  H. Arikan Biology Department, Ege University, 35100 Bornova, Izmir, Turkey e-mail: [email protected]

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extreme selective pressures induced by substantial fluctuation in population size. Although the establishment of genetic diversity in such populations is one of the controversial aspects of speciation research, different founder-effect concepts are credited for some of the chief agents in attaining the degree of reproductive isolation necessary for speciation (Carson 1968, 1982, 1985; Carson and Templeton 1984; Gavrilets 2000; Gavrilets et al. 2000; King 1995; Mayr 1954, 1982; Nei 2005; Templeton 1980, 1982; Wright 1932, 1982a, b). These peripheral populations are typically viewed as incipient species that may continue to diverge (Mayr 1963; Mayr and Ashlock 1991) and represent an opportunity to investigate current evolutionary processes. These populations are also considered to have a high value for conservation (Lesica and Allendorf 1995). The fire-bellied toad, Bombina bombina L., has a wide distribution in eastern, northern, and central Europe. It is also found in northwest Anatolia. B. bombina is the only representative of the Bombinatoridae living in Turkey, where it is confined to small areas in Turkish Thrace and northwestern Anatolia (Fig. 1). Both Thracian and Anatolian populations of B. bombina are located as peripheral isolates (sensu Mayr 1963) on the southeast of the southern margin of the species’ range. Anatolian populations of B. bombina, which are isolated geographically by the Sea of ¨ zeti and Marmara, have been described as a subspecies, B. bombina arifiyensis O ¨ Yılmaz 1987 (Baran and Atatu¨r 1998; Ozeti and Yılmaz 1987; Yılmaz 1984, 1986). Nevertheless, the magnitude and pattern of genetic differentiation of Anatolian B. bombina from the European and Thracian populations have not been shown. Inferences on Anatolian and Thracian fire-bellied toads are currently based ¨ zeti and Yılmaz 1987; Yılmaz 1984, 1986), but primarily on morphological data (O no cytogenetic or genetic data are available. The mtDNA data (Szymura et al. 2000) obtained from only four toads from Sakarya are too scarce for a detailed conclusion. Furthermore, the origins of the geographically isolated Anatolian and the Thracian populations have not been discussed and remain obscure. It was mentioned only briefly that the fire-bellied toad penetrated into the lowlands of Sakarya during the Wurm period (Arntzen 1978; Hofman et al. 2007). Although a low level of allopatric differentiation was found among European populations of B. bombina, it was reported that European B. bombina were separated into two groups (Szymura et al. 2000; Vo¨ro¨s et al. 2006). The southern group occupies an area south of the Sudety and Carpathian Mountains, and the northern group consists of populations to the north and east of them (Szymura et al. 2000). The sample from Anatolia, although geographically distant, was found to be more similar to the northern group than the southern B. bombina (Szymura et al. 2000). Despite their parapatric distribution, differences in habitat preference, and a large number of diagnostic traits that differentiate the two species, it is well-known that the European B. bombina hybridize with the related species B. variegata in Central Europe (Szymura 1993). Extensive studies on European Bombina have generally focused on genetic analysis of hybrid zones, rangewide mtDNA divergence, and their phylogenetic relationships (Hofman and Szymura 2007; Hofman et al. 2007; Pabijan et al. 2008; Szymura et al. 2000; Szymura and Barton 1986, 1991; Vo¨ro¨s et al. 2006). A previous study (Szymura et al. 2000), which also includes an

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Fig. 1 Pie graphs of eight typical loci in B. bombina, showing geographic changes in allele frequencies. a Ldh-2, Gd-1, Aat-2, and G6pd-1 loci. b Sod-2, Ada-1, Ada-2, and Pgm-2 loci. Large circles indicate populations with male and female samples; small circles are only males (sample number over 15). Sampling localities: 1 Do¨s¸ emebataklıg˘ı, 2 Durusu, and 3 Bu¨yu¨kdo¨llu¨k

Anatolian sample of this species, attempted to analyze mtDNA variation in firebellied toads but did not include the Thracian populations. Therefore, elucidation of the details of geographic variation in Thracian and Anatolian populations of B. bombina is also required to understand the biogeographic history of this species. In the present study, we used information from 20 allozyme loci to test the hypothesis that geographically isolated Anatolian populations of fire-bellied toad

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show genetic differentiation when compared with Thracian populations located on the other side of the Bosporus. Our main objective was to investigate the current genetic variation among Thracian and Anatolian populations of B. bombina on the basis of allozyme patterns of relatively larger sample sizes and to discuss the historical biogeography of the species in those regions.

Materials and Methods The study was conducted in Turkish Thrace and northwestern Anatolia. The firebellied toads, B. bombina (Linnaeus 1761), were sampled over a 2-year period (2004–2005) and captured as breeding adults in the spring. We analyzed 53 individuals (33 males, 20 females) from Do¨semebataklıg˘ı (Sakarya), Durusu (Istanbul), and Bu¨yu¨kdo¨llu¨k (Edirne) at 4–10 m above sea level (Fig. 1). The specimens were brought to the laboratories alive, and the tissue samples were taken within 3 days of their capture. Allozyme Assay Liver, heart, gonads, and skeletal muscle samples were used for enzyme electrophoresis. Tissue samples were homogenized in cold extraction buffer (100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM potassium HEPES, 50 mM sucrose, pH 7.7), centrifuged at 14,000 rpm, and supernatants were stored at -22°C until use. Native gels were prepared with 7% acrylamide–0.5% agarose. The electrophoretic separation was performed at a constant 30 mA and 4°C for 4–6 h using an SE Ruby 600 apparatus (Amersham Biosciences, USA) with gels of 18 9 16 9 0.15 cm. The enzymes were visualized by standard procedures described in Manchenko (1994), with slight modifications, and 20 presumptive loci encoding 12 allozymes were scored. The enzymes and the buffer systems are presented in Appendix 1. Gels were photographed, and the polymorphic loci count and frequency were submitted to appropriate statistical treatment. Genetic interpretations of allozyme data were made following Buth (1990). For individual genotypes, all the electromorphs from different tissues were scored together. The nomenclature of enzymes and EC numbers mainly followed Murphy et al. (1996). Statistical Analysis Genetic variation at allozyme loci was evaluated in terms of expected heterozygosity (HE), proportion of polymorphic loci (95% polymorphism criterion P), allelic richness (A), and gene diversity (GD). Allelic richness and gene diversity were calculated using Fstat 2.9.3 (Goudet 2001). Allele frequency and expected (HE) and observed (HO) heterozygosity were estimated with Genepop 3.4 (Raymond and Rousset 1995). This program was also used to test for Hardy–Weinberg equilibrium, linkage disequilibrium, and heterogeneity in allele frequency distribution for all loci and all pairwise comparisons using the Markov chain method with 1,000 iterations.

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Levels of population divergence at allozyme loci were investigated by computing FST (Weir and Cockerham 1984) with the program Fstat 2.9.3 (Goudet 2001). The significance of FST for all loci and pairwise population comparisons was assessed by permutating the values 1,000 times. When doing multiple simultaneous comparisons, we used the sequential Bonferroni procedure (Rice 1989), with a = 0.05, to adjust the statistical significance levels. The heterozygosity analysis was carried out using Bottleneck software version 1.2.02 (Piry et al. 1999) to assess whether the Thracian and Anatolian fire-bellied toads fit the criterion for a recent bottleneck (Cornuet and Luikart 1996; Luikart et al. 1999). This program estimates the expected distribution of the heterozygosity from the observed number of alleles at each polymorphic locus and populations assuming mutation-drift equilibrium. The probability of significant heterozygosity excess was calculated using the Wilcoxon sign-rank test. Computations were based on both the infinite allele model (Kimura and Crow 1964) and the stepwise mutation model (Kimura and Ohta 1978). A qualitative graphical method using methods described by Luikart and Cornuet (1998) was also utilized to determine whether there was a mode-shift distortion in allele frequencies. To estimate the degree of genetic differentiation across the populations, we calculated Nei’s (1972) genetic distance. The distance matrix was clustered by the UPGMA method (Sneath and Sokal 1973) using the GDA 1.1. Computer Program (Lewis and Zaykin 2002).

Results Genetic Variability, Hardy–Weinberg Equilibrium, and Linkage Disequilibrium For the 12 enzyme systems assayed, 20 gene loci were recorded, encoding 35 putative alleles. Of the 20 loci scored, 15 were polymorphic, and five loci (Mdhp, Pgd, Glyd, Atpase-1, and Atpase-2) were fixed in all the samples examined. These loci provided no information about population heterogeneity, but they were scored for computing average heterozygosity and genetic distance. All samples showed high genetic variability for most of the allozyme loci. Percentages of polymorphic loci were 0.75 in all samples (Appendix 2), mean expected heterozygosity (HE) varied from 0.302 to 0.321, and mean observed heterozygosity (HO) ranged among samples from 0.356 to 0.400 (Table 1). The amount of genetic variability was not substantially different among samples and at all polymorphic loci; in all populations, two alleles were observed. Allele frequencies for allozyme loci are shown in Appendix 2. Ldh-2, Gd-1, Aat-2, G6pd-1, Sod-2, Ada-1, Ada-2, and Pgm-2 loci demonstrated geographic changes in allele frequency (Fig. 1). No significant deviations from Hardy–Weinberg equilibrium were observed for individual populations (p [ 0.001) or loci (p [ 0.001) after adjusting for multiple comparisons (Table 1), but FIS values were lower than zero in most of the loci and

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Table 1 Genetic variation at 15 polymorphic allozyme loci in peripheral Anatolian and Thracian B. bombina populations Locus

Population, N Anatolian Do¨s¸ emebataklıg˘ı, 18

Thracian Durusu, 15

HE

HO

GD

dHWE

HE

Adh-1

0.424

0.611

0.431

ns

0.358 0.467 0.367 ns

0.499 0.450 0.513 ns

Adh-2

0.444

0.667

0.451

ns

0.444 0.533 0.457 ns

0.439 0.650 0.445 ns

Ldh-1

0.424

0.611

0.431

ns

0.231 0.267 0.238 ns

0.320 0.400 0.326 ns

Ldh-2

0.375

0.500

0.382

ns

0.124 0.133 0.129 ns

0.320 0.400 0.326 ns

G-6-Pd 0.494

0.778

0.500

ns

0.464 0.733 0.471 ns

0.469 0.350 0.484 ns

Sod-1

0.444

0.667

0.451

ns

0.500 0.733 0.51

ns

0.420 0.600 0.426 ns

Sod-2

0.375

0.167

0.392

ns

0.500 0.467 0.519 ns

0.499 0.150 0.521 ns

Ada-1

0.401

0.444

0.412

ns

0.498 0.933 0.500 ns

0.469 0.450 0.482 ns

Ada-2

0.375

0.389

0.386

ns

0.480 0.400 0.500 ns

0.499 0.650 0.508 ns

Aat-1

0.486

0.278

0.507

ns

0.491 0.467 0.510 ns

0.495 0.600 0.505 ns

Aat-2

0.105

0.111

0.108

ns

0.480 0.667 0.49

ns

0.500 0.400 0.516 ns

Pgm-1

0.500

0.556

0.513

ns

0.491 0.600 0.505 ns

0.455 0.500 0.466 ns

Pgm-2

0.198

0.222

0.203

ns

0.464 0.733 0.471 ns

0.095 0.100 0.097 ns

Gd-1

0.494

0.667

0.503

ns

0.231 0.267 0.238 ns

0.489 0.850 0.492 ns

Gd-2

0.500

0.444

0.516

ns

0.500 0.600 0.514 ns

0.455 0.700 0.461 ns

Mean

0.302

0.356

0.309

ns

0.313 0.400 0.320 ns

0.321 0.363 0.328 ns

SD

0.203

0.274

0.208

0.213 0.299 0.218

0.212 0.275 0.217

HO

GD

Thracian Bu¨yu¨kdo¨llu¨k, 20 dHWE HE

HO

GD

dHWE

N Number of genotypes assayed for each population at each locus HE Expected heterozygosity. HO Observed heterozygosity. GD Gene diversity. dHWE Deviation from Hardy–Weinberg expectation (ns no significant deviation after Bonferroni correction)

all of the samples. Although these negative FIS values were not significant (p [ 0.01), the mean of FIS is -0.159 (p \ 0.01), indicating a general excess of heterozygote (Table 2). Fisher’s exact test for linkage disequilibrium revealed no significant locus pair/population comparisons at the 1% confidence level. The G-test gave us a compelling reason to reject the hypothesis that genotypic distribution is identical across the populations (p \ 0.001). Evidence for Recent Population Bottlenecks We analyzed the allele frequencies using the program Bottleneck (Cornuet and Luikart 1996; Piry et al. 1999), which computes the expected distribution of the heterozygosity from the observed number of alleles at each polymorphic locus and populations and compares it to the observed heterozygosity under the assumption of mutation-drift equilibrium. All the samples showed a very significant heterozygosity excess at most of the loci for the infinite allele and stepwise mutation models (p \ 0.001). The assumption that all 15 polymorphic loci fit the two models of

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Table 2 Fixation index for polymorphic loci in B. bombina populations Locus

Overall FST

Fixation index (FIS) Anatolian Do¨s¸ emebataklıg˘ı

Thracian Durusu

Thracian Bu¨yu¨kdo¨llu¨k

Total

Adh-1

0.044

-0.417

0.123

-0.273

-0.147

Adh-2

0.000

-0.478

-0.462

-0.167

-0.382

Ldh-1

0.022

-0.417

-0.226

-0.120

-0.287

Ldh-2

0.034

-0.308

-0.226

-0.037

-0.238

G-6-Pd

0.025

-0.556

0.277

-0.556

-0.242

Sod-1

0.030

-0.478

-0.407

-0.439

-0.441

Sod-2

0.054

0.575

0.712

0.101

0.485

Ada-1

0.043

-0.079

0.066

-0.867

-0.263 -0.057

Ada-2

0.093

-0.008

-0.280

0.200

Aat-1

0.000

0.452

-0.188

0.084

0.106

Aat-2

0.219

-0.030

0.224

-0.359

-0.019

Pgm-1

0.001

-0.083

-0.073

-0.189

-0.110

Pgm-2

0.168

-0.097

-0.027

-0.556

-0.342

Gd-1

0.158

-0.325

-0.727

-0.120

-0.468

Gd-2

0.010

0.139

-0.520

-0.167

-0.182

Mean

0.057

-0.149ns

-0.104ns

-0.246ns

-0.159**

ns No significant deviation from Hardy–Weinberg expectation after Bonferroni correction ** p \ 0.001, when all populations and all loci analyzed together

mutation equilibrium could be rejected for all three samples. Because the genetic diversity excess (He [ Heq) at 15 polymorphic loci in Thracian and Anatolian B. bombina populations was similar under both mutation models, only the infinite allele procedure is presented in Table 3. Likewise the mode-shift indicator of allele frequency distribution was not a normal L-shaped distribution for all samples as expected under mutation-drift equilibrium. Therefore, significant statistical evidence of recent bottlenecks in all three populations analyzed was proved. Genetic Divergence Among Populations Samples of B. bombina had low FST with a mean of 0.057 ± 0.018 and range of 0.00–0.219 (Table 2). Although the mean FST value showed a moderate level of differentiation, significant values were obtained for four of the loci examined, and significant differences were detected over all loci for FST (p \ 0.01). Significant heterogeneity was also found in allelic distribution for the Aat-2, Pgm-2, Gd-1, and Ada-2 loci (Fisher’s exact test, p \ 0.01). The large differences in allele frequency distribution resulted in a high level of population differentiation: all FST population pairwise comparisons were significant after Bonferroni correction (p \ 0.01; Table 4). Allozyme pairwise FST matrices indicated some geographic signals: Thracian Durusu and Bu¨yu¨kdo¨llu¨k samples showed a very low FST value (0.0358),

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Table 3 Infinite allele model analysis for genetic diversity excessa at 15 polymorphic loci in Thracian and Anatolian B. bombina populations Locus

Anatolian Do¨s¸ emebataklıg˘ı (n = 36) p = 0.00008

Thracian Bu¨yu¨kdo¨llu¨k (n = 40) p = 0.00008

Thracian Durusu (n = 30) p = 0.00015

He

Heq

p

He

Heq

p

He

Heq

p

Adh-1

0.437

0.236

0.208

0.370

0.249

0.3100

0.512

0.233

0.0250

Adh-2

0.457

0.231

0.173

0.460

0.246

0.1790

0.450

0.230

0.1860

Ldh-1

0.437

0.232

0.205

0.239

0.251

0.4970

0.328

0.228

0.3310

Ldh-2

0.386

0.231

0.254

0.129

0.253

0.4070

0.328

0.224

0.3070

G6Pd

0.508

0.236

0.061

0.480

0.239

0.1370

0.481

0.225

0.1160

Sod-1

0.457

0.236

0.170

0.517

0.247

0.0100

0.431

0.228

0.2020

Sod-2

0.386

0.235

0.279

0.517

0.244

0.0140

0.512

0.227

0.0380

Ada-1

0.413

0.235

0.234

0.515

0.240

0.0450

0.481

0.223

0.1080

Ada-2

0.386

0.243

0.297

0.497

0.260

0.1310

0.512

0.229

0.0390

Aat-1

0.500

0.231

0.086

0.508

0.242

0.0890

0.508

0.238

0.0570

Aat-2

0.108

0.239

0.396

0.497

0.251

0.1260

0.513

0.233

0.0190

Pgm-1

0.514

0.239

0.010

0.508

0.240

0.0640

0.467

0.219

0.1340

Pgm-2

0.203

0.228

0.505

0.480

0.240

0.1590

0.097

0.221

0.4070

Gd-1

0.508

0.227

0.048

0.239

0.235

0.4550

0.501

0.235

0.0840

Gd-2

0.514

0.238

0.015

0.517

0.241

0.0150

0.467

0.222

0.1400

a

He [ Heq, assuming that populations were in mutation-drift equilibrium. Heq (expected equilibrium heterozygosity) and He (gene diversity) are computed according to Nei (1987) n Number of haploid genome. p Probability, one tail for H excess. p Probability He [ Heq

Table 4 FST values between pairs of populations and pairwise Nei’s (1972) original genetic distance among B. bombina populations Sample pair

Pairwise FST

Nei’s D

Do¨s¸ emebataklıg˘ı/Durusu Do¨s¸ emebataklıg˘ı/Bu¨yu¨kdo¨llu¨k

0.0863**

0.0556

0.0513**

0.0364

Bu¨yu¨kdo¨llu¨k/Durusu

0.0358**

0.0298

Significance of FST after sequential Bonferroni correction is denoted as follows: ** p \ 0.01

pairwise, whereas the highest genetic differentiation value was obtained between Anatolian Do¨s¸ emebataklıg˘ı and Thracian Durusu samples (Table 4). Nei’s D ranged from 0.0298 to 0.0556, with the largest D value between Thracian Durusu and Anatolian Do¨s¸ emebataklıg˘ı samples. Thracian populations showed the lowest genetic distance value (DNei = 0.0298), and these two samples were well grouped together in the UPGMA phenogram based on Nei’s distance (Fig. 2). The Anatolian population of B. bombina was clearly separated from the Thracian populations. UPGMA of DNei confirmed the divergence between the Anatolian and Thracian populations.

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Fig. 2 UPGMA phenogram constructed from Nei’s (1972) original genetic distance. Population 1 Do¨s¸ emebataklıg˘ı, 2 Durusu, and 3 Bu¨yu¨kdo¨llu¨k

Discussion Our samples verified a high degree of genetic variation, contrary to the conventional expectation of small isolated populations. Although the degree of genetic variability was similar among samples, variations in the Ldh-2, Gd-1, Aat-2 and G6pd-1, Sod-2, Ada-1, Ada-2, and Pgm-2 loci differentiated the Anatolian and Thracian populations. In the Anatolian sample, the allele frequency b was dominant at the Gd-1 locus, and a was dominant at G6pd-1; whereas in Thracian populations, a was dominant at the Gd-1 locus and b was dominant at G6pd-1 (Fig. 1). Furthermore, the Aat-2 locus (b allele) in the Anatolian Do¨s¸ emebataklıg˘ı sample and the Pgm-2 locus in the Thracian Bu¨yu¨kdo¨llu¨k sample (a allele) were nearly fixed for one allele, and heterozygosity was reduced. The Aat-2 and Sod-2 loci also showed geographically clinal changes in allele frequencies (Fig. 1) and have the highest FST values (Table 2). Some of the loci showed differences from European B. bombina; it was noted that Ldh-1, Aat-1, and G6pd were fixed for one allele (Hofman and Szymura 2000), but our samples were clearly polymorphic for these loci. Negative FIS values usually indicate an excess of heterozygosity, consistent with outbreeding. However, the presence of a physical salt barrier between the Anatolian and Thracian populations, combined with the genic differentiation obtained in the Aat-2, Pgm-2, Gd-1, and Ada-2 loci and significant pairwise FST values, implies that gene flow between populations is unlikely. Negative FIS values are also expected for small populations that are subdivided into completely isolated units (Cockerham 1973). It was noted that inbreeding subdivisions or isolates, heterozygote advantage, differential fertility and unequal male and female gametic gene frequencies, disassortative mating, and mixtures of subdivisions are all important factors producing negative FIS values (Balloux 2004; Cheeser 1991; Cockerham 1973; Nei 1973; Pudovkin et al. 1996). On the other hand, the Aat-2 locus showed a nearly fixed allele in Anatolia and two common alleles in Thrace, producing a locus FST of 0.219 (Table 2). The FST value of Aat-2 is much higher than that found for other allozyme loci, suggesting that some selection events could be operating at the Aat-2 locus in the Anatolian population. Our results on allozyme variation between populations from Thrace and Anatolia indicated their very close genetic relationship, with Nei’s genetic distance values ranging from 0.0298 to 0.0556. Although Nei’s distance may not necessarily provide precise estimates of divergence time, these extremely low levels of genetic differentiation reflect their relatively recent formation. Assuming a divergence rate

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for fire-bellied toad allozyme loci of either 5 9 106 (Nei 1975) and 2.1–11.5 9 106 (Fromhage et al. 2004) or 1.4 9 106 years (molecular clock calibration for the closest relative, Alytes; Garcia-Paris and Jockusch 1999), the mean genetic distance values between Thracian and Anatolian populations indicate a Middle or Upper Pleistocene lineage separation. This divergence time suggests that these two lineages were separated from each other before the formation of the Bosporus as an isolating geographic barrier. Therefore, it is possible to assume that Thracian and Anatolian populations had different ancestral origins and that their founders represent different fractions of the variability of the species. The peripheral and isolated locations of Anatolian and Thracian B. bombina populations and the time of divergence estimated here also suggest that these populations were most likely founded from a northern ancestral population (sensu Szymura et al. 2000). This point of view also explains why Anatolian B. bombina was more similar to the northern group, despite their geographic distance. Over the past 3 million years, the Black Sea alternated from a completely isolated interior lake to a brackish marine environment several times (Bahr 2005; Major et al. 2006; Ryan et al. 1997). It has been estimated that the Black Sea was lacustrine for 90% of its Pleistocene history (Major et al. 2006; Ross 1978). During these lacustrine periods, a connection was repeatedly reformed between Europe and Anatolia. It is possible that river systems of the emerged shelf zone and also the Bosporus allowed the dispersion of B. bombina to Anatolia (Fig. 3). At the beginning of the last deglaciation period, the reconnection of the Black Sea to the global oceans (*9.4 ka BP cal) via the Bosporus and the Dardanelles straits separated the Anatolian B. bombina from their European relatives. On the other hand, it was suggested that European B. bombina was probably isolated in the Black Sea region during the Last Glacial Maximum and subsequently recolonized Central Europe during interglacial periods (Arntzen 1978; Hewitt 2004; Spolsky et al. 2006; Szymura 1993; Szymura et al. 2000). It is therefore likely that the present European, Thracian, and Anatolian B. bombina groups have different colonization histories; some specific geographic signals should be reflected either in the allozyme frequencies or in their chromosomes. A possible model for invasion, colonization, geographic isolation, and differentiation processes of Thracian and Anatolian B. bombina populations is presented in Fig. 3. There is some evidence, especially from the number of alleles at polymorphic loci, that the populations experienced bottlenecks that accompanied migration to Anatolia during the Pleistocene glacial range expansion of this species and subsequent isolation. As predicted by Nei et al. (1975), bottlenecks have a stronger and more immediate effect on allelic diversity than heterozygosity. Whereas heterozygosity is still high, the number of alleles of these peripheral populations is reduced, most likely caused in large part by the loss of rare alleles during recent changes in effective population size. The loss of alleles as the species expanded following the last glaciations was also reported for northern populations of B. bombina (Szymura et al. 2000). It was observed that most of the loci examined in the present study showed a substantial heterozygosity excess for all populations (one tail: p \ 0.001; Table 3), and allele frequencies were not a normal L-shaped distribution for all samples as expected under mutation-drift equilibrium. A significant excess of heterozygosity can be taken as a strong indicator of recent

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Fig. 3 A model for invasion, colonization, geographic isolation, and differentiation processes of Thracian and Anatolian B. bombina populations. a and b dispersion routes of European B. bombina to Thrace and Anatolia (*230 to 64 ka BP cal). Dotted line margins of the founding populations, geographic isolation (*10 ka BP cal). Arrows bottlenecking

change in effective population size but also of a selective advantage of heterozygotes (Maruyama and Fuerst 1985; Allendorf 1986; Lande and Barrowclaugh 1987; Nei et al. 1975; Leberg 1992; Cornuet and Luikart 1996; Nei 2005). On the contrary, Aat-2 and Pgm-2 loci in Anatolia and Ldh-1, Ldh-2, and Pgm-2 loci in Thracian populations showed a deficit in heterozygotes, probably reflecting an earlier bottleneck event. Therefore, it is possible to assume that these peripheral populations experienced two bottlenecks; one was induced possibly during the range expansion during the Middle or Late Pleistocene, and the second was related to recent habitat reductions due to human actions after geographic isolation. Genetic variation revealed by FST indicated moderate but significant levels of genetic variability among Anatolian and Thracian populations (mean FST = 0.057, p \ 0.01). This structure is probably due not only to historical isolation in geographically disconnected environments but also to a combination of subsequent selection, genetic drift, and present environmental pressures, which may account for the pattern of structure among peripheral populations of this species. These small, isolated peripheral populations of B. bombina also have a special conservation value. We propose that these populations must be conserved separately by relevant landscape restoration projects at either a national or international scale because they are genetically different and loss of any one population would lead to a dramatic loss of genetic variation.

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In conclusion, genetic variability at the allozyme level differed between Thracian and Anatolian B. bombina. In advance of more definitive testing of the major forces underlying this genetic variation, we consider that incipient speciation is occurring in Anatolian populations of B. bombina. Analysis of genetic structures of peripheral and central populations of fire-bellied toads by means of DNA polymorphism, especially sequence analysis of markers near or within functionally important genes involved in reproductive isolation (fast evolving markers such as gamete recognition proteins and skin secretion peptides) will provide more effective data sets to establish a more detailed genetic model. Furthermore, a detailed cytogenetic analysis of the present lineages should be conducted. Acknowledgments We wish to thank Assoc. Prof. Dr. U. Kaya, Dr. U. C. Eris¸ mis¸ , Dr. F. Turgay, and Dr. F. Sayım for valuable help with collection of the specimens. We are further sincerely grateful to Prof. Dr. A. Kence, Prof. Dr. B. Falakalı Mutaf, and Prof. Dr. M. K. Atatu¨r for constructive comments on the manuscript. We should also like to thank Prof. Dr. D. Sperlich and three anonymous reviewers for critical suggestions and comments on earlier versions of this paper. We are also grateful to Dr. B. Keskin for reproduction of distribution maps. This study was supported by the Scientific and Technological Research Council of Turkey (Tubitak Project No. TBAG 103T073).

Appendix

Appendix 1 Enzymes and buffer systems used in the analysis of genetic variation among B. bombina populations Enzyme

EC No.

Locus

Tissue

Buffer system

Alcohol dehydrogenase

1.1.1.1

Adh-1

L, M, H, G

TG, 8.5

Alcohol dehydrogenase

1.1.1.1

Adh-2

L, M, H, G

TG, 8.5

Lactate dehydrogenase

1.1.1.27

Ldh-1

L, M, H, G

TG, 9.5

Lactate dehydrogenase

1.1.1.27

Ldh-2

L, M, H, G

TG, 9.5

Malic enzyme

1.1.1.40

Mdhp

L, M, H, G

TG, 8.5

Phosphogluconate dehydrogenase

1.1.1.44

Pgd

L, M, H, G

TBE, 8.0

Glucose dehydrogenase

1.1.1.47

Gd-1

L, M, H, G

TG, 8.3

Glucose dehydrogenase

1.1.1.47

Gd-2

L, M, H, G

TG, 8.3

Glucose-6-phosphate dehydrogenase

1.1.1.49

G-6-Pd

L, M, H, G

TG, 8.3

Glycerol dehydrogenase

1.1.1.72

Glyd

L, M, H, G

TG, 8.5

Superoxide dismutase

1.15.1.1

Sod-1

L, M, H, G

TG, 8.3

Superoxide dismutase

1.15.1.1

Sod-2

L, M, H, G

TG, 8.3

Aspartate aminotransferase

2.6.1.1

Aat-1

L, M, H, G

TG, 8.3

Aspartate aminotransferase

2.6.1.1

Aat-2

L, M, H, G

TG, 8.3

Adenosine deaminase

3.5.4.4

Ada-1

L, M, H, G

TG, 8.3

Adenosine deaminase

3.5.4.4

Ada-2

L, M, H, G

TG, 8.3

Adenosine triphosphatase

3.6.1.3

Atpase-1

L, M, H, G

TC, 7.0

Adenosine triphosphatase

3.6.1.3

Atpase-2

L, M, H, G

TC, 7.0

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137

Appendix 1 continued Enzyme

EC No.

Locus

Tissue

Buffer system

Phosphoglucomutase

5.4.2.2

Pgm-1

L, M, H, G

TG, 8.3

Phosphoglucomutase

5.4.2.2

Pgm-2

L, M, H, G

TG, 8.3

L Liver; M Skeletal muscle; H Heart muscle; G Gonads TG Tris–Glycine, pH 8.3, 8.5, 9.5; TBE Tris–Borate–EDTA, pH 8.0; TC Tris–Citrate, pH 7.0

Appendix 2 Allele frequencies at 20 allozyme loci in B. bombina populations

Locus

Allele

Allele frequency Do¨s¸ emebataklıg˘ı

Durusu

Bu¨yu¨kdo¨llu¨k

a

0.306

0.233

0.475

b

0.694

0.767

0.525

a

0.667

0.667

0.675

b

0.333

0.333

0.325

a

0.306

0.133

0.200

b

0.694

0.867

0.800

a

0.250

0.067

0.200

b

0.750

0.933

0.800

Mdhp

a

1.000

1.000

1.000

Pgd-

a

1.000

1.000

1.000

Gd-1

a

0.444

0.867

0.575

b

0.556

0.133

0.425

a

0.500

0.500

0.350

b

0.500

0.500

0.650

a

0.556

0.367

0.375

b

0.444

0.633

0.625

Glyd

a

1.000

1.000

1.000

Sod-1

a

0.667

0.500

0.700

b

0.333

0.500

0.300

a

0.750

0.500

0.475

b

0.250

0.500

0.525

a

0.583

0.567

0.450

b

0.417

0.433

0.550

a

0.056

0.400

0.500

b

0.944

0.600

0.500

a

0.278

0.533

0.375

b

0.722

0.467

0.625

a

0.750

0.400

0.525

b

0.250

0.600

0.475

Atpase-1

a

1.000

1.000

1.000

Atpase-2

a

1.000

1.000

1.000

Adh-1 Adh-2 Ldh-1 Ldh-2

Gd-2 G-6-Pd-1

Sod-2 Aat-1 Aat-2 Ada-1 Ada-2

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138 Appendix 2 continued

Biochem Genet (2010) 48:125–140

Locus

Pgm-1 Pgm-2 P 95%

Allele

Allele frequency Do¨s¸ emebataklıg˘ı

Durusu

Bu¨yu¨kdo¨llu¨k

a

0.500

0.433

0.350

b

0.500

0.567

0.650

a

0.889

0.633

0.950

b

0.111

0.367

0.050

750,000

750,000

750,000

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