Biochemical Systematics and Ecology 39 (2011) 266–276

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Biochemical Systematics and Ecology

journal homepage: www.elsevier .com/locate/biochemsyseco

Genetic structure of loggerhead turtle (Caretta caretta) populationsin Turkey

Can Yilmaz, Oguz Turkozan, Fevzi Bardakci*

Department of Biology, Faculty of Arts and Science, Adnan Menderes University, 09010 Aydın, Turkey

a r t i c l e i n f o

Article history:Received 26 April 2011Accepted 28 August 2011Available online 18 September 2011

Keywords:Caretta carettaMitochondrial DNAMicrosatellitePopulation geneticsTurkeyMediterranean

* Corresponding author. Tel.: þ90 5334481991; faE-mail addresses: ylmzcn77@gmail.com (C. Yilm

0305-1978/$ – see front matter � 2011 Published bdoi:10.1016/j.bse.2011.08.018

a b s t r a c t

This study investigates genetic structure of loggerhead turtle (Caretta caretta) for conser-vation purposes using both mitochondrial and nuclear DNA markers. To evaluate overallgenetic structure of C. caretta at the Turkish nesting beaches and to compare it withpreviously published data from other nesting sites of the Mediterranean Sea, we havestudied 256 hatchlings from 18 different nesting beaches. Seven distinct haplotypes weredetected, of which three have previously been reported from the Mediterranean and onefrom the Atlantic. The remaining three haplotypes are described for the first time for C.caretta in the present study. Distribution of these haplotypes among the nesting sitesshowed a significant genetic structuring, indicating that females are philopatric and thatgene flow among populations is restricted. Both mtDNA and microsatellite analysesdetermined genetic structuring (mtDNA: gst ¼ 0.214, p < 0.01; nDNA Fst ¼ 0.0004 p < 0.05)among nesting aggregates of C. caretta throughout the study area and enabled thedetection of the different haplotypes to inform conservation strategies.

� 2011 Published by Elsevier Ltd.

1. Introduction

Three species of marine turtles (Caretta caretta, Chelonia mydas and Dermochelys coriacea) occur in the Mediterranean.Among these, C. caretta is the most common breeder within the Mediterranean (Broderick et al., 2002). The species isregarded as endangered globally but its status needs updating (IUCN, 2011). A recent review of sea turtles in the Mediter-ranean by Casale (2008) proposed that as many as 150,000 turtles are estimated to be caught yearly as bycatch in the basin,probably leading to over 50,000 deaths.

Greece, Turkey, Libya and Cyprus are the countries with the densest C. caretta nesting activity in the region (Margaritouliset al., 2003; Casale and Margaritoulis, 2010). Egypt, Lebanon, Israel, Italy, Syria and Tunisia have also been reported as minornesting grounds (Margaritoulis et al., 2003). In Turkey, between 769 and 3521 nests per year were reported during the years1979–2006 (Türkozan and Kaska, 2010).

Previous studies on the genetic structure of C. caretta in the Mediterranean (Bowen et al., 1993; Encalada et al., 1998)reported that these rookeries were isolated from the Atlantic populations at the beginning of Holocene and representa functionally independent management unit (MU). Within the Mediterranean, Turkish colonies have been shown to begenetically distinct from the others based on limited sampling from East and West of the Mediterranean Beaches of Turkey(Laurent et al., 1998; Carreras et al., 2007; Garofalo et al., 2009). The population differentiation for different nesting sites ofTurkey was also shown by mitochondrial and nuclear DNA study (Schroth et al., 1996). Furthermore, Turkish rookeries foundto contribute to distant foraging areas in the western and central Mediterranean (Laurent et al., 1998; Carreras et al., 2006)

x: þ90 256 213 53 79.az), turkozan@adu.edu.tr (O. Turkozan), fevzi.bardakci@gmail.com (F. Bardakci).

y Elsevier Ltd.

C. Yilmaz et al. / Biochemical Systematics and Ecology 39 (2011) 266–276 267

and central Mediterranean African shelf (Casale et al., 2008). However, all these studies included limited sampling from a fewTurkish nesting beaches. In the Mediterranean, a total of 31 haplotypes have been recorded (Laurent et al., 1998; Carreraset al., 2006, 2007; Casale et al., 2008; Garofalo et al., 2009). Of these, 14 were found only in foraging grounds of the Medi-terranean Sea and have not been previously recorded from any nesting beaches. These data indicate that our information onnesting beaches is still incomplete.

The aim of this study is to provide comprehensive information on the genetic structure of C. caretta in Turkey by using bothmitochondrial and nuclear DNA markers with fine scale sampling from almost every nesting beach with a large number ofsamples per beach. Evaluation of population structure of loggerhead turtle nesting in the Turkish beaches could providevaluable information for the conservation of this endangered species due to the anthropogenic reasons.

2. Material and methods

2.1. Collection and DNA extraction

During the nesting seasons of 2003–2008, dead hatchlings of C. Carettawere sampled from 256 different nests located on18 Turkish nesting beaches (Dalyan, Dalaman, Fethiye, Kale, Patara, Kumluca, Tekirova, Çıralı, Belek, Kızılot, Gazipasa, Anamur,Göksu Deltası, Alata, Kazanlı, Akyatan, A�gyatan ve Samanda�g) (Fig. 1 and Table 1). In order to prevent the sampling ofhatchlings from multiple nests of the same females, we collected the samples only from the clutches laid within a 10 daywindow as females do not nest at intervals shorter than this period. There is a possibility that wemay have sampled the samefemale during different nesting seasons however, using three very conservative filtering methods no significant effect ofpseudoreplication was found in the mtDNA haplotypes frequencies (Garofalo et al., 2009).

2.2. Mitochondrial DNA analysis

A fragment of 859 base-pair (bp) of the mtDNA d-loop region was amplified by polymerase chain reaction (PCR Mas-tercycler Personel, Eppendorf, Germany) using the primer pair LCM15382 and H950 (Abreu-Grobois et al., 2006). The PCRprotocol was carried out 35 cycles at 94 �C for 30 s, 55 �C for 1 min and 72 �C for 1 min. PCR products were visualized inagarose gel and purified with the GenElute PCR Clean-Up Kit, (Sigma, Germany). Purified PCR products were sequenced inboth forward and reverse directions using a 3730xl capillary system automatic sequencer (Macrogen Inc., S. Korea). Sequenceswere aligned by eye using the program BioEdit ver 7.0.9 (Hall, 1999) and compared with previously described haplotypesrecorded in the Archie Carr Center for Sea Turtle Research database (http://accstr.ufl.edu/). In addition, previous data from theMediterranean were also included for a general assessment of Mediterranean populations.

Genetic differentiation between different samples from the same locality was assessed with the Zs* (Hudson et al., 1992)and Chi-square tests. These analyses were implemented in the programs CHIRXC (Zaykin and Pudovkin, 1993) and DNAsp4.50 (Rozas et al., 2003). Later, some nesting beaches with small sampling sizes were combinedwith neighboring beaches andafter this grouping UPGMA trees were constructed based on gst values of pairwise comparisons. After grouping, to determinehow the total variationwas partitionedwithin and among populations a hierarchical Analysis of Molecular Variance (AMOVA)(Excoffier et al., 1992), implemented by the program ARLEQUIN ver. 2.000 (Schneider et al., 2000) was conducted. Variance

Fig. 1. The nesting grounds of marine turtles in Turkey.

Table 1Sampling sites, dates, number of individuals (n) and frequencies of haplotypes.

Beach Abbreviations Year of sampling No of specimens CC-A2.1 CC-A3.1 CC-A13.1 CC-A43.1 CC-A52.1 CC-A53.1 CC-A3.2

Dalyan DY 2004 15 8 7 – – – – –

2005 15 10 52008 10 7 3

Dalaman DL 2007 15 5 10 – – – – –

2008 5 – 5Fethiye FT 2003 15 11 4 – – – – –

2008 15 12 3Patara PT 2007 2 2 – – – – – –

2008 3 2 1Kale KL 2007 4 2 2 – – – – –

Kumluca KM 2007 15 13 2 – – – – –

Çıralı ÇR 2007 14 11 3 – – – – –

2008 8 7 1Tekirova TK 2006 2 2 – – – – – –

Belek BL 2007 15 15 – – – – –

2008 10 9 1Kızılot KZ 2007 4 4 – – – – – –

2008 8 8Gazipasa GZ 2007 9 8 – 1 – – – –

Anamur AN 2007 14 12 1 – – 1 – –

2008 10 9 1G.Deltası GD 2005 15 14 1 – – – –

2008 12 8 2 1 1Alata AL 2007 6 6 – – – – – –

Kazanlı KZ 2007 2 2 – – – – – –

Akyatan AK 2006 2 1 1 – – – – –

2007 2 22008 2 2

A�gyatan A�G 2006 1 1 – – – – – –

Samanda�g SM 2007 6 3 2 – 1 – – –

Total 256 196 54 1 1 1 2 1

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components were expressed at three hierarchic levels: (i) among individuals within groups, (ii) among populations withingroups, and (iii) among groups.

Haplotype diversity (h), nucleotide diversity (p) and genetic distance (gst) (Nei, 1982) between each pair of populationswere calculated using the program DNAsp 4.50 (Rozas et al., 2003). Differentiation among population pairs within theMediterranean was also assessed by Chi-square and Zs* tests. Effective population size (Ne) was estimated with the equationp ¼ 2Ne � m (Chen and Hebert, 1999) using average mutation rate (m ¼ 2 � 10�8) as recommended for sea turtles (Encaladaet al., 1998) The number of migrants (Nm) between each population pair was calculated from genetic distances through theequation Nm ¼ 0.5 (1/Gammast � 1) (Takahata and Palumbi, 1985). A haplotype network was constructed using the TCS v1.02package (Clement et al., 2000), which implements \z statistical parsimony (SP) described by Templeton et al. (1992).Comparisons between the Turkish and other Mediterranean populations (Carreras et al., 2007; Encalada et al., 1998; Garofaloet al., 2009; Laurent et al., 1998) were limited to short sequences as these were the only sequences available for the Medi-terranean populations. The sequential Bonferroni correction (Rice, 1989) was implemented for the multiple tests.

2.3. Microsatellite analysis

Six previously described microsatellite loci for sea turtles were used in this study: Cm72, Cm84, and Cc117 (FitzSimmonset al., 1995), Cc7 and Cc141 (Bowen et al., 2005) and Ccar176 (Moore and Ball, 2002; Carreras et al., 2007). Each primer pairwas fluorescently labeled with DY549, 6-FAM and NED. Each locus was amplified using 32 cycles at 94 �C for 45 s, 55 �C for1 min and 72 �C for 1 min. Allele length was determined on an ABI 3730 automated DNA analyzer (Macrogen Inc., SouthKorea). Allele sizes were assigned using the program Genemaker v1.8 (SoftGenetics LLC�).

Number of alleles (k), gene diversity (He) and observed heterozygosity (Ho) in each population and each locus werecalculated. A pairwise test was carried out for population differentiation (Fst) and departure from Hardy–Weinberg equi-librium as well as for detecting linkage disequilibrium between loci. P values for population differentiation were calculatedwith Markov chain randomization (Guo and Thompson, 1992). All these statistical analyses were carried out by using Gen-epop ver. 4.0 (Rousset, 2008). The sequential Bonferroni correction (Rice, 1989) was used for the multiple tests to adjustsignificance levels. Moreover, the program Bottleneck ver 1.2 (Cornuet and Luikart,1996) was used to detect recent bottleneckin populations.

Effective population size (Ne) was estimated using the average dinucleotide repeat mutation rate of the sea turtle C. mydas(2 � 10�3) calculated by Ellegren (2000) using the values obtained by FitzSimmons (1998). To estimate the effective pop-ulation size we tested two models: the infinite-allele model (IAM; Kimura and Crow, 1964, that predicts

C. Yilmaz et al. / Biochemical Systematics and Ecology 39 (2011) 266–276 269

H ¼ 4Nem=ð1þ 4NemÞ, and the stepwise mutation model (SMM) Ohta and Kimura, 1973 that predictsH ¼ 1� ð1=

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1þ 8Nem

pÞ since the mutation model most probably followed bymicrosatellites falls within them Pascual et al.,

2001). Finally, values of effective population size obtained from mtDNA and nDNA data were compared with the actualreproductive population size. The number of migrants between each population pair (Nm) was calculated from the Fst valuesusing the formula: Nm ¼ 1/4(1/Fst � 1) (Wright, 1951). We also estimated nuclear gene flow using a maximum-likelihoodmethod based on a coalescent approach (Beerli and Felsenstein, 1999) implemented in MIGRATE-n vers. 3.0.3 (Beerli,2006). Gene flow (Nm) obtained from mtDNA was compared with that obtained from nDNA using a Wilcoxon signed ranktest.

Population structure was examined using the software STRUCTURE ver 2.3 (Pritchard et al., 2000), which employsa Bayesian clustering method to estimate the most likely number of populations (K) without using prior information on thegeographic location of sampled individuals. The program assumes linkage equilibrium among loci within populations.Twenty runs were carried out by setting the number of clusters (K) from 1 to 18 (number of populations) with 10,000 burn-inperiod followed by 100,000 MCMC (Monte Carlo Markov Chain) replicates, assuming an admixture model and correlatedallele frequencies. The choice of the most likely number of clusters (K) was carried out by comparing log probabilities of data[Pr(XjK)] for each value of K, as well as by calculating an ad hoc statisticDK based on the rate of change in the log probability ofdata between successive K values, as described by Evanno et al. (2005).

3. Results

3.1. Mitochondrial DNA

Among the 256 samples analyzed, six polymorphic sites were observed defining seven haplotypes including threepreviously undiscovered ones. CC-A2.1 and CC-A3.1 were the most dominant haplotypes with frequencies 76.56% and 21.09%,respectively. The former was detected in all nesting sites while the latter was dominant in the nesting sites on the Aegean Seaand western Mediterranean beaches. The geographical distribution of the haplotypes and their frequencies are given in Table1. CC-A43.1, previously reported from Juno Beach (Florida) (Shamblin et al., 2011), was recorded from the Mediterraneannesting ground for the first time. Haplotypes CC-A52.1 (GeneBank accession number, HM366724), CC-A53.1 (GeneBankaccession number, HM179462) and a variant of the CC-A3, here designated CC-A3.2 (GeneBank accession number,HM179461), are described for first time in the present work.

The relationship among the seven mtDNA haplotypes obtained from 18 C. caretta nesting sites was summarized byconstructing an unrooted parsimony network (Fig. 2). Haplotypes CC-A13.1, CC-A43.1 and CC-A53.1 differed by one substi-tution from haplotypes CC-A2.1 while CC-A52.1 and CC-A3.2 separated from CC-A3.1 by one substitution. Haplotype CC-A2.1was predominant in all nesting sites except Dalaman with a frequency of 0.25. The second most common haplotype CC-A3.1was found in all but not in six nesting sites, Tekirova, Belek, Kızılot, Gazipasa, Alata and Kazanlı. Apart from these two mostcommon haplotypes, others were specific to the population in which they were detected (Table 1). We detected no temporalvariation where we had data for more than one year.

Although all populations shared haplotype CC-A2.1, pairwise genetic distances revealed highly significant differencesbetween some of the nesting sites (overall gst ¼ 0.213, p < 0.001). For further analysis some nesting beaches with smallnesting sizes were combined with neighboring beaches based on gst values of pairwise comparisons and after this groupinga UPGMA tree was constructed (Fig. 3). This analysis showed the existence of five management units in Turkey namely,Dalyan, Dalaman, western Turkey (WTR), middle Turkey (MTR) and eastern Turkey (ETR). However, WTR and ETR are notdifferent from each other. After adjustment of significance levels for gst values based on mtDNA using sequential Bonferronicorrection procedure, Dalyan/WTR and MTR/ETR pairs were not significant (see Fig. 3 and Table 2). We separated them sincethey are geographically distant nesting sites. It seems that the presence/absence and frequency of CC-A3.1 responsible for thegeneral pattern of these management units.

AMOVA analysis was used to determine the level of genetic structuring among and between nesting populations. Resultsof AMOVA analysis showed that 23.4% of total variationwas calculated among groups (FCT: 0.234),�3.30% among populationswithin groups (FSC: �0.043), and 79.89% within populations (FST: 0.201). Hierarchical AMOVA results indicated significantgenetic population structure at the first (within populations) and second (among groups) levels (p < 0.01).

Measures of within-population variation, as determined by haplotype and nucleotide diversity, are given in Table 3.Genetic diversity was highly different among nesting sites both in terms of haplotypes diversity (h ¼ 0.083–0.481) andnucleotide diversity (p ¼ 0.00010–0.00056). The highest haplotype and nucleotide diversity values were found in the Dalyanpopulation, which was the westernmost nesting site. Furthermore, effective population size (Ne) ranged from 2.50 to 14.07Estimates of gene flow (Nm) for mtDNAwere also highly variable and varied between 0.7 and 28.85 (Table 4). The gene flowwas the lowest between Dalaman and ETR while the highest between ETR and WTR.

3.2. Microsatellites

All loci were polymorphic with overall allele numbers ranging from 8 (Cm72) to 17 (Ccar176) with a mean of 12.83 allelesper locus (Table 5). In total, 77 alleles were found across six loci examined. None of the investigated nesting beaches werefixed for any specific private allele. Total mean allele richness estimated for each locus across all nesting beaches studied were

C. Yilmaz et al. / Biochemical Systematics and Ecology 39 (2011) 266–276270

the lowest for the cm72 locus (3.30) and the highest for the cc7 locus (10.12) (Table 5). No departure from Hardy–Weinbergequilibrium was detected in loci from any populations except Dalyan (chi-square, p ¼ 0.01), MTR (chi-square, p ¼ 0.00) andETR (chi-square, p ¼ 0.00). Furthermore, no linkage disequilibriumwas found between loci pair (chi-square, p > 0.05) (Table5). Therefore, all loci were assumed to be independent. Observed heterozygosity ranged from Ho ¼ 0.09 (Cm72; Dalyan) toHo ¼ 0.89 (Cc7; East). Gene diversity varied between He ¼ 0.09 (Cm72; Dalyan) and He ¼ 0.98 (Cc7; East). None of thebottleneck tests yielded a significant outcome under the TPMmodel. Effective population size (Ne) ranged from 188 (Dalyan)to 278 (WTR) under the IAM model while it was 328 (Dalyan) and 588 (WTR) under the SMM model (Table 3).

Comparisons of all pairwise Fst values of the nesting beaches showed significant genetic structuring (overall Fst ¼ 0.0004p < 0.05); but genetic differentiation between population pairs was only statistically significant for 3 out of 10 pair-wisecomparisons (Table 2) and none after sequential Bonferroni correction. Gene flow (Nm) between the populations washighly variable (Table 3). Gene flow estimated from nDNA was always higher than those calculated from mtDNA (Wilcoxonsigned rank test, Z ¼ �4.544, p < 0.0001).

Bayesian clustering analysis implemented in the computer software STRUCTURE (Pritchard et al., 2000) were usedwithoutusing prior information on the sampling locations of C. caretta hatchlings. Since the increment in the mean ln likelihoodvalues was very low (K ¼ 2, mean ln likelihood ¼ �4611.24 SD ¼ 78.05; K ¼ 3, mean ln likelihood ¼ �4640.48, SD ¼ 39.44;K¼ 4, mean ln likelihood¼�4750.29, SD¼ 112.42; K¼ 5, mean ln likelihood¼ 4972.1, SD¼ 340.7), the DK and ad hoc statistic

Fig. 2. Estimated parsimony network of mtDNA haplotypes. Each pie graph represents one haplotype and its frequency in each population. The size of each piegraph depends on its absolute frequency.

Fethiye

Grup A

Cirali

Anamur

G Deltasi

Grup C

Dalyan

Grup B

Tekirova

Belek

Dalaman

0.000.050.100.15

Western Turkey

Eastern Turkey

Middle Turkey

Fig. 3. UPGMA tree based on the gst values of pairwise comparisons for nesting beaches (Group A: Patara, Kale ve Kumluca; Group B: Gazipasa ve Kızılot; GroupC: Alata, Kazanlı, Akyatan, A�gyatan ve Samanda�g).

C. Yilmaz et al. / Biochemical Systematics and Ecology 39 (2011) 266–276 271

(Evanno et al., 2005) suggested themost probable number of populations is three (K¼ 2, DK¼ 1.379; K¼ 3, DK¼ 3.389; K¼ 4,DK¼ 2.034). Nevertheless, the three clusters did not correspond to any nesting sites and each individual was assigned to eachof the three clusters with approximately equal probability. Results of the Hierarchical AMOVA analysis supported this resultbecause, unlike mtDNA, there was no significant genetic structuring: (1) among groups (percent of variation ¼ �0.13486,FCT¼�0.0014) (2) among populations within groups (percent of variation¼ 0.39423, FSC¼ 0.0039) and (3) among individualswithin populations (percent of variation ¼ 2.02702, FIS ¼ 0.02032) (4) within individuals (percent of variation ¼ 97.71,FIT ¼ 0.02286).

4. Discussion

The Mediterranean and the Aegean beaches of Turkey represent most of the major nesting areas of C. caretta. Therefore,a comprehensive investigation on the genetic structuring and differentiation among nesting populations of C. caretta acrossthe TurkishMediterranean and Aegean Sea beaches has been carried out using bothmtDNA and nDNAmarkers and datawerecompared with those reported for other nesting sites in theMediterranean (Encalada et al., 1998; Laurent et al., 1998; Carreraset al., 2007; Garofalo et al., 2009). To date, a total of 55 mtDNA haplotypes (including 3 novel haplotypes from this study) forthe Atlantic and Mediterranean C. caretta have been reported by the Archie Carr Center for Sea Turtle Research (ACCSTR)mtDNA database (http://accstr.ufl.edu/ccmtdna.html). Thirty one (56%) of these haplotypes were found in the foraging andnesting grounds of the Mediterranean (Laurent et al., 1998; Carreras et al., 2006, 2007; Casale et al., 2008; Garofalo et al.,2009). Of these haplotypes, 14 were found only in foraging grounds of the Mediterranean Sea. Previous studies reporteda total of two haplotypes from the nesting aggregations of Turkey to date (Laurent et al., 1998; Carreras et al., 2007). In thecurrent study, the number of haplotypes known from Turkey increased to seven, the highest among other Mediterraneancountries although only short sequences were done in almost all previous studies. Schroth et al. (1996) found that thefrequency of the haplotype CC-A3, previously reported from the foraging grounds in the western (Carreras et al., 2006) andeastern Mediterranean (Laurent et al., 1998), declined fromwest to east. If we compare our results to theirs we find a similarpattern if only the beaches they sampled are considered. It appears that CC-A3 declines fromwest to east. However, with ouradditional sampled beaches, this pattern completely changes. This case shows the importance of thorough sampling andprevious inference of a cline of CC-A3 may have been in error. Carreras et al. (2007) found the frequency of CC-A3 to be 6% in

Table 2Genetic structure of grouping beaches based on mtDNA and nuclear DNA. Cells below the diagonal show genetic distances based on mtDNA (gst values) andthose above the diagonal show genetic distances based on nDNA (Fst value) (left: Z test; right: chi-square) a: P< 0.05, b: P < 0.01, c: P< 0.001 (WTR: Fethiye,Patara, Kale, Kumluca and Çıralı; MTR: Tekirova, Belek, Kızılot, and Gazipasa; ETR: Anamur, Göksu Delta, Alata, Kazanlı, Akyatan, A�gyatan and Samanda�g).Asterisks indicate values that are significant at P < 0.05 following sequential Bonferroni corrections.

Dalyan Dalaman WTR MTR ETR

Dalyan – �0.0049 �0.0005 a0.0039 �0.0053Dalaman *a0.125b* – �0.0002 �0.0021 �0.0022WTR a0.031 *c0.220c* – a0.0060 �0.0031MTR *c0.214c* *c0.582c* *c0.084b* – a0.0034ETR *b0.062a *c0.265c* 0.008 a0.039 –

Table 3Genetic variability of Turkish nesting beaches after grouping. Haplotype diversity (h) nucleotide diversity (p), gene diversity (He) observed heterozygosity(Ho) IAM (Kimura and Crow, 1964) and SMM (Ohta and Kimura, 1973).

Beach mtDNA nDNA

h p Ne He Ho NeIAM (range) NeSMM (range)

Dalyan 0.481 0.00056 14,075 0.60 0.65 188 (39–658) 328 (68–1151)Dalaman 0.395 0.00046 11,55 0.65 0.68 232 (48–815) 448 (93–1571)WTR 0.339 0.00039 9750 0.69 0.68 278 (58–976) 588 (112–2063)MTR 0.083 0.00010 2500 0.68 0.69 274 (55–932) 548 (114–1922)ETR 0.297 0.00041 10,250 0.65 0.67 232 (48–815) 448 (93–1571)

C. Yilmaz et al. / Biochemical Systematics and Ecology 39 (2011) 266–276272

western Turkey (Fethiye Beach) while our value was 23% for the same beach. The frequency of haplotype CC-A3 was found tobe higher in the western nesting sites than those in the eastern in contrast to a previous study (Laurent et al., 1998) based ona few number of nesting beaches and small samples size from Turkey.

The low nucleotide divergences observed among the Mediterranean haplotypes compared with those of Atlanticpopulations (Encalada et al., 1998; Pearce, 2001; Bowen et al., 2005) give support to Bowen et al. (1993) suggestion of therecent origin of the Mediterranean populations founded by a few migrants from the Atlantic. Carreras et al. (2007)proposed that CC-A2 is one of the first haplotypes that invaded the Mediterranean since it is the most dominant one.They also proposed that CC-A3 was one of the colonizing haplotypes but they were uncertain about the origin of thishaplotype, or whether it has evolved independently in the Mediterranean. Haplotype CC-A3 is also present at higherfrequencies across Turkish nesting populations, especially in the Western Mediterranean. The parsimonious networkrelationships of mtDNA haplotypes of the Turkish C. caretta determined in this study suggests that CC-A3 is one of thefounder haplotype along with CC-A2 which is different only by one mutation step. It is evident that CC-A2 is the originalhaplotype and the others are those derived from it by at least two mutation steps indicating recent isolation of theMediterranean C. caretta population.

The presence of CC-A3 haplotype only in the Turkish nesting sites can be explained by philopatry of C. caretta females.On the other hand, haplotypes CC-A13 and CC-A43 were previously reported from the Atlantic (Florida) nesting sites(Bowen et al., 2004). Although these haplotypes may have evolved from CC-A2, they might also be among the founderhaplotypes that invaded the Mediterranean and then became rare due to founder effect. A comparison of haplotypes andtheir frequencies among Turkish C. caretta nesting sites with those from throughout the Mediterranean showed thathaplotype CC-A3 is specific to Turkish C. caretta nesting sites along with the rare haplotypes CC-A52, CC-A53 and CC-A3.2(Table 6).

As summarized in Table 6, a comparison mtDNA haplotype distributions in the Mediterranean showed that C. caretta fromdifferent regions of theMediterranean have specific haplotypes. Pairwise comparisons X2 and Z* test results of mtDNA geneticdistance (gst) values showed that C. caretta from Dalyan, Dalaman and WTR are differentiated from those belong to otherregions of the Mediterranean (Table 7) (Lebanon for WTR). Significant deviations between pairs of localities after Bonferronicorrection are given in Table 7. Overall analysis of mtDNA data from all Mediterranean C. caretta suggest that Calabria(southern Italy, Garofalo et al., 2009) and Dalyan have the highest haplotype (H) and nucleotide diversity (p) in the wholeMediterranean (Table 8). Garofalo et al. (2009) have comparedmtDNA haplotypes of Calabriawith those from the Eastern andthe Western Mediterranean coast of Turkey and found a highest genetic divergence between Calibrian and Turkish nestingpopulations.

Genetic distance values between nesting sites based on the nDNA have been found lower than those estimated frommtDNA sequence data. These results are well in accord with those reported for C. caretta from North Atlantic nesting sites

Table 4Estimates of gene flow between nesting beaches based onmtDNA or nDNA using differentmethods (Results are presented as themigration rate from the firstpopulation to second M1–2, from the second population to the first population M2–1 and total migration rate (M).

mtDNA nDNA

gst Fst Migrate

Nm Nm M1–2 M2–1 M

Dalyan-Dalaman 1.75 – 50.74 48.80 49.77Dalyan-WTR 7.76 – 25.85 105.65 65.75Dalyan-MTR 0.92 63.85 32.76 52.08 42.42Dalyan-ETR 3.77 – 43.74 100.50 72.12Dalaman-WTR 0.89 – 12.59 93.56 53.08Dalaman-MTR 0.18 – 39.56 48.04 43.80Dalaman-ETR 0.7 – 20.47 33.37 26.92West-MTR 2.74 41.42 89.33 43.19 66.26West-ETR 28.85 – 64.88 35.49 50.19Middle-ETR 6.19 73.28 41.59 44.99 43.29

Table 5Sample size (N) number of alleles (NA) allelic richness (AR), expected heterozygosity (Ho), observed heterozygosity (Ho), FIS and P value of Hardy–Weinbergequilibrium analyses estimated for five microsatellite loci. Statistically significant values are shown bold. Asterisks indicate values that are significant atP < 0.05 following sequential Bonferroni corrections.

Locus Dalyan Dalaman WTR MTR ETR All

Cc-117N 66 40 140 90 88 424NA 9 7 11 10 8 11AR 7.68 7.00 8.50 8.43 6.53 7.97He 0.61 0.60 0.81 0.78 0.68 0.73Ho 0.74 0.67 0.78 0.74 0.73 0.74FIS 0.18 0.10 �0.04 �0.05 0.06 �0.004P value 0.07 0.23 0.14 0.09 0.07 0.26Cm-72N 68 40 140 90 88 424NA 3 4 4 5 6 8AR 2.42 4.00 2.92 3.17 3.77 3.30He 0.09 0.20 0.17 0.11 0.11 0.14Ho 0.09 0.24 0.16 0.13 0.15 0.15FIS �0.02 0.15 �0.06 0.14 0.26 0.00004P value 1.00 0.15 1.00 0.04 0.05 0.08Cm-84N 68 40 140 90 88 424NA 9 9 11 10 9 11AR 8.22 9.00 7.95 8.72 7.98 8.58He 0.65 0.70 0.66 0.71 0.66 0.68Ho 0.81 0.82 0.81 0.84 0.82 0.82FIS 0.20 0.15 0.19 0.16 0.20 0.0016P value 0.04 0.68 0.00 0.00 0.00 0.01*Cc-141N 68 40 140 90 88 424NA 10 8 11 10 10 14AR 9.01 8.00 8.63 9.07 8.65 8.82He 0.79 0.85 0.91 0.78 0.82 0.84Ho 0.82 0.83 0.84 0.81 0.83 0.83FIS 0.03 �0.02 �0.09 0.04 0.02 0.00007P value 0.00 0.97 0.38 0.30 0.15 0.37Cc-7N 66 40 138 90 88 420NA 11 10 13 12 13 16AR 9.73 10.00 9.66 10.23 10.83 10.12He 0.88 0.85 0.91 0.93 0.98 0.92Ho 0.88 0.84 0.87 0.88 0.89 0.88FIS 0.00 �0.01 �0.05 �0.06 �0.10 0.00008P value 0.29 0.29 0.45 0.36 0.47 0.49Ccar-176N 68 40 138 86 86 418NA 9 10 13 11 7 17AR 7.24 10.00 8.29 8.45 5.63 7.85He 0.59 0.70 0.65 0.77 0.63 0.67Ho 0.56 0.69 0.60 0.72 0.59 0.63FIS �0.06 �0.02 �0.08 �0.06 �0.06 0.005P value 0.66 0.56 0.93 0.72 0.55 0.11AllNA 51 48 63 58 53 77AR 7.38 8.00 7.66 8.01 7.23 –

He 0.60 0.65 0.69 0.68 0.65 –

Ho 0.65 0.68 0.68 0.69 0.67 –

FIS 0.08 0.05 �0.01 0.01 0.04 –

P value 0.01 0.51 0.07 0.00 0.00 –

C. Yilmaz et al. / Biochemical Systematics and Ecology 39 (2011) 266–276 273

(Pearce, 2001). Evaluation of both nDNA and mtDNA results give support to previous studies reporting stronger femalephilopatry than male of C. caretta. Results of gene flow estimates based on both mtDNA and nDNA (Table 4) show higher Nmvalues of nDNA between nesting sites than those of mtDNA. Effective populations size estimates based on microsatellite arefound to be higher than those based on mtDNA sequence data. A reasonable explanation for lower genetic diversity deter-mined in mtDNA than nDNA of C. caretta in this study is higher fixation rate of mtDNA due to its maternal inheritance.

A grouping of neighboring nesting sites showed a significant differentiation of mtDNA (gst) between Dalaman and others(Table 7), probably due to the different frequencies of CC-A2 and CC-A3 haplotypes as well as dominance of the latter allele inthe western nesting sites. The DK and ad hoc statistics (Evanno et al., 2005) of nDNA data suggested the most probable

Table 6Haplotype frequencies in the Mediterranean.

Sampling site N Haplotypes (%) Source

CC-A2 CC-A3 CC-A6 CC-A10 CC-A13 CC-A20 CC-A29 CC-A31 CC-A32 CC-A43 CC-A52 CC-A53 CC-A3.2

Zakynthos 20 85 – 5 – – – – – 10 – – – – Carreras et al., 2007Kyparissia 21 90 – 10 – – – – – – – – – – Encalada et al., 1998Lakonikos 19 95 – 5 – – – – – – – – – – Carreras et al., 2007Greece 10 90 – – 10 – – – – – – – – – Laurent et al., 1998Crete 19 100 – – – – – – – – – – – – Carreras et al., 2007Cyprus 10 100 – – – – – – – – – – – – Carreras et al., 2007Cyprus 35 100 – – – – – – – – – – – – Encalada et al., 1998Lebanon 9 100 – – – – – – – – – – – – Carreras et al., 2007Israel 19 84 – – – – – 16 – – – – – – Carreras et al., 2007Western Turkey 16 94 6 – – – – – – – – – – – Carreras et al., 2007Eastern Turkey 32 59 41 – – – – – – – – – – – Laurent et al., 1998Calabria 47 59.6 – – – – 36.2 – 4.2 – – – – – Garofalo et al., 2009Dalyan 40 62.5 37.5 – – – – – – – – – – – Present studyDalaman 20 25 75 – – – – – – – – – – – Present studyWTR 76 78.95 21.05 – – – – – – – – – – – Present studyMTR 48 95.83 – – – 2.083 – – – – – – 2.083 – Present studyETR 72 83.33 11.11 – – – – – – – 1.39 1.39 1.39 1.39 Present study

C. Yilmaz et al. / Biochemical Systematics and Ecology 39 (2011) 266–276274

number of populations is three. Comparisons of Fst values between four groups produced by joining close neighboring nestingsites displayed lower values between first-second, first-third and second–third pairs of groups of which last two weresignificantly different that might explain the results of the DK and ad hoc statistics suggesting three populations despite thefact that hierarchical AMOVA analysis did not support this grouping.

Genetic structuring of C. caretta in the Mediterranean is explained by female philopatry. The mechanism behind femalenatal homing behavior is not yet understood. Carreras et al. (2006) proposed that genetic structuring in the westernMediterranean feeding grounds could be explained by the pattern of sea surface currents and water masses. This hypothesisalso supported by results from Cape Verde C. caretta in the Mediterranean exclusively in the feeding grounds surrounded byAtlantic currents (Monzón-Argüello et al., 2008). According to Schroeder et al. (2003), C. caretta mating takes place nearnesting sites where a male meets more females allowing male mediated gene flow among nesting sites. Based on the resultsof our current investigation, we suggest that hatchlings from different nesting sites possibly move to certain foraging groundsand stay there until they reach maturity then return to their natal home for their first breeding. After their first breedingexperiences, young mature females are more able to move long distances for feeding or nesting. This hypothesis could betested through a detailed investigation on the size of nesting females laying eggs and its relationship with their mtDNAhaplotypes, genetic structure of nesting populations, and foraging habitats.

From a conservation perspective, there are several implications of our study. First, in addition to being divergent, Turkish C.caretta have a high genetic diversity in the Mediterranean. Furthermore, pairwise comparisons of Fst values suggest thatTurkish nesting sites could be distinguished as five management units. Mapping of genetic diversity within and amongnesting sites would allow us to define specific nesting populations that could be used as sources for recovering geneticdiversity in nesting sites that have been disturbed by human activities. Second, microsatellite data suggest that there is highallelic richness in C. caretta nesting across Turkish beaches, which is evidence for possible foraging grounds near nesting areasof Turkish coasts. To enhance conservation, a more comprehensive study including samples collected annually from allnesting sites, neritic, and foraging habitats would allow us to identify the connections between each region and understandtheir origin, migration routes, and life histories.

Table 7Pairwise comparison of mtDNA in the Mediterranean. Cells below the diagonal show genetic distances based on mtDNA (gst values) and those above thediagonal show migration rates (left: Z test; right: chi-square) a: P < 0.05, b: P < 0.01, c: P < 0.001 (WTR: Western Turkey, MTR: Middle Turkey, ETR: EasternTurkey GRE: Greece, CRE: Crete, CYP: Cyprus, LEB: Lebanon, ISR: Israel, CLB: Calabria. Asterisks indicate values that are significant at P < 0.05 followingsequential Bonferroni corrections. The data from other Mediterranean nesting beaches was compiled from Carreras et al. (2007) and Garofalo et al. (2009).The analysis was carried out only with short sequences. The data of Laurent et al. (1998) from eastern Turkey were not included in the analysis.

Dalyan Dalaman WTR MTR ETR GRE CRE CYP ISR LBN CLB

Dalyan 1.75 5.76 0.92 3.62 1.37 1.29 0.73 1.32 2.27 0.94Dalaman a0.125b 0.84 0.18 0.65 0.37 0.17 0.11 0.26 0.27 0.42WTR a0.042a *c0.230c* 3.58 58.62 4.38 6.44 2.98 4.39 12.38 1.28MTR *c0.214c* *c0.582c* *c0.065b 5.83 9.86 41.43 22.19 3.87 74.42 1.16ETR b0.065a *c0.276c* 0.004 a0.041 6.93 9.68 4.72 5.46 18.20 1.40GRE *c0.154c* *c0.403c* *b0.054c* 0.025 a0.035b 16.82 8.25 6.10 31.53 1.34CRE b0.162b *c0.594c* a0.037a 0.006 0.025 0.015 – 3.42 – 1.78CYP *c0.256c* *c0.686c* *c0.077c* 0.011 b0.050a a0.029 – 2.23 – 0.96ISR *c0.159c* *c0.490c* 0.054c* 0.061 0.044a 0.039a 0.068 a0.101b 5.45 1.69LBN a0.099a *c0.482c* 0.020 0.003 0.014 0.008 – – 0.044 3.19CLB *c0.211c* *c0.371c* *c0.163c* *c0.177c* *c0.151c* *c0.158c* b0.123b *c0.207c* b0.129c* a0.073

Table 8Genetic variability (short haplotypes) in the Mediterranean (Haplotype diversity (h), nucleotide diversity (p), Effectivepopulation size (Ne). The data from other Mediterranean nesting beaches was compiled from (Carreras et al., 2007) and(Garofalo et al., 2009). The analysis was carried out only with short sequences. The data of Laurent et al. (1998) fromeastern Turkey were not included in the analysis.

h p Ne

Dalyan 0.481 0.00127 31,750Dalaman 0.395 0.00104 26,000WTR 0.305 0.00080 20,000MTR 0.083 0.00022 5500ETR 0.293 0.00086 21,500GRE 0.188 0.00064 1600CRE – – –

CYR – – –

ISR 0.247 0.00065 16,250LBN – –

CLB 0.524 0.00146 36,500

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Acknowledgments

This study is financially supported by The Scientific and Technological Research Council of Turkey (TÜB_ITAK) with a projectnumber 106T248. The authors would like to thank their colleagues Serap Ergene, A. Fuat Canbolat, Yakup Kaska, HakanDurmus, Bektas Sönmez, Askın Uçar and Cemil Aymak for supporting tissue sampling. The authors would like to thank Dr.James Ford Parham of Alabama Natural History Museum and Dr. Carlos Carreras for commenting on an earlier form of themanuscript. Dr. Brian Shamblin and Dr. Alberto-Abreu Grobois gave insightful comments on the draft of the manuscript. Lastbut not least authors would like to thank Dr. Aaron Bauer of Villanova University for his help for the improvement of thelanguage of the paper.

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