Miguel A. Carretero3, Claudia Corti4, D. James Harris3, Uwe Fritz1,**
Abstract. Using 10 polymorphic microsatellite loci and sequences of the mitochondrial cytochrome b gene, we examinegene flow in more than 100 spur-thighed tortoises from Transcaucasia and compare our findings with previously publishedAFLP and mtDNA data. While mtDNA sequences correspond to three deeply divergent clades and AFLP data suggesttwo distinct groups, microsatellite data indicate weak differentiation and extensive gene flow. We conclude that eachmarker system reflects a distinct episode in the evolutionary history of the Testudo graeca complex, corresponding tophases of vicariance and extensive gene flow. We hypothesize that the reciprocally monophyletic and deeply divergentmtDNA lineages reflect old vicariance events, while the conflicting nuclear markers are the legacy of younger episodesof extensive gene flow. The differentiation pattern found in the AFLP markers, with AFLP groups matching allopatricallyor parapatrically distributed mitochondrial lineages, is likely to be older than the microsatellite differentiation, which couldcorrespond to Holocene range expansions into Caucasian valleys and lower altitudes. Owing to the largely mutually exclusivedistribution ranges of the deeply divergent mtDNA lineages, their identification with distinct subspecies is a reasonable andstraightforward classification that facilitates communication and acknowledges the conspecifity of the involved evolutionaryunits. Consequently, Transcaucasia constitutes an intergradation zone of T. g. armeniaca, T. g. buxtoni and T. g. ibera.
The taxonomy of spur-thighed tortoises (Tes-tudo graeca complex) has been in a stateof flux during the past 25 years. While thisgroup had traditionally been regarded as awidely distributed polytypic species (Wermuthand Mertens, 1961, 1977; Anderson, 1979;Pritchard, 1979), many subspecies were later el-evated to species level and additional specieswere described or resurrected from synonymy
1 - Museum of Zoology (Museum für Tierkunde), Senck-enberg Dresden, A.B. Meyer Building, 01109 Dresden,Germany
2 - Faculty of Biology, Yerevan State University, AlekManoogian 1, Yerevan, 0025, Armenia
3 - CIBIO, Centro de Investigação em Biodiversidade eRecursos Genéticos, Universidade do Porto, CampusAgrário de Vairão, 4485-661 Vairão, Portugal
4 - Museo di Storia Naturale dell’Università di Firenze,Sezione di Zoologia “La Specola”, Via Romana, 17,50125 Firenze, Italy*These authors contributed equally to this publication.**Corresponding author;e-mail: [email protected]
based on morphological evidence (Chkhikvadzeand Tuniyev, 1986; Weissinger, 1987; High-field and Martin, 1989a, b, c; Highfield, 1990;Chkhikvadze and Bakradze, 1991, 2002; Perälä,1996, 2002; Pieh, 2001; Pieh and Perälä, 2002,2004; Chkhikvadze, Mazanaeva and Sham-makov, 2011). As a result, some authors claimedthat the T. graeca complex consists of ap-proximately 20 distinct species with mutu-ally exclusive allopatric or parapatric ranges inthe western Mediterranean region, the Balkanpeninsula and the Near and Middle East (for areview, see Guyot-Jackson, 2004; Fritz et al.,2007). However, molecular genetic investiga-tions found much less differentiation (van derKuyl et al., 2002; Harris et al., 2003; van derKuyl, Ballasina and Zorgdrager, 2005; Parhamet al., 2006; Fritz et al., 2007, 2009), withonly six deeply divergent mitochondrial lin-eages identified. These results suggested thatmany of the morphologically defined taxa areinvalid and that external morphology of spur-thighed tortoises is heavily impacted by envi-ronmental factors (Carretero et al., 2005; Fritz et
Figure 1. Mitochondrial phylogeny of the Testudo graeca complex as inferred from ML analyses of a 1216-bp-long alignment(cyt b gene plus adjacent DNA coding for tRNAs) of 140 ingroup sequences (data set of Fritz et al., 2007 combined with46 sequences generated for the present study; two sequences of North African tortoises corrected according to Vambergeret al., 2011). Terminal clades collapsed to cartoons; grey cartoons indicate that haplotypes of the respective clade wererepresented among the samples sequenced for the present study. Numbers at nodes are ML bootstrap values and posteriorprobabilities from Bayesian inference. Root length shortened by 80%. On the right, the correlation of mtDNA clades withAFLP groups (Mikulicek et al., 2013) and microsatellite clusters (K = 3, this study) is shown. Note that mtDNA cladesA and E represent one and the same AFLP cluster, although they are not sister groups. Merging colours for microsatellitedata symbolize admixed individuals. The letter C in the Caucasian AFLP group indicates the occurrence of a mitochondrialhaplotype of clade C in a tortoise from Katekh, Azerbaijan, belonging to the Caucasian AFLP group (Mikulicek et al., 2013).
al., 2007). In the following, we use the terminol-ogy of Fritz et al. (2007) and label the six majormitochondrial clades by the upper-case lettersA-F (fig. 1).
Using nuclear-genomic ISSR fingerprinting,a method useful in discriminating the tradi-tionally recognized Testudo species T. graeca,T. hermanni, T. horsfieldii, T. kleinmanni andT. marginata, Fritz et al. (2007) found onlynegligible differentiation among tortoises rep-resenting these six mitochondrial clades andconcluded that they are best regarded as con-specific. Acknowledging the deep mitochon-drial divergences, Fritz et al. (2007) proposedto identify each clade with a distinct sub-species, while other authors preferred to recog-nize no subspecies at all (Parham et al., 2006).
In a follow-up study, Mikulicek et al. (2013)re-examined the differentiation within the T.graeca complex using more sensitive AFLPfingerprints and found four well-differentiatedAFLP groups. Two of these groups, the west-ern Mediterranean AFLP group and the central-eastern Iranian AFLP group (Mikulicek et al.,2013), correspond to the completely or largelyallopatric mitochondrial lineages from the west-ern Mediterranean (mtDNA clade B) and east-ern Iran (mtDNA clade F; fig. 1). Each of theremaining two AFLP clusters includes two ge-ographically neighbouring mtDNA clades, sug-gestive of extensive gene flow among tortoisesharbouring the respective mitochondrial lin-eages. The Caucasian AFLP group comprisesthe mtDNA clades A and E. These two clades
Gene flow in Testudo graeca 339
are not sister groups in phylogenetic analy-ses (fig. 1). The Balkans-Middle Eastern AFLPgroup embraces the mtDNA clades C and D,which are sister groups. The ranges of these twodistinct AFLP groups abut in the central Cauca-sus, and in one site (Katekh, Azerbaijan) twotortoises were found that belonged to each ofthe two distinct AFLP groups, without any in-dication of nuclear genomic admixture. On theother hand, both tortoises yielded haplotypes ofmtDNA clade C, indicating recent or past geneflow between the Caucasian and the Balkans-Middle Eastern AFLP groups. Since mtDNAis known to be transferred relatively easy fromone species to another as the result of inter-specific hybridization (Mallet, 2005; Currat etal., 2008), this complicated situation raises thequestion of whether distinct reproductively iso-lated species might be involved (Mikulicek etal., 2013), which hybridize only rarely.
In the present study we focus exactly on thisquestion: Could some of the distinct genetic lin-eages of Caucasian spur-thighed tortoises rep-resent reproductively isolated units qualifyingas Biological Species sensu Mayr (1942, 1963)or do they alternatively represent one and thesame Biological Species? For testing these hy-potheses, we use rapidly evolving microsatel-lite markers, which are an ideal tool for as-sessing gene flow. We genotyped more than100 tortoises from Armenia, Georgia, Iran andNagorno Karabakh using 10 polymorphic mi-crosatellite loci. The sampling region is knownto harbour three distinct mtDNA clades corre-
sponding to two AFLP clusters (Fritz et al.,2007; Mikulicek et al., 2013). We analyze ourdata set with an unsupervised Bayesian clus-tering approach as implemented in STRUCTURE
2.3.3 (Pritchard, Stephens and Donnelly, 2000;Hubisz et al., 2009) and compare the resultswith mtDNA sequence variation and the AFLPgroups of Mikulicek et al. (2013). If distinct re-productively isolated species were representedamong our samples, they should correspondto clearly differentiated microsatellite clusterswith little or no evidence of gene flow. By con-trast, if only one more-or-less panmictic specieswere concerned, the expectation is little nucleargenomic differentiation and, in secondary con-tact zones of distinct microsatellite clusters, ex-tensive gene flow among clusters.
Materials and methods
Sampling and laboratory procedures
DNA of 121 ethanol-preserved samples of tortoises from26 sites in Armenia, Georgia, Iran and Nagorno Karabakh(Appendix) was isolated using the InnuPREP DNA MiniKit (Analytik Jena AG, Jena, Germany) or the NucleoSpinTissue Kit (Macherey-Nagel, Düren, Germany). FollowingSalinas et al. (2011), the samples were genotyped using10 microsatellite loci; three or four loci each were com-bined in multiplex PCRs (table 1). The final volume of eachmultiplex PCR was 10 μl containing 0.5 units Taq poly-merase (Bioron, Ludwigshafen, Germany) with the bufferrecommended by the supplier and a final concentration of1.5 mM MgCl2 (Bioron), 0.2 mM of each dNTP (Thermo-Scientific, St. Leon-Rot, Germany), 2 μg Bovine Serum Al-bumin (Thermo-Scientific), approximately 10-20 ng of to-tal DNA and primer concentrations ranging from 0.2 μMto 0.8 μM. Forward primers were fluorescent-labelled. The
Table 1. Microsatellite loci, multiplex sets, allele size ranges and number of alleles of the individual loci.
Locus Original reference Multiplex set Fluorescent label Allele size range [bp] Number of alleles
GmuB08 King and Julian (2004) 1 ATTO 565 195-249 17Goag6 Edwards et al. (2003) 1 6-FAM 282-404 19Test76 Forlani et al. (2005) 1 HEX 112-138 4GmuD51 King and Julian (2004) 2 ATTO 550 132-224 17Test56 Forlani et al. (2005) 2 HEX 192-274 31Test71 Forlani et al. (2005) 2 6-FAM 119-141 10Gp61 Schwartz et al. (2003) 3 6-FAM 179-193 6Gp81 Schwartz et al. (2003) 3 HEX 361-363 2Test10 Forlani et al. (2005) 3 HEX 179-241 26Test21 Forlani et al. (2005) 3 ATTO 565 193-237 13
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PCR cycling conditions were as follows: 43 cycles with de-naturation at 94°C for 60 s but for 11 min in the first cy-cle, annealing at 58°C for 45 s and extension at 72°C for45 s but for 30 min in the final cycle. Fragment lengthswere determined on an ABI 3130xl Genetic Analyzer usingthe GeneScanTM-600 LIZ® Size Standard and the softwareGENEMAPPER 4.0 (Applied Biosystems, Foster City, USA).For 14 samples no microsatellite data could be produced,because the respective sample was used up or PCR failedconsistently.
In addition, the mitochondrial cyt b gene was se-quenced for 46 samples representing all collecting sites (Ap-pendix). Two mtDNA fragments overlapping by approxi-mately 300 bp were amplified using the primer pairs CytbGplus mt-E-Rev2 (Spinks et al., 2004; Fritz et al., 2006) andmt-c-For2 plus mt-f-na (Fritz et al., 2006). PCR was per-formed in a final volume of 20 μl using 1 unit Taq poly-merase (Bioron) with the buffer recommended by the sup-plier and a final concentration of 0.25 mM of each dNTP(Thermo-Scientific), 0.5 μM of each primer and approxi-mately 10-40 ng of total DNA. The PCR cycling conditionswere as follows: 35-40 cycles with denaturation at 94°C for45 s but for 5 min in the first cycle, annealing at 50°C for30 s, and extension at 72°C for 60 s but for 10 min in the fi-nal cycle. PCR products were purified using the ExoSAP-ITenzymatic cleanup (USB Europe GmbH, Staufen, Germany;1:20 dilution; modified protocol: 30 min at 37°C, 15 minat 80°C) and sequenced on an ABI 3130xl Genetic Ana-lyzer using the BigDye Terminator v3.1 Cycle SequencingKit (Life Technologies, Darmstadt, Germany) and the PCRprimers. DNA sequences were aligned in BIOEDIT 7.1.11(Hall, 1999) with previously published data (Fritz et al.,2007), yielding a 1216-bp-long alignment of 140 sequencescomprising the complete cyt b gene plus adjacent DNA cod-ing for tRNAs. Two erroneous North African sequences ofFritz et al. (2007) were corrected according to Vamberger etal. (2011). GenBank accession numbers for sequences gen-erated for the present study are HF954117-HF954162.
Data analysis
Pairwise linkage disequilibrium between microsatellite lociand Hardy-Weinberg equilibrium were tested in ARLEQUIN
3.11 (Excoffier, Laval and Schneider, 2005) using tortoisesfrom three populations or groups of neighbouring popula-tions with sufficient sample sizes (collecting sites 8, 9 andsites 19-24 lumped together; see Appendix). There was noevidence for linkage disequilibrium, and microsatellite dataof all tortoises were then subjected to unsupervised clusteranalysis using STRUCTURE 2.3.3 (Pritchard, Stephens andDonnelly, 2000; Hubisz et al., 2009). However, many lociwere found to be not in Hardy-Weinberg equilibrium, andMICRO-CHECKER 2.2.3 (van Oosterhout et al., 2004) sug-gested the presence of null alleles (table 2). Therefore, thedata set was corrected for null alleles according to Falush,Stephens and Pritchard (2007). The optimal number of clus-ters was determined by the �K method of Evanno, Regnautand Goudet (2005). Considering the number of collectingsites for which microsatellite data were available (n = 26),population structure was modelled using an upper bound of
Table 2. Testing for Hardy-Weinberg equilibrium (HWE)using tortoises from three populations or groups of neigh-bouring populations with sufficient sample sizes (collectingsites 8, 9 and sites 19-24 lumped together; see Appendix).Yes = in HWE, no = not in HWE. Asterisks indicate pres-ence of null alleles.
30 and an admixture scenario with allele frequencies cor-related, allowing the individuals to have mixed ancestries.The burn-in was set to 25 × 104 and the number of fur-ther MCMC runs to 75 × 104. Calculations were repeated10 times for each K; convergence of likelihood values wasreached after the burn-in. Clustering results and individualadmixture were visualized using bar plots with the soft-ware DISTRUCT 1.1 (Rosenberg, 2004). In a conservativeapproach following Randi (2008), individuals with propor-tions for cluster membership below 80% were treated ashaving mixed ancestries.
Genetic differentiation among different subsets of themicrosatellite data was inferred by FST values and analy-ses of molecular variance (AMOVA) using ARLEQUIN 3.11(10 000 permutations). For comparing number and size ofmicrosatellite alleles, a frequency table was produced us-ing CONVERT 1.31 (Glaubitz, 2004) and locus-specific ob-served (HO ) and expected heterozygosities (HE ) were es-timated in ARLEQUIN. Locus-specific allelic richness (AR)values were obtained in FSTAT 2.9.3.2 (Goudet, 1995).
Phylogenetic relationships of mtDNA sequences wereinferred by Maximum Likelihood (ML) analyses usingRAxML 7.2.6 (Stamatakis, 2006) and the implemented evo-lutionary model GTR + G. Five independent ML searcheswere performed using different starting conditions and thefast bootstrap algorithm to explore the robustness of thephylogenetic trees by comparing the best trees. Subse-quently, 1000 non-parametric thorough bootstrap replicateswere calculated and the values plotted against the besttree. In addition, analyses using MRBAYES 3.2.1 (Ronquistet al., 2012) were run. The best evolutionary model wasestablished in JMODELTEST 0.1.1 (Posada, 2008) by theBayesian Information Criterion, resulting in the TPM1uf +G model. Phylogenetic analyses were performed using twoparallel runs (each with four chains) and default parameters.Both chains ran for 10 million generations with every 100th
generation sampled. Using a burn-in of 2.5 million genera-tions, only the plateau of the most likely trees was sampled
Gene flow in Testudo graeca 341
for generating a 50% majority rule consensus. The posteriorprobability of any individual clade in this consensus treecorresponds to the percentage of all trees containing thatclade, and is a measure of clade frequency and credibility.In both approaches, a sequence of Testudo hermanni (Gen-Bank accession number AJ888362) served for tree-rooting.
Additionally, uncorrected p distances of mtDNA cladeswere calculated using MEGA 5.05 (Tamura et al., 2011) andthe pairwise deletion option.
Results
The 10 studied microsatellite loci are in parthighly polymorphic, with allele numbers rang-ing from 2 to 31 per locus (table 1) and a to-tal allele number of 145. The �K method ofEvanno, Regnaut and Goudet (2005) suggestedfor the STRUCTURE results four as the optimalnumber of clusters. When K = 4 is used asa framework, there is a high degree of admix-ture evident. However, in some collecting sitesmore or less pure genotypes are detected be-sides admixed individuals (fig. 2). The phyloge-netic analyses of mtDNA sequences of tortoisesrepresenting all sites found three deeply diver-gent mitochondrial lineages among the studiedsamples (figs 1 and 2), corresponding to themtDNA clades A, C and E of Fritz et al. (2007).Mean uncorrected p distances of the cyt b genebetween these and the remaining three othermtDNA clades of the Testudo graeca complexamount to 2.50-5.70%. In particular, the diver-gences between clades A, C and E range from4.15% to 4.90% (table 3). Haplotypes of clade Aare widely distributed in the Araxes valley andNagorno Karabakh, while haplotypes of clade Care largely confined to the northernmost Arme-nian sites. However, in two sites in the Araxesvalley haplotypes of clades A and C occur syn-topically. Haplotypes of clade E are only foundin northwestern Iran (fig. 2; Appendix). Noneof the three mtDNA clades corresponds to anyof the four clusters revealed by the microsatel-lite analyses. Also when barplots for K = 3 arecompared to mtDNA clades, the general picturedoes not change and the incongruity betweenmitochondrial haplotypes and nuclear-genomicgenotypes remains (fig. 2).
When genetic diversity indices for the dis-tinct STRUCTURE clusters (K = 3 and K =4) are compared and tortoises with mixed an-cestries are excluded, the red cluster with theleast amount of admixture has the lowest val-ues, with just two private alleles, despite moreor less even sample sizes (table 4). Fixation in-dices (FST values) for K = 3 range from 0.12to 0.21 (table 5), and according to an AMOVA,17% of the molecular variance occurs amongclusters and 83% within clusters. For K = 4,FST values range from 0.15 to 0.26 (table 5),with 22% of the molecular variance occurringamong clusters and 78% within clusters.
From a morphological point of view, our tor-toise samples represent two principal pheno-types, individuals with depressed shells, as orig-inally described as typical for Testudo graecaarmeniaca (Chkhikvadze and Bakradze, 1991;Pieh, Fritz and Berglas, 2002), and tortoiseswith domed shells, the normal character state inthe T. graeca complex. When morphologicallysimilar tortoises (M. Arakelyan, V. Mashkaryan,M.A. Carretero, C. Corti, unpubl.) are lumpedtogether, three geographically coherent groupsemerge (fig. 2: top). However, in the two north-ern groups (Araxes valley) there are also someindividuals whose morphological assignment tothe domed vs. flat phenotype is ambiguous. Inany case, using microsatellite data these threegroups differ by FST values ranging from 0.10to 0.15 and they do very roughly correspond tothe three microsatellite clusters under K = 3,including admixed individuals. According to anAMOVA, 11% of the observed global molec-ular variance occurs among the three morpho-logical groups and 89% within the groups (tor-toises with mixed genetic ancestry included).
Discussion
In agreement with a previous paper (Fritz etal., 2007), our present investigation confirmeddeep mitochondrial divergences in Transcau-casian tortoises of the Testudo graeca complex(table 3). In our study area occur three of the
342 V. Mashkaryan et al.
Gene flow in Testudo graeca 343
six mitochondrial clades of the T. graeca com-plex. Compared to mtDNA variation, Mikuliceket al. (2013) found less differentiation usingAFLP data (152 markers), with only four AFLP
Table 3. Mean uncorrected p distances (percentages) be-tween and within the six major mtDNA clades A-F of theTestudo graeca complex based on the data set of Fritz et al.(2007), merged with the sequences generated in the presentstudy (1144 bp cyt b). Two erroneous sequences of westernMediterranean tortoises of Fritz et al. (2007) were replacedfor the corrected sequences (see Vamberger et al., 2011).Below the diagonal, divergences between clades are givenand on the diagonal in boldface, divergences within the re-spective clade.
Table 4. Genetic diversity of Testudo graeca, based on 10microsatellite loci (individuals with mixed ancestries ex-cluded). Cluster names (colours) refer to fig. 2. Abbrevia-tions: n, number of individuals; nA, number of alleles; nA,average number of alleles; nP , number of private alleles;AR, allelic richness; HO , average observed heterozygosity;HE , average expected heterozygosity.
groups. Two of these groups perfectly corre-spond to mitochondrial clades in the westernMediterranean and in central and eastern Iran(mtDNA clade B and F, respectively). Due totheir completely allopatric or parapatric distri-bution ranges, and hence the impossibility ofany recent gene flow, such a pattern is not toosurprising. Some other findings by Mikuliceket al. (2013) require more attention: (1) One ofthe two remaining AFLP groups, the so-calledBalkans-Middle Eastern AFLP group, embracestortoises harbouring mtDNA clades C and D,which are sister groups. (2) The fourth AFLPgroup, the so-called Caucasian AFLP group, in-cludes mtDNA clades A and E, which are notsister groups (Fritz et al., 2007). This suggestsin both cases extensive gene flow within eachAFLP group. (3) The distribution ranges of theCaucasian and Balkans-Middle Eastern AFLPgroups abut in Transcaucasia, and Mikuliceket al. (2013) found two tortoises representingeach of these AFLP groups syntopically at one
Table 5. Fixation indices (FST values) for microsatellitedata under K = 3 and K = 4 (individuals with mixed an-cestries excluded). All FST values are significantly differentfrom zero.
Figure 2. Cluster assignment of 107 spur-thighed tortoises from 26 collecting sites in Transcaucasia using STRUCTURE 2.3.3,based on 10 polymorphic microsatellite loci. Shown are the STRUCTURE runs with the highest probability values for each K .In the barplots, the genotype of an individual tortoise is represented by a vertical column. Below a threshold of 80% for clustermembership, distinct colours within one column indicate mixed ancestry. The white lines in the barplots represent 20% andthe numbers below the barplots refer to collecting sites. Different collecting sites are separated by black vertical lines. Coloursof sampling sites in the maps correspond to STRUCTURE clusters; slices indicate tortoises with mixed ancestries or conflictingcluster assignment. Top: Results for K = 3. Encircled collecting sites in the map denote morphological groups (for otherlocalities, no morphological data were available); the black bar approximately indicates the Lesser Caucasus. Bottom: Resultsfor K = 4. For encircled collecting sites, mtDNA data were available as indicated. At locality 4, four tortoises harbouredhaplotypes of clade A and two of clade C. At locality 13, five tortoises yielded haplotypes of clade A and one of clade C.Inset shows location of map section.
344 V. Mashkaryan et al.
site (Katekh, Azerbaijan), without any admix-ture. This could indicate that the two involvedAFLP groups represent reproductively isolatedunits, and hence, that each of these units couldqualify as a full species under the BiologicalSpecies Concept (Mayr, 1942, 1963). However,both tortoises from Katekh harboured mtDNAhaplotypes representing the same clade (cladeC), providing evidence either for intraspecificgene flow or interspecific introgression.
If two reproductively largely isolated specieswere involved, and the shared mitochondrialhaplotypes of the tortoises from Katekh werethe result of interspecific hybridization or in-trogression, it should be expected that ourmicrosatellite markers would reveal largelydistinct nuclear gene pools for the involvedspecies – as is the case, for instance, even inbiological species which lost their original mi-tochondrial genome by introgressive hybridiza-tion (e.g. Lissotriton montandoni, Zielinski etal., 2013). Such distinct gene pools should cor-respond to highly distinct clusters in unsuper-vised STRUCTURE analyses and should be char-acterized by pronounced genetic differences, asreflected by many private alleles and highly dis-tinct fixation indices of microsatellite loci. Onthe other hand, if the observed mismatches be-tween nuclear genomic and mitochondrial dif-ferentiation result rather from recent secondarycontact of two reproductively fully compatibleevolutionary lineages, the differentiation of dis-tinct clusters should be rather weak and geneflow should be extensive as indicated by manyindividuals with mixed ancestries. Moreover,private alleles should be rare, and fixation in-dices should be low.
In agreement with the latter, the STRUCTURE
analyses of our microsatellite data provide ev-idence for a generally high degree of admix-ture (fig. 2), with relatively few individualsassigned to more or less pure clusters underK = 4. This high degree of admixture reflectsthe difficulties to find data partitions in Hardy-Weinberg equilibrium, one of the two maincriteria of STRUCTURE for cluster delineation
(Pritchard, Stephens and Donnelly, 2000). How-ever, Hardy-Weinberg equilibrium is not ex-pected in recently admixed populations.
STRUCTURE revealed the most pronounceddifferentiation for the red cluster, which corre-sponds – with some exceptions – to flat-shelledtortoises matching the original description of T.g. armeniaca Chkhikvadze and Bakradze, 1991.Such tortoises occur in a geographically quiteisolated situation in the valley of the AraxesRiver, embedded between the eastern Anato-lian mountains and the Lesser Caucasus. Pos-sibly their flat-shelled morphotype is associatedwith burrow-digging, while the domed tortoisescorresponding to the other microsatellite clus-ters generally do not dig deep burrows. How-ever, neither fixation and diversity indices ofmicrosatellites (tables 4 and 5) nor AFLP dataor mtDNA sequences (figs 1 and 2) supporta genetic isolation of the flat-shelled tortoises.The flat-shelled tortoises harbour mitochondrialhaplotypes of clades A and C, which do also oc-cur in tortoises with domed shells, and the Cau-casian AFLP group embraces flat-shelled anddomed tortoises. Even if tortoises with mixedancestry are completely disregarded, FST val-ues between the red cluster and the other clus-ters are not extraordinarily high for microsatel-lites, ranging from 0.19 to 0.21 for K = 3and from 0.24 to 0.26 for K = 4 (table 5).When topotypic individuals of mixed ancestryare included, FST values are for obvious rea-sons lower and amount only to 0.10-0.15. Alsowithin other tortoise species, similar or evendistinctly higher FST values have been reported(Chelonoidis chilensis, based on 10 microsatel-lite loci: 0.10-0.21, Fritz et al., 2012a; Gopherusagassizii, based on 20 microsatellite loci: 0.01-0.13, Hagerty and Tracy, 2010; G. polyphe-mus, based on 9 microsatellite loci: 0.06-0.51,Schwartz and Karl, 2005; Testudo marginata,based on 11 microsatellite loci: 0.05-0.16, Perezet al., 2012). Most notably, based on seven mi-crosatellite loci FST values of up to 0.24 werefound within one and the same subspecies of T.
Gene flow in Testudo graeca 345
graeca, the western Mediterranean T. g. graeca(Graciá et al., 2013).
In addition, the red cluster is characterizedby the lowest number of private alleles of all(table 4). This, together with the evidence fora high degree of admixture and FST valuesmatching the differentiation within other tor-toise species, supports the view that the T.graeca complex represents only one Biologi-cal Species sensu Mayr (1942, 1963), despitedeeply divergent mitochondrial lineages (ta-ble 3). The three mtDNA clades A, C and Eoccurring in our study region differ by 4.15%to 4.90% in uncorrected p distances of thecyt b gene. These values exceed uncorrected p
distances as observed among some other con-generic tortoise species (3.7% to 12.7%; Fritzet al., 2012a; Kindler et al., 2012), underliningthat species delineation in chelonians should notrely on genetic distances alone (Vargas-Ramírezet al., 2010; Praschag et al., 2011; Stuckas andFritz, 2011; Fritz et al., 2012a, b; Kindler et al.,2012).
In phylogenetic analyses of mtDNA se-quences, one of the Caucasian clades, cladeA, constitutes the sister group of the west-ern Mediterranean clade B (Fritz et al., 2007,2009; fig. 1), and another clade occurring inthe Caucasus region, clade C, is distributedover a highly disjunct range including parts ofthe Balkan peninsula, Turkey, the Russian andGeorgian Black Sea coast and the Caucasus.The fossil record suggests that this patchy rangeis a consequence of Pleistocene extinction (Fritzet al., 2007). These observations provide evi-dence that the mitochondrial clades within T.graeca are very old. Using a fossil-calibratedmolecular clock, Fritz et al. (2009) estimatedthat the six major clades of T. graeca haveevolved approximately 4.2-1.8 million yearsago. Despite these old and deep divergences,microsatellite differentiation is rather weak anddifferentiation patterns of AFLP and microsatel-lite markers are not congruent. This indicatesrepeated phases of extensive gene flow and vi-cariance, which is not surprising when the Pleis-
tocene history is considered. We hypothesizethat the deeply divergent mtDNA lineages re-flect old vicariance events, while the conflict-ing nuclear markers are the legacy of youngerepisodes of extensive gene flow. The differenti-ation pattern found in the AFLP markers, withAFLP groups matching allopatrically or para-patrically distributed mitochondrial lineages, islikely to be older than the microsatellite differ-entiation, which could correspond to Holocenerange expansions into Caucasian valleys andlower altitudes. It is expected that genealogiesof some nuclear genes will also deviate, id-iosyncratically reflecting individual gene trees,gene flow and recombination. This suggests thatcoalescent-based species delineation attempts(e.g. Yang and Rannala, 2010) will be fraughtwith difficulties in sexually reproducing organ-isms which experienced repeated switches be-tween vicariance and extensive gene flow, suchas the T. graeca complex. Any resulting classi-fication based on genetic coalescence along thetime axis necessarily has therefore to conflictwith population genetic differentiation and geneflow in the here and now.
Owing to the largely mutually exclusive dis-tribution ranges of the mtDNA lineages (fig. 3),the approach of Fritz et al. (2007, 2009), whohave identified distinct mtDNA lineages withsubspecies, is a reasonable and straightforwardclassification that facilitates communication andacknowledges on the one hand the deep mi-tochondrial divergences and on the other theconspecifity of the involved evolutionary units.Consequently, Transcaucasia constitutes an in-tergradation zone of Testudo graeca armeni-aca sensu Fritz et al. (2007), corresponding tomtDNA clade A, T. g. ibera sensu Fritz et al.(2007), corresponding to mtDNA clade C, andT. g. buxtoni sensu Fritz et al. (2007), corre-sponding to mtDNA clade E.
The observed differences among the threemarker systems (fig. 1) do not only provideinsights in biogeography and taxonomy. Theyalso underscore that the different modes of in-
346 V. Mashkaryan et al.
Figure 3. Distribution ranges of eastern Testudo graeca subspecies according to Fritz et al. (2007, 2009) and the present study.The distribution of T. graeca is shown in grey. Subspecies ranges are indicated, as far as known, by hatching; cross-hatching,secondary intergradation zones; question marks, unknown subspecies allocation.
heritance of mtDNA, AFLP and microsatellitemarkers may contribute to distinct differentia-tion patterns. Mitochondrial DNA is generallyinherited only through the maternal line (Bal-lard and Whitlock, 2004; Currat et al., 2008),while AFLP and microsatellite markers are bi-parentally inherited with dominant or codom-inant modes of inheritance, respectively (Bee-bee and Rowe, 2008). Mitochondrial DNA typ-ically experiences less gene flow, but intro-gresses more easily than nuclear DNA (Curratet al., 2008), and sex-specific differences in dis-persal may further contribute to mismatches be-tween the marker systems. This is in line withthe observation that males of T. graeca have dis-tinctly larger home ranges than females (Díaz-Paniagua, Keller and Andreu, 1995), suggestingthat gene flow is mainly male-mediated and thatthe persistence of deeply divergent mtDNA lin-eages is fostered by smaller home ranges of fe-males.
Acknowledgements. Christian Kehlmaier, Anke Müllerand Anja Rauh helped in the laboratory and ChristianKehlmaier genotyped and sequenced some of the sam-ples. Field work in Armenia and Georgia and part of thelaboratory work was funded by the Gulbenkian Founda-tion, Portugal (program ‘Preserving Armenian biodiver-sity: Joint Portuguese-Armenian program for training inmodern conservation biology’) and by the Fundação paraa Ciência e a Tecnologia, FCT, Portugal (PTDC/BIA-BEC/101256/2008). Melita Vamberger was funded by aPhD fellowship of the German Academic Exchange Service(DAAD; A/09/91179).
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Appendix. Studied samples of the Testudo graeca complex.
Population Lab code Collection site N E K = 3 K = 4 mtDNA clade
1 AC558 Georgia: Bolnisi 41.44 44.53 yellow admixed n/a2 AC073 Armenia: Tavush: Kokhb 40.90 45.39 yellow green C2 AC519 Armenia: Tavush: Kokhb 40.90 45.39 admixed admixed n/a2 AC520 Armenia: Tavush: Kokhb 40.90 45.39 blue blue n/a3 R-3 Armenia: Noyemberyan 41.18 45.01 n/a n/a C3 T1975 Armenia: Noyemberyan 41.18 45.01 n/a n/a C3 AC038 Armenia: Noyemberyan 41.18 45.01 yellow green C4 AC041 Armenia: Armavir 40.15 43.84 n/a n/a A4 AC042 Armenia: Armavir 40.15 43.84 n/a n/a A4 AC044 Armenia: Armavir 40.15 43.84 red red n/a4 AC045 Armenia: Armavir 40.15 43.84 yellow admixed n/a4 AC213 Armenia: Armavir 40.15 43.84 red red n/a4 AC064 Armenia: Armavir 40.15 43.84 yellow admixed n/a4 AC200 Armenia: Armavir 40.15 43.84 red red A4 AC201 Armenia: Armavir 40.15 43.84 yellow green n/a4 AC202 Armenia: Armavir 40.15 43.84 admixed blue A4 AC203 Armenia: Armavir 40.15 43.84 yellow green C4 AC204 Armenia: Armavir 40.15 43.84 yellow green C5 AC074 Armenia: Garni 40.12 44.74 red red A5 AC076 Armenia: Garni 40.12 44.74 red red n/a5 AC075 Armenia: Garni 40.12 44.74 n/a n/a A6 AC001 Armenia: Armavir: Vanand 40.11 43.82 red red A6 AC002 Armenia: Armavir: Vanand 40.11 43.82 red red n/a6 AC003 Armenia: Armavir: Vanand 40.11 43.82 red red n/a6 AC005 Armenia: Armavir: Vanand 40.11 43.82 red red n/a6 AC006 Armenia: Armavir: Vanand 40.11 43.82 n/a n/a A6 AC007 Armenia: Armavir: Vanand 40.11 43.82 red red n/a6 AC008 Armenia: Armavir: Vanand 40.11 43.82 red red n/a6 AC160 Armenia: Armavir: Vanand 40.11 43.82 red red n/a7 AC039 Armenia: Gorovan 39.89 44.71 red red A8 AC061 Armenia: Ararat: Urtsadzor 39.91 44.81 n/a n/a A8 AC063 Armenia: Ararat: Urtsadzor 39.91 44.81 n/a n/a A8 Ac065 Armenia: Ararat: Urtsadzor 39.91 44.81 n/a n/a A8 AC066 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a8 AC067 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a8 AC068 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a8 AC069 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a
350 V. Mashkaryan et al.
Appendix. (Continued.)
Population Lab code Collection site N E K = 3 K = 4 mtDNA clade
8 AC071 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a8 AC072 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a8 AC142 Armenia: Ararat: Urtsadzor 39.91 44.81 red admixed n/a8 AC143 Armenia: Ararat: Urtsadzor 39.91 44.81 red admixed n/a8 AC146 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a8 AC147 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a8 AC149 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a8 AC151 Armenia: Ararat: Urtsadzor 39.91 44.81 admixed admixed n/a8 AC153 Armenia: Ararat: Urtsadzor 39.91 44.81 blue blue n/a8 AC156 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a8 AC157 Armenia: Ararat: Urtsadzor 39.91 44.81 red red n/a8 AC158 Armenia: Ararat: Urtsadzor 39.91 44.81 admixed admixed n/a8 AC205 Armenia: Ararat: Urtsadzor 39.91 44.81 red red A8 AC206 Armenia: Ararat: Urtsadzor 39.91 44.81 n/a n/a A9 T-2 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 n/a n/a A9 AC009 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 admixed admixed n/a9 AC010 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue admixed A9 AC011 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue admixed n/a9 AC012 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue blue n/a9 AC013 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue yellow n/a9 AC014 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 admixed admixed n/a9 AC015 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue admixed n/a9 AC017 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue admixed A9 AC018 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue yellow n/a9 AC019 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue blue A9 AC034 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue admixed n/a9 AC207 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue blue A9 AC208 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue blue A9 AC209 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue blue A9 AC210 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue blue n/a9 DB7821 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue blue A9 AC211 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 admixed admixed A9 AC212 Nagorno Karabakh: Kashatagh: Tcobi 39.02 46.65 blue blue n/a
