Mitochondrial diversity of the widespread Central Asian …...Yoshan Moodley7, Attaullah Pindrani8,...
Transcript of Mitochondrial diversity of the widespread Central Asian …...Yoshan Moodley7, Attaullah Pindrani8,...
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Amphibia-Reptilia 30 (2009): 245-257
Mitochondrial diversity of the widespread Central Asian steppetortoise (Testudo horsfieldii Gray, 1844): implications for taxonomy
and relocation of confiscated tortoises
Uwe Fritz1,*, Markus Auer1, Marina A. Chirikova2, Tatyana N. Duysebayeva2,
Valery K. Eremchenko3, Haji Gholi Kami4, Roman D. Kashkarov5, Rafaqat Masroor6,
Yoshan Moodley7, Attaullah Pindrani8, Pavel Široký9, Anna K. Hundsdörfer1
Abstract. Using a nearly range-wide sampling, we investigated phylogeographic differentiation and mitochondrial diversityof Testudo horsfieldii, the only tortoise species confined to Central Asia. We identified three major haplotype clades withmainly parapatric distribution that do not correspond well to the currently recognized three subspecies. One clade is restrictedto the Fergana Valley and seems to represent a previously overlooked evolutionarily significant unit. Another clade, consistingof several largely parapatrically distributed haplotypes, occurs in the north and the central southern part of the species’ range.The third clade, likewise comprising several largely parapatrically distributed haplotypes, was identified from the south-eastern corner of the Caspian Sea in the west, from Afghanistan and Pakistan in the east and from two more northerlysites in western and south-eastern Uzbekistan. It is possible that this clade also occurs in eastern Turkmenistan and adjacentAfghanistan, regions not sampled for the present study. The generally parapatric distribution of individual haplotypes, evenwithin each of the three major clades, suggests advanced lineage sorting, either due to limited dispersal abilities, glacialisolation in distinct local microrefuges or both acting in accord. The localized distribution of endemic haplotypes in thenorthern and central plains as well as in the mountainous eastern and southern parts of the distribution range supports theexistence of multiple microrefuges there. Records of haplotypes of distinct clades in sympatry or close geographic proximityare likely the result of Holocene range expansions. In recent years, thousands of confiscated steppe tortoises were released intothe wild. The detected mitochondrial differentiation offers a powerful tool for nature conservation, as a means of determiningthe geographic origin of confiscated tortoises and selecting suitable reintroduction regions.
Keywords: Central Asia, phylogeography, subspecies, Testudo horsfieldii horsfieldii, Testudo horsfieldii kazachstanica,Testudo horsfieldii rustamovi.
1 - Museum of Zoology (Museum für Tierkunde), NaturalHistory State Collections Dresden, A.B. Meyer Build-ing, D-01109 Dresden, Germany
2 - Institute of Zoology, Ministry of Education and Science,Al-Faraby av. 93, Almaty, 050060, Kazakhstan
3 - NABU Central Asia, Tabachnaya Street 24, Bishkek,720011, Kyrgyz Republic
4 - Department of Biology, Faculty of Sciences, GorganUniversity of Agricultural Sciences and Natural Re-sources, Gorgan, Golestan Province, Iran
5 - Department of Zoology, Biological Faculty, Na-tional University of Uzbekistan, Vuzgorodok, Tashkent,100174, Uzbekistan
6 - Pakistan Museum of Natural History, Shakarparian,Garden Avenue, Islamabad, Pakistan
7 - Max Planck Institute for Infection Biology, Departmentof Molecular Biology, Charitéplatz 1, D-10117 Berlin,Germany
8 - Sustainable Use Specialist Group Central Asia (SUSG-CAsia), Habitat and Species Conservation Project,BRSP House, 5-A Saryab Road, PO Box 216, Quetta,Pakistan
Introduction
The small to medium-sized Testudo horsfieldiiGray, 1844 (maximum shell length approx. 30cm) is the only tortoise species confined toCentral Asia. Its range encompasses easternIran, Afghanistan, Turkmenistan, Uzbekistan,Tajikistan, Kyrgyzstan, southern Kazakhstan,the westernmost part of Xinjiang (China),and in Pakistan part of the region borderingAfghanistan (Bannikov et al., 1977; Iverson,1992; Kuzmin, 2002; Fritz and Havaš, 2007).This is an area of about 2500 km in an east-west
9 - Department of Biology and Wildlife Diseases, Facultyof Veterinary Hygiene and Ecology, University of Vet-erinary and Pharmaceutical Sciences, Palackého 1-3,CZ-612 42 Brno, Czech Republic∗Corresponding author; e-mail:[email protected]
© Koninklijke Brill NV, Leiden, 2009. Also available online - www.brill.nl/amre
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direction and 1500 km in a north-south direc-tion. The natural history of the steppe tortoisediffers significantly from that of its Mediter-ranean congeners. Testudo horsfieldii is a spe-cialized burrow-dwelling steppicolous and de-serticolous species with a very short activity pe-riod to cope with extreme summer heat and win-ter cold (Kami, 1999; Kuzmin, 2002; Lagardeet al., 2002). For instance, steppe tortoises areactive for less than three months in southernUzbekistan (Lagarde et al., 2002). As requiredby their burrow-digging mode of life, steppetortoises have a distinctive morphology. Theirstrong fore-feet are shovel-like with reduceddigits and phalanges; the shell is almost as longas broad and conspicuously depressed (Ban-nikov et al., 1977; Ernst et al., 2000; Kuzmin,2002; Hitschfeld et al., 2008). Due to the mor-phological distinctiveness of T. horsfieldii, itsgeneric allocation to Testudo has repeatedlybeen challenged. However, removal from thisgenus seems unwarranted since all phylogeneticanalyses using a five-gene data set (mtDNA:12S rRNA, 16S rRNA, cyt b; nDNA: C-mos,Rag2) of approximately two-thirds of all testu-dinid species consistently found the five Testudospecies monophyletic, albeit with moderate sup-port values (Fritz and Bininda-Emonds, 2007).
In contrast to many Mediterranean species(e.g., Weiss and Ferrand, 2006; Schmitt, 2007),little is known about the phylogeography ofCentral Asian species (Fritz et al., 2008), mak-ing T. horsfieldii an attractive object for suchinvestigations. In the Mediterranean, phylogeo-graphic structures of widespread reptiles gen-erally correlate with their glacial refugia (e.g.,Joger et al., 2007; Schmitt 2007; Weiss andFerrand, 2007). Therefore, we expect that thephylogeography of a widely distributed CentralAsian species like T. horsfieldii could be help-ful in locating its refuges as well. Moreover, ob-served phylogeographic structures should cor-respond to subspecific differentiation, whereasconflicts between subspecies delineations andphylogeographic patterns often provide evi-dence for the need of taxonomic revisions
(Avise, 2000; Zink and Barrowclough, 2008).Currently, three subspecies of T. horsfieldii arerecognized (Fritz and Havaš, 2007), and thesediffer mainly in shell shape, size and col-oration (Chkhikvadze, 1988; Chkhikvadze etal., 1999; Kuzmin, 2002). However, such char-acters proved to be of little help for reveal-ing evolutionarily significant units in other tes-tudinids (Testudo: Carretero et al., 2005; Fritzet al., 2005, 2006, 2007; Attum et al., 2007;Široký and Fritz, 2007; Indotestudo: Ives etal., 2008). Although the exact distribution ofthe three putative T. horsfieldii subspecies isnot entirely clear, T. h. kazachstanica (Chkhik-vadze, 1988) is thought to occur in the north-ern part of the range; T. h. rustamovi (Chkhik-vadze, Amiranashvili and Ataev, 1990) in south-western Turkmenistan, adjacent Iran and south-western Kazakhstan; and T. h. horsfieldii Gray,1844 in the rest of the species’ range (Fritzand Havaš, 2007; Vasilyev et al., 2008). A fewyears ago, it was proposed that these three sub-species, as well as Testudo baluchiorum An-nandale, 1906, described from what is now theAfghan-Pakistani border region and previouslyconsidered a junior synonym of T. h. horsfieldii,represent distinct species (Perälä, 2002; Vet-ter, 2002). But a recent investigation using 12SrRNA sequences of 59 samples from Iran, east-ern Uzbekistan and eastern Kazakhstan foundlittle variation and concluded that the observedvariation supports conspecificity of the studiedpopulations (Vasilyev et al., 2008). In this pa-per, only four haplotypes differing by 1-3 siteswere identified. One haplotype occurred in allnorthern sites of Uzbekistan and Kazakhstan;the other three were found in four localities inIran and south-easternmost Uzbekistan. How-ever, the 12S rRNA gene is slowly evolving andonly moderately informative in revealing phy-logeographic differentiation of chelonians. Forinstance, low variation was found among 12SrRNA sequences of T. graeca (van der Kuyl etal., 2002; Harris et al., 2003), while the morevariable mitochondrial cytochrome b gene (cytb) yielded a much more detailed and clearly
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structured phylogeographic pattern (Fritz et al.,2007, 2009).
In the present paper, we use cyt b sequencevariation in a nearly range-wide sampling of thesteppe tortoise to address the following ques-tions: (i) is T. horsfieldii a phylogeographi-cally structured species, and if so, (ii) does thephylogeographic pattern correlate with putativeglacial refuges, and (iii) does it match the rangesof the currently recognized subspecies?
Beyond science, a better knowledge of thephylogeography of T. horsfieldii could alsodirectly improve future conservation strate-gies. In the former Central Asian Soviet Re-publics, the species is massively harvested forthe pet-trade. As corollary of the implementa-tion of CITES, thousands of confiscated tor-toises have been released into the wild, of-ten without knowledge of their geographic ori-gin (M.A. Chirikova, T.N. Duysebayeva, V.K.Eremchenko, R.D. Kashkarov, own observ.; T.Harder, pers. comm.) and risking ‘genetic pol-lution’ of native populations. Given that a clearphylogeographic structure exists, mitochondrialhaplotyping of confiscated tortoises would helpto determine their geographic origin and to se-lect suitable reintroduction regions.
Materials and methods
Sampling
Eighty blood or saliva samples of Testudo horsfieldii werefield-collected, representing populations of most of thespecies’ range and all three currently recognized subspecies(samples were assigned to subspecies according to their ge-ographic origin). In addition, nine cyt b sequences fromGenBank and 11 tissue samples of pet-trade tortoises fromthe collection of the Museum of Zoology Dresden wereused. This total sample corresponds to 58 sampling sites(Appendix).
DNA extraction, PCR and sequencing
Total genomic DNA was extracted by overnight incubationat 55◦C in lysis buffer (6% DTAB, 1.125 M NaCl, 75 mMTris-HCl, 37.5 mM EDTA, pH 8.0) including 0.5 mg of pro-teinase K (Merck, Whitehouse Station, NJ) and subsequentpurification following the DTAB method (Gustincich et al.,1991). DNA was precipitated from the supernatant with 0.2
volumes of 4 M LiCl and 0.8 volumes of isopropanol, cen-trifuged, washed, dried and resuspended in TE buffer.
Our target sequence was an mtDNA fragment contain-ing the complete cyt b gene and approximately 20 bp ofthe adjacent tRNA-Thr gene. For polymerase chain reaction(PCR) and sequencing, the primers CytbG (Spinks et al.,2004), mt-c-For2, mt-f-na3, and mt-E-Rev2 (Praschag et al.,2007) were used. PCR amplification, PCR product purifi-cation and sequencing followed Praschag et al. (2007). Se-quencing was performed on an ABI 3130 (Applied Biosys-tems, Foster City, CA). None of the sequences contained in-ternal stop codons, and nucleotide frequencies correspondedto those of coding mtDNA; therefore we conclude to haveamplified and sequenced mtDNA and not nuclear copies ofmitochondrial genes.
Sequence analyses
The 100 Testudo horsfieldii sequences were manually col-lapsed into haplotypes, resulting in 26 distinct haplotypes.Six of these haplotypes are GenBank sequences and 15 hap-lotypes were found only once (Appendix). Five of the Gen-Bank haplotypes were represented by 429-bp-long to 1063-bp-long sequences only, while the sixth GenBank haplo-type and our own sequences were 1167 bp long. Phyloge-netic analyses were run for two data sets, one including all26 haplotypes and another with the shorter GenBank hap-lotypes removed. Data were analysed under the optimalitycriteria Maximum Parsimony (MP; equal weighting, com-mand: hs add = cl) and Maximum Likelihood (ML) usingPAUP*4.0b10 (Swofford, 2002) and under Bayesian infer-ence of phylogeny (BA) using MrBayes 3.1 (Ronquist andHuelsenbeck, 2003; settings: ngen = 10 000 000 nchains =4 nrun = 1 sample = 500 temp = 0.2 savebrlens = yes start-ingtree = random; burn-in set to sample only the plateauof most likely trees). Two Genbank sequences of a closelyand a distantly related testudinid species served as out-groups (AM230496, Testudo h. hermanni; DQ497301, Stig-mochelys p. pardalis). Testudo hermanni is the sister speciesof T. horsfieldii (Fritz and Bininda-Emonds, 2007). For thedata set of 26 haplotypes, 1103 of 1167 aligned characterswere constant in the ingroup sequences; 31 characters werevariable and parsimony-informative; 33 variable characterswere singletons. In the smaller data set, 1122 sites wereconstant; 25 were variable and parsimony-informative and20 variable characters were parsimony-uninformative. Thebest evolutionary model was established using Modeltest3.06 (Posada and Crandall, 1998; AIC best-fit model: TrN +I). Bootstrap support values were obtained in PAUP*4.0b10(MP: nreps = 1000, maxtree = 100; ML: nreps = 100, max-tree = 1). Uncorrected p distances were calculated usingMEGA 3.1 (Kumar et al., 2004).
Within the same species or between closely relatedspecies, relationships of haplotypes are likely to be retic-ulate and ancestral haplotypes may persist, which is why in-traspecific gene evolution may be only imperfectly reflectedby dichotomous trees (Posada and Crandall, 2001). There-fore, we also calculated a parsimony network using TCS1.21 (Clement et al., 2000). This software is based on sta-tistical parsimony and connects haplotypes via a minimal
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number of mutational steps and allows for alternative path-ways. A further advantage of network analyses is that infor-mation about the age of haplotypes may be obtained. Interi-orly located haplotypes, having more than one mutationalconnection, are thought to be ancestral to and older thantip haplotypes (Posada and Crandall, 2001). To avoid am-biguities caused by much shorter GenBank sequences, weincluded in network analysis only the 91 sequences of 1167bp length produced in this investigation, the correspondingfragment of the complete mitochondrial genome from Gen-Bank (DQ080045), and in the last position of the data set,the 1140-bp-long GenBank sequence DQ497322.
Further, population-based analyses were performed forthe 1167-bp-long sequences to better elucidate past demo-graphic events as well as population structure. The softwareDnaSP 4.1 (Rozas et al., 2003) was used to carry out mis-match distribution analyses (Rogers and Harpending, 1992)as well as Fu and Li’s (1993) tests for neutrality. A Man-tel regression of a pairwise geographic distance matrix (thatis, great-arc distances obtained from Cartesian co-ordinates)with a pairwise genetic matrix of uncorrected p distances,
obtained in MEGA 3.1, was performed to ascertain if thedistribution of genetic diversity across the species was spa-tially correlated.
Results
For both data sets, all phylogenetic analyses re-vealed the same three major clades (indicatedin red, blue and black in fig. 1). The basalbranching pattern of these three major cladesvaried between the tree-building methods, how-ever. While ML suggested, with weak support,that the ‘red clade’ forms the sister group of aclade comprising the ‘blue clade’ and the ‘blackclade’, Bayesian analysis placed all of the threemajor clades in a basal polytomy. Under MP,
Figure 1. One of nine ML trees for 26 mtDNA haplotypes of Testudo horsfieldii (1167 bp, cyt b and partial tRNA-Thrgenes; − ln L = 2990.5257). The other eight ML trees differ only in the branching pattern within the ‘red clade’. Majorhaplotype clades indicated on the right. Individual known-locality haplotypes are indicated by letters; haplotypes identifiedin more than one individual in upper case and bold; haplotypes identified only once, lower case. For haplotypes of unknowngeographic origin, GenBank accession numbers or voucher numbers are given (MTD T = Museum of Zoology Dresden,tissue collection). AJ888365, AY678410, AY678438, ‘r’, and ‘s’ are haplotypes represented by shorter GenBank sequences(429-1063 bp). Numbers above nodes are ML bootstrap values; below nodes, Bayesian posterior probabilities and MPbootstraps. The large asterisk indicates this branch was not found under BA and MP (see text). For the data set of 21haplotypes, one ML tree with the same major topology was obtained (− ln L = 2815.0805). Support values were for this dataset (ML/BA/MP): red clade, 72/0.85/70; blue clade, 92/0.93/92, black clade, 95/1.0/95; under ML: (blue clade + blackclade), 52; under MP: (red clade + black clade), 53.
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Table 1. Uncorrected p distances (percentages) of the three Testudo hors-fieldii clades (all 26 haplotypes). Below diagonal, distances between clades;on the diagonal, within-clade distances in bold (average and ranges).
Red Blue Black
Red 0.50 (0.09-1.37)Blue 1.01 (0.60-1.65) 0.09 (–)Black 1.57 (0.72-2.79) 1.31 (0.94-2.23) 1.02 (0.09-2.03)
24 equally parsimonious trees were obtained forthe 26 haplotype data set (tree length = 294;CI = 0.9252, RI = 0.8866); the 21 haplotypedata set resulted in two equally parsimonioustrees (tree length = 272; CI = 0.8105, RI =0.8896). In all MP trees, a basal position ofthe ‘blue clade’ and a sister group relationshipof the ‘red clade’ and the ‘black clade’ werefavoured, although with weak bootstrap support(
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Figure 2. Geographic distribution of mtDNA haplotypes of Testudo horsfieldii. Colours correspond to other figures. Noteoccurrence of haplotypes of the ‘red clade’ and the ‘black clade’ in the same sites or in close geographic proximity. Simplifiedrange borders of the three currently recognized subspecies indicated. Stars represent type localities: 1 – T. h. rustamovi(Chkhikvadze, Amiranashvili and Ataev, 1990); 2 – T. h. kazachstanica (Chkhikvadze, 1988); 3 – T. h. horsfieldii Gray, 1844;4 – ‘T. baluchiorum Annandale, 1906’.
population is expected to result in a peak inthe distribution of pairwise differences (plottedred lines) between sequences (Rogers and Harp-ending, 1992), whereas a population maintain-ing a constant size through time is expected tofollow a trend similar to that plotted by bluelines. Analyses of the entire data set, as wellas for each clade, showed a large proportion ofclosely related haplotypes. However, a past sud-den population expansion is most likely to haveoccurred in the ‘black clade’, while the ‘redclade’ shows moderate signs of demographicexpansion. The ‘blue clade’ perfectly fits themodel of constant population size. Fu and Li’s(1993) D* and F* indices of neutrality were allslightly negative for all tested groups (fig. 4),and in the case of a neutral marker such as mi-tochondrial DNA, this indicates a greater num-ber of pairwise differences relative to segregat-ing sites and a possibly expanding population.
However, none of these indices were significant.A Mantel regression found that only 24% of thevariation in the genetic data could be attributedto geographic variation, precluding the assump-tion of a clinal population structure of isolation-by-distance.
Discussion
Although we found only moderate variation inthe studied mtDNA fragment, our results pro-vide evidence that Testudo horsfieldii is a phylo-geographically structured species. We identifiedthree major clades of haplotypes with mainlyparapatric ranges. Even within each of the threemajor clades, the more frequent haplotypes havelargely parapatric distributions, suggestive ofadvanced lineage sorting, either due to lim-ited dispersal abilities, glacial isolation in dis-
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Figure 3. Parsimony network of 93 mtDNA sequencesof Testudo horsfieldii (1167 bp, cyt b and partial tRNA-Thr genes; spring tree). Colours correspond to other fig-ures; symbol size, approximate haplotype frequency; miss-ing node haplotypes, open circles. Each line joining hap-lotypes indicates one substitution except when hatchmarksacross lines are present; then each mark indicates one step.Only known-locality haplotypes bear letters. Haplotype fre-quencies are B: n = 4; D: n = 14; E: n = 3; F: n = 27; I:n = 12; K: n = 7; M: n = 2; O: n = 6; P: n = 6; T: n = 2.Other haplotypes were identified only once.
tinct local microrefuges or both acting in con-cert. The average uncorrected p distances of thethree clades (table 1) fall within the range ob-served between North African subspecies of T.graeca, arguing for Early to Middle Pleistocenedifferentiation in both cases and in favour ofrepeated glacial fragmentation (cf. Fritz et al.,2009). Furthermore, the localized distributionof endemic haplotypes in the northern and cen-tral plains as well as in the mountainous easternand southern parts of the distribution range ofT. horsfieldii supports the existence of multiplemicrorefuges. This is in line with our results of
the Mantel test, indicating discontinuous diver-gence in geographic distribution.
The three major clades of T. horsfieldii do notagree well with its currently recognized threesubspecies and also do not support the validityof T. baluchiorum Annandale, 1906, describedfrom the Afghan-Pakistani border region (fig.2). One of our three major clades, the ‘blueclade’ from the Fergana Valley within the rangeof T. h. kazachstanica, seems to represent apreviously overlooked evolutionarily significantunit. Its occurrence in the Fergana Valley sug-gests that microrefuges were located not onlywithin the extant southern range, but also fairlyfar north. The ‘blue clade’, isolated in the Fer-gana Valley, has maintained a constant popu-lation size throughout its demographic history,while the other clades show at least some evi-dence of recent population expansion that seemsto be related to Holocene warming and range ex-tension.
Except the Fergana Valley, the distribution ofour ‘red clade’ in the north matches the rangeof T. h. kazachstanica and the distribution ofthe northern 12S rRNA haplotype of Vasilyev etal. (2008). However, in the south the same ‘redclade’ penetrates deep into the putative rangesof T. h. horsfieldii and T. h. rustamovi, propos-ing a distinctly larger range of T. h. kazachstan-ica than thought before. The other haplotypesidentified from the ranges of T. h. horsfieldii andT. h. rustamovi represent another major clade(‘black clade’), suggesting weak differentiationand synonymy of both subspecies. In some sitesin Iran and western and south-eastern Uzbek-istan, haplotypes of the ‘black clade’ were re-corded together with or in close proximity tohaplotypes of the ‘red clade’. This, as well asthe record of a southern 12S rRNA haplotypein south-eastern Uzbekistan (Vasilyev et al.,2008), may indicate the existence of secondarycontact zones due to Holocene range expansionsand a wider distribution of the ‘black clade’in Turkmenistan and northern Afghanistan, re-gions not sampled for the present study.
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Figure 4. Mismatch distributions and demographic indices for the entire data set (panel A) and for each of the threephylogenetic clades (panels B-D). Observed mismatch distributions are graphed in grey columns, whereas line distributionscorrespond to those expected under a model of constant population size (blue) and a model of population expansion (red).NS, not significant.
In the former Central Asian Soviet Republics,steppe tortoises have been heavily exploited forthe pet-trade for decades. From 1976 to 1993,more than one million steppe tortoises werecollected in Kazakhstan alone (United NationsEnvironment Programme, World ConservationMonitoring Centre, 2004). With the implemen-tation of CITES, collection and export quo-tas were introduced (Rhodin, 2003; Bykova etal., 2007). For instance, in 2002 Kazakhstan,Tajikistan and Uzbekistan had export quotasof 40 000, 20 000 and 30 000 live tortoises, re-spectively (United Nations Environment Pro-gramme, World Conservation Monitoring Cen-tre, 2004). Because of such harvest quotas andpoaching, the number of confiscated tortoisesincreased considerably and thousands of confis-cated tortoises were released into the wild, of-ten without knowledge of their geographic ori-
gin (M.A. Chirikova, T.N. Duysebayeva, V.K.Eremchenko, R.D. Kashkarov, own observ.; T.Harder, pers. comm.). While we excluded fromsampling any sites that could harbour such re-leased tortoises, it is obvious that the currentrepatriation practice bears the risk of admixtureof distinct genetic lineages and should not becontinued.
Conclusions
Testudo horsfieldii is a phylogeographicallystructured species. Its differentiation patternstrongly suggests that the current diversity wasshaped by glacial range interruptions. Refugesand microrefuges were most probably not onlylocated in the south of the extant range, butalso fairly far north. The weak agreement be-tween subspecies and phylogroups of T. hors-
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fieldii suggests that the morphological char-acters used for subspecies delineation are oflimited taxonomic value, as in other Testudospecies (Carretero et al., 2005; Fritz et al., 2005,2006, 2007, 2009; Attum et al., 2007; Širokýand Fritz, 2007). This is also underlined bythe fact that some allegedly diagnostic osteo-logical characters of T. horsfieldii subspecies(Perälä, 2002) were found to be ontogeneticvariation (Hitschfeld et al., 2008). One of thethree mtDNA clades identified in the present pa-per seems to represent a previously overlookedevolutionarily significant unit. Future investiga-tions should re-analyze morphological variationin the light of phylogeographic differentiation tounravel the impact of ecological constraints andancestry.
The phylogeographic pattern of T. horsfieldiiargues for great caution with further releasesof confiscated steppe tortoises without knowl-edge of their origin. However, our study of-fers a highly conservation-relevant applicationof phylogeography. Mitochondrial haplotypingof confiscated tortoises would allow for a muchbetter selection of suitable releasing sites, basedon the largely allopatric distribution of individ-ual haplotypes.
Acknowledgements. Thorsten Harder (NABU CentralAsia) helped with field work in Kyrgyzstan and providedvaluable information. Thanks go to the local nature conser-vation authorities and the German Agency for Nature Con-servation (BfN) for permits. Lab work was done by AnkeMüller (Museum of Zoology Dresden).
References
Attum, O., Baha el Din, S., Carranza, S., Earley, R., Arnold,E.N., Kingsbury, B. (2007): An evaluation of the tax-onomic validity of Testudo werneri. Amphibia-Reptilia28: 393-401.
Avise, J.C. (2000): Phylogeography. The History and For-mation of Species. Cambridge, MA and London, Har-vard University Press.
Bannikov, A.G., Darevskii, I.S., Ishchenko, V.G., Rustamov,A.K., Shcherbak, N.N. (1977): Opredelitel’ zemnovod-nykh i presmykayushchikhsya fauny SSSR. Moscow,Prosveshcheniye.
Bykova, E.V., Sorochinsky, V.G., Golenkevich, A.V., Pere-gontsev, E.A., Sorochinskaya, I.N., Sorochinsky, G.Y.(2007): Ranching as a method of conservation of thewild population of Horsfield’s tortoise, Agrionemyshorsfieldii. Russ. J. Herpetol. 14: 232-236.
Carretero, M.A., Znari, M., Harris, D.J., Macé, J.C. (2005):Morphological divergence among populations of Tes-tudo graeca from west-central Morocco. Animal Biol.55: 259-279.
Chkhikvadze, V.M. (1988): O sistematicheskom polozheniisovremennykh sukhoputnykh cherepakh Srednei Azii iKazakhstana. Izv. Akad. Nauk GSSR, Ser. biol. 14: 110-113.
Chkhikvadze, V.M., Amiranashvili, N.G., Ataev, Ch.(1990): Novyi podvid sukhoputnoi cherepakhi iz yugo-zapadnogo Turkmenistana. Izv. Akad. Nauk Turkmensk.SSR, Ser. biol. 1: 72-75.
Clement, M., Posada, D., Crandall, K.A. (2000): TCS: acomputer program to estimate gene genealogies. Mol.Ecol. 9: 1657-1660.
Ernst, C.H., Altenburg, R.G.M., Barbour, R.W. (2000): Tur-tles of the World. World Biodiversity Database, CD-ROM Series, Windows Version 1.2. Amsterdam, Biodi-versity Center of ETI.
Fritz, U., Bininda-Emonds, O.R.P. (2007): When genesmeet nomenclature: Tortoise phylogeny and the shiftinggeneric concepts of Testudo and Geochelone. Zoology110: 298-307.
Fritz, U., Havaš, P. (2007): Checklist of chelonians of theworld. Vertebr. Zool. 57: 149-368.
Fritz, U., Široký, P., Kami, H., Wink, M. (2005): Environ-mentally caused dwarfism or a valid species – Is Testudoweissingeri Bour, 1996 a distinct evolutionary lineage?New evidence from mitochondrial and nuclear genomicmarkers. Mol. Phylogenet. Evol. 37: 389-401.
Fritz, U., Auer, M., Bertolero, A., Cheylan, M., Fattizzo, T.,Hundsdörfer, A.K., Martín Sampayo, M., Pretus, J.L.,Široký, P., Wink, M. (2006): A rangewide phylogeog-raphy of Hermann’s tortoise, Testudo hermanni (Rep-tilia: Testudines: Testudinidae): implications for taxon-omy. Zool. Scr. 35: 531-543.
Fritz, U., Hundsdörfer, A.K., Široký, P., Auer, M., Kami,H., Lehmann, J., Mazanaeva, L.F., Türkozan, O., Wink,M. (2007): Phenotypic plasticity leads to incongruencebetween morphology-based taxonomy and genetic dif-ferentiation in western Palaearctic tortoises (Testudograeca complex; Testudines, Testudinidae). Amphibia-Reptilia 28: 97-121.
Fritz, U., Ayaz, D., Buschbom, J., Kami, H.G., Mazanaeva,L.F., Aloufi, A.A., Auer, M., Rifai, L., Šilić, T., Hunds-dörfer, A.K. (2008): Go east: Phylogeographies ofMauremys caspica and M. rivulata – Discordance ofmorphology, mitochondrial and nuclear genomic mark-ers and rare hybridization. J. Evolution. Biol. 21: 527-540.
Fritz, U., Harris, D.J., Fahd, S., Rouag, R., Graciá Martínez,E., Giménez Casalduero, A., Široký, P., Kalboussi, M.,Jdeidi, T.B., Hundsdörfer, A.K. (2009): Mitochondr-ial phylogeography of Testudo graeca in the WesternMediterranean: Old complex divergence in North Africaand recent arrival in Europe. Amphibia-Reptilia 30: 63-80.
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254 U. Fritz et al.
Fu, Y.-X., Li, W.-H. (1993): Statistical tests of neutrality ofmutations. Genetics 133: 693-709.
Gustincich, S., Manfioletti, G., del Sal, G., Schneider, C.,Carninci, C. (1991): A fast method for high-qualitygenomic DNA extraction from whole human blood.BioTechniques 11: 298-302.
Harris, D.J., Znari, M., Macé, J.-C., Carretero, M.A. (2003):Genetic variation in Testudo graeca from Morocco esti-mated using 12S rRNA sequencing. Rev. Esp. Herpetol.17: 5-9.
Hitschfeld, E., Auer, M., Fritz, U. (2008): Phalangeal for-mulae and ontogenetic variation of carpal morphology inTestudo horsfieldii and T. hermanni. Amphibia-Reptilia29: 93-99.
Iverson, J.B. (1992): A Revised Checklist with DistributionMaps of the Turtles of the World. Richmond, IN, Pri-vately Printed.
Ives, I., Spinks, P.Q., Shaffer, H.B. (2008): Morphologicaland genetic variation in the endangered Sulawesi tortoiseIndotestudo forstenii. Conserv. Genet. 9: 709-713.
Joger, U., Fritz, U., Guicking, D., Kalyabina-Hauf, S.,Nagy, Z.T., Wink, M. (2007): Phylogeography of west-ern Palaearctic reptiles – Spatial and temporal speciationpatterns. Zool. Anz. 246: 293-313.
Kami, H.G. (1999): On the biology of the Afghan Tortoise,Testudo horsfieldi, in north-eastern Iran. Zool. MiddleEast 19: 43-54.
Kumar, S., Tamura, K., Nei, M. (2004): MEGA3: Integratedsoftware for Molecular Evolutionary Genetics Analysisand sequence alignment. Brief. Bioinform. 5: 150-163.
van der Kuyl, A.C., Ballasina, D.L.P., Dekker, J.T., Maas,H., Willemsen, R.E., Goudsmit, J. (2002): Phylogeneticrelationships among the species of the genus Testudo(Testudines: Testudinidae) inferred from mitochondrial12S rRNA gene sequences. Mol. Phylogenet. Evol. 22:174-183.
Kuzmin, S.L. (2002): The Turtles of Russia and Other Ex-Soviet Republics. Frankfurt am Main, Chimaira.
Lagarde, F., Bonnet, X., Nagy, K., Henen, B., Corbin, J.,Naulleau, G. (2002): A short spring before a long jump:the ecological challenge in the steppe tortoise (Testudohorsfieldi). Can. J. Zool. 80: 493-502.
Perälä, J. (2002): Biodiversity in relatively neglected taxa ofTestudo L., 1758 s. l. Chelonii 3: 40-53.
Posada, D., Crandall, K.A. (1998): Modeltest: testing themodel of DNA substitution. Bioinformatics 14: 817-818.
Posada, D., Crandall, K.A. (2001): Intraspecific gene ge-nealogies: trees grafting into networks. Trends Ecol.Evol. 16: 37-45.
Praschag, P., Hundsdörfer, A.K., Fritz, U. (2007): Phy-logeny and taxonomy of endangered South and South-east Asian freshwater turtles elucidated by mtDNA se-quence variation (Testudines: Geoemydidae: Batagur,Callagur, Hardella, Kachuga, Pangshura). Zool. Scr.36: 429-442.
Rhodin, A.G.J. (2003): Conservation and Trade of AsianFreshwater Turtles and Tortoises: Updated Reviewof Status and Threats with Recommendations forCITES Listings. Geneva, CITES. Available at http://www.cites.org/eng/com/ac/19/E19-15-1.doc.
Rogers, A.R., Harpending, H. (1992): Population growthmakes waves in the distribution of pairwise geneticdifferences. Mol. Biol. Evol. 9: 552-569.
Ronquist, F., Huelsenbeck, J.P. (2003): MrBayes 3: Bayes-ian phylogenetic inference under mixed models. Bioin-formatics 19: 1572-1574.
Rozas, J., Sanchez-DelBarrio, J.C., Messeguer, X., Rozas,R. (2003): DnaSP, DNA polymorphism analyses by thecoalescent and other methods. Bioinformatics 19: 2496-2497.
Schmitt, T. (2007): Molecular biogeography of Europe:Pleistocene cycles and postglacial trends. Front. Zool.4 (11): 1-12.
Široký, P., Fritz, U. (2007): Is Testudo werneri a distinctspecies? Biologia 62: 228-231.
Spinks, P.Q., Shaffer, H.B., Iverson, J.B., McCord, W.P.(2004): Phylogenetic hypotheses for the turtle familyGeoemydidae. Mol. Phylogenet. Evol. 32: 164-182.
Swofford, D.L. (2002): PAUP*. Phylogenetic Analysis Us-ing Parsimony (*and Other Methods), Version 4. Sun-derland, MA, Sinauer Associates.
United Nations Environment Programme, World Conser-vation Monitoring Centre (2004): Review of Signifi-cant Trade. Analysis of Trade Trends with Notes onthe Conservation Status of Selected Species. AnnexC: Reptiles and Amphibians. Geneva, CITES. Avail-able at http://www.cites.org/common/com/ac/20/E20-inf-12.pdf.
Vasilyev, V.A., Bondarenko, D.A., Peregontsev, E.A.,Voronov, A.S., Ryskov, A.P., Semenova, S.K. (2008):Polimorfism gena 12S rRNK i filogeografiya sredneazi-atskoy cherepakhi Agrionemys horsfieldii Gray, 1844.Genetika 55: 784-788.
Vetter, H. (2002): Terralog: Turtles of the World, Vol. 1.Africa, Europe and Western Asia. Frankfurt am Main,Chimaira.
Weiss, N., Ferrand, N. (2007): Phylogeography of South-ern European Refugia. Berlin, Heidelberg, New York,Springer.
Zink, R.M., Barrowclough, G.F. (2008): MitochondrialDNA under siege in avian phylogeography. Mol. Ecol.17: 2107-2121.
Received: September 10, 2008. Accepted: December 22,2008.
-
Diversity of Testudo horsfieldii 255A
ppen
dix.
Sam
ples
and
Gen
Ban
kse
quen
ces
ofTe
stud
oho
rsfie
ldii
and
thei
rha
plot
ypes
.Sam
ple
num
bers
are
eith
erM
TD
Tnu
mbe
rs(M
useu
mof
Zoo
logy
Dre
sden
,tis
sue
colle
ctio
n)or
Gen
Ban
kac
cess
ion
num
bers
.Hap
loty
pes
iden
tified
inm
ore
than
one
case
inbo
ld.A
cces
sion
num
bers
ofha
plot
ypes
iden
tified
inth
isst
udy
are
FM88
3673
-FM
8836
92.
Sam
ple
Subs
peci
esL
ocal
ityL
atitu
deL
ongi
tude
Hap
loty
pe
5034
hors
field
iiA
fgha
nist
an:G
hazn
i33
◦ 33′
N68
◦ 26′
En
3049
hors
field
iiIr
an:K
hora
san:
Sarb
ishe
h32
◦ 35′
N59
◦ 48′
EK
3048
hors
field
iiIr
an:K
hora
san:
vici
nity
ofB
irja
nd32
◦ 53′
N59
◦ 12′
EK
5362
hors
field
iiPa
kist
an:H
azar
ganj
i30
◦ 02.
820′
N66
◦ 52.
945′
El
5359
hors
field
iiPa
kist
an:H
azar
ganj
i30
◦ 02.
994′
N66
◦ 52.
872′
EO
5361
hors
field
iiPa
kist
an:H
azar
ganj
i30
◦ 03.
033′
N66
◦ 53.
983′
EO
5355
hors
field
iiPa
kist
an:K
arkh
asa
30◦ 1
0.94
2′N
66◦ 5
7.25
1′E
O53
56ho
rsfie
ldii
Paki
stan
:Kuc
hlac
k30
◦ 22.
489′
N66
◦ 58.
416′
EM
5358
hors
field
iiPa
kist
an:Q
uetta
30◦ 0
8.95
7′N
66◦ 5
6.93
1′E
O53
60ho
rsfie
ldii
Paki
stan
:Que
tta30
◦ 08.
957′
N66
◦ 56.
931′
EO
5357
hors
field
iiPa
kist
an:T
orgh
ar31
◦ 11.
888′
N68
◦ 27.
359′
EM
AJ8
8836
5ka
zach
stan
ica
Kaz
akhs
tan
––
AJ8
8836
554
16ka
zach
stan
ica
Kaz
akhs
tan:
Kyz
ylku
msa
nds,
WN
WC
hard
ara
villa
ge41
◦ 14′
N67
◦ 42′
EF
5418
kaza
chst
anic
aK
azak
hsta
n:K
yzyl
kum
sand
s,W
NW
Cha
rdar
avi
llage
41◦ 1
5′N
67◦ 4
2′E
F54
15ka
zach
stan
ica
Kaz
akhs
tan:
SA
rys
sand
s42
◦ 09′
N68
◦ 25′
EF
5414
kaza
chst
anic
aK
azak
hsta
n:A
rys
sand
s42
◦ 13′
N68
◦ 19′
ED
5417
kaza
chst
anic
aK
azak
hsta
n:A
rys
sand
s42
◦ 13′
N68
◦ 19′
ED
5413
kaza
chst
anic
aK
azak
hsta
n:A
rys
sand
s42
◦ 13′
N68
◦ 19′
Eh
5412
kaza
chst
anic
aK
azak
hsta
n:E
Kyz
ylku
msa
nds;
197
mel
evat
ion
42◦ 1
6′N
67◦ 5
6′E
F54
09ka
zach
stan
ica
Kaz
akhs
tan:
Mt.
Kar
atau
43◦ 4
0′N
69◦ 0
1′E
F30
64ka
zach
stan
ica
Kaz
akhs
tan:
Jam
bylr
egio
n:M
erke
Dis
tric
t:vi
cini
tyof
Ken
esh
villa
ge43
◦ 57.
666′
N73
◦ 34.
217′
ED
3062
kaza
chst
anic
aK
azak
hsta
n:Ja
mby
lreg
ion:
Mer
keD
istr
ict:
vici
nity
ofK
enes
hvi
llage
43◦ 5
7.66
6′N
73◦ 3
4.21
7′E
F54
07ka
zach
stan
ica
Kaz
akhs
tan:
Zhu
sand
ala
valle
y,S
Tauk
umsa
nds
(sou
ther
nL
ake
Bal
khas
hre
gion
);37
7m
elev
atio
n44
◦ 30.
266′
N75
◦ 03.
697′
EF
5408
kaza
chst
anic
aK
azak
hsta
n:Z
husa
ndal
ava
lley,
STa
ukum
sand
s(s
outh
ern
Lak
eB
alkh
ash
regi
on);
377
mel
evat
ion
44◦ 3
0.26
6′N
75◦ 0
3.69
7′E
F54
05ka
zach
stan
ica
Kaz
akhs
tan:
coas
tof
Ala
kull
ake
(sou
ther
nm
argi
nof
Lak
eB
alkh
ash)
;340
mel
evat
ion
44◦ 5
0.24
9′N
74◦ 1
2.77
8′E
g54
06ka
zach
stan
ica
Kaz
akhs
tan:
10km
SEK
ashk
aten
izvi
llage
(wes
tern
coas
tof
Lak
eB
alkh
ash)
;359
mel
evat
ion
45◦ 4
2.88
3′N
73◦ 2
4.30
5′E
F54
19ka
zach
stan
ica
Kaz
akhs
tan:
SWco
astL
ake
Bal
khas
h,5
kmto
Min
aral
villa
ge45
◦ 24.
067′
N73
◦ 38.
280′
EF
5411
kaza
chst
anic
aK
azak
hsta
n:A
ralS
ea:B
arsa
kelm
esis
land
45◦ 4
0′N
59◦ 5
5′E
F54
10ka
zach
stan
ica
Kaz
akhs
tan:
Eco
astA
ralS
ea,3
0km
SEK
arat
eren
villa
ge45
◦ 47′
N61
◦ 19′
EF
3060
kaza
chst
anic
aK
yrgy
zsta
n:Fe
rgan
aV
alle
y:Ja
lalA
bad:
Kyz
yk-K
iyya
Dis
tric
t,40
◦ 23′
N72
◦ 15′
EI
18km
NE
Kyz
yl-K
iyya
tow
n,vi
cini
tyof
Kha
uzvi
llage
3061
kaza
chst
anic
aK
yrgy
zsta
n:Fe
rgan
aV
alle
y:Ja
lalA
bad:
Kyz
yk-K
iyya
Dis
tric
t,40
◦ 23′
N72
◦ 15′
EI
18km
NE
Kyz
yl-K
iyya
tow
n,vi
cini
tyof
Kha
uzvi
llage
3056
kaza
chst
anic
aK
yrgy
zsta
n:Fe
rgan
aV
alle
y:Ja
lalA
bad:
May
li-Su
uca
nyon
,7km
from
Kok
-Tas
hvi
llage
41◦ 1
4′N
72◦ 2
7′E
I
-
256 U. Fritz et al.A
ppen
dix.
(Con
tinue
d).
Sam
ple
Subs
peci
esL
ocal
ityL
atitu
deL
ongi
tude
Hap
loty
pe
3057
kaza
chst
anic
aK
yrgy
zsta
n:Fe
rgan
aV
alle
y:Ja
lalA
bad:
May
li-Su
uca
nyon
,7km
from
Kok
-Tas
hvi
llage
41◦ 1
4′N
72◦ 2
7′E
I30
58ka
zach
stan
ica
Kyr
gyzs
tan:
Ferg
ana
Val
ley:
Jala
lAba
d:M
ayli-
Suu
cany
on,7
kmfr
omK
ok-T
ash
villa
ge41
◦ 14′
N72
◦ 27′
EI
3059
kaza
chst
anic
aK
yrgy
zsta
n:Fe
rgan
aV
alle
y:Ja
lalA
bad:
May
li-Su
uca
nyon
,7km
from
Kok
-Tas
hvi
llage
41◦ 1
4′N
72◦ 2
7′E
j30
54ka
zach
stan
ica
Kyr
gyzs
tan:
Ferg
ana
Val
ley:
Jala
lAba
d:12
kmSW
Tash
-Kom
urto
wn
41◦ 1
8′N
72◦ 0
8′E
I30
55ka
zach
stan
ica
Kyr
gyzs
tan:
Ferg
ana
Val
ley:
Jala
lAba
d:12
kmSW
Tash
-Kom
urto
wn
41◦ 1
8′N
72◦ 0
8′E
I30
50ka
zach
stan
ica
Kyr
gyzs
tan:
Ferg
ana
Val
ley:
Jala
lAba
d:W
Ala
-Buk
avi
llage
41◦ 2
4′N
71◦ 2
8′E
I30
51ka
zach
stan
ica
Kyr
gyzs
tan:
Ferg
ana
Val
ley:
Jala
lAba
d:W
Ala
-Buk
avi
llage
41◦ 2
4′N
71◦ 2
8′E
I30
63ka
zach
stan
ica
Kyr
gyzs
tan:
Ferg
ana
Val
ley:
Jala
lAba
d:W
Ala
-Buk
avi
llage
41◦ 2
4′N
71◦ 2
8′E
I30
52ka
zach
stan
ica
Kyr
gyzs
tan:
Ferg
ana
Val
ley:
Jala
lAba
d:vi
cini
tyof
Ker
ben
(Kar
avan
)vi
llage
41◦ 2
9′N
71◦ 4
5′E
I30
53ka
zach
stan
ica
Kyr
gyzs
tan:
Ferg
ana
Val
ley:
Jala
lAba
d:vi
cini
tyof
Ker
ben
(Kar
avan
)vi
llage
41◦ 2
9′N
71◦ 4
5′E
I30
65ka
zach
stan
ica
Kyr
gyzs
tan:
Chu
yre
gion
:7.5
kmN
Tele
kvi
llage
,lef
tban
kof
Ak-
Suu
rive
r43
◦ 14′
N74
◦ 03′
NF
3066
kaza
chst
anic
aK
yrgy
zsta
n:C
huy
regi
on:7
.5km
NTe
lek
villa
ge,l
eftb
ank
ofA
k-Su
uri
ver
43◦ 1
4′N
74◦ 0
3′N
F35
32ka
zach
stan
ica
Uzb
ekis
tan:
Eco
cent
er“D
jeir
an”;
223
mel
evat
ion
39◦ 3
4.73
7′N
64◦ 4
1.29
6′E
E35
28ka
zach
stan
ica
Uzb
ekis
tan:
Zar
avsh
anN
atur
eR
eser
ve;7
20m
elev
atio
n39
◦ 40.
292′
N67
◦ 05.
492′
ED
3536
kaza
chst
anic
aU
zbek
ista
n:10
kmS
Tur
kmen
villa
ge;8
00m
elev
atio
n39
◦ 53.
520′
N68
◦ 30.
000′
EO
3533
kaza
chst
anic
aU
zbek
ista
n:20
kmW
Ora
zjan
villa
ge;3
73m
elev
atio
n40
◦ 31.
773′
N65
◦ 10.
315′
ED
3537
kaza
chst
anic
aU
zbek
ista
n:25
kmE
cros
sroa
dsJa
ngig
azga
n-Z
afar
abad
(sou
ther
nK
yzyl
kum
);35
7m
elev
atio
n40
◦ 36.
414′
N65
◦ 16.
705′
ED
3525
kaza
chst
anic
aU
zbek
ista
n:w
este
rnpa
rtof
Aja
kagi
tma
depr
essi
on;1
00m
elev
atio
n40
◦ 39.
672′
N64
◦ 26.
847′
ED
3529
kaza
chst
anic
aU
zbek
ista
n:20
kmE
Jang
igaz
gan
villa
ge;1
95m
elev
atio
n40
◦ 43.
344′
N64
◦ 52.
290′
ED
3531
kaza
chst
anic
aU
zbek
ista
n:K
arak
ata
depr
essi
on:v
icin
ityof
Azn
ekw
ell;
193
mel
evat
ion
40◦ 4
3.38
4′N
64◦ 5
2.21
9′E
D35
34ka
zach
stan
ica
Uzb
ekis
tan:
2km
WK
yzyl
cha
villa
ge(n
orth
ern
foot
hills
ofN
urat
auri
dge)
;410
mel
evat
ion
40◦ 4
4.27
1′N
66◦ 0
4.03
4′E
D35
35ka
zach
stan
ica
Uzb
ekis
tan:
10km
NA
jakk
uduk
villa
ge(n
orth
ern
foot
hills
ofSa
ngru
ntau
ridg
e);4
50m
elev
atio
n41
◦ 19.
170′
N65
◦ 05.
416′
ED
3538
kaza
chst
anic
aU
zbek
ista
n:40
kmS
Uzu
nkud
ukvi
llage
;357
mel
evat
ion
41◦ 2
3.69
4′N
63◦ 1
0.80
9′E
F35
23ka
zach
stan
ica
Uzb
ekis
tan:
Buc
hara
regi
on:K
ara-
kyr
lake
;98
mel
evat
ion
41◦ 2
4.10
2′N
63◦ 1
4.42
5′E
F35
24ka
zach
stan
ica
Uzb
ekis
tan:
wes
tern
part
ofN
urat
auri
dge:
20km
NN
urat
aci
ty;4
01m
elev
atio
n41
◦ 24.
102′
N65
◦ 47.
695′
ED
3526
kaza
chst
anic
aU
zbek
ista
n:15
kmN
Tam
dy-t
ruba
villa
ge;9
9m
elev
atio
n41
◦ 31.
885′
N64
◦ 01.
906′
EF
3527
kaza
chst
anic
aU
zbek
ista
n:D
jam
anku
msa
nds:
vici
nity
ofA
ktak
yrvi
llage
;398
mel
evat
ion
41◦ 4
4.20
4′N
63◦ 1
9.24
4′E
F35
30ka
zach
stan
ica
Uzb
ekis
tan:
Buk
anta
um
ount
ain
ridg
e:C
hing
ildy
gorg
e;41
7m
elev
atio
n42
◦ 41.
514′
N63
◦ 25.
550′
EF
3521
kaza
chst
anic
aU
zbek
ista
n:U
styu
rtpl
atea
u;24
0m
elev
atio
n43
◦ 19.
201′
N58
◦ 09.
710′
EF
3522
kaza
chst
anic
aU
zbek
ista
n:U
styu
rtpl
atea
u:K
arak
alka
n,10
0km
SEK
ungr
ad;2
46m
elev
atio
n43
◦ 30.
017′
N58
◦ 10.
006′
EF
3520
kaza
chst
anic
aU
zbek
ista
n:U
styu
rtpl
atea
u:vi
cini
tyof
Chu
ruk
wel
l;21
4m
elev
atio
n44
◦ 59.
853′
N56
◦ 54.
800′
EP
1429
rust
amov
iIr
an:S
emna
n:M
arko
uh,6
0km
SD
amgh
an35
◦ 38′
N54
◦ 23′
EB
2671
rust
amov
iIr
an:K
hora
san:
Nis
habo
or(N
eysh
abou
r)36
◦ 03′
N59
◦ 07′
EB
3027
rust
amov
iIr
an:K
hora
san:
Anj
irB
elag
,35
kmSE
Bes
anga
n,S
Shar
akhs
;834
mel
evat
ion
36◦ 0
5.59
2′N
60◦ 3
9.24
0′E
a30
26ru
stam
ovi
Iran
:Kho
rasa
n:A
njir
Bel
ag,3
5km
SEB
esan
gan,
SSh
arak
hs;8
34m
elev
atio
n36
◦ 05.
592′
N60
◦ 39.
240′
Eq
-
Diversity of Testudo horsfieldii 257A
ppen
dix.
(Con
tinue
d).
Sam
ple
Subs
peci
esL
ocal
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atitu
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2274
rust
amov
iIr
an:K
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Bes
anga
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EM
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N59
◦ 00′
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2275
rust
amov
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an:K
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san:
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anga
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ke,5
0km
EM
assh
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◦ 15′
N59
◦ 00′
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2276
rust
amov
iIr
an:K
hora
san:
Bes
anga
nla
ke,5
0km
EM
assh
ad36
◦ 15′
N59
◦ 00′
EK
5241
rust
amov
iIr
an:K
hora
san:
Bes
anga
nla
ke,5
0km
EM
assh
ad36
◦ 15′
N59
◦ 00′
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3025
rust
amov
iIr
an:K
hora
san:
Ab
Bel
utch
,17
kmSE
Bes
anga
n,S
Shar
akhs
36◦ 1
6′N
60◦ 3
9′E
P30
30ru
stam
ovi
Iran
:Kho
rasa
n:K
hang
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,Cha
hA
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ian,
near
Tur
kmen
ian
bord
er,W
Shar
akhs
;152
mel
evat
ion
36◦ 3
4.85
5′N
60◦ 4
5.63
0′E
B26
70ru
stam
ovi
Iran
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esta
n:H
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pich
villa
gene
arG
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◦ 46′
N54
◦ 28′
EB
1419
rust
amov
iIr
an:G
oles
tan:
vici
nity
ofG
orga
nci
ty36
◦ 51′
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◦ 28′
EP
AJ8
8836
6ru
stam
ovi
Iran
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esta
n:vi
cini
tyof
Gor
gan
city
36◦ 5
1′N
54◦ 2
8′E
r22
79ru
stam
ovi
Iran
:Gol
esta
n:M
iank
aleh
peni
nsul
a36
◦ 54.
337′
N54
◦ 00.
413′
EP
AJ8
8836
7ru
stam
ovi
Iran
:Gol
esta
n:M
iank
aleh
peni
nsul
a36
◦ 54.
337′
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◦ 00.
413′
Es
1422
rust
amov
iIr
an:G
oles
tan:
Mia
nkal
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nins
ula
36◦ 5
4.33
7′N
54◦ 0
0.41
3′E
T14
23ru
stam
ovi
Iran
:Gol
esta
n:M
iank
aleh
peni
nsul
a36
◦ 54.
337′
N54
◦ 00.
413′
ET
2674
rust
amov
iIr
an:K
hora
san:
Baj
gira
n37
◦ 36′
N58
◦ 24′
EK
1424
rust
amov
iIr
an:G
oles
tan:
Mor
aveh
Tapp
eh37
◦ 55′
N55
◦ 57′
EP
1425
rust
amov
iIr
an:G
oles
tan:
Mor
aveh
Tapp
eh37
◦ 55′
N55
◦ 57′
EP
DQ
0800
45ru
stam
ovi
Tur
kmen
ista
n:K
opet
Dag
37◦ 5
0′N
58◦ 1
5′E
c
AY
6784
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AY
6784
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6784
38?
unkn
own
––
AY
6784
38D
Q44
5847
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know
n–
–E
DQ
4973
22?
unkn
own
––
F66
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know
n–
–M
TD
T66
83?
unkn
own
––
F84
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know
n–
–F
941
?un
know
n–
–D
1296
?un
know
n–
–M
TD
T12
9616
95?
unkn
own
––
F22
56?
unkn
own
––
F34
35?
unkn
own
––
E34
36?
unkn
own
––
D44
24?
unkn
own
––
F44
25?
unkn
own
––
MT
DT
4425