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Amphibia-Reptilia 30 (2009): 245-257 Mitochondrial diversity of the widespread Central Asian steppe tortoise (Testudo horsfieldii Gray, 1844): implications for taxonomy and relocation of confiscated tortoises Uwe Fritz 1,* , Markus Auer 1 , Marina A. Chirikova 2 , Tatyana N. Duysebayeva 2 , Valery K. Eremchenko 3 , Haji Gholi Kami 4 , Roman D. Kashkarov 5 , Rafaqat Masroor 6 , Yoshan Moodley 7 , Attaullah Pindrani 8 , Pavel Široký 9 , Anna K. Hundsdörfer 1 Abstract. Using a nearly range-wide sampling, we investigated phylogeographic differentiation and mitochondrial diversity of Testudo horsfieldii, the only tortoise species confined to Central Asia. We identified three major haplotype clades with mainly parapatric distribution that do not correspond well to the currently recognized three subspecies. One clade is restricted to the Fergana Valley and seems to represent a previously overlooked evolutionarily significant unit. Another clade, consisting of 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 northerly sites in western and south-eastern Uzbekistan. It is possible that this clade also occurs in eastern Turkmenistan and adjacent Afghanistan, regions not sampled for the present study. The generally parapatric distribution of individual haplotypes, even within each of the three major clades, suggests advanced lineage sorting, either due to limited dispersal abilities, glacial isolation in distinct local microrefuges or both acting in accord. The localized distribution of endemic haplotypes in the northern and central plains as well as in the mountainous eastern and southern parts of the distribution range supports the existence of multiple microrefuges there. Records of haplotypes of distinct clades in sympatry or close geographic proximity are likely the result of Holocene range expansions. In recent years, thousands of confiscated steppe tortoises were released into the wild. The detected mitochondrial differentiation offers a powerful tool for nature conservation, as a means of determining the 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), Natural History 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, Gorgan University 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, Department of 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 horsfieldii Gray, 1844 (maximum shell length approx. 30 cm) is the only tortoise species confined to Central Asia. Its range encompasses eastern Iran, Afghanistan, Turkmenistan, Uzbekistan, Tajikistan, Kyrgyzstan, southern Kazakhstan, the westernmost part of Xinjiang (China), and in Pakistan part of the region bordering Afghanistan (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, Faculty of 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

Mitochondrial diversity of the widespread Central Asian …...Yoshan Moodley7, Attaullah Pindrani8, Pavel Široký9, Anna K. Hundsdörfer1 Abstract. Using a nearly range-wide sampling,

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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

  • 246 U. Fritz et al.

    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

  • Diversity of Testudo horsfieldii 247

    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

  • 248 U. Fritz et al.

    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.

  • Diversity of Testudo horsfieldii 249

    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(

  • 250 U. Fritz et al.

    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-

  • Diversity of Testudo horsfieldii 251

    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.

  • 252 U. Fritz et al.

    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-

  • Diversity of Testudo horsfieldii 253

    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).

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  • Diversity of Testudo horsfieldii 255A

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    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

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    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

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    arak

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    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

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    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:

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    ,35

    kmSE

    Bes

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    n,S

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    ;834

    mel

    evat

    ion

    36◦ 0

    5.59

    2′N

    60◦ 3

    9.24

    0′E

    a30

    26ru

    stam

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    Iran

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    SEB

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    arak

    hs;8

    34m

    elev

    atio

    n36

    ◦ 05.

    592′

    N60

    ◦ 39.

    240′

    Eq

  • Diversity of Testudo horsfieldii 257A

    ppen

    dix.

    (Con

    tinue

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    Sam

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    rust

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    san:

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    assh

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    ◦ 15′

    N59

    ◦ 00′

    EK

    2275

    rust

    amov

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    hora

    san:

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    anga

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    ◦ 15′

    N59

    ◦ 00′

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    2276

    rust

    amov

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    san:

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    anga

    nla

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    ◦ 15′

    N59

    ◦ 00′

    EK

    5241

    rust

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    anga

    nla

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    assh

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    ◦ 15′

    N59

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    3025

    rust

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    Bes

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    36◦ 1

    6′N

    60◦ 3

    9′E

    P30

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    stam

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    ;152

    mel

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    36◦ 3

    4.85

    5′N

    60◦ 4

    5.63

    0′E

    B26

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    stam

    ovi

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    :Gol

    esta

    n:H

    ezar

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    n36

    ◦ 46′

    N54

    ◦ 28′

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    1419

    rust

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    ◦ 51′

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    esta

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    36◦ 5

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    a36

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    ◦ 54.

    337′

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    413′

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    36◦ 5

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    a36

    ◦ 54.

    337′

    N54

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    413′

    ET

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    rust

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    ◦ 36′

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    rust

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    ◦ 55′

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    rust

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    ◦ 55′

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    37◦ 5

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    ––

    MT

    DT

    4425