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HERPETOLOGICAL JOURNAL, Vol. 14, pp. 35-45 (2004)

THE INTER- AND INTRASPECIFIC STATUS OF AEGEAN MA UR RJVULA TA (CHELONIA, BATAGURIDAE) AS INFERRED BY MITOCHONDRIAL DNA SEQUENCES

NIKOS POULAKAKIS1 PETROS LYMBERAKIS1, EFSTRATlOS VALAKOS3 GEORGIA MANTZIOU'·2, , AND MOYSIS MYLONAS1•2

'Natural History Museum of , University of Crete

2 Department of Biology, University of Crete

3Section of and Human Physiology, Departmenl of Biology, University of Athens

The (Chelonia, Bataguridae) is widely distributed throughout Asia, Europe and NW Africa. Three (Mauremys caspica, Mauremys rivulata and Mauremys leprosa) are di scant inuously distributed around the Mediterranean region. Present distributions are much smaller than those documented within the fossil record of Mauremys in the Mediterranean region. All three extant species are identified on the basis of morphology. In the present study we compare partial mitochondrial DNA sequences of cyt-b from J 6 populations of Mauremys rivulata from Greece, one from Jordan (M. rivulata), two from Syria (M. caspica) and one from Morocco (M. leprosa). Comparison of cyt-b partial sequences supports the monophyly of the three species considered, as well as their proposed taxonomic status (i.e. separation at the species level). Mauremys leprosa is the most differentiated of the three, M. caspica and M. rivulata being more closely related. Climatic changes during the Pleistocene influenced the distribution of M. rivulata and resulted in a latitudinal oscillation of the populations in a north - south direction in Greece, and consequently in a mixing of their genetic material. This hypothesis is confirmed by the absence of corre lation between genetic distances and geographical origin oft he spec imens studied. Key words: Aegean region, cytochrome b, Mediterranean, phylogeography INTRODUCTION Melentis ( 1966) places its origin earlier in the Eocene. Prior to the Pleistocene, it was widely distributed in Eu­ Mauremys caspica (G e li n, J 774), Mauremys m rope, North Africaand the Arabian Peninsula. rivulata (Valenciennes, 833) and Mauremys leprosa I Until very recently rivulata and M. leprosa were (Schweigger, 1812) are the sole European representa­ M. . treated as subspecies of caspica. Mauremys leprosa tives of the diverse fa mily Bataguridae. The M. was the first to be raised to species level. According to consists of 23 genera, distributed in the Palearctic re­ Fritz (2001), this elevation had been considered by gion, with the exception of the genus , Boulenger ( 1889) and later by Siebenrock ( 1909), but which is distributed in Centra 1 and South America was not widely aclrnowledged w1til after l 980 (Busack (Pough al. , 1998; Fritz, 2001 ). The systematic posi­ et Ernst, l980). lt was based on morphometric studies as tion of the familyis very problematic (McDowell, 1964; & well as on the biochemical studies of Merkle (1975). Gaffney & MeyJan, 1988; Pough et al. , 1998; Fritz, Mauremys leprosa is geographically separated fromthe 2001 ). It has been traditionally considered to be a sub­ other two species by a gap in the present distribution of family of (Hirayama, I 984; I 1erson, 1992), the genus (Fig. I). According to Busack & Ernst( 1980), or a separate paraphyletic family (Gaffney & Meylan, geographic isolation, which led to cessation of gene 1988; Pough et al., 1998), but nowadays the opinion of the flow, started in the Pliocene. o111er authors (Shaffer et al. , 1997; Fritz, 2001) - who Mauremys rivulata was raised to the species level consider Bataguridae as a separate monophyletic family more recently (Fritz Wischuf, 1997). The authors - prevails, although they all agree that to resolve this is­ & stated that morphometric featurescould not separate the sue, additional data from more complete sampling are two species, but suggested species status on the basis of required. The family Bataguridae is known at least from the colour pattern of the carapace and plastron. Al­ the Eocene in the Old World and the Nearctic. It is pos­ though the two species are separated by geographic sible that the Paleocene "Emydidae", described from barriers (Fritz Wischuf, 1997) and occupy ecologi­ China, actually belong to the Bataguridae family (Fritz, & cally different habitats (Busack & Ernst, 1980), a 2001). The genus Mauremys is known in the Western narrow contact zone exists, from which Fritz Wischuf Palearctic from the Oligocene (Fritz, 200 I), but & (1997) report two hybrids. Apart from the morphological studies (Busack & Correspondence: G. Mantziou, Natural History Museum of Ernst, 1980; Fritz & Wisclmf, 1997; Tok, 1999), Merkle Crete, University of Crete, Knossou Av., P. 0. Box 2208, GR71409 lrakleio, Crete, Greece. E-mail : (1975) examined these species by protein electrophore­ [email protected]; [email protected] sis. He examined 17 proteins and found that M. caspica 36 G. MANTZIOU ET AL.

and M. rivulata were identical; M. leprosa shared only evolution and extensive intraspecific polymorphism 13 of these proteins with the other two taxa. (Avise et al., 1987). Mitochondrial DNA evolution rate Fritz and W ischuf ( 1997) noted that the coloration of appears significantly slower in Testudines, relative to specimens preserved in ethanol degrades and is not as other groups of vertebrates (Avise et al. , 1992; Lamb et clear in old as it is in young animals. Moreover, al. , I 994; Lenk et al., 1999). Nevertheless, mtDNA can ofthe genus Mauremys exhibit great intraspecific be very infomwtive in cases where morphological data variation in colour patterns (Schleich et al. , 1996). seem to be inconclusive (Lenk et al., 1998), which is the There are no molecular studies on the Mediterranean case with Mauremys species (Fritz & Wischuf, 1997). Mauremys species. We thereforeconsidered that the use of a molecular approach to independently test the MA TERlALS AND METHODS present would be of great interest. Jn the present study we investigate the intraspecific relation­ SAMPLES ships of Aegean M. rivulata and their relation to the other two Mediterranean Mauremys species. We ad­ Twenty-eight specimens of Mauremys were collected dress questions on genetic distances among the species from20 localities (Table I). Of these localities, 24 con­ and attempt a reconstruction of their phylogenetic rela­ tained M. rivulata, two contained M. caspica and two tionships. contained M. leprosa. Two published sequences of other We use partial sequences ofcytochrome-b (cyt-b) of Batagurids were used in the analysis database: the mitochondrial DNA (mtDNA), a gene already used sp inosa (GenBank U8 1362, Shaffer et al., in several similar studies (Lamb et al. , 1994; Lamb & 1997) and Cuora aurocapitata (Gen.Bank AF043262, Lydeard, 1994; Lenk et al., 1998; Lenk et al. , 1999). Wu et al., unpublished). Mitochondrial DNA is a very useful tool in detecting ge­ Homologous sequences of the emydid Emys netic differences and phylogeographic patterns at the orbicularis (Linnaeus, 1758) and the intraspecific level, or in closely related species, due to marginata (Schoepff, 1795) were included in the study its non-recombining mode of inheritance, rapid pace of as outgroups.

TABLE 1. Museum number, exact locality and accession number of each specimen, used in the analysis (code is used in Figs. I, 3, 4, 5 and Table 2)

Species Museum number Locality Code Accession number

Mauremys rivulata NHMC 80.3.15.41 Greece, Crete, Almyros River 1 AF487640 Mauremys rivulata NHMC 80.3.15.26 Greece, Crete, Almyros River 2 AF487638 Mauremys rivulata NHMC 80.3.15.16 Greece, Crete, Almyros River 3 AF487639 Mauremys rivulata NHMC 80.3. 15.125 Greece, Crete, Preveli 4 AF487633 Mauremys rivulata NHMC 80.3. 15.71 Greece, Crete, Gavdos 5 AF487637 Mauremys rivulata NHMC 80.3.15.66 Greece, Crete, Gavdos 6 AF487641 Mauremys rivulata NHMC 80.3.15.93 Greece, Crete, Krya Yrysi 7 AF487632 Mauremys rivulata NHMC 80.3. 1 5.45 Greece, Crete, Zakros 8 AF487627 Mauremys rivulata NHMC 80.3.1 5.79 Greece, Crete, Georgioupoli 9 AF487629 Mauremys rivulata NHMC 80.3. 15.77 Greece, Crete, Georgioupoli 10 AF487628 Mauremys rivulata NHMC 80.3.15.75 Greece, Crete, Akrotiri 11 AF487625 Mauremys rivulata NHMC 80.3.15. 128 Greece, Chios is!. 12 AF487630 Mauremys rivulata NHMC 80.3.15. J 27 Greece, Chios is!. 13 AF48763 1 Mauremys rivu/ala NHMC 80.3.1 5.98 Greece, Lesvos is!. 14 AF487636 Mauremys rivulata NHMC 80.3.15.53 Greece, Kyklades, Naxos is!. 15 AF487634 Mauremys rivulata NHMC 80.3.15.83 Greece, Dodekanisa, Rodos isl. 16 AF487635 Mauremys rivulata NHMC 80.3.15.100 Greece, Dodekanisa, Jkaria is!. 17 AF4876 I 9 Mauremys rivulata NHMC 80.3.15.103 Greece, Samos is!. 18 AF487622 Mauremys rivulata NHMC 80.3.15.104 Greece, Samos is!. 19 AF487620 Mauremys rivulata NHMC 80.3. 15.132 Greece, Dodekanisa, Kos isl. 20 AF487621 Mauremys rivulata NHMC 80.3. l 5.135 Greece, Thessalia, Larisa 21 AF487623 Mauremys rivulata NHMC 80.3. 15.136 Greece, Thessalia, Larisa 22 AF487626 Mauremys rivulata NHMC 80.3.15.137 Greece, Peloponnisos, Kakkavas 23 AF487624 Mauremys rivu/ata NHMC 80.3.1 5.56 Jordan, 25 km south of Jarash 24 AF487642 Mauremys caspica NHMC 80.3.112.I Syria, 5 km afterAs Suwar 25 AF487644 towards Marqadah Mauremys caspica NHMC 80.3.1 12.62 Syria, lake Al Asad 26 AF487643 Mauremys /eprosa NHMC 80.3.1 l3. l 19 Morocco, Qued Tensift 27 AF487645 Mauremys leprosa NHMC 80.3.113.120 Morocco, Qued Tensift 28 AF487646 Emys orbicularis NHMC 80.3. l 6.6 Greece, Thessalia, Larisa 29 AF487648 Emys orbicularis NHMC 80.3.16.7 Greece, Thessalia, Larisa 30 AF487649 Testudo marginala NHMC 80.3.22.6 Greece, Kythira is!. 31 AF487647 AEGEAN MA UREMYS RIVULA TA 37

)

FIG. I. Present distribution of the three Mediterranean Mauremys species and geographic origin of the studied specimens (numbers correspond to the code listed in Table I).

This materia I was collected from J 999 to 200 J dur­ Research) thermocycler. The cycle programme com­ ing several field trip . The geographical origin of the prised of an injtial denaturation step of 2 min at 94°C, studied specimens is shown in Fig. I. followed by 35 cycles of denat11Jationfor l m.iJ1 at 94°C, Blood samples were obtained from every animal, ex­ annealing for l nun at 50°C, extension for l mm at 72°C cept from animals with numbers NHMC 80.3. I 12. 62, and a final extension at 72°C for 10 nun. NHMC 80.3.15.98 and NHMC 80.3.22.6, which died . Sequencing of double-strnnded DNA was performed and from which tissue sample was isolated and kept in in both directions in a PE-ABI377 sequencer (using dye­ ethanol (95%). The blood was collected by coccygeal terminator chemistry) . The primers in the sequencing vein punctme as described by Haskell & Parkas (1994 ), reactions were the same as in the amplification proce­ preserved in ethanol and stored at 4"C. After blood sam­ dme. pling the animals were released. Museum numbers were given to each animal that was captured and although SEQUENCES ALIGNMENT AND PJ-JY LOGENETJC ANALYSIS they were released the numbers still correspond to the Multiple sequence alignment was perfonned using a tissue samples taken fromeac h one (Table l ) . ClustalX program package (version 1.8: Thompson et al., 1997), using the default parameters, alternative gap DNA EXTRACTJON, AMPLIFICATION AND SEQUENCING opening and gap extension penalties, with minor modifi­ Blood samples were first centrifuged at 13 OOO rprn cations made by eye. for 4 min. Ethanol was removed and samples were left Pairwise sequence comparisons were made for the at 37°C for 1 ham. Total genomic DNA wa s extracted cyt-b data set using MEGA (v .2, Kumar et al., 2001) in fo llowing standard proteinase-k protocol, with standard to determine the number, nature, distance and dis­ salt extraction method (Sambrook et al. , 1989). tribution of base substitutions. Genetic distance was PCR amplification, targeting a segment of the cyt-b estimated using the Kimura two-parameter model gene of the mitochondrial genome (mtDNA) was done (Kimura, 1980). on all extractions. The wliversal primers L14724 and Evolutionary relationships, which result from DNA HJ517 5 (Palumbi, 1996) were used to amplifya 451 bp sequence data, are reliable only if sites are not satmated fragment oftbe nlitochondrial cyt-b gene. by multiple substitutions (Swofford et al., 1996). To as­ Amplifications were performed in a 1 Oµl total reac­ sess potential saturation of substitutions of the cyt-b tion volume, where 1 µl of template DNA was mixed sequences, the numbers of transitions (Ts) and with 0.2 mM dNTPs, 1 .5 mM MgCl2, 4 pmol of each transversions (Tv) were plotted against the correspond­ primer and 0.5 units of Taq Polymerase (GibcoBRL). ing W1Corrected ?-distances for all pairwise Thennocycling was then performed in a PTC- 100 (MJ- comparisons. 38 G. MANTZIOU ET AL.

Phylogenetic relationships among specimens were groups have not yet been fully identified, and many infened via neighbour-joining (NJ, Saitou & Nei 1987), studies have shown considerable rate heterogeneity maximum parsimony (MP, Swofford et al. , 1996), and (Hillis et al. , I 996). Nevertheless, the use of clock as­ maximwn likelihood methods (ML, Felsenstein 1981 ). sumptions for closely related taxa is generally NJ trees were implemented by MEGA (v. 2.0, Kumar et considered to be more reliable than for distantly related al., 2001) using Kimura 's ( 1980) two-parameter dis­ taxa (Caccone et al., 1997), which stems from the tance estinuite, even though the distance metric used in premise that rates of evolution of a particular gene are NJ had no effect on topology. MP and ML trees were likely to be stable in closely related taxonomic groups, constructed using PAUP (Windows Version 4.0b8a, with similar life histories, metabolic rates, and genera­ Swofford,2002). tion times. In this respect, the estimation of "local" Nucleotides were used as discrete, unordered charac­ rates forclosely related taxa might be preferable over a ters. The shortest tree was looked forwith the branch "universal" rate (Hillis et al., I 996). In the present and bound search. When more than one rnil1imal length study we use an evolutionary rate suggested for turtle tree was found, the strict consensus tree was presented. mtDNA (Avise et al., 1992; Lamb et al., 1994; Lenk et Confidence estimates were obtained via bootstrapping al., 1999), instead of the universal rate. with 1 OOO replicates (Felsenstein, 1985). The published sequence used in the relative rate test For maxinmm likelihood (ML) analysis (Felsestein, as outgroup (one specimen), was that of 1981 ), the best fit model of DNA substitution and the tn:porcatus (GenBank U81 349, Shafferet al., 1997). parameter estinuites usedfor tree cons truction were cho­ The sequence data fromth is study were deposited iJ1 sen by perfom1ing hierarchical likelihood ratio tests the GenBank Data library under the accession numbers (Huelsenbeck & Crandall, 1997) using Modeltest 3.06 AF487619-AF487649. (Posada & Crandall, 1998). Heuristic ML searches were RESULTS performed with 10 replicates ofrandom sequence addi­ tion and tree bisection-reconnection (TBR) branch BASE COM POSITION swapping. ML bootstraps employed 1 OOO iterations. A total of I l unique haplotypes from the 28 speci­ The model parameters (substitution rate matrix, gamma mens of Mauremys were obtained in this study (Table disnibution approximation with four rate classes, and 2), the lengths of which ranged from 365 to 427 bp. empirical nucleotide frequencies) were estimated ini­ Within the cyt-b gene of the presented sequences, no in­ tially from the starting tTees generated by the approach sertions, no deletions and no premature stop codons described above (Huelsenbeck & Crandall, 1997). were encountered. These estinUlteswere used in a ML analysis to produce a A total of 435 base pairs were aligned, of which 42 tree from which the parameters were then reestimated. sites (9.65%) were variable among the Mauremys spe­ In an iterative fashion, these steps were repeated until cies (26.66% including outgroups) and 25 (5.75%) were the ML score converged to its maximum value parsimony informative (I 6. 78% including outgroups ). (Swofford et al. 1996). Nine (2 1 .43%) of the 42 variable positions represent A minimum spanning network was constructed changes in the first codon position, 8 (19.05%) in the among M. rivulata haplotypes, by using Arlequin second and 25 (59.52%) in the third. v.2000 (Schneider et al. , 2000). Mean base composition of the fragment of cyt-b of Tajima's relative rate test (Tajima, 1993; Nei & the three codon positions is provided in Table 3. There Kumar, 2000) was canied out using MEGA (v.2.0) iJ1 is a strong bias iJ1 base composition (Bias C of lrwiJ1 et order to assess differences in rates among separate liJ1e­ al. , 1991 ), a feature characteristic of cyt-band other mi­ ages. Statistical estimation of the validity of the tochondrial protein-coding genes in mammals and molecular clock hypothesis was performedusing the x2 . This fact supports the authenticity of the mito­ test proposed by Fitch ( 1976). In addition, the maximwn chondrial sequences (Irwin et al. , 1991; McGuire & likelihood model was used to test the null hypothesis Heang, 2001; Lenk et al., 2001; Surget-Groba et al., that the sequences were evolving at constant rates and 200 l ). As expected, the abundance of G's was low therefore fit a molecular clock (Muse & Weir, 1992). (12.9%), whereas the percentages of A, T and C were This hypothesis may be tested once we have chosen one quite similar (26.9-3 1.0%). However, a significant of the models of evolution, simply calculating the log compositional bias exists at the second and especially likelihood score of the chosen model with the molecular the thfrd codon position. The frequencyof guanine at the clock enforced and comparing it vviththe log likelihood firstposition is 21.1%, while a marked under-presenta­ previously obtained without enforcing the molecular tion of guanine was observed at both second (I 6.2%) clock. In this case, the molecular clock is the null hy­ and third position (1.8%). pothesis. The number of degrees of freedom is the number of OTUs - 2. It should be mentioned that in this GENETIC DTVERGENCE AND SA TURA TJON analysis we used not only the unique haplotypes, but all 33 sequences. Sununarized Kimura two-parameter distances be­ Clock assumptions must be treated cautiously since tween all pairs are given in Table 4. Sequence the differences in mtDNA evolution in higher vertebrate divergence ranged from 0.24% within M. rivulata AEGEAN MA UREMYS RJVULA TA 39

TABLE 2. Grouping of selected Mauremys samples into 11 TABLE 3. Percentage base composition at first, second and unique haplotypes (numbers in parenthesis correspond to the third codon position for all 33 specimens. Compositional code listed in Table I) bias index (CBI) is calculated as C=(2/3):Elc, - 0.251 where C is the compositional bias index and c, the frequency of ith Haplotype Samples Frequency base (Irwin et al. , 1991 ).

N c eotide Position Mean M. rivulata u l Samos (19) 25% h I First Second Third Akrotiri (1 1) Kos (20) A 30.0 21.4 40.8 31.0 Gavdos (5) T 26.8 37.5 16.6 26.9 Gavdos (6) Krya Vrysi (7) c 22. l 24.9 40.8 29.2 G 21.1 16.2 1.8 h2 lkaria ( l 7) 29. 16% 12.9 Samos (18) CBI 0.091 0. 167 Larissa (21) 0.421 Larissa (22) Peloponi ssos (23) Jordan (24) 40

Preveli (4) 36 h3 Chios (l3) 8.33% 32 Lesvos ( l 4) 6 tra nsitions Zakros (8) 2.50% 28 h4 1 . transverslons Georgioupoli (9) 24 Georgioupoli (I0) i'! 0 20 h5 Naxos (J5) 8.33% J!l Chios (12) 16 h6 Rodos (l6) 4. 16% 12 h7 Almyros (l) 12.50% 8 A lmyros (2) A lmyros (3) M. caspica h8 Syria (25) 50% 0 0.02 0.04 0.06 0.08 0.1 0.12 0 14 0 16 0.18 h9 Syria (26) 50% p..

haplotypes to 16.97% between E. orbicu/aris and M. · mately linear relationship with distances, which indi­ leprosa. If we consider only Mauremys species, se­ cates that saturation bas not occurred. quence divergence ranged from 0.24 % (within M. PHYLOGENETIC RELATIONSH IPS rivu/ata) to 7.45% between M. /eprosa and M. caspica. The results of saturation analysis are presented in Fig For the phylogenetic analyses, a data set of 16 cyt-b 2. Both transitions and transversions show an approxi- sequences (I I unique haplotypes of Mauremys spp.,

TABLE 4. Genetic distances (Kimura two-parameter) between the different tax a. In-group sequence divergence is given in diagonal. The range ofge netic distances is given in the parentheses.

M. rivu/ata M. caspica M. leprosa C. aurocapitata H. sp inosa T. marginata E. orbicularis

M. rivu/ata 0.7 (0.24- 1.78) M. casp ica 3.36 1.83 (2.45-3.95) M. /eprosa 6. 16 7. 14 0.28 (5.9-6.66) (6.83-7.45) C. aurocapitata 9.56 8.94 10.49 n/c (8.41-10.26) (8.81 -9.07) (l 0.34-1 0.63) J-1. sp inosa I 1.49 11.03 12.53 12.91 n/c (I 0.23-12.37) ( l 0.56-1 1 .5) (12.36-12.71) T. marginata 12.88 13.06 14.5 1 15.42 13.91 n/c (12.4 1-13.89) (12.36-l 3. 76) (14.34-14.68) E. orbicularis 14.64 14.54 16.23 16.44 12.87 15.47 0.25 (12.72-16.31) (12. 70-16.63) (15.49-16.97) 40 G. MANTZlOU ET AL.

h4 .. ------h1

.. h7 "' ,....:B.._1-1------h3 .. ,,

... h2 .. h2 i------

h1 =E

h6 M. cupfca (25) ------h3 83

.. �-----..ll'°�o h10 Af. Jeprosa (27) 63 h6 ... h11 M. loprosa (22) 57

C. aurocspitAta h4

T. marglt18IB (31)

------H. spinosa h5 100 E. orbicufarls (29)

E. orblcularis (30) h8 M. casp/ce (25)

0.02 63

h9 M. caspica (26) FIG. 3. Phylogenetic analysis of the mitochondrial cyt-b gene of Mauremys. Tree inferred by the neighbour-joining (NJ) 82 method, based on replicates. h10 M. leprosa (27) I OOO 100

two of Emys orbicularis and one of Testudo marginata h11 M. leprosa (28) from this study, two of other Batagurid species fromthe 54 literature) was used. Tree length distribution, deter­ .....______c. BUfOCBpltara

mined from random sampling of 106 unweighted trees, 100 was significantly skewed to the left (gl=-0.471), sug­ gesting strong phylogenetic signal in the data (P

TABLE 5. Test of hypotheses relating to the model of evolution appropriate for phylogeny reconstruction (Huel senbeck & Crandall, 1997). P- values were obtained with Modeltest (Posada & Crandall, 1998).

Null hypothesis Models compared df p -JnLO -htl.,l Equal nucletide frequencies H0:JC69, H1 :F81 1513.1719 1481.9972 3 0.0000 Equal Ti and Tv rates H0:8I, H 1 :HKY85 1481.9972 1425.8398 0.0000 Equal Ti rates H0:HKY85, H1 :TrN 1425.8398 1417.3188 0.0000 Equal Tv rates H0:TrN, H 1 :TIM 1417.3188 1416.5848 0.2257 Equal rates among sites H0:TrN, H1 :TrN+G 1417.3189 1387.3484 0.0000 Proportion of invariable sites H :TrN+G, H :TrN+I+G 1387.3484 1387.2443 0.3241 0 1 AEGEAN MA UREM YS RJVULA TA 41

67

..

... 56

55

h4

h5

65 h8 M. cssplca (25) 70

h9 M. casplca (26)

h10 M. leprou (27) 100

h11 M. lepross (28).

67

FIG. 7. Minimum spanning network for the 7 M. rivu/ata

100 haplotypes. Size of each circle is proportional to the frequency of the haplotypes.

TABLE 6. Estimated splitting time between clades. The splitting time is estimated using a divergence rate of 0.3%- 0.4% per Myr. A: M. rivulata clade, B: M. caspica clade, C: M. /eprosa clade.

Clades Da Splitting Time

Rate: 0.3% Rate: 0.4% FIG. 5. A Maximum Likelihood (ML) tree derived from cyt-b. per Myr per Myr sequences of Mauremys species. Probability percentages of bootstrap replicates supporting each branching pattern are (A&B) vs. C 5.88% 19.6 Myr ago 14.7 Myr ago given beside the corresponding nodes (numbers correspond A vs. B 2. 1% 7 Myr ago 5.3 Myr ago to the code listed in Table J). Bootstrap values >50% are shown. Tajima 's relative rate test was carried out for many different pair-combinations of the examined clades and resulted that all these clades evolve with the same rate (X2 <3 .84, P>0,05). The likelihood ratio test was em­ ployed to investigate the rate of homogeneity for the analysed species (Huelsenbeck & Crandall, 1997). Be­ cause the simpler ( clocklike) tree cannot be rejected at a significance level of 5% (LRT=17.66, df=3 1, X =l 9.28), we do not reject the application of a mo­ criucai lecular clock to the species used in the analysis. Since our results are compatible with the molecular clock hypothesis, we can use the suggested evolving rate for mtDNA ofEmydidae (0.3% -0.4% per Myr) (Lenk et al., 1999). The resulting estimated splitting times be­

tween clades are summarized in Table 6.

DISCUSSION

PHYLOGENETIC RELA TIONSHJPS

Our results indicate that M. rivulata, M. caspica and FIG. 6. Geographic distribution of M. rivulata haplotypes. M. leprosa are indeed genetically isolated taxa. These Note the absence of geographic patterns. three taxa constitute a monophyletic group, which splits 42 G. MANTZIOU ET AL.

HYPOTHESIS I H YPOTHESJS 2 paleontological data with the results we present, perm.its a preliminary interpretationof the phylogeographic pat­ j J f l � 'i tern of M. rivulata in the Eastern MediteJTanean and especially in the area of the Aegean Sea. ]------�--�-i---t-1 According to Lenk et al. ( 1999), the climate change J 1 1 1 in Europe 3.2 Myr ago triggered a sudden radiation of _ J I Emys orbicularis. During the climatic oscillations of the _ Pleistocene, tbe range of E. orbicularis probably frag­ Q) ! • ___ _ _ r __ '.'; 1--1----t--r mented recurrently, with isolated populations along a I slender belt throughout southern Europe. This belt has i' � � . . been shaped by cold climates to the north and by barriers _ _ _ _ _ � _ _ 2:_3t�r- _ � . :- I • � _ y of inappropriate habitat to the south. Thus, southern .� - _ _ _ } � i 7Myr1 Italy and Greece sen1ed as refugiafor the populations of E. orbicularis. ::.: -� !. J :. : -c ;,, -- --- · .� Kotsakis (1980) claimed that the lower Pleistocene ;.... : ...... -14.7 M�•r · · · · · �: • • •, •,, • • •' ""'I . .... glaciations, which provoked the southward shift of dis­ · · <. : ... .. - � : 19.6 Myr tribution of E. orbicularis in Italy, drove Mauremys °"' populations to extinction in the peninsula. --:>. '�------Thereby, the main factor that influenced the present ...l.� j • I distribution of Mauremys in the Mediterranean region, and particularly the distribution of M. rivulata in the

------�------Balkans, is climatic change, which prevailed during the ., � � c glacial periods of the Pleistocene. The cold periods re­ w� sulted in the latitudinal shift of Mauremys populations in . ? . the Italian Peninsula towards the south, and ultimately FIG. 8. Alternative hypothesis on the phylogenetic Jed to their extinction. Something similar occurred in the relationships of the three species in question. By applying Balkan Peninsula with M rivulata, resulting in a latitu­ mtDNA evolutionary rate of 0.4% per Myr or of 0.3% per dinal shift of the populations towards the south and east, Myr, we result in hypothesis I or hypothesis 2, respectively. but it did not result in extinction since they found ref­ uges on the coast of Asia Minor and in the Aegean into two genealogical lineages. The first conesponds to islands. the lineage leading to M. leprosa, whereas the second Consequently, the shiftingand rearrangements of M. corresponds respectively to the lineage from which the rivulata populations led to their mixing and the probable caspica-rivulata group emerged. The very small within­ establishment ofnew populations on islands, where they group divergence of M. rivulata and the multifold did not exist before and where populations of E. orbicu­ between-group divergence (3.36% between M. caspica laris were not favoured due to ecological factors. and M. rivulata) support the elevation of M. rivulata to According to Lenk et al. ( 1999), E. orbicularis is more the species level, as suggested by Fritz & Wischuf favoured by continental climate conditions. (1997), although furthersampling in the area of contact On the other hand, the genetic distances observed is necessary to fully resolve this issue. among M. . rivulata haplotypes, and tJ1eir relationships The elevation of M. leprosa, which was based on based on minimum spamung network, are not related to electrophoretic and morphometric data (Merkle, 1975; respective geograpluc distances. This fact cannot be ex­ Busack & Ernst, 1980), is furthersupported fromour re­ plained by the vicariance approach of the distributional sults. pattern of M. rivulata, but can be better explained by a Specimens of M. rivulata (from the Middle East to dispersal model. To propose a dispersal model we need Greece) cluster with a small intraspecific differentiation to have evidence that M. rivulata is easily dispersed. ranging from 0.24%to 1 .78% (mean=0.7%). This diver­ Accord..ing to Lenk et al. ( 1999), marine straits repre­ gence is not related to the geographic origin of the sent no absolute baniers forE. orbicularis and coastal specimens (see Fig. 6, Fig. 7 and Appendix 1). The corridors could have promoted genetic exchange. This small intraspecific divergence observed in M rivulata, is probably also the case for M. rivulata since this spe­ is comparable to results reported by Lenk et al. ( 1999) cies inhabits also brackish waters (Gasith & Sidis, 1983; for Emys orbicularis. The authors compared a great Sid.is & Gasith, 1985; Engelmann et al., 1993). Conse­ number of populations from Europe and N. Africa, quently, the easily accessible marine straits, which which split to seven groups ofhaplotypes without being appeared in the Aegean area repeatedly during Pliocene separated geographically. and Pleistocene, and the great dispersal capacity of M. In general, phylogeograph..ic patterns are considered rivulata through coastal corridors, are supporting the to be the result of a multifactorial process, which is dispersal model. The extant populations of M. rivulata somewhat arbitrary and variable among different spe­ are probably still under the influence of this dispersal cies (Taberlet et al., 1998). The combination of existing procedure. AEGEAN MA UREMYS RJ VULA TA 43

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APPENDIX J

Genetic distances (Kimura two-parameter) between the Mauremys haplotypes and other tax a used in the analysis. M. r. , M. rivulata; M.c. , M. caspica; M.l. , M. leprosa; Ca., C. aurocapitata; H.s., H. sp inosa; T.m.,T. marginata; E. o. , E. orbicularis.

hi h2 h3 h4 h5 h6 h7 h8 h9 hlO hl I Ca. H.s. Tm. E.o. (M. r. ) (M. r. ) (M. r. ) (M. r. ) (M.r. ) (M. r. ) (M.r. ) (M.c. ) (M.c. ) (M. /. ) (M.c. ) (30)

hi (M.r. )

h2 (M.r. ) 0.24

h3 (M.r. ) 1.31 I.78 h4 (M.r.) 0.84 135 0.79 h5 (M. r. ) 0.55 1.07 0.53 0.53 h6 (M.r.) 0.28 0.80 0.26 0.53 0.26 h7 (M.r. ) 0.27 0.79 0.52 0.80 0.53 0.53 h8 (M. r. ) 3.54 3.54 3.28 337 3.07 3.06 3.83 h9 (M.c.) 2.39 2.66 3.79 3.73 3.40 3.08 3.02 1.81 h!O (M.I.) 6.07 6 16 6.04 5.77 5.79 5.79 6.32 7.00 6.45 hi I (M. /. ) 5.62 5.98 5.87 5.61 5.61 5.61 5.87 6.80 6.60 0.28 C. a. 8.76 9.04 9.03 9.33 8.94 8.58 7.77 8.39 8.19 9.72 9.46 H. s. 10.73 11.02 l I .2 1 10.33 10.26 10.23 9.39 9.68 l 0.45 11.46 1 l.15 11.58 T 111. 11.62 11.64 12.40 11.48 11.41 I 1 .38 11.17 12.26 11.20 12.75 12.95 13.6 12.46 E. o. (30) 11.58 11.82 13.56 14.08 13.73 13.4 1 12.64 14.33 11.53 13.74 14.53 14.53 11.68 13.58 E. o. (29) 11.88 12.13 13.90 14.45 14.08 13.76 12.98 14.68 11.53 14.12 14.90 14.53 11.68 13.90 0.25