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The parasitic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause the lethal disease chytridiomycosis in amphibians and therefore may play a role in population declines. The yellow-bellied toad Bombina variegata suffered strong declines throughout western and northwestern parts of its range and is therefore listed as highly endangered for Germany and the federal state of Hesse. Whether chytridiomycosis may play a role in the observed local declines of this strictly protected anuran species has never been tested. We investigated 19 Hessian yellow-bellied toad populations for Bd infection rates, conducted capture-mark-recapture studies in 4 of them over 2 to 3 yr, examined survival histories of recaptured infected individuals, and tested whether multi-locus heterozygosity of individuals as well as expected heterozygosity and different environmental variables of populations affect probabilities of Bd infection. Our results show high prevalence of Bd infection in Hessian yellow-bellied toad populations, but although significant decreases in 2 populations could be observed, no causative link to Bd as the reason for this can be established. Mass mortalities or obvious signs of disease in individuals were not observed. Conversely, we show that growth of Bd-infected populations is possible under favorable habitat conditions and that most infected individuals could be recaptured with improved body indices. Neither genetic diversity nor environmental variables appeared to affect Bd infection probabilities. Hence, genetically diverse amphibian specimens and populations may not automatically be less susceptible for Bd infection.

Members of the Euphorbiaceae are ecologically important elements of Southeast Asian forests. Species of the pioneer tree genus Macaranga, which is also known for its association with ants, are often abundant in disturbed areas. Phylogenetic studies suggested a recent radiation of section Pachystemon which comprises the majority of obligate myrmecophytes within Macaranga. In the present study, we analyzed the genetic structure of three closely related species of this section (M. constricta, M. griffithiana, and M. motleyana) with the aim of resolving their controversial taxonomy and historical biogeography. Chloroplast DNA haplotypes proved to be species-specific and showed a strong phylogeographic pattern. Nuclear microsatellite data supported the taxonomic distinctness of M. griffithiana and M. motleyana, but gave ambiguous results for M. constricta. Genetic differentiation was stronger each within M. griffithiana and M. motleyana than between M. constricta and M. griffithiana, highlighting problems of defining species boundaries. We found no indication for introgression or hybridization events. The high intraspecific morphological variation of the Bornean endemic M. motleyana was partly reflected by similar patterns of genetic variation. The pronounced genetic structure indicates a comparatively long diversification for this species. In contrast, the weak genetic differentiation within M. griffithiana, as well as the widespread distribution of its most common chloroplast haplotype from peninsular Malaysia up to Indochina, indicates a recent expansion in this area. Despite their morphological similarity and close relatedness, all species possess their own specific ant-partners with a corresponding distribution pattern.

Myrmekophyten sind standig von Ameisen besiedelte Pflanzen. Die Ameisen erhalten eine Wohnstatt und Futter von der Pflanze, im Gegenzug beschutzen sie ihren Wirt vor Herbivoren und pflanzlichen Konkurrenten. Die vielfaltigen Beziehungen zwischen tropischen Pionierbaumen der Gattung Macaranga und ihren Partnerameisen der Gattung Crematogaster stellen die bedeutendste und artenreichste mutualistische Ameisen-Pflanzen-Assoziation der Altwelttropen dar. Mit Hilfe vergleichender DNA-Sequenzierung und molekularer Marker-Technologien analysieren wir die Verwandtschaftsverhaltnisse innerhalb der etwa 30 myrmekophytischen Macaranga-Arten und ihre Co-Evolution mit Ameisen sowie die Populationsgenetik und historische Biogeografie einzelner Artengruppen. Ausmas und Strukturierung der genetischen Diversitat dieser Pionierbaumarten zeigen interessante Korrelationen mit ihrer Reproduktionsbiologie und der Besiedlung von zunehmend fragmentierten Pionierhabitaten im tropischen Regenwald. Molecular systematic studies in Southeast Asian ant-plants Myrmecophytes are plants that are permanently inhabited by ants. They provide nesting space and feed their partners, whereas the ants protect their hosts from herbivores and competitors such as climbers and lianas. The manifold relationships between tropical pioneer trees of the genus Macaranga and their partner ants of the genus Crematogaster constitute the most important and species-rich mutualistic ant-plant relationship of the Paleotropics. We use comparative DNA sequencing and molecular marker technologies to evaluate phylogenetic relationships among the about 30 myrmecophytic Macaranga species as well as their co-evolution with ants. We also study the population genetics and historical biogeography of particular Macaranga species groups. Patterns of genetic diversity and differentiation in these pioneer tree species show interesting correlations with their reproductive biology and with characteristics of the increasingly fragmented pioneer habitats in the rainforests of Sundaland.

As a result of intensive exploitation, disturbed forests now dominate large areas of lowland tropical rainforest in South-East Asia. The genus Macaranga comprises some of the most important pioneer tree species of the region, namong them M. beccariana and M. hypoleuca , two closely related obligate ant-plants pollinated by thrips. We used nuclear and plastid DNA markers to address questions of genetic diversity and population structure. Twelve plastid haplotypes were detected among 281 samples, three of which were shared between the two study species. Hybrids between the two species appear to be rare. Overall, genetic diversity in both species was moderate to high, with low levels of population differentiation, consistent with other tropical pioneer trees. Genetic structure was generally more pronounced in plastid than in nuclear data, indicating that gene flow via pollen may be more efficient than via seeds. Thrips apparently also serve as efficient pollinators over long distances, perhaps through a combination of passive dispersal by wind and active search for inflorescences in the target area. Our results indicate that M. beccariana and M. hypoleuca populations from recently disturbed habitats do not yet suffer from reduced genetic diversity or increased inbreeding.

A population genetic analysis based on eight genomic SSR markers and three EST-SSR (expressed sequence tags) markers developed in peach (Prunus persica (L.) Batsch) and Japanese plum (Prunus salicina Lindl.) was carried out in 12 wild populations of cherry plum (Prunus divaricata Ledeb.) sampled along the Iranian coast of the Caspian Sea. A total of 184 alleles (3–31 per locus) were detected with a mean value of 16.7 alleles per locus. None of the loci or populations showed deviation from Hardy–Weinberg equilibrium, and all markers proved to be unlinked. The mean values for the observed and the expected heterozygosity were 0.66 and 0.73, respectively. There was very little genetic differentiation among populations, as was indicated by low overall values of Wright’s FST (0.03) and Nei’s GST (0.08). An analysis of molecular variance (AMOVA) showed that 96.8% of the total variance was attributable to differences between individuals within populations. Genetic and geographic distances were nevertheless positively correlated, as evidenced by a Mantel test. The high level of genetic diversity and the apparent lack of genetic structure in wild P. divaricata may be attributed to frequent long distance gene flow through frugivorous birds and possibly humans, as has been documented for other Prunus species.

In the present study, we use mtDNA sequence data (cyt b gene) in combination with nuclear DNA sequences (C-mos, Rag2 genes, R35 intron), nuclear genomic fingerprints (ISSR) and morphological data to reveal species diversity within the Southeast Asian leaf turtle genus Cyclemys, a morphologically difficult group comprising cryptic species. Two morphologically distinct major groupings exist, a yellow-bellied species group with three taxa (Cyclemys atripons, C. dentata, C. pulchristriata) and a dark-bellied species group. The latter contains besides the morphologically variable C. oldhamii three additional new species (C. enigmatica n. sp., C. fusca n. sp., C. gemeli n. sp.). According to mtDNA data, C. fusca and C. gemeli constitute with high support the sister group of a clade comprising all other species, indicating that the dark-bellied species are not monophyletic, despite morphological similarity. mtDNA sequences of C. enigmatica, being highly distinct in nuclear genomic markers, do not differ from the sympatric C. dentata, suggesting that the original mitochondrial genome of C. enigmatica was lost due to introgressive hybridization. Morphological discrimination of Cyclemys species is possible using multivariate methods. However, gross morphology of most dark-bellied species on the one hand and of C. atripons and C. pulchristriata on the other is so similar that reliable species determination is only possible when genetic markers are used. The high diversity within Cyclemys requires revision of the IUCN Red List Categories for leaf turtles because the former assessment was based on the wrong assumption that in the entire range of the genus occurs only a single species.

The molecular phylogeography of the viperine snake, Natrix maura (Linnaeus, 1758), was investigated using complete sequences of the mitochondrial cytochrome b gene and genomic ISSR-PCR fingerprinting. In a total of 120 samples, 44 unique cytochrome b haplotypes were found which defined three major genetic lineages associated with samples from Morocco, Tunisia and Europe, respectively. The same lineages were supported by nuclear data. A possible fourth lineage exists in southern Spain. Genetic distances of cytochrome b sequences between the three main lineages were in the range of 3.9–5.6%, suggesting independent evolution since the early Pliocene. Distinction of the three major lineages at the subspecies or species level is discussed to account taxonomically for the high intraspecific variation in the viperine snake. A more detailed analysis of the European samples based on genetic diversity indices and a network reconstruction suggests a complex Pleistocene history for the viperine snake in Europe. Clear differentiation was found between populations south and north of the central Iberian mountain ranges, suggesting Pleistocene glacial refugia both in the southern and northern Iberian peninsula. In the south, genetic diversity was associated with the main river valleys, whereas northern haplotypes were more broadly distributed, indicating gene flow or postglacial range expansions. Unexpectedly high levels of genetic variation in southeastern France and northwestern Italy would be compatible with the hypothesis of a glacial refugium north of the Pyrenees or in Italy. However, due to the dependence of N. maura on warm climates, the assumption of a northern refugium seems unwarranted. We believe that further sampling in northern Spain is likely to reveal genetically diverse populations which could have served as sources for postglacial recolonization of France and Italy.

A phylogeographic analysis of eight species complexes of European reptiles was performed using different molecular methods. While mitochondrial genes (mainly cytochrome b sequences) enabled conclusions about phylogeography and differentiation, additional application of bisexually inherited markers provided information about speciation stages. As species with similar distribution patterns in southern and Central Europe were selected, matching phylogeographic patterns are useful for drawing general conclusions: (1) The species complexes are in different stages of speciation. In some cases, cryptic species were detected. (2) Highest genetic diversity occurs in southern Europe, the Near East and the Caucasus, regions corresponding with glacial refuges in the Iberian, Apennine and Balkan Peninsulas as well as in Turkey and the Caucasus. Often, several microrefugia must have existed in close neighbourhood. Additional microrefugia were located in southern France and in the Carpathian Basin. (3) North Africa and the Middle East did not serve as glacial refuges for Central or northern European lineages and are typically inhabited by independent clades. (4) Evidence for multiple range retractions and expansions, which were postulated for the times of Pleistocene climatic oscillations, could be found in the Balkans, but in Central Europe their traces have been wiped out by the last glacial. Only the Holocene invasion has left imprints in the genomes from this area. (5) Central and northern Europe were recolonized from Balkan and Pontic refugia in the Holocene.

We developed primer sequences for five polymorphic microsatellite loci in the tropical ant-plant genus Macaranga (Euphorbiaceae). Population genetic parameters were determined on the basis of 30 individuals from each of two Macaranga species in Borneo. Allele numbers per locus ranged from three to 13. Expected and observed heterozygosities ranged from 0.160 to 0.850 and from 0.130 to 0.700, respectively. Four of the five primer pairs cross-amplify polymorphic PCR products in a wide range of Macaranga species.

Some aspects of the natural history of snakes of the colubrid genus Natrix have been well studied. With their extensive European distribution and relative abundance, their ecology, reproduction and behaviour are well known. Yet other facets of their biology remain poorly understood. These include knowledge of Natrix phylogeny, hypotheses explaining the current distribution of the three extant members of the genus, and their evolution and relationships. In this study we used molecular data, the nucleotide sequences of four protein-coding mitochondrial genes (3806 bp total), to provide a well-supported phylogeny for the genus Natrix. With these molecular data, evidence from the fossil record, and knowledge of palaeogeological events, we used two approaches in designing a time scale which we used to date the major events in Natrix speciation and intraspecific variation. Our data strongly support a phylogeny for the genus in which N. maura is basal with N. natrix and N. tessellata being sister species. The calibrated molecular clock suggests that N. maura diverged from the common ancestor of the three species 18–27 mya and that N. natrix and N. tessellata diverged 13–22 mya. Although the ranges of these estimates are large they support an early Miocene to late Oligocene origin for the three species. Intraspecific divergence is estimated to have commenced 5.3, 6.0 and 6.7 mya with evolutionary rates of 1 : 1.25 : 1.35% per million years for N. maura, N. natrix and N. tessellata, respectively. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87, 127–143.

The viperine snake (Natrix maura) is an important agent of decline of the threatened midwife toad (Alytes muletensis) of Mallorca. However, there is a paucity of biological data to support the notion that the viperine snake is an introduced species to the island. Here we compare mitochondrial cytochrome b gene sequences and genomic ISSR-PCR fingerprints from Mallorcan and mainland European viperine snakes. Identical or nearly identical haplotypes and very similar ISSR-PCR profiles provide strong evidence that N. maura arrived only recently to Mallorca. There is no indication of a recent natural colonization of the island by transmarine dispersal. The data therefore support historical information that N. maura was introduced to Mallorca by human agency comparatively recently, and that management measures to reduce the impact of the snake on toad populations are justified.

Abstract The European pond turtle, Emys orbicularis, is a wide ranging species, distributed from Northwest Africa over a large part of Europe and Asia Minor to the Caspian and Aral Seas. For 106 pond turtles from France mtDNA sequence variation has been assessed, using a 1031 bp portion of the mitochondrial cytochrome b gene. Three of nine haploclades currently known from the entire species' range were found in France. One clade (II) is represented with four very similar haplotypes, differing by one mutation, and the two other clades (V, VI) are represented with one haplotype each. A syntopic occurrence of clades II and V is reported for the first time for the Camargue. Besides, clade II occurs in the French regions Aquitaine, Centre-Val de Loire, and Rhône-Alpes. Outside of France, it is found mainly in the catchment areas of the Danube and Oder rivers and in the Balkans. Haploclade V, which is also known from the Apennine peninsula, Sardinia, and the northern Mediterranean coast of Spain, is restricted in France to Corsica and the Provence-Alpes-Côte d'Azur region. A single individual bearing a haplotype of an Iberian and North African clade (VI) was found in Aquitaine near Pau. This could indicate gene flow between the Iberian peninsula and West France, if the specimen is native. The distribution of the distinct haploclades in France probably reflects Holocene range expansions, especially of haploclade II turtles. In the postglacial, haploclade II terrapins arrived from the east and spread over the Rhône corridor to the Mediterranean coast. In the southern Rhône area they met and hybridized with haploclade V turtles. Further research is needed to clarify whether this hybridization is a locally restricted phenomenon.

We present morphometric data for the pink-footed shearwater (Puffinus creatopus) from both Chilean breeding colonies on Isla Mocha and Juan Fernandez: weight, total length, wing length, tail length, head length, three bill and three tarsus size measurements and length of middle toe. Significant differences were found for most measurements between males and females as well as between birds from the two different breeding localities. In general, males were bigger than females, and birds from Juan Fernandez were bigger than those from Isla Mocha. Geographic differences most likely reflect some kind of ecomorphological adaptation. Some variables, especially the total length of head, seem to be useful for sex determination in the field.

We present a simple method to screen for DNA sequence variation in microsatellite- and indel-rich regions of the chloroplast genome. The single nucleotide sequence (SNS) analysis provides a trade-off between the time- and cost-effective, but less informative and homoplasy-sensitive electrophoretic detection of microsatellite and indel size variation on the one hand, and more costly, but also more accurate methods like DNA sequencing on the other. The principle of the SNS method is to sequence one instead of all four nucleotides of a target region amplified by polymerase chain reaction. By careful selection of the respective nucleotide, almost the same amount of information can be retrieved from these partial sequences as could be from complete sequences; however, only a third to a fourth of the money and time resources are needed.

We provide primer sequences for 11 new polymorphic microsatellite markers developed in the tropical ant-plant genus Macaranga (Euphorbiaceae), after enrichment cloning of Macaranga tanarius and Macaranga hypoleuca. Allele numbers per locus ranged from two to 16 among 20 accessions of M. tanarius, and from three to 10 among 22 accessions of M. hypoleuca. Observed and expected heterozygosities ranged from 0.150 to 0.900 and from 0.375 to 0.894 in M. tanarius, and from 0.545 to 1.000 and from 0.434 to 0.870 in M. hypoleuca, respectively. Six of the 11 primer pairs successfully cross-amplified polymorphic polymerase chain reaction products in Macaranga winkleri.

The palaeotropic genus Macaranga (Euphorbiaceae) is an excellent model system to analyze co-evolutionary processes associated with myrmecophytism, a mutualistic interaction between plants and ants. Ant-plants like Macaranga provide nesting space and feed their partners, whereas the ants protect the plants from herbivores and competitors such as lianas. We used genome-based evidence to investigate speciation mechanisms in Macaranga ant-plants, and their co-evolution with ants from the genus Crematogaster. Our previous work had shown that myrmecophytic Macaranga species show little genetic differentiation, suggesting an adaptive radiation. We hypothesized that the obligatory symbiosis with ants may reduce gene flow among plant populations, eventually enhancing allopatric speciation. To test this hypothesis, we verified the monophyly of the investigated plant lineages by phylogenetic analyses, reconstructed parsimony networks based on chloroplast DNA (cpDNA) variation, and assessed population genetic parameters using nuclear microsatellites and cpDNA haplotypes. Our data provided evidence for vicariant events as well as for hybridization and cpDNA introgression among closely related Macaranga species. The extent of population differentiation within myrmecophytic versus non-myrmecophytic species proved to be in a similar range, indicating that our working hypothesis of enhanced allopatric speciation in myrmecophytes cannot be sustained by the present evidence. Nevertheless, the mutualistic interactions of Macaranga and associated ants may be a key innovation that opened an adaptive zone putatively exploited by the divergence of Macaranga.

The phylogeographic history of eight species complexes of West Palearctic reptiles was reconstructed using mitochondrial and nuclear markers. Cryptic endemic taxa were detected in the Southern European peninsulas (Iberia, Southern Italy/Sicily, and Greece) as well as in North Africa, Anatolia, Iran, and the Caucasus. These endemics are mainly of Tertiary or early Pleistocene age. Only part of them can be categorized as relicts of a former, more widely distributed taxon, having survived in ice age refugia; others have probably always remained in a restricted area, close to their origin.

Abstract Based on more than 1100 samples of Emys orbicularis and E. trinacris, data on mtDNA diversity and distribution of haplotypes are provided, including for the first time data for Armenia, Georgia, Iran, and the Volga, Ural and Turgay River Basins of Russia and Kazakhstan. Eight mitochondrial lineages comprising 51 individual haplotypes occur in E. orbicularis, a ninth lineage with five haplotypes corresponds to E. trinacris. A high diversity of distinct mtDNA lineages and haplotypes occurs in the south, in the regions where putative glacial refuges were located. More northerly parts of Europe and adjacent Asia, which were recolonized by E. orbicularis in the Holocene, display distinctly less variation; most refuges did not contribute to northern recolonizations. Also in certain southern European lineages a decrease of haplotype diversity is observed with increasing latitude, suggestive of Holocene range expansions on a smaller scale.

Geographic variation in the mtDNA haplotypes (cytochrome b gene) of 127 European pond turtles from Italy was investigated. Thirty-eight of the Italian samples were also studied by nuclear fingerprinting (ISSR PCR) and compared with samples from other parts of the range representing all nine currently known mtDNA lineages of Emys orbicularis. Our genetic findings were compared against morphological data sets (measurements, colour pattern) for 109 adult turtles from southern Italy. Italy is displaying on a small geographical scale the most complicated variation known over the entire distributional area of Emys (North Africa over Europe and Asia Minor to the Caspian and Aral Seas). The Tyrrhenic coast of the Apennine Peninsula, the Mt. Pollino area and Basilicata are inhabited by Emys orbicularis galloitalica, a subspecies harbouring a distinct mtDNA lineage. The same lineage is also found in Sardinia. Along the Adriatic coast of Italy and on the Salentine Peninsula (Apulia, southern Italy), another morphologically distinctive subspecies (Emys orbicularis hellenica) occurs, which also bears a different mtDNA lineage. A higher diversity of mtDNA haplotypes in the south of the Apennine Peninsula suggests that the glacial refugia of E. o. galloitalica and E. o. hellenica were located here. A further refuge of E. o. hellenica probably existed in the southern Balkans. The west coasts of the Balkans and Corfu have probably been colonized from Italy and not from the geographically closer southern Balkanic refuge. In Sicily, a third mtDNA lineage is distributed, which is sister to all other known lineages of Emys. Morphologically, Sicilian pond turtles resemble E. o. galloitalica. However, nuclear fingerprinting revealed a clear distinctiveness of the Sicilian taxon, whereas no significant divergence was detected between representatives of the other eight mtDNA lineages of Emys. Furthermore, nuclear fingerprinting provided no evidence for current or past gene flow between the Sicilian taxon and the mainland subspecies of E. orbicularis. Therefore, Sicilian pond turtles are described here as a species new to science. Some populations in Calabria and on the Salentine Peninsula comprise individuals of different mtDNA lineages. We interpret this as a natural contact. However, we cannot exclude that these syntopic occurrences are the result of human activity. For example, in other parts of Italy, the natural distribution pattern of Emys is obscured by allochthonous turtles. This could also be true for southern Italy. The discovery of the complex taxonomic differentiation in southern Italy requires reconsidering conservation strategies.

Macaranga (Euphorbiaceae) has received much ecological and evolutionary research attention as a genus that includes some of the most conspicuous pioneer trees of Southeast Asian tropical rainforests and because of its manifold associations with ants, including about 30 species that are obligate ant-plants (myrmecophytes). We used sequence data from three chloroplast DNA loci (ccmp5, ccmp6, atpB-rbcL) to assess phylogeographical patterns in species of Macaranga, section Pruinosae, sampled from various regions of Borneo and the Malay Peninsula. Forty-nine chloroplast DNA haplotypes (HT) were identified among 768 specimens from five species, Macaranga gigantea (N = 329; 23 HT), Macaranga pearsonii (N = 347; 21 HT), Macaranga puberula (N = 24; 4 HT), Macaranga hosei (N = 48; 6 HT), and Macaranga pruinosa (N = 20; 5 HT). Forty-one haplotypes were species-specific, whereas eight haplotypes were shared by two, three, or four species and occupied internal positions in a parsimony network. Population genetic parameters based on haplotype frequencies proved to be in a similar range in the non-myrmecophytic M. gigantea and in the ant-associated M. pearsonii, which have overlapping distributions in northern and eastern Borneo. A comparison of GST and NST values revealed a strong phylogeographic structure in both species, whereas colonization pathways suggested by the network topology were different. Both species exhibited similar levels of haplotypic diversity and moderate to high levels of population differentiation. There were no obvious indications for an influence of the symbiotic ant partners on the population structure of their host plants.

Satellite tracking was used to examine movements of the threatened Pink-footed Shearwater (Puffinus creatopus) from Isla Mocha, Chile, during the chick-rearing period in 1998. We were able to track one breeding bird, one bird of unknown status and one prospector. Whereas the first two performed foraging flights and returned to the colony after about a two weeks absence, the last traveled north for four weeks, suggesting that it had started migration towards the wintering grounds in the northern hemisphere. Faster travel speeds during the day than during the night were found for all three birds. A major foraging zone was located about 250-300 km north of Isla Mocha and close to the Chilean mainland. The preference of Pink-footed Shearwaters for waters close to the coast when feeding, as well as when migrating, has implications for the conservation of the species. Received 5July 2000, accepted 26 August 2000.

The European pond turtle, Emys orbicularis (Linnaeus, 1758), is one of the world's most widely distributed chelonian species. We investigated its mitochondrial phylogeography and demography using ∼1300cyt b sequences from the entire range, with a focus on the eastern part, in particular on Turkey, where the species currently suffers massive losses. Coloration data from >1450 turtles were compared with mtDNA differentiation to assess the validity of the currently accepted subspecies from Turkey, the Black Sea Region, the Transcaucasus, and Iran. Our study region harbors considerable part of the mtDNA diversity of Emys, including a newly discovered lineage and 16 new haplotypes. In this area corresponding to approximately one-third of the entire distribution range, six out of the ten mitochondrial lineages and about half of all 72 haplotypes occur. Two mitochondrial lineages (VIII, X) are confined to small ranges along the southern coast of Turkey, another lineage (I) occupies the remainder of Turkey, the entire Black Sea Region, and the north-eastern part of the species’ range. In the south-western corner of the Black Sea and in the Aegean Region, two lineages (II, IV) occur that have their main distribution areas farther west. In the Transcaucasus and northern Iran, another endemic lineage (VII) is found. Lineage I is the largest and most diverse of all lineages and has its greatest diversity in Anatolia. Phylogeographic and demographic data suggest Anatolia as an ancient glacial refuge for turtles harboring mitochondrial lineages I, VIII and X, and that Anatolia and the Black Sea coasts constitute a hotspot for a younger burst of diversification within lineage I. These two regions correspond to the glacial refuge from which lineage I turtles recolonized more northerly parts of the range in the Holocene; lineage II represents an off-shoot of lineage I that became isolated in a westward-located refuge in the south-eastern Balkans during a previous Pleistocene glacial. Our data on coloration indicate that such characters have only limited value for delineating evolutionarily significant units. We propose to reduce the number of subspecies using mtDNA lineages as arbiter, and to recognize three subspecies as valid in Turkey, the Black Sea Region, the Transcaucasus and Iran: Emys orbicularis orbicularis (mtDNA lineage I); E. o. eiselti Fritz et al., 1998 (X); and E. o. persica Eichwald, 1831 (VII). However, the southern Turkish lineage VIII most probably represents an additional undescribed subspecies. Both southern Turkish endemics are critically endangered, with only three surviving populations of fewer than 30 adults each. We recommend establishing sanctuaries for them, and including them in the IUCN Red List.

The dice snake, Natrix tessellata (Laurenti, 1768), is a suitable study organism to address questions of Eurasian phylogeography due to its wide Palearctic distribution. We analysed complete mitochondrial cytochrome b sequences and nuclear ISSR-PCR fingerprints of more than 300 specimens representing nearly the entire geographic range. Nine major mitochondrial lineages were discovered based on mtDNA sequences. The three most basal lineages comprised populations from Iran, Jordan–Egypt, and Greece, respectively. Other lineages were associated with samples from the Turkish peninsula, the Caucasus, the Aral Sea, and eastern Kazakhstan. A sister-group relationship was found between two lineages from Crete and the European mainland. Assuming an evolutionary rate of 1.35% sequence divergence per million years, among-lineage p-distances of 1.7–8.4% suggest that intraspecific differentiation might date back as far as the Miocene/Pliocene transition 5–6 million years ago. The pattern of genetic differentiation in mitochondrial phylogeny with regard to Asia Minor and the region of the Aral Sea was not congruent with the results of the nuclear ISSR-PCR analyses, and suggests admixing within some mtDNA clades at contact zones. The taxonomic implications of the high intraspecific variation in the dice snake are discussed.