Markus Auer

Phenotypic plasticity leads to incongruence between morphology-based taxonomy and genetic differentiation in western Palaearctic tortoises (Testudo graeca complex; Testudines, Testudinidae)

Tortoises of the Testudo graeca complex inhabit a patchy range that covers part of three continents (Africa, Europe, Asia). It extends approximately 6500 km in an east-west direction from eastern Iran to the Moroccan Atlantic coast and about 1600 km in a north-south direction from the Danube Delta to the Libyan Cyrenaica Peninsula. Recent years have seen a rapid increase of recognized taxa. Based on morphological investigations, it was suggested that this group consists of as many as 20 distinct species and is paraphyletic with respect to T. kleinmanni sensu lato and T. marginata . Based on samples from representative localities of the entire range, we sequenced the mitochondrial cytochrome b gene and conducted nuclear genomic fingerprinting with ISSR PCR. The T. graeca complex is monophyletic and sister to a taxon consisting of T. kleinmanni sensu lato and T. marginata . The T. graeca complex comprises six well-supported mtDNA clades (A-F). Highest diversity is found in the Caucasian Region, where four clades occur in close neighbourhood. This suggests, in agreement with the fossil record, the Caucasian Region as a radiation centre. Clade A corresponds to haplotypes from the East Caucasus. It is the sister group of another clade (B) from North Africa and western Mediterranean islands. Clade C includes haplotypes from western Asia Minor, the southeastern Balkans and the western and central Caucasus Region. Its sister group is a fourth, widely distributed clade (D) from southern and eastern Asia Minor and the Levantine Region (Near East). Two further clades are distributed in Iran (E, northwestern and central Iran; F, eastern Iran). Distinctness of these six clades and sister group relationships of (A + B) and (C + D) are well-supported; however, the phylogeny of the resulting four clades (A + B), (C + D), E and F is poorly resolved. While in a previous study (Fritz et al., 2005a) all traditionally recognized Testudo species were highly distinct using mtDNA sequences and ISSR fingerprints, we detected within the T. graeca complex no nuclear genomic differentiation paralleling mtDNA clades. We conclude that all studied populations of the T. graeca complex are conspecific under the Biological Species Concept. There is major incongruence between mtDNA clades and morphologically defined taxa. Morphologically well-defined taxa, like T. g. armeniaca or T. g. floweri , nest within clades comprising also geographically neighbouring, but morphologically distinctive populations of other taxa (clade A: T. g. armeniaca , T. g. ibera , T. g. pallasi ; clade D: T. g. anamurensis , T. g. antakyensis , T. g. floweri , T. g. ibera , T. g. terrestris ), while sequences of morphologically similar tortoises of the same subspecies ( T. g. ibera sensu stricto or T. g. ibera sensu lato) scatter over two or three genetically distinct clades (A, C or A, C, D, respectively). This implies that pronounced morphological plasticity, resulting in phenotypes shaped by environmental pressure, masks genetic differentiation. To achieve a more realistic taxonomic arrangement reflecting mtDNA clades, we propose reducing the number of T. graeca subspecies considerably and regard in the eastern part of the range five subspecies as valid ( T. g. armeniaca , T. g. buxtoni , T. g. ibera , T. g. terrestris , T. g. zarudnyi ). As not all North African taxa were included in the present study, we refrain from synonymizing North African taxa with T. g. graeca (mtDNA clade B) that represents a further valid subspecies.

Mitochondrial phylogeography of European pond turtles ( Emys orbicularis , Emys trinacris ) – an update

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.

A rangewide phylogeography of Hermann's tortoise, Testudo hermanni (Reptilia: Testudines: Testudinidae): implications for taxonomy

Hermanns tortoise (Testudo hermanni), the best‐known western Palaearctic tortoise species, has a rare natural distribution pattern comprising the Mediterranean areas of the Iberian, Apennine, and Balkan Peninsulas, as well as Sicily, Corsica and Sardinia. The western part of this range is traditionally considered habitat for T. h. hermanni, while T. h. boettgeri occurs in the Balkans. Taxonomy of this tortoise has been challenged in recent years, with the two subspecies being considered full species and the central Dalmatian populations of T. h. boettgeri being considered a third species, T. hercegovinensis. Using an mtDNA fragment approximately 1150 bp long (cytochrome b gene and adjacent portion of tRNA‐Thr gene), we investigated mtDNA diversity with regard to contrasting concepts of two subspecies or three species. Seven closely related haplotypes were identified from the western Mediterranean and 15 different, in part much‐differentiated, haplotypes from the Balkans. Western Mediterranean haplotypes differ from Balkan haplotypes in 16–42 mutation steps. One to seven mutation steps occur within western Mediterranean populations. Balkan haplotypes, differing in 1−37 nucleotides, group in parsimony network analysis into three major assemblages that display, in part, a similar degree of differentiation to that of western Mediterranean haplotypes relative to Balkan haplotypes. Rates of sequence evolution are different in both regions, and low divergence, palaeogeography and the fossil record suggest a slower molecular clock in the western Mediterranean. While monophyly in western Mediterranean haplotypes is well‐supported, conflicting evidence is obtained for Balkan haplotypes; maximum parsimony supports monophyly of Balkan haplotypes, but other phylogenetic analyses (Bayesian, ML, ME) indicate Balkan haplotypes could be paraphyletic with respect to the western Mediterranean clade. These results imply a process of differentiation not yet complete despite allopatry in the western Mediterranean and the Balkans, and suggest all populations of T. hermanni are conspecific. In the western Mediterranean no clear geographical pattern in haplotype distribution is found. Distribution of Balkan haplotypes is more structured. One group of similar haplotypes occurs in the eastern Balkans (Bulgaria, Republic of Macedonia, Romania and the Greek regions Evvia, Macedonia, Peloponnese, Thessaly and Thrace). Two distinct haplotypes, differing in eight to nine mutation steps from the most common haplotype of the first group, are confined to the western slope of the Taygetos Mts. in the Peloponnese. Yet another group, connected over between four and 23 mutation steps with haplotypes of the eastern Balkan group, occurs along the western slope of the Dinarid and Pindos Mts. (Istria, Dalmatia, western Greece). Taygetos haplotypes are nested within other haplotypes in all phylogenetic analyses and support for monophyly of the other Balkan groups is at best weak. We conclude that using the traditional two subspecies model should be continued for T. hermanni. Phylogeographies of T. hermanni and Emys orbicularis, another codistributed chelonian, are markedly different, but share a few similarities. Both were forced to retreat to southern refuges during Pleistocene glaciations. With the advent of Holocene warming, E. orbicularis underwent rapid range expansion and temperate regions of Europe and adjacent Asia were recolonized from refuges in the Balkans and the northern Black Sea Region. By contrast, T. hermanni remained more or less confined to refuges and nearby regions, resulting in a much smaller range, and allopatric and parapatric distribution of haplotype groups and clades. MtDNA lineages are more diverse in E. orbicularis than they are in T. hermanni on southern European peninsulas, indicating several distinct glacial refuges in close proximity and extensive intergradation during Holocene range expansion for E. orbicularis. In T. hermanni it is likely that only on the Balkan Peninsula was more than one refuge located, corresponding to the parapatric ranges of haplotype groups currently there. On the old western Mediterranean islands Corsica and Sardinia no differentiated (E. orbicularis) or only weakly differentiated haplotypes (T. hermanni) occur, even though there is evidence for the presence of both species on Corsica since at least the Middle Pleistocene. High mountain chains constitute major barriers separating distinct mtDNA clades or groups in each species.

Go east: phylogeographies of Mauremys caspica and M. rivulata– discordance of morphology, mitochondrial and nuclear genomic markers and rare hybridization

In recent years many cases of hybridization and introgression became known for chelonians, requiring a better understanding of their speciation mechanisms. Phylogeographic investigations offer basic data for this challenge. We use the sister species Mauremys caspica and M. rivulata, the most abundant terrapins in the Near and Middle East and South‐east Europe, as model. Their phylogeographies provide evidence that speciation of chelonians fits the allopatric speciation model, with both species being in the parapatric phase of speciation, and that intrinsic isolation mechanisms are developed during speciation. Hybridization between M. caspica and M. rivulata is very rare, suggesting that the increasing numbers of hybrids in other species are caused by human impact on environment (breakdown of ecological isolation). Genetic differentiation within M. caspica and M. rivulata resembles the paradigm of southern genetic richness and northern purity of European biota. However, in west Asia this pattern is likely to reflect dispersal and vicariance events older than the Holocene. For M. caspica three distinct Pleistocene refuges are postulated (Central Anatolia, south coast of Caspian Sea, Gulf of Persia). Morphologically defined subspecies within M. caspica are not supported by genetic data. This is one of the few studies available about the phylogeography of west and central Asian species.

Diversity of the Southeast Asian leaf turtle genus Cyclemys: how many leaves on its tree of life?

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.

Osteology in the Cuora galbinifrons complex suggests conspecifity of C. bourreti and C. galbinifrons, with notes on shell osteology and phalangeal formulae within the Geoemydidae

Thirty-six complete skeletons, three shells, and x-rays of the extremities of 32 additional turtles of the Cuora galbinifrons complex have been compared with 38 other geoemydid species from 19 genera. Cuora bourreti differs from C. galbinifrons and C. picturata by a lost phalanx in the fourth finger and fourth toe. Individuals with a shell shape intermediate between C. bourreti and C. galbinifrons, as found on Hainan Island (China), have either the reduced phalangeal formula of C. bourreti (manus: 2-3-3-2-2, pes: 2-3-3-2-1) or the complete number of phalanges (manus: 2-3-3-3-2, pes: 2-3-3-3-1). Only in C. flavomarginata did we also register a lost phalanx in the fourth digit of manus and pes; in the pes of C. mouhotii the same character state may occur. In C. flavomarginata the fifth digit of the pes is also lacking. Some other terrestrial and semiterrestrial geoemydids (Cuora mccordi, Heosemys spinosa, and in part C. mouhotii and Leucocephalon yuwonoi) display a similar pattern of phalangeal reduction, resulting in the loss of the fifth digit of the pes. Likewise, in tortoises (Testudinidae), a further group of terrestrial chelonians, and the terrestrial turtle genus Terrapene (Emydidae) the loss of phalanges or complete digits is known to occur. Malayemys subtrijuga, Morenia petersi, Pangshura smithii and Siebenrockiella crassicollis differ from all other studied geoemydid taxa by an additional phalanx in the fifth digit of the manus (2-3-3-3-3); one P. smithii has on one body side three phalanges in the fifth digit of the pes (2-3-3-3-3). These are highly aquatic turtles with extensive toe webbing. Probably, longer digits (and thus a higher phalangeal number) are a favorable prerequisite for swimming while phalangeal loss seems to be the consequence of walking. Cuora bourreti and C. picturata have consistently in the bony carapace a very rare character state regarding the articulation of the rib tips with the peripheral plates. In both species the rib tips are intercalated between two peripheral plates in the bridge region. Intercalated rib tips like those in C. bourreti and C. picturata were found only in C. m. mouhotii, but not in the southern subspecies C. mouhotii obsti .I nC. galbinifrons and all other geoemydid taxa studied the rib tips articulate on the bridge within the underlying peripheral and not between two peripherals. In turtles morphologically intermediate between C. bourreti and C. galbinifrons both characters states are found. This suggests that such individuals are hybrids or intergrades. Three known- locality specimens from Hainan Island display both extremes and an intermediate character state. This, together with external morphology and the occurrence of both phalangeal formulae in approximately the same frequency on that island, argues for genetic introgression of C. bourreti on the Hainan population of C. galbinifrons. We conclude that our findings qualify C. galbinifrons and C. bourreti under the Biological Species Concept as conspecific.

Phalangeal formulae and ontogenetic variation of carpal morphology in Testudo horsfieldii and T. hermanni

We compared variation in phalangeal and carpal morphology of the Central Asian Testudo horsfieldii, a burrow- digging tortoise species, with its sister taxon T. hermanni, a Mediterranean species without extensive digging behaviour. Thirty-two Testudo horsfieldii kazachstanica shared the same phalangeal formula (0-2-2-1-1). The distal carpal 1 and metacarpal I as well as the pisiform were consistently lacking, sometimes also the medial centrale. Phalangeal morphology was more variable in Testudo hermanni hermanni. In 29 specimens four phalangeal formulae were found that differed with respect to the reduction of digit 1 (1-2-2-2-1, M-2-2-2-1, D-2-2-2-1, 0-2-2-2-1). The pisiform develops late in ontogeny and is lacking in very most juveniles and subadults. In contrast to T. h. kazachstanica, the medial centrale is always present. In both T. h. kazachstanica and T. h. hermanni carpalia increasingly fuse with age and size. The underlying morphological patterns differ however. The extreme character state in aged T. h. kazachstanica is one large solid bone element, formed by the fused intermedium, ulnare and both centralia. Aged T. h. hermanni have, in contrast, two separate larger carpal elements, one formed by the fused lateral and medial centralia and the other by the fused distal carpalia 1 and 2; the intermedium and ulnare never fuse with one another or with other carpalia. While a partial or complete loss of digit 1 seems to be characteristic for all Testudo species, we propose that the extensive fusion of carpal elements in T. horsfieldii is correlated with its natural history because a rigid manus could be advantageous for burrow-digging. Also the reduction of digit 4 (one phalanx present), a rare character among testudinids and not occurring in other Testudo species, could be linked with its mode of life.

Variation of hyoid morphology in geoemydid terrapins

The hyoid apparati of 25 geoemydid species in 16 genera (including aquatic, semiterrestrial and terrestrial taxa) are compared, and for Cuora galbinifrons bourreti ontogenetic development of the hypoid apparatus is described. Generally, ossification of the hyoid apparatus increases with age. The majority of terrestrial species maintain cartilaginous or partially cartilaginous hyoid bodies and second branchial horns throughout life however. Also in tortoises (Testudinidae) hyoid body and second branchial horns remain cartilaginous, suggesting a positive correlation between mode of life and hyoid morphology (terrestrial species with partly cartilaginous hyoid apparatus vs. aquatic species with fully ossified hyoid apparatus).

Phylogeography of the endangered black-breasted leaf turtle ( Geoemyda spengleri ) and conservation implications for other chelonians

Known-locality samples of Geoemyda spengleri from three Chinese provinces (Guangdong, Guangxi, Hainan) and northern Vietnam provide clear evidence for phylogeographic structure in a southern Chinese chelonian species. Within southern China two clades of mitochondrial haplotypes from east and west of the Xi Jiang together form the sister group to haplotypes from northern Vietnam. Turtles from Hainan Island harbour haplotypes of the same clade as turtles from Guangxi, 400 km distant. These findings suggest that phylogeographic differentiation may have once existed in other co-distributed, highly endangered chelonian species. The possibility of phylogeographic structure should be considered in any in-situ and ex-situ conservation program for Chinese chelonians.

Phalangeal formulae of geoemydid terrapins (Batagur, Callagur, Hardella, Heosemys, Kachuga, Orlitia, Pangshura, Rhinoclemmys) reflect distinct modes of life

Using radiographies of manus and pes of 133 geoemydid terrapins of nine highly aquatic species (genera Batagur, Callagur, Hardella, Kachuga, Orlitia, Pangshura), four aquatic Rhinoclemmys species, three semiterrestrial Heosemys species, and three terrestrial Rhinoclemmys species, we confirm a correlation between mode of life and phalangeal formulae. Terrestrial geoemydid species tend to have lost phalanges or entire digits, while highly aquatic species with extensive toe webbing generally have retained the full phalangeal number in manus and pes. Phalangeal formulae of species that are not so strictly adapted to aquatic environments, and having less extensive toe webbing, are intermediate, like in the studied semiterrestrial species. In a recent paper, Fritz et al. (2006) proposed that phalangeal formulae in geoemydid terrap- ins are correlated with the mode of life. Ter- restrial species tend to have reduced phalangeal formulae of manus and pes, while in aquatic species no loss of phalanges or digits occurs. However, only few individuals of highly aquatic species were available in that study (one spec- imen each of Malayemys subtrijuga, Morenia petersi, Pangshura smithii, and Siebenrockiella crassicollis). In the present paper, we test this hypothesis by comparing phalangeal formulae of nine highly aquatic geoemydid species of the genera Batagur, Callagur, Hardella, Kachuga, Orlitia, and Pangshura with seven Rhinoclem- mys and three Heosemys species. Three of the Rhinoclemmys species are fully terrestrial; the other four represent freshwater turtles that are less adapted to aquatic lifestyle and have less extensive toe webbing than the other previ- ously mentioned aquatic geoemydids. Among the three semiterrestrial Heosemys species, two prefer terrestrial habitats as adults (H. depressa,