Nikolay A. Poyarkov

Phylogeography of two European newt species — discordance between mtDNA and morphology

The newts Triturus vulgaris and Triturus montandoni are sister species that exhibit contrasting levels of intraspecific morphological variation. Triturus vulgaris has a broad Eurasiatic distribution encompassing both formerly glaciated and unglaciated areas and shows substantial morphological differentiation in the southern part of its range, while T. montandoni, confined to the Carpathians, is morphologically uniform. We analysed sequence variation of two mtDNA fragments of the total length of c. 1850 bp in 285 individuals of both species collected from 103 localities. Phylogenetic analysis of 200 unique haplotypes defined 12 major clades, their age estimated at c. 4.5–1.0 million years (Myr). Most of the older clades were found in the southern part of the range, and also in central Europe, mainly in Romania. The distribution of mtDNA clades points to the existence of several glacial refugia, located in the Caucasus region, Anatolia, the Balkan Peninsula, Italy, and more to the north in central Europe. The concordance between mtDNA based phylogeny and the distribution of T. vulgaris subspecies was weak. Triturus montandoni haplotypes did not form a monophyletic group. Instead they were found in six clades, in five of them mixed with T. vulgaris haplotypes, most likely as a result of past or ongoing hybridization and multiple introgression of mtDNA from T. vulgaris to T. montandoni. Patterns of sequence variation within clades suggested long‐term demographic stability in the southern groups, moderate and relatively old demographic growth in the populations inhabiting central Europe, and high growth in some of the groups that colonized northern parts of Europe after the last glacial maximum.

Cryptic crested newt diversity at the Eurasian transition : the mitochondrial DNA phylogeography of near Eastern Triturus newts

Crested newts of the Triturus karelinii group occur in a phylogeographically understudied region: the Near East. Controversy surrounds the systematic position of these newts within the complete crested newt assemblage (the Triturus cristatus superspecies). We explore the situation using mitochondrial sequence data (ND2 and ND4, approximately 1.7kb) and employing different methods of phylogenetic inference (Bayesian inference and Maximum Likelihood using mixed models) and molecular dating (r8s and BEAST). The T. karelinii group is monophyletic and constitutes one of four main lineages in the T. cristatus superspecies. The separation of the T. karelinii group from the remaining crested newts around 9Ma is related to the formation of the Mid-Aegean Trench, which separated the Balkan and Anatolian landmasses. The T. karelinii group comprises three geographically structured clades (eastern, central and western). The genetic divergence shown by these clades is comparable to that among recognized crested newt species. We suggest the uplift of the Armenian Plateau to be responsible for the separation of the eastern clade around 7Ma, and the re-establishment of a marine connection between the Black Sea and the Mediterranean at the end of the Messinian Salinity Crisis to have caused the split between the central and western clade around 5.5Ma. Genetic structuring within the three clades dates to the Quaternary Ice Age (<2.59Ma) and is associated with alternating periods of isolation and reconnection caused by periodic changes in sea level and surface runoff.

Multilocus species tree analyses resolve the radiation of the widespread Bufo bufo species group (Anura, Bufonidae)

New analytical methods are improving our ability to reconstruct robust species trees from multilocus datasets, despite difficulties in phylogenetic reconstruction associated with recent, rapid divergence, incomplete lineage sorting and/or introgression. In this study, we applied these methods to resolve the radiation of toads in the Bufo bufo (Anura, Bufonidae) species group, ranging from the Iberian Peninsula and North Africa to Siberia, based on sequences from two mitochondrial and four nuclear DNA regions (3490 base pairs). We obtained a fully-resolved topology, with the recently described Bufo eichwaldi from the Talysh Mountains in south Azerbaijan and Iran as the sister taxon to a clade including: (1) north African, Iberian, and most French populations, referred herein to Bufo spinosus based on the implied inclusion of populations from its type locality and (2) a second clade, sister to B. spinosus, including two sister subclades: one with all samples of Bufo verrucosissimus from the Caucasus and another one with samples of B. bufo from northern France to Russia, including the Apennine and Balkan peninsulas and most of Anatolia. Coalescent-based estimations of time to most recent common ancestors for each species and selected subclades allowed historical reconstruction of the diversification of the species group in the context of Mediterranean paleogeography and indicated a long evolutionary history in this region. Finally, we used our data to delimit the ranges of the four species, particularly the more widespread and historically confused B. spinosus and B. bufo, and identify potential contact zones, some of which show striking parallels with other co-distributed species.

Cold Code: the global initiative to DNA barcode amphibians and nonavian reptiles

DNA barcoding facilitates the identification of species and the estimation of biodiversity by using nucleotide sequences, usually from the mitochondrial genome. Most studies accomplish this task by using the gene encoding cytochrome oxidase subunit I (COI; Entrez COX1). Within this barcoding framework, many taxonomic initiatives exist, such as those specializing in fishes, birds, mammals, and fungi. Other efforts center on regions, such as the Arctic, or on other topics, such as health. DNA barcoding initiatives exist for all groups of vertebrates except for amphibians and nonavian reptiles. We announce the formation of Cold Code, the international initiative to DNA barcode all species of these ‘cold‐blooded’ vertebrates. The project has a Steering Committee, Coordinators, and a home page. To facilitate Cold Code, the Kunming Institute of Zoology, Chinese Academy of Sciences will sequence COI for the first 10 specimens of a species at no cost to the steward of the tissues.

Molecular differentiation and taxonomy of the sunwatcher toad-headed agama species complex Phrynocephalus superspecies helioscopus (Pallas 1771) (Reptilia: Agamidae)

Lizards of the sunwatcher toad-headed agama species complex Phrynocephalus superspecies helioscopus, mostly distributed in Central Asia and Middle East, were examined using analysis of variation at the mitochondrial cytochrome oxidase c subunit I gene fragment and fingerprint analysis of nuclear DNA (inter-SINE PCR technique). A total of 86 individual tissue samples from 53 populations, to the full extent representing different parts of the species complex range, were subjected to molecular genetic examination, and surprisingly deep differentiation was revealed. The populations analyzed split into 12 isolated phylogroups, many of which were characterized by a narrow range and genetic isolation. Monophyly of sunwatcher (Ph. helioscopus) and Persian (Ph. persicus) toad-headed agamas was confirmed. However, both of these species probably represent the species complexes. Zoogeography of Central Asiais discussed.

Phylogeography of the Siberian newt Salamandrella keyserlingii by mitochondrial DNA sequence analysis

Differentiation of geographical populations of the Siberian newt Salamandrella keyserlingii throughout the species range was analyzed using a fragment of the cytochrome b gene. The population of the Primorye region (Russian Far East) is separated to the greatest extent; the Japanese and South Kuril populations are the next most separate. These populations are possibly subspecies. Geographical differentiation of populations in the Siberian part of the species range is lower, lacks a clinal variation, and is irregular. The molecular variation of S. keyserlingii supports the hypothesis that several primary vicarious refugia of pre-Pleistocene differentiation of a common ancestor of Salamandrella occurred in the southeastern part of its current distribution range and that northern and western regions were gradually colonized via repeated steps of expansion and retreat in the Siberian part of the modern species range.

Red River barrier and Pleistocene climatic fluctuations shaped the genetic structure of Microhyla fissipes complex (Anura: Microhylidae) in southern China and Indochina

South China and Indochina host striking species diversity and endemism. Complex tectonic and climatic evolutions appear to be the main drivers of the biogeographic patterns. In this study, based on the geologic history of this region, we test 2 hypotheses using the evolutionary history of Microhyla fissipes species complex. Using DNA sequence data from both mitochondrial and nuclear genes, we first test the hypothesis that the Red River is a barrier to gene flow and dispersal. Second, we test the hypothesis that Pleistocene climatic cycling affected the genetic structure and population history of these frogs. We detect 2 major genetic splits that associate with the Red River. Time estimation suggests that late Miocene tectonic movement associated with the Red River drove their diversification. Species distribution modeling (SDM) resolves significant ecological differences between sides of the Red River. Thus, ecological divergence also probably promoted and maintained the diversification. Genogeography, historical demography, and SDM associate patterns in southern China with climate changes of the last glacial maximum (LGM), but not Indochina. Differences in geography and climate between the 2 areas best explain the discovery. Responses to the Pleistocene glacial–interglacial cycling vary among species and regions.

Diversity of the snail-eating snakes Pareas (Serpentes, Pareatidae) from Taiwan

Pareatidae are a group of mollusc‐eating snakes widely distributed in South‐eastern Asia. Due to their dietary specialization, the asymmetric dentition of pareatids has recently become an interesting issue in evolutionary biology. However, phylogenetic relationships and species diversity of pareatids are still poorly studied. A total of three Pareas species, P. formosanus (Van Denburgh 1909), P. compressus (Oshima 1910) and P. komaii (Maki 1931), have been reported for Taiwan. However, only P. formosanus is currently regarded as a valid species. Using mitochondrial sequence phylogeny, nuclear c‐mos haplotype network, as well as multivariate morphometrics, we re‐evaluated the taxonomic status of Pareas from Taiwan, the Ryukyus and adjacent regions. These lines of evidence showed congruent results for the coexistence of three Pareas species in Taiwan, with prominent genetic and morphological differentiation and differing level of dentition asymmetry. The currently used name P. formosanus should be applied only to the snakes with red iris, comparatively short lower jaw and totally smooth dorsal scales. An examination of the type material indicated that the name P. compressus should be regarded as a junior synonym of P. formosensis sensu stricto. Pareas komaii (Oshima 1910) is confirmed as a valid taxon with yellow iris, elongated lower jaw and strongly keeled dorsals. The third clade is characterized by a yellow iris, elongated lower jaw and weakly keeled dorsals. Despite their sympatric occurrence, every examined individual showed consistent grouping from mitochondrial, nuclear and morphological markers, indicating there is no gene flow among these three clades. Here, we describe the third clade as a new specie, Pareas atayal sp. nov.

Pecularities of Bony Skeleton Development in Asian Clawed Salamanders (Onychodactylus, Hynobiidae) Related to Embryonization

We studied skull, vertebral column, and limb skeleton development in Japanese clawed salamander Onychodactylus japonicus (Hynobiidae). The study is based on the ontogenetic series of embryos and larvae obtained from wild-captured adults by artificial induction of breeding using hormonal stimulation. The first stages of the skeleton formation in O. japonicus are shifted to the late embryonic period and hatching larvae already possess a well-ossified vertebral column, large number of skull ossifications and show signs of ossification in the forelimb skeleton. Compared to the primitive pattern of the skeleton development typical for other hynobiid salamanders, O. japonicus shows a number of heterochronies related to embryonization. In particular, this species is characterized by an earlier ossification of the vertebral column compared to that of the skull and by the delayed development and early reduction of the coronoid. Our results, along with the previously reported data on the skeleton development in the Fischer’s clawed salamander O. fischeri (Smirnov and Vassilieva, 2002), indicate that the genus Onychodactylus is characterized by the loss or reduction of several skeletal features typically found at early larval stages in other Hynobiidae species. In particular, provisional bones (especially the coronoid) and their dentition are underdeveloped. In addition, it is corroborated that the first tooth generation is absent in Onychodactylus, whereas such monocuspid nonpedicellate tooth generation normally develops at the early larval stages of other caudate amphibians. Since similar patterns of skeleton ontogeny are observed in other caudate groups with different extent of embryonization, it is proposed that, in different lineages of Urodela, the evolution of ontogeny followed similar pathways and was accompanied by the same changes in skeletogenesis.

Phylogenetic relationships and subgeneric taxonomy of toad-headed agamas Phrynocephalus (Reptilia, Squamata, Agamidae) as determined by mitochondrial DNA sequencing

119 Toaddheaded agamas (Phrynocephalus) is an essenn tial element of arid biotopes throughout the vast area spanning the countries of Middle East and Central Asia. They constitute one of the most diverse genera of the agama family (Agamidae), variously estimated to comprise 26 to 40 species [1]. The subgeneric Phrynoo cephalus taxonomy is poorly studied: recent taxoo nomic revision have been conducted without analysis of the entire genus diversity [1]; therefore, its phylogee netic position within Agamidae family remains unclear [2–5]. There were several attempts to reconn struct the phylogeny of this genus [4–10]. Reconn struction on the basis of the morphological traits is diff ficult because of high intraspecific morphological variation of the toaddheaded agamas and because of prevalence of substrate races, the morphs confined to specific substrates [11]. At the same time, the phylogee netic schemes on the basis of molecular data [4, 5, 10] are incomplete: they have been obtained on a small sample of species, and the genetic markers used were insufficiently informative. To clarify the main patterns of herpetofauna differr entiation in arid Eurasian areas, we studied phylogee netic relationships between 37 agama taxa encompasss ing about 80% of the known Phrynocephalus species and all the main species groups. The resultant scheme makes it possible to revise the subgeneric taxonomy of toaddheaded agamas and to clarify the positions of some morphs. Tissue samples of 52 Phrynocephalus specimens and eight closely related agamas from the Agaminae subfamily (the genera Laudakia, Paralaudakia, Trapelus, and Stellagama) were used in molecular genetic analysis. In total, 69 sequences from the Genn Bank were studied, 28 of which served as outgroups (the members of Agamidae, Chamaeleonidae, Iguanidae, and Lacertidae). The fragment sequences of the following four mitochondrial DNA genes were used in phylogenetic analysis: the genes of subunit I of cytochrome c oxii dase (COI), of subunits II and IV of NADHHdehydroo genase (ND2 and ND4), and of cytochrome b (cyt b). The overall length of alignment was 2703 bp (Genn Bank numbers HQ919083; HM915020; and KF6916166KF691738, see table). The algorithms of the maximum likelihood (ML), maximum parsimony (MP), and Bayesian analysis (UBA) for the concatee nated sequences of the four genes were used in analyy sis. To assess the level of genetic differentiation between taxa, the average uncorrected pdistances were calculated. The figure shows the dendrograms obtained. There is considerable topological similarity between the phylogenetic schemes for nucleotide and amino …