Leo J. Borkin

Mitochondrial phylogeography of the moor frog, Rana arvalis

The moor frog Rana arvalis is a lowland species with a broad Eurasiatic distribution, from arctic tundra through forest to the steppe zone. Its present‐day range suggests that glacial refugia of this frog were located outside southern European peninsulas. We studied the species‐wide phylogeographical pattern using sequence variation in a 682 base pairs fragment of mtDNA cytochrome b gene; 223 individuals from 73 localities were analysed. Two main clades, A and B, differing by c. 3.6% sequence divergence were detected. The A clade is further subdivided into two subclades, AI and AII differing by 1.0%. All three lineages are present in the Carpathian Basin (CB), whereas the rest of the species range, including huge expanses of Eurasian lowlands, are inhabited solely by the AI lineage. We infer that AII and B lineages survived several glacial cycles in the CB but did not expand, at least in the present interglacial, to the north. The geographical distribution and genealogical relationships between haplotypes from the AI lineage indicate that this group had two glacial refugia, one located in the eastern part of the CB and the other probably in southern Russia. Populations from both refugia contributed to the colonization of the western part of the range, whereas the eastern part was colonized from the eastern refugium only. The effective population size as evidenced by θML is an order of magnitude higher in the AI lineage than in the AII and B lineages. Demographic expansion was detected in all three lineages.

Fossorial but widespread: the phylogeography of the common spadefoot toad (Pelobates fuscus), and the role of the Po Valley as a major source of genetic variability

Pelobates fuscus is a fossorial amphibian that inhabits much of the European plain areas. To unveil traces of expansion and contraction events of the species’ range, we sequenced 702 bp of the mitochondrial cytochrome b gene. To infer the population history we applied phylogeographical methods, such as nested clade phylogeographical analysis (NCPA), and used summary statistics to analyse population structure under a neutral model of evolution. Populations were assigned to different drainage systems and we tested hypotheses of explicit refugial models using information from analysis of molecular variance, nucleotide diversity, effective population size estimation, NCPA, mismatch distribution and Bayesian dating. Coalescent simulations were used as post hoc tests for plausibility of derived or a priori assumed biogeographical hypotheses. Our combination of all approaches enabled the reconstruction of the colonization history and phylogeography of P. fuscus and confirmed a previous assumption of the existence of two major genetic lineages within P. fuscus. Using the Afro‐European vicariance of Pelobates cultripes and Pelobates varaldii and applying Bayesian dating we estimated the divergence of these phylogeographical lineages to the Pliocene. We suggest the existence of three different glacial refugia: (i) the area between the Caspian and Black Seas as the origin for the expansion of the ‘eastern lineage’; (ii) the Danube system as a centre of diversity for part of the ‘western lineage’; (iii) the Po Valley, the largest centre of genetic variability. This fits the hypothesis that climatic fluctuation was a key event for differentiation processes in P. fuscus.

Radically different phylogeographies and patterns of genetic variation in two European brown frogs, genus Rana

We reconstruct range-wide phylogeographies of two widespread and largely co-occurring Western Palearctic frogs, Rana temporaria and R. dalmatina. Based on tissue or saliva samples of over 1000 individuals, we compare a variety of genetic marker systems, including mitochondrial DNA, single-copy protein-coding nuclear genes, microsatellite loci, and single nucleotide polymorphisms (SNPs) of transcriptomes of both species. The two focal species differ radically in their phylogeographic structure, with R. temporaria being strongly variable among and within populations, and R. dalmatina homogeneous across Europe with a single strongly differentiated population in southern Italy. These differences were observed across the various markers studied, including microsatellites and SNP density, but especially in protein-coding nuclear genes where R. dalmatina had extremely low heterozygosity values across its range, including potential refugial areas. On the contrary, R. temporaria had comparably high range-wide values, including many areas of probable postglacial colonization. A phylogeny of R. temporaria based on various concatenated mtDNA genes revealed that two haplotype clades endemic to Iberia form a paraphyletic group at the base of the cladogram, and all other haplotypes form a monophyletic group, in agreement with an Iberian origin of the species. Demographic analysis suggests that R. temporaria and R. dalmatina have genealogies of roughly the same time to coalescence (TMRCA ~3.5 mya for both species), but R. temporaria might have been characterized by larger ancestral and current effective population sizes than R. dalmatina. The high genetic variation in R. temporaria can therefore be explained by its early range expansion out of Iberia, with subsequent cycles of differentiation in cryptic glacial refugial areas followed by admixture, while the range expansion of R. dalmatina into central Europe is a probably more recent event.

Correlations of geographic distribution and temperature of embryonic development with the nuclear DNA content in the Salamandridae (Urodela, Amphibia)

We used flow cytometry to measure the nuclear DNA content in erythrocytes of 27 salamandrid species. Across these species, diploid genome size varied more than 2 fold (51.3-104.4 pg). According to genome size and geographic distribution, 3 groups of newt species were recognized: West Palearctics with smaller amounts of nuclear DNA; Nearctic, with intermediate values; and East Asiatic, with higher genome sizes. Viviparous West Palearctic salamanders differed from most of the oviparous West Palearctic newts in possessing larger genome sizes. The nuclear DNA content strongly correlates with species range limits. At the same temperature, embryos of salamandrid species with larger genome sizes have a markedly longer developmental time than those with smaller genomes. We present an analysis of the relationships between the amount of nuclear DNA and water temperature at the breeding sites.

Bearing of Chromosome C-banding patterns on the Classification of Eurasian Toads of the Bufo bufo Complex

Distribution of NORs and C-spots in the karyotypes was compared among six forms of the Bufo bufo complex, i.e. B. j. japonicus, B. j. formosus, B. torrenticola and B. gargarizans miyakonis from Japan, and B. b. bufo and B. b. verrucosissimus from USSR. All forms invariably possessed NORs on 6q. All the 11 pairs of homologous chromosomes had constitutive heterochromatin on the centromere region in every form of toad examined. Further, each form had pericentric heterochromatin on 1p and telomeric one on 6q. Pairs 8 and 9 lacked C-bands except the centromeric spot in every form. Other chromosomes revealed unique C-spots specific to each form, and each form could be characterized from others by the banding pattern. Comparisons of the C-banding patterns of the three forms obtained with those of the published data revealed several discrepancies, but most of them were attributed to the unlike standard in recognizing spots by the different authors. Although the C-banding pattern is suggested to have some taxonomic value, systematic relationships among the six forms cannot be directly estimated by the analyses of banding patterns.

The Occurrence of Polymely and Polydactyly in Natural Populations of Anurans of the USSR

Various cases of isolated and mass polymely and polydactyly in anurans of the USSR fauna are summarized. Mass polydactylies in frogs Rana arvalis arvalis and Rana lessonae-Rana esculenta as well as in toad Bufo bufo bufo are reported. Classiication of polydactyly types based on X-ray analysis is proposed. Species-specificity and lozalized appearance of mass polydactyly factor(s) are discussed.

Cytological maps of lampbrush chromosomes of European water frogs (Pelophylax esculentus complex) from the Eastern Ukraine

BackgroundHybridogenesis (hemiclonal inheritance) is a kind of clonal reproduction in which hybrids between parental species are reproduced by crossing with one of the parental species. European water frogs (Pelophylax esculentus complex) represent an appropriate model for studying interspecies hybridization, processes of hemiclonal inheritance and polyploidization. P. esculentus complex consists of two parental species, P. ridibundus (the lake frog) and P. lessonae (the pool frog), and their hybridogenetic hybrid – P. esculentus (the edible frog). Parental and hybrid frogs can reproduce syntopically and form hemiclonal population systems. For studying mechanisms underlying the maintenance of water frog population systems it is required to characterize the karyotypes transmitted in gametes of parental and different hybrid animals of both sexes.ResultsIn order to obtain an instrument for characterization of oocyte karyotypes in hybrid female frogs, we constructed cytological maps of lampbrush chromosomes from oocytes of both parental species originating in Eastern Ukraine. We further identified certain molecular components of chromosomal marker structures and mapped coilin-rich spheres and granules, chromosome associated nucleoli and special loops accumulating splicing factors. We recorded the dissimilarities between P. ridibundus and P. lessonae lampbrush chromosomes in the length of orthologous chromosomes, number and location of marker structures and interstitial (TTAGGG)n-repeat sites as well as activity of nucleolus organizer. Satellite repeat RrS1 was mapped in centromere regions of lampbrush chromosomes of the both species. Additionally, we discovered transcripts of RrS1 repeat in oocytes of P. ridibundus and P. lessonae. Moreover, G-rich transcripts of telomere repeat were revealed in association with terminal regions of P. ridibundus and P. lessonae lampbrush chromosomes.ConclusionsThe constructed cytological maps of lampbrush chromosomes of P. ridibundus and P. lessonae provide basis to define the type of genome transmitted within individual oocytes of P. esculentus females with different ploidy and from various population systems.

Alai! Alai! - a new species of the Gloydius halys (Pallas, 1776) complex (Viperidae, Crotalinae), including a brief review of the complex

During a scientific field expedition to the Alai-Pamir range five specimens of the genus Gloydius have been collected in the larger Alai. A morphological and genetical examination of the specimens has shown that they are part of the G. halys complex, but represent a new taxon which is characterized by the following unique character combination: It is a slender and moderately stout small snake, up to 479 mm total length. The head has nine symmetrical plates on the upper head, 7 supralabial and 8-9 infralabial scales. Body scales in 20-22 rows around midbody, 143-156 ventral and 35-45 usually paired subcaudal scales. The cloacal plate not divided. The general coloration consists of various different tones of olive, tan and brown, having a distinct head, but an indistinct body pattern with, excluding the tail, 26-29 transverse crossbands, which are not extending to the sides of the body. The haplotype network shows the new species within the G. halys complex and close related to both, G. h. halys and G. h. caraganus. So far the new described species is only known from the Alai range. However, various Gloydius specimens are found in Kyrgyzstan and because of the complicated taxonomy those specimens have to re-identified to clarify their status and the status of the new species.

Obituary: Valery K. Eremchenko (1949-2014)

On April 14th of 2014 a leading herpetologist of his country, Dr. Valery Konstantinovich Eremchenko passed away in Bishkek, Kyrgyzstan, after several years of severe illness. We lost a remarkable expert of the herpetofauna of Middle Asia, a friend and a collaborator.