Dan Cogălniceanu

Phylogeography of the fire-bellied toads Bombina: independent Pleistocene histories inferred from mitochondrial genomes.

The fire‐bellied toads Bombina bombina and Bombina variegata, interbreed in a long, narrow zone maintained by a balance between selection and dispersal. Hybridization takes place between local, genetically differentiated groups. To quantify divergence between these groups and reconstruct their history and demography, we analysed nucleotide variation at the mitochondrial cytochrome b gene (1096 bp) in 364 individuals from 156 sites representing the entire range of both species. Three distinct clades with high sequence divergence (K2P = 8–11%) were distinguished. One clade grouped B. bombina haplotypes; the two other clades grouped B. variegata haplotypes. One B. variegata clade included only Carpathian individuals; the other represented B. variegata from the southwestern parts of its distribution: Southern and Western Europe (Balkano–Western lineage), Apennines, and the Rhodope Mountains. Differentiation between the Carpathian and Balkano–Western lineages, K2P ∼ 8%, approached interspecific divergence. Deep divergence among European Bombina lineages suggests their preglacial origin, and implies long and largely independent evolutionary histories of the species. Multiple glacial refugia were identified in the lowlands adjoining the Black Sea, in the Carpathians, in the Balkans, and in the Apennines. The results of the nested clade and demographic analyses suggest drastic reductions of population sizes during the last glacial period, and significant demographic growth related to postglacial colonization. Inferred history, supported by fossil evidence, demonstrates that Bombina ranges underwent repeated contractions and expansions. Geographical concordance between morphology, allozymes, and mtDNA shows that previous episodes of interspecific hybridization have left no detectable mtDNA introgression. Either the admixed populations went extinct, or selection against hybrids hindered mtDNA gene flow in ancient hybrid zones.

Nuclear and mitochondrial phylogeography of the European fire-bellied toads Bombina bombina and Bombina variegata supports their independent histories

Exact location and number of glacial refugia still remain unclear for many European cold‐blooded terrestrial vertebrates. We performed a fine‐scaled multilocus phylogeographic analysis of two Bombina species combining mitochondrial variation of 950 toads from 385 sites and nuclear genes (Rag‐1, Ncx‐1) from a subset of samples to reconstruct their colonization and contemporary variation patterns. We identified the lowlands northwest of the Black Sea and the Carpathians to be important refugial areas for B. bombina and B. variegata, respectively. This result emphasizes the importance of Central European refugia for ectothermic terrestrial species, far north of the Mediterranean areas regarded as exclusive glacial refugia for the animals. Additional refugia for B. variegata have been located in the southern Apennines and Balkans. In contrast, no evidence for the importance of other east European plains as refugial regions has been found. The distribution of mtDNA and Ncx‐1 variation suggests the presence of local refugia near the Black Sea for B. bombina; however, coalescent simulations did not allow to distinguish whether one or two refugia were present in the region. Strong genetic drift apparently accompanied postglacial expansions reducing diversity in the colonization areas. Extended sampling, coupled with the multilocus isolation with migration analysis, revealed a limited and geographically restricted gene flow from the Balkan to Carpathian populations of B. variegata. However, despite proximity of inferred B. bombina and B. variegata refugia, gene exchange between them was not detected.

Comparing three body condition indices in amphibians: a case study of yellow-bellied toad Bombina variegata

Body condition is important because it is correlated with population and habitat quality parameters. Since the direct measurements are either lethal or unreliable, a wide range of non-lethal body condition indices has been proposed. The aim of our study was to apply and compare three body condition indices (Fultons index, relative body condition mass index and residual index) using body size indicator - body mass data for 24 populations of the yellow bellied toad (Bombina variegata). The condition index should be independent of body size indicator, in this case snout vent length (SVL). Therefore we tested all three indices for the statistical independence of SVL and for the normality of distribution. Fultons index violated the independence assumption, whereas the relative body condition mass index did not have a normal distribution. Residual index was found both independent of SVL and normally distributed. Moreover, the residual index highlighted biological significant differences on the basis of altitude and season. Our results recommend the residual index as a useful tool in amphibian monitoring and conservation.

Diversity and distribution of amphibians in Romania

Abstract Nineteen species of amphibians inhabit Romania, 9 of which reach their range limit on this territory. Based on published occurrence reports, museum collections and our own data we compiled a national database of amphibian occurrences. We georeferenced 26779 amphibian species occurrences, and performed an analysis of their spatial patterns, checking for hotspots and patterns of species richness. The results of spatial statistic analyses supported the idea of a biased sampling for Romania, with clear hotspots of increased sampling efforts. The sampling effort is biased towards species with high detectability, protected areas, and large cities. Future sampling efforts should be focused mostly on species with a high rarity score in order to accurately map their range. Our results are an important step in achieving the long-term goals of increasing the efficiency of conservation efforts and evaluating the species range shifts under climate change scenarios.

Diversity and distribution of reptiles in Romania

Abstract The reptile fauna of Romania comprises 23 species, out of which 12 species reach here the limit of their geographic range. We compiled and updated a national database of the reptile species occurrences from a variety of sources including our own field surveys, personal communication from specialists, museum collections and the scientific literature. The occurrence records were georeferenced and stored in a geodatabase for additional analysis of their spatial patterns. The spatial analysis revealed a biased sampling effort concentrated in various protected areas, and deficient in the vast agricultural areas of the southern part of Romania. The patterns of species richness showed a higher number of species in the warmer and drier regions, and a relatively low number of species in the rest of the country. Our database provides a starting point for further analyses, and represents a reliable tool for drafting conservation plans.

Using connectivity metrics and niche modelling to explore the occurrence of the northern crested newt Triturus cristatus (Amphibia, Caudata) in a traditionally managed landscape

SUMMARY Spatial models are increasingly employed to help understand the distribution of organisms and establish conservation priorities. Classic patch- orientated models may have limited power to accurately predict the organismsdistributions. Pond breeding amphibians are appropriate study organisms because of their complex life cycle, low dispersal and sensitivity to environmental conditions. Here connectivity metrics and niche modelling were used to predict the occurrence of the northern crested newt in a rural landscape from central Romania. Pond-related variables, such as macrophyte cover and the presence of predatory fish, were the most important predictors of newt occurrence, followed by one landscape-related variable (urbanization) and a connectivity metric (nearest neighbouring occupied pond).Mostofthelandscapeandconnectivityvariables were not adequate predictors, presumably because most of the terrestrial habitats in this traditionally used rural landscape are ecologically optimal for amphibians. Conservation measures for the northern crested newt should promote the preservation of traditional extensive agricultural practices and discourage stocking of ponds with predatory fish.

Effect of habitat drying on the development of the Eastern spadefoot toad ( Pelobates syriacus ) tadpoles

Amphibians exhibit plasticity in the timing of metamorphosis, and tadpoles of many species respond to pond drying by accelerating their development. In the present study we investigated the phenotypic plasticity of the developmental response to water volume reduction in tadpoles of Eastern spadefoot toad Pelobates syriacus. The response of tadpoles to the simulated drying conditions was evaluated by gradually reducing the water level in the experimental containers under controlled laboratory conditions. Four water level treatments were used: constant high, slow decrease, fast decrease and constant low level. We tested if (i) tadpoles can speed up their development in a drying aquatic habitat, and (ii) if the accelerated development causes a reduced body size at metamorphosis. Our results showed that P. syriacus tadpoles were able to respond to pond drying by speeding up their metamorphosis and that metamorphosis was not influenced by water level, but by water level decrease rate. The accelerated development caused by the decreasing water level resulted in smaller body size at metamorphosis. The smallest size at metamorphosis was in tadpoles raised in constant low water level treatments and was probably induced by the crowding effect. We compared our results to similar studies which show that the response of the Eastern spadefoot toad tadpoles to pond drying is less impressive, especially if compared to the response of the North American spadefoot toads inhabiting desert environments.

Moving into protected areas? Setting conservation priorities for Romanian reptiles and amphibians at risk from climate change.

Rapid climate change represents one of the top threats to biodiversity, causing declines and extinctions of many species. Range shifts are a key response, but in many cases are incompatible with the current extent of protected areas. In this study we used ensemble species distribution models to identify range changes for 21 reptile and 16 amphibian species in Romania for the 2020s and 2050s time horizons under three emission scenarios (A1B = integrated world, rapid economic growth, A2A = divided world, rapid economic growth [realistic scenario], B2A = regional development, environmentally-friendly scenario) and no- and limited-dispersal assumptions. We then used irreplaceability analysis to test the efficacy of the Natura 2000 network to meet conservation targets. Under all scenarios and time horizons, 90% of the species suffered range contractions (greatest loses under scenarios B2A for 2020s, and A1B for 2050s), and four reptile species expanded their ranges. Two reptile and two amphibian species are predicted to completely lose climate space by 2050s. Currently, 35 species do not meet conservation targets (>40% representation in protected areas), but the target is predicted to be met for 4 - 14 species under future climate conditions, with higher representation under the limited-dispersal scenario. The Alpine and Steppic-Black Sea biogeographic regions have the highest irreplaceability value, and act as climate refugia for many reptiles and amphibians. The Natura 2000 network performs better for achieving herpetofauna conservation goals in the future, owing to the interaction between drastic range contractions, and range shifts towards existing protected areas. Thus, conservation actions for herpetofauna in Romania need to focus on: (1) building institutional capacity of protected areas in the Alpine and Steppic-Black Sea biogeographic regions, and (2) facilitating natural range shifts by improving the conservation status of herpetofauna outside protected areas, specifically in traditionally-managed landscapes and abandoned cropland.

Evolutionary history of Ichthyosaura alpestris (Caudata, Salamandridae) inferred from the combined analysis of nuclear and mitochondrial markers

Widespread species with morphologically and ecologically differentiated populations are key to understand speciation because they allow investigating the different stages of the continuous process of population divergence. The alpine newt, Ichthyosaura alpestris, with a range that covers a large part of Central Europe as well as isolated regions in all three European Mediterranean peninsulas, and with strong ecological and life-history differences among populations, is an excellent system for such studies. We sampled individuals across most of the range of the species, and analyzed mitochondrial (1442 bp) and nuclear (two nuclear genes -1554 bp- and 35 allozyme loci) markers to produce a time-calibrated phylogeny and reconstruct the historical biogeography of the species. Phylogenetic analyses of mtDNA data produced a fully resolved topology, with an endemic, Balkan clade (Vlasina) which is sister to a clade comprising an eastern and a western group. Within the former, one clade (subspecies I. a. veluchiensis) is sister to a clade containing subspecies I. a. montenegrina and I. a. serdara as well as samples from southern Romania, Bosnia-Herzegovina, Serbia and Bulgaria (subspecies I. a. reiseri and part of I. a. alpestris). Within the western group, populations from the Italian peninsula (subspecies I. a. apuana and I. a. inexpectata) are sister to a clade containing samples from the Iberian Peninsula (subspecies I. a. cyreni) and the remainder of the samples from subspecies I. a. alpestris (populations from Hungary, Austria, Poland, France, Germany and the larger part of Romania). Results of (∗)BEAST analyses on a combined mtDNA and nDNA dataset consistently recovered with high statistical support four lineages with unresolved inter-relationships: (1) subspecies I. a. veluchiensis; (2) subspecies I. a. apuana+I. a. inexpectata; (3) subspecies I. a. cyreni+part of subspecies I. a. alpestris (the westernmost populations, plus most Romanian populations); and (4) the remaining populations, including subspecies I. a. serdara, I. a. reiseri and I. a. montenegrina and part of subspecies I. a. alpestris, plus samples from Vlasina. Our time estimates are consistent with ages based on the fossil record and suggest a widespread distribution for the I. alpestris ancestor, with the split of the major eastern and western lineages during the Miocene, in the Tortonian. Our study provides a solid, comprehensive background on the evolutionary history of the species based on the most complete combined (mtDNA+nDNA+allozymes) dataset to date. The combination of the historical perspective provided by coalescent-based analyses of mitochondrial and nuclear DNA variation with individual-based multilocus assignment methods based on multiple nuclear markers (allozymes) also allowed identification of instances of discordance across markers that highlight the complexity and dynamism of past and ongoing evolutionary processes in the species.

Genetic diversity and distribution patterns of diploid and polyploid hybrid water frog populations (Pelophylax esculentus complex) across Europe

Polyploidization is a rare yet sometimes successful way for animals to rapidly create geno‐ and phenotypes that may colonize new habitats and quickly adapt to environmental changes. In this study, we use water frogs of the Pelophylax esculentus complex, comprising two species (Pelophylax lessonae, genotype LL; Pelophylax ridibundus, RR) and various diploid (LR) and triploid (LLR, LRR) hybrid forms, summarized as P. esculentus, as a model for studying recent hybridization and polyploidization in the context of speciation. Specifically, we compared the geographic distribution and genetic diversity of diploid and triploid hybrids across Europe to understand their origin, maintenance and potential role in hybrid speciation. We found that different hybrid and parental genotypes are not evenly distributed across Europe. Rather, their genetic diversity is structured by latitude and longitude and the presence/absence of parental species but not of triploids. Highest genetic diversity was observed in central and eastern Europe, the lowest in the northwestern parts of Europe. This gradient can be explained by the decrease in genetic diversity during postglacial expansion from southeastern glacial refuge areas. Genealogical relationships calculated on the basis of microsatellite data clearly indicate that hybrids are of multiple origin and include a huge variety of parental genomes. Water frogs in mixed‐ploidy populations without any parental species (i.e. all‐hybrid populations) can be viewed as evolutionary units that may be on their way towards hybrid speciation. Maintenance of such all‐hybrid populations requires a continuous exchange of genomes between diploids and triploids, but scenarios for alternative evolutionary trajectories are discussed.