Additional information on publications by Sylvain Ursenbacher

Aim
Ecological niche-based models (ENM) frequently rely on bioclimatic variables (BioV) to reconstruct biogeographic scenarios for species evolution, ignoring mechanistic relations. We tested if climatic predictors relevant to species hydric and thermal physiology better proximate distribution patterns and support location of Pleistocene refugia derived from phylogeographic studies.
Location
The Western Palaearctic.
Taxon
Vipera berus and Zootoca vivipara, two cold-adapted species.
Methods
We used two sets of variables, that is physiologically meaningful climatic variables (PMV) and BioV, in a multi-algorithm ENM approach, to compare their ability to predict current and Last Glacial Maximum (LGM) species ranges. We estimated current and LGM permafrost extent to address spatially the cold hardiness dissimilarity between both species.
Results
PMV explained more accurately the current distribution of these two cold-adapted species and identified the importance of summer temperature and solar radiation that constrain activity in cold habitats. PMV also provide a better insight than BioV predictors on LGM distribution. By including notably, the permafrost extent, PMV-based models gave parsimonious putative arrangement and validity of refugia for each clade and subclade in accordance with phylogeographic data. Northern refugia were also identified from 48 to 52 degrees N for V. berus and from 50 to 54 degrees N for Z. vivipara.
Main Conclusions
Our hybrid approach based on PMV generated more realistic predictions for both current (biogeographical validation) and past distributions (phylogeographic validation). By combining constraints during the activity period (summer climatic niche) and those inherent to the wintering period (freeze tolerance), we managed to identify glacial refuges in agreement with phylogeographic hypotheses concerning post-glacial routes and colonization scenarios.

Keywords: climatic refugia - cold-climate specialist - ecological niche-based models - hybrid models - permafrost - phylogeography - Pleistocene - squamates

Most of Switzerland is inhabited by the nominotypical subspecies of the barred grass snake (Natrix helvetica helvetica), which is characterized by mitochondrial DNA lineage E. Only in the northeast of the country, the common grass snake (N. natrix) occurs and hybridizes with N. h. helvetica in a narrow contact zone. However, we discovered that in southern and western Switzerland barred grass snakes representing another mtDNA lineage (lineage C) are widely distributed. Lineage C is typical for Alpine populations of the southern subspecies N. h. sicula. Our microsatellite analyses of the Swiss samples revealed differences between the two subspecies and also a substructure with two clusters in each subspecies. Furthermore, we discovered a contact and hybrid zone of N. h. helvetica and N. h. sicula along the northern shore of Lake Geneva and also confirm that interbreeding with alien common grass snakes (N. n. moreotica, mtDNA lineage 7) occurs there. This finding is of concern for nature conservation and measures should be taken to prevent further genetic pollution. Using morphometrics, we found no differences between the two subspecies of N. helvetica, while N. natrix was slightly distinct from N. helvetica.

Keywords: microsatellites - mitochondrial DNA - morphometrics - nuclear DNA - taxonomy

Asp vipers (Vipera aspis) and adders (Vipera berus) coexist in the Loire-Atlantique department in France where the two species reach their respective range limits. This contact zone is of special interest since hybridization has been recently discovered there. We carried out extensive sampling to further investigate the hybrid status of morphologically typical individuals and to evaluate the proportion of genetically admixed individuals in this area. Using microsatellite markers, no hybrids with typical morphological traits of either asp vipers or adders were detected. All recently investigated individuals with intermediate morphological traits were shown to be hybrids. A rather low proportion of genetically admixed individuals (1.5-3%) and a very small number of supposed second-generation hybrids suggest reduced fertility of first-generation hybrids or low viability of their progeny. The investigation of mtDNA of newly sampled hybrids support the finding that hybridization only occurs between female V. aspis and male V. berus. Several possible explanations for the unidirectional hybridization are discussed and consequent future studies suggested.

Keywords: contact zone - Eurasian vipers - interspecific hybridization - Viperidae

Many male reptiles maximize their reproductive success by mating with multiple females. This behaviour has often been observed in female reptiles as well, although one mating would usually be sufficient to fertilize the available female eggs. Using 13 microsatellite markers, multiple paternity (MP) was confirmed for the first time in the asp viper (Vipera aspis) in 47% of the tested clutches, with only up to two different fathers. Phenotypic characteristics (length, weight) of the mother did not influence the number of sires, and the number of young did not dif- fer between litters with one or two sires. Males that sired offspring were larger than males that did not successfully reproduce. Excluding an exceptionally long- distance movement (2464 m), the distance between fathers and mothers was limited (127 m
162). Although males exhibit notable combat behaviour during the breeding season, there was no sexual size dimorphism in the study population.

Keywords: Vipera aspis - multiple paternity - sexual size dimorphism - microsatellite - European vipers - reproductive success - reproductive tactics

High?throughput sequencing has become an accurate method for the identification of species present in soil, water, faeces, gut or stomach contents. However, information at the species level is limited due to the choice of short barcodes and based on the idea that DNA is too degraded to allow longer sequences to be amplified. We have therefore developed a long DNA metabarcoding method based on the sequencing of short reads followed by de novo assembly, which can precisely identify the taxonomic groups of organisms associated with complex diets, such as omnivorous individuals. The procedure includes 11 different primer pairs targeting the COI gene, the large subunit of the ribulose?1,5?bisphosphate carboxylase gene, the maturase K gene, the 28S rRNA and the trnL?trnF chloroplastic region. We validated this approach using 32 faeces samples from an omnivorous reptile, the European pond turtle (Emys orbicularis, L. 1758). This metabarcoding approach was assessed using controlled experiments including mock communities and faecal samples from captive feeding trials. The method allowed us to accurately identify prey DNA present in the diet of the European pond turtles to the species level in most of the cases (82.4%), based on the amplicon lengths of multiple markers (168–1,379 bp, average 546 bp), and produced by de novo assembly. The proposed approach can be adapted to analyse various diets, in numerous conservation and ecological applications. It is consequently appropriate for detecting fine dietary variations among individuals, populations and species as well as for the identification of rare food items.

Keywords: de novo assembly - diet analysis - European pond turtle - faecal analysis - long metabarcoding

Spatial autocorrelation (SAC) is a common feature of ecological data where observations tend to be more similar at some geographic distance(s) than expected by chance. Despite the implications of SAC for data dependencies, its impact on the performance of species distribution models (SDMs) remains controversial, with reports of both strong and negligible impacts on inference. Yet, no study has compre- hensively assessed the prevalence and the strength of SAC in the residuals of SDMs over entire geographic areas. Here, we used a large-scale spatial inventory in the western Swiss Alps to provide a thorough assess- ment of the importance of SAC for (1) 850 species belonging to nine taxonomic groups, (2) six predictors commonly used for modeling species distributions, and (3) residuals obtained from SDMs fitted with two algorithms with the six predictors included as covariates. We used various statistical tools to evaluate (1) the global level of SAC, (2) the spatial pattern and spatial extent of SAC, and (3) whether local clusters of SAC can be detected. We further investigated the effect of the sampling design on SAC levels. Overall, while environmental predictors expectedly displayed high SAC levels, SAC in biodiversity data was rather low overall and vanished rapidly at a distance of ~5–10 km. We found low evidence for the existence of local clusters of SAC. Most importantly, model residuals were not spatially autocorrelated, suggesting that inferences derived from SDMs are unlikely to be affected by SAC. Further, our results suggest that the influence of SAC can be reduced by a careful sampling design. Overall, our results suggest that SAC is not a major concern for rugged mountain landscapes.

Keywords: correlograms - Mantel - Moran - mountains - spatial autocorrelation - species distribution models - western Swiss Alps

Mountain areas are biodiversity hotspots and provide a multitude of ecosystem services of irreplaceable socio-economic value. In the European Alps, air temperature has increased at a rate of about 0.36 degrees C decade(-1) since 1970, leading to glacier retreat and significant snowpack reduction. Due to these rapid environmental changes, this mountainous region is undergoing marked changes in spring phenology and elevational distribution of animals, plants and fungi. Long-term monitoring in the European Alps offers an excellent natural laboratory to synthetize climate-related changes in spring phenology and elevational distribution for a large array of taxonomic groups. This review assesses the climatic changes that have occurred across the European Alps during recent decades, spring phenological changes and upslope shifts of plants, animals and fungi from evidence in published papers and previously unpublished data. Our review provides evidence that spring phenology has been shifting earlier during the past four decades and distribution ranges show an upwards trend for most of the taxonomic groups for which there are sufficient data. The first observed activity of reptiles and terrestrial insects (e.g. butterflies) in spring has shifted significantly earlier, at an average rate of -5.7 and -6.0 days decade(-1), respectively. By contrast, the first observed spring activity of semi-aquatic insects (e.g. dragonflies and damselflies) and amphibians, as well as the singing activity or laying dates of resident birds, show smaller non-significant trends ranging from -1.0 to +1.3 days decade(-1). Leaf-out and flowering of woody and herbaceous plants showed intermediate trends with mean values of -2.4 and -2.8 days decade(-1), respectively. Regarding species distribution, plants, animals and fungi (N = 2133 species) shifted the elevation of maximum abundance (optimum elevation) upslope at a similar pace (on average between +18 and +25 m decade(-1)) but with substantial differences among taxa. For example, the optimum elevation shifted upward by +36.2 m decade(-1) for terrestrial insects and +32.7 m decade(-1) for woody plants, whereas it was estimated to range between -1.0 and +11 m decade(-1) for semi-aquatic insects, ferns, birds and wood-decaying fungi. The upper range limit (leading edge) of most species also shifted upslope with a rate clearly higher for animals (from +47 to +91 m decade(-1)) than for plants (from +17 to +40 m decade(-1)), except for semi-aquatic insects (-4.7 m decade(-1)). Although regional land-use changes could partly explain some trends, the consistent upward shift found in almost all taxa all over the Alps is likely reflecting the strong warming and the receding of snow cover that has taken place across the European Alps over recent decades. However, with the possible exception of terrestrial insects, the upward shift of organisms seems currently too slow to track the pace of isotherm shifts induced by climate warming, estimated at about +62 to +71 m decade(-1) since 1970. In the light of these results, species interactions are likely to change over multiple trophic levels through phenological and spatial mismatches. This nascent research field deserves greater attention to allow us to anticipate structural and functional changes better at the ecosystem level.

Keywords: biodiversity redistribution - climate change velocity - disequilibrium dynamics - global warming - migration - mountain ecosystems - phenological mismatches - upslope shift

The description of Vipera walser from the Northern Italian Alps as a new species (Ghielmi et al., 2016, Journal of Zoological Systematics and Evolutionary Research, 54, 161) was one of the most unexpected surprises of European herpetology in the 21st century. In mitochondrial (mt) DNA, it is closely related to a group of vipers only present in the Caucasus region and Northeastern Anatolia. However, its morphology is similar to the V. berus populations that inhabit nearby mountains in the Swiss-Italian Alps, which raises questions on its relationships and status. We thus sequenced five nuclear (nu) genes to determine the position of V. walser relative to V. berus and to the Caucasian/Northeastern Anatolian vipers in nuDNA. We also reanalyzed five previously sequenced mtDNA fragments. NuDNA markers recovered V. walser as closely related to Italian populations of V. berus and not to the Caucasian/Anatolian species, thus contradicting the mtDNA phylogeny. We checked that each of the five mtDNA fragments independently amplified by Ghielmi et al. (2016, Journal of Zoological Systematics and Evolutionary Research, 54, 161) produced individual gene trees compatible with the concatenated mtDNA phylogeny, thus excluding the hypothesis that NUMTs sequencing generated the mtDNA relationships reported by Ghielmi et al. (2016, Journal of Zoological Systematics and Evolutionary Research, 54, 161). Given the low level of nuclear differentiation between V. walser and the Italian population of V. berus, we argue that ancient admixture between V. berus and the ancestral population of V. walser is the most likely explanation for this case of cyto-nuclear discordance and we discuss the consequences of these results on the systematic status of V. walser.

Keywords: cyto-nuclear conflicts - reptiles - snakes - systematics - vipers

Aim: To understand the role of climate in fostering diversification, this study reconstructs the historical biogeography of Western Mediterranean vipers.
Location: Western Mediterranean Basin.
Taxon: Vipera aspis and Vipera latastei-monticola complex.
Methods: Phylogeographic analyses were performed over mitochondrial (three genes) and nuclear sequences (two genes), extensively covering species ranges. A total of 4,056 records were assigned to genetic units, using interpolations of genetic data, to test phylogenetic niche conservatism, under a 3D hypervolume approach, and to perform pal- aeoclimatic reconstructions for main lineages that diversified during the Pleistocene.
Results: Bayesian inferences based on mtDNA recovered three Miocene clades and nine Pliocene lineages that distinctly diversified during the Pleistocene. Diversification until late Pliocene was mostly restricted to southern regions of Iberian and Italian Peninsulas, and north-western African mountains, expanding northwards during the Pleistocene. Accordingly, genetic diversity was higher in southern regions. Ecological niche tests support a general mode of allopatric diversification with niche conservatism, although niche shift was found for two divergence events. Palaeoclimatic models identified particular requirements for the distribution of main lineages and distinct responses to the cooling and warm events of the Pleistocene. Areas of climatic stability during Pleistocene were identified for main lineages; however, climatic stability was weakly correlated with haplotype diversity.
Main conclusions: Our integrative approach enhances the understanding of the evolutionary and ecological dynamics that occurred in the Western Mediterranean region, evidencing the palaeo-tectonic and -climatic factors driving diversification since the Miocene in this biodiversity hotspot. Comparisons among patterns of diversification, haplotype diversity and climatic stability suggest that southern Iberian and Italian Peninsulas, and North-West African Mountains acted as refugia since the Pliocene, while some northern areas favoured population persistence during the Pleistocene.

Keywords: Mediterranean Basin - palaeoclimatic models - phylogenetic niche conservatism - phylogeography - Pleistocene - Viperinae

The fire-bellied toad Bombina bombina has recently been introduced in Moselle, north-eastern France, in an area where the yellow-bellied toad Bombina variegata occurs naturally. Both species hybridize in a wide area throughout Europe where their distribution overlaps. Therefore, there is a risk of introgression regarding the Bombina variegata population in north-eastern France. In order to assess the status of the introduced population of Bombina bombina and its origin, we investigated its genetic characteristics and structure using both mitochondrial (cytochrome b) and nuclear DNA (microsatellites markers). The results demonstrated a lack of introgression in the Bombina variegata population. Though experiencing a bottleneck effect, the introduced Bombina bombina population displays a high genetic diversity. If a propensity for expansion is found within the introduced population of Bombina bombina, it could be considered as a potential invasive species in France, and thus threaten the native species.

Keywords: invasive species - population genetics - conservation - cytochrome b.

Molecular technologies, such as metabarcoding, have become powerful tools for conservation purposes. Here, we present a non-invasive study analyzing the diet of one population of European pond turtle (Emys orbicularis) during its whole activity period and of four other populations during the same period, based on faecal sample, and using for the first time on this species, a long metabarcoding approach. Emys orbicularis is an emblematic freshwater species of wetlands in Europe. In several countries, this species is endangered and, in Switzerland, Emys orbicularis is ranked as critically endangered on the Swiss Red List. A national conservation program was created to reintroduce this species and raised the question if this reintroduced species could be a threat for other endangered species. We developed a new method of long metabarcoding analysis, using universal PCR primers to determine prey species occurrence in the faeces. The analysis conducted on 174 faeces collected on 142 individuals revealed 1153 preys from 270 species. Emys orbicularis consumed plants throughout the year with a more diverse diet during the reproduction period (April-June). This study therefore not only determines precisely the omnivorous and opportunistic diet of the Emys orbicularis, but also shows that this species is not a threat to its environment, as 85.5% of the consumed species were not list on the Swiss Red List. Moreover, it also demonstrated that the genetic analyses of faeces could be an efficient tool to determine trophic interaction with a high level of precision, yielding promising perspectives for food web ecology.

Keywords: omnivorous diet - endangered species - faecal analyses - long metabarcoding - Emys orbicularis - biodiversity - conservation

As anthropogenic degradation of biodiversity and ecosystems increases, so does the potential threat to the supply of ecosystem services, a key contribution of nature to people. Biodiversity has often been used in spatial conservation planning and has been regarded as one among multiple services delivered by ecosystems. Hence, biodiversity conservation planning should be integrated in a framework of prioritizing services in order to inform decision-making. Here, we propose a prioritization approach based on scenarios maximising both the provision of ecosystem services and the conservation of biodiversity hotspots. Different weighting scenarios for the _-diversity in four taxonomic groups and 10 mapped ecosystem services were used to simulate varying priorities of policymakers in a mountain region. Our results illustrate how increasing priorities to ecosystem services can be disadvantageous to biodiversity. Moreover, the analysis to identify priority areas that best compromise the conservation of _-diversity and ecosystem services are predominantly not located within the current protected area network. Our analyses stress the need for an appropriate weighting of biodiversity within decision making that seek to integrate multiple ecosystem services. Our study paves the way toward further integration of multiple biodiversity groups and components, ecosystem services and various socio-economic scenarios, ultimately fuelling the development of more informed, evidence-based spatial planning decisions for conservation.

Keywords: economic valuation - _-diversity - human-nature trade-off - spatial prioritization - conservation planning - decision support tool - zonation software

Protected areas (PAs) play a critical role in conserving biodiversity and maintaining viable populations of threatened species. Yet, as global change could reduce the future effectiveness of existing PAs in covering high species richness, updating the boundaries of existing PAs or creating new ones might become necessary to uphold conservation goals. Modelling tools are increasingly used by policymakers to support the spatial prioritization of biodiversity conservation, enabling the inclusion of scenarios of environmental changes to achieve specific targets. Here, using the Western Swiss Alps as a case study, we show how integrating species richness derived from species distribution model predictions for four taxonomic groups under present and future climate and land-use conditions into two conservation prioritization schemes can help optimize extant and future PAs. The first scheme, the “Priority Scores Method” identified priority areas for the expansion of the existing PA network. The second scheme, using the zonation software, allowed identifying priority conservation areas while incorporating global change scenarios and political costs. We found that existing mountain PAs are currently not situated in the most environmentally nor politically suitable locations when maximizing alpha diversity for the studied taxonomic groups and that current PAs could become even less optimum under the future climate and land-use change scenarios. This analysis has focused on general areas of high species richness or species of conservation concern and did not account for special habitats or functional groups that could have been used to create the existing network. We conclude that such an integrated framework could support more effective conservation planning and could be similarly applied to other landscapes or other biodiversity conservation indices.

Keywords: conservation prioritization - decision support tools - land costs - species distribution models - Switzerland - zonation

The traditional southern Pleistocene refugia hypothesis in Europe has lately been challenged for several animal and plant species, and importance of more northern areas has been pointed out. The Carpathian Basin is one of the recently recognized biodiversity hotspots in Europe, representing rich fauna especially at the marginal regions. Marginal populations are prone to have lower genetic diversity and higher genetic differentiation than central populations. Here we examined one mitochondrial DNA fragment and nine nuclear (microsatellite) loci to describe the genetic diversity and phylogeographical pattern of fire salamander (Salamandra salamandra) populations in the Carpathian Basin with focusing on the southern margins of the Western Carpathians, where the species is present with isolated populations. Analyses of microsatellites indicated reduced genetic diversity for most of the isolated populations. While the analyses of the mitochondrial DNA showed the homogeneity of Carpathian Basin populations, the analyses with the nuclear markers revealed a more recent genetic split between western (Alpine) and eastern (Carpathian) populations, and separated the Apuseni Mountains population. Using Approximate Bayesian Computation analyses we identified the most probable colonization scenario for the isolated North Hungarian populations. Split between isolated salamander populations from the central populations in the Carpathian Mountains dates back to the beginning of the Late Pleistocene, while split between most of the isolated Hungarian populations can be associated with the Last Glacial Maximum. We found evidence for long-time isolation between the marginal Carpathian Basin and central fire salamander populations. Our results also proved the glacial survival of S. salamandra in the temperate forests of northeast Pannonia (North Hungarian Mountains), confirming that the Carpathian Basin served as important northerly refugia during the Pleistocene climatic oscillations.

Keywords: Salamandra salamandra - Carpathian Mountains - northerly refugia - phylogeography

Genetic introgression has recently become an important concern for conservation genetics as it can occur between rare and related common species, between various genetic groups and between individuals from different origins. Our aims were: i) to determine whether hybridisation or introgression occurs between Emys orbicularis subspecies in a natural contact zone in France; ii) to determine the geographic extent of the contact zone between distinct subspecies in France; iii) to evaluate at which level introgression occurs, and finally; iv) to evaluate whether combining mitochondrial and nuclear genetic markers reflects the same contact zone as when only one genetic marker is used. Introgression was evaluated by genotyping biparentally inherited microsatellites and sequencing the maternally inherited cytochrome b gene of French populations. We demonstrated strong introgression between subspecies under natural conditions in the old contact zone in southern and eastern France. Our results corroborated that introgression reflects past natural events, but also demonstrated that human impact has altered these patterns. We finally confirmed that the combination of mitochondrial and nuclear genetic markers is more appropriate to reveal introgression than the use of only one genetic marker

Keywords: conservation genetics - France - hybridization - introgression

We report the occurrence of alien grass snakes (Natrix natrix ssp.) in Western Switzerland (Lausanne), at a locality where striped grass snakes, presumably from Croatia (N. n. persa), escaped from outdoor enclosures of a reptile park in the 1970s, within the natural range of the threatened barred grass snake (N. n. helvetica). Three gravid females were captured at the introduction site in 2016: two exhibited colour patterns typical of native N. n. helvetica and the third had an intermediate phenotype. Yet, their neonates featured diverse colour patterns, several typical to N. n. persa. Genetic analyses identified two distinct mitochondrial alien lineages: one specific to N. n. persa/cypriaca and one specific to south-Alpine N. n. helvetica (genetically unrelated to native Western Swiss N. n. helvetica). These results suggest that alien grass snakes of multiple origins have established sustainable populations at the introduction site for the past 40 years, and potentially hybridized and introgressed with the local taxa. Hence, it alarms on the captive breeding practices of potentially invasive reptiles in animal parks, and adds to a long list of uncontrolled herps’ introductions, a major threat to biodiversity in Western Europe.

We report on a new population of Vipera anatolica from the Geyik Mountain Range in eastern Antalya Province, Turkey. It represents only the second known location, and is situated in a valley about 200 km east from the terra typica at Kohu Da_ in western Antalya Province. We compare both populations and, based on marked differences in morphology, habitat, genetics, and its geographically isolated location, we describe the recently discovered population as a new subspecies. Aspects of ecology, threats, and conservation needs are discussed.

Keywords: conservation - distribution - food - genetics - habitat - morphology - Turkey - Vipera anatolica

Background
Sperm size and quality are key factors for fertilization success. There is increasing empirical evidence demonstrating that sperm form and function are influenced by selective pressures. Theoretical models predict that sperm competition could favour the evolution of longer sperm. In hermaphrodites, self-fertilizing species are expected to have shorter sperm than cross-fertilizing species, which use sperm stored from several mating partners for the fertilization of their eggs and thus are exposed to intense sperm competition. We tested this hypothesis by comparing original data on sperm length in 57 species of simultaneously hermaphroditic stylommatophoran gastropods from Europe and South America with respect to the species’ breeding system. We used 28S rRNA nuclear and COI mitochondrial sequence data to construct a molecular phylogeny. Phylogenetic generalized linear models were applied to examine the potential influence of morphological and life-history characters.
Results
The best-fit model revealed that the breeding system and age at sexual maturity influence sperm length in gastropods. In general, species with predominant cross-fertilization had longer sperm than species with predominant self-fertilization or a mixed breeding system. Across species with shells (snails), sperm length also increased with shell size.
Conclusions
Our study provides evidence that sperm length in stylommatophoran gastropods is influenced by the risk of sperm competition, as well as by age at sexual maturity and shell size. This finding extends present knowledge of sperm evolution to a group of so far poorly studied simultaneous hermaphrodites.

Keywords: comparative approach - gastropods - phylogeny - sexual selection - sperm competition - sperm evolution

Environmental DNA (or eDNA) is a novel method to detect the presence of species in lentic and lotic aquatic environments. Small volumes of water (15 ml) can be sufficient to detect a species in a pond. In the laboratory, PCR is used to detect the DNA of the study species. Several studies compare detection probabilities of eDNA and methods commonly used by field herpetologists. Using the Crested newt (Triturus cristatus) as a model species, these studies showed that eDNA has higher detection probabilities than other methods. Environmental DNA is often cheaper than other methods when all costs of a survey are considered. Because there is a relationship between the concentration of eDNA and abundance, eDNA may in the future be used to estimate abundance.

Keywords: environmental DNA - eDNA - detection probability - survey - monitoring - amphibian - pond - stream - Triturus cristatus

Contact zones of closely related and ecologically similar species constitute rare opportunities to study the evolutionary consequences of past speciation processes. They represent natural laboratories where strong competition could lead to the exclusion of one species, or the various species may switch into distinct ecological niches. Alternatively, if reproductive isolation has not been achieved yet, they may hybridize. We elucidate the degree of taxon integrity by comparing genetics and habitat use of three similar-sized congeneric viper species, Vipera ammodytes, V. aspis, and V. berus, of Nadiza Valley in western Slovenia. No hybridization was detected both for mitochondrial and nuclear genomes. Similarly, external intermediacy by a single pre-study viper (likely V. ammodytes x V. aspis) indicates that hybridization occasionally occurs, but should be very rare. Populations of the three related viperids are partially allopatric in Nadiza Valley, but they also coexist in a narrow contact zone in the montane grassland along the south-exposed slope of Mount Stol (1673 m a.s.l.). Here, the three species that occupy areas in or near patches of rocky microhabitats (e.g., stone piles, slides and walls) live in syntopy. However, fine-scaled measurements of structural components show a partial habitat segregation, in which V. berus becomes more dominant at elevations above 1400 m and occupies mostly the mountain ridge and north-exposed slopes of Mount Stol, V. aspis occurs below 1300 m and is the only species to inhabit stoneless patches of grass and bushes around 1000 m and lower, and V. ammodytes occurs at all elevations up to 1500 m, but is restricted to a rocky microhabitat. We suggest that a high degree of microstructure divergence, slightly different environmental niches, and a generally favourable habitat for all three viper species keep  the pressure for mis-mating and hybridization low, although mechanisms, such as reduced hybrid inferiority and temporal mating segregation can not be excluded yet.

Keywords: Alps - ecology - genetics - microhabitat - snakes

Understanding the impact of postglacial recolonisation on genetic diversity is essential in explaining current patterns of genetic variation. The central-marginal hypothesis (CMH) predicts a reduction in genetic diversity from the core of the distribution to peripheral populations, as well as reduced connectivity between peripheral populations. While the CMH has received considerable empirical support, its broad applicability is still debated and alternative hypotheses predict different spatial patterns of genetic diversity. Using microsatellite markers, we analysed the genetic diversity of the adder (Vipera berus) in Western Europe to reconstruct postglacial recolonisation. Approximate Bayesian Computation (ABC) analyses suggested a postglacial recolonisation from two routes: a western route from the Atlantic Coast up to Belgium and a central route from the Massif Central to the Alps. This cold-adapted species likely used two isolated glacial refugia in Southern France, in permafrost-free areas during the last glacial maximum. Adder populations further from putative glacial refugia had lower genetic diversity and reduced connectivity; therefore, our results support the predictions of the CMH. Our study also illustrates the utility of highly variable nuclear markers, such as microsatellites, and ABC to test competing recolonisation hypotheses.

Keywords: population genetic structure - microsatellite - snake - glacial refugia - Approximate Bayesian Computation

Colour polymorphism is widespread among vertebrates and plays important roles in prey-predator interactions, thermoregulation, social competition, and sexual selection. However, the genetic mechanisms involved in colour variation have been studied mainly in domestic mammals and birds, whereas information on wild animals remains scarce. Interestingly, the pro-opiomelanocortin gene (POMC) gives rise to melanocortin hormones that trigger melanogenesis (by binding the melanocortin-1-receptor; Mc1r) and other physiological and behavioural functions (by binding the melanocortin receptors Mc1-5rs). Owing to its pleiotropic effect, the POMC gene could therefore account for the numerous covariations between pigmentation and other phenotypic traits. We screened the POMC and Mc1r genes in 107 wild asp vipers (Vipera aspis) that can exhibit four discrete colour morphs (two unpatterned morphs: concolor or melanistic; two patterned morphs: blotched or lined) in a single population. Our study revealed a correlation between a single nucleotide polymorphism situated within the 3-untranslated region of the POMC gene and colour variation, whereas Mc1r was not found to be polymorphic. To the best of our knowledge, we disclose for the first time a relationship between a mutation at the POMC gene and coloration in a wild animal, as well as a correlation between a genetic marker and coloration in a snake species. Interestingly, similar mutations within the POMC 3-untranslated region are linked to human obesity and alcohol and drug dependence. Combined with our results, this suggests that the 3-untranslated region of the POMC gene may play a role in its regulation in distant vertebrates.

Keywords: colour polymorphism - melanogenesis - melanocortin one receptor gene - POMC - snake - Vipera aspis

The coloration of ectotherms plays an important role in thermoregulation processes. Dark individuals should heat up faster and be able to reach a higher body temperature than light individuals and should therefore have benefits in cool areas. In central Europe, montane local populations of adder (Vipera berus) and asp viper (Vipera aspis) exhibit a varying proportion of melanistic individuals. We tested whether the presence of melanistic V. aspis and V. berus could be explained by climatic conditions. We measured the climatic niche position and breadth of monomorphic (including strictly patterned individuals) and polymorphic local populations, calculated their niche overlap and tested for niche equivalency and similarity. In accordance with expectations, niche overlap between polymorphic local populations of both species is high, and even higher than that of polymorphic versus monomorphic montane local populations of V. aspis, suggesting a predominant role of melanism in determining the niche of ectothermic vertebrates. However, unexpectedly, the niche of polymorphic local populations of both species is narrower than that of monomorphic ones, indicating that colour polymorphism does not always enable the exploitation of a greater variability of resources, at least at the intraspecific level. Overall, our results suggest that melanism might be present only when the thermoregulatory benefit is higher than the cost of predation.

Keywords: reptile - melanism - thermoregulation - niche

The Asp viper (Vipera aspis) is a highly endangered species in the Plateau and in the Jura Mountains of Switzerland. In the past, numerous populations disappeared due to human activities, but, currently, the trend is inverted and several locations are favourable for this species. However, it is not known if the remaining populations are still genetically variable and viable. In this study, 12 new microsatellite markers were developed for Vipera aspis using Next Generation Sequencing (pyrosequencing) techniques. All markers proved to be polymorphic with 2 - 9 alleles. Additionally, cross- amplification tests for two Eurasian viper species (V. berus and V. ammodytes) showed the applicability of some of these markers for the most widespread European vipers.

1. The use of environmental DNA (eDNA) to detect species in aquatic environments such as ponds and streams is a powerful new technique with many benefits. However, species detection in eDNA-based surveys is likely to be imperfect, which can lead to underestimation of the distribution of a species.
2. Site occupancy models account for imperfect detection and can be used to estimate the proportion of sites where a species occurs from presence/absence survey data, making them ideal for the analysis of eDNA-based surveys. Imperfect detection can result from failure to detect the species during field work (e.g. by water samples) or during laboratory analysis (e.g. by PCR).
3. To demonstrate the utility of site occupancy models for eDNA surveys, we reanalysed a data set estimating the occurrence of the amphibian chytrid fungus Batrachochytrium dendrobatidis using eDNA. Our reanalysis showed that the previous estimation of species occurrence was low by 5–10%. Detection probability was best explained by an index of the number of hosts (frogs) in ponds.
4. Per-visit availability probability in water samples was estimated at 0.45 (95% CRI 0.32, 0.58) and per-PCR detection probability at 0.85 (95% CRI 0.74, 0.94), and six water samples from a pond were necessary for a cumulative detection probability >95%. A simulation study showed that when using site occupancy analysis, researchers need many fewer samples to reliably estimate presence and absence of species than without use of site occupancy modelling.
5. Our analyses demonstrate the benefits of site occupancy models as a simple and powerful tool to estimate detection and site occupancy (species prevalence) probabilities despite imperfect detection. As species detection from eDNA becomes more common, adoption of appropriate statistical methods, such as site occupancy models, will become crucial to ensure that reliable inferences are made from eDNA-based surveys.

Keywords: environmental DNA - survey - monitoring - detection probability - site occupancy model

Males can enhance their reproductive success through mating with multiple females. For females, however, one mating is usually sufficient to inseminate all of their ova. Females may benefit from multiple mating by producing genetically more diverse offspring, and by having the opportunity to choose sperm of the genetically most compatible male. We used five microsatellite loci to investigate the occurrence and frequency of multiple paternity in 11 clutches of the grass snake (Natrix natrix) in Switzerland. Using a very conservative estimate (program GERUD), two or more fathers were found in 27% of the clutches. However, based on the maximum likelihood estimate (program COLONY), multiple paternity occurred in 91% of the clutches with 2-5 contributing males per female. This is the first investigation demonstrating multiple paternity in a European natricine, with a frequency similar to those found in new world natricines.

Keywords: COLONY - GERUD - microsatellite DNA - Natricinae - paternity analysis

In this study, sexual dimorphism and dorsal patterns were investigated in Latifi's viper (Montivipera latifii) from Iran. Sexual dimorphism was evaluated in 13 males and 15 females using 12 morphological characteristics. Despite the low sample size, the results showed that both sexes significantly differ in the number of subcaudal scales, the number of outer circumocular scales and tail length. In a limited area, the Lar National Park, three different dorsal patterns were observed (N=26 specimens): about 50% displayed a complete zigzag dorsal pattern, 15% of the individuals displayed a striped dorsal pattern, and about 35% had an incomplete zigzag dorsal pattern. These findings confirmed partially results from former published studies. Finally, we hypothesised that the four pattern described in M. latifii could be a combination of only two genetically define dorsal marks.

Keywords: Montivipera latifii - dorsal pattern - sexual dimorphism - genetic basis

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Die fortschreitende Industrialisierung führte im Elsass zu einer starken Ausdehnung des Strassennetzes. Ebenso wurden grosse, vorher naturbelassene Flächen in Weinbaugebiete und Ackerland umgewandelt. Dies bedeutete für die meisten Arten eine Fragmentierung ihrer Habitate. Dieser Artikel befasst sich mit den Auswirkungen der Lebensraumfragmentierung auf die Populationen der Schlingnatter und der Wechselkröte. Die beiden Arten unterscheiden sich in den Ergebnissen. In den untersuchten Populationen der Schlingnatter konnte keine genetische Differenzierung festgestellt werden. Bei der Wechselkröte wurden hingegen drei unterscheidbare Gruppen von Populationen gefunden. Trotzdem scheint das Strassennetz, entgegen zahlreicher früherer Studien bei verschiedenen Tierarten, keinen oder einen sehr geringen Einfluss auf den Austausch von Individuen zwischen Teilpopulationen dieser beiden Arten zu haben.

The Next Generation Sequencing (pyrosequencing) technique allows rapid, low-cost development of microsatellite markers. We have used this technology to develop 14 polymorphic loci for the endangered meadow viper (Vipera ursinii). Based on 37,000 reads, we developed primers for 66 microsatellite loci and found that 14 were polymorphic. The number of alleles per locus varies from 1 to 12 (for 30 individuals tested). At a cost of about 1/3 that of a normal microsatellite development, we were able to define enough microsatellite markers to conduct population genetic studies on a non-model species.

Keywords: conservation genomics - microsatellite - Vipera ursinii - 454 sequencing

In Switzerland all eight snake species are threatened and have been added to the Swiss Red List with different levels of concern. The most threatened is the viperine snake, Natrix maura, a semi-aquatic snake, which is considered as “Critically Endangered” (CR) in Switzerland. Only one population is presently living on the shores of Lake Geneva, mainly located between St-Saphorin and Treytorrens (Canton Vaud). But the population size estimation trends show a drastic reduction of this species. Alteration of the lake shore structure and the introduction of the ecologically very similar dice snake, Natrix tessellata, since the 1920s could be the causes of the observed decline of the viperine snake in the last 15 years. A higher fecundity and a larger body size render the alien species probably more competitive and possibly have a key role on the population decrease of the native species. In order to test the hypothesis of competition between both species, their habitats between St-Saphorin and Treytorrens were described in detail in order to detect differential interspecific use. In total 23 environmental variables were measured at the study area and data were subsequently analysed using tests of proportion. Results indicate that the dice snake prefers slopes with limited vegetation height (0.5–1 m), but inhabits also slopes with light vegetation cover and relatively narrow littoral zones. In contrast, the habitat use of the viperine snake relates to littoral zones with less steep slopes, but abundant vegetation. In addition, wide littoral zones seem to be preferred by the native species, such as the region near Treytorrens where the viperine snake was observed to be most numerous. Results obtained in this study were used to suggest particular shore management action, in particular to promote the native species.

Keywords: habitat partitioning - introduction - interspecific competition - Natrix tessellata - Natrix maura - Lake Geneva

Understanding patterns of genetic structure is fundamental for developing successful management programmes for isolated populations of threatened species. Trochulus caelatus is a small terrestrial snail endemic to calcareous rock cliffs in the Northwestern Swiss Jura Mountains. Eight microsatellite loci were used to assess the effect of habitat isolation on genetic population structure and gene flow among nine populations occurring on distinct cliffs. We found a high genetic differentiation among populations (mean F ST = 0.254) indicating that the populations are strongly isolated. Both allelic richness and effective population size were positively correlated with the size of the cliffs. Our findings support the hypothesis that T. caelatus survived on ice-free cliffs during the Pleistocene glacier advancements from the Alps. Due to the establishment of beech and pine forest under recent, temperate climate conditions, dispersal between cliffs is no longer possible for rock-dwelling snails such as T. caelatus. Our results provide basic data for developing a conservation action plan for this endangered gastropod species

Keywords: Helicoidea - Land snails - microsatellites - population genetic structure Trochulus caelatus

The survival of threatened species as the European tree frog (Hyla arborea) is strongly dependent on the genetic variability within the populations, as well as gene flow between them. In Switzerland, only two sectors in its western portion still harbour metapopulations, the first characterised by a very heterogeneous and urbanized landscape; and the second, on the opposite, characterised by a uninterrupted landscape of suitable habitats. In this study, six microsatellite loci were used to establish levels of genetic differentiation among the populations from the two different locations. The results show that the populations have: (i) weak levels of genetic differentiation (FST within metapopulation c. 0.04), (ii) no difference in structuring between the two metapopulations, (iii) a significantly lower genetic diversity (Hs) and observed heterozygozity (Ho) within the metapopulation situated in a disturbed landscape. Our results suggest that even if the dispersal of H. arborea among contiguous ponds seems to be efficient in areas of heterogeneous landscape, a loss of genetic diversity can occur.

Keywords: amphibians - conservation - Hylidae - microsatellite markers - fragmentation

Despite the increase of animal and plant introductions worldwide and the strong augmentation of the reptile trade, few invasive snake populations have been studied. Dice snakes (Natrix tessellata) were introduced to the shores of Lake Geneva (Switzerland) in the early 1920s, and are now well established. This region of introduction was previously inhabited by Viperine snakes (N. maura). Ever since these two species have been under monitoring (which began in 1996) the Viperine snake population has shown drastic decline. We examine here the possibility of trophic competition by analysing diet composition, prey size and trophic niche overlap. Spatial distribution is also assessed in order to address the question of spatial competitive exclusion. We found very similar diets, and thus a high trophic niche overlap, indicating no partitioning of the trophic resource. No arguments in favour of spatial competitive exclusion were found. Our study suggests that trophic competition may occur between the two natricines and that it may give an explanation for the drastic decline of the Viperine snake in this area. Other pathways potentially playing a role in the exclusion of the Viperine snake are discussed.

Keywords: Natricinae - competition - alien species - diet - foraging - colubrids

Although the adder (Vipera berus) has a large distribution area, this species is particularly threatened in Western Europe due to high habitat fragmentation and human persecution. We developed 13 new microsatellite markers in order to evaluate population structure and genetic diversity in the Swiss and French Jura Mountains, where the species is limited to only a few scattered populations. We found that V. berus exhibits a considerable genetic differentiation among populations (global Fst = 0.269), even if these are not geographically isolated. Moreover, the genetic diversity within populations in the Jura Mountains and in the less perturbed Swiss Alps is significantly lower than in other French populations, possibly due to post-glacial recolonisation processes. Finally, in order to minimize losses of genetic diversities within isolated populations, suggestions for the conservation of this species in fragmented habitats are proposed.

Keywords: population genetic structure - Jura Mountains - microsatellite - snake - Vipera berus

The nose-horned viper (Vipera ammodytes) occurs in a large part of the south-eastern Europe and Asia Minor. Phylogenetic relationships were reconstructed for a total of 59 specimens using sequences from three mitochondrial regions (16S and cytochrome b genes, and control region, totalling 2308 bp). A considerable number of clades were observed within this species, showing a large genetic diversity within the Balkan peninsula. Splitting of the basal clades was evaluated to about 4 million years ago. Genetic results are in contradiction with presently accepted taxonomy based on morphological characters: V. a. gregorwallneri and V. a. ruffoi do not display any genetic difference compared with the nominotypic subspecies (V. a. ammodytes), involving that these subspecies can be regarded as synonyms. High genetic divergence in the central part of the Balkan peninsula is not concordant with low morphological differentiation. Finally, the extensive genetic diversity within the Balkan peninsula and the colonisation routes are discussed.

Keywords: mitochondrial DNA - phylogeography - snake - nose-horned viper - glacial refugia - Balkan Peninsula - Vipera ammodytes

In order to contribute to the debate about southern glacial refugia used by temperate species and more northern refugia used by boreal or cold-temperate species, we examined the phylogeography of a widespread snake species (Vipera berus) inhabiting Europe up to the Arctic Circle. The analysis of the mitochondrial DNA (mtDNA) sequence variation in 1043 bp of the cytochrome b gene and in 918 bp of the noncoding control region was performed with phylogenetic approaches. Our results suggest that both the duplicated control region and cytochrome b evolve at a similar rate in this species. Phylogenetic analysis showed that V. berus is divided into three major mitochondrial lineages, probably resulting from an Italian, a Balkan and a Northern (from France to Russia) refugial area in Eastern Europe, near the Carpathian Mountains. In addition, the Northern clade presents an important substructure, suggesting two sequential colonization events in Europe. First, the continent was colonized from the three main refugial areas mentioned above during the Lower-Mid Pleistocene. Second, recolonization of most of Europe most likely originated from several refugia located outside of the Mediterranean peninsulas (Carpathian region, east of the Carpathians, France and possibly Hungary) during the Mid-Late Pleistocene, while populations within the Italian and Balkan Peninsulas fluctuated only slightly in distribution range, with larger lowland populations during glacial times and with refugial mountain populations during interglacials, as in the present time. The phylogeographical structure revealed in our study suggests complex recolonization dynamics of the European continent by V. berus, characterized by latitudinal as well as altitudinal range shifts, driven by both climatic changes and competition with related species.

Keywords: colonization history - mitochondrial DNA - molecular phylogeography - snake - Vipera berus

A five year study of an adder population (Vipera berus) in the Swiss Jura mountains: number of animals and discussion of population estimate methods. In order to estimate the number of adults in an isolated population of adder (Vipera berus) in the Swiss Jura Mountains, several population estimate methods were compared. 81 periods of capture were done during the five years study with the number of periods varing from 8 to 26 each year. The methods for estimating closed populations give more reliable results than estimations for open populations. The software CAPTURE (Otis et al. 1978) seems to give the most convincing estimations and appears to be the prime method for estimating viper populations. The selected site accommodates about fifty adults and the density is approximately 1 adult per hectare, a relatively low level compared to the density in the Alps.

Keywords: adder, Vipera berus, population size estimates, Swiss Jura mountains, density