Aim To infer the phylogenetic relationships and biogeography of Hydromantes, with special emphasis on the European taxa. In particular, we aimed to test: (1) the monophyly of the European species and current views on their interrelationships; and (2) previously proposed timings of the separation of European and American Hydromantes, and of biogeographically important events within Europe.Location California and the Western Mediterranean Basin, specifically southeast France, Italy, and the island of Sardinia.Methods Partial sequences of mitochondrial genes (cytochrome b and 12S rRNA) were obtained from 45 specimens of Hydromantes, including all European extant species and subspecies, and two species from California. In addition, a fragment of the mitochondrial 16S rRNA gene was amplified for 16 specimens. Data sets were aligned using ClustalX, and well-supported phylogenetic trees were produced using maximum-likelihood, Bayesian and maximum-parsimony methods. Estimates of divergence times were obtained with the program r8s, the molecular clock being calibrated using the opening of the Strait of Gibraltar, the final event in the Messinian Salinity Crisis of 5.3 Ma.Results Separation between the American and European clades occurred approximately 13.5 Ma, most probably before or after westward dispersal across the Bering Land Bridge. In Europe, divergence started in the late Miocene, when Hydromantes (A.) genei separated from other members of the genus 9 Ma and colonized south-west Sardinia. Movement between the European mainland and Sardinia, by a member of the subgenus Speleomantes, occurred in the Messinian Salinity Crisis, after the Mediterranean Basin desiccated almost completely 5.96 Ma. Subsequent widespread aridification fragmented the geographical ranges of Hydromantes, which live in cool and humid conditions, resulting in the origin of the six species in the subgenus Speleomantes. In contrast, a second period of diversification, in continental Europe 2-1.3 Ma, was probably caused by very cold interludes during the climatic oscillations that characterized the Pleistocene. Main conclusionsThe molecular clock used here indicates that the separation of Californian and European Hydromantes occurred more recently than previously believed, and the same is true of some subsequent phylogenetic divergences within Europe. Estimated dates for these divergence events are consistent with known geophysical and climatic events that could have caused or facilitated them.

ABSTRACT:The chytridiomycete fungus Batrachochytrium dendrobatidis is known to be focally distributed across Europe, but has only been linked to chytridiomycosis at a few locations in Spain. Here we report the second occurrence of chytridiomycosis in European amphibians. We found a population of endangered Sardinian newts (Euproctus platycephalus) exhibiting clinical signs of disease including loss of digits and patchy, discolored skin. Molecular examination of skin samples tested positive for B. dendrobatidis. The population of E. platycephalus has been in decline on a timescale consistent with the global emergence of chytridiomycosis, and the ecology of this salamander suggests that the disease in this species warrants concern.

Batrachochytrium dendrobatidis (Bd), the causative agent of the amphibian disease chytridiomycosis, is an important factor in the global decline of amphibians. Within Europe, animals that exhibit clinical signs of the disease have only been reported in Spain despite the pathogen's wide, but patchy, distribution on the continent. Recently, another occurrence of chytridiomycosis was reported in Euproctus platycephalus, the Sardinian brook newt, on the Mediterranean island of Sardinia, but without any evidence of fatal disease. We report further evidence of the emergence of Bd on Sardinia and the first evidence of lethal chytridiomycosis outside of Spain. Unusual mortalities of the Tyrrhenian painted frog (Discoglossus sardus) were found at three sites in the Limbara mountains of northern Sardinia. Molecular and histological screens of corpses, frogs, and tadpoles from these sites revealed infection with Bd. Infection and mortality occurred at locations that are unusual in terms of the published habitat requirements of the pathogen. Given the endemicity, the IUCN Red List status of the amphibian species on Sardinia, and the occurrence of infection and mortality caused by chytridiomycosis, there is serious reason for concern for the impact that disease emergence may have on the conservation of the amphibians of the island.

Aim In a highly endemic, threatened amphibian assemblage, we measure and describe the geographic and taxonomic distribution of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), with a view to identifying those sites and species most at risk of infection and its negative consequences. Additionally, we aimed to determine the potential for direct transmission events between two known carriers of Bd infection.Location The island of Sardinia.Methods We collected swab and tissue samples of amphibians from a wide range of geographic sites and species. We used epidemiological and statistical techniques to quantify deviations from a random distribution. We used random forests to investigate habitat use and overlap in two species known to be infected by Bd to quantify a surrogate measure of the contact rates between these two species.Results Both geographic and taxonomic distributions of Bd were highly nonrandom: we identified a cluster of infections in the north of the island and found that two species, Euproctus platycephalus and Discoglossus sardus, had a relatively high prevalence of infection within this cluster. Our analyses suggest that, on the basis of their fine-scale habitat use, they have relatively little opportunity for direct transmission and could maintain Bd infection independently.Main conclusions Our results illustrate how obtaining detailed information on the geographic and taxonomic distribution of infection is a useful first step in assessing the risk of infection for species within the region. Attempting to quantify possible routes of transmission amongst species further aids us in identifying mechanisms of pathogen persistence within the host community. Within this assemblage, we have identified two hosts that carry infection and may be at risk. Further, our research suggests that these two species may be able to maintain infection independently of one another, which has implications for attempts to mitigate this parasite.

Estimating the genetic variation and demographic trends of species in decline is of major concern in conservation genetics. This study contributes to understanding how historical and anthropogenic factors shape the distribution of current genetic diversity in one of the most endangered reptiles in Western Europe, the Hermann's tortoise Testudo hermanni hermanni. We used 17 microsatellite loci, chosen from a pyrosequencing library specifically developed for the subspecies to genotype eight populations distributed over about 30 sample localities covering almost the entire geographic distribution of the sub-species. The population genetic results reflect a very strong genetic structure and identify three major clusters among the Hermann's tortoise in the occidental Mediterranean basin: a continental cluster (Albera in Spain, Var in France and continental Italy), an insular cluster (Corsica, Sardinia and Sicily) and a cluster of mixed lineage (Minorca). Each of the eight studied populations is characterized by only one lineage except for Minorca, in which two lineages were identified. In contrast to what most empirical studies predict, the genetic diversity of the Hermann's tortoise is greater on islands than on the continent. Islands can therefore be considered as genetic sanctuaries with high conservation potential for this species, particularly in Corsica. Historical demographic patterns inferred with a generalized stepwisemutation model (GSM) using maximum likelihood showed significant past demographic changes in only two of the eight sampled populations: a demographic bottleneck was detected in the Albera population (Spain) and a demographic expansion in Corsica (France). In contrast to what was argued in previous studies, these results indicate that the Hermann's tortoise lineage found in Corsica is autochthonous. The origin of both lineages found in Minorca remains speculative. Lastly, our study identifies the six most relevant management units (sensu Moritz, 1994) for conservation purposes on the basis that they represent a significant part of the evolutionary legacy of the species. Some conservation recommendations were proposed, in particular for the most threatened population in Albera.