Different patterns of introgression in a three species hybrid zone among European cave salamanders.

Hybrid zones occur where genetically distinct populations meet, mate and produce offspring with mixed ancestry. In Plethodontid salamanders, introgressive hybridization is a common phenomenon, where hybrids backcross with parental populations leading to the spread of new alleles into the parental genomes. Whereas many hybrid zones have been reported in American Plethodontid salamanders, only a single hybrid zone has been documented in European plethodontids so far, which is located at the Apuan Alps in the Italian Peninsula. Here, we describe a previously unreported hybrid zone in the Northern Apennines involving all the three Plethodontid salamander species inhabiting the Italian Peninsula. We found 21 new Speleomantes sites of occurrence, from a hitherto unexplored area located at the boundaries between three Speleomantes species ranges. Using mitochondrial (Cytb and ND2 genes) and nuclear markers (two diagnostic SNPs at the NCX1 gene), we revealed a three-way contact zone where all the three mainland species hybridize: S. strinatii, S. ambrosii and S. italicus. We observed a strong mitonuclear discordance, with mitochondrial markers showing a conspicuous geographic pattern, while diagnostic nuclear SNPs coexisted in both the same populations and individuals, providing evidence of hybridization in many possible combinations. The introgression is asymmetric, with S. italicus mitogenome usually associated with S. a. ambrosii and, to a lesser extent, to S. strinatii nuclear alleles. This finding confirms that Plethodontid are a group of choice to investigate hybridization mechanisms and suggests that behavioural, genetic and ecological components may concur in determining the direction and extent of introgression.

Evolution of personality and locomotory performance traits during a late Pleistocene island colonization in a tree frog.

Recent empirical and theoretical studies suggest that personality and locomotory performance traits linked to dispersal abilities are crucial components of the dispersal syndromes, and that they can evolve during range expansions and colonization processes. Island colonization is one of the best characterized processes in dispersal biogeography, and its implication in the evolution of phenotypic traits has been investigated over a wide range of temporal scales. However, the effect of island colonization on personality and performance traits of natural populations, and how these traits could drive island colonization, has been little explored. Noteworthy, no studies have addressed these processes in the context of late Pleistocene range expansions. Here, we investigated the contribution of island colonization triggered by postglacial range expansions to intraspecific variation in personality and locomotory performance traits. We compared boldness, exploration, jumping performance, and stickiness abilities in populations from 3 equidistant areas of the Tyrrhenian tree frog Hyla sarda, 2 from the main island (Corsica Island), and 1 from the recently colonized island of Elba. Individuals from Elba were significantly bolder than individuals from Corsica, as they emerged sooner from a shelter (P = 0.028), while individuals from Corsica showed markedly higher jumping and stickiness performance (both P < 0.001), resulting as more performing than those of Elba. We discuss these results in the context of the major microevolutionary processes at play during range expansion, including selection, spatial sorting, founder effects, and their possible interaction with local adaptation processes.

De novo transcriptome assembly and annotation for gene discovery in Salamandra salamandra at the larval stage.

Dispersal is a key process in ecology and evolutionary biology, as it shapes biodiversity patterns over space and time. Attitude to disperse is unevenly distributed among individuals within populations, and that individual personality can have pivotal roles in the shaping of this attitude. Here, we assembled and annotated the first de novo transcriptome of the head tissues of Salamandra salamandra from individuals, representative of distinct behavioral profiles. We obtained 1,153,432,918 reads, which were successfully assembled and annotated. The high-quality of the assembly was confirmed by three assembly validators. The alignment of contigs against the de novo transcriptome led to a mapping percentage higher than 94%. The homology annotation with DIAMOND led to 153,048 (blastx) and 95,942 (blastp) shared contigs, annotated on NR, Swiss-Prot and TrEMBL. The domain and site protein prediction led to 9850 GO-annotated contigs. This de novo transcriptome represents reliable reference for comparative gene expression studies between alternative behavioral types, for comparative gene expression studies within Salamandra, and for whole transcriptome and proteome studies in amphibians.

Unveiling a hotspot of genetic diversity in southern Italy for the endangered Hermann's tortoise Testudo hermanni.

BACKGROUND: Hotspots of intraspecific genetic diversity represent invaluable resources for species to cope with environmental changes, and their identification is increasingly recognized as a major goal of conservation ecology research. However, even for iconic and endangered species, conservation strategies are often planned without thorough information on the geographic patterns of genetic variation. Here, we investigated the spatial patterns of genetic variation of the endangered Hermann's tortoise Testudo hermanni in the Italian Peninsula by genotyping 174 individuals at 7 microsatellite loci, with the aim to contribute to planning effective conservation strategies. RESULTS: Ordination-based and Bayesian clustering analyses consistently identified three main genetic clusters, one spread in the central and northern part of the peninsula, and two restricted to southern Italy and Sicily, respectively. The highest levels of genetic diversity were found in populations of the southern cluster and, in particular, at the northern edges of its distribution (He > 0.6, Ar > 2.8 ), that correspond to areas of putative secondary contact and admixture between distinct lineages. Our results clearly identify a hotspot of genetic diversity for the Hermann's tortoise in southern Italy. CONCLUSION: We inferred the evolutionary history and the spatial patterns of genetic variation of the Hermann's tortoise in the Italian Peninsula. We identified three main genetic clusters along the peninsula and a hotspot of intraspecific diversity in southern Italy. Our results underline the urgent need for conservation actions to warrant the long-term persistence of viable tortoise populations in this area. Furthrmore, these data add further evidence to the role of southern Italy as a biodiversity hotspot for temperate fauna, claiming for higher consideration of this area in large scale conservation programs.

Brain de novo transcriptome assembly of a toad species showing polymorphic anti-predatory behavior.

Understanding the genomic underpinnings of antipredatory behaviors is a hot topic in eco-evolutionary research. Yellow-bellied toad of the genus Bombina are textbook examples of the deimatic display, a time-structured behavior aimed at startling predators. Here, we generated the first de novo brain transcriptome of the Apennine yellow-bellied toad Bombina pachypus, a species showing inter-individual variation in the deimatic display. Through Rna-Seq experiments on a set of individuals showing distinct behavioral phenotypes, we generated 316,329,573 reads, which were assembled and annotated. The high-quality assembly was confirmed by assembly validators and by aligning the contigs against the de novo transcriptome with a mapping percentage higher than 91.0%. The homology annotation with DIAMOND (blastx) led to 77,391 contigs annotated on Nr, Swiss Prot and TrEMBL, whereas the domain and site protein prediction made with InterProScan led to 4747 GO-annotated and 1025 KEGG-annotated contigs. The B. pachypus transcriptome described here will be a valuable resource for further studies on the genomic underpinnings of behavioral variation in amphibians.

From STRs to SNPs via ddRAD-seq: Geographic assignment of confiscated tortoises at reduced costs.

Assigning individuals to their source populations is crucial for conservation research, especially for endangered species threatened by illegal trade and translocations. Genetic assignment can be achieved with different types of molecular markers, but technical advantages and cost saving are recently promoting the shift from short tandem repeats (STRs) to single nucleotide polymorphisms (SNPs). Here, we designed, developed, and tested a small panel of SNPs for cost-effective geographic assignment of individuals with unknown origin of the endangered Mediterranean tortoise Testudo hermanni. We started by performing a ddRAD-seq experiment on 70 wild individuals of T. hermanni from 38 locations. Results obtained using 3182 SNPs are comparable to those previously obtained using STR markers in terms of genetic structure and power to identify the macro-area of origin. However, our SNPs revealed further insights into the substructure in Western populations, especially in Southern Italy. A small panel of highly informative SNPs was then selected and tested by genotyping 190 individuals using the KASP genotyping chemistry. All the samples from wild populations of known geographic origin were genetically re-assigned with high accuracy to the original population. This reduced SNPs panel represents an efficient molecular tool that enables individuals to be genotyped at low cost (less than €15 per sample) for geographical assignment and identification of hybrids. This information is crucial for the management in-situ of confiscated animals and their possible re-allocation in the wild. Our methodological pipeline can easily be extended to other species.

Paso doble: A two-step Late Pleistocene range expansion in the Tyrrhenian tree frog Hyla sarda.

The Tyrrhenian tree frog, Hyla sarda, is an amphibian endemic to the Tyrrhenian islands (Western Mediterranean). Previous investigations of its Pleistocene evolutionary history suggested that it colonised the northern portion of its current range, through a spatial diffusion process from the Sardinia island, during the last glaciation. However, southern and northern portions of the species' range experienced markedly different climatic conditions during the Late Pleistocene, suggesting the possibility of an unusual two-step process of demographic expansion. Here, we use Bayesian phylogeographic approaches to locate the ancestral area in Sardinia and to characterise better the demographic component of this expansion event. These analyses located the ancestral area for H. sarda populations along the central-eastern coast of the Sardinia island, within an area previously shown to host suitable bioclimatic conditions for H. sarda populations throughout the Late Pleistocene. Historical demographic reconstructions clearly showed that a two-step process of demographic growth fits well the data, with northern populations expanding later than Sardinia populations. The harsher climatic conditions occurred in northern islands during the glacial epoch, as compared to Sardinia, likely delayed tree frog colonisation of northern territories, and the associated demographic growth.

Reconstructing hotspots of genetic diversity from glacial refugia and subsequent dispersal in Italian common toads (Bufo bufo).

Genetic diversity feeds the evolutionary process and allows populations to adapt to environmental changes. However, we still lack a thorough understanding of why hotspots of genetic diversity are so 'hot'. Here, we analysed the relative contribution of bioclimatic stability and genetic admixture between divergent lineages in shaping spatial patterns of genetic diversity in the common toad Bufo bufo along the Italian peninsula. We combined population genetic, phylogeographic and species distribution modelling (SDM) approaches to map ancestral areas, glacial refugia, and secondary contact zones. We consistently identified three phylogeographic lineages, distributed in northern, central and southern Italy. These lineages expanded from their ancestral areas and established secondary contact zones, before the last interglacial. SDM identified widespread glacial refugia in peninsular Italy, sometimes located under the present-day sea-level. Generalized linear models indicated genetic admixture as the only significant predictor of the levels of population genetic diversity. Our results show that glacial refugia contributed to preserving both levels and patterns of genetic diversity across glacial-interglacial cycles, but not to their formation, and highlight a general principle emerging in Mediterranean species: higher levels of genetic diversity mark populations with substantial contributions from multiple genetic lineages, irrespective of the location of glacial refugia.

Publisher Correction: Quaternary history, population genetic structure and diversity of the cold-adapted Alpine newt Ichthyosaura alpestris in peninsular Italy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Quaternary history, population genetic structure and diversity of the cold-adapted Alpine newt Ichthyosaura alpestris in peninsular Italy.

Mediterranean peninsulas are major biodiversity hotspots, and cold-adapted species are an important component of this biodiversity. However, cold-adapted species contributed surprisingly little to our knowledge of the intimate links between Quaternary environmental changes, species' responses to these changes, and current patterns of intraspecific biodiversity. Here, we investigated the genetic structure and evolutionary history of a cold-adapted amphibian, the Alpine newt Ichthyosaura alpestris, within the Italian peninsula. Nuclear and mitochondrial markers consistently identified three distinct genetic lineages, whose divergence dates to the Early Pleistocene (1.9 and 0.8 million years ago). Our results show that the Italian peninsula provided multiple Pleistocene refugia to this cold-adapted species, and suggest that allopatric fragmentation followed by secondary admixture have been key events in the formation of its current pattern of genetic diversity. Indeed, estimates of population genetic diversity clearly identified contact populations as those achieving the highest levels of diversity. Such concordance among cold-adapted and temperate species in terms of processes triggering the formation of regional patterns of genetic diversity provides strong support for the hypothesis that gene exchange between divergent lineages, rather than long-term stability of refugial populations, has been the main step toward the formation of hotspots of intraspecific biodiversity.

Climate change promotes hybridisation between deeply divergent species.

Rare hybridisations between deeply divergent animal species have been reported for decades in a wide range of taxa, but have often remained unexplained, mainly considered chance events and reported as anecdotal. Here, we combine field observations with long-term data concerning natural hybridisations, climate, land-use, and field-validated species distribution models for two deeply divergent and naturally sympatric toad species in Europe (Bufo bufo and Bufotes viridis species groups). We show that climate warming and seasonal extreme temperatures are conspiring to set the scene for these maladaptive hybridisations, by differentially affecting life-history traits of both species. Our results identify and provide evidence of an ultimate cause for such events, and reveal that the potential influence of climate change on interspecific hybridisations goes far beyond closely related species. Furthermore, climate projections suggest that the chances for these events will steadily increase in the near future.

Geographic distribution of the chytrid pathogen Batrachochytrium dendrobatidis among mountain amphibians along the Italian peninsula.

The amphibian chytrid pathogen Batrachochytrium dendrobatidis (Bd) is considered a major cause of amphibian population declines, particularly in montane areas. Here, we investigated the presence and distribution of Bd among populations of 3 mid- to high-altitude species spanning the entire Italian peninsula (486 individuals from 39 sites overall): the stream frog Rana italica, the fire salamander Salamandra salamandra gigliolii, and the alpine newt Mesotriton alpestris apuanus. We found Bd in all of the analyzed species. Despite the widespread distribution of the pathogen, its overall prevalence (6, 9 and 19%, respectively) was lower than previously reported for the endangered Apennine yellow-bellied toad Bombina pachypus (62.5%). Moreover, several populations of the species studied here were not infected, even at sites where Bd has been detected in other host species. When coupled with the lack of evidence for Bd-related mortalities in these species in peninsular Italy, these results suggest that mechanisms of resistance and/or tolerance are protecting populations of these species from the pathogenic activity of Bd. Nevertheless, in light of the dynamic pattern of Bd-host interactions reported in other studies, of Bd-related mortalities in at least 1 study species (S. s. salamandra) in other areas, and the ongoing climate changes in montane environments, we suggest that the occurrence of Bd should be considered a potential threat to the long-term persistence of these species, and urge the implementation of monitoring and conservation plans.