Leon Blaustein, Ori Segev, Valentina Rovelli, Shirli Bar-David, Lior Blank, Antonina Polevikov, Nadav Pezaro, Tamar Krugman, Simona Showstack, Avi Koplovich, Lital Ozeri and Alan R. Templeton

The Near Eastern fire salamander, Salamandra infraimmaculata, is considered an endangered species in Israel and is near-threatened regionally. For 25 years, our laboratory has sought ethical sampling methods to protect individuals and populations of Salamandra. To “mark” individuals for estimating dispersal and population size, we use non-invasive individual-specific markings from photographs of larvae and adults. We demonstrated through mesocosm experiments (which are less mortality-driven than in nature) that exotic Gambusia affinis have extreme negative mortality effects on Salamandra larvae. From a compassionate conservation aspect, G. affinis should not be killed and placed in habitats where amphibians are not in danger and mosquitoes can be controlled. We identified breeding-site characteristics demonstrating that permanent breeding sites support larger adult populations than temporary breeding sites. For population genetics studies, we take minimal sized tail tips from adults (which have no adverse effects) for microsatellite data. For gene expression studies, rather than sacrifice entire bodies, we demonstrated that by taking only small larval tail tips, we could follow gene expression. We additionally demonstrated that tail tip removal does not affect survival, time to or size at metamorphosis. We documented high road kill rates at a specific breeding site. To prevent potential disease spread, we sterilize boots and sampling gear. We use results for implementing or recommending conservation of individuals and populations – e.g., identifying: movement corridors for breeding site dispersal; roadkill hotspots for under-road tunnels; suitable habitat for pool construction for more effective conservation; utilizing population genetics for recommending management units; information on demography and genetic diversity to identify hotspots for conservation; removal of Gambusia for amphibian protection.

Valentina Rovelli, Aritz Ruiz-González, Leonardo Vignoli, Daniele Macale, Vincenzo Buono, Francesca Davoli, David R. Vieites, Nadav Pezaro and Ettore Randi

Abstract

Next Generation Sequencing (NGS) and related technologies have revolutionized the field of conservation and population genetics, providing novel tools and the capacity to discover thousands of new Single Nucleotide Polymorphisms (SNPs) for the analysis of population parameters. However, gathering NGS data for organisms with very large genomes, such as amphibians, remains challenging because it is still unclear how the current methods perform. Here, we use the Genotyping-by-Sequencing (GBS) approach to generate SNP data for the genotyping of two amphibian species that are of conservation concern, the Sardinian brook salamander (Euproctus platycephalus) and the Italian stream frog (Rana italica). Both E. platycephalus and R. italica have very large genomes (5.53 Gb and >20 Gb, respectively) so genomic data are not available for either of them. We used 95 individual samples and one Illumina lane for each species, with an additional lane for E. platycephalus. After filtering, we obtained 961 and 854 high-coverage SNPs for E. platycephalus and R. italica, respectively. Our results suggest that GBS can serve as a reliable and cost-effective method for genotyping large amphibian genomes, including non-model species.