Novel tools for conservation genetics in marine fish: population structure and evolution of the European hake (Merluccius merluccius) inferred by SNP variation and applications to traceability

The research presented in my PhD thesis is part of a wider European project, FishPopTrace, focused on traceability of fish populations and products. My work was aimed at developing and analyzing novel genetic tools for a widely distributed marine fish species, the European hake (Merluccius merluccius), in order to investigate population genetic structure and explore potential applications to traceability scenarios. A total of 395 SNPs (Single Nucleotide Polymorphisms) were discovered from a massive collection of Expressed Sequence Tags, obtained by high-throughput sequencing, and validated on 19 geographic samples from Atlantic and Mediterranean. Genome-scan approaches were applied to identify polymorphisms on genes potentially under divergent selection (outlier SNPs), showing higher genetic differentiation among populations respect to the average observed across loci. Comparative analysis on population structure were carried out on putative neutral and outlier loci at wide (Atlantic and Mediterranean samples) and regional (samples within each basin) spatial scales, to disentangle the effects of demographic and adaptive evolutionary forces on European hake populations genetic structure. Results demonstrated the potential of outlier loci to unveil fine scale genetic structure, possibly identifying locally adapted populations, despite the weak signal showed from putative neutral SNPs. The application of outlier SNPs within the framework of fishery resources management was also explored. A minimum panel of SNP markers showing maximum discriminatory power was selected and applied to a traceability scenario aiming at identifying the basin (and hence the stock) of origin, Atlantic or Mediterranean, of individual fish. This case study illustrates how molecular analytical technologies have operational potential in real-world contexts, and more specifically, potential to support fisheries control and enforcement and fish and fish product traceability.

Conservation genetics of European wildcat (Felis silvestris silvestris): a wide and integrating analysis protocol for admixture inferences and population structure

Introgression of domestic cat genes into European wildcat (Felis silvestris silvestris) populations and reduction of wildcats’ range in Europe, leaded by habitat loss and fragmentation, are considered two of the main conservation problems for this endangered feline. This thesis addressed the questions related with the artificial hybridization and populations’ fragmentation, using a conservation genetics perspective. We combined the use of highly polymorphic loci, Bayesian statistical inferences and landscape analyses tools to investigate the origin of the geographic-genetic substructure of European wildcats (Felis silvestris silvestris) in Italy and Europe. The genetic variability of microsatellites evidenced that European wildcat populations currently distributed in Italy differentiated in, and expanded from two distinct glacial refuges during the Last Glacial Maximum. The genetic and geographic substructure detected between the eastern and western sides of the Apennine ridge, resulted by adaptation to specific ecological conditions of the Mediterranean habitats. European wildcat populations in Europe are strongly structured into 5 geographic-genetic macro clusters corresponding to: the Italian peninsular & Sicily; Balkans & north-eastern Italy; Germany eastern; central Europe; and Iberian Peninsula. Central European population might have differentiated in the extra-Mediterranean Würm ice age refuge areas (Northern Alps, Carpathians, and the Bulgarian mountain systems), while the divergence among and within the southern European populations might have resulted by the Pleistocene bio geographical framework of Europe, with three southern refugia localized in the Balkans, Italian Peninsula and Iberia Peninsula. We further combined the use of most informative autosomal SNPs with uniparental markers (mtDNA and Y-linked) for accurately detecting parental genotypes and levels of introgressive hybridization between European wild and domestic cats. A total of 11 hybrids were identified. The presence of domestic mitochondrial haplotypes shared with some wild individuals led us to hypnotize the possibility that ancient introgressive events might have occurred and that further investigation should be recommended.

Ecological genetics and conservation genomics of wolf (Canis lupus)

In the present work, we apply both traditional and Next Generation Sequencing (NGS) tools to investigate some of the most important adaptive traits of wolves (Canis lupus). In the first part, we analyze the variability of three Major Histocompatibility Complex (MHC) class II genes in the Italian wolf population, also studying their possible role in mating choice and their influence on fitness traits. In the second section, as part of a larger canid genome project, we will exploit NGS data to investigate the transcript-level differences between the wolf and the dog genome that can be correlated to domestication.

Landscape ecology and genetics of the wolf in Italy

This PhD Thesis includes five main parts on diverse topics. The first two parts deal with the trophic ecology of wolves in Italy consequently to a recent increase of wild ungulates abundance. Data on wolf diet across time highlighted how wild ungulates are important food resource for wolves in Italy. Increasing wolf population, increasing numbers of wild ungulates and decreasing livestock consume are mitigating wolf-man conflicts in Italy in the near future. In the third part, non-invasive genetic sampling techniques were used to obtain genotypes and genders of about 400 wolves. Thus, wolf packs were genetically reconstructed using diverse population genetic and parentage software. Combining the results on pack structure and genetic relatedness with sampling locations, home ranges of wolf packs and dispersal patterns were identified. These results, particularly important for the conservation management of wolves in Italy, illustrated detailed information that can be retrieved from genetic identification of individuals. In the fourth part, wolf locations were combined with environmental information obtained as GIS-layers. Modern species distribution models (niche models) were applied to infer potential wolf distribution and predation risk. From the resulting distribution maps, information pastures with the highest risk of depredation were derived. This is particularly relevant as it allows identifying those areas under danger of carnivore attack on livestock. Finally, in the fifth part, habitat suitability models were combined with landscape genetic analysis. On one side landscape genetic analyses on the Italian wolves provided new information on the dynamics and connectivity of the population and, on the other side, a profound analysis of the effects that habitat suitability methods had on the parameterization of landscape genetic analyses was carried out to contributed significantly to landscape genetic theory.