Determining the conservation status of the mountain hare (Lepus timidus) in the Italian Alps using novel epigenetic, metataxonomic and genomic approaches

For this PhD thesis epigenetics, genomics and metataxonomics techniques were applied to better understand how the mountain hare (Lepus timidus) respond to direct and indirect consequences of climate change. The thesis is indeed divided into four main chapters. I) Epigenetic response of mountain hare (Lepus timidus) in different climate zones. Epigenetic analysis were performed on mountain hare tissue samples from different European populations through Methylation-Capture technique. Bioinformatic analysis allowed to identify DMR (Differentially Methyl-ated Regions) on genes encoding for thermoregulation and coat color change, explaining the adaptation of the studied populations to different environmental conditions (mean winter temperature and snow cover duration condition). II) Sympatric Lepus spp. in the central Italian Alps have significantly different gut microbiotas, where analysis of 16S rRNA for prokaryotes, and ITS for fungal communities on faecal samples were performed for describing the composition of intestinal microbiota of the mountain hare (Lepus timidus) and the European brown hare (Lepus europaeus) living in sympatry. Bioinformatics techniques for metataxonomic definition and statistical analyses allowed to assess the impact of host species (mountain hare and European brown hare) and different altitudinal ranges in shaping the gut microbiota composition. III) A new set of Multi-locus Intron Polymorphism Loci (MIPs) for species and hybrid identification in mammals. A set of 192 well conserved exonic regions was selected by aligning 161 different mammalian genomes, and primers were designed for the amplification of intronic regions among them. I then set up and developed a new investigation technique concerning the intronic loci, named ‘MIPs - Multi-Intronic Polymorphism’. I tested this panel on tissue samples obtained from individuals belonging to six different mammalian families and proved it is a valid instrument for species and hybrids definition. IV) Low levels of hybridization and isolation-by-distance detected in two Alpine hare species (Lepus timidus, L. europaeus) using a novel panel of Multi-locus Intron Polymorphism (MIP) loci. Here the newly developed MIPs panel was applied for species identification, individuals’ genetic structure definition and to test Isolation-by-distance in the mountain hare and European brown hare populations in the Province of Sondrio. I then proved the applicability of this panel to perform population genomics analysis. Although climate warming is reshaping Alpine ecosystems, the mountain hare shows notable resilience, maintaining adaptive potential through both genetic and epigenetic mechanisms and preserving distinct gut microbiota despite sympatry with the expanding European brown hare. However, human-driven pressures—such as restocking, habitat alteration, and increasing hybridization risk—still pose significant threats, highlighting the need for careful management practices and modern conservation tools, including epigenetic monitoring, microbiome assessments, and genetic surveillance