Sympatric Lepus spp. in the central Italian Alps host significantly different gut microbiotas.

The mountain hare (Lepus timidus) is an arctic-alpine species with relictual populations in the Italian Alps, typically occurring at elevations above 2000 m a.s.l. This species is threatened by habitat loss and fragmentation, and declining snow cover due to climate warming. Moreover, as treelines shift upward, the European brown hare (L. europaeus) is expanding its distribution into areas previously dominated by the mountain hare, potentially leading to resource competition, and loss of local adaptation through hybridization and inter-specific gene flow. In particular, the consequences of sympatry on diversity and composition of prokaryote and fungal communities of the gut microbiota, which are critical to individual health, are currently unknown. Here, we compared the gut microbiota of these two hare species in an area of overlap in the central Alps by analysing fresh faecal pellets collected from Val Mazia/Matschertal, Italy along an elevational gradient (1000 to 2500 m a.s.l.). For the first time, we describe the prokaryote diversity and composition of L. timidus, and the fungal gut communities (mycobiota) of both Lepus species. Species identity was confirmed for 95 samples via mtDNA barcoding, while gut microbiota richness and composition were investigated using amplicon sequencing, targeting the V3-V4 region of the prokaryote 16S rRNA gene and fungal ITS2 regions. Distinct prokaryote and fungal communities were observed for each species, even where their distributions overlap, indicating differences in their functional diversity. Interestingly, for both Lepus species, elevation influenced fungal but not prokaryote diversity. Therefore, sympatry appears to have had minimal impact on gut microbiota composition of either species thus far. Given the expected upward range shift of L. europaeus under climate warming and its continued restocking for hunting, our findings provide an important baseline for assessing the health and adaptability of L. timidus as well as the effectiveness of conservation efforts aimed at protecting this species. However, expanding this research to other areas of sympatry will be essential to understand if gut microbiota composition is indicative of L. timidus conservation status across its range.