Sympatric Lepus spp. in the 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 above 2000 m a.s.l. It is threatened by habitat loss and fragmentation, as well as by the upward expansion of the European brown hare (L. europaeus), which may result in resource competition and loss of local adaptation through hybridization and interspecific gene flow. Gut microbiota diversity and composition play a key role in animal health and survival; therefore, investigating gut microbial communities in species exposed to rapid biotic and abiotic environmental changes may improve our understanding of their conservation status and extinction risk. However, the effects of sympatry on gut microbiota diversity and composition remain largely unexplored. In this study, we compared the gut microbiota of the two hare species within a zone of overlap in the central Alps (Val Mazia/Matschertal, Italy) by analysing fresh faecal pellets collected along an elevational gradient (1000–2500 m a.s.l.). Species identity was confirmed using mtDNA barcoding, while gut microbiota was characterised through amplicon sequencing targeting prokaryotic 16S rRNA and fungal ITS2 marker genes. Our results confirm the effectiveness of non-invasive sampling for monitoring gut microbial diversity and composition in elusive species such as Lepus spp. Distinct prokaryotic and fungal communities were detected for each species, even in areas where their distributions overlapped. Faecal samples of L. europaeus were primarily dominated by Firmicutes and enriched in Bacteroidota and Spirochaetota, whereas L. timidus showed a predominance of Proteobacteria, followed by Bacteroidota and Acidobacteriota. Moreover, predicted functional profiles derived from taxonomic composition revealed differences in biosynthetic and metabolic potential between the host-associated microbiotas of the two species. Elevation influenced fungal, but not prokaryotic, diversity in both species. Overall, our findings suggest that sympatry has so far had a limited impact on the gut microbiota composition of either species. In light of the expected upward range expansion of L. europaeus under climate warming and its continued restocking for hunting purposes, this study provides an important baseline for assessing the health and adaptability of L. timidus, as well as the effectiveness of conservation measures. Extending this research to additional areas of sympatry will be essential to determine whether gut microbiota composition can serve as an indicator of L. timidus conservation status across its range.