Full-Length 16S and 18S rRNA Long-Read Sequencing Reveals Gut Microbiome Diversity in the European Brown Hare (Lepus europaeus)

The European brown hare (Lepus europaeus) is a declining wildlife species of ecological and epidemiological importance, yet its intestinal microbiome remains poorly characterized. Here, Oxford Nanopore long-read sequencing was used to analyse full-length 16S and 18S rRNA genes from pooled large-intestine contents of 30 healthy hares divided into three groups. Comparative taxonomic assignment at 95% and 80% sequence identity thresholds revealed striking differences in diversity estimates, with the lower threshold uncovering up to ten-fold more taxa. Across all samples, 40 phyla, 360 families, 1027 genera, and 3373 species were identified, including 30 taxa not previously reported in lagomorphs. These included Monoglobus pectinilyticus, Ruminococcus champanellensis, Odoribacter splanchnicus, Butyricimonas virosa, and Akkermansia muciniphila, associated with pectin degradation, cellulose hydrolysis, butyrate production, mucin degradation, bile acid transformation, and nitrogen recycling. Several taxa relevant to both animal and human health were also detected, supporting hares as sentinels of environmental microbiota within a One Health framework. These findings show that analytical parameter selection strongly shapes microbiome interpretation and provide the most comprehensive gut microbiome profile of the European brown hare to date. The study expands lagomorph microbial ecology and highlights long-read sequencing as a valuable tool for wildlife microbiome surveillance in undercharacterized host species globally.