Long-Read Amplicon Sequencing Uncovers Complex Microbial Communities in the European Brown Hare Gut: Methodological Implications for Wildlife Microbiome Research

The declining European brown hare ( Lepus europaeus ) population necessitates comprehensive microbiome studies to better understand factors affecting their health. This study employed Oxford Nanopore third-generation sequencing to characterize the hare gut microbiome through full-length 16S and 18S rRNA gene analysis. Intestinal contents from 30 hunted brown hares were pooled into three composite samples for sequencing. Comparative analysis using 80% and 95% sequence matching thresholds revealed dramatic differences in detected diversity. The 80% threshold identified substantially more taxa across all taxonomic levels (up to 10-fold more species) and revealed previously unreported microbiome components, including Spirochaetota (25.2% at 80%) and Ascomycota (4.3% at 95%). Statistical analysis confirmed significant differences between samples at genus and species levels for both thresholds (p < 0.05). Our approach identified 28 unique phyla, 360 unique families, 1,027 unique genera, and 3,373 unique species not reported in previous studies. These findings demonstrate how threshold selection fundamentally alters microbiome characterization and highlights the potential of long-read sequencing for expanding our understanding of wildlife microbiomes, which may contribute to improved conservation strategies for declining species.