Hidden Subterranean Landscape Fragmentation Influences Species Distribution in Proteus scopolii and P. xanthostictus in the Classical Karst Sector

Subterranean environments are characterised by high spatial fragmentation and limited connectivity, which can strongly influence genetic structure and species distributions in troglobitic organisms. Here, we investigated the occurrence and spatial distribution of Proteus scopolii and P. xanthostictus within the Italian sector of the Classical Karst, the only Italian area where the two species naturally coexist. Species distributions were assessed using the mitochondrial cytochrome c oxidase subunit I (COI) marker, combined with morphological traits and the Scaled Mass Index (SMI), in individuals collected from multiple caves along the hydrological gradient of the Classical Karst. Results revealed sympatric occurrence of the two species, with a clear upstream–downstream distributional gradient. A sharp transition was detected between the Trebiciano and Luftloch caves, approximately 1 km apart, suggesting the presence of hydrogeological barriers limiting species distribution. Further downstream, in the Reka–Timavo and Soča–Isonzo aquifer systems, only P. scopolii was detected. This study represents the largest sampling effort to date across olm populations at their westernmost natural range, highlighting the role of subterranean hydrological structure and local barriers in shaping species distributions and genetic patterns, and underscoring the ecological complexity and conservation importance of karst aquifers.