Hidden connections: tracing subterranean biodiversity of the Reka–Timavo River through eDNA

The Reka–Timavo hydrological system, extending nearly 40 km underground, remains only partially explored, with approximately half of its course documented within the transboundary karst region of Italy and Slovenia. Environmental DNA (eDNA) has transformed biodiversity research by enabling the detection of species distributions even in habitats that are otherwise inaccessible. In this study, we examined metazoan communities of the Reka–Timavo system using eDNA from water samples collected at two subterranean sites. At the ponor zone of the Škocjan Caves Regional Park (Slovenia) and at Luftloch Cave (Italy), located 13 km downstream in a straight line and newly discovered in March 2024; this site lies approximately 20 km upstream from the river resurgences.Our results revealed both shared taxa, reflecting the hydrological connectivity within the system, and distinct assemblages, highlighting ecological differences among the sampling sites. Slovenian samples were dominated by rotifers, particularly members of the genus Ploima, whereas Italian was characterized by arthropods, including copepods (Acanthocyclops) and dipteran insects. Notably, hydrozoans - rarely recorded in subterranean environments - were detected. The DNA of the freshwater jellyfish Craspedacusta sowerbii, native to East Asia but now widespread in European inland waters, was detected at all sites. Its inconspicuous polyp stage often hampers direct observation, and its presence here corroborates a recent record by the Museum of Natural Sciences in the upper part of the Reka-Timavo River, where the species was found. Additionally, DNA of Hydra vulgaris, already known from the region, was identified in Slovenian samples. Of particular interest is the detection of Velkovrhia enigmatica, the only known stygobiotic freshwater hydrozoan endemic to the Dinaric karst. This species has never been reported in Italy before, and its DNA detection highlights the need for further ecological and biogeographical investigations of the Reka-Timavo system.