Beneath Borders: Tracing Hidden Waters and Subterranean Life in the Reka-Timavo System Through Environmental DNA

The Reka-Timavo hydrological system, with an underground course extending approximately 90 km, represents a still partially unexplored environment in the transboundary area between Italy and Slovenia. The environmental DNA (eDNA) approach has significantly enhanced the ability to collect data on species distribution across a wide range of ecosystems. In this study, we investigated the metazoan community present in the waters of the Reka-Timavo system through eDNA analysis. Sampling was conducted at two sites within the Škocjan Caves Park (Slovenia) and one site in the Luftloch Cave (Italy) between late July and the first week of August 2024. Environmental DNA was extracted, amplified and purified using commercial kits, and sequenced with Illumina technology (NovaSeq6000 SP) at a depth of 2×250 bp. The three analysed sites displayed dis􀆟nct biological assemblages. In the Slovenian locations, most of the zooplanktonic community belonged to the phylum Ro􀆟fera, par􀆟cularly the genus Ploima. In contrast, the dominant taxa in the Italian site (Luftloch Cave) were from the phylum Arthropoda, primarily the class Copepoda (Acanthocyclops spp.) and class Insecta (order Diptera). Hydrozoan detections in subterranean environments are rare; thus, we highlight findings related to this taxonomic group. DNA from Craspedacusta sowerbii, a freshwater jellyfish native to East Asia, was identified in all sampled caves. Although considered common in Western European freshwater systems, this species is often underreported due to its inconspicuous polyp stage. Its detection in the Reka-Timavo system supports a recent report by the Natural History Museum, confirming its presence as an alien species in the upper Timavo (Slovenia). Additionally, DNA from Hydra vulgaris, a species already known in the region, was found in the Slovenian samples. Of particular interest was the detec􀆟on of Velkovrhia enigmatica (Matjašič & Sket, 1971), the only known stygobiotic freshwater hydrozoan, endemic to the Dinaric Karst. This finding is especially noteworthy and warrants further investigation due to its ecological significance. These preliminary results lay a solid and promising foundation for the application of eDNA methodologies in the study of subterranean environments. The data collected so far suggest that this approach could become a powerful and innova􀆟ve tool for biodiversity assessment in underground ecosystems, paving the way for more detailed and targeted future research.