Tracking pathogens with eDNA in natural areas: how environmental gradients shape surveillance strategies.

The wildlife-livestock-environment interface is a complex system with significant implications for biodiversity and disease dynamics. The episystem concept integrates interconnections among hosts, pathogens, and the environment across time and space. Environmental nucleic acid detection (ENAD) is a non-invasive, PCR-based method for monitoring pathogens via DNA/RNA in soil, water, feces, and surfaces. It works in both aquatic and terrestrial settings and offers cost-effective surveillance. However, how environmental variables influence ENAD performance remains poorly explored, particularly in heterogeneous geographic contexts. In this study, we evaluated 18 sites in Iberian Peninsula within an integrated wildlife monitoring network, collecting 10 surface sponge samples per site and a total of 146 environmental fecal samples from wild boar and wild ruminants. We assessed differences in pathogen marker detection among sponge sampling methods and compared detection rates between sponge and fecal samples. Additionally, we investigated the relationship between ENAD results and environmental variables, mammal community composition and wildlife health indicators. Our results show that habitat and climate characteristics have significant influence on pathogen ENAD at larger geographic scales. Notably, greater pathogen diversity and richness were observed in sponges located at higher latitudes. While most markers found in feces are also detectable in surface sponges, making them the most effective method, combining different sponge sampling methodologies provides the best overall coverage of detectable pathogen markers. Additionally, we developed a predictive map linking pathogen marker detection in sponges to habitat and climate factors across the Iberian Peninsula, providing a useful tool for optimizing future sampling efforts. Keywords: Environmental DNA; zoonosis; disease surveillance; non-invasive; integrated wildlife monitoring; episystem; natural areas.