Tracking pathogen-related markers with eDNA in natural areas: how environmental factors shape surveillance strategies

The wildlife-livestock-environment interface is a complex system with implications for biodiversity and diseases. Environmental nucleic acid detection (ENAD) is a non-invasive method for monitoring DNA/RNA from microorganisms, meiofauna, and hosts. However, how environmental variables influence ENAD in heterogeneous geographic contexts remains poorly explored. In this study, 18 sites were evaluated in Iberian Peninsula, collecting 10 surface sponge samples per site and a total of 146 environmental fecal samples to investigate how environmental, mammal community and wildlife health variables may influence ENAD, using different pathogen-related markers as the study model. Differences in ENAD were assessed among sponge sampling methods and between sponge and fecal samples. The results show that environmental characteristics influence ENAD, at least for pathogen-related markers and at larger geographic scales. Greater pathogen marker diversity and richness was observed at higher latitudes. Most markers found in feces were also detectable in surface sponges. Combining different sponge sampling methodologies provided the best overall coverage of detectable environmental DNA. A predictive map linking ENAD in sponges to environmental factors was developed.