Airborne DNA and Spider Webs Outperform Other eDNA Sources for Monitoring Terrestrial Vertebrates

Understanding the strengths and limitations of different environmental DNA substrates is essential for optimising terrestrial vertebrate surveys and monitoring. However, the performance of newly explored substrates (airborne eDNA, vegetation swabs, spiderwebs) compared to longstanding eDNA sources (water and soil) is uncertain. Using a metabarcoding approach, we assessed vertebrate eDNA diversity across seven substrates: three airborne DNA collection methods (a powered “active” fan system and two passive collection methods), spider webs, vegetation swabs (including swabbing tree trunks and leaves), water, and soil at Perth Zoo and Karakamia Wildlife Sanctuary. Active air sampling and spider webs yielded the highest taxonomic richness (Zoo: 83 and 62 taxa; Karakamia: 44 and 40, respectively), with no significant difference in the community composition, suggesting they capture eDNA from similar sources; however, all substrates contributed unique taxa detections. Passive airborne DNA collection, though less efficient than active samplers (mean taxonomic richness per sample: Zoo: 14.8 vs. 5.8; Karakamia: 6.9 vs. 2.7), showed potential as their low cost and simplicity may enable increased replication or longer deployment times, potentially improving detections. Our direct comparison of terrestrial eDNA substrates shows that airborne DNA sampling offers a genuine advance for terrestrial vertebrate biomonitoring. However, substrate-specific biases were evident, with vegetation swabs favouring arboreal mammals, while water was dominated by aquatic and semi-aquatic species, highlighting the influence of species ecology on DNA deposition. eDNA studies targeting terrestrial vertebrates must consider the heterogeneity of vertebrate DNA distribution across ecosystems and the need for careful selection of eDNA substrates.