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 optimizing 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 fan system, and two passive collection methods), spider webs, vegetation swabs, water, and soil at two sites. The highest species richness was detected by powered air samples and spider webs (powered air: 83 at Perth Zoo, 44 at Karakamia; spider webs: 62 and 40, respectively), with no significant difference in the community composition, suggesting they capture eDNA from similar sources however, all substrates contributed unique detections. Passive airborne DNA collection, though less efficient than powered devices, (mean species richness per sample: 14.8 vs. 5.8 at Perth Zoo; 6.9 vs. 2.7 at Karakamia), demonstrated great potential as their low cost and simplicity may enable increased replication or longer deployment times, potentially increasing the probability of species detection. Our direct comparison of terrestrial eDNA substrates shows that airborne DNA is a step forward and not just hype. 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.