A Breath of Fresh Air: A Novel Passive Airborne eDNA Approach for Scalable Terrestrial Biodiversity Monitoring.

Terrestrial biodiversity's rapid decline demands expansion of high-resolution biomonitoring to support science-based policy. Environmental DNA (eDNA) analysis has proven effective in aquatic systems but remains underexplored for terrestrial habitats, partly due to point-source sampling bias and difficulty in upscaling. Air has emerged as a promising substrate, yet most studies use active samplers which tend to be expensive, bulky, and require power, potentially hindering broad deployment. We present a newly developed, inexpensive, reusable, and easy-to-use passive approach (Nutshell eDNA sampler) to capture eDNA suspended in air across time (6-96 h) within Rotterdam Zoo, the Netherlands. Its performance was compared with two commonly used active airborne eDNA samplers for vertebrate diversity detection. In total, 88 species were detected, including 24 zoo residents. The Nutshell eDNA sampler was the most effective at detecting zoo residents, surpassing active samplers in species richness within 48 h and continuing to accumulate new species beyond 96 h, including detections of both patchy and singleton signals. It also detected the furthest species signal (515 m). Zoo airborne eDNA further demonstrated a positive correlation to species total biomass, suggesting larger vertebrates or populations release proportionately more DNA into the air. Our findings indicate that for long, unsupervised biomonitoring, passive airborne eDNA sampling presents a promising approach for assessing vertebrate communities and putatively reduces detection noise in stochastic air eDNA signals due to its signal integration. While deeper investigations into airborne eDNA sampling strategies are needed, passive methods can offer a much-needed logistically flexible, low-maintenance approach compared with short-burst collection strategies employed by many active samplers.