Active Filtration of Airborne eDNA for Vertebrate Surveys in Tropical Caves

Hidden from sunlight, caves are remarkable subterraneous ecosystems that host a wide range of animals, including unique species. Conventional cave biodiversity surveys can be physically challenging, disturb sensitive vertebrate communities, or require animal collection. Here we evaluate the use of metabarcoding of airborne environmental DNA (eDNA) as a non-invasive method for surveying cave vertebrates. We collected 55 samples by filtering air for 24 h at three light zones (outside, and in twilight and dark zones inside the cave) in three tropical caves in Amazonian Brazil. Using two metabarcoding primer sets targeting mammal and vertebrate DNA, we detected 123 wild vertebrate taxa encompassing mammals, birds, amphibians, and a reptile, of which 74 were identified to genus level. Genus richness in the twilight zone was significantly higher than outside the caves. Vertebrate diversity was also highest in the twilight zone, and community composition differed significantly between light zones and caves. Mammals and amphibians were mostly detected in the twilight and dark zones, whereas birds were more frequently detected outside the cave and in the twilight zone. While preliminary, our results show that caves with smaller entrances yielded more homogeneous communities in the twilight and dark zones. We detected eight of the 13 bat species and one of the four amphibian species recorded in previous surveys, while also detecting taxa not previously recorded in the caves, demonstrating complementarity with conventional surveys. As airborne eDNA requires minimal human presence in cave interiors, it reduces disturbance to sensitive fauna, lowers safety risks to surveyors, and offers a scalable method to complement conventional cave biodiversity surveys in tropical regions.