Comparative Efficiency of Multiple eDNA Sampling Methods for Monitoring Vertebrate Diversity in Mountainous Region

Monitoring biodiversity in mountain ecosystem biodiversity is vital for preserving these rapidly changing ecosystems, yet it remains challenging due to complex terrain and climate. DNA‐based methods, such as environmental DNA (eDNA) and invertebrate‐derived DNA (iDNA), offer rapid, cost‐effective, and non‐invasive solutions to biodiversity monitoring. This study compared the detection efficiency of vertebrate species for three types of eDNA sampling strategies (water eDNA, air eDNA, and iDNA from leeches) in the Gaoligong Mountain area of southwest China. Utilizing five primer sets (12SV05, 16Smam, and VertU primers set) for metabarcoding, we detected 235 vertebrate species, with water eDNA performing the best (173 species), followed by iDNA (102 species) and air eDNA (56 species). Each method identified a different number of unique species: 100 unique with water eDNA, 37 with leeches, and 20 with air eDNA (particularly small birds). Meanwhile, multi‐species occupancy modeling validated the feasibility of eDNA‐based species detection and recovered the distribution patterns of vertebrate taxa in the study area. Our multi‐modal survey underscores the importance of integrating multiple eDNA sampling methods and primer sets to enhance detection efficiency and accuracy for vertebrate species in complex mountain ecosystems.