Detection of Vertebrate Diversity Using Fly- and Leech-Derived iDNA

Developing cost-effective monitoring tools that cover a broad range of taxonomic groups is urgently needed to implement effective conservation strategies. One promising approach involves collecting genetic samples from hematophagous, sarcophagous, or coprophagous invertebrates, followed by amplification and sequencing of residual vertebrate DNA to reveal vertebrate diversity. In invertebrate-derived DNA (iDNA) monitoring, species-specific life histories, physiological traits, and behavioral characteristics introduce distinct biases, depending on the invertebrate taxon used. In this study, we assessed terrestrial vertebrate diversity by simultaneously employing iDNA derived from leeches and flies, together with camera trapping, in a species-poor temperate forest ecosystem in Yakushima, Japan. Our aim was to evaluate the detection biases of each method and to identify optimal combinations of monitoring tools. We detected a greater number of vertebrate species using fly-derived iDNA (15 species) than using leech-derived iDNA (5 species). Fly-derived iDNA detected mammals, birds, reptiles, and amphibians, whereas leech-derived iDNA detected only mammals and amphibians. This difference was partly due to the greater ease of collecting flies compared with leeches, which resulted in different sample sizes. However, even when accounting for sample size, fly-derived iDNA captured a broader range of species. Camera trapping detected a comparable number of mammal species to fly-derived iDNA but provided lower taxonomic resolution. Notably, we detected all non-volant terrestrial mammal species known to inhabit in this ecosystem of low species diversity by combining fly-derived iDNA with camera trapping. However, these methods were less effective in detecting avian and reptilian diversity.