Assessment of leaf-litter invertebrate biodiversity using high throughput sequencing

Leaf litter ecosystems and their fauna are largely understudied, despite their critical ecological roles. Here, we investigate challenges associated with estimating biodiversity in terrestrial leaf litter. Current methodologies for biodiversity assessment are fraught with limitations; amongst the most significant is a decline in taxonomic expertise, complicating the process of species identification and the significant costs associated with species-level morphological identifications. DNA barcoding employs molecular markers to identify animal species, and the mitochondrial gene cytochrome c oxidase I (COI) is the agreed upon standard for animals. DNA metabarcoding facilitates the identification of multiple species without necessitating taxonomic expertise. Recent studies indicate that environmental DNA (eDNA) may exhibit greater sensitivity compared to taxonomic identifications completed on animals collected using traditional methods (e.g., pitfall traps, pan traps). To test whether eDNA methodology works in a real-world scenario, we sampled leaf litter across a temperate forest/field ecotone. Leaf litter was dried, ground and processed to extract environmental DNA. We evaluated multiple DNA extraction protocols to test their relative efficacy. We found that the Qiagen Blood and Tissue Kit was the most effective at recovering invertebrate diversity and found that there were notable differences in the biodiversity recovered between forest and field habitats. Temperature emerged as a significant factor influencing the composition of the communities observed. Our methodology is applicable across various environments for efficient biodiversity assessment and would be particularly beneficial for monitoring pests and invasive species. Our approach offers a cost-effective and timely alternative to conventional biodiversity assessment methods and underscores the significance of accurate assessment methodologies for leaf litter communities.