What are the best practices for curating eDNA custom barcode reference libraries? A case study using Australian subterranean fauna

Identification of species for environmental assessment and monitoring is essential for understanding anthropogenic impacts on biodiversity, but for subterranean fauna this task is frequently difficult and time consuming. The implementation of environmental DNA (eDNA) metabarcoding for biodiversity discovery and assessment offers considerable promise for improving the rate, accuracy and efficiency of species detection in ecosystems both above and below the ground. Importantly, for a better understanding of the biodiversity and ecology of organisms detected using eDNA, a custom library of known reference sequences with associated correct taxonomic metadata—i.e., a barcode reference library (BRL)—is required. Yet, minimal guidance is currently available on how an effective (i.e. shareable, multi-sequence, that permits metadata and has a unified nomenclature) and accurate (i.e. verified) custom BRL can be achieved. Here, we present a detailed roadmap for curation of a BRL for subterranean fauna. To do this, we (1) curated a custom sequence database of subterranean fauna at an environmentally sensitive location, Bungaroo Creek in the Pilbara region of Western Australia, for four gene loci useful for eDNA metabarcoding ( COI , 18S rRNA, 12S rRNA and 16S rRNA); (2) addressed major gaps in taxonomy and disparate nomenclature of subterranean fauna by estimating 17–29 putative new species with standard delimitation methods, including 34 Barcode Index Numbers (BINs) in BOLD, and (3) summarised a best practice workflow for curation of a custom BRL that has broad applicability and can be applied to any taxa.