Ants are often the most ubiquitous and ecologically influential components of terrestrial systems, exhibiting an exceptionally high level of endemic diversity. Simultaneously, exotic ants can be amongst the most environmentally and economically devastating biological invaders. Distinguishing between exotic and related natives is essential for early detection and to reduce the chance of establishment, but at the species-level there remains a great deal of uncertainty among several of the most diverse, widespread and ecologically dominant genera. This taxonomic impediment negatively impacts the use of molecular techniques relying on reference databases, due to the poor state of ant DNA sequences in publicly available repositories, with many species not represented at all and others incorrectly identified. As a result, biosecurity screening for targeted exotic ants and assigning ant species-level identifications for biodiversity remain two separate areas of focus in Australia.<br> Here we propose a workflow for the identification of ants that enables simultaneous processing of large numbers of “bulk” ant samples each containing many individuals, increasing resolution for taxonomic assignment, and generating a curated database linked to voucher specimens. We use a non-destructive DNA extraction method and compare two high throughput sequencing (HTS) metabarcoding platforms – MiSeq (Illumina) and MinION (Oxford Nanopore Technologies) – processing up to 180 bulk mixed ant samples per run. This approach allowed for the acquisition of curated DNA sequences from voucher specimens morphologically examined by expert taxonomists. This work highlights the advantages and current limitations of DNA-based identifications, the needs for standardisation, as well as the importance of a taxonomy-based curation of DNA database for both biodiversity and biosecurity.
