Genomics‐Based Approaches to Ant Monitoring in Land Management: Validation of COI Metabarcoding Primers

Ants (Hymenoptera: Formicidae) are globally dominant invertebrates and widely used as bioindicators of ecosystem change. However, their application in biomonitoring is constrained by the time required to process samples and the need for ever diminishing taxonomic expertise. DNA metabarcoding potentially offers an alternative approach, but validated ant‐specific assays essential for accurate assessment of ant diversity are lacking. In this study, using a hierarchical framework aligned with recommended validation guidelines, we assessed primer performance in silico, in vitro and in situ to identify assays that maximize detection while minimizing bias for detecting ant species in Australian tropical savanna. In silico analyses involved evaluating 14 COI primer sets, including three newly designed ant‐specific primers (AntF1, AntR1 and EPTDr2n_Ant) and 17 general invertebrate primers previously validated for terrestrial arthropods, against a custom reference database of 19 Australian ant genera with PrimerMiner. AntF1/AntR1, fwhF2/EPTDr2n_Ant and fwhF2/fwhR2n had the lowest penalty scores. In vitro metabarcoding of mock communities recovered 68% and 70% of genera with AntF1/AntR1 and fwhF2/fwhR2n, respectively, but only 0.05% for fwhF2/EPTDr2n_Ant. Spike‐in assays revealed strong inhibitory effects of primer‐template mismatches and soil environmental matrix on amplification efficiency. In situ comparisons of five surface soil eDNA samples recovered a subset of genera previously detected from far more extensive pitfall‐trapping, with the ant‐specific AntF1/AntR1 outperforming the general invertebrate primer fwhF2/fwhR2n for genus‐level detection. We recommend AntF1/AntR1 as the primary assay for ant biomonitoring in Australia (and likely elsewhere in the world), suitable for both soil eDNA surveys and specimen metabarcoding, and fwhF2/fwhR2n for specimen metabarcoding. This study provides the first targeted validation of metabarcoding primers specifically for ants in Australia, providing a robust foundation for incorporating eDNA approaches into ant biomonitoring in land management.