Barcoding Gaps and Sequencing Prioritisation in a Global Biodiversity Stronghold

Effective ecological monitoring increasingly depends on eDNA metabarcoding, yet the accuracy of these approaches hinges on the completeness and quality of reference databases. In tropical biodiversity strongholds, where conservation urgency is greatest, systematic gaps and biases in these databases remain poorly characterised. Here, we develop an integrated framework to quantify, explain, and prioritise barcode reference gaps for mammals from Brazil – the world’s largest tropical forest country. Evaluating barcode presence for 731 species across ten primer combinations spanning four mitochondrial loci, we find that fewer than half of species are represented in any single primer database, with interspecific barcode sharing reducing effective taxonomic resolution by up to 50% in recently radiated clades. Database gaps are strongly phylogenetically structured: coverage is highest in charismatic, species-poor lineages and lowest in species-rich clades dominated by small-bodied taxa. Critically, conservation status does not predict barcode inclusion, revealing a fundamental misalignment between molecular infrastructure and conservation need. Using hierarchical Bayesian models, we show that range size and time since scientific description are the strongest predictors of inclusion, and that gaps are driven primarily by clade-level research traditions rather than individual species traits - implying that closing them requires targeted investment across entire neglected lineages. We integrate these drivers with evolutionary distinctiveness and extinction risk into a Barcoding Priority Score that identifies sequencing priorities at global and national scales, and map these priorities across Brazilian biomes. Our transferable framework provides a scalable roadmap for guiding sequencing investments in hyperdiverse regions where biodiversity loss is outpacing molecular documentation.