Accounting for Intra‐ and Intergenomic Sequence Variation in Reference Barcodes Improves eDNA Metabarcoding Biodiversity Assessment

Environmental DNA (eDNA) metabarcoding can rapidly characterise biodiversity, yet its accuracy and effectiveness are limited by incomplete DNA barcode reference databases. We evaluated how comprehensive reference databases that include sequence variation within genomes (intragenomic) and across individuals and species (intergenomic) improve eDNA-based biodiversity assessments. We collected coral tissue and water samples at deep sites offshore Puerto Rico for reference barcoding and eDNA metabarcoding. Genome skimming coral specimens yielded 28S barcodes for 314 of 346 samples (90.8%) and revealed divergent intragenomic 28S lineages in multiple octocoral families. Incorporating local reference barcodes substantially changed ASV taxonomic classifications: 22 ASVs (8.9%) gained genus-level resolution, 19 ASVs (7.7%) were reassigned to different genera, and 14 ASVs (5.7%) lost incorrect genus-level classifications. Thus, incomplete reference databases produce not only unclassified ASVs but also false positive detections and ecologically meaningful misclassifications. When intragenomic 28S lineages were excluded from the reference database, 18 ASVs (7.4%) could not be classified to family or genus, demonstrating that unrecognised intragenomic variation can be mistaken for unsampled taxa. Integrating reference genome skimming and eDNA metabarcoding expanded known coral family richness by 36% at depths shallower than 1000 m and by 181% at depths greater than 1000 m. eDNA also detected two coral families previously unknown off Puerto Rico and nearby islands, underscoring its potential for biodiversity discovery.