A Museum‐Curated, Voucher‐Backed 12S Barcode Reference Library for Fishes of the Ecuadorian Amazon

ABSTRACT

Environmental DNA (eDNA) metabarcoding is an emerging method that promises to expedite and improve biodiversity surveys by identifying species from the genetic material they shed into the environment. eDNA methods offer many advantages over conventional specimen‐based surveys, but their effectiveness hinges on reference libraries, which for highly biodiverse regions such as the Ecuadorian Amazon are too often incomplete, inconsistently curated and disconnected from morphological voucher specimens needed to reliably identify and revise taxa. To address this gap, we collected approximately 5000 fish specimens and 1500 tissues of an estimated 311 morphologically defined species from Ecuador's Aguarico, Napo and Pastaza river basins, and built a new, rigorously curated, museum voucher–backed 12S reference library spanning 748 barcodes from over 303 species across 12 orders, 46 families and 171 genera. When tested against unknown haplotypes from concurrently collected eDNA samples, the library nearly doubled the proportion identified to species (40.0%, vs. 21.3% using GenBank). It also enabled the empirical estimation of new species‐level barcode thresholds: 0.4% for the full‐length barcode, 0.9% for a general vertebrate mini‐barcode, and 0.6% for a fish‐specific mini‐barcode. By linking museum vouchers, updated taxonomy, and operational scalability, this reference library helps transform eDNA metabarcoding from an exciting possibility to a practical tool for understanding, monitoring and safeguarding Amazonian freshwater biodiversity.