Integrative eDNA Assessment of Freshwater Biodiversity

Abstract

Environmental DNA (eDNA) metabarcoding -- the extraction, amplification, and sequencing of organismal DNA shed into water -- has transformed freshwater biodiversity assessment by enabling comprehensive, non-invasive species detection from bulk water samples, yet the performance of eDNA metabarcoding relative to conventional survey methods and its suitability for regulatory bioassessment under the EU Water Framework Directive remain incompletely validated across taxonomic groups and aquatic habitats. This study conducted a systematic comparison of eDNA metabarcoding (COI and 12S rRNA markers; Illumina MiSeq 2x250 bp; 2,840 water samples from 284 river and lake sites across Sweden, Spain, and Austria, 2019-2021) against concurrent conventional surveys (kick-net macroinvertebrates, electrofishing for fish, amphibian call surveys, dip-netting) at the same 284 sites. eDNA metabarcoding detected a mean 84.4 +/- 6.4% of species recorded by conventional methods, plus 28.4 +/- 8.4% additional species not detected in any conventional survey (predominantly rare, cryptic, nocturnal, or elusive taxa). Across all taxa, the eDNA-conventional method agreement for species presence/absence was 88.4% (Cohen's kappa = 0.74). eDNA-derived biological quality element (BQE) indices for fish and macroinvertebrates showed Spearman rs = +0.84 with conventional BQE indices from the same sites, meeting the minimum threshold (rs > 0.80) for WFD regulatory compliance proposed by the DNAqua-Net European network. An eDNA-BQE validation framework integrating sampling design, reference database completeness, and bioinformatics pipeline validation achieved 88.4% correct ecological status classification compared to conventional assessment -- exceeding the 80% target for operational WFD bioassessment deployment.