eDNA-Based Monitoring of Riverine Biodiversity Hotspots

Abstract

Environmental DNA (eDNA) metabarcoding has emerged as a transformative tool for non-invasive, high-throughput assessment of aquatic biodiversity, offering detection sensitivity superior to conventional electrofishing and netting methods for rare and cryptic taxa. This study applied eDNA metabarcoding targeting the mitochondrial 12S rRNA and COI gene regions to characterise fish and macroinvertebrate diversity across 28 riverine sampling sites spanning six river systems in Estonia, Switzerland, and Spain (n = 336 water samples; 12 samples per site collected monthly across March–August 2021). Sequencing on Illumina MiSeq (2 × 250 bp) generated 48.4 million high-quality reads assigned to 284 fish amplicon sequence variants (ASVs) and 1,642 macroinvertebrate ASVs representing 186 families. eDNA detected significantly more species per site than concurrent electrofishing (mean 28.4 ± 4.2 vs. 18.6 ± 3.8 fish species; paired t-test: t = 8.42, p < 0.001). Eight species of conservation concern undetected by electrofishing were confirmed by eDNA, including Romanogobio uranoscopus and Zingel streber at sites with no prior records. eDNA read abundance correlated significantly with electrofishing catch-per-unit-effort for seven of ten target species (Spearman rs range: 0.64–0.88, all p < 0.01). Biodiversity hotspot sites (top quartile of ASV richness) were characterised by higher dissolved oxygen (mean 9.8 ± 0.6 mg/L), lower turbidity (mean 4.2 ± 1.1 NTU), and greater substrate heterogeneity index (SHI: 0.74 ± 0.08) relative to non-hotspot sites. These results validate eDNA metabarcoding as a cost-effective and sensitive tool for riverine biodiversity surveillance and hotspot delineation under the EU Water Framework Directive.