Increased power of repetitive nuclear DNA for species detection and eDNA-biomass estimation: performance of an ultra-high copy marker of northern pike

Abstract Environmental DNA (eDNA) is a non-invasive tool for aquatic biodiversity monitoring that primarily proves qualitative information on species presence but is increasingly used to obtain semi-quantitative estimates of abundance and biomass. Mitochondrial DNA has, due to its cellular abundance, dominated eDNA assay development while the potential of highly repetitive nuclear regions remains largely unexplored. We developed and evaluated an eDNA assay for northern pike ( Esox lucius ) targeting the 5S ribosomal DNA region, repeated in extremely high numbers (> 20,000 copies) within the pike genome and compared its performance against an established mitochondrial COI marker. Specificity was evaluated in vitro against co-occurring freshwater species and performance was assessed across two controlled experiments on adult and juvenile pike. The repetitive nuclear assay amplified four to seven cycles earlier than the mitochondrial assay in pike tissue, and two to five cycles earlier in the controlled experiments, demonstrating substantially increased signal strength. While highly repetitive nuclear DNA increased eDNA signal strength, a trade-off emerged in terms of specificity, and questions remain regarding interspecies variation of such regions. Highly repetitive nuclear eDNA markers show considerable promise for increasing signal intensity and detection probabilities, offering a promising approach for improving sensitivity of species-specific eDNA monitoring.