A novel degenerate primer set for eDNA metabarcoding of amphibians, turtles and fish

Metabarcoding of environmental DNA (eDNA) has revolutionized the detection of aquatic species across large geographic scales. However, the effectiveness of eDNA metabarcoding across taxa is marker-dependent, often requiring multiple markers to detect divergent taxonomic groups. Here, we introduce a novel metabarcoding marker designed to simultaneously detect amphibians, turtles, and fish from eDNA samples. We initially optimized this marker to match species from central and southern California, USA, and conducted validation on 525 field-collected eDNA samples from central California, while also testing an existing published amphibian marker from the literature. Additionally, we assessed the global applicability of the marker with in-silico analysis through comparison with 11,350 amphibian, turtle, and fish mitochondrial genomes downloaded from GenBank. Field validation demonstrated the markers robustness in detecting aquatic vertebrates, outperforming the published marker in sensitivity, particularly for turtles and fish. In-silico evaluation demonstrated the potential for global use of this marker, indicating broad applicability for amplifying eDNA from amphibians, turtles, and fish worldwide. Our findings highlight the utility of this novel marker for monitoring of aquatic vertebrate communities, with implications for conservation, management, and ecological research.