ABSTRACT Environmental DNA (eDNA) metabarcoding is a powerful tool for monitoring elusive marine species, but its effectiveness is constrained by incomplete reference databases. This limitation is especially evident for epaulette sharks ( Hemiscyllium ), a genus of small benthic sharks endemic to the Indo‐Australian Archipelago. Six of the nine species occur in eastern Indonesia and are fully protected under national law, yet monitoring their distribution remains challenging using traditional methods. To address this gap, we developed the first genus‐specific eDNA metabarcoding assay for Hemiscyllium , targeting the mitochondrial NADH4 gene. Laboratory validation confirmed that the new ES‐200ND4 primer amplified Hemiscyllium DNA in simulated eDNA experiments, even at low concentrations (< 11 ng/μL). Field application in Raja Ampat detected the endemic H. freycineti at six of the seven sampling locations. The ES‐200ND4 primer demonstrated taxonomic specificity, with all 55 ASVs generated from field samples assigned exclusively to H. freycineti (mean genetic distance: 0.78%), while the universal 12S marker (elas02) exhibited non‐specific amplification but detected a broader range of marine taxa, including H. freycineti (17 ASVs), Carcharhinus sp. (1 ASV), and numerous phytoplankton groups. Notably, eDNA detection at Dayan, where daytime visual surveys recorded no sharks, underscores the method's capacity to reveal species presence when traditional approaches fall short. Overall, this study presents the first genus‐specific eDNA metabarcoding assay for Hemiscyllium spp. and the first application of NADH4 as a metabarcoding marker in marine environments. Finally, this scalable assay supports Indonesia's protective legislation for Hemiscyllium and offers a transferable framework for monitoring other data‐limited, cryptic, or threatened marine taxa.
Prabowo et al. (Fri,) studied this question.