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.