Context. DNA recovered from predator faeces (scats) can be used to determine the presence of fauna and shed light on their life histories and inter-species interactions. DNA metabarcoding, which involves concurrent amplification and sequencing of DNA from multiple taxa, represents an important advance by enabling the simultaneous detection of multiple species from such samples. Although an attractive proposition, metabarcoding requires ‘universally’ applicable genetic markers that can discriminate among a broad range of taxa, while also targeting sequences that are sufficiently short to be amplified from degraded DNA.

Aims. To identify, evaluate, and test metabarcoding DNA markers suitable for the detection of marsupials and other Australian fauna from terrestrial predator scats found in nature.

Methods. We apply a bioinformatic approach using publicly available DNA databases and a locally derived and marker-specific reference-DNA database to evaluate the diagnostic ability and likelihood of amplification of candidate metabarcoding markers for marsupials and other taxa that may be consumed by predators.

Key results. We identify two markers (12SV5 and 16SMam) that are suitable for use and successfully identify marsupial sequences at a high level of resolution. These markers work best in combination because they bring complementary levels of primer specificity and diagnostic ability in detecting multiple prey species as well as the predator. We also show that these samples work well in predator scats sampled from the wild in Tasmania.

Conclusions. These markers provide a useful tool for surveying mammalian predators and their prey and could also be applied to eDNA analyses from other sample types. Improvements to the reference database and further development of markers targeting different taxonomic groups will improve the resolution and usefulness of this approach.

Implications. Metabarcoding of predator scats provides a potent approach to non-invasive wildlife survey that offers the opportunity for the detection of multiple species across all vertebrates.