Complementary broad-range mitochondrial markers improve eDNA characterization of marine metazoan diversity

Environmental DNA (eDNA) metabarcoding leverages genetic material present in the habitat for detection of species and has great potential for rapid biodiversity assessment. Comprehensive eDNA characterization of marine metazoan biodiversity requires broad amplification of genetic markers or complementary loci to detect a wide diversity of taxa. In this study, we tested the effectiveness of the universal cytochrome c oxidase subunit I (COI) and marine vertebrate 16S ribosomal RNA markers to recover coastal marine taxa from eDNA samples collected along the urban coast of Singapore. We recovered 260 molecular operational taxonomic units (MOTUs) from a total of 12 L of water (triplicates of 1 L at each of four sites) with no overlap of MOTUs observed between the two assays despite the expectation that the COI primer set could potentially amplify a broad range of metazoans. Notably, our COI assay identified mainly invertebrates, while the 16S assay primarily recovered vertebrates. For the relatively new 16S barcoding marker, we applied two species delimitation programs on a curated, comprehensive vertebrate dataset to determine the distance threshold between intra- and interspecific comparisons (2%). Community analyses revealed that metazoan communities were distinct between sites for both assays. In addition, DNA metabarcoding was shown to be helpful for the monitoring of non-indigenous and threatened marine species, such as Mytella strigata and Aetobatus ocellatus, respectively. Our findings highlight the potential of a complementary multi-marker approach for biomonitoring invertebrates and vertebrates comprehensively across distinct habitats.