Environmental DNA (eDNA) metabarcoding, a technique for retrieving multi-species DNA from environmental samples, can detect a diverse array of marine species from filtered seawater samples. There is a growing potential to integrate eDNA alongside existing monitoring methods in order to establish or improve the assessment of species diversity. Remote island reefs are increasingly vulnerable to climate-related threats and as such there is a pressing need for efficient whole-ecosystem surveying approaches to baseline biodiversity, study assemblage changes and implement appropriate conservation protections. In this study, we investigated the utility of eDNA metabarcoding as a high-resolution, multi-trophic biomonitoring tool at the Cocos (Keeling) Islands, Australia (CKI) – a remote tropical coral reef atoll situated within the eastern Indian Ocean. Metabarcoding assays targeting the mitochondrial 16S rRNA and CO1 genes, as well as the 18S rRNA nuclear gene, were applied to 252 surface seawater samples collected from 42 sites within a 140 km² area. Our assays successfully detected a wide range of bony fish and elasmobranchs (244 taxa), crustaceans (88), molluscs (37) and echinoderms (7). Site composition varied significantly, reflecting habitat partitioning across the island ecosystem and demonstrating the localisation of eDNA signals despite extensive tidal and oceanic movements. In addition, we document putative new occurrence records for 46 taxa and compare the efficiency of our eDNA approach to visual survey techniques at CKI. Our study demonstrates the utility of a multi-marker metabarcoding approach in capturing multi-trophic biodiversity across an entire coral reef atoll and sets an important baseline for ongoing monitoring and management.

Six one-litre seawater replicates were sampled from 42 sites in and around the main Cocos (Keeling) Island atoll, Australia, in April 2017, totalling 252 samples across a 140 km² area. Each sample was individually filtered across Pall 0.2ml Supor® polyethersulfone membranes using a Pall Sentino® Microbiology pump (Pall Corporation, Port Washington, USA), within three hours of collection. DNA was extracted from the filter membranes, within two weeks of collection, using a DNeasy Blood and Tissue Kit (Qiagen; Venlo, Netherlands) with the following modifications: 540ml of ATL lysis buffer and 60ml of Proteinase K during the cell digestion phase. Previously published primers were sourced to amplify bony fish, elasmobranchs, crustaceans and other eukaryotes (including molluscs and echinoderms), respectively, from mixed environmental samples. We applied four PCR assays, herein referred to as 16S Fish, COI Elasmobranch, 16S Crustacean and 18S Universal; primer information can be sourced from Table 1 of the manuscript. Fusion-tagged amplicons were sequenced on either a 300 cycle (for unidirectional sequencing) or 500 cycle (for paired-end sequencing) MiSeq® V2 Standard Flow Cell on an Illumina MiSeq platform (Illumina, San Diego, USA), housed in the TrEnD Laboratory at Curtin University. Sequences were merged, demultiplexed and filtered using a combination of AdapterRemoval (v2; Schubert, Lindgreen, & Orlando, 2016), Geneious® (10.0.6; Kearse et al., 2012), USEARCH (v9.0.2132; Edgar, 2010) and OBITools (v1.2.9; Boyer, Mercier, Bonin, Taberlet, & Coissac, 2014); see supplementary information of manuscript for more details. We provide here the demultiplexed (unfiltered) data for each sample per assay and subsampled presence/absence matrices for each taxonomic group, i.e. fish/elasmobranchs, crustaceans, molluscs and echinoderms.