Since the release of the MinION sequencer in 2014, it has been applied to great effect in the remotest and harshest of environments, and even in space. One of the most common applications of MinION is for nanopore-based DNA barcoding in situ for species identification and discovery, yet the existing sample capability is limited (n ≤ 10). Here, we assembled a portable sequencing setup comprising the BentoLab and MinION and developed a workflow capable of processing 32 samples simultaneously. We demonstrated this enhanced capability out at sea, where we collected samples and barcoded them onboard a dive vessel moored off Sisters’ Islands Marine Park, Singapore. In under 9 h, we generated 105 MinION barcodes, of which 19 belonged to fresh metazoans processed immediately after collection. Our setup is thus viable and would greatly fortify existing portable DNA barcoding capabilities. We also tested the performance of the newly released R10.3 nanopore flow cell for DNA barcoding, and showed that the barcodes generated were ~99.9% accurate when compared to Illumina references. A total of 80% of the R10.3 nanopore barcodes also had zero base ambiguities, compared to 50–60% for R9.4.1, suggesting an improved homopolymer resolution and making the use of R10.3 highly recommended.

Synchronous multispecific coral spawning generally occurs annually and forms an integral part of the coral life cycle. Apart from spawning times and species participation, however, much else remains unknown. Here, we applied environmental DNA (eDNA) metabarcoding to study two tropical reef sites of contrasting coral cover before, during and after coral spawning. Using coral‐ITS2 and vertebrate‐12S markers, we evaluated eDNA as an alternative monitoring tool by assessing its capabilities in detecting spawning species and tracking relative abundances of coral and fish eDNA. Over 3 years, elevated eDNA coral signals during the event (proportional read increase of up to five‐fold) were observed, detecting a total of 38 coral and 133 fish species with all but one of the coral species visually observed to be spawning. This is also the first demonstration that eDNA metabarcoding can be used to infer the diurnal partitioning of night‐ and day‐time spawning, spawning in coral species overlooked by visual surveys, and the associated changes in fish trophic structures as an indicator of spawning events. Our study paves the way for applied quantitative eDNA metabarcoding approaches to better study ephemeral and important biological events.

Background: Sharks and rays are some of the most threatened marine taxa due to the high levels of bycatch and significant demand for meat and fin-related products in many Asian communities. At least 25% of shark and ray species are considered to be threatened with extinction. In particular, the density of reef sharks in the Pacific has declined to 3–10% of pre-human levels. Elasmobranchs are thought to be sparse in highly urbanised and turbid environments. Low visibility coupled with the highly elusive behaviour of sharks and rays pose a challenge to diversity estimation and biomonitoring efforts as sightings are limited to chance encounters or from carcasses ensnared in nets. Here we utilised an eDNA metabarcoding approach to enhance the precision of elasmobranch diversity estimates in urbanised marine environments.Results: We applied eDNA metabarcoding on seawater samples to detect elasmobranch species in the hyper-urbanised waters off Singapore. Two genes—vertebrate 12S and elasmobranch COI—were targeted and amplicons subjected to Illumina high-throughput sequencing. With a total of 84 water samples collected from nine localities, we found 47 shark and ray molecular operational taxonomic units, of which 16 had species-level identities. When data were compared against historical collections and contemporary sightings, eDNA detected 14 locally known species as well as two potential new records.Conclusions: Local elasmobranch richness uncovered by eDNA is greater than the seven species sighted over the last two decades, thereby reducing phantom diversity. Our findings demonstrate that eDNA metabarcoding is effective in detecting shark and ray species despite the challenges posed by the physical environment, granting a more consistent approach to monitor these highly elusive and threatened species.

Environmental DNA (eDNA) with metabarcoding or metagenomics will likely become a major biomonitoring tool in the 21st century, perhaps even more so in the face of increased coastal urbanization and its associated effects such as pollution, land reclamation, and seabed dredging. Together, these impacts and the consequent high turbidity pose severe challenges to traditional survey techniques that rely heavily on visual observations. We here demonstrate that eDNA can be used for biomonitoring in turbid waters, using Singapore as a case study. The cytochrome c oxidase subunit I (COI) locus was used to detect 525 metazoan molecular operational taxonomic units (MOTUs) based on eDNA obtained from 52 L of filtered seawater. Of these, 130 MOTUs (24.7%) were identifiable to species, including ecologically significant species that in some cases were invasive or rarely observed. Metazoan signals also enabled discrimination of discrete, but connected, environments from intertidal and subtidal zones. Taxa with known habitat preferences were found to have left trace eDNA at sites that matched putatively suitable habitats. Moreover, no coast‐specific signals were detected in open water samples, which suggest that intermixing of water was limited. The study confirms that eDNA metabarcoding is a viable biomonitoring tool for coastal areas with highly sedimented waters.