Recent years have seen an increase in the number of studies that use DNA sequence information in addition to morphological methods, as the latter alone can be inadequate for morphologically similar, cryptic species. Marine onchidiid slugs (Mollusca: Gastropoda: Onchidiidae), which commonly inhabit intertidal environments and have a near-global distribution, comprise 11 genera and 86 described species. Singapore has 19 recorded species of onchidiids with Peronia verruculata (Cuvier, 1830) being the most abundant. Here we present mitochondrial DNA sequence data from 63 P. verruculata-like specimens to show that they constitute two closely-related clades with distinct sequence signatures and a clear barcode gap. Intraclade distances are 0–1.8% while interclade distances range from 4.7 to 6.0%. Scanning electron microscopy of internal copulation organs reveals that the two clades have distinct penial and accessory gland spine structures, which also differ in size. Using an integrative taxonomic approach, we propose that what was initially thought to be P. verruculata in Singapore, actually are two different species of Peronia.

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.

Autonomous Reef Monitoring Structure (ARMS) are standardized devices for sampling biodiversity in complex marine benthic habitats such as coral reefs. When coupled with DNA sequencing, these devices greatly expand our ability to document marine biodiversity. Unfortunately, the existing workflow for processing macrofaunal samples (>2-mm) in the ARMS pipeline—which involves Sanger sequencing—is expensive, laborious, and thus prohibitive for ARMS researchers. Here, we propose a faster, more cost-effective alternative by demonstrating a successful application of the MinION-based barcoding approach on the >2 mm-size fraction of ARMS samples. All data were available within 3.5–4 h, and sequencing costs relatively low at approximately US$3 per MinION barcode. We sequenced the 313-bp fragment of the cytochrome c oxidase subunit I (COI) for 725 samples on both MinION and Illumina platforms, and retrieved 507–584 overlapping barcodes. MinION barcodes were highly accurate (∼99.9%) when compared with Illumina reference barcodes. Molecular operational taxonomic units inferred between MinION and Illumina barcodes were consistently stable, and match ratios demonstrated highly congruent clustering patterns (≥0.96). Our method would make ARMS more accessible to researchers, and greatly expedite the processing of macrofaunal samples; it can also be easily applied to other small-to-moderate DNA barcoding projects (<10,000 specimens) for rapid species identification and discovery.

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.