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Background: DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads. Results: We show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12–15 h of sequencing. Conclusion: Our results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications. [ABSTRACT FROM AUTHOR]

Identification of fish larvae based on morphology is typically limited to higher taxonomic ranks (e.g., family or order), as larvae possess few morphological diagnostic characters for precise discrimination to species. When many samples are presented at any one time, the use of morphology to identify such specimens can be laborious and time‐consuming. Using a reverse workflow for specimen sorting and identification leveraging high‐throughput DNA sequencing, thousands of fish larvae can be DNA barcoded and sorted into molecular operational taxonomic units (mOTUs) in a single sequencing run with the nanopore sequencing technology (e.g., MinION). This process reduces the time and financial costs of morphology‐based sorting and instead deploys experienced taxonomists for species taxonomic work where they are needed most. In this study, a total of 3022 fish larval specimens from plankton tows across four sites in Singapore were collected and sorted based on this workflow. Eye tissue from individual samples was used for DNA extraction and sequencing of cytochrome c oxidase subunit I. We generated a total of 2746 barcodes after quality filtering (90.9% barcoding success), identified 2067 DNA barcodes (75.3% identification success), and delimited 256 mOTUs (146 genera, 52 families). Our analyses identified specific challenges to species assignment, such as the potential misidentification of publicly available sequences used as reference barcodes. We highlighted how the conservative application and comparison of a local sequence database can help resolve identification conflicts. Overall, this proposed approach enables and expedites taxonomic identification of fish larvae, contributing to the enhancement of reference barcode databases and potentially better understanding of fish connectivity. [ABSTRACT FROM AUTHOR]

Gerromorpha Popov, 1971 is a fascinating and diverse insect lineage that evolved about 200 Mya to spend their entire life cycle on the air–water interface and have since colonized all types of aquatic habitats. The subfamily Halobatinae Bianchi, 1896 is particularly interesting because some species have adapted to life on the open ocean—a habitat where insects are very rarely found. Several attempts have been made to reconstruct the phylogenetic hypotheses of this subfamily, but the use of a few partial gene sequences recovered only a handful of well-supported relationships, thus limiting evolutionary inferences. Fortunately, the emergence of high-throughput sequencing technologies has enabled the recovery of more genetic markers for phylogenetic inference. We applied genome skimming to obtain mitochondrial and nuclear genes from low-coverage whole-genome sequencing of 85 specimens for reconstructing a well-supported phylogeny, with particular emphasis on Halobatinae. Our study confirmed that Metrocorini Matsuda, 1960, is paraphyletic, whereas Esakia Lundblad, 1933, and Ventidius Distant, 1910, are more closely related to Halobatini Bianchi, 1896, than Metrocoris Mayr, 1865, and Eurymetra Esaki, 1926. We also found that Ventidius is paraphyletic and in need of a taxonomic revision. Ancestral state reconstruction suggests that Halobatinae evolved progressively from limnic to coastal habitats, eventually attaining a marine lifestyle, especially in the genus Halobates Eschscholtz, 1822, where the oceanic lifestyle evolved thrice. Our results demonstrate that genome skimming is a powerful and straightforward approach to recover genetic loci for robust phylogenetic analysis in non-model insects. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Coral reefs are among the richest marine ecosystems on Earth, but there remains much diversity hidden within cavities of complex reef structures awaiting discovery. While the abundance of corals and other macroinvertebrates are known to influence the diversity of other reef‐associated organisms, much remains unknown on the drivers of cryptobenthic diversity. A combination of standardized sampling with 12 units of the Autonomous Reef Monitoring Structure (ARMS) and high‐throughput sequencing was utilized to uncover reef cryptobiome diversity across the equatorial reefs in Singapore. DNA barcoding and metabarcoding of mitochondrial cytochrome c oxidase subunit I, nuclear 18S and bacterial 16S rRNA genes revealed the taxonomic composition of the reef cryptobiome, comprising 15,356 microbial ASVs from over 50 bacterial phyla, and 971 MOTUs across 15 metazoan and 19 non‐metazoan eukaryote phyla. Environmental factors across different sites were tested for relationships with ARMS diversity. Differences among reefs in diversity patterns of metazoans and other eukaryotes, but not microbial communities, were associated with biotic (coral cover) and abiotic (distance, temperature and sediment) environmental variables. In particular, ARMS deployed at reefs with higher coral cover had greater metazoan diversity and encrusting plate cover, with larger‐sized non‐coral invertebrates influencing spatial patterns among sites. Our study showed that DNA barcoding and metabarcoding of ARMS constitute a valuable tool for quantifying cryptobenthic diversity patterns and can provide critical information for the effective management of coral reef ecosystems. [ABSTRACT FROM AUTHOR]

Accurate identification and association of larval specimens with adults is a major challenge in insect taxonomy. Fortunately, it is now possible for nonexperts to sort collections of bulk samples with DNA barcodes rapidly and cost-effectively. We demonstrate this process using nanopore barcoding of 757 marine insects (Insecta: Gerromorpha), of which 81% were nymphs and many samples did not have co-occurring adult males for specific identification. We successfully associated 738 specimens (97%) to nine gerromorphan species, which would have been impossible to identify using morphological characters alone. This improved ability to incorporate information from all life-history stages has led to greater precision of species distributional ranges—knowledge that will be crucial for a more complete understanding of marine insects. We also highlighted two distinct, nonoverlapping Gerromorpha COI sequence databases on GenBank—a consequence of using two different primer sets to amplify different regions of COI. This issue inevitably hinders species identification with DNA-based methods, particularly for poorly represented groups such as marine insects. We bridged these databases by analyzing full-length COI sequences. We believe this will inspire future studies to incorporate DNA-based methods for more adult–larval association studies and for enhancing existing genetic resources, especially in understudied groups. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The mitochondrial genetic code is much more varied than the standard genetic code. The invertebrate mitochondrial code, for instance, comprises six initiation codons, including five alternative start codons. However, only two initiation codons are known in the echinoderm and flatworm mitochondrial code, the canonical ATG and alternative GTG. Here, we analyzed 23 Asteroidea mitogenomes, including ten newly sequenced species and unambiguously identified at least two other start codons, ATT and ATC, both of which also initiate translation of mitochondrial genes in other invertebrates. These findings underscore the diversity of the genetic code and expand upon the suite of initiation codons among echinoderms to avoid erroneous annotations. Our analyses have also uncovered the remarkable conservation of gene order among asteroids, echinoids, and holothuroids, with only an interchange between two gene positions in asteroids over ∼500 Ma of echinoderm evolution. [ABSTRACT FROM AUTHOR]