Environmental DNA (eDNA) metabarcoding offers an effective solution to determine fish species compositions in communities across diverse environments. However, it is not clear how different metabarcoding primers perform in terms of recovering fish species and community diversity in the Indo-Pacific bioregion. In this study, we compared the relative performance of five metabarcoding primers (Berry 16S, Riaz 12S, MiFish E 12S, MiFish U 12S, and Leray CO1) in recovering Indo-Pacific fish taxa. We tested the primers using template DNA from three different environments: (1) a controlled mock community composed of tissue-based DNA extractions from 96 species, (2) a semi-controlled Indo-Pacific reef fish community from a public aquarium tank, and (3) a natural tropical coral reef lagoon. In the mock community sample, each primer recovered a distinct subset of the community, and no single primer recovered all taxa. Of the 65 distinct genera included in the mock community, all but six were recovered by at least one primer, representing 91% of genera. Fifty-nine of the 96 included species (61%) were identified to species level using at least one primer set. From the aquarium community, 17 of the 20 known genera were recovered (85%), and 13 out of 24 (54%) censused species were identified by at least one primer. In the coral reef lagoon, 48 genera were identified, and 47 species-level identifications were made, including 87% endemic and established species. Overall, we find Riaz 12S performed better than other fish-specific markers, although there were differences in the specific fish taxa recovered. While all markers performed well in the mock community in terms of the relative proportion of fish sequences recovered, this did not accurately predict how they would perform under natural conditions. Caution is therefore urged in using a mock community alone to evaluate metabarcoding primer performance for studies in natural environments.

ABSTRACT Variability in larval transport has long been hypothesized to drive recruitment fluctuations in fishes, yet evidence for these hypotheses is often lacking. Further, the origins of many of these hypotheses come from temperate to subpolar regions, leaving such questions largely underexplored for other regions, especially the tropics. To assess drivers of larval transport in a tropical archipelago, we simulated the dispersal of a culturally and commercially important bottomfish, uku ( Aprion virescens ), from Penguin Bank, its most prominent spawning location in the main Hawaiian islands. We used Lagrangian particle tracking models forced by a regional ocean model to assess the degree of interisland potential connectivity from this spawning location and the drivers of interannual variability in this potential connectivity across 13 years from 2008 to 2020. Simulated uku larvae released from Penguin Bank primarily reached Maui Nui and Oʻahu, the nearest potential settlement areas, with lower potential connectivity to more distant regions. Interannual variability in overall number of connections was pronounced and linked both to local wind speed and direction, with increased particle loss occurring during higher overall wind speeds and more northerly winds. Recruitment deviations from the stock assessment of uku showed a similar pattern, with recruitment estimates significantly decreasing during years of strong northerly winds. Our results provide evidence, derived from a simulation model integrating ecological and physical components, of larval dispersal patterns contributing to the recruitment of a socioeconomically important species and sensitivity of these patterns to local wind forcing. Understanding how these wind patterns will change in a warming climate may be essential to understanding patterns in uku recruitment in coming years.

Understanding connectivity between populations is key to identifying hotspots of diversity, dispersal sinks and sources, and effective management units for natural resources. Multi-species connectivity seeks to overcome species-specific idiosyncrasies to identify shared patterns that are most critical to spatial management. The linear Hawaiian archipelago provides an excellent platform to assess multi-species connectivity patterns, with shared boundaries to gene flow identified across a majority of the 41 coral reef species surveyed to date. Here, we evaluate genome-scale data by comparing consistency and resolution to previous connectivity studies using far fewer loci. We used pool-seq to genotype 22,503–232,730 single nucleotide polymorphisms per species (625,215 SNPs total) from the same individuals published in previous studies of two fishes, two corals, and two lobsters. Additionally, one coral species (Pocillopora meandrina) without previous archipelago-wide population genetic data was included. With greater statistical power, most genetic differences between pairwise comparisons of islands were significant (250 of 308), consistent with the most recent larval dispersal models for the Hawaiian Archipelago. These data reveal significant differentiation at a finer scale than previously reported using single-marker studies, yet did not overturn any of the conclusions or management implications drawn from previous studies. We confirm that population genomic datasets are consistent with previously reported patterns of multispecies connectivity but add a finer layer of population resolution that is pertinent to management.

Island communities, like the Republic of the Marshall Islands (RMI), depend on marine resources for food and economics, so plastic ingestion by those resources is a concern. The gastrointestinal tracts of nine species of reef fish across five trophic groups (97 fish) were examined for plastics >1 mm. Over 2100 putative plastic particles from 72 fish were identified under light microscopy. Only 115 of these from 47 fish passed a plastic screening method using Fourier-transform infrared microspectroscopy (μFTIR) in reflectance mode. All of these were identified as natural materials in a final confirmatory analysis, attenuated total reflectance FTIR. The high false-positive rate of visual and μFTIR methods highlight the importance of using multiple polymer identification methods. Limited studies on ingested plastic in reef fish present challenging comparisons because of different methods used. No plastic >1 mm were found in the RMI reef fish, reassuring human consumers.

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. Graphical Abstract

Despite the dramatic decrease in high-throughput sequencing costs over time, sequencing the ideal number of individuals for population genetic inference remains prohibitively expensive. When research questions require only population-level resolution, pooling individual samples before sequencing (pool-seq) can substantially reduce costs while still providing allele frequencies of Single Nucleotide Polymorphisms (SNPs). However, analyzing pooled data is comparatively difficult and less standardized than individual-based analyses. Although several programs have been developed to handle pool-seq data, most require extensive formatting or programming skills to operate. Here we introduce assessPool, an open-source R and Bash pipeline for pool-seq analyses with a focus on population structure. AssessPool accepts a Variant-Call Format (VCF) file and a FASTA-formatted reference, providing a straightforward transition from commonly used pipelines such as Stacks or dDocent. AssessPool handles varying numbers of pools and utilizes PoPoolation2 to generate locus-by-locus pairwise F ST values and associated Fisher T-test values as measures of population structure. Starting with a VCF file containing all identified SNPs, assessPool facilitates several key functionalities for population genetic analyses: i) filtering SNPs based on adjustable criteria with parameter suggestions for pool-seq data, ii) organizing data structures for analysis based on input pools, iii) creating customizable run scripts for FST calculations using PoPoolation2 and/or the {poolfstat} R package, for all pairwise comparisons, iv) calculating locus-specific F ST values using PoPoolation2 and/or {poolfstat}, v) importing F ST output into a format compatible with R, vi) producing population genomic summary statistics, and vii) generating interactive plots to visualize and explore data. A pooled dataset generated from wild populations is used here to showcase the features of the assessPool pipeline for population genomic analyses. ### Competing Interest Statement The authors have declared no competing interest.

Background Reef manta rays ( Mobula alfredi ) are globally distributed in tropical and subtropical seas. Their life history traits (slow growth, late maturity, low reproductive output) make them vulnerable to perturbations and therefore require informed management strategies. Previous studies have reported wide-spread genetic connectivity along continental shelves suggesting high gene flow along continuous habitats spanning hundreds of kilometers. However, in the Hawaiian Islands, tagging and photo-identification evidence suggest island populations are isolated despite proximity, a hypothesis that has not yet been evaluated with genetic data. Results This island-resident hypothesis was tested by analyzing whole mitogenome haplotypes and 2048 nuclear single nucleotide polymorphisms (SNPs) between M. alfredi (n = 38) on Hawaiʻi Island and Maui Nui (the 4-island complex of Maui, Molokaʻi, Lānaʻi and Kahoʻolawe). Strong divergence in the mitogenome ( Φ ST = 0.488) relative to nuclear genome-wide SNPs (neutral F ST = 0.003; outlier F ST = 0.186), and clustering of mitochondrial haplotypes among islands provides robust evidence that female reef manta rays are strongly philopatric and do not migrate between these two island groups. Combined with restricted male-mediated migration, equivalent to a single male moving between islands every 2.2 generations (~ 64 years), we provide evidence these populations are significantly demographically isolated. Estimates of contemporary effective population size ( N e ) are 104 (95% CI: 99–110) in Hawaiʻi Island and 129 (95% CI: 122–136) in Maui Nui. Conclusions Concordant with evidence from photo identification and tagging studies, these genetic results indicate reef manta rays in Hawaiʻi have small, genetically-isolated resident island populations. We hypothesize that due to the Island Mass Effect, large islands provide sufficient resources to support resident populations, thereby making crossing deep channels separating island groups unnecessary. Small effective population size, low genetic diversity, and k-selected life history traits make these isolated populations vulnerable to region-specific anthropogenic threats, which include entanglement, boat strikes, and habitat degradation. The long-term persistence of reef manta rays in the Hawaiian Islands will require island-specific management strategies.

The west coast of HawaiModified Letter Turned Commai Island hosts elevated primary production compared to offshore waters and an abundance of large pelagic animals for reasons that are not entirely understood. Here we show that the nearshore environment off the west coast of HawaiModified Letter Turned Commai exhibits an increased biomass and abundance of mesopelagic micronekton. Acoustic surveys from this study and prior work show a higher nautical area scattering coefficient (as a proxy for biomass) in a deep non-migratory layer in nearshore sites compared to offshore sites that is persistent over five years. Cobb trawl samples taken at the depths of the deep scattering layer (similar to 450 to similar to 550 m) in 2016 and 2017 showed 1.3 to 2.2 times higher biomass and 2.7 times higher abundance nearshore (similar to 4 km from shore) compared to offshore (similar to 24 km from shore). Fishes dominated the trawl catches and a large fraction of the nearshore enhancements were due to Sternoptychidae and Serrivomeridae across both years. In contrast, Melamphaeidae consistently were more abundant and had greater biomass offshore. This deep scattering assemblage contrasts with the mesopelagic boundary layer assemblage by being nonmigratory and taxonomically different in composition. These nearshore mesopelagic enhancements could occur as the result of increased nearshore food supplies deriving from Island Mass Effect enhanced primary production or from advection and concentration in a complex flow environment in the lee of the island. Regardless of mechanism, this temporally persistent, high biomass, largely nonmigratory layer of mesopelagic micronekton is different from the better known migratory mesopelagic boundary layer assemblage and may provide food to deeper diving marine mammals and pelagic fishes possibly explaining the aggregation of large pelagic animals in this region.

We provide the complete mitochondrial genome of the reef manta ray, Mobula alfredi, using an ezRAD approach. The total length of the mitogenome was 18,166 bp and contained 13 protein-coding genes, 22 transfer RNAs genes, two ribosomal RNA genes, and one non-coding control region. The gene organization and length are similar to other Mobula species. This reference mitogenome that includes the control region is expected to be a valuable resource for molecular-based species identification, population genomics, and phylogeography.

The Republic of the Marshall Islands (RMI) has been affected by marine pollution from militarization and urbanization. To address concerns raised by the Marshall Islands Marine Resources Authority, this study examined concentrations of dissolved contaminants in reef and pelagic fishes in the RMI and assessed potential associated risks. Metals, organochlorine pesticides, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) were examined in reef and pelagic fishes from six atolls: Kwajalein, Majuro, Jaluit, Utirik, Rongelap, and Wotje. Clear trophic patterns emerged for metals. Total arsenic was highest in higher trophic level reef fishes, particularly in the camouflage grouper (Epinephelus polyphekadion) (>100 μg g-1 total As), but inorganic arsenic was negligible in higher trophic levels and showed an inverse trend with the highest percentages present in parrotfishes and herbivores. Copper and mercury were elevated in higher trophic level reef and pelagic fishes, respectively, and the maximum mercury concentrations (6.45 μg g-1 in Gymnosarda unicolor) were among the highest reported in the Pacific. Conversely, cadmium and lead were highest in lower trophic levels, like surgeonfishes and parrotfishes. PCBs were more clearly linked to locations and were highest at two atolls with military history (Kwajalein and Jaluit) (>U.S. EPA Screening Value of 2.5 ppb). PAHs were ubiquitous across taxa (detected in 97% of samples), but the highest concentrations were in lower trophic levels. Organochlorine pesticides were detected at very low concentrations that do not likely pose a risk. We compare concentrations to established thresholds for human health and find that - for specific locations and species - contaminant concentrations may pose a risk to fish and other marine taxa, as well as human consumers. This study provides baseline information that aids the development of marine conservation and public health recommendations and addresses a data gap that persists for marine pollution throughout the Pacific Islands.

DNA barcoding is critical to conservation and biodiversity research, yet public reference databases are incomplete. Existing barcode databases are biased toward cytochrome oxidase subunit I (COI) and frequently lack associated voucher specimens or geospatial metadata, which can hinder reliable species assignments. The emergence of metabarcoding approaches such as environmental DNA (eDNA) has necessitated multiple marker techniques combined with barcode reference databases backed by voucher specimens. Reference barcodes have traditionally been generated by Sanger sequencing, however sequencing multiple markers is costly for large numbers of specimens, requires multiple separate PCR reactions, and limits resulting sequences to targeted regions. High-throughput sequencing techniques such as genome skimming enable assembly of complete mitogenomes, which contain the most commonly used barcoding loci (e.g., COI, 12S, 16S), as well as nuclear ribosomal repeat regions (e.g., ITS1&2, 18S). We evaluated the feasibility of genome skimming to generate barcode references databases for marine fishes by assembling complete mitogenomes and nuclear ribosomal repeats. We tested genome skimming across a taxonomically diverse selection of 12 marine fish species from the collections of the National Museum of Natural History, Smithsonian Institution. We generated two sequencing libraries per species to test the impact of shearing method (enzymatic or mechanical), extraction method (kit-based or automated), and input DNA concentration. We produced complete mitogenomes for all non-chondrichthyans (11/12 species) and assembled nuclear ribosomal repeats (18S-ITS1-5.8S-ITS2-28S) for all taxa. The quality and completeness of mitogenome assemblies was not impacted by shearing method, extraction method or input DNA concentration. Our results reaffirm that genome skimming is an efficient and (at scale) cost-effective method to generate all mitochondrial and common nuclear DNA barcoding loci for multiple species simultaneously, which has great potential to scale for future projects and facilitate completing barcode reference databases for marine fishes.

Estimating stomatopod species diversity using morphology alone has long been difficult; though over 450 species have been described, new species are still being discovered regularly despite the cryptic behaviors of adults. However, the larvae of stomatopods are more easily obtained due to their pelagic habitat, and have been the focus of recent studies of diversity. Studies of morphological diversity describe both conserved and divergent traits in larval stomatopods, but generally cannot be linked to a particular species. Conversely, genetic studies of stomatopod larvae using DNA barcoding can be used to estimate species diversity, but are generally not linked to known species by analyses of morphological characters. Here we combine these two approaches, larval morphology and genetics, to estimate stomatopod species diversity in the Hawaiian Islands. Over 22 operational taxonomic units (OTUs) were identified genetically, corresponding to 20 characterized morphological types. Species from three major superfamilies of stomatopod were identified: Squilloidea (4 OTUs, 3 morphotypes), Gonodactyloidea (9, 8), and Lysiosquilloidea (6, 7). Among these, lysiosquilloids were more diverse based on larval morphotypes and OTUs as compared to previously documented Hawaiian species (3), while squilloids had a lower diversity of species represented by collected larvae as compared to the seven species previously documented. Two OTUs / morphotypes could not be identified to superfamily as their molecular and morphological features did not closely match any available information, suggesting they belong to poorly sampled superfamilies. The pseudosquillid, Pseudosquillana richeri, was discovered for the first time from Hawai'i. This study contributes an updated estimate for Hawaiian stomatopod diversity for a total of 24 documented species, provides references for identification of larval stomatopods across the three major superfamilies, and emphasizes the lack of knowledge of species diversity in more cryptic stomatopod superfamilies, such as Lysiosquilloidea.

Preassembly of Gymnothorax fimbriatus (UNSM 395396) based on SRA data (SRR14433904), derived using SPAdes 3.15.2. All contigs less than 200 bp removed.

Lines of smooth ocean surface water known as "slicks" appear in coastal waters worldwide and can be caused by a number of natural mechanisms. Off the western coast of the island of Hawai'i, USA, internal waves, submarine groundwater discharge, tidal fronts, and headland fronts were all observed to cause the formation of slicks. Neuston net sampling showed slicks to contain enhanced densities of phytoplankton, zooplankton, larval invertebrates, larval fish, and plastic fragments when compared to ambient water. In particular, internal wave slicks were observed to be both the most frequently occurring type of slick and the overall strongest accumulator of neustonic organisms and plastics when compared to the other types of slicks. Satellite imagery illustrated that slicks are visible along most of the West Hawai'i coastline more than 75% of the time. Furthermore, acoustic backscatter observations showed that the accumulation associated with slicks frequently extended from the surface to depths of 5 m. These findings highlight the important roles internal wave slicks can have in coastal ecosystems.

Most marine animals have a pelagic larval phase that develops in the coastal or open ocean. The fate of larvae has profound effects on replenishment of marine populations that are critical for human and ecosystem health. Larval ecology is expected to be tightly coupled to oceanic features, but for most taxa we know little about the interactions between larvae and the pelagic environment. Here, we provide evidence that surface slicks, a common coastal convergence feature, provide nursery habitat for diverse marine larvae, including > 100 species of commercially and ecologically important fishes. The vast majority of invertebrate and larval fish taxa sampled had mean densities 2–110 times higher in slicks than in ambient water. Combining in-situ surveys with remote sensing, we estimate that slicks contain 39% of neustonic larval fishes, 26% of surface-dwelling zooplankton (prey), and 75% of floating organic debris (shelter) in our 1000 km 2 study area in Hawai‘i. Results indicate late-larval fishes actively select slick habitats to capitalize on concentrations of diverse prey and shelter. By providing these survival advantages, surface slicks enhance larval supply and replenishment of adult populations from coral reef, epipelagic, and deep-water ecosystems. Our findings suggest that slicks play a critically important role in enhancing productivity in tropical marine ecosystems.

Preassembly of Forcipiger flavissimus (USNM 411089) based on SRA data (SRR14433901), derived using SPAdes 3.15.2. All contigs less than 200 bp removed.

Preassembly of Tylosurus crocodilus (USNM 442362) based on SRA data (SRR14433895), derived using SPAdes 3.15.2. All contigs less than 200 bp removed.

While ocean sunfishes hold the vertebrate record for having the most ova in a single female, information on the natural history of their larvae and juveniles has yet to be collated. This chapter includes the most comprehensive compilation of larval molid records to date, and places the records within a global spatial context. Gathered from natural history museum collections, modern ichthyoplankton sampling efforts and historical records dating back to the 1800s, a total of 452 records were collected comprising 9,770 larvae in total: 285 Masturus, 84 Mola spp., 61 unspecified Molidae and 9,340 Ranzania (340 of which were eggs). Ranzania was primarily reported from the Sargasso Sea, Northern Brazil, Hawai'i and southern Polynesia. Masturus, were reported from the Sargasso Sea and at scattered locations across both Atlantic and Pacific Ocean basins. Lastly, for Mola spp., records existed for Eastern Australia and the Eastern USA. Larval stages of early development for each molid genera are identified, described, and illustrated with original drawings. Larval records also include larvae extracted from the guts of 26 different predator species: 17 large pelagic piscivorous fishes from six families, and nine seabirds species from five families. While this chapter cannot be considered an exhaustive compilation of all molid larval knowledge, it does provide a foundation on which to build a more extensive molid larval database. It also underscores the importance of collecting hydrographic metadata to accompany each record. We hope that future studies can use this review to help locate additional spawning sites and achieve the critical mass of data required for habitat suitability modelling. Such a vantage point would allow an assessment of how spawning sites may be impacted by ongoing environmental and anthropogenic changes and inform management plans.