Search

Next-generation sequencing technologies, such as Nanopore MinION, Illumina Hiseq and Novaseq, and PacBio Sequel II, hold immense potential for advancing genomic research on non-model organisms, including the vast majority of marine species. However, application of these technologies to marine invertebrate species is often impeded by challenges in extracting and purifying their genomic DNA due to high polysaccharide content and other secondary metabolites. In this study, we help resolve this issue by developing and testing DNA extraction protocols for Kellet's whelk ( Kelletia kelletii ), a subtidal gastropod with ecological and commercial importance, by comparing four DNA extraction methods commonly used in marine invertebrate studies. In our comparison of extraction methods, the Salting Out protocol was the least expensive, produced the highest DNA yields, produced consistent high DNA quality, and had low toxicity. We validated the protocol using an independent set of tissue samples, then applied it to extract high-molecular-weight (HMW) DNA from over three thousand Kellet's whelk tissue samples. The protocol demonstrated scalability and, with added clean-up, suitability for RAD-seq, GT-seq, as well as whole genome sequencing using both long read (ONT MinION) and short read (Illumina NovaSeq) sequencing platforms. Our findings offer a robust and versatile DNA extraction and clean-up protocol for supporting genomic research on non-model marine organisms, to help mediate the under-representation of invertebrates in genomic studies., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ., (©2023 Daniels et al.)

Coral reefs are declining worldwide primarily because of bleaching and subsequent mortality resulting from thermal stress. Currently, extensive efforts to engage in more holistic research and restoration endeavors have considerably expanded the techniques applied to examine coral samples. Despite such advances, coral bleaching and restoration studies are often conducted within a specific disciplinary focus, where specimens are collected, preserved, and archived in ways that are not always conducive to further downstream analyses by specialists in other disciplines. This approach may prevent the full utilization of unexpended specimens, leading to siloed research, duplicative efforts, unnecessary loss of additional corals to research endeavors, and overall increased costs. A recent US National Science Foundation-sponsored workshop set out to consolidate our collective knowledge across the disciplines of Omics, Physiology, and Microscopy and Imaging regarding the methods used for coral sample collection, preservation, and archiving. Here, we highlight knowledge gaps and propose some simple steps for collecting, preserving, and archiving coral-bleaching specimens that can increase the impact of individual coral bleaching and restoration studies, as well as foster additional analyses and future discoveries through collaboration. Rapid freezing of samples in liquid nitrogen or placing at -80 °C to -20 °C is optimal for most Omics and Physiology studies with a few exceptions; however, freezing samples removes the potential for many Microscopy and Imaging-based analyses due to the alteration of tissue integrity during freezing. For Microscopy and Imaging, samples are best stored in aldehydes. The use of sterile gloves and receptacles during collection supports the downstream analysis of host-associated bacterial and viral communities which are particularly germane to disease and restoration efforts. Across all disciplines, the use of aseptic techniques during collection, preservation, and archiving maximizes the research potential of coral specimens and allows for the greatest number of possible downstream analyses., Competing Interests: Robert J. Toonen is a PeerJ Section Editor. The authors declare that they have no competing interests.

The global decline of coral reefs has driven considerable interest in active coral restoration. Despite their importance and dominance on mature reefs, relatively few coral restoration projects use slower growth forms like massive and encrusting coral species. Micro-fragmentation can increase coral cover by orders of magnitude faster than natural growth, which now allows cultivation of slow growing massive forms and shows promise and flexibility for active reef restoration. However, the major causes of variation in growth and survival of outplanted colonies remain poorly understood. Here, we report simple outplanting assays to aid in active reef restoration of slower growing species and increase the likelihood of restoration success. We used two different micro-fragmentation assays. Pyramid assays were used to examine variation associated with fragment size (ranging from ≈1-9 cm 2 ), nursery residence time (for both in-situ and ex-situ nurseries), and 2D vs . 3D measurements of growth. Block assays were used to examine spatial variation among individual performance at outplanting sites in the field. We found 2D and 3D measurements correlated well, so measured survivorship and growth using top-down planar images for two of the main Hawaiian reef building corals, the plating Montipora capitata and the massive Porites compressa . Pyramid assays housed and outplanted from the in-situ nursery showed no effect of residence time or size on overall survivorship or growth for either species. Results from the ex-situ nursery, however, varied by species, with P. compressa again showing no effect of nursery residence time or size on survivorship or growth. In contrast, nursery culture resulted in improved survivorship of small M. capitata fragments, but net growth showed a weak positive effect of nursery time for medium fragments. Small fragments still suffered higher mortality than either medium or large fragments. Due to their lower mortality, medium fragments (≈3 cm 2 ) appear to be the best compromise between growth and survivorship for outplanting. Likewise, given weak positive gains relative to the investment, our results suggest that it could be more cost-effective to simply outplant medium fragments as soon as possible, without intermediate culture in a nursery. Furthermore, the block assay revealed significant differences in survivorship and growth among sites for individuals of both species, emphasizing the importance of considering spatial variation in coral survival and growth following outplanting. These results highlight the value of using short-term micro-fragmentation assays prior to outplanting to assess size, and location specific performance, optimizing the efficiency of active reef restoration activities and maximizing the probability of success for active coral restoration projects., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ and specifically the special issue “The State of Active Coral Reef Conservation and Restoration”., (© 2022 Knapp et al.)

Elevated seawater temperatures associated with climate change lead to coral bleaching. While the ultimate causes of bleaching are well understood, the proximate physiological mechanisms underlying the bleaching response are not as well defined. Here we measured nitric oxide synthase activity, oxidative stress, and cell death in algal symbionts (Symbiodinaceae) freshly isolated from the reef-building coral Pocillopora acuta collected in the field under natural non-bleaching conditions and from corals experimentally exposed to elevated temperatures. Nitric oxide synthase activity in the algal symbionts was >3 orders of magnitude higher than that of the host and increased dramatically with increasing temperature and time of exposure (up to 72 h), consistent with the onset of bleaching for these corals. Oxidative stress and cell death among the algal symbionts were highest in coral holobionts exposed to intermediate as opposed to maximal temperatures, suggesting that these mechanisms are not proximal triggers for bleaching in this species. Our results point to nitric oxide production by the algal symbionts, rather than symbiont dysfunction, as a more important driver of coral bleaching under acute thermal stress in this coral., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ., (© 2022 Jury et al.)

The drastic decline in coral coverage has stimulated an interest in reef restoration, and various iterations of coral nurseries have been used to augment restoration strategies. Here we examine the growth of two species of Hawaiian Montipora that were maintained in mesocosms under either ambient or warmed annual bleaching conditions for two consecutive years prior to outplanting to determine whether preconditioning aided coral restoration efforts. Using coral trees to create a nearby ocean nursery, we examined whether: (1) previous ex situ mesocosm growth would mirror in situ coral tree nursery growth; and (2) thermal ex situ stress-hardening would predict future success during natural warming events in situ for corals moved from tanks to trees. For Montipora capitata , we found that variation in growth was explained primarily by genotype; growth rates in the mesocosms were similar to those in situ , irrespective of preconditioning. Variation in M. flabellata growth, however, was explained by both genotype and culture method such that an individual M. flabellata colony that grew well in the tanks did not necessarily perform as well on the coral trees. For both species, previous exposure to elevated temperatures in the mesocosms provided no benefit to either growth or survival during a warming event in the coral tree nursery compared to those grown in ambient temperatures. Overall, M. capitata performed better in the tree nursery with higher net growth, lower mortality, and was subject to less predation than M. flabellata . Our results show little benefit of the additional cost and time of stress-hardening these corals prior to outplanting because it is unlikely to aid resilience to future warming events. These results also suggest that selecting corals for restoration based on long-term genotype growth performance may be more effective for optimal outcomes but should be weighed against other factors, such as coral morphology, in situ nursery method, location, and other characteristics., Competing Interests: Coauthor Robert J. Toonen is an editor for PeerJ., (© 2022 Henley et al.)

Background: Understanding region-wide patterns of larval connectivity and gene flow is crucial for managing and conserving marine biodiversity. Dongsha Atoll National Park (DANP), located in the northern South China Sea (SCS), was established in 2007 to study and conserve this diverse and remote coral atoll. However, the role of Dongsha Atoll in connectivity throughout the SCS is seldom studied. In this study, we aim to evaluate the role of DANP in conserving regional marine biodiversity., Methods: In total, 206 samples across nine marine species were collected and sequenced from Dongsha Atoll, and these data were combined with available sequence data from each of these nine species archived in the Genomic Observatories Metadatabase (GEOME). Together, these data provide the most extensive population genetic analysis of a single marine protected area. We evaluate metapopulation structure for each species by using a coalescent sampler, selecting among panmixia, stepping-stone, and island models of connectivity in a likelihood-based framework. We then completed a heuristic graph theoretical analysis based on maximum dispersal distance to get a sense of Dongsha's centrality within the SCS., Results: Our dataset yielded 111 unique haplotypes across all taxa at DANP, 58% of which were not sampled elsewhere. Analysis of metapopulation structure showed that five out of nine species have strong regional connectivity across the SCS such that their gene pools are effectively panmictic (mean pelagic larval duration (PLD) = 78 days, sd = 60 days); while four species have stepping-stone metapopulation structure, indicating that larvae are exchanged primarily between nearby populations (mean PLD = 37 days, sd = 15 days). For all but one species, Dongsha was ranked within the top 15 out of 115 large reefs in the South China Sea for betweenness centrality. Thus, for most species, Dongsha Atoll provides an essential link for maintaining stepping-stone gene flow across the SCS., Conclusions: This multispecies study provides the most comprehensive examination of the role of Dongsha Atoll in marine connectivity in the South China Sea to date. Combining new and existing population genetic data for nine coral reef species in the region with a graph theoretical analysis, this study provides evidence that Dongsha Atoll is an important hub for sustaining connectivity for the majority of coral-reef species in the region., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ., (© 2021 Liu et al.)

Phylogenomic studies can provide insights into speciation, adaptation, and extinction, while providing a roadmap for conservation. Hawaiian tree snails are a model system for an adaptive radiation facing an extinction crisis. In the last 5 years, nearly all populations of Hawaiian tree snails across the 30 remaining species in the subfamily Achatinellinae (Achatinellidae) have declined from hundreds or thousands in the wild down to undetectable levels. Nearly 100 species historically occurred across dramatic environmental gradients on five of the Hawaiian Islands, but habitat loss, overcollection, and predation by invasive species have decimated populations. As such, this system offers the opportunity to integrate efforts to conserve evolutionary potential into conservation planning for a rapidly declining subfamily. Here, we used genome-wide, restriction-site associated DNA sequencing (RADseq), along with mitochondrial genome reconstruction, to resolve evolutionary relationships to inform conservation efforts. Phylogenetic analysis of nearly 400k genome-wide SNPs from 59 populations and 25 species across six genera in the family Achatinellidae, was generally concordant with taxonomy, geography, and mtDNA with several notable exceptions; mtDNA was unable to resolve some deeper nodes (e.g., the monophyly of Achatinella ), while SNP data did not resolve as many shallow nodes. Both phylogenetic and coalescent analysis revealed deep divergences between populations within Achatinella mustelina that were consistent with species-level differences. Given cryptic species-level divergence within populations that are geographically proximate, they are at higher risk of extirpation from invasive predators and climate change than previously assumed. This study clarifies evolutionary relationships within this model system for adaptive radiation, forming the basis for conservation strategies such as translocation, captive rearing, and hybridization trials to prevent the loss of capacity to adapt to rapidly changing environmental conditions., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ., (© 2021 Price et al.)

Conservation genetic approaches for elasmobranchs have focused on regions of the mitochondrial genome or a handful of nuclear microsatellites. High-throughput sequencing offers a powerful alternative for examining population structure using many loci distributed across the nuclear and mitochondrial genomes. These single nucleotide polymorphisms are expected to provide finer scale and more accurate population level data; however, there have been few genomic studies applied to elasmobranch species. The desire to apply next-generation sequencing approaches is often tempered by the costs, which can be offset by pooling specimens prior to sequencing (pool-seq). In this study, we assess the utility of pool-seq by applying this method to the same individual silky sharks, Carcharhinus falciformis , previously surveyed with the mtDNA control region in the Atlantic and Indian Oceans. Pool-seq methods were able to recover the entire mitochondrial genome as well as thousands of nuclear markers. This volume of sequence data enabled the detection of population structure between regions of the Atlantic Ocean populations, undetected in the previous study (inter-Atlantic mitochondrial SNPs F ST values comparison ranging from 0.029 to 0.135 and nuclear SNPs from 0.015 to 0.025). Our results reinforce the conclusion that sampling the mitochondrial control region alone may fail to detect fine-scale population structure, and additional sampling across the genome may increase resolution for some species. Additionally, this study shows that the costs of analyzing 4,988 loci using pool-seq methods are equivalent to the standard Sanger-sequenced markers and become less expensive when large numbers of individuals (>300) are analyzed., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ., (© 2020 Kraft et al.)

Atlantia is described as a new genus pertaining to the family Dendrophylliidae (Anthozoa, Scleractinia) based on specimens from Cape Verde, eastern Atlantic. This taxon was first recognized as Enallopsammia micranthus and later described as a new species, Tubastraea caboverdiana , which then changed the status of the genus Tubastraea as native to the Atlantic Ocean. Here, based on morphological and molecular analyses, we compare fresh material of T. caboverdiana to other dendrophylliid genera and describe it as a new genus named Atlantia in order to better accommodate this species. Evolutionary reconstruction based on two mitochondrial and one nuclear marker for 67 dendrophylliids and one poritid species recovered A. caboverdiana as an isolated clade not related to Tubastraea and more closely related to Dendrophyllia cornigera and Leptopsammia pruvoti . Atlantia differs from Tubastraea by having a phaceloid to dendroid growth form with new corallites budding at an acute angle from the theca of a parent corallite. The genus also has normally arranged septa (not Portualès Plan), poorly developed columella, and a shallow-water distribution all supporting the classification as a new genus. Our results corroborate the monophyly of the genus Tubastraea and reiterate the Atlantic non-indigenous status for the genus. In the light of the results presented herein, we recommend an extensive review of shallow-water dendrophylliids from the Eastern Atlantic., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ., (©2020 Capel et al.)

We examined genetic structure in the lobe coral Porites lobata among pairs of highly variable and high-stress nearshore sites and adjacent less variable and less impacted offshore sites on the islands of Oahu and Maui, Hawaii. Using an analysis of molecular variance framework, we tested whether populations were more structured by geographic distance or environmental extremes. The genetic patterns we observed followed isolation by environment, where nearshore and adjacent offshore populations showed significant genetic structure at both locations (AMOVA F ST = 0.04∼0.19, P  < 0.001), but no significant isolation by distance between islands. Strikingly, corals from the two nearshore sites with higher levels of environmental stressors on different islands over 100 km apart with similar environmentally stressful conditions were genetically closer ( F ST = 0.0, P = 0.73) than those within a single location less than 2 km apart ( F ST = 0.04∼0.08, P  < 0.01). In contrast, a third site with a less impacted nearshore site (i.e., less pronounced environmental gradient) showed no significant structure from the offshore comparison. Our results show much stronger support for environment than distance separating these populations. Our finding suggests that ecological boundaries from human impacts may play a role in forming genetic structure in the coastal environment, and that genetic divergence in the absence of geographical barriers to gene flow might be explained by selective pressure across contrasting habitats., Competing Interests: Robert J. Toonen is an Academic Editor and Section Editor for Aquatic Biology for PeerJ., (©2020 Tisthammer et al.)

Seabirds in the order of Procellariiformes have one of the highest proportions of threatened species of any avian order. Species undergoing recovery may be predicted to have a genetic signature of a bottleneck, low genetic diversity, or higher rates of inbreeding. The Hawaiian Band-rumped Storm Petrel ('Akē'akē; Hydrobates castro ), a long-lived philopatric seabird, suffered massive population declines resulting in its listing under the Endangered Species Act in 2016 as federally Endangered. We used high-throughput sequencing to assess patterns of genetic diversity and potential for inbreeding in remaining populations in the Hawaiian Islands. We compared a total of 24 individuals, including both historical and modern samples, collected from breeding colonies or downed individuals found on the islands of Kaua'i, O'ahu, Maui, and the Big Island of Hawai'i. Genetic analyses revealed little differentiation between breeding colonies on Kaua'i and the Big Island colonies. Although small sample sizes limit inferences regarding other island colonies, downed individuals from O'ahu and Maui did not assign to known breeding colonies, suggesting the existence of an additional distinct breeding population. The maintenance of genetic diversity in future generations is an important consideration for conservation management. This study provides a baseline of population structure for the remaining nesting colonies that could inform potential translocations of the Endangered H. castro ., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ., (© 2020 Antaky et al.)

Octopus oliveri is a widespread and common rocky intertidal cephalopod that mates readily in the laboratory, but for which mating behavior has not been reported previously. Four sets of behavioral experiments were recorded wherein three males, small, medium & large in varying order, were introduced to each of six females, for a total of 24 individual females and 12 individual males utilized in the experiments. Video analysis shows that successful mating occurred in each of the mount, reach and beak-to-beak positions. Mating was observed for all males, regardless of size relative to the female, or order of introduction. Females showed preference for the first male to which they were introduced in experimental pairings rather than any specific male trait, and mating time increased significantly with increasing female size. Five novel microsatellite markers were developed and used to test paternity in the eleven broods resulting from these experimental pairings. We found skewed paternity in each brood, with early male precedence and male size being the best predictors of parentage. Multiple paternity was observed in every experimental cross but was estimated to be comparatively low in the field, suggesting that sperm limitation might be common in this species. We saw no evidence of direct sperm competition in Octopus oliveri , but larger males produced significantly more offspring. This study contributes to the growing research on cephalopod mating systems and indicates that octopus mating dynamics might be more variable and complex than thought previously., Competing Interests: Robert J. Toonen is an Academic Editor and the Section Editor for Aquatic Biology at PeerJ.

The seagrass, Zostera capensis , occurs across a broad stretch of coastline and wide environmental gradients in estuaries and sheltered bays in southern and eastern Africa. Throughout its distribution, habitats are highly threatened and poorly protected, increasing the urgency of assessing the genomic variability of this keystone species. A pooled genomic approach was employed to obtain SNP data and examine neutral genomic variation and to identify potential outlier loci to assess differentiation across 12 populations across the ∼9,600 km distribution of Z. capensis . Results indicate high clonality and low genomic diversity within meadows, which combined with poor protection throughout its range, increases the vulnerability of this seagrass to further declines or local extinction. Shared variation at outlier loci potentially indicates local adaptation to temperature and precipitation gradients, with Isolation-by-Environment significantly contributing towards shaping spatial variation in Z. capensis . Our results indicate the presence of two population clusters, broadly corresponding to populations on the west and east coasts, with the two lineages shaped only by frequency differences of outlier loci. Notably, ensemble modelling of suitable seagrass habitat provides evidence that the clusters are linked to historical climate refugia around the Last Glacial Maxi-mum. Our work suggests a complex evolutionary history of Z. capensis in southern and eastern Africa that will require more effective protection in order to safeguard this important ecosystem engineer into the future., Competing Interests: Robert Toonen is an Academic Editor for PeerJ.

Neopetrosia proxima (Porifera: Demospongiae: Haplosclerida) is described as a morphologically variable sponge common on shallow reefs of the Caribbean. However, the range of morphological and reproductive variation within putative N. proxima led us to hypothesize that such variability may be indicative of cryptic species rather than plasticity. Using DNA sequences and morphological characters we confirmed the presence of three previously undescribed species of Neopetrosia . Morphological differences of each new congener were best resolved by partial gene sequences of the mitochondrial cytochrome oxidase subunit 1 over nuclear ones (18S rRNA and 28S rRNA). Several new characters for Neopetrosia were revealed by each new species. For example, N. dendrocrevacea sp. nov. and N. cristata sp. nov. showed the presence of grooves on the surface of the sponge body that converge at the oscula, and a more disorganized skeleton than previously defined for the genus. N. sigmafera sp. nov. adds the (1) presence of sigma microscleres, (2) significantly wider/longer oxeas (>200 μm), and (3) the presence of parenchymella larvae. Sampling of conspecifics throughout several locations in the Caribbean revealed larger spicules in habitats closer to the continental shelf than those in remote island locations. Our study highlights the importance of integrating molecular and morphological systematics for the discrimination of new Neopetrosia spp. despite belonging to one of several polyphyletic groups (families, genera) within the current definition of the order Haplosclerida., Competing Interests: Robert J. Toonen serves as an Academic Editor for PeerJ.

We use a novel individual-based model (IBM) to simulate larval dispersal around the island of Moloka'i in the Hawaiian Archipelago. Our model uses ocean current output from the Massachusetts Institute of Technology general circulation model (MITgcm) as well as biological data on four invertebrate and seven fish species of management relevance to produce connectivity maps among sites around the island of Moloka'i. These 11 species span the range of life history characteristics of Hawaiian coral reef species and show different spatial and temporal patterns of connectivity as a result. As expected, the longer the pelagic larval duration (PLD), the greater the proportion of larvae that disperse longer distances, but regardless of PLD (3-270 d) most successful dispersal occurs either over short distances within an island (<30 km) or to adjacent islands (50-125 km). Again, regardless of PLD, around the island of Moloka'i, connectivity tends to be greatest among sites along the same coastline and exchange between northward, southward, eastward and westward-facing shores is limited. Using a graph-theoretic approach to visualize the data, we highlight that the eastern side of the island tends to show the greatest out-degree and betweenness centrality, which indicate important larval sources and connectivity pathways for the rest of the island. The marine protected area surrounding Kalaupapa National Historical Park emerges as a potential source for between-island larval connections, and the west coast of the Park is one of the few regions on Moloka'i that acts as a net larval source across all species. Using this IBM and visualization approach reveals patterns of exchange between habitat regions and highlights critical larval sources and multi-generational pathways to indicate priority areas for marine resource managers., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ.

The Gila robusta species complex in the Lower Colorado River Basin has a complicated taxonomic history. Recent authors have separated this group into three nominal taxa, G. robusta , G. intermedia , and G. nigra , however aside from location, no reliable method of distinguishing individuals of these species currently exists. To assess relationships within this group, we examined morphology of type specimens and fresh material, and used RADseq methods to assess phylogenetic relationship among these nominal species. Maximum likelihood and Bayesian inference tree building methods reveal high concordance between tree topologies based on the mitochondrial and nuclear datasets. Coalescent SNAPP analysis resolved a similar tree topology. Neither morphological nor molecular data reveal diagnostic differences between these species as currently defined. As such, G. intermedia and G. nigra should be considered synonyms of the senior G. robusta . We hypothesize that climate driven wet and dry cycles have led to periodic isolation of population subunits and subsequent local divergence followed by reestablished connectivity and mixing. Management plans should therefore focus on retaining genetic variability and viability of geographic populations to preserve adaptability to changing climate conditions., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ.

The banded coral shrimp, Stenopus hispidus (Crustacea: Decapoda: Stenopodidea) is a popular marine ornamental species with a circumtropical distribution. The planktonic larval stage lasts ∼120-253 days, indicating considerable dispersal potential, but few studies have investigated genetic connectivity on a global scale in marine invertebrates. To resolve patterns of divergence and phylogeography of S. hispidus , we surveyed 525 bp of mitochondrial cytochrome c oxidase subunit I (COI) from 198 individuals sampled at 10 locations across ∼27,000 km of the species range. Phylogenetic analyses reveal that S. hispidus has a Western Atlantic lineage and a widely distributed Indo-Pacific lineage, separated by sequence divergence of 2.1%. Genetic diversity is much higher in the Western Atlantic ( h = 0.929; π  = 0.004) relative to the Indo-Pacific ( h = 0.105; π  < 0.001), and coalescent analyses indicate that the Indo-Pacific population expanded more recently (95% HPD (highest posterior density) = 60,000-400,000 yr) than the Western Atlantic population (95% HPD = 300,000-760,000 yr). Divergence of the Western Atlantic and Pacific lineages is estimated at 710,000-1.8 million years ago, which does not readily align with commonly implicated colonization events between the ocean basins. The estimated age of populations contradicts the prevailing dispersal route for tropical marine biodiversity (Indo-Pacific to Atlantic) with the oldest and most diverse population in the Atlantic, and a recent population expansion with a single common haplotype shared throughout the vast Indian and Pacific oceans. In contrast to the circumtropical fishes, this diminutive reef shrimp challenges our understanding of conventional dispersal capabilities of marine species., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ.

Species within the scleractinian genus Pocillopora Lamarck 1816 exhibit extreme phenotypic plasticity, making identification based on morphology difficult. However, the mitochondrial open reading frame (mtORF) marker provides a useful genetic tool for identification of most species in this genus, with a notable exception of P. eydouxi and P. meandrina . Based on recent genomic work, we present a quick and simple, gel-based restriction fragment length polymorphism (RFLP) method for the identification of all six Pocillopora species occurring in Hawai'i by amplifying either the mtORF region, a newly discovered histone region, or both, and then using the restriction enzymes targeting diagnostic sequences we unambiguously identify each species . Using this approach, we documented frequent misidentification of Pocillopora species based on colony morphology. We found that P. acuta colonies are frequently mistakenly identified as P. damicornis in Kāne'ohe Bay, O'ahu. We also found that P. meandrina likely has a northern range limit in the Northwest Hawaiian Islands, above which P. ligulata was regularly mistaken for P. meandrina ., Competing Interests: Robert J. Toonen Is an Academic Editor for PeerJ.

Although the invasive azooxanthellate corals Tubastraea coccinea and T. tagusensis are spreading quickly and outcompeting native species in the Atlantic Ocean, there is little information regarding the genetic structure and path of introduction for these species. Here we present the first data on genetic diversity and clonal structure from these two species using a new set of microsatellite markers. High proportions of clones were observed, indicating that asexual reproduction has a major role in the local population dynamics and, therefore, represents one of the main reasons for the invasion success. Although no significant population structure was found, results suggest the occurrence of multiple invasions for T. coccinea and also that both species are being transported along the coast by vectors such as oil platforms and monobouys, spreading these invasive species. In addition to the description of novel microsatellite markers, this study sheds new light into the invasive process of Tubastraea ., Competing Interests: Robert J. Toonen is an Academic Editor for PeerJ.

Information on connectivity is becoming increasingly in demand as marine protected areas are being designed as an integral part of a network to protect marine resources at the ecosystem level. Larval dispersal and population structure, however, remain very difficult to assess. Here, we tested the predictions of a detailed oceanographic connectivity model of larval dispersal and coral recruitment within Palau and between Palau and Yap, which was developed to support the review of the existing network of marine protected areas in Palau. We used high throughput microsatellite genotyping of the coral Acropora hyacinthus to characterize population genetic structure. Pairwise F' ST values between Palau and Yap (0.10), Palau and Ngulu (0.09) and Yap and Ngulu (0.09) were all significant and similar to pairwise F' ST values of sites within Palau (0.02-0.12) and within Yap (0.02-0.09) highlighting structure at island scale and indicating that recruitment may be even more localized than previously anticipated. A bottleneck test did not reveal any signs of a founder effect between Yap and Palau. Overall, the data supports the idea that recovery of A. hyacinthus in Palau did not come exclusively from a single source but most likely came from a combination of areas, including sites within Palau. In light of these results there seems to be very little connectivity around the barrier reef and management recommendation would be to increase the number or the size of MPAs within Palau., Competing Interests: Rob Toonen is an Academic Editor for PeerJ.

Predictive habitat suitability models are powerful tools for cost-effective, statistically robust assessment of the environmental drivers of species distributions. The aim of this study was to develop predictive habitat suitability models for two genera of scleractinian corals (Leptoserisand Montipora) found within the mesophotic zone across the main Hawaiian Islands. The mesophotic zone (30-180 m) is challenging to reach, and therefore historically understudied, because it falls between the maximum limit of SCUBA divers and the minimum typical working depth of submersible vehicles. Here, we implement a logistic regression with rare events corrections to account for the scarcity of presence observations within the dataset. These corrections reduced the coefficient error and improved overall prediction success (73.6% and 74.3%) for both original regression models. The final models included depth, rugosity, slope, mean current velocity, and wave height as the best environmental covariates for predicting the occurrence of the two genera in the mesophotic zone. Using an objectively selected theta ("presence") threshold, the predicted presence probability values (average of 0.051 for Leptoseris and 0.040 for Montipora) were translated to spatially-explicit habitat suitability maps of the main Hawaiian Islands at 25 m grid cell resolution. Our maps are the first of their kind to use extant presence and absence data to examine the habitat preferences of these two dominant mesophotic coral genera across Hawai'i.

Fusion is an important life history strategy for clonal organisms to increase access to shared resources, to compete for space, and to recover from disturbance. For reef building corals, fragmentation and colony fusion are key components of resilience to disturbance. Observations of small fragments spreading tissue and fusing over artificial substrates prompted experiments aimed at further characterizing Atlantic and Pacific corals under various conditions. Small (∼1-3 cm(2)) fragments from the same colony spaced regularly over ceramic tiles resulted in spreading at rapid rates (e.g., tens of square centimeters per month) followed by isogenic fusion. Using this strategy, we demonstrate growth, in terms of area encrusted and covered by living tissue, of Orbicella faveolata, Pseudodiploria clivosa, and Porites lobata as high as 63, 48, and 23 cm(2) per month respectively. We found a relationship between starting and ending size of fragments, with larger fragments growing at a faster rate. Porites lobata showed significant tank effects on rates of tissue spreading indicating sensitivity to biotic and abiotic factors. The tendency of small coral fragments to encrust and fuse over a variety of surfaces can be exploited for a variety of applications such as coral cultivation, assays for coral growth, and reef restoration.

We investigate the survivorship, growth and diet preferences of hatchery-raised juvenile urchins, Tripneustes gratilla, to evaluate the efficacy of their use as biocontrol agents in the efforts to reduce alien invasive algae. In flow-through tanks, we measured urchin growth rates, feeding rates and feeding preferences among diets of the most common invasive algae found in Kāne'ohe Bay, Hawai'i: Acanthophora spicifera, Gracilaria salicornia, Eucheuma denticulatum and Kappaphycus clade B. Post-transport survivorship of outplanted urchins was measured in paired open and closed cages in three different reef environments (lagoon, reef flat and reef slope) for a month. Survivorship in closed cages was highest on the reef flat (∼75%), and intermediate in the lagoon and reef slope (∼50%). In contrast, open cages showed similar survivorship on the reef flat and in the lagoon, but only 20% of juvenile urchins survived in open cages placed on the reef slope. Urchins grew significantly faster on diets of G. salicornia (1.58 mm/week ± 0.14 SE) and Kappaphycus clade B (1.69 ± 0.14 mm/wk) than on E. denticulatum (0.97 ± 0.14 mm/wk), with intermediate growth when fed on A. spicifera (1.23 ± 0.11 mm/wk). Interestingly, urchins display size-specific feeding preferences. In non-choice feeding trials, small urchins (17.5-22.5 mm test diameter) consumed G. salicornia fastest (6.08 g/day ± 0.19 SE), with A. spicifera (4.25 ± 0.02 g/day) and Kappaphycus clade B (3.83 ± 0.02 g/day) intermediate, and E. denticulatum was clearly the least consumed (2.32 ± 0.37 g/day). Medium-sized (29.8-43.8 mm) urchins likewise preferentially consumed G. salicornia (12.60 ± 0.08 g/day), with less clear differences among the other species in which E. denticulatum was still consumed least (9.35 ± 0.90 g/day). In contrast, large urchins (45.0-65.0 mm) showed no significant preferences among the different algae species at all (12.43-15.24 g/day). Overall consumption rates in non-choice trials were roughly equal to those in the choice trials, but differences among feeding rates on each species were not predictive of feeding preferences when urchins were presented all four species simultaneously. In the choice feeding trials, both small and medium urchins clearly preferred A. spicifera over all other algae (roughly twice as much consumed as any other species). Again, however, differences were less pronounced among adult urchins, with adults showing a significant preference for A. spicifera and Kappaphycus clade B compared to the other two algal species. These findings indicate that outplanted urchins are surviving on the reef flats and eating a variety of alien invasive algae as intended. Although juvenile urchins show stronger feeding preferences, these animals grow quickly, and adult urchins are more generalist herbivores that consume all four alien invasive algae.

Cryptic species are widespread across the phylum Porifera making the identification of non-indigenous species difficult, an issue not easily resolved by the use of morphological characteristics. The widespread order Haplosclerida is a prime example due to limited and plastic morphological features. Here, we study the reported introduction of Haliclona (Soestella) caerulea from the Caribbean to Palmyra Atoll via Hawai'i using morphological characteristics and genetic analyses based on one nuclear (18s rDNA) and three mitochondrial (COI, the barcoding COI extension (COI ext.) and rnl rDNA) markers. Despite no clear division in lengths of the oxea spicules between the samples, both mtDNA and nDNA phylogenetic trees supported similar topologies resolving two distinct clades. Across the two clades, the concatenated mtDNA tree resolved twelve subclades, with the COI ext. yielding most of the variability between the samples. Low sequence divergence values (0.68%) between two of the subclades indicate that the same species is likely to occur at Palmyra, Hawai'i and the Caribbean, supporting the hypothesis that H. caerulea was introduced to Palmyra from the Caribbean, although whether species came directly from the Caribbean to Palmyra or from Hawai'i remains unresolved. Conversely, the pattern of highly divergent cryptic species supports the notion that traditionally used spicule measurements are taxonomically unreliable in this group. This study illustrates how understanding the scale of within- as opposed to between-species level genetic variation is critical for interpreting biogeographic patterns and inferring the origins of introduced organisms.

Kāne'ohe Bay, which is located on the on the NE coast of O'ahu, Hawai'i, represents one of the most intensively studied estuarine coral reef ecosystems in the world. Despite a long history of anthropogenic disturbance, from early settlement to post European contact, the coral reef ecosystem of Kāne'ohe Bay appears to be in better condition in comparison to other reefs around the world. The island of Moku o Lo'e (Coconut Island) in the southern region of the bay became home to the Hawai'i Institute of Marine Biology in 1947, where researchers have since documented the various aspects of the unique physical, chemical, and biological features of this coral reef ecosystem. The first human contact by voyaging Polynesians occurred at least 700 years ago. By A.D. 1250 Polynesians voyagers had settled inhabitable islands in the region which led to development of an intensive agricultural, fish pond and ocean resource system that supported a large human population. Anthropogenic disturbance initially involved clearing of land for agriculture, intentional or accidental introduction of alien species, modification of streams to supply water for taro culture, and construction of massive shoreline fish pond enclosures and extensive terraces in the valleys that were used for taro culture. The arrival by the first Europeans in 1778 led to further introductions of plants and animals that radically changed the landscape. Subsequent development of a plantation agricultural system led to increased human immigration, population growth and an end to traditional land and water management practices. The reefs were devastated by extensive dredge and fill operations as well as rapid growth of human population, which led to extensive urbanization of the watershed. By the 1960's the bay was severely impacted by increased sewage discharge along with increased sedimentation due to improper grading practices and stream channelization, resulting in extensive loss of coral cover. The reefs of Kāne'ohe Bay developed under estuarine conditions and thus have been subjected to multiple natural stresses. These include storm floods, a more extreme temperature range than more oceanic reefs, high rates of sedimentation, and exposure at extreme low tides. Deposition and degradation of organic materials carried into the bay from the watershed results in low pH conditions such that according to some ocean acidification projections the rich coral reefs in the bay should not exist. Increased global temperature due to anthropogenic fossil fuel emmisions is now impacting these reefs with the first "bleaching event" in 1996 and a second more severe event in 2014. The reefs of Kāne'ohe Bay have developed and persist under rather severe natural and anthropogenic perturbations. To date, these reefs have proved to be very resilient once the stressor has been removed. A major question remains to be answered concerning the limits of Kāne'ohe Bay reef resilience in the face of global climate change.

Morphological variation in the geographically widespread coral Porites lobata can make it difficult to distinguish from other massive congeneric species. This morphological variation could be attributed to geographic variability, phenotypic plasticity, or a combination of such factors. We examined genetic and microscopic morphological variability in P. lobata samples from the Galápagos, Easter Island, Tahiti, Fiji, Rarotonga, and Australia. Panamanian P. evermanni specimens were used as a previously established distinct outgroup against which to test genetic and morphological methods of discrimination. We employed a molecular analysis of variance (AMOVA) based on ribosomal internal transcribed spacer region (ITS) sequence, principal component analysis (PCA) of skeletal landmarks, and Mantel tests to compare genetic and morphological variation. Both genetic and morphometric methods clearly distinguished P. lobata and P. evermanni, while significant genetic and morphological variance was attributed to differences among geographic regions for P. lobata. Mantel tests indicate a correlation between genetic and morphological variation for P. lobata across the Pacific. Here we highlight landmark morphometric measures that correlate well with genetic differences, showing promise for resolving species of Porites, one of the most ubiquitous yet challenging to identify architects of coral reefs.

Here, we introduce ezRAD, a novel strategy for restriction site-associated DNA (RAD) that requires little technical expertise or investment in laboratory equipment, and demonstrate its utility for ten non-model organisms across a wide taxonomic range. ezRAD differs from other RAD methods primarily through its use of standard Illumina TruSeq library preparation kits, which makes it possible for any laboratory to send out to a commercial genomic core facility for library preparation and next-generation sequencing with virtually no additional investment beyond the cost of the service itself. This simplification opens RADseq to any lab with the ability to extract DNA and perform a restriction digest. ezRAD also differs from others in its flexibility to use any restriction enzyme (or combination of enzymes) that cuts frequently enough to generate fragments of the desired size range, without requiring the purchase of separate adapters for each enzyme or a sonication step, which can further decrease the cost involved in choosing optimal enzymes for particular species and research questions. We apply this method across a wide taxonomic diversity of non-model organisms to demonstrate the utility and flexibility of our approach. The simplicity of ezRAD makes it particularly useful for the discovery of single nucleotide polymorphisms and targeted amplicon sequencing in natural populations of non-model organisms that have been historically understudied because of lack of genomic information.

The effects of tides on littoral marine habitats are so ubiquitous that shorelines are commonly described as 'intertidal', whereas waves are considered a secondary factor that simply modifies the intertidal habitat. However mean significant wave height exceeds tidal range at many locations worldwide. Here we construct a simple sinusoidal model of coastal water level based on both tidal range and wave height. From the patterns of emergence and submergence predicted by the model, we derive four vertical shoreline benchmarks which bracket up to three novel, spatially distinct, and physically defined zones. The (1) emergent tidal zone is characterized by tidally driven emergence in air; the (2) wave zone is characterized by constant (not periodic) wave wash; and the (3) submergent tidal zone is characterized by tidally driven submergence. The decoupling of tidally driven emergence and submergence made possible by wave action is a critical prediction of the model. On wave-dominated shores (wave height ≫ tidal range), all three zones are predicted to exist separately, but on tide-dominated shores (tidal range ≫ wave height) the wave zone is absent and the emergent and submergent tidal zones overlap substantially, forming the traditional "intertidal zone". We conclude by incorporating time and space in the model to illustrate variability in the physical conditions and zonation on littoral shores. The wave:tide physical zonation model is a unifying framework that can facilitate our understanding of physical conditions on littoral shores whether tropical or temperate, marine or lentic.

Widespread polyphyly in stony corals (order Scleractinia) has prompted efforts to revise their systematics through approaches that integrate molecular and micromorphological evidence. To date, these approaches have not been comprehensively applied to the dominant genera in mesophotic coral ecosystems (MCEs) because several species in these genera occur primarily at depths that are poorly explored and from which sample collections are limited. This study is the first integrated morphological and molecular systematic analysis of the genera Leptoseris and Pavona to examine material both from shallow-water reefs (<30 m) and from mid- to lower-MCEs (>60 m). Skeletal and tissue samples were collected throughout the Hawaiian Archipelago between 2-127 m. A novel mitochondrial marker (cox1-1-rRNA intron) was sequenced for 70 colonies, and the micromorphologies of 94 skeletons, plus selected type material, were analyzed. The cox1-1-rRNA intron resolved 8 clades, yet Leptoseris and Pavona were polyphyletic. Skeletal micromorphology, especially costal ornamentation, showed strong correspondence and discrete differences between mitochondrial groups. One putative new Leptoseris species was identified and the global depth range of the genus Pavona was extended to 89 m, suggesting that the diversity of mesophotic scleractinians has been underestimated. Examination of species' depth distributions revealed a pattern of depth zonation: Species common in shallow-water were absent or rare >40 m, whereas others occurred only >60 m. These patterns emphasize the importance of integrated systematic analyses and more comprehensive sampling by depth in assessing the connectivity and diversity of MCEs.