Towards reproducible metabarcoding data: Lessons from an international cross-laboratory experiment.

Advances in high-throughput sequencing (HTS) are revolutionizing monitoring in marine environments by enabling rapid, accurate and holistic detection of species within complex biological samples. Research institutions worldwide increasingly employ HTS methods for biodiversity assessments. However, variance in laboratory procedures, analytical workflows and bioinformatic pipelines impede the transferability and comparability of results across research groups. An international experiment was conducted to assess the consistency of metabarcoding results derived from identical samples and primer sets using varying laboratory procedures. Homogenized biofouling samples collected from four coastal locations (Australia, Canada, New Zealand and the USA) were distributed to 12 independent laboratories. Participants were asked to follow one of two HTS library preparation workflows. While DNA extraction, primers and bioinformatic analyses were purposefully standardized to allow comparison, many other technical variables were allowed to vary among laboratories (amplification protocols, type of instrument used, etc.). Despite substantial variation observed in raw results, the primary signal in the data was consistent, with the samples grouping strongly by geographical origin for all data sets. Simple post hoc data clean-up by removing low-quality samples gave the best improvement in sample classification for nuclear 18S rRNA gene data, with an overall 92.81% correct group attribution. For mitochondrial COI gene data, the best classification result (95.58%) was achieved after correction for contamination errors. The identified critical methodological factors that introduced the greatest variability (preservation buffer, sample defrosting, template concentration, DNA polymerase, PCR enhancer) should be of great assistance in standardizing future biodiversity studies using metabarcoding.

Zooplankton biogeographic boundaries in the California Current System as determined from metabarcoding.

Within the southern California Current ecosystem there are two well-documented breaks in marine community structure at Point Conception and Punta Eugenia. We explored the presence of similar breaks in a diverse zooplankton community through metabarcoding of mixed net tow tissue samples collected during an expedition from Monterey to Baja California in February of 2012. We recovered a high diversity of species as well as patterns of species presence that align with their previously documented ranges in this region. We found a clear break at Punta Eugenia in overall zooplankton community structure, while Point Conception was weakly linked to changes in community structure. We analyzed this dataset through two parallel bioinformatic pipelines to examine the robustness of these results. Our overall conclusions were consistent across both pipelines, however there were differences in species detection. This study illustrates the utility of metabarcoding analysis on mixed tissue samples for recovering known patterns of diversity, as well as allowing elucidation of broad patterns of community differentiation across many groups of organisms.

A database of metazoan cytochrome c oxidase subunit I gene sequences derived from GenBank with CO-ARBitrator.

The Cytochrome C Oxidase subunit I gene ("COI") is the de facto standard for animal DNA barcoding. Organism identification based on COI requires an accurate and extensive annotated database of COI sequences. Such a database can also be of value in reconstructing evolutionary history and in diversity studies. Two COI databases are currently available: BOLD and Midori. BOLD's submissions conform to stringent sequence and metadata requirements; BOLD is specific to COI but makes no attempt to be comprehensive. Midori, derived from GenBank, has more sequences but less stringent standards than BOLD, resulting in higher error rates. To address the need for a comprehensive and accurate COI database, we adapted the ARBitrator algorithm, which classifies based only on sequence properties and has successfully auto-curated bacterial genes mined from GenBank. The adapted algorithm, which we call CO-ARBitrator, built a database of over a million metazoan COI sequences. Sensitivity and specificity are significantly higher than Midori. Specificity is comparable to what BOLD achieves with data quality prerequisites. Results and software are publicly available.

Partial Mitochondrial Genome Sequences of Two Abyssal Sponges (Porifera: Hexactinellida), Bathydorus laniger and Docosaccus maculatus.

We announce the nearly complete mitochondrial genome sequences of two hexactinellid sponges, Bathydorus laniger and Docosaccus maculatus A contiguous region of over 15,000 bp was sequenced from each genome. An uncommon structural element was identified as a series of repetitive elements with sequences matching cob in the genome of D. maculatus.

Transoceanic dispersal of the mussel Mytilus galloprovincialis on Japanese tsunami marine debris: An approach for evaluating rafting of a coastal species at sea.

Biofouled debris from the 2011 Great East Japan earthquake and tsunami has landed in the Northeast Pacific and along the Hawaiian Islands since 2012. As of 2017, >630 biofouled debris items with >320 living species of algae, invertebrates, and fish have been examined. The invasive mussel Mytilus galloprovincialis was present on >50% of those items. Size, reproduction, and growth of this filter-feeding species were examined to better understand long-distance rafting of a coastal species. The majority of mussels (79%) had developing or mature gametes, and growth rates averaged 0.075±0.018 SE mm/day. Structural and elemental (barium/calcium) analysis of mussel shells generated estimates of growth in coastal waters (mean=1.3 to 25mm total length), which provides an indication of residence times in waters along North America and the Hawaiian Islands prior to landing. Detailed studies of individual species contribute to our understanding of debris as a transport vector and aid efforts to evaluate potential risks associated with marine debris.

Tsunami-driven rafting: Transoceanic species dispersal and implications for marine biogeography.

The 2011 East Japan earthquake generated a massive tsunami that launched an extraordinary transoceanic biological rafting event with no known historical precedent. We document 289 living Japanese coastal marine species from 16 phyla transported over 6 years on objects that traveled thousands of kilometers across the Pacific Ocean to the shores of North America and Hawai'i. Most of this dispersal occurred on nonbiodegradable objects, resulting in the longest documented transoceanic survival and dispersal of coastal species by rafting. Expanding shoreline infrastructure has increased global sources of plastic materials available for biotic colonization and also interacts with climate change-induced storms of increasing severity to eject debris into the oceans. In turn, increased ocean rafting may intensify species invasions.

The importance of standardization for biodiversity comparisons: A case study using autonomous reef monitoring structures (ARMS) and metabarcoding to measure cryptic diversity on Mo'orea coral reefs, French Polynesia.

The advancement of metabarcoding techniques, declining costs of high-throughput sequencing and development of systematic sampling devices, such as autonomous reef monitoring structures (ARMS), have provided the means to gather a vast amount of diversity data from cryptic marine communities. However, such increased capability could also lead to analytical challenges if the methods used to examine these communities across local and global scales are not standardized. Here we compare and assess the underlying biases of four ARMS field processing methods, preservation media, and current bioinformatic pipelines in evaluating diversity from cytochrome c oxidase I metabarcoding data. Illustrating the ability of ARMS-based metabarcoding to capture a wide spectrum of biodiversity, 3,372 OTUs and twenty-eight phyla, including 17 of 33 marine metazoan phyla, were detected from 3 ARMS (2.607 m2 area) collected on coral reefs in Mo'orea, French Polynesia. Significant differences were found between processing and preservation methods, demonstrating the need to standardize methods for biodiversity comparisons. We recommend the use of a standardized protocol (NOAA method) combined with DMSO preservation of tissues for sessile macroorganisms because it gave a more accurate representation of the underlying communities, is cost effective and removes chemical restrictions associated with sample transportation. We found that sequences identified at ≥ 97% similarity increased more than 7-fold (5.1% to 38.6%) using a geographically local barcode inventory, highlighting the importance of local species inventories. Phylogenetic approaches that assign higher taxonomic ranks accrued phylum identification errors (9.7%) due to sparse taxonomic coverage of the understudied cryptic coral reef community in public databases. However, a ≥ 85% sequence identity cut-off provided more accurate results (0.7% errors) and enabled phylum level identifications of 86.3% of the sequence reads. With over 1600 ARMS deployed, standardizing methods and improving databases are imperative to provide unprecedented global baseline assessments of understudied cryptic marine species in a rapidly changing world.

Mitochondrial genome rearrangements in the scleractinia/corallimorpharia complex: implications for coral phylogeny.

Corallimorpharia is a small Order of skeleton-less animals that is closely related to the reef-building corals (Scleractinia) and of fundamental interest in the context of understanding the potential impacts of climate change in the future on coral reefs. The relationship between the nominal Orders Corallimorpharia and Scleractinia is controversial-the former is either the closest outgroup to the Scleractinia or alternatively is derived from corals via skeleton loss. This latter scenario, the "naked coral" hypothesis, is strongly supported by analyses based on mitochondrial (mt) protein sequences, whereas the former is equally strongly supported by analyses of mt nucleotide sequences. The "naked coral" hypothesis seeks to link skeleton loss in the putative ancestor of corallimorpharians with a period of elevated oceanic CO2 during the Cretaceous, leading to the idea that these skeleton-less animals may be harbingers for the fate of coral reefs under global climate change. In an attempt to better understand their evolutionary relationships, we examined mt genome organization in a representative range (12 species, representing 3 of the 4 extant families) of corallimorpharians and compared these patterns with other Hexacorallia. The most surprising finding was that mt genome organization in Corallimorphus profundus, a deep-water species that is the most scleractinian-like of all corallimorpharians on the basis of morphology, was much more similar to the common scleractinian pattern than to those of other corallimorpharians. This finding is consistent with the idea that C. profundus represents a key position in the coral <-> corallimorpharian transition.

The founding charter of the Genomic Observatories Network.

The co-authors of this paper hereby state their intention to work together to launch the Genomic Observatories Network (GOs Network) for which this document will serve as its Founding Charter. We define a Genomic Observatory as an ecosystem and/or site subject to long-term scientific research, including (but not limited to) the sustained study of genomic biodiversity from single-celled microbes to multicellular organisms.An international group of 64 scientists first published the call for a global network of Genomic Observatories in January 2012. The vision for such a network was expanded in a subsequent paper and developed over a series of meetings in Bremen (Germany), Shenzhen (China), Moorea (French Polynesia), Oxford (UK), Pacific Grove (California, USA), Washington (DC, USA), and London (UK). While this community-building process continues, here we express our mutual intent to establish the GOs Network formally, and to describe our shared vision for its future. The views expressed here are ours alone as individual scientists, and do not necessarily represent those of the institutions with which we are affiliated.

Assessing mercury exposure and biomarkers in largemouth bass (Micropterus salmoides) from a contaminated river system in California.

We evaluated mercury (Hg) exposure and two biomarkers, metallothionein (MT) gene expression and histopathological alterations in a wild fish species, largemouth bass (Micropterus salmoides), collected from the Sacramento-San Joaquin Delta, CA, a region polluted with Hg from historic mining activities. Hg is highly toxic and can disrupt multiple physiological systems in vertebrate species, including the immune system. Total mercury (THg) concentration in muscle tissue ranged from 0.12 to 0.98 ppm (wet weight) and was not related to body condition (r (2) = 0.005, p = 0.555). Using linear regression analysis, we found a positive relationship between MT gene expression (as determined using quantitative polymerase chain reaction) and copper, zinc, manganese, aluminum, and nickel (decreased to one variable by way of principal component analysis) (r (2) = 0.379, p = 0.044), a negative relationship with selenium (r (2) = 0.487, p = 0.017), and a weak, negative relationship with THg concentrations (r (2) = 0.337, p = 0.061). Juvenile largemouth bass collected from Hg-contaminated areas displayed histopathological features of immunosuppression compared with those collected from less contaminated areas as evidenced by significantly lower macrophage density in kidney and liver tissue (p = 0.018 and 0.020, respectively), greater trematode density in liver tissue (p = 0.014), and a greater number of adult trematodes. Our results suggest that largemouth bass may be experiencing sublethal effects from chronic Hg exposure. Furthermore, our findings illustrate the utility of examining multiple sublethal markers of effect to assess the impacts of contaminant exposure on physiological function in wild species.

Bathydorus laniger and Docosaccus maculatus (Lyssacinosida; Hexactinellida): Two new species of glass sponge from the abyssal eastern North Pacific Ocean.

Two new species of glass sponge were discovered from the abyssal plain 200 km west of the coast of California (Station M). The sponges have similar gross morphology--an unusual plate-like form with basalia stilling the body above soft abyssal sediments. Bathydorus laniger sp. n. differs from its congeners by the presence of dermal and atrial stauractins; it is also supported by smooth hypodermal pentactins and hypoatrial hexactins. Microscleres include oxyhexasters and oxyhemihexasters. Docosaccus maculatus sp. n. contains large hexactins (>1 cm), characteristic of the genus. Megascleres include dermal hexactins, atrial pentactins, and choanosomal hexactins and diactins. Microscleres include oxytipped hemihexasters and floricomes. Several features serve to differentiate this species from its only known congener.

Ecology of cryptic invasions: latitudinal segregation among Watersipora (Bryozoa) species.

Watersipora is an invasive genus of bryozoans, easily dispersed by fouled vessels. We examined Cytochrome c oxidase subunit I haplotypes from introduced populations on the US Pacific coastline to investigate geographic segregation of species and/or haplotypes. In California, the W. subtorquata group fell into three major sub-groups: W. subtorquata clades A and B, and W. "new sp.". W. subtorquata clades A and B were common in southern California south of Point Conception, a recognized biogeographic boundary, whereas further north, W. subtorquata clade A and W. n. sp. were frequent. The southern California region also had colonies of a morphologically distinct species, W. arcuata, also found in southern Australia and Hawaii; COI variation indicates a common ancestral source(s) in these introductions. The distribution of Watersipora-complex lineages on different coastlines is shown to be temperature correlated. Accordingly, pre-exisitng temperature-based adaptations may play a key role in determining invasion patterns.

Genetic perspectives on marine biological invasions.

The extent to which the geographic distributions of marine organisms have been reshaped by human activities remains underappreciated, and so does, consequently, the impact of invasive species on marine ecosystems. The application of molecular genetic data in fields such as population genetics, phylogeography, and evolutionary biology have improved our ability to make inferences regarding invasion histories. Genetic methods have helped to resolve longstanding questions regarding the cryptogenic status of marine species, facilitated recognition of cryptic marine biodiversity, and provided means to determine the sources of introduced marine populations and to begin to recover the patterns of anthropogenic reshuffling of the ocean's biota. These approaches stand to aid materially in the development of effective management strategies and sustainable science-based policies. Continued advancements in the statistical analysis of genetic data promise to overcome some existing limitations of current approaches. Still other limitations will be best addressed by concerted collaborative and multidisciplinary efforts that recognize the important synergy between understanding the extent of biological invasions and coming to a more complete picture of both modern-day and historical marine biogeography.

Genetic patterns across multiple introductions of the globally invasive crab genus Carcinus.

The European green crab Carcinus maenas is one of the world's most successful aquatic invaders, having established populations on every continent with temperate shores. Here we describe patterns of genetic diversity across both the native and introduced ranges of C. maenas and its sister species, C. aestuarii, including all known non-native populations. The global data set includes sequences from the mitochondrial cytochrome c oxidase subunit I gene, as well as multilocus genotype data from nine polymorphic nuclear microsatellite loci. Combined phylogeographic and population genetic analyses clarify the global colonization history of C. maenas, providing evidence of multiple invasions to Atlantic North America and South Africa, secondary invasions to the northeastern Pacific, Tasmania, and Argentina, and a strong likelihood of C. maenas x C. aestuarii hybrids in South Africa and Japan. Successful C. maenas invasions vary broadly in the degree to which they retain genetic diversity, although populations with the least variation typically derive from secondary invasions or from introductions that occurred more than 100 years ago.

Fine scale endemism on coral reefs: archipelagic differentiation in turbinid gastropods.

The perceived wide geographic range of organisms in the sea, facilitated by ready dispersal of waterborne dispersal stages, is a challenge for hypotheses of marine speciation but a boon to efforts of marine conservation. Wide species ranges are especially striking in the reef-rich Indo-west Pacific, the largest and most diverse marine biogeographic region, extending across half the planet. The insular marine biota of the tropical Pacific is characterized by wide-ranging species and provides the most striking examples of long distance dispersal, with endemism largely confined to the most remote island groups. Here we show that the gastropod Astralium "rhodostomum" has developed endemic clades on almost every Pacific archipelago sampled, a pattern unprecedented in marine biogeography, and reminiscent of the terrestrial biota of oceanic islands. Mitochondrial DNA sequences indicate that this species-complex is comprised of at least 30 geographically isolated clades, separated by as little as 180 km. Evidence suggests that such fine scale endemism and high diversity is not exceptional, but likely characterizes a substantial fraction of the reef biota. These results imply that (1) marine speciation can regularly occur over much finer spatial scales than generally accepted, (2) the diversity of coral reefs is even higher than suggested by morphology-based estimates, and (3) conservation efforts need to focus at the archipelagic level in the sea as on land.

Fission in sea anemones: integrative studies of life cycle evolution.

Sea anemones (Phylum Cnidaria; Class Anthozoa, Order Actiniaria) exhibit a diversity of developmental patterns that include cloning by fission. Because natural histories of clonal and aclonal sea anemones are quite different, the gain and loss of fission is an important feature of actiniarian lineages. We have used mitochondrial DNA and nuclear intron DNA phylogenies to investigate the evolution of longitudinal fission in sixteen species in the genus Anthopleura, and reconstructed an aclonal ancestor that has given rise at least four times to clonal descendents. For A. elegantissima from the northeastern Pacific Ocean, a transition to clonality by fission was associated with an up-shore habitat shift, supporting prior hypotheses that clonal growth is an adaptation to the upper shore. Fission in Actiniaria likely precedes its advent in Anthopleura, and its repeated loss and gain is perplexing. Field studies of the acontiate sea anemone Aiptasia californica provided insight to the mechanisms that regulate fission: subtidal Aiptasia responded to experimentally destabilized substrata by increasing rates of pedal laceration. We put forth a general hypothesis for actiniarian fission in which sustained tissue stretch (a consequence of substratum instability or intrinsic behavior) induces tissue degradation, which in turn induces regeneration. The gain and loss of fission in Anthopleura lineages may only require the gain and loss of some form of stretching behavior. In this view, tissue stretch initiates a cascade of developmental events without requiring complex gene regulatory linkages.