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The frequency and intensity of cyanobacterial blooms is increasing worldwide. Multiple factors are implicated, most of which are anthropogenic. New Zealand provides a useful location to study the impacts of human settlement on lake ecosystems. The first humans (Polynesians) arrived about 750 years ago. Following their settlement, there were marked landscape modifications which intensified after European settlement about 150 years ago. The aims of this study were to reconstruct cyanobacterial communities in six lakes over the last 1000 years and explore key drivers of change. Cyanobacterial environmental DNA was extracted from sediment cores and analysed using metabarcoding and droplet digital PCR. Cyanobacteria, including potentially toxic or bloom forming species, were already present in these lakes prior to human arrival, however their overall abundance was low. Total cyanobacteria abundance and richness increased in all lakes after European settlement but was very pronounced in four lakes, where bloom-forming taxa became dominant. These shifts occurred concomitant with land-use change. The catchment of one deteriorated lake is only moderately modified, thus the introduction of non-native fish is posited as the key factor driving this change. The paleolimnological approach used in this study has enabled new insights into timing and potential causes of changes in cyanobacterial communities., (© 2022. The Author(s).)

Symbiodiniaceae are a diverse group of dinoflagellates, the majority of which are free-living and/or associated with a variety of protists and other invertebrate hosts. Maintenance of isolated cultures is labour-intensive and expensive, and cryopreservation provides an excellent avenue for their long-term storage. We aimed to cryopreserve 15 cultured isolates from six Symbiodiniaceae genera using dimethyl sulfoxide (DMSO) as the cryoprotectant agent (CPA). Under 15% DMSO, 10 isolates were successfully cryopreserved using either rapid freezing or controlled-rate freezing. Cultures that failed or had low survival, were subjected to (1) a reduction of CPA to 10%, or (2) increased salinity treatment before freezing. At 10% DMSO, three further isolates were successfully cryopreserved. At 15% DMSO there were high cell viabilities in Symbiodinium pilosum treated with 44 parts per thousand (ppt) and 54 ppt culture medium. An isolate of Fugacium sp. successfully cryopreserved after salinity treatments of 54 ppt and 64 ppt. Fatty acid (FA) analyses of S. pilosum after 54 ppt salinity treatment showed increased saturated FA levels, whereas Fugacium sp. had low poly-unsaturated FAs compared to normal salinity (34 ppt). Understanding the effects of salinity and roles of FAs in cryopreservation will help in developing protocols for these ecologically important taxa., (© 2022. The Author(s).)

Fish disease surveillance methods can be complicated and time consuming, which limits their value for timely intervention strategies on aquaculture farms. Novel molecular-based assays using droplet digital Polymerase Chain Reaction (ddPCR) can produce immediate results and enable high sample throughput with the ability to multiplex several targets using different fluorescent dyes. A ddPCR tetraplex assay was developed for priority salmon diseases for farmers in New Zealand including New Zealand Rickettsia -like organism 1 (NZ-RLO1), NZ-RLO2, Tenacibaculum maritimum , and Yersinia ruckeri . The limit of detection in singleplex and tetraplex assays was reached for most targets at 10 -9  ng/μl with, respectively, NZ-RLO1 = 0.931 and 0.14 copies/μl, NZ-RLO2 = 0.162 and 0.21 copies/μl, T. maritimum = 0.345 and 0.93 copies/μl, while the limit of detection for Y. ruckeri was 10 -8 with 1.0 copies/μl and 0.7 copies/μl. While specificity of primers was demonstrated in previous studies, we detected cross-reactivity of T. maritimum with some strains of Tenacibaculum dicentrarchi and Y. ruckeri with Serratia liquefaciens , respectively. The tetraplex assay was applied as part of a commercial fish disease surveillance program in New Zealand for 1 year to demonstrate the applicability of tetraplex tools for the salmonid aquaculture industry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 von Ammon, Averink, Kumanan, Brosnahan, Pochon, Hutson and Symonds.)

Lakes and their catchments have been subjected to centuries to millennia of exploitation by humans. Efficient monitoring methods are required to promote proactive protection and management. Traditional monitoring is time consuming and expensive, which limits the number of lakes monitored. Lake surface sediments provide a temporally integrated representation of environmental conditions and contain high microbial biomass. Based on these attributes, we hypothesized that bacteria associated with lake trophic states could be identified and used to develop an index that would not be confounded by non-nutrient stressor gradients. Metabarcoding (16S rRNA gene) was used to assess bacterial communities present in surface sediments from 259 non-saline lakes in New Zealand encompassing a range of trophic states from alpine microtrophic lakes to lowland hypertrophic lakes. A subset of lakes (n = 96) with monitoring data was used to identify indicator amplicon sequence variants (ASVs) associated with different trophic states. A total of 10,888 indicator taxa were identified and used to develop a Sediment Bacterial Trophic Index (SBTI), which signficantly correlated (r 2  = 0.842, P < 0.001) with the Trophic Lake Index. The SBTI was then derived for the remaining 163 lakes, providing new knowledge of the trophic state of these unmonitored lakes. This new, robust DNA-based tool provides a rapid and cost-effective method that will allow a greater number of lakes to be monitored and more effectively managed in New Zealand and globally. The SBTI could also be applied in a paleolimnological context to investigate changes in trophic status over centuries to millennia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Contaminants are often at low concentrations in ecosystems and their effects on exposed organisms can occur over long periods of time and across multiple generations. Alterations to subcellular mechanistic pathways in response to exposure to contaminants can provide insights into mechanisms of toxicity that methods measuring higher levels of biological may miss. Analysis of the whole transcriptome can identify novel mechanisms of action leading to impacts in exposed biota. The aim of this study was to characterise how exposures to copper, benzophenone and diclofenac across multiple generations altered molecular expression pathways in the marine copepod Gladioferens pectinatus. Results of the study demonstrated differential gene expression was observed in cultures exposure to diclofenac (569), copper (449) and benzophenone (59). Pathways linked to stress, growth, cellular and metabolic processes were altered by exposure to all three contaminants with genes associated with oxidative stress and xenobiotic regulation also impacted. Protein kinase functioning, cytochrome P450, transcription, skeletal muscle contraction/relaxation, mitochondrial phosphate translocator, protein synthesis and mitochondrial methylation were all differentially expressed with all three chemicals. The results of the study also suggested that using dimethyl sulfoxide as a dispersant influenced the transcriptome and future research may want to investigate it's use in molecular studies. Data generated in this study provides a first look at transcriptomic response of G. pectinatus exposed to contaminants across multiple generations, future research is needed to validate the identified biomarkers and link these results to apical responses such as population growth to demonstrate the predictive capacity of molecular tools., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© 2021 John Wiley & Sons Ltd.)

Understanding the historical onset of cyanobacterial blooms in freshwater bodies can help identify their potential drivers. Lake sediments are historical archives, containing information on what has occurred in and around lakes over time. Paleolimnology explores these records using a variety of techniques, but choosing the most appropriate method can be challenging. We compared results obtained from a droplet digital PCR assay targeting a cyanobacterial-specific region of the 16S rRNA gene in sedimentary DNA and cyanobacterial pigments (canthaxanthin, echinenone, myxoxanthophyll and zeaxanthin) analysed using high-performance liquid chromatography in four sediment cores. There were strong positive relationships between the 16S rRNA gene copy concentrations and individual pigment concentrations, but relationships differed among lakes and sediment core depths within lakes. The relationships were more consistent when all pigments were summed, which we attribute to different cyanobacteria species, in different lakes, at different times producing different suites of pigments. Each method had benefits and limitations, which should be taken into consideration during method selection and when interpreting paleolimnological data. We recommend this biphasic approach when making inferences about changes in the entire cyanobacterial community because they yielded complementary information. Our results support the view that molecular methods can yield results similar to traditional paleolimnological proxies when caveats are adequately addressed.

Characterization of microbial assemblages via environmental DNA metabarcoding is increasingly being used in routine monitoring programs due to its sensitivity and cost-effectiveness. Several programs have recently been developed which infer functional profiles from 16S rRNA gene data using hidden-state prediction (HSP) algorithms. These might offer an economic and scalable alternative to shotgun metagenomics. To date, HSP-based methods have seen limited use for benthic marine surveys and their performance in these environments remains unevaluated. In this study, 16S rRNA metabarcoding was applied to sediment samples collected at 0 and ≥1,200 m from Norwegian salmon farms, and three metabolic inference approaches (Paprica, Picrust2 and Tax4Fun2) evaluated against metagenomics and environmental data. While metabarcoding and metagenomics recovered a comparable functional diversity, the taxonomic composition differed between approaches, with genera richness up to 20× higher for metabarcoding. Comparisons between the sensitivity (highest true positive rates) and specificity (lowest true negative rates) of HSP-based programs in detecting functions found in metagenomic data ranged from 0.52 and 0.60 to 0.76 and 0.79, respectively. However, little correlation was observed between the relative abundance of their specific functions. Functional beta-diversity of HSP-based data was strongly associated with that of metagenomics (r ≥ 0.86 for Paprica and Tax4Fun2) and responded similarly to the impact of fish farm activities. Our results demonstrate that although HSP-based metabarcoding approaches provide a slightly different functional profile than metagenomics, partly due to recovering a distinct community, they represent a cost-effective and valuable tool for characterizing and assessing the effects of fish farming on benthic ecosystems., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Opportunities to study community-level responses to extreme natural pulse disturbances in unaltered ecosystems are rare. Lake sediment records that span thousands of years can contain well-resolved sediment pulses, triggered by earthquakes. These palaeorecords provide a means to study repeated pulse disturbances and processes of resistance (insensitivity to disturbance) and ecological resilience (capacity to regain structure, function and process). In this study, sedimentary DNA was extracted from a sediment core from Lake Paringa (New Zealand) that is situated in a near natural catchment. Metabarcoding and inferred functions were used to assess the lake microbial community over the past 1100 years - a period that included four major earthquakes. Microbial community composition and function differed significantly between highly perturbed (postseismic, ~50 years) phases directly after the earthquakes and more stable (interseismic, ~250 years) phases, indicating a lack of community resistance. Although community structure differed significantly in successive postseismic phases, function did not, suggesting potential functional redundancy. Significant differences in composition and function in successive interseismic phases demonstrate that communities are not resilient to large-scale natural pulse disturbances. The clear difference in structure and function, and high number of indicator taxa (responsible for driving differences in communities between phases) in the fourth interseismic phase probably represents a regime shift, possibly due to the two-fold increase in sediment and terrestrial biospheric organic carbon fluxes recorded following the fourth earthquake. Large pulse disturbances that enhance sediment inputs into lake systems may produce an underappreciated mechanism that destabilises lake ecosystem processes., (© 2021 John Wiley & Sons Ltd.)

Freshwater eels are ecologically, and culturally important worldwide. The New Zealand long-finned eel ( Anguilla dieffenbachii ) and short-finned eel ( Anguilla australis ) are apex predators, playing an important role in ecosystem functioning of rivers and lakes. Recently, there has been a national decline in their populations due to habitat destruction and commercial harvest. The emergence of targeted environmental DNA detection methodologies provides an opportunity to enhance information about their past and present distributions. In this study we successfully developed species-specific droplet digital Polymerase Chain Reaction (ddPCR) assays to detect A. dieffenbachii and A. australis DNA in water and sediment samples. Assays utilized primers and probes designed for regions of the mitochondrial cytochrome b and 16S ribosomal RNA genes in A. dieffenbachii and A. australis , respectively. River water samples ( n = 27) were analyzed using metabarcoding of fish taxa and were compared with the ddPCR assays. The presence of A. dieffenbachii and A. australis DNA was detected in a greater number of water samples using ddPCR in comparison to metabarcoding. There was a strong and positive correlation between gene copies (ddPCR analyses) and relative eel sequence reads (metabarcoding analyses) when compared to eel biomass. These ddPCR assays provide a new method for assessing spatial distributions of A. dieffenbachii and A. australis in a range of environments and sample types., Competing Interests: Xavier Pochon is an Academic Editor in the Aquatic Biology section of PeerJ., (©2021 Thomson-Laing et al.)

The Pacific oyster Crassostrea gigas is the world's most cultivated oyster and seed supply is heavily reliant on hatchery production where recurring mass mortality events are a major constraint. Outbreaks of bacterial infection via microalgal feed are frequently implicated in these mortalities. This study assessed the effects of feeding compromised microalgae to developing oyster larvae. Intentionally 'stressed' (high pH) or non-stressed microalgae were fed to 11 day-old oyster larvae at two feeding rations for 96 h, followed by a recovery period. Biological endpoints of larval performance were measured following the 96 h exposure and subsequent recovery. Bacterial communities associated with the microalgae feed, rearing seawater, and the oyster larvae, were characterized and correlated with effects on oyster fitness parameters. Feeding stressed algae to oyster larvae for 96 h increased the occurrence of deformities (>70% vs. 20% in control), reduced feeding and swimming ability, and slowed development. Following the recovery period, fewer larvae reached pediveliger stage (2.7% vs. 36% in control) and became spat (1.5% vs. 6.6% in control). The quantity of stressed algae supplied to oyster larvae also influenced overall larval performance, with high feeding rations generally causing greater impairment than low rations. Bacterial profiling using 16S rRNA showed that most bacterial families characterized in larval tissue were also present in larval rearing seawater and in the microalgae feed (98%). The rearing seawater showed the highest bacterial richness compared to the larval and the microalgal compartments, regardless of feeding regime. In larval tissue, bacterial richness was highest in stressed and high-feed treatments, and negatively correlated with larval fitness parameters. These results suggest significant dysbiosis induced by compromised feed and/or increased feed ration. Several bacterial genera (e.g., Halomonas, Marinomonas ) were strongly associated with impaired larval performance while the presence of genera in larvae including Vibrio was closely associated with overfeeding. Our research demonstrated that metabarcoding can be effectively used to identify microbiota features associated with larval fitness., Competing Interests: PW was employed by company Moana New Zealand Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vignier, Laroche, Rolton, Wadsworth, Kumanan, Trochel, Pochon and King.)

A decade after environmental scientists integrated high-throughput sequencing technologies in their toolbox, the genomics-based monitoring of anthropogenic impacts on the biodiversity and functioning of ecosystems is yet to be implemented by regulatory frameworks. Despite the broadly acknowledged potential of environmental genomics to this end, technical limitations and conceptual issues still stand in the way of its broad application by end-users. In addition, the multiplicity of potential implementation strategies may contribute to a perception that the routine application of this methodology is premature or "in development", hence restraining regulators from binding these tools into legal frameworks. Here, we review recent implementations of environmental genomics-based methods, applied to the biomonitoring of ecosystems. By taking a general overview, without narrowing our perspective to particular habitats or groups of organisms, this paper aims to compare, review and discuss the strengths and limitations of four general implementation strategies of environmental genomics for monitoring: (a) Taxonomy-based analyses focused on identification of known bioindicators or described taxa; (b) De novo bioindicator analyses; (c) Structural community metrics including inferred ecological networks; and (d) Functional community metrics (metagenomics or metatranscriptomics). We emphasise the utility of the three latter strategies to integrate meiofauna and microorganisms that are not traditionally utilised in biomonitoring because of difficult taxonomic identification. Finally, we propose a roadmap for the implementation of environmental genomics into routine monitoring programmes that leverage recent analytical advancements, while pointing out current limitations and future research needs., (© 2020 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

The dinoflagellate family Symbiodiniaceae comprises numerous divergent genera containing species whose ecologies range from endosymbiotic to free-living. While many associate with invertebrates including corals, sea anemones, jellyfish, giant clams, and flatworms, others occur within the cytoplasm of large protists, most notably benthic foraminifera in the sub-family Soritinae. Recent systematic revisions to the Symbiodiniaceae left out formal naming of some divergent lineages because each lacked a representative type species to erect new genus names. Here we provide genetic, morphological and ecological evidence to describe a new genus and species. Miliolidium n. gen. is closely related to the genus Durusdinium and contains several genetically divergent ecologically distinct lineages found in distant geographic locations indicating an Indo-Pacific wide distribution. One of these, Miliolidium leei n. sp., is represented by an isolate cultured from Amphisorus sp. originally collected in the Gulf of Eilat, northern Red Sea. Its peripheral chloroplast extensions are uniquely petal- or lobe-shaped, and cells possess a pyrenoid with three stalks connecting to chloroplasts, and without thylakoid intrusions. It is related to an isolate cultured from an azooxanthellate sponge from Palau and another that is commonly harbored by the soritid Marginopora vertebralis in shallow reef habitats from Guam. Research on Symbiodiniaceae diversity including free-living species in benthic habitats and those mutualistic with soritid foraminifera remains extremely limited as does our knowledge of their diversity, physiology, biogeography, and ecology., (© 2021 International Society of Protistologists.)

The analysis of benthic bacterial community structure has emerged as a powerful alternative to traditional microscopy-based taxonomic approaches to monitor aquaculture disturbance in coastal environments. However, local bacterial diversity and community composition vary with season, biogeographic region, hydrology, sediment texture, and aquafarm-specific parameters. Therefore, without an understanding of the inherent variation contained within community complexes, bacterial diversity surveys conducted at individual farms, countries, or specific seasons may not be able to infer global universal pictures of bacterial community diversity and composition at different degrees of aquaculture disturbance. We have analyzed environmental DNA (eDNA) metabarcodes (V3-V4 region of the hypervariable SSU rRNA gene) of 138 samples of different farms located in different major salmon-producing countries. For these samples, we identified universal bacterial core taxa that indicate high, moderate, and low aquaculture impact, regardless of sampling season, sampled country, seafloor substrate type, or local farming and environmental conditions. We also discuss bacterial taxon groups that are specific for individual local conditions. We then link the metabolic properties of the identified bacterial taxon groups to benthic processes, which provides a better understanding of universal benthic ecosystem function(ing) of coastal aquaculture sites. Our results may further guide the continuing development of a practical and generic bacterial eDNA-based environmental monitoring approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Frühe, Dully, Forster, Keeley, Laroche, Pochon, Robinson, Wilding and Stoeck.)

The introduction and spread of marine non-indigenous species (NIS) and pathogens into new habitats are a major threat to biodiversity, ecosystem services, human health, and can have substantial economic consequences. Shipping is considered the main vector for marine biological invasions; less well understood is the increased spread of marine NIS and pathogens rafting on marine plastic debris (MPD). Despite an increasing research interest and recent progress in characterizing the plastisphere, this manuscript highlights critical knowledge gaps and research priorities towards a better understanding of the biosecurity implications of MPD. We advocate for future research to (i) investigate plastisphere community succession and the factors influencing NIS propagules and pathogens recruitment through robust experimental investigations; (ii) combine microscopy and molecular approaches to effectively assess the presence of specific taxa; (iii) include additional genetic markers to thoroughly characterize the biodiversity associated with MPD and explore the presence of specific marine pests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Molecular-based approaches can provide timely biodiversity assessments, showing an immense potential to facilitate decision-making in marine environmental management. However, the uptake of molecular data into environmental policy remains minimal. Here, we showcase a selection of local to global scale studies applying molecular-based methodologies for environmental management at various stages of implementation. Drawing upon lessons learned from these case-studies, we provide a roadmap to facilitate applications of DNA-based methods to marine policies and to overcome the existing challenges. The main impediment identified is the need for standardized protocols to guarantee data comparison across spatial and temporal scales. Adoption of Translational Molecular Ecology - the sustained collaboration between molecular ecologists and stakeholders, will enhance consensus with regards to the objectives, methods, and outcomes of environmental management projects. Establishing a sustained dialogue among stakeholders is key to accelerating the adoption of molecular-based approaches for marine monitoring and assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Marine sediments contain a high diversity of micro- and macro-organisms which are important in the functioning of biogeochemical cycles. Traditionally, anthropogenic perturbation has been investigated by identifying macro-organism responses along gradients. Environmental DNA (eDNA) analyses have recently been advocated as a rapid and cost-effective approach to measuring ecological impacts and efforts are underway to incorporate eDNA tools into monitoring. Before these methods can replace or complement existing methods, robustness and repeatability of each analytical step has to be demonstrated. One area that requires further investigation is the selection of sediment DNA extraction method. Environmental DNA sediment samples were obtained along a disturbance gradient adjacent to a Chinook ( Oncorhynchus tshawytscha ) salmon farm in Otanerau Bay, New Zealand. DNA was extracted using four extraction kits (Qiagen DNeasy PowerSoil, Qiagen DNeasy PowerSoil Pro, Qiagen RNeasy PowerSoil Total RNA/DNA extraction/elution and Favorgen FavorPrep Soil DNA Isolation Midi Kit) and three sediment volumes (0.25, 2, and 5 g). Prokaryotic and eukaryotic communities were amplified using primers targeting the 16S and 18S ribosomal RNA genes, respectively, and were sequenced on an Illumina MiSeq. Diversity and community composition estimates were obtained from each extraction kit, as well as their relative performance in established metabarcoding biotic indices. Differences were observed in the quality and quantity of the extracted DNA amongst kits with the two Qiagen DNeasy PowerSoil kits performing best. Significant differences were observed in both prokaryotes and eukaryotes ( p < 0.001) richness among kits. A small proportion of amplicon sequence variants (ASVs) were shared amongst the kits (~3%) although these shared ASVs accounted for the majority of sequence reads (prokaryotes: 59.9%, eukaryotes: 67.2%). Differences were observed in the richness and relative abundance of taxonomic classes revealed with each kit. Multivariate analysis showed that there was a significant interaction between "distance" from the farm and "kit" in explaining the composition of the communities, with the distance from the farm being a stronger determinant of community composition. Comparison of the kits against the bacterial and eukaryotic metabarcoding biotic index suggested that all kits showed similar patterns along the environmental gradient. Overall, we advocate for the use of Qiagen DNeasy PowerSoil kits for use when characterizing prokaryotic and eukaryotic eDNA from marine farm sediments. We base this conclusion on the higher DNA quality values and richness achieved with these kits compared to the other kits/amounts investigated in this study. The additional advantage of the PowerSoil Kits is that DNA extractions can be performed using an extractor robot, offering additional standardization and reproducibility of results., Competing Interests: Xavier Pochon is an Academic Editor for PeerJ. John K Pearman, Susanna A Wood, Anastasija Zaiko, Georgia Thomson-Laing, Laura Biessy, Javier Atalah, and Xavier Pochon are employed by Cawthron Institute., (© 2020 Pearman et al.)

Incidental detection of species of concern (e.g., invasive species, pathogens, threatened and endangered species) during biodiversity assessments based on high-throughput DNA sequencing holds significant risks in the absence of rigorous, fit-for-purpose data quality and reporting standards. Molecular biodiversity data are predominantly collected for ecological studies and thus are generated to common quality assurance standards. However, the detection of certain species of concern in these data would likely elicit interest from end users working in biosecurity or other surveillance contexts (e.g., pathogen detection in health-related fields), for which more stringent quality control standards are essential to ensure that data are suitable for informing decision-making and can withstand legal or political challenges. We suggest here that data quality and reporting criteria are urgently needed to enable clear identification of those studies that may be appropriately applied to surveillance contexts. In the interim, more pointed disclaimers on uncertainties associated with the detection and identification of species of concern may be warranted in published studies. This is not only to ensure the utility of molecular biodiversity data for consumers, but also to protect data generators from uncritical and potentially ill-advised application of their science in decision-making.

In this study, the evolution of ballast water (BW) assemblages across different trophic levels was characterized over a 21 day cross-latitudinal vessel transit using a combination of molecular methods. Triplicate BW samples were collected every second day and size-fractionated (<2.7, 10, >50 μm). Measurements of adenosine triphosphate (ATP) and metabarcoding of environmental nucleic acid (DNA and RNA) analyses, complemented by microscopy and flow cytometry, were performed on each sample. Measured ATP concentrations exhibited high variance between replicates and a strong negative trend in the large (≥50 μm) fraction over the voyage. In concert with microscopy, the metabarcoding data indicated a die-off of larger metazoans during the first week of study and gradual reductions in dinoflagellates and ochrophytes. The ATP and metabarcoding data signaled persistent or increased cellular activity of heterotrophic bacteria and protists in the BW, which was supported by flow cytometry. The metabarcoding showed the presence of active bacteria in all size fractions, suggesting that the sequential filtration approach does not ensure taxonomical differentiation, which has implications for BW quality assessment. Although our data show that ATP and metabarcoding have potential for indicative BW screening for BW compliance monitoring, further research and technological development is needed to improve representativeness of sampling and deliver the unequivocal response criteria required by the international Ballast Water Management Convention.

Dimethylsulfoniopropionate (DMSP) is a key compound in the marine sulfur cycle, and is produced in large quantities in coral reefs. In addition to Symbiodiniaceae, corals and associated bacteria have recently been shown to play a role in DMSP metabolism. Numerous ecological studies have focused on DMSP concentrations in corals, which led to the hypothesis that increases in DMSP levels might be a general response to stress. Here we used multiple species assemblages of three common Indo-Pacific holobionts, the scleractinian corals Pocillopora damicornis and Acropora cytherea, and the giant clam Tridacna maxima and examined the DMSP concentrations associated with each species within different assemblages and thermal conditions. Results showed that the concentration of DMSP in A. cytherea and T. maxima is modulated according to the complexity of species assemblages. To determine the potential importance of symbiotic dinoflagellates in DMSP production, we then explored the relative abundance of Symbiodiniaceae clades in relation to DMSP levels using metabarcoding, and found no significant correlation between these factors. Finally, this study also revealed the existence of homologs involved in DMSP production in giant clams, suggesting for the first time that, like corals, they may also contribute to DMSP production. Taken together, our results demonstrated that corals and giant clams play important roles in the sulfur cycle. Because DMSP production varies in response to specific species-environment interactions, this study offers new perspectives for future global sulfur cycling research.

Lake surface sediments are dominated by microorganisms that play significant roles in biogeochemical cycling within lakes. There is limited knowledge on the relative importance of local environmental factors and altitude on bacterial and microeukaryotic community richness and composition in lake sediments. In the present study, surface sediment samples were collected from 40 lakes along an altitude gradient (2-1215 m). Microbial communities were characterized using 16S (bacteria) and 18S (microeukaryotes) rRNA gene metabarcoding. Bacterial and microeukaryotic richness were not correlated with altitude but instead to environmental variables (e.g. area of water in the catchment (bacteria: R = -0.43). For both bacteria and microeukaryotes, dissimilarity in the community structure had a higher correlation to combined environmental variables (without altitude) (bacteria: R = 0.53; microeukaryotes: R = 0.55) than altitude alone (bacteria: R = 0.34; microeukaryotes: R = 0.47). Sediment sulfur and productive grassland were important variables in determining the relative abundance of sulfate reducing bacteria. Nitrospira, was positively related to altitude but negatively to water column total organic carbon and the proportion of productive grassland in the catchment. Little overlap in amplicon sequence variants was shown amongst lakes. This has important considerations for management decisions, suggesting that to protect biodiversity, conservation of numerous lakes and lake types is required., (© FEMS 2020.)

Background: Giant clams and scleractinian (reef-building) corals are keystone species of coral reef ecosystems. The basis of their ecological success is a complex and fine-tuned symbiotic relationship with microbes. While the effect of environmental change on the composition of the coral microbiome has been heavily studied, we know very little about the composition and sensitivity of the microbiome associated with clams. Here, we explore the influence of increasing temperature on the microbial community (bacteria and dinoflagellates from the family Symbiodiniaceae) harbored by giant clams, maintained either in isolation or exposed to other reef species. We created artificial benthic assemblages using two coral species (Pocillopora damicornis and Acropora cytherea) and one giant clam species (Tridacna maxima) and studied the microbial community in the latter using metagenomics., Results: Our results led to three major conclusions. First, the health status of giant clams depended on the composition of the benthic species assemblages. Second, we discovered distinct microbiotypes in the studied T. maxima population, one of which was disproportionately dominated by Vibrionaceae and directly linked to clam mortality. Third, neither the increase in water temperature nor the composition of the benthic assemblage had a significant effect on the composition of the Symbiodiniaceae and bacterial communities of T. maxima., Conclusions: Altogether, our results suggest that at least three microbiotypes naturally exist in the studied clam populations, regardless of water temperature. These microbiotypes plausibly provide similar functions to the clam host via alternate molecular pathways as well as microbiotype-specific functions. This redundancy in functions among microbiotypes together with their specificities provides hope that giant clam populations can tolerate some levels of environmental variation such as increased temperature. Importantly, the composition of the benthic assemblage could make clams susceptible to infections by Vibrionaceae, especially when water temperature increases. Video abstract.

Over the last decade, there has been growing interest in the analysis of environmental DNA (eDNA) to infer the presence of organisms in aquatic environments. The efficacy of eDNA/eRNA based tools are highly depend on the turnover rate of the molecule (their release and degradation). Environmental DNA has been shown to persist for days, weeks or years in environmental samples. Environmental RNA (eRNA) is thought to degrade faster than eDNA, however to our knowledge, no experimental studies have explored this. Here we present an aquarium study to investigate eDNA and eRNA shedding rates and degradation for two sessile marine invertebrates. The copy numbers for eDNA and eRNA were assessed using droplet digital PCR targeting the mitochondrial Cytochrome c Oxidase subunit 1 (COI) gene. Environmental RNA persisted after organism removal for much longer than expected with detections for up to 13 h. In contrast, eDNA was detected is samples collected up to 94 h after organism removal. There was no evidence that the decay rates constants for eDNA and eRNA were different (p = 0.6, Kruskal-Wallis tests). Both eDNA and eRNA was detected in biofilms collected at the end of the experiment (day 21). This suggests binding with organic or inorganic compounds or stabilization of these molecules in the biofilm matrix. The finding of the prolonged persistence of eRNA may provide new opportunities for improved biodiversity surveys through reducing false positives caused by legacy DNA and could also facilitate new research on environmental transcriptomics., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Fish farms are increasingly situated in strong current sites above or near to mixed-bottom habitats that include organisms not normally considered in the context of organic enrichment. This study takes a holistic view of the benthic enrichment process by combining different survey techniques on complimentary spatial scales: conventional macrofaunal cores, larger-scale visual quantification of epibiota and environmental-DNA metabarcoding of microbial communities. A large tube forming polychaete (Arenicola marina), normally found intertidally and living too deep for conventional sampling, was observed occupying an opportunistic niche in areas of high deposition and in very close association with Capitellid worm complexes. The surface-dwelling brittlestar, Ophiocomina nigra, was abundant at distances of 250-1000 m from Farm-B, suggesting a positive response to enrichment, but was displaced where sedimentation exceed 5 g m 2 d -1 . A corresponding gradient was evident within the sediment microbial communities, supporting established theories about ecosystem engineering and multi-species synergies for organic waste assimilation. Many of the bacteria present in the near-farm sediments were linked to the farmed fish and fish health issues suggesting one or two-way inoculation pressures. These functionally different benthic organisms are intrinsically linked and the resulting synergy has the potential to assimilate significant quantities of anthropogenically produced organic waste contributing to environmental sustainability., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Targeted species-specific and community-wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R 2  = 0.81, p < .001, biofouling R 2  = 0.68, p < .001); however, qPCR copy numbers were on average 125-fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data., (© 2019 John Wiley & Sons Ltd.)

Environmentally induced epigenetic modifications have been proposed as one mechanism underlying rapid adaptive evolution of invasive species. Didemnum vexillum is an invasive colonial ascidian that has established in many coastal waters worldwide. Phylogenetic analyses have revealed that D. vexillum populations consist of two distinct clades; clade B appears to be restricted to the native range (Japan), whereas clade A is found in many regions throughout the world, including New Zealand. The spread of D. vexillum clade A suggests that it might be intrinsically more invasive than clade B, despite low levels of genetic diversity compared to populations from the native region. This study investigated whether D. vexillum clade A exhibits epigenetic signatures (specifically differences in DNA methylation) associated with invasiveness. Global DNA methylation patterns were significantly different between introduced clade A colonies, and both clades A and B in the native range. Introduced colonies also showed a significant reduction in DNA methylation levels, which could be a mechanism for increasing phenotypic plasticity. High levels of DNA methylation diversity were maintained in the introduced population, despite reduced levels of genetic diversity, which may allow invasive populations to respond quickly to changes in new environments. Epigenetic changes induced during the invasion process could provide a means for rapid adaptation despite low levels of genetic variation in introduced populations.

One of the mechanisms of rapid adaptation or acclimatization to environmental changes in corals is through the dynamics of the composition of their associated endosymbiotic Symbiodiniaceae community. The various species of these dinoflagellates are characterized by different biological properties, some of which can confer stress tolerance to the coral host. Compelling evidence indicates that the corals' Symbiodiniaceae community can change via shuffling and/or switching but the ecological relevance and the governance of these processes remain elusive. Using a qPCR approach to follow the dynamics of Symbiodiniaceae genera in tagged colonies of three coral species over a 10-18 month period, we detected putative genus-level switching of algal symbionts, with coral species-specific rates of occurrence. However, the dynamics of the corals' Symbiodiniaceae community composition was not driven by environmental parameters. On the contrary, putative shuffling event were observed in two coral species during anomalous seawater temperatures and nutrient concentrations. Most notably, our results reveal that a suit of permanent Symbiodiniaceae genera is maintained in each colony in a specific range of quantities, giving a unique 'Symbiodiniaceae signature' to the host. This individual signature, together with sporadic symbiont switching may account for the intra-specific differences in resistance and resilience observed during environmental anomalies.

High-throughput sequencing is revolutionizing our ability to comprehensively characterize free-living and symbiotic Symbiodiniaceae, a diverse dinoflagellate group that plays a critical role in coral reef ecosystems. Most studies however, focus on a single marker for metabarcoding Symbiodiniaceae, potentially missing important ecological traits that a combination of markers may capture. In this proof-of-concept study, we used a small set of symbiotic giant clam ( Tridacna maxima ) samples obtained from nine French Polynesian locations and tested a dual-index sequence library preparation method that pools and simultaneously sequences multiple Symbiodiniaceae gene amplicons per sample for in-depth biodiversity assessments. The rationale for this approach was to allow the metabarcoding of multiple genes without extra costs associated with additional single amplicon dual indexing and library preparations. Our results showed that the technique effectively recovered very similar proportions of sequence reads and dominant Symbiodiniaceae clades among the three pooled gene amplicons investigated per sample, and captured varying levels of phylogenetic resolution enabling a more comprehensive assessment of the diversity present. The pooled Symbiodiniaceae multi-gene metabarcoding approach described here is readily scalable, offering considerable analytical cost savings while providing sufficient phylogenetic information and sequence coverage., Competing Interests: Xavier Pochon is an Academic Editor for PeerJ.

Molecular techniques may provide effective tools to enhance marine biosecurity surveillance. Prior to routine implementation, evidence-based consideration of their benefits and limitations is needed. In this study, we assessed the efficiency and practicality of visual diver surveys and real-time PCR assays (targeting DNA and RNA) for detecting two marine invasive species whose infestation levels varied between species and location: Sabella spallanzanii and Styela clava . Filtered water samples ( n = 171) were collected in parallel with dive surveys at two locations as part of the New Zealand Marine High Risk Site Surveillance programme: Nelson Harbour (27 sites) and Waitemata Harbour (30 sites). Diver surveys resulted in a greater number of detections compared to real-time PCR: S. clava - 21 versus 5 sites in Nelson, 6 versus 1 in Auckland; S. spallanzanii - 18 versus 10 in Auckland, no detections in Nelson. Occupancy modelling derived detection probabilities for the real-time PCR for S. clava were low (14%), compared to S. spallanzanii (66%). This could be related to abundances, or species-specific differences in DNA shedding. Only one RNA sample was positive, suggesting that most detections were from extracellular DNA or non-viable fragments. While molecular methods cannot yet replace visual observations, this study shows they provide useful complementary information.

To prevent the settlement and/or the growth of fouling organisms (i.e. bacteria, fungi or microalgae), benthic sessile species have developed various defense mechanisms among which the production of chemical molecules. While studies have mostly focused on the release of chemical compounds by single species, there exist limited data on multi-species assemblages. We used an integrative approach to explore the potential interactive effects of distinct assemblages of two corals species and one giant clam species on biofouling appearance and composition. Remarkably, we found distinct biofouling communities suggesting the importance of benthic sessile assemblages in biofouling control. Moreover, the assemblage of 3 species led to an inhibition of biofouling, likely through a complex of secondary metabolites. Our results highlight that through their different effect on their near environment, species assemblages might be of upmost importance for their survival and therefore, should now be taken into account for sustainable management of coral reefs.

The dinoflagellate microalga Symbiodinium sustains coral reefs, one of the most diverse ecosystems of the biosphere, through mutualistic endosymbioses with a wide diversity of benthic hosts [1]. Despite its ecological and economic importance, the presence of Symbiodinium in open oceanic waters remains unknown, which represents a significant knowledge gap to fully understand the eco-evolutionary trajectory and resilience of endangered Symbiodinium-based symbioses. Here, we document the existence of Symbiodinium (i.e., now the family Symbiodiniaceae [2]) in tropical- and temperate-surface oceans using DNA and RNA metabarcoding of size-fractionated plankton samples collected at 109 stations across the globe. Symbiodinium from clades A and C were, by far, the most prevalent and widely distributed lineages (representing 0.1% of phytoplankton reads), while other lineages (clades B, D, E, F, and G) were present but rare. Concurrent metatranscriptomics analyses using the Tara Oceans gene catalog [3] revealed that Symbiodinium clades A and C were transcriptionally active in the open ocean and expressed core metabolic pathways (e.g., photosynthesis, carbon fixation, glycolysis, and ammonium uptake). Metabarcodes and expressed genes of clades A and C were detected in small and large plankton size fractions, suggesting the existence of a free-living population and a symbiotic lifestyle within planktonic hosts, respectively. However, high-resolution genetic markers and microscopy are required to confirm the life history of oceanic Symbiodinium. Overall, the previously unknown, metabolically active presence of Symbiodinium in oceanic waters opens up new avenues for investigating the potential of this oceanic reservoir to repopulate coral reefs following stress-induced bleaching., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Marine infrastructure can favor the spread of non-indigenous marine biofouling species by providing a suitable habitat for them to proliferate. Cryptic organisms or those in early life stages can be difficult to distinguish by conventional morphological taxonomy. Molecular tools, such as metabarcoding, may improve their detection. In this study, the ability of morpho-taxonomy and metabarcoding (18S rRNA and COI) using three reference databases (PR2, BOLD and NCBI) to characterize biodiversity and detect non-indigenous species (NIS) in biofouling was compared on 60 passive samplers deployed over summer and winter in a New Zealand marina. Highest resolution of metazoan taxa was identified using 18S rRNA assigned to PR2. There were higher assignment rates to NCBI reference sequences, but poorer taxonomic identification. Using all methods, 48 potential NIS were identified. Metabarcoding detected the largest proportion of those NIS: 77% via 18S rRNA/PR2 and NCBI and 35% via COI/BOLD and NCBI. Morpho-taxonomy detected an additional 14% of all identified NIS comprising mainly of bryozoan taxa. The data highlight several on-going challenges, including: differential marker resolution, primer biases, incomplete sequence reference databases, and variations in bioinformatic pipelines. Combining morpho-taxonomy and molecular analysis methods will likely enhance the detection of NIS from complex biofouling.

Taxonomic and functional community structures may respond differently to anthropogenic stressors. Used in combination they can provide an estimate of functional redundancy, a key component of ecosystem resilience. In this study, the utility of incorporating functional community structure and co-occurrence network properties into impact assessments of offshore oil and gas (O&G) operations on benthic bacterial communities was investigated. Sediment samples and physico-chemical data were collected along a transect at increasing distances from one exploratory drilling (ED), and one gas production and drilling (GPD) field. Bacterial community composition was determined by 16S rRNA metabarcoding. A hidden-state prediction method (PAPRICA) was used to characterize bacterial metabolic community functions. At both sites, diversity differed significantly between near-field (impacted) and far-field (non-impacted) stations, with both taxonomic and functional alpha-diversity positively affected in near-field stations at the GPD site. The opposite pattern was observed in the near-field samples of ED with lower and higher values respectively. Overall, impacted stations displayed a distinct network signature, with a lower ratio of positive interactions and signs of higher community cohesion. Community profiles from metabolic inference and co-occurrence network topology provided complementary information to taxonomic indices, which may assist with assessing the effects of O&G activities on benthic communities.

Metabarcoding and metabolomics were used to explore the taxonomic composition and functional diversity of eukaryotic biofouling communities on plates with antifouling paints at two French coastal sites: Lorient (North Eastern Atlantic Ocean; temperate and eutrophic) and Toulon (North-Western Mediterranean Sea; mesotrophic but highly contaminated). Four distinct coatings were tested at each site and season for one month. Metabarcoding showed biocidal coatings had less impact on eukaryotic assemblages compared to spatial and temporal effects. Ciliophora, Chlorophyceae or Cnidaria (mainly hydrozoans) were abundant at Lorient, whereas Arthropoda (especially crustaceans), Nematoda, and Ochrophyta dominated less diversified assemblages at Toulon. Seasonal shifts were observed at Lorient, but not Toulon. Metabolomics also showed clear site discrimination, but these were associated with a coating and not season dependent clustering. The meta-omics analysis enabled identifications of some associative patterns between metabolomic profiles and specific taxa, in particular those colonizing the plates with biocidal coatings at Lorient.

Environmentally induced epigenetic changes may contribute to phenotypic plasticity, increase adaptive potential in changing environments, and play a key role in the establishment and spread of invasive species in new habitats. In this study, we used methylation-sensitive amplified polymorphism (MSAP) to assess environmentally induced DNA methylation changes in a globally invasive clonal ascidian, Didemnum vexillum . We tested the effect of increasing temperature (19, 25 and 27 °C) and decreasing salinity (34, 32, 30, 28 and 26 practical salinity units (PSU)) on global DNA methylation, growth and survival rates. Exposure to 27 °C resulted in significant changes in DNA methylation over time. Growth also decreased in colonies exposed to high temperatures, suggesting they were under thermal stress. In contrast, no differences in growth nor DNA methylation patterns were observed in colonies exposed to a decreasing salinity gradient, potentially due to prior adaptation. The results of this study show that environmental stress can induce significant global DNA methylation changes in an invasive marine invertebrate on very rapid timescales, and that this response varies depending on the type, magnitude, and duration of the stressor. Changes in genomic DNA methylation and the rate of growth may act to 'buy survival time' under stressful conditions, expanding the distribution limits of this globally invasive species., Competing Interests: Xavier Pochon and Andrew E. Fidler are Academic Editors for PeerJ.

The Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of Symbiodinium , a dinoflagellate genus of algal endosymbionts that is pervasively associated with marine invertebrates, and notably reef-building corals. Here we tested three commonly used ITS2 primer pairs (SYM&#95;VAR&#95;5.8S2/SYM&#95;VAR&#95;REV, ITSintfor2/ITSReverse, and ITS-DINO/ITS2Rev2) with regard to amplification specificity and sensitivity towards Symbiodinium , as well as sub-genera taxonomic bias. We tested these primers over a range of sample types including three coral species, coral surrounding water, reef surface water, and open ocean water to assess their suitability for use in large-scale next generation sequencing projects and to develop a standardised PCR protocol. We found the SYM&#95;VAR&#95;5.8S2/SYM&#95;VAR&#95;REV primers to perform superior to the other tested ITS2 primers. We therefore used this primer pair to develop a standardised PCR protocol. To do this, we tested the effect of PCR-to-PCR variation, annealing temperature, cycle number, and different polymerase systems on the PCR efficacy. The Symbiodinium ITS2 PCR protocol developed here delivers improved specificity and sensitivity towards Symbiodinium with apparent minimal sub-genera taxonomic bias across all sample types. In particular, the protocol's ability to amplify Symbiodinium from a range of environmental sources will facilitate the study of Symbiodinium populations across biomes., Competing Interests: Xavier Pochon is an Academic Editor for PeerJ.

Standardized ecosystem-based monitoring surveys are critical for providing information on marine ecosystem health. Environmental DNA/RNA (eDNA/eRNA) metabarcoding may facilitate such surveys by quickly and effectively characterizing multi-trophic levels. In this study, we assessed the suitability of eDNA/eRNA metabarcoding to evaluate changes in benthic assemblages of bacteria, Foraminifera and other eukaryotes along transects at three offshore oil and gas (O&G) drilling and production sites, and compared these to morphologically characterized macro-faunal assemblages. Bacterial communities were the most responsive to O&G activities, followed by Foraminifera, and macro-fauna (the latter assessed by morphology). The molecular approach enabled detection of hydrocarbon degrading taxa such as the bacteria Alcanivorax and Microbulbifer at petroleum impacted stations. Most identified indicator taxa, notably among macro-fauna, were highly specific to site conditions. Based on our results we suggest that eDNA/eRNA metabarcoding can be used as a stand-alone method for biodiversity assessment or as a complement to morphology-based monitoring approaches., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vessel hulls and underwater infrastructure can be severely impacted by marine biofouling. Knowledge on which abiotic conditions of artificial structures influence bacterial and eukaryotic community composition is limited. In this study, settlement plates with differing surface texture, orientation and copper-based anti-fouling coatings were deployed in a marina. After three months, biofouling samples were collected and bacterial and eukaryotic communities characterised using DNA metabarcoding. The copper anti-fouling coating treatments incurred the most significant compositional changes (p ≤ 0.001) within both domains. Bacterial diversity decreased, with Gammaproteobacteria becoming the dominant phylum. In contrast, protist diversity increased as well as opportunist nematodes and bryozoans; urochordates and molluscs became less abundant. Network analyses displayed complex relationships on untreated plates, while revealing a simpler, but disturbed and unstable community composition on the anti-fouling coated plates. These networks of copper treatments displayed opportunist taxa that appeared as key organisms in structuring the bacterial and eukaryotic communities., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This corrects the article DOI: 10.1038/sdata.2016.17.

High-throughput sequencing metabarcoding studies in marine biosecurity have largely focused on targeting environmental DNA (eDNA). DNA can persist extracellularly in the environment, making discrimination of living organisms difficult. In this study, bilge water samples (i.e., water accumulating on-board a vessel during transit) were collected from 15 small recreational and commercial vessels. eDNA and eRNA molecules were co-extracted and the V4 region of the 18S ribosomal RNA gene targeted for metabarcoding. In total, 62.7% of the Operational Taxonomic Units (OTUs) were identified at least once in the corresponding eDNA and eRNA reads, with 19.5% unique to eDNA and 17.7% to eRNA. There were substantial differences in diversity between molecular compartments; 57% of sequences from eDNA-only OTUs belonged to fungi, likely originating from legacy DNA. In contrast, there was a higher percentage of metazoan (50.2%) and ciliate (31.7%) sequences in the eRNA-only OTUs. Our data suggest that the presence of eRNA-only OTUs could be due to increased cellular activities of some rare taxa that were not identified in the eDNA datasets, unusually high numbers of rRNA transcripts in ciliates, and/or artefacts produced during the reverse transcriptase, PCR and sequencing steps. The proportions of eDNA/eRNA shared and unshared OTUs were highly heterogeneous within individual bilge water samples. Multiple factors including boat type and the activities performed on-board, such as washing of scientific equipment, may play a major role in contributing to this variability. For some marine biosecurity applications analysis, eDNA-only data may be sufficient, however there are an increasing number of instances where distinguishing the living portion of a community is essential. For these circumstances, we suggest only including OTUs that are present in both eDNA and eRNA data. OTUs found only in the eRNA data need to be interpreted with caution until further research provides conclusive evidence for their origin.

The Mediterranean fanworm, Sabella spallanzanii Gmelin 1791, was first detected in the Southern Hemisphere in the 1990s and is now abundant in many parts of southern Australia and in several locations around northern New Zealand. Once established, it can proliferate rapidly, reaching high densities with potential ecological and economic impacts. Early detection of new S. spallanzanii incursions is important to prevent its spread, guide eradication or control efforts and to increase knowledge on the species' dispersal pathways. In this study, we developed a TaqMan probe real-time polymerase chain reaction assay targeting a region of the mitochondrial cytochrome oxidase I gene. The assay was validated in silico and in vitro using DNA from New Zealand and Australian Sabellidae with no cross-reactivity detected. The assay has a linear range of detection over seven orders of magnitude with a limit of detection reached at 12.4 × 10 -4  ng/μL of DNA. We analysed 145 environmental (water, sediment and biofouling) samples and obtained positive detections only from spiked samples and those collected at a port where S. spallanzanii is known to be established. This assay has the potential to enhance current morphological and molecular-based methods, through its ability to rapidly and accurately identify S. spallanzanii in environmental samples.

Sequencing environmental DNA (eDNA) is increasingly being used as an alternative to traditional morphological-based identification to characterize biological assemblages and monitor anthropogenic impacts in marine environments. Most studies only assess eDNA which, compared to eRNA, can persist longer in the environment after cell death. Therefore, eRNA may provide a more immediate census of the environment due to its relatively weaker stability, leading some researchers to advocate for the use of eRNA as an additional, or perhaps superior proxy for portraying ecological changes. A variety of pre-treatment techniques for screening eDNA and eRNA derived operational taxonomic units (OTUs) have been employed prior to statistical analyses, including removing singleton taxa (i.e., OTUs found only once) and discarding those not present in both eDNA and eRNA datasets. In this study, we used bacterial (16S ribosomal RNA gene) and eukaryotic (18S ribosomal RNA gene) eDNA- and eRNA-derived data from benthic communities collected at increasing distances along a transect from an oil production platform (Taranaki, New Zealand). Macro-infauna (visual classification of benthic invertebrates) and physico-chemical data were analyzed in parallel. We tested the effect of removing singleton taxa, and removing taxa not present in the eDNA and eRNA libraries from the same environmental sample (trimmed by shared OTUs), by comparing the impact of the oil production platform on alpha- and beta-diversity of the eDNA/eRNA-based biological assemblages, and by correlating these to the morphologically identified macro-faunal communities and the physico-chemical data. When trimmed by singletons, presence/absence information from eRNA data represented the best proxy to detect changes on species diversity for both bacteria and eukaryotes. However, assessment of quantitative beta-diversity from read abundance information of bacteria eRNA did not, contrary to eDNA, reveal any impact from the oil production activity. Overall, the data appeared more robust when trimmed by shared OTUs, showing a greater effect of the platform on alpha- and beta-diversity. Trimming by shared OTUs likely removes taxa derived from legacy DNA and technical artefacts introduced through reverse transcriptase, polymerase-chain-reaction and sequencing. Findings from our scoping study suggest that metabarcoding-based biomonitoring surveys should, if funds, time and expertise allow, be assessed using both eDNA and eRNA products., Competing Interests: Dr. Xavier Pochon is an Academic Editor for PeerJ.

Cyanobacteria are commonly associated with eutrophic lakes, where they often form blooms and produce toxins. However, they are a ubiquitous component of phytoplankton in lakes of widely varying trophic status. We hypothesised that cyanobacterial diversity would vary among lakes of differing trophic status, but that the relative importance of geographical and hydromorphological characteristics driving these patterns would differ across trophic groups. DNA from 143 New Zealand lakes that spanned a range of geographic, hydromorphological and trophic gradients was analysed using automated rRNA intergenic spacer analysis and screened for genes involved in cyanotoxin production. Statistical analysis revealed significant delineation among cyanobacterial communities from different trophic classes. Multivariate regression indicated that geographical features (latitude, longitude and altitude) were significant in driving cyanobacterial community structure; however, partitioning of their effects varied among trophic categories. High-throughput sequencing was undertaken on selected samples to investigate their taxonomic composition. The most abundant and diverse (71 operational taxonomic units) taxon across all lake types was the picocyanobacteria genus Synechococcus Cyanotoxins (microcystins n = 23, anatoxins n = 1) were only detected in eutrophic lowland lakes. Collectively, these data infer that increasing eutrophication of lakes will have broad-scale impacts on planktonic cyanobacteria diversity and the prevalence of cyanotoxins., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

At present, environmental impacts from offshore oil and gas activities are partly determined by measuring changes in macrofauna diversity. Morphological identification of macrofauna is time-consuming, expensive and dependent on taxonomic expertise. In this study, we evaluated the applicability of using foraminiferal-specific metabarcoding for routine monitoring. Sediment samples were collected along distance gradients from two oil platforms off Taranaki (New Zealand) and their physico-chemical properties, foraminiferal environmental DNA/RNA, and macrofaunal composition analyzed. Macrofaunal and foraminiferal assemblages showed similar shifts along impact gradients, but responded differently to environmental perturbations. Macrofauna were affected by hypoxia, whereas sediment grain size appeared to drive shifts in foraminifera. We identified eight foraminiferal molecular operational taxonomic units that have potential to be used as bioindicator taxa. Our results show that metabarcoding represents an effective tool for assessing foraminiferal communities near offshore oil and gas platforms, and that it can be used to complement current monitoring techniques., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Recent studies have advocated biomonitoring using DNA techniques. In this study, two high-throughput sequencing (HTS)-based methods were evaluated: amplicon metabarcoding of the cytochrome C oxidase subunit I (COI) mitochondrial gene and gene enrichment using MYbaits (targeting nine different genes including COI). The gene-enrichment method does not require PCR amplification and thus avoids biases associated with universal primers. Macroinvertebrate samples were collected from 12 New Zealand rivers. Macroinvertebrates were morphologically identified and enumerated, and their biomass determined. DNA was extracted from all macroinvertebrate samples and HTS undertaken using the illumina miseq platform. Macroinvertebrate communities were characterized from sequence data using either six genes (three of the original nine were not used) or just the COI gene in isolation. The gene-enrichment method (all genes) detected the highest number of taxa and obtained the strongest Spearman rank correlations between the number of sequence reads, abundance and biomass in 67% of the samples. Median detection rates across rare (<1% of the total abundance or biomass), moderately abundant (1-5%) and highly abundant (>5%) taxa were highest using the gene-enrichment method (all genes). Our data indicated primer biases occurred during amplicon metabarcoding with greater than 80% of sequence reads originating from one taxon in several samples. The accuracy and sensitivity of both HTS methods would be improved with more comprehensive reference sequence databases. The data from this study illustrate the challenges of using PCR amplification-based methods for biomonitoring and highlight the potential benefits of using approaches, such as gene enrichment, which circumvent the need for an initial PCR step., (© 2015 John Wiley & Sons Ltd.)

In this experimental study the patterns in early marine biofouling communities and possible implications for surveillance and environmental management were explored using metabarcoding, viz. 18S ribosomal RNA gene barcoding in combination with high-throughput sequencing. The community structure of eukaryotic assemblages and the patterns of initial succession were assessed from settlement plates deployed in a busy port for one, five and 15 days. The metabarcoding results were verified with traditional morphological identification of taxa from selected experimental plates. Metabarcoding analysis identified > 400 taxa at a comparatively low taxonomic level and morphological analysis resulted in the detection of 25 taxa at varying levels of resolution. Despite the differences in resolution, data from both methods were consistent at high taxonomic levels and similar patterns in community shifts were observed. A high percentage of sequences belonging to genera known to contain non-indigenous species (NIS) were detected after exposure for only one day.

Symbiotic partnerships between heterotrophic hosts and intracellular microalgae are common in tropical and subtropical oligotrophic waters of benthic and pelagic marine habitats. The iconic example is the photosynthetic dinoflagellate genus Symbiodinium that establishes mutualistic symbioses with a wide diversity of benthic hosts, sustaining highly biodiverse reef ecosystems worldwide. Paradoxically, although various species of photosynthetic dinoflagellates are prevalent eukaryotic symbionts in pelagic waters, Symbiodinium has not yet been reported in symbiosis within oceanic plankton, despite its high propensity for the symbiotic lifestyle. Here we report a new pelagic photosymbiosis between a calcifying ciliate host and the microalga Symbiodinium in surface ocean waters. Confocal and scanning electron microscopy, together with an 18S rDNA-based phylogeny, showed that the host is a new ciliate species closely related to Tiarina fusus (Colepidae). Phylogenetic analyses of the endosymbionts based on the 28S rDNA gene revealed multiple novel closely related Symbiodinium clade A genotypes. A haplotype network using the high-resolution internal transcribed spacer-2 marker showed that these genotypes form eight divergent, biogeographically structured, subclade types that do not seem to associate with any benthic hosts. Ecological analyses using the Tara Oceans metabarcoding data set (V9 region of the 18S rDNA) and contextual oceanographic parameters showed a global distribution of the symbiotic partnership in nutrient-poor surface waters. The discovery of the symbiotic life of Symbiodinium in the open ocean provides new insights into the ecology and evolution of this pivotal microalga and raises new hypotheses about coastal pelagic connectivity.

Trait-based approaches advance ecological and evolutionary research because traits provide a strong link to an organism's function and fitness. Trait-based research might lead to a deeper understanding of the functions of, and services provided by, ecosystems, thereby improving management, which is vital in the current era of rapid environmental change. Coral reef scientists have long collected trait data for corals; however, these are difficult to access and often under-utilized in addressing large-scale questions. We present the Coral Trait Database initiative that aims to bring together physiological, morphological, ecological, phylogenetic and biogeographic trait information into a single repository. The database houses species- and individual-level data from published field and experimental studies alongside contextual data that provide important framing for analyses. In this data descriptor, we release data for 56 traits for 1547 species, and present a collaborative platform on which other trait data are being actively federated. Our overall goal is for the Coral Trait Database to become an open-source, community-led data clearinghouse that accelerates coral reef research.