Combining environmental DNA and visual surveys can inform conservation planning for coral reefs.

Environmental DNA (eDNA) metabarcoding has the potential to revolutionize conservation planning by providing spatially and taxonomically comprehensive data on biodiversity and ecosystem conditions, but its utility to inform the design of protected areas remains untested. Here, we quantify whether and how identifying conservation priority areas within coral reef ecosystems differs when biodiversity information is collected via eDNA analyses or traditional visual census records. We focus on 147 coral reefs in Indonesia's hyper-diverse Wallacea region and show large discrepancies in the allocation and spatial design of conservation priority areas when coral reef species were surveyed with underwater visual techniques (fishes, corals, and algae) or eDNA metabarcoding (eukaryotes and metazoans). Specifically, incidental protection occurred for 55% of eDNA species when targets were set for species detected by visual surveys and 71% vice versa. This finding is supported by generally low overlap in detection between visual census and eDNA methods at species level, with more overlap at higher taxonomic ranks. Incomplete taxonomic reference databases for the highly diverse Wallacea reefs, and the complementary detection of species by the two methods, underscore the current need to combine different biodiversity data sources to maximize species representation in conservation planning.

Environmental DNA: The next chapter.

Molecular tools are an indispensable part of ecology and biodiversity sciences and implemented across all biomes. About a decade ago, the use and implementation of environmental DNA (eDNA) to detect biodiversity signals extracted from environmental samples opened new avenues of research. Initial eDNA research focused on understanding population dynamics of target species. Its scope thereafter broadened, uncovering previously unrecorded biodiversity via metabarcoding in both well-studied and understudied ecosystems across all taxonomic groups. The application of eDNA rapidly became an established part of biodiversity research, and a research field by its own. Here, we revisit key expectations made in a land-mark special issue on eDNA in Molecular Ecology in 2012 to frame the development in six key areas: (1) sample collection, (2) primer development, (3) biomonitoring, (4) quantification, (5) behaviour of DNA in the environment and (6) reference database development. We pinpoint the success of eDNA, yet also discuss shortfalls and expectations not met, highlighting areas of research priority and identify the unexpected developments. In parallel, our retrospective couples a screening of the peer-reviewed literature with a survey of eDNA users including academics, end-users and commercial providers, in which we address the priority areas to focus research efforts to advance the field of eDNA. With the rapid and ever-increasing pace of new technical advances, the future of eDNA looks bright, yet successful applications and best practices must become more interdisciplinary to reach its full potential. Our retrospect gives the tools and expectations towards concretely moving the field forward.

Environmental DNA unveils deep phylogeographic structure of a freshwater fish.

Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using an environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach Barbatula toni, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from B. toni in 189 rivers. A total of 51 samples, representing the entire island, were then selected from the B. toni eDNA-positive sample set for the subsequent analyses. To elucidate the phylogeographic structure of B. toni, newly developed eDNA metabarcoding primers (Barba-cytb-F/R) were applied to these samples, specifically targeting their haplotypic variation in cytochrome b. After a bioinformatic processing to mitigate haplotypic false positives, a total of 50 eDNA haplotypes were identified. Two regionally restricted, genetically distinct lineages of the species were revealed as a result of phylogeographic analyses on the haplotypes and tissue-derived DNA from B. toni. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonisation of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography.

The Tree of Life eDNA metabarcoding reveals a similar taxonomic richness but dissimilar evolutionary lineages between seaports and marine reserves.

Coastal areas host a major part of marine biodiversity but are seriously threatened by ever-increasing human pressures. Transforming natural coastlines into urban seascapes through habitat artificialization may result in loss of biodiversity and key ecosystem functions. Yet, the extent to which seaports differ from nearby natural habitats and marine reserves across the whole Tree of Life is still unknown. This study aimed to assess the level of α and ß-diversity between seaports and reserves, and whether these biodiversity patterns are conserved across taxa and evolutionary lineages. For that, we used environmental DNA (eDNA) metabarcoding to survey six seaports on the French Mediterranean coast and four strictly no-take marine reserves nearby. By targeting four different groups-prokaryotes, eukaryotes, metazoans and fish-with appropriate markers, we provide a holistic view of biodiversity on contrasted habitats. In the absence of comprehensive reference databases, we used bioinformatic pipelines to gather similar sequences into molecular operational taxonomic units (MOTUs). In contrast to our expectations, we obtained no difference in MOTU richness (α-diversity) between habitats except for prokaryotes and threatened fishes with higher diversity in reserves than in seaports. However, we observed a marked dissimilarity (ß-diversity) between seaports and reserves for all taxa. Surprisingly, this biodiversity signature of seaports was preserved across the Tree of Life, up to the order. This result reveals that seaports and nearby marine reserves share few taxa and evolutionary lineages along urbanized coasts and suggests major differences in terms of ecosystem functioning between both habitats.

eDNA-based detection reveals invasion risks of a biofouling bivalve in the world's largest water diversion project.

Environmental DNA (eDNA) has increasingly been used to detect rare species (e.g., newly introduced nonindigenous species) in both terrestrial and aquatic ecosystems, often with distinct advantages over traditional methods. However, whether water eDNA signals can be used to inform invasion risks remains debatable owing to inherent uncertainties associated with the methods used and the varying conditions among study systems. Here, we sampled eDNA from canals of the central route of the South-to-North Water Diversion Project (hereafter SNWDP) in China to investigate eDNA distribution and efficacy to inform invasion risks in a unique lotic system. We first conducted a total of 16 monthly surveys in this system (two sites in the source reservoir and four sites in the main canal) to test if eDNA could be applied to detect an invasive, biofouling bivalve, the golden mussel Limnoperna fortunei. Second, we initiated a one-time survey in a sub-canal of the SNWDP using refined sampling (12 sites in ~22 km canal) and considered a few environmental predictors. We found that detection of target eDNA in the main canal was achieved up to 1100 km from the putative source population but was restricted to the warmer months (May-November). Detection probability exhibited a significant positive relationship with average daily minimum air temperature and with water temperature, consistent with the expected spawning season. eDNA concentration in the main canal generally fluctuated across months and sites and was generally higher in warmer months. Golden mussel eDNA concentration in the sub-canal decreased significantly with distance from the source and with increasing water temperature and became almost undetectable at ~22 km distance. Given the enormity of the SNWDP, golden mussels may eventually expand their distribution in the main canal, with established "bridgehead" populations facilitating further spread. Our findings suggest an elevated invasion risk of golden mussels in the SNWDP in warm months, highlighting the critical period for spread and, possibly, management.

Real-Time Monitoring on the Chinese Giant Salamander Using RPA-LFD.

The Chinese giant salamander (Andrias davidianus), listed as an endangered species under "secondary protection" in China, faces significant threats due to ecological deterioration and the expansion of human activity. Extensive field investigations are crucial to ascertain the current status in the wild and to implement effective habitat protection measures to safeguard this species and support its population development. Traditional survey methods often fall short due to the elusive nature of the A. davidianus, presenting challenges that are time-consuming and generally ineffective. To overcome these obstacles, this study developed a real-time monitoring method that uses environmental DNA (eDNA) coupled with recombinase polymerase amplification and lateral flow strip (RPA-LFD). We designed five sets of species-specific primers and probes based on mitochondrial genome sequence alignments of A. davidianus and its close relatives. Our results indicated that four of these primer/probe sets accurately identified A. davidianus, distinguishing it from other tested caudata species using both extracted DNA samples and water samples from a tank housing an individual. This method enables the specific detection of A. davidianus genomic DNA at concentrations as low as 0.1 ng/mL within 50 min, without requiring extensive laboratory equipment. Applied in a field survey across four sites in Huangshan City, Anhui Province, where A. davidianus is known to be distributed, the method successfully detected the species at three of the four sites. The development of these primer/probe sets offers a practical tool for field surveying and monitoring, facilitating efforts in population recovery and resource conservation for A. davidianus.

Environmental DNA and remote sensing datasets reveal the spatial distribution of aquatic insects in a disturbed subtropical river system.

Biodiversity datasets with high spatial resolution are critical prerequisites for river protection and management decision-making. However, traditional morphological biomonitoring is inefficient and only provides several site estimates, and there is an urgent need for new approaches to predict biodiversity on fine spatial scales throughout the entire river systems. Here, we combined the environmental DNA (eDNA) and remote sensing (RS) technologies to develop a novel approach for predicting the spatial distribution of aquatic insects with high spatial resolution in a disturbed subtropical Dongjiang River system of southeast China. First, we screened thirteen RS-based vegetation indices that significantly correlated with the eDNA-inferred richness of aquatic insects. In particular, the green normalized difference vegetation index (GNDVI) and normalized difference red-edge2 (NDRE2) were closely related to eDNA-inferred richness. Second, using the gradient boosting decision tree, our data showed that the spatial pattern of eDNA-inferred richness could achieve a high spatial resolution to 500 m reach and accurate prediction of more than 80%, and the prediction efficiency of the headwater streams (Strahler stream order = 1) was slightly higher than the downstream (Strahler stream order >1). Third, using the random forest algorithm, the spatial distribution of aquatic insects could reach a prediction rate of over 70% for the presence or absence of specific genera. Overall, this study provides a new approach to achieving high spatial resolution prediction of the distribution of aquatic insects, which supports decision-making on river diversity protection under climate changes and human impacts.

Reconstruction of plasmids by shotgun sequencing from environmental DNA: which bioinformatic workflow?

Plasmids play important roles in microbial evolution and also in the spread of antibiotic resistance. Plasmid sequences are extensively studied from clinical isolates but rarely from the environment with a metagenomic approach focused on the plasmid fraction referred to as the plasmidome. A clear challenge in this context is to define a workflow for discriminating plasmids from chromosomal contaminants existing in the plasmidome. For this purpose, we benchmarked existing tools from assembly to detection of the plasmids by reference-free methods (cBar and PlasFlow) and database-guided approaches. Our simulations took into account short-reads alone or combined with moderate long-reads like those actually generated in environmental genomics experiments. This benchmark allowed us to select the best tools for limiting false-positives associated to plasmid prediction tools and a combination of reference-guided methods based on plasmid and bacterial databases.

Environment and shipping drive environmental DNA beta-diversity among commercial ports.

The spread of nonindigenous species by shipping is a large and growing global problem that harms coastal ecosystems and economies and may blur coastal biogeographical patterns. This study coupled eukaryotic environmental DNA (eDNA) metabarcoding with dissimilarity regression to test the hypothesis that ship-borne species spread homogenizes port communities. We first collected and metabarcoded water samples from ports in Europe, Asia, Australia and the Americas. We then calculated community dissimilarities between port pairs and tested for effects of environmental dissimilarity, biogeographical region and four alternative measures of ship-borne species transport risk. We predicted that higher shipping between ports would decrease community dissimilarity, that the effect of shipping would be small compared to that of environment dissimilarity and shared biogeography, and that more complex shipping risk metrics (which account for ballast water and stepping-stone spread) would perform better. Consistent with our hypotheses, community dissimilarities increased significantly with environmental dissimilarity and, to a lesser extent, decreased with ship-borne species transport risks, particularly if the ports had similar environments and stepping-stone risks were considered. Unexpectedly, we found no clear effect of shared biogeography, and that risk metrics incorporating estimates of ballast discharge did not offer more explanatory power than simpler traffic-based risks. Overall, we found that shipping homogenizes eukaryotic communities between ports in predictable ways, which could inform improvements in invasive species policy and management. We demonstrated the usefulness of eDNA metabarcoding and dissimilarity regression for disentangling the drivers of large-scale biodiversity patterns. We conclude by outlining logistical considerations and recommendations for future studies using this approach.

Plumbing the depths with environmental DNA (eDNA): Metabarcoding reveals biodiversity zonation at 45-60 m on mesophotic coral reefs.

Mesophotic coral ecosystems (MCEs) are tropical reefs found at depths of ~30-150 m, below the region most heavily impacted by heat stress and other disturbances. Hence, MCEs may serve as potential refugia for threatened shallow reefs, but they also harbour depth-endemic fauna distinct from shallow reefs. Previous studies have characterized biodiversity patterns along depth gradients, but focussed primarily on conspicuous taxa (fishes, corals, etc.). Environmental DNA (eDNA) metabarcoding offers a more holistic approach to assess biodiversity patterns across the tree of life. Here, we use three metabarcoding assays targeting fishes (16S rRNA), eukaryotes (18S rDNA) and metazoans (COI) to assess biodiversity change from the surface to ~90 m depth across 15-m intervals at three sites within the Hawaiian Archipelago. We observed significant community differences between most depth zones, with distinct zonation centred at 45-60 m for eukaryotes and metazoans, but not for fishes. This finding may be attributable to the higher mobility of reef fishes, although methodological limitations are likely a contributing factor. The possibility for MCEs to serve as refugia is not excluded for fishes, but invertebrate communities >45 m are distinct, indicating limited connectivity for the majority of reef fauna. This study provides a new approach for surveying biodiversity on MCEs, revealing patterns in a much broader context than the limited-taxon studies that comprise the bulk of our present knowledge.

Groundwater environmental DNA metabarcoding reveals hidden diversity and reflects land-use and geology.

Despite being the most important source of liquid freshwater on the planet, groundwater is severely threatened by climate change, agriculture, or industrial mining. It is thus extensively monitored for pollutants and declines in quantity. The organisms living in groundwater, however, are rarely the target of surveillance programmes and little is known about the fauna inhabiting underground habitats. The difficulties accessing groundwater, the lack of expertise, and the apparent scarcity of these organisms challenge sampling and prohibit adequate knowledge on groundwater fauna. Environmental DNA (eDNA) metabarcoding provides an approach to overcome these limitations but is largely unexplored. Here, we sampled water in 20 communal spring catchment boxes used for drinking water provisioning in Switzerland, with a high level of replication at both filtration and amplification steps. We sequenced a portion of the COI mitochondrial gene, which resulted in 4917 ASVs, yet only 3% of the reads could be assigned to a species, genus, or family with more than 90% identity. Careful evaluation of the unassigned reads corroborated that these sequences were true COI sequences belonging mostly to diverse eukaryotic groups, not present in the reference databases. Principal component analyses showed a strong correlation of the community composition with the surface land-use (agriculture vs. forest) and geology (fissured rock vs. unconsolidated sediment). While incomplete reference databases limit the assignment of taxa in groundwater eDNA metabarcoding, we showed that taxonomy-free approaches can reveal large hidden diversity and couple it with major land-use drivers, revealing their imprint on chemical and biological properties of groundwater.

Through the eDNA looking glass: Responses of fjord benthic foraminiferal communities to contrasting environmental conditions.

The health of coastal marine environments is severely declining with global changes. Proxies, such as those based on microeukaryote communities, can record biodiversity and ecosystem responses. However, conventional studies rely on microscopic observations of limited taxonomic range and size fraction, missing putatively ecologically informative community components. Here, we tested molecular tools to survey foraminiferal biodiversity in a fjord system (Sweden) on spatial and temporal scales: Alpha and beta diversity responses to natural and anthropogenic environmental trends were assessed and variability of foraminiferal environmental DNA (eDNA) compared to morphology-based data. The identification of eDNA-obtained taxonomic units was aided by single-cell barcoding. Our study revealed wide diversity, including typical morphospecies recognized in the fjords, and so-far unrecognized taxa. DNA extraction method impacted community composition outputs significantly. DNA extractions of 10 g sediment more reliably represented present diversity than of 0.5-g samples and, thus, are preferred for environmental assessments in this region. Alpha- and beta diversity of 10-g extracts correlated with bottom-water salinity similar to morpho-assemblage diversity changes. Sub-annual environmental variability resolved only partially, indicating damped sensitivity of foraminiferal communities on short timescales using established metabarcoding techniques. Systematically addressing the current limitations of morphology-based and metabarcoding studies may strongly improve future biodiversity and environmental assessments.

An Environmental DNA Primer for Microbial and Restoration Ecology.

Environmental DNA (eDNA) sequencing-DNA collected from the environment from living cells or shed DNA-was first developed for working with microbes and has greatly benefitted microbial ecologists for decades since. These tools have only become increasingly powerful with the advent of metabarcoding and metagenomics. Most new studies that examine diverse assemblages of bacteria, archaea, protists, fungi, and viruses lean heavily into eDNA using these newer technologies, as the necessary sequencing technology and bioinformatic tools have become increasingly affordable and user friendly. However, eDNA methods are rapidly evolving, and sometimes it can feel overwhelming to simply keep up with the basics. In this review, we provide a starting point for microbial ecologists who are new to DNA-based methods by detailing the eDNA methods that are most pertinent, including study design, sample collection and storage, selecting the right sequencing technology, lab protocols, equipment, and a few bioinformatic tools. Furthermore, we focus on how eDNA work can benefit restoration and what modifications are needed when working in this subfield.

Extracellular DNA in Environmental Samples: Occurrence, Extraction, Quantification, and Impact on Microbial Biodiversity Assessment.

Environmental DNA, i.e., DNA extracted directly from environmental samples, has been used to understand microbial communities in the environment and to monitor contemporary biodiversity in the conservation context. Environmental DNA often contains both intracellular DNA (iDNA) and extracellular DNA (eDNA). eDNA can persist in the environment and complicate environmental DNA sequencing-based analyses of microbial communities and biodiversity. Although several studies acknowledged the impact of eDNA on DNA-based profiling of environmental communities, eDNA is still being neglected or ignored in most studies dealing with environmental samples. In this article, we summarize key findings on eDNA in environmental samples and discuss the methods used to extract and quantify eDNA as well as the importance of eDNA on the interpretation of experimental results. We then suggested several factors to consider when designing experiments and analyzing data to negate or determine the contribution of eDNA to environmental DNA-based community analyses. This field of research will be driven forward by (i) carefully designing environmental DNA extraction pipelines by taking into consideration technical details in methods for eDNA extraction/removal and membrane-based filtration and concentration; (ii) quantifying eDNA in extracted environmental DNA using multiple methods, including qPCR and fluorescent DNA binding dyes; (iii) carefully interpreting the effect of eDNA on DNA-based community analyses at different taxonomic levels; and (iv) when possible, removing eDNA from environmental samples for DNA-based community analyses.

Fishing for fish environmental DNA: Ecological applications, methodological considerations, surveying designs, and ways forward.

Vast global declines of freshwater and marine fish diversity and population abundance pose serious threats to both ecosystem sustainability and human livelihoods. Environmental DNA (eDNA)-based biomonitoring provides robust, efficient, and cost-effective assessment of species occurrences and population trends in diverse aquatic environments. Thus, it holds great potential for improving conventional surveillance frameworks to facilitate fish conservation and fisheries management. However, the many technical considerations and rapid developments underway in the eDNA arena can overwhelm researchers and practitioners new to the field. Here, we systematically analysed 416 fish eDNA studies to summarize research trends in terms of investigated targets, research aims, and study systems, and reviewed the applications, rationales, methodological considerations, and limitations of eDNA methods with an emphasis on fish and fisheries research. We highlighted how eDNA technology may advance our knowledge of fish behaviour, species distributions, population genetics, community structures, and ecological interactions. We also synthesized the current knowledge of several important methodological concerns, including the qualitative and quantitative power eDNA has to recover fish biodiversity and abundance, and the spatial and temporal representations of eDNA with respect to its sources. To facilitate ecological applications implementing fish eDNA techniques, recent literature was summarized to generate guidelines for effective sampling in lentic, lotic, and marine habitats. Finally, we identified current gaps and limitations, and pointed out newly emerging research avenues for fish eDNA. As methodological optimization and standardization improve, eDNA technology should revolutionize fish monitoring and promote biodiversity conservation and fisheries management that transcends geographic and temporal boundaries.

Can nuclear aquatic environmental DNA be a genetic marker for the accurate estimation of species abundance?

Environmental DNA (eDNA) analysis is a promising tool for the sensitive and effective monitoring of species distribution and abundance. Traditional eDNA analysis has targeted mitochondrial DNA (mtDNA) fragments due to their abundance in cells; however, the quantification may vary depending on cell type and physiology. Conversely, some recent eDNA studies have targeted multi-copy nuclear DNA (nuDNA) fragments, such as ribosomal RNA genes, in water, and reported a higher detectability and more rapid degradation than mitochondrial eDNA (mt-eDNA). These properties suggest that nuclear eDNA (nu-eDNA) may be useful for the accurate estimation of species abundance relative to mt-eDNA, but which remains unclear. In this study, we compiled previous studies and re-analyzed the relationships between mt- and nu-eDNA concentration and species abundance by comparing the R2 values of the linear regression. We then performed an aquarium experiment using zebrafish (Danio rerio) to compare the relationships across genetic regions, including single-copy nuDNA. We found more accurate relationships between multi-copy nu-eDNA and species abundance than mt-eDNA in these datasets, although the difference was not significant upon weighted-averaging the R2 values. Moreover, we compared the decay rate constants of zebrafish eDNA across genetic regions and found that multi-copy nu-eDNA degraded faster than mt-eDNA under pH 7, implying a quick turnover of multi-copy nu-eDNA in the field. Although further empirical studies of nu-eDNA applications are necessary to support our findings, this study provides the groundwork for improving the estimation accuracy of species abundance via eDNA analysis.

Biodiversity exploration in autumn using environmental DNA in the South China sea.

The South China Sea (SCS) is an important part of the Indo-Pacific convergence zone, with high biodiversity and abundant marine resources. Traditional methods are primarily used to monitor biodiversity. However, a few studies have used environmental DNA (eDNA) metabarcoding to research the assemblage structure of the SCS. This study used eDNA metabarcoding to survey the SCS assemblage and its relationship with environmental factors over a month-long time-series (August 30th to September 30th, 2020) of seawater samples from the central part of the SCS (9°-20°86' N, 113°-118°47' E). 32 stations were divided into six groups (A, B, C, D, E, F) according to longitude. We collected water samples, extracted eDNA, and amplified 18S rRNA gene V4 region (18S V4), 18S rRNA gene V9 region (18S V9), and 12S rRNA gene (12S). Krona diagrams were used to show species composition. We identified 192 phytoplankton, 104 invertebrate, and 61 fish species from 18S V4, 18S V9, and 12S, respectively. Generally, the three assemblage structures exhibited an increase in species diversity with increasing longitude. Group E had the highest fish diversity. Groups F and C had the highest phytoplankton and invertebrate diversity, respectively. Canonical correspondence analysis showed that four factors (chlorophyll a, depth, salinity, and temperature) were correlated with assemblage structure. Chlorophyll a was the main environmental factor that affected fish, phytoplankton, and invertebrate assemblage structures; salinity was strongly correlated with fish and invertebrate assemblage structures; temperature was a key factor that impacted fish and invertebrate assemblage structures; and depth was strongly correlated with invertebrate assemblage structure. Our results revealed that eDNA metabarcoding is a powerful tool for improving detection rate and using multiple markers is an effective approach for monitoring biodiversity. This study provided information that can be used to enhance biodiversity protection efforts in the SCS.

Chemical and microbial diversity covary in fresh water to influence ecosystem functioning.

Invisible to the naked eye lies a tremendous diversity of organic molecules and organisms that make major contributions to important biogeochemical cycles. However, how the diversity and composition of these two communities are interlinked remains poorly characterized in fresh waters, despite the potential for chemical and microbial diversity to promote one another. Here we exploited gradients in chemodiversity within a common microbial pool to test how chemical and biological diversity covary and characterized the implications for ecosystem functioning. We found that both chemodiversity and genes associated with organic matter decomposition increased as more plant litterfall accumulated in experimental lake sediments, consistent with scenarios of future environmental change. Chemical and microbial diversity were also positively correlated, with dissolved organic matter having stronger effects on microbes than vice versa. Under our experimental scenarios that increased sediment organic matter from 5 to 25% or darkened overlying waters by 2.5 times, the resulting increases in chemodiversity could increase greenhouse gas concentrations in lake sediments by an average of 1.5 to 2.7 times, when all of the other effects of litterfall and water color were considered. Our results open a major new avenue for research in aquatic ecosystems by exposing connections between chemical and microbial diversity and their implications for the global carbon cycle in greater detail than ever before.

Detection of fish species from marine protected areas of the North Sea using environmental DNA.

This report describe the first application of environmental DNA-metabarcoding approach for the assessment of fish species diversity in two marine protected areas of the North Sea: the Doggerbank and the Sylt Outer Reef. We collected 64 water samples and detected 24 fish species. We discuss qualitative differences between MPAs and compare the results with those obtained from bottom-trawl surveys in the same areas. We found three additional species to those documented in the same year with trawls, including the critically endangered European eel.

Depicting Temporal, Functional, and Phylogenetic Patterns in Estuarine Diazotrophic Communities from Environmental DNA and RNA.

Seasonally nitrogen-limited and phosphorus-replete temperate coastal waters generally host dense and diverse diazotrophic communities. Despite numerous studies in marine systems, little is known about diazotrophs and their functioning in oligohaline estuarine environments. Here we applied a combination of nifH transcript and metagenomic shotgun sequencing approaches to investigate temporal shifts in taxonomic composition and nifH activity of size-fractionated diazotrophic communities in a shallow and mostly freshwater coastal lagoon. Patterns in active nifH phylotypes exhibited a clear seasonal succession, which reflected their different tolerances to temperature change and nitrogen (N) availability. Thus, in spring, heterotrophic diazotrophs (Proteobacteria) dominated the nifH phylotypes, while increasing water temperature and depletion of inorganic N fostered heterocystous Cyanobacteria in summer. Metagenomic data demonstrated four main N-cycling pathways and three of them with a clear seasonal pattern: denitrification (spring) → N2 fixation (summer) → assimilative NO3- reduction (fall), with NH4+ uptake into cells occurring across all seasons. Although a substantial denitrification signal was observed in spring, it could have originated from the re-suspended benthic rather than planktonic community. Our results contribute to a better understanding of the realized genetic potential of pelagic N2 fixation and its seasonal dynamics in oligohaline estuarine ecosystems, which are natural coastal biogeochemical reactors.