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Faecal contamination of freshwater and marine environments represents a significant risk for public health, recreational activity and food safety, and tools for evaluating complex multi-source contamination remain largely in the development phase. We evaluated the efficacy of the Fast Expectation Maximization (FEAST) microbial source tracking (MST) algorithm to apportion sources of faecal contamination among four mammalian species of interest in coastal waters in New Zealand. Using 16S ribosomal DNA metabarcoding of faecal samples from cows, fur seals, and sheep, as well as human wastewater, we aimed to differentiate and quantify the contribution of these sources in mixed faecal samples. Multivariate analysis confirmed significant differences in the microbial communities associated with each mammalian source, with specific bacterial classes indicative of different sources. The FEAST algorithm was tested using mixed DNA and mixed faecal samples, and we found that the algorithm correctly assigned the dominant source from all samples, but underestimated the dominant source's proportional contribution. This underestimation suggests the need for further refinement and validation to ensure accurate source apportionment in environmental samples where the faecal signal is likely to be a minor component. Despite these limitations, the findings of our study, in combination with the evidence from others who have tested the FEAST algorithm in environmental settings, indicates that it represents an advance on existing tools for microbial source tracking and may become a useful addition to the toolbox for environmental management., 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 © 2024 Elsevier Ltd. All rights reserved.)

Tetrodotoxin (TTX) is a potent neurotoxin causing human intoxications from contaminated seafood worldwide and is of emerging concern in Europe. Shellfish have been shown to contain varying TTX concentrations globally, with concentrations typically higher in Pacific oysters Crassostrea gigas in Europe. Despite many decades of research, the source of TTX remains unknown, with bacterial or algal origins having been suggested. The aim of this study was to identify potential source organisms causing TTX contamination in Pacific oysters in French coastal waters, using three different techniques. Oysters were deployed in cages from April to September 2021 in an estuary where TTX was previously detected. Microscopic analyses of water samples were used to investigate potential microalgal blooms present prior or during the peak in TTX. Differences in the bacterial communities from oyster digestive glands (DG) and remaining flesh were explored using metabarcoding, and lastly, droplet digital PCR assays were developed to investigate the presence of Cephalothrix sp., one European TTX-bearing species in the DG of toxic C. gigas. Oysters analysed by liquid chromatography-tandem mass spectrometry contained quantifiable levels of TTX over a three-week period (24 June-15 July 2021), with concentrations decreasing in the DG from 424 μg/kg for the first detection to 101 μg/kg (equivalent to 74 to 17 μg/kg of total flesh), and trace levels being detected until August 13, 2021. These concentrations are the first report of the European TTX guidance levels being exceeded in French shellfish. Microscopy revealed that some microalgae bloomed during the TTX peak, (e.g., Chaetoceros spp., reaching 40,000 cells/L). Prokaryotic metabarcoding showed increases in abundance of Rubritaleaceae (genus Persicirhabdus) and Neolyngbya, before and during the TTX peak. Both phyla have previously been described as possible TTX-producers and should be investigated further. Droplet digital PCR analyses were negative for the targeted TTX-bearing genus Cephalothrix., 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 © 2024 Elsevier Ltd. All rights reserved.)

The Arabian Peninsula accounts for approximately 6% of the world's coral reefs. Some thrive in extreme environments of temperature and salinity. Using 51 Autonomous Reef Monitoring Structure (ARMS), a standardized non-destructive monitoring device, we investigated the spatial patterns of coral reef cryptobenthic diversity in four ecoregions around the Arabian Peninsula and analyzed how geographical and/or environmental drivers shape those patterns. The mitochondrial cytochrome c oxidase subunit I (COI) gene was used to identify Amplicon Sequence Variants and assign taxonomy of the cryptobenthic organisms collected from the sessile and mobile fractions of each ARMS. Cryptobenthic communities sampled from the two ecoregions in the Red Sea showed to be more diverse than those inhabiting the Arabian (Persian) Gulf and the Gulf of Oman. Geographic distance revealed a stronger relationship with beta diversity in the Mantel partial correlation than environmental distance. However, the two mobile fractions (106-500 µm and 500-2000 µm) also had a significant correlation between environmental distance and beta diversity. In our study, dispersal limitations explained the beta diversity patterns in the selected reefs, supporting the neutral theory of ecology. Still, increasing differences in environmental variables (environmental filtering) also had an effect on the distribution patterns of assemblages inhabiting reefs within short geographic distances. The influence of geographical distance in the cryptofauna assemblages makes these relevant, yet usually ignored, communities in reef functioning vulnerable to large scale coastal development and should be considered in ecosystem management of such projects., (© 2024. The Author(s).)

Freshwater fish biodiversity and abundance are decreasing globally. The drivers of decline are primarily anthropogenic; however, the causative links between disturbances and fish community change are complex and challenging to investigate. We used a suite of sedimentary DNA methods (droplet digital PCR and metabarcoding) and traditional paleolimnological approaches, including pollen and trace metal analysis, ITRAX X-ray fluorescence and hyperspectral core scanning to explore changes in fish abundance and drivers over 1390 years in a small lake. This period captured a disturbance trajectory from pre-human settlement through subsistence living to intensive agriculture. Generalized additive mixed models explored the relationships between catchment inputs, internal drivers, and fish community structure. Fish community composition distinctly shifted around 1350 CE, with the decline of a sensitive Galaxias species concomitant with early land use changes. Total fish abundance significantly declined around 1950 CE related to increases in ruminant bacterial DNA (a proxy for ruminant abundance) and cadmium flux (a proxy for phosphate fertilizers), implicating land use intensification as a key driver. Concurrent shifts in phytoplankton and zooplankton suggested that fish communities were likely impacted by food web dynamics. This study highlights the potential of sedDNA to elucidate the long-term disturbance impacts on biological communities in lakes., 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 © 2024. Published by Elsevier B.V.)

An essential component of the coral reef animal diversity is the species hidden in crevices within the reef matrix, referred to as the cryptobiome. These organisms play an important role in nutrient cycling and provide an abundant food source for higher trophic levels, yet they have been largely overlooked. Here, we analyzed the distribution patterns of the mobile cryptobiome (>2000 μm) along the latitudinal gradient of the Saudi Arabian coast of the Red Sea. Analysis was conducted based on 54 Autonomous Reef Monitoring Structures. We retrieved a total of 5273 organisms, from which 2583 DNA sequences from the mitochondrially encoded cytochrome c oxidase I were generated through sanger sequencing. We found that the cryptobiome community is variable over short geographical distances within the basin. Regression tree models identified sea surface temperature (SST), percentage cover of hard coral and turf algae as determinant for the number of operational taxonomic units present per Autonomous Reef Monitoring Structures (ARMS). Our results also show that the community structure of the cryptobiome is associated with the energy available (measured as photosynthetic active radiation), sea surface temperature, and nearby reef habitat characteristics (namely hard corals, turf and macroalgae). Given that temperature and reef benthic characteristics affect the cryptobiome, current scenarios of intensive climate change are likely to modify this fundamental biological component of coral reef functioning. However, the trajectory of change is unknow and can be site specific, as for example, diversity is expected to increase above SST of 28.5°C, and with decreasing hard coral and turf cover. This study provides a baseline of the cryptobenthic community prior to major coastal developments in the Red Sea to be used for future biodiversity studies and monitoring projects. It can also contribute to better understand patterns of reef biodiversity in a period where Marine Protected Areas are being discussed in the region., Competing Interests: NO authors have competing interests, (Copyright: © 2024 Villalobos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Two gene regions commonly used to characterise the diversity of eukaryotic communities using metabarcoding are the 18S ribosomal DNA V4 and V9 gene regions. We assessed the effectiveness of these two regions for characterising diverisity of coastal eukaryotic microalgae communities (EMCs) from tropical and temperate sites. We binned amplicon sequence variants (ASVs) into the high level taxonomic groups: dinoflagellates, pennate diatoms, radial centric diatoms, polar centric diatoms, chlorophytes, haptophytes and 'other microalgae'. When V4 and V9 generated ASV abundances were compared, the V9 region generated a higher number of raw reads, captured more diversity from all high level taxonomic groups and was more closely aligned with the community composition determined using light microscopy. The V4 region did resolve more ASVs to a deeper taxonomic resolution within the dinoflagellates, but did not effectively resolve other major taxonomic divisions. When characterising these communities via metabarcoding, the use of multiple gene regions is recommended, but the V9 gene region can be used in isolation to provide high-level community biodiversity to reflect relative abundances within groups. This approach reduces the cost of sequencing multiple gene regions whilst still providing important baseline ecosystem function information., (© 2024. The Author(s).)

The Symbiodinium genus is ancestral among other Symbiodiniaceae lineages with species that are both symbiotic and free living. Changes in marine ecosystems threaten their existence and crucial ecological roles. Cryopreservation offers an avenue for their long-term storage for future habitat restoration after coral bleaching. In our previous study we demonstrated that high salinity treatments of Symbiodiniaceae isolates led to changes in their fatty acid (FA) profiles and higher cell viabilities after cryopreservation. In this study, we investigated the role of increased salinity on FA production and the genes involved in FA biosynthesis and degradation pathways during the cryopreservation of Symbiodinium pilosum. Overall, there was a twofold increase in mass of FAs produced by S. pilosum after being cultured in medium with increased salinity (54 parts per thousand; ppt). Dimethyl sulfoxide (Me 2 SO) led to a ninefold increase of FAs in standard salinity (SS) treatment, compared to a fivefold increase in increased salinity (IS) treatments. The mass of the FA classes returned to baseline during recovery. Transcriptomic analyses showed an acyl carrier protein gene was significantly upregulated after Me 2 SO treatment in the SS cultures. Cytochrome P450 reductase genes were significantly down regulated after Me 2 SO addition in SS treatment preventing FA degradation. These changes in the expression of FA biosynthesis and degradation genes contributed to more FAs in SS treated isolates. Understanding how increased salinity changes FA production and the roles of specific genes in regulating FA pathways will help improve current freezing protocols for Symbiodiniaceae and other marine microalgae., Competing Interests: Declarations of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cyanobacterial blooms are one of the most significant threats to global water security and freshwater biodiversity. Interactions among multiple stressors, including habitat degradation, species invasions, increased nutrient runoff, and climate change, are key drivers. However, assessing the role of anthropogenic activity on the onset of cyanobacterial blooms and exploring response variation amongst lakes of varying size and depth is usually limited by lack of historical records. In the present study we applied molecular, paleolimnological (trace metal, Itrax-µ-XRF and hyperspectral scanning, chronology), paleobotanical (pollen) and historical data to reconstruct cyanobacterial abundance and community composition and anthropogenic impacts in two dune lakes over a period of up to 1200 years. Metabarcoding and droplet digital PCR results showed very low levels of picocyanobacteria present in the lakes prior to about CE 1854 (1839-1870 CE) in the smaller shallow Lake Alice and CE 1970 (1963-1875 CE) in the larger deeper Lake Wiritoa. Hereafter bloom-forming cyanobacteria were detected and increased notably in abundance post CE 1984 (1982-1985 CE) in Lake Alice and CE 1997 (1990-2007 CE) in Lake Wiritoa. Currently, the magnitude of blooms is more pronounced in Lake Wiritoa, potentially attributable to hypoxia-induced release of phosphorus from sediment, introducing an additional source of nutrients. Generalized linear modelling was used to investigate the contribution of nutrients (proxy = bacterial functions), temperature, redox conditions (Mn:Fe), and erosion (Ti:Inc) in driving the abundance of cyanobacteria (ddPCR). In Lake Alice nutrients and erosion had a statistically significant effect, while in Lake Wiritoa nutrients and redox conditions were significant., 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 © 2023 Elsevier B.V. All rights reserved.)

Anthropogenic eutrophication is one of the most pressing issues facing lakes globally. Our ability to manage lake eutrophication is hampered by the limited spatial and temporal extents of monitoring records, stemming from the time-consuming and expensive nature of physiochemical and biological monitoring. Diatom-based biomonitoring presents an alternative to traditional eutrophication monitoring, yet it is restricted by the high degree of taxonomic expertise required. Environmental DNA metabarcoding, while providing a promising substitute for diatom community enumeration, is plagued by inadequate taxonomic coverage of reference databases and methodological bias, limiting its use for biomonitoring. Here we show that taxonomy-free diatom-biomonitoring, in which environmental DNA metabarcoding data is utilised but not assigned to specific taxonomic classes, presents an accurate, fast, and relatively automated alternative to taxonomically assigned eutrophication biomonitoring. Our taxonomy-free index accounted for 85% of trophic level variability across 89 lakes and had the lowest average prediction error of the three approaches tested. By not relying on taxonomic identification or metabarcoding reference databases, taxonomy-free biomonitoring maintains diatom diversity that is lost in taxonomic assignment using molecular approaches. Furthermore, by utilising lake sediments, the approach outlined here presents a time-integrated estimation of lake trophic level and thus does not require time-consuming seasonal sampling. Taxonomy-free biomonitoring addresses the limitations of traditional physicochemical eutrophication monitoring and taxonomic biomonitoring alternatives and can be used to extend the spatial and temporal extents of eutrophication monitoring., 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 © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Managing marine nonindigenous species (mNIS) is challenging, because marine environments are highly connected, allowing the dispersal of species across large spatial scales, including geopolitical borders. Cross-border inconsistencies in biosecurity management can promote the spread of mNIS across geopolitical borders, and incursions often go unnoticed or unreported. Collaborative surveillance programs can enhance the early detection of mNIS, when response may still be possible, and can foster capacity building around a common threat. Regional or international databases curated for mNIS can inform local monitoring programs and can foster real-time information exchange on mNIS of concern. When combined, local species reference libraries, publicly available mNIS databases, and predictive modeling can facilitate the development of biosecurity programs in regions lacking baseline data. Biosecurity programs should be practical, feasible, cost-effective, mainly focused on prevention and early detection, and be built on the collaboration and coordination of government, nongovernment organizations, stakeholders, and local citizens for a rapid response., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Institute of Biological Sciences.)

Cyanobacterial blooms are increasing in frequency and intensity globally, impacting lake ecosystem health and posing a risk to human and animal health due to the toxins they can produce. Cyanobacterial pigments preserved in lake sediments provide a useful means of understanding the changes that have led to cyanobacterial blooms in lakes. However, there is some uncertainty as to whether specific carotenoids are unique to certain genera or types of cyanobacteria. To fill this knowledge gap, we analyzed pigments in 34 cyanobacteria cultures and applied the findings to sediments from three New Zealand lakes. The cyanobacterial carotenoids canthaxanthin, echinenone and zeaxanthin were detected in all cultures, whereas myxoxanthophyll was only detected in ten cultures (Microcoleus, Planktothrix and the picocyanobacteria cultures; Synechococcaceae). The sum of the individual carotenoid concentrations provided the strongest relationship with cyanobacterial biomass (R 2  = 0.58) and could be used in paleolimnology studies to evaluate general cyanobacterial abundance. Ratios of canthaxanthin, zeaxanthin and myxoxanthophyll relative to echinenone indicated that carotenoid ratios could be used to differentiate picocyanobacteria and bloom-forming cyanobacteria, to some degree. High zeaxanthin/echinenone ratios were measured in picocyanobacteria and low zeaxanthin/echinenone ratios were measured in bloom-forming cyanobacteria. The zeaxanthin/echinenone ratio was applied to sediment core samples where the cyanobacterial community was also evaluated by 16S rRNA gene metabarcoding, with the zeaxanthin/echinenone ratios showing similar patterns to those observed in the cultures. The preliminary assessment described here suggests that zeaxanthin/echinenone ratios could provide a valuable paleoecological proxy for evaluating historical shifts in cyanobacterial communities and warrants further exploration., 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 © 2023 Elsevier B.V. All rights reserved.)

Advances in high-throughput sequencing (HTS) technologies and their increasing affordability have fueled environmental DNA (eDNA) metabarcoding data generation from freshwater, marine and terrestrial ecosystems. Research institutions worldwide progressively employ HTS for biodiversity assessments, new species discovery and ecological trend monitoring. Moreover, even non-scientists can now collect an eDNA sample, send it to a specialized laboratory for analysis and receive in-depth biodiversity record from a sampling site. This offers unprecedented opportunities for biodiversity assessments across wide temporal and spatial scales. The large volume of data produced by metabarcoding also enables incidental detection of species of concern, including non-indigenous and pathogenic organisms. We introduce an online app-Pest Alert Tool-for screening nuclear small subunit 18S ribosomal RNA and mitochondrial cytochrome oxidase subunit I datasets for marine non-indigenous species as well as unwanted and notifiable marine organisms in New Zealand. The output can be filtered by minimum length of the query sequence and identity match. For putative matches, a phylogenetic tree can be generated through the National Center for Biotechnology Information's BLAST Tree View tool, allowing for additional verification of the species of concern detection. The Pest Alert Tool is publicly available at https://pest-alert-tool-prod.azurewebsites.net/., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Lakes provide crucial ecosystem services and harbour unique and rich biodiversity, yet despite decades of research and management focus, cultural eutrophication remains a predominant threat to their health. Our ability to manage lake eutrophication is restricted by the lack of long-term monitoring records. To circumvent this, we developed a bio-indicator approach for inferring trophic level from lake diatom communities and applied this to sediment cores from two lakes experiencing eutrophication stress. Diatom indicators strongly predicted observed trophic levels, and when applied to sediment cores, diatom predicted trophic level reconstructions were consistent with monitoring data and land-use histories. However, there were significant recent shifts in diatom communities not captured by the diatom-based index or monitoring data, suggesting that conventional trophic level indices obscure important ecological change. New approaches, such as the one in this study, are critical to detect early changes in water quality and prevent the decline of lake ecosystems worldwide., 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 © 2023. Published by Elsevier B.V.)

Interactions among multiple stressors, legacies of past perturbations, and the lack of historical information make it difficult to determine the influence of individual anthropogenic impacts on lakes and separate them from natural ecosystem variability. In the present study, we coupled paleolimnological approaches, historical data, and ecological experiments to disentangle the impacts of multiple long-term stressors on lake ecosystem structure and function. We found that the lake structure and function remained resistant to the impacts of catchment deforestation and erosion, and the introduction of several exotic fish species. Changes in ecosystem structure and function were consistent, with nutrient enrichment being the primary driver of change. Significant and sustained changes in the lake diatom community structure (and their nutrient requirements), bacterial community function, and paleolimnological proxies of ecosystem function coincided with nitrogen and phosphorus fertilizers in the catchment. The results highlight that the effects of increased nutrient inputs are much stronger than the influence of other, potentially significant, drivers of ecosystem change, and that the degree of nutrient impact can be underestimated by environmental monitoring due to its diffuse and accumulative nature. Delineating the effects of multiple anthropogenic drivers requires long-term records of both impacts and lake ecosystem change across multiple trophic levels.

Lakes provide habitat for a diverse array of species and offer a wide range of ecosystem services for humanity. However, they are highly vulnerable as they are not only impacted by adverse actions directly affecting them, but also those on the surrounding environment. Improving knowledge on the processes responsible for community assembly in different biotic components will aid in the protection and restoration of lakes. Studies to date suggested a combination of deterministic (where biotic/abiotic factors act on fitness differences amongst taxa) and stochastic (where dispersal plays a larger factor in community assembly) processes are responsible for structuring biotic communities, but there is no consensus on the relative roles these processes play, and data is lacking for lakes. In the present study, we sampled different biotic components in 34 lakes located on the South Island of New Zealand. To obtain a holistic view of assembly processes in lakes we used metabarcoding to investigate bacteria in the sediment and surface waters, and eukaryotes in the sediment and two different size fractions of the water column. Physicochemical parameters were collected in parallel. Results showed that deterministic processes dominated the assembly of lake communities although the relative importance of variable and homogeneous selection differed among the biotic components. Variable selection was more important in the sediment (SSbact and SSeuks) and for the bacterioplankton (Pbact) while the assembly of the eukaryotic plankton (SPeuks, LPeuks) was driven more by homogeneous selection. The ease of human access to the lakes had a significant effect on lake communities. In particular, clade III of SAR11 and Daphnia pulex were only present in lakes with public access. This study provides insights into the distribution patterns of different biotic components and highlights the value in understanding the drivers of different biological communities within lakes., Competing Interests: Susanna A Wood is an Academic Editor for PeerJ. John K. Pearman, Georgia Thomson-Laing, Lucy Thompson, Sean Waters and Susanna A. Wood are employed by the Cawthron Institute. Marcus J. Vandergoes is employed by GNS Science., (© 2022 Pearman et al.)

The combination of molecular tools, standard surveying techniques, and long-term monitoring programs are relevant to understanding environmental and ecological changes in coral reef communities. Here we studied temporal variability in cryptobenthic coral reef communities across the continental shelf in the central Red Sea spanning 6 years (three sampling periods: 2013-2019) and including the 2015 mass bleaching event. We used a combination of molecular tools (barcoding and metabarcoding) to assess communities on Autonomous Reef Monitoring Structures (ARMS) as a standardized sampling approach. Community composition associated with ARMS for both methodologies (barcoding and metabarcoding) was statistically different across reefs (shelf position) and time periods. The partition of beta diversity showed a higher turnover and lower nestedness between pre-bleaching and post-bleaching samples than between the two post-bleaching periods, revealing a community shift from the bleaching event. However, a slight return to the pre-bleaching community composition was observed in 2019 suggesting a recovery trajectory. Given the predictions of decreasing time between bleaching events, it is concerning that cryptobenthic communities may not fully recover and communities with new characteristics will emerge. We observed a high turnover among reefs for all time periods, implying a homogenization of the cryptobiome did not occur across the cross shelf following the 2015 bleaching event. It is possible that dispersal limitations and the distinct environmental and benthic structures present across the shelf maintained the heterogeneity in communities among reefs. This study has to the best of our knowledge presented for the first time a temporal aspect into the analysis of ARMS cryptobenthic coral reef communities and encompasses a bleaching event. We show that these structures can detect cryptic changes associated with reef degradation and provides support for these being used as long-term monitoring tools., (© 2022. The Author(s).)

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).)

Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Lake sediments accumulate information on biological communities thus acting as natural archives. Traditionally paleolimnology has focussed on fossilized remains of organisms, however, many organisms do not leave fossil evidence, meaning major ecosystem components are missing from environmental reconstructions. Many paleolimnology studies now incorporate molecular methods, including investigating microbial communities using environmental DNA (eDNA), but there is uncertainty about the contribution of living organisms to molecular inventories. In the present study, we obtained DNA and RNA inventories from sediment spanning 700 years to investigate the contribution of past and active communities to the molecular signal from sedimentary archives. Additionally, a droplet digital PCR (ddPCR) targeting the 16S ribosomal RNA (16S rRNA) gene of the photosynthetic cyanobacterial genera Microcystis was used to explore if RNA signals were from legacy RNA. We posit that the RNA signal is a mixture of legacy RNA, dormant cells, living bacteria and modern-day trace level contaminants that were introduced during sampling and preferentially amplified. The presence of legacy RNA was confirmed by the detection of Microcystis in sediments aged to ~200 years ago. Recent comparisons between 16S rRNA gene metabarcoding and traditional paleo proxies showed that past changes in bacterial communities can be reconstructed from sedimentary archives. The recovery of RNA in the present study has provided new insights into the origin of these signals. However, caution is required during analysis and interpretation of 16S rRNA gene metabarcoding data especially in recent sediments were there are potentially active bacteria., (© 2021 John Wiley & Sons Ltd.)

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.)

Bacteria are vital components of lake systems, driving a variety of biogeochemical cycles and ecosystem services. Bacterial communities have been shown to have a skewed distribution with a few abundant species and a large number of rare species. The contribution of environmental processes or geographic distance in structuring these components is uncertain. The discrete nature of lakes provides an ideal test case to investigate microbial biogeographical patterns. In the present study, we used 16S rRNA gene metabarcoding to examine the distribution patterns on local and regional scales of abundant and rare planktonic bacteria across 167 New Zealand lakes covering broad environmental gradients. Only a few amplicon sequence variants (ASVs) were abundant with a higher proportion of rare ASVs. The proportion of locally abundant ASVs was negatively correlated with the percentage of high productivity grassland in the catchment and positively with altitude. Regionally rare ASVs had a restricted distribution and were only found in one or a few lakes. In general, regionally abundant ASVs had higher occupancy rates, although there were some with restricted occupancy. Environmental processes made a higher contribution to structuring the regionally abundant community, while geographic distances were more important for regionally rare ASVs. A better understanding of the processes structuring the abundance and distribution of bacterial communities within lakes will assist in understand microbial biogeography and in predicting how these communities might shift with environmental change., 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 Pearman, Thomson-Laing, Thomson-Laing, Thompson, Waters, Reyes, Howarth, Vandergoes and Wood.)

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.)

Picocyanobacteria (Pcy) are important yet understudied components of lake foodwebs. While phylogenetic studies of isolated strains reveal a high diversity of freshwater genotypes, little is known about abiotic drivers associated with Pcy in different lakes. Due to methodological limitations, most previous studies assess potential drivers using total cell abundances as a response, with often conflicting and inconsistent results. In the present study, we explored how picocyanobacterial communities respond to environmental change using a combination of epifluorescence microscopy and community data determined using 16S rRNA gene metabarcoding. Temporal shifts in picocyanobacterial abundance, diversity and community dynamics were assessed in relation to potential environmental drivers in five contrasting lakes over 1year. Cell abundances alone were not consistently related to environmental variables across lakes. However, the addition of metabarcoding data revealed diverse picocyanobacterial communities that differed significantly between lakes, driven by environmental variables related to trophic state. Within each lake, communities were temporally dynamic and certain amplicon sequence variants (ASVs) were strongly associated with specific environmental drivers. Rapid shifts in community structure and composition were often related to environmental changes, indicating that lacustrine Pcy can persist at high abundances through collective community adaptation. These results demonstrate that a combination of microscopy and metabarcoding enables an in-depth characterisation of picocyanobacterial communities and reveals strain-specific drivers. We recommend that future studies cease referring to picocyanobacterial as one functional group and take strain specific variability into consideration., 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 Schallenberg, Pearman, Burns and Wood.)

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.)

Environmental genomics is a promising field for monitoring biodiversity in a timely fashion. Efforts have increasingly been dedicated to the use of bacteria DNA derived data to develop biotic indices for benthic monitoring. However, a substantial debate exists about whether bacteria-derived data using DNA metabarcoding should follow, for example, a taxonomy-based or a taxonomy-free approach to marine bioassessments. Here, we showcase the value of DNA-based monitoring using the impact of fish farming as an example of anthropogenic disturbances in coastal areas and compare the performance of taxonomy-based and taxonomy-free approaches in detecting environmental alterations. We analysed samples collected near to the farm cages and along distance gradients from two aquaculture installations, and at control sites, to evaluate the effect of this activity on bacterial assemblages. Using the putative response of bacterial taxa to stress we calculated the taxonomy-based biotic index microgAMBI. The distribution of individual amplicon sequence variants (ASVs), as a function of a gradient in sediment acid volatile sulphides, was then used to derive a taxonomy-free bacterial biotic index specific for this data set using a de novo approach based on quantile regression splines. Our results show that microgAMBI revealed a organically enriched environment along the gradient. However, the de novo biotic index outperformed microgAMBI by providing a higher discriminatory power in detecting changes in abiotic factors directly related to fish production, whilst allowing the identification of new ASVs bioindicators. The de novo strategy applied here represents a robust method to define new bioindicators in regions or habitats where no previous information about the response of bacteria to environmental stressors exists., (© 2021 John Wiley & Sons Ltd.)

Freshwater picocyanobacteria (Pcy) are important yet understudied components of lake ecosystems. Most previous studies have relied on cell abundances to assess Pcy dynamics in largely oligotrophic lakes, while little is known about spatial diversity and dynamics across different lake types. In the present study we assessed the horizontal-spatial abundance and community structure of Pcy in two contrasting (oligotrophic and hypertrophic) New Zealand lakes using epifluorescence microscopy and 16S rRNA metabarcoding. Pcy abundance and community composition differed significantly both between and within the oligotrophic and hypertrophic lakes. While spatial variability was observed in both study lakes, these differences were particularly pronounced in the oligotrophic, morphometrically complex Lake Wanaka where cell abundances were typically higher in bays than open-water sites and community structure differed significantly between sites. Community structuring appeared to be driven by localised environmental conditions, with different factors influencing each lake. These results suggest that single spot-samples are insufficient to gain an understanding of Pcy dynamics and consequently, phytoplankton dynamics in lakes., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Lake sediments are natural archives that accumulate information on biological communities and their surrounding catchments. Paleolimnology has traditionally focussed on identifying fossilized organisms to reconstruct past environments. In the last decade, the application of molecular methodologies has increased in paleolimnological studies, but further research investigating factors such as sample heterogeneity and DNA degradation are required. In the present study we investigated bacterial community heterogeneity (16S rRNA metabarcoding) within depth slices (1-cm width). Sediment cores were collected from three lakes with differing sediment compositions. Samples were collected from a variety of depths which represent a period of time of approximately 1,200 years. Triplicate samples were collected from each depth slice and bacterial 16S rRNA metabarcoding was undertaken on each sample. Accumulation curves demonstrated that except for the deepest (oldest) slices, the combination of three replicate samples were insufficient to characterise the entire bacterial diversity. However, shared Amplicon Sequence Variants (ASVs) accounted for the majority of the reads in each depth slice (max. shared proportional read abundance 96%, 86%, 65% in the three lakes). Replicates within a depth slice generally clustered together in the Non-metric multidimensional scaling analysis. There was high community dissimilarity in older sediment in one of the cores, which was likely due to the laminae in the sediment core not being horizontal. Given that most paleolimnology studies explore broad scale shifts in community structure rather than seeking to identify rare species, this study demonstrates that a single sample is adequate to characterise shifts in dominant bacterial ASVs., Competing Interests: MJS works for GNS Science while JKP, GTL, LT and SAW work for Cawthron Institute. The funders provided support in the form of salaries for the authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. The commercial affiliations do not alter our adherence to PLOS ONE policies on sharing data and materials.

Autonomous Reef Monitoring Structures (ARMS) have been applied worldwide to characterize the critical yet frequently overlooked biodiversity patterns of marine benthic organisms. In order to disentangle the relevance of environmental factors in benthic patterns, here, through standardized metabarcoding protocols, we analyse sessile and mobile (<2 mm) organisms collected using ARMS deployed across six regions with different environmental conditions (3 sites × 3 replicates per region): Baltic, Western Mediterranean, Adriatic, Black and Red Seas, and the Bay of Biscay. A total of 27,473 Amplicon Sequence Variants (ASVs) were observed ranging from 1,404 in the Black Sea to 9,958 in the Red Sea. No ASVs were shared among all regions. The highest number of shared ASVs was between the Western Mediterranean and the Adriatic Sea (116) and Bay of Biscay (115). Relatively high numbers of ASVs (103), mostly associated with the genus Amphibalanus, were also shared between the lower salinity seas (Baltic and Black Seas). We found that compositional differences in spatial patterns of rocky-shore benthos are determined slightly more by dispersal limitation than environmental filtering. Dispersal limitation was similar between sessile and mobile groups, while the sessile group had a larger environmental niche breadth than the mobile group. Further, our study can provide a foundation for future evaluations of biodiversity patterns in the cryptobiome, which can contribute up to 70% of the local biodiversity., (© 2020 John Wiley & Sons Ltd.)

Nutrient loading is a major threat to estuaries and coastal environments worldwide, therefore, it is critical that we have good monitoring tools to detect early signs of degradation in these ecologically important and vulnerable ecosystems. Traditionally, bottom-dwelling macroinvertebrates have been used for ecological health assessment but recent advances in environmental genomics mean we can now characterize less visible forms of biodiversity, offering a more holistic view of the ecosystem and potentially providing early warning signals of disturbance. We carried out a manipulative nutrient enrichment experiment (0, 150 and 600 g N fertilizer m -2 ) in two estuaries in New Zealand to assess the effects of nutrient loading on benthic communities. After seven months of enrichment, environmental DNA (eDNA) metabarcoding was used to examine the response of eukaryotic (18S rRNA), diatom only (rbcL) and bacterial (16S rRNA) communities. Multivariate analyses demonstrated changes in eukaryotic, diatom and bacterial communities in response to nutrient enrichment at both sites, despite differing environmental conditions. These patterns aligned with changes in macrofaunal communities identified using traditional morphological techniques, confirming concordance between disturbance indicators detected by eDNA and current monitoring approaches. Clear shifts in eukaryotic and bacterial indicator taxa were seen in response to nutrient loading while changes in diatom only communities were more subtle. Community changes were discernible between 0 and 150 g N m -2 treatments, suggesting that estuary health assessment tools could be developed to detect early signs of degradation. Increasing variation in community structure associated with nutrient loading could also be used as an indicator of stress or approaching tipping points. This work represents a first step towards the development of molecular-based estuary monitoring tools, which could provide a more holistic and standardized approach to ecosystem health assessment with faster turn-around times and lower costs., 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 Ltd. 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.)

We investigated the influence of seagrass canopies on the benthic biodiversity of bacteria and macroinvertebrates in a Red Sea tropical lagoon. Changes in abundance, number of taxa and assemblage structure were analyzed in response to seagrass densities (low, SLD; high, SHD; seagrasses with algae, SA), and compared with unvegetated sediments. Biological and environmental variables were examined in these four habitats (hereafter called treatments), both in the underlaying sediments and overlaying waters, at three randomly picked locations in March 2017. Differences between treatments were more apparent in the benthic habitat than in the overlaying waters. The presence of vegetation (more than its cover) and changes in sedimentary features (grain size and metals) at local scales influenced the observed biological patterns, particularly for macroinvertebrates. Of note, the highest percentage of exclusive macroinvertebrate taxa (18% of the gamma diversity) was observed in the SHD treatment peaking in the SA for bacteria. Benthic macroinvertebrates and bacteria shared a generally low number of taxa across treatments and locations; approximately, 25% of the gamma diversity was shared among all treatments and locations for macrofauna, dropping to 11% for bacteria. Given the low overlap in the species distribution across the lagoon, sustaining the connectivity among heterogeneous soft sediment habitats appears to be essential for maintaining regional biodiversity. This study addresses a current scientific gap related to the relative contributions of vegetated and unvegetated habitats to biodiversity in tropical regions.

Tetrodotoxin (TTX) is one of the most potent naturally occurring compounds and is responsible for many human intoxications worldwide. Paphies australis are endemic clams to New Zealand which contain varying concentrations of TTX. Research suggests that P. australis accumulate the toxin exogenously, but the source remains uncertain. The aim of this study was to identify potential bacterial TTX-producers by exploring differences in bacterial communities in two organs of P. australis : the siphon and digestive gland. Samples from the digestive glands of a non-toxic bivalve Austrovenus stutchburyi that lives amongst toxic P. australis populations were also analyzed. Bacterial communities were characterized using 16S ribosomal RNA gene metabarcoding in P. australis sourced monthly from the Hokianga Harbor, a site known to have TTX-bearing clams, for 1 year, from ten sites with varying TTX concentrations around New Zealand, and in A. stutchburyi from the Hokianga Harbor. Tetrodotoxin was detected in P. australis from sites all around New Zealand and in all P. australis collected monthly from the Hokianga Harbor. The toxin averaged 150 μg kg -1 over the year of sampling in the Hokianga Harbor but no TTX was detected in the A. stutchburyi samples from the same site. Bacterial species diversity differed amongst sites ( p < 0.001, F = 5.9) and the diversity in siphon samples was significantly higher than in digestive glands ( p < 0.001, F = 65.8). Spirochaetaceae (4-60%) and Mycoplasmataceae (16-78%) were the most abundant families in the siphons and the digestive glands, respectively. The bacterial communities were compared between sites with the lowest TTX concentrations and the Hokianga Harbor (site with the highest TTX concentrations), and the core bacterial communities from TTX-bearing individuals were analyzed. The results from both spatial and temporal studies corroborate with previous hypotheses that Vibrio and Bacillus could be responsible for the source of TTX in bivalves. The results from this study also indicate that marine cyanobacteria, in particular picocyanobacteria (e.g., Cyanobium , Synechococcus , Pleurocapsa , and Prochlorococcu s), should be investigated further as potential TTX producers., (Copyright © 2020 Biessy, Pearman, Smith, Hawes and Wood.)

Prochlorococcus and Synechococcus are pico-sized cyanobacteria that play a fundamental role in oceanic primary production, being particularly important in warm, nutrient-poor waters. Their potential response to nutrient enrichment is expected to be contrasting and to differ from larger phytoplankton species. Here, we used a metagenomic approach to characterize the responses to nutrient enrichment in the community of picocyanobacteria and to analyze the cyanophage response during a mesocosms experiment in the oligotrophic Red Sea. Natural picoplankton community was dominated by Synechococcus clade II, with marginal presence of Prochlorococcus (0.3% bacterial reads). Increased nutrient input triggered a fast Synechococcus bloom, with clade II being the dominant, with no response of Prochlorococcus growth. The largest bloom developed in the mesocosms receiving a single initial input of nutrients, instead of daily additions. The relative abundances of cyanophage sequences in cellular metagenomes increased during the experiment from 12.6% of total virus reads up to 40% in the treatment with the largest Synechococcus bloom. The subsequent collapse of the bloom pointed to a cyanophage infection on Synechococcus that reduced its competitive capacity, and was then followed by a diatom bloom. The cyanophage attack appears to have preferentially affected the most abundant Synechococcus clade II, increasing the evenness within the host population. Our results highlight the relevance of host-phage interactions on determining population dynamics and diversity of Synechococcus populations., (Copyright © 2020 Coello-Camba, Diaz-Rua, Duarte, Irigoien, Pearman, Alam and Agusti.)

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.)

Global ocean expeditions have provided minimum estimates of ocean's prokaryote diversity, supported by apparent asymptotes in the number of prokaryotes with sampling effort, of about 40,000 species, representing <1% of the species cataloged in the Earth Microbiome Project, despite being the largest habitat in the biosphere. Here we demonstrate that the abundance of prokaryote OTUs follows a scaling that can be represented by a power-law distribution, and as a consequence, we demonstrate, mathematically and through simulations, that the asymptote of rarefaction curves is an apparent one, which is only reached with sample sizes approaching the entire ecosystem. We experimentally confirm these findings using exhaustive repeated sampling of a prokaryote community in the Red Sea and the exploration of global assessments of prokaryote diversity in the ocean. Our findings indicate that, far from having achieved a thorough sampling of prokaryote species abundance in the ocean, global expeditions provide just a start for this quest as the richness in the global ocean is much larger than estimated.

Autonomous Reef Monitoring Structures (ARMS) have been applied worldwide to describe eukaryotic cryptic reef fauna. Conversely, bacterial communities, which are critical components of coral reef ecosystem functioning, remain largely overlooked. Here we deployed 56 ARMS across the 2,000-km spread of the Red Sea to assay biodiversity, composition and inferred underlying functions of coral reef-associated bacterial communities via 16S rRNA gene sequencing. We found that bacterial community structure and diversity aligned with environmental differences. Indeed, sea surface temperature and macroalgae cover were key in explaining bacterial relative abundance. Importantly, taxonomic and functional alpha diversity decreased under more extreme environmental conditions (e.g., higher temperatures) in the southern Red Sea. This may imply a link between bacterial community diversity and functional capabilities, with implications for conservation management. Our study demonstrates the utility of ARMS to investigate the response of coral reef-associated bacterial communities to environmental change., (© 2019 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

We investigated the validity of Autonomous Reef Monitoring Structures (ARMS) as monitoring tools for hard bottoms across a wide geographic and environmental range. We deployed 36 ARMS in the northeast Atlantic, northwest Mediterranean, Adriatic and Red Sea at 7-17 m depth. After 12-16 months, community composition was inferred from photographs, in six plate-faces for each ARMS. Overall, we found a highly significant effect of sea region, site (within seas), and plate-face on community composition. Plate-faces thus represent distinct micro-habitats and provide pseudo-replicates, increasing statistical power. Within each sea region taken individually, there was also a highly significant effect of site and plate-face. Because strong effects were obtained despite the fusion of taxonomic categories at high taxonomic ranks (to ensure comparability among biogeographic provinces), ARMS photo-analysis appears a promising monitoring tool for each sea region. We recommend keeping three ARMS per site and analyzing more numerous sites within a sea region to investigate environmental effects., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In an era of coral reef degradation, our knowledge of ecological patterns in reefs is biased towards large conspicuous organisms. The majority of biodiversity, however, inhabits small cryptic spaces within the framework of the reef. To assess this biodiverse community, which we term the 'reef cryptobiome', we deployed 87 autonomous reef monitoring structures (ARMS), on 22 reefs across 16 degrees latitude of the Red Sea. Combining ARMS with metabarcoding of the mitochondrial cytochrome oxidase I gene, we reveal a rich community, including the identification of 14 metazoan phyla within 10 416 operational taxonomic units (OTUs). While mobile and sessile subsets were similarly structured along the basin, the main environmental driver was different (particulate organic matter and sea surface temperature, respectively). Distribution patterns of OTUs showed that only 1.5% were present in all reefs, while over half were present in a single reef. On both local and regional scales, the majority of OTUs were rare. The high heterogeneity in community patterns of the reef cryptobiome has implications for reef conservation. Understanding the biodiversity patterns of this critical component of reef functioning will enable a sound knowledge of how coral reefs will respond to future anthropogenic impacts.

Microbial planktonic communities are critical components of marine biogeochemical pathways. Despite this, there is still limited knowledge on the dynamics of this group in warm and oligotrophic waters. We used high-throughput sequencing to characterise the bacterial (16S rRNA) and eukaryotic (18S rRNA) microbial plankton communities in two regions under the influence of anthropogenic impacts (a port and sewage outflow) and a coastal region with no direct anthropogenic disturbances in the central Red Sea. Overall, bacterial and eukaryotic components responded in a similar way to the environmental conditions. Community composition and structure were more sensitive than alpha diversity measures to environmental impacts. With the exception of eukaryotes, for which the number of OTU differed significantly between sampling periods in all the regions, environmental changes associated with anthropogenic pressures seem to be better reflected by variations in the relative dominance of microbial groups. For example, elevated proportional abundances of nitrifying and sewage-/faecal-related bacteria at the impacted sites were observed compared with the coastal region. The recently developed microgAMBI also appeared to correlate well with the level of anthropogenic impact the regions experienced, showing the potential to be applied in oligotrophic waters.

In a world of declining biodiversity, monitoring is becoming crucial. Molecular methods, such as metabarcoding, have the potential to rapidly expand our knowledge of biodiversity, supporting assessment, management, and conservation. In the marine environment, where hard substrata are more difficult to access than soft bottoms for quantitative ecological studies, Artificial Substrate Units (ASUs) allow for standardized sampling. We deployed ASUs within five regional seas (Baltic Sea, Northeast Atlantic Ocean, Mediterranean Sea, Black Sea, and Red Sea) for 12-26 months to measure the diversity and community composition of macroinvertebrates. We identified invertebrates using a traditional approach based on morphological characters, and by metabarcoding of the mitochondrial cytochrome oxidase I (COI) gene. We compared community composition and diversity metrics obtained using the two methods. Diversity was significantly correlated between data types. Metabarcoding of ASUs allowed for robust comparisons of community composition and diversity, but not all groups were successfully sequenced. All locations were significantly different in taxonomic composition as measured with both kinds of data. We recovered previously known regional biogeographical patterns in both datasets (e.g., low species diversity in the Black and Baltic Seas, affinity between the Bay of Biscay and the Mediterranean). We conclude that the two approaches provide complementary information and that metabarcoding shows great promise for marine monitoring. However, until its pitfalls are addressed, the use of metabarcoding in monitoring of rocky benthic assemblages should be used in addition to classical approaches rather than instead of them.

Coral reefs harbor diverse assemblages of organisms yet the majority of this diversity is hidden within the three dimensional structure of the reef and neglected using standard visual surveys. This study uses Autonomous Reef Monitoring Structures (ARMS) and amplicon sequencing methodologies, targeting mitochondrial cytochrome oxidase I and 18S rRNA genes, to investigate changes in the cryptic reef biodiversity. ARMS, deployed at 11 sites across a near- to off-shore gradient in the Red Sea were dominated by Porifera (sessile fraction), Arthropoda and Annelida (mobile fractions). The two primer sets detected different taxa lists, but patterns in community composition and structure were similar. While the microhabitat of the ARMS deployment affected the community structure, a clear cross-shelf gradient was observed for all fractions investigated. The partitioning of beta-diversity revealed that replacement (i.e. the substitution of species) made the highest contribution with richness playing a smaller role. Hence, different reef habitats across the shelf are relevant to regional diversity, as they harbor different communities, a result with clear implications for the design of Marine Protected Areas. ARMS can be vital tools to assess biodiversity patterns in the generally neglected but species-rich cryptic benthos, providing invaluable information for the management and conservation of hard-bottomed habitats over local and global scales.

An important aspect of population dynamics for coral reef fishes is the input of new individuals from the pelagic larval pool. However, the high biodiversity and the difficulty of identifying larvae of closely related species represent obstacles to more fully understanding these populations. In this study, we combined morphology and genetic barcoding (Cytochrome Oxidase I gene) to characterize the seasonal patterns of the larval fish community at two sites in close proximity to coral reefs in the central-north Red Sea: one shallower inshore location (50 m depth) and a nearby site located in deeper and more offshore waters (~ 500 m depth). Fish larvae were collected using oblique tows of a 60 cm-bongo net (500 μm mesh size) every month for one year (2013). During the warmer period of the year (June-November), the larval fish stock was comparable between sampling sites. However, during the colder months, abundances were higher in the inshore than in the offshore waters. Taxonomic composition and temporal variation of community structure differed notably between sites, potentially reflecting habitat differences, reproductive patterns of adults, and/or advective processes in the area. Eleven out of a total of 62 recorded families comprised 69-94% of the fish larval community, depending on sampling site and month. Richness of taxa was notably higher in the inshore station compared to the offshore, particularly during the colder period of the year and especially for the gobiids and apogonids. Two mesopelagic taxa (Vinciguerria sp. and Benthosema spp.) comprised an important component of the larval community at the deeper site with only a small and sporadic occurrence in the shallower inshore waters. Our data provide an important baseline reference for the larval fish communities of the central Red Sea, representing the first such study from Saudi Arabian waters.

The semi-enclosed nature of the Red Sea (20.2°N-38.5°N) makes it a natural laboratory to study the influence of environmental gradients on microbial communities. This study investigates the composition and structure of microbial prokaryotes and eukaryotes using molecular methods, targeting ribosomal RNA genes across different regions and seasons. The interaction between spatial and temporal scales results in different scenarios of turbulence and nutrient conditions allowing for testing of ecological theory that categorizes the response of the plankton community to these variations. The prokaryotic reads are mainly comprised of Cyanobacteria and Proteobacteria (Alpha and Gamma), with eukaryotic reads dominated by Dinophyceae and Syndiniophyceae. Periodic increases in the proportion of Mamiellophyceae and Bacillariophyceae reads were associated with alterations in the physical oceanography leading to nutrient increases either through the influx of Gulf of Aden Intermediate Water (south in the fall) or through water column mixing processes (north in the spring). We observed that in general dissimilarity amongst microbial communities increased when nutrient concentrations were higher, whereas richness (observed OTUs) was higher in scenarios of higher turbulence. Maximum abundance models showed the differential responses of dominant taxa to temperature giving an indication how taxa will respond as waters become warmer and more oligotrophic.

Coral reefs harbor the most diverse assemblages in the ocean, however, a large proportion of the diversity is cryptic and, therefore, undetected by standard visual census techniques. Cryptic and exposed communities differ considerably in species composition and ecological function. This study compares three different coral reef assessment protocols: i) visual benthic reef surveys: ii) visual census of Autonomous Reef Monitoring Structures (ARMS) plates; and iii) metabarcoding techniques of the ARMS (including sessile, 106-500 μm and 500-2000 μm size fractions), that target the cryptic and exposed communities of three reefs in the central Red Sea. Visual census showed a dominance of Cnidaria (Anthozoa) and Rhodophyta on the reef substrate, while Porifera, Bryozoa and Rhodophyta were the most abundant groups on the ARMS plates. Metabarcoding, targeting the 18S rRNA gene, significantly increased estimates of the species diversity (p < 0.001); revealing that Annelida were generally the dominant phyla (in terms of reads) of all fractions and reefs. Furthermore, metabarcoding detected microbial eukaryotic groups such as Syndiniophyceae, Mamiellophyceae and Bacillariophyceae as relevant components of the sessile fraction. ANOSIM analysis showed that the three reef sites showed no differences based on the visual census data. Metabarcoding showed a higher sensitivity for identifying differences between reef communities at smaller geographic scales than standard visual census techniques as significant differences in the assemblages were observed amongst the reefs. Comparison of the techniques showed no similar patterns for the visual techniques while the metabarcoding of the ARMS showed similar patterns amongst fractions. Establishing ARMS as a standard tool in reef monitoring will not only advance our understanding of local processes and ecological community response to environmental changes, as different faunal components will provide complementary information but also improve the estimates of biodiversity in coral reef benthic communities. This study lays the foundations for further studies looking at integrating traditional reef survey methodologies with complementary approaches, such as metabarcoding, which investigate other components of the reef community., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The present study aims to characterize the benthic eukaryotic biodiversity patterns at a coarse taxonomic level in three areas of the central Red Sea (a lagoon, an offshore area in Thuwal and a shallow coastal area near Jeddah) based on extracellular DNA. High-throughput amplicon sequencing targeting the V9 region of the 18S rRNA gene was undertaken for 32 sediment samples. High levels of alpha-diversity were detected with 16,089 operational taxonomic units (OTUs) being identified. The majority of the OTUs were assigned to Metazoa (29.2%), Alveolata (22.4%) and Stramenopiles (17.8%). Stramenopiles (Diatomea) and Alveolata (Ciliophora) were frequent in a lagoon and in shallower coastal stations, whereas metazoans (Arthropoda: Maxillopoda) were dominant in deeper offshore stations. Only 24.6% of total OTUs were shared among all areas. Beta-diversity was generally lower between the lagoon and Jeddah (nearshore) than between either of those and the offshore area, suggesting a nearshore-offshore biodiversity gradient. The current approach allowed for a broad-range of benthic eukaryotic biodiversity to be analysed with significantly less labour than would be required by other traditional taxonomic approaches. Our findings suggest that next generation sequencing techniques have the potential to provide a fast and standardised screening of benthic biodiversity at large spatial and temporal scales., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The diversity of microbial plankton has received limited attention in the main basin of the Red Sea. This study investigates changes in the community composition and structure of prokaryotes and eukaryotes at the extremes of the Red Sea along cross-shelf gradients and between the surface and deep chlorophyll maximum. Using molecular methods to target both the 16S and 18S rRNA genes, it was observed that the dominant prokaryotic classes were Acidimicrobiia, Alphaproteobacteria and Cyanobacteria, regardless of the region and depth. The eukaryotes Syndiniophyceae and Dinophyceae between them dominated in the north, with Bacillariophyceae and Mamiellophyceae more prominent in the southern region. Significant differences were observed for prokaryotes and eukaryotes for region, depth and distance from shore. Similarly, it was noticed that communities became less similar with increasing distance from the shore. Canonical correspondence analysis at the class level showed that Mamiellophyceae and Bacillariophyceae correlated with increased nutrients and chlorophyll a found in the southern region, which is influenced by the input of Gulf of Aden Intermediate Water., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The composition of zooplankton in the water column has received limited attention in the main body of the Red Sea and this study investigates the change in the community both spatially and temporally across 11 stations in the central Red Sea. Using molecular methods to target the v9 region of the 18S rRNA gene a total of approximately 11.5 million reads were sequenced resulting in 2528 operational taxonomic units (OTUs) at 97% similarity. The phylum Arthropoda dominated in terms of reads accounting for on average 86.2% and 65.3% for neuston nets and vertical multinets respectively. A reduction in the number of OTUs was noticed with depth for both total metazoa and Maxillopoda whilst there was also a significant change in the composition of the Maxillopoda community. The genus Corycaeus had a higher proportion of reads in the epipelagic zone with Pleuromamma becoming increasingly dominant with depth. No significant difference was observed in the community between night and day sampling however there was a significant difference in the zooplankton community between two sampling periods separated by 10 days.