The challenge of implementing environmental DNA metabarcoding to detect elasmobranchs in a resource-limited marine protected area.

Elasmobranchs are threatened and eDNA metabarcoding is a powerful tool that can help efforts to better understand and conserve them. Nevertheless, the inter-calibration between optimal methodological practices and its implementation in resource-limited situations is still an issue. Based on promising results from recent studies, the authors applied a cost-effective protocol with parameters that could be easily replicated by any conservationist. Nonetheless, the results with fewer elasmobranchs detected than expected reveal that endorsed primers and sampling strategies still require further optimization, especially for applications in resource-limited conservation programmes.

Ecosystems monitoring powered by environmental genomics: A review of current strategies with an implementation roadmap.

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

Insights from the Genomes of Microbes Thriving in Uranium-Enriched Sediments.

Elevated uranium dose (4 g kg-1) causes a shift in billabong sediment communities that result in the enrichment of five bacterial species. These taxa include Geobacter, Geothrix and Dyella species, as well as a novel-potentially predatory-Bacteroidetes species, and a new member of class Anaerolineae (Chloroflexi). Additionally, a population of methanogenic Methanocella species was also identified. Genomic reconstruction and metabolic examination of these taxa reveal a host of divergent life strategies and putative niche partitioning. Resistance-nodulation-division heavy metal efflux (RND-HME) transporters are implicated as potential uranium tolerance strategies among the bacterial taxa. Potential interactions, uranium tolerance and ecologically relevant catabolism are presented in a conceptual model of life in this environment.

Effects of uranium concentration on microbial community structure and functional potential.

Located in the Northern Territory of Australia, Ranger uranium mine is directly adjacent to the UNESCO World Heritage listed Kakadu National Park, with rehabilitation targets needed to ensure the site can be incorporated into the park following the mine's closure in 2026. This study aimed to understand the impact of uranium concentration on microbial communities, in order to identify and describe potential breakpoints in microbial ecosystem services. This is the first study to report in situ deployment of uranium-spiked sediments along a concentration gradient (0-4000 mg U kg-1 ), with the study design maximising the advantages of both field surveys and laboratory manipulative studies. Changes to microbial communities were characterised through the use of amplicon and shotgun metagenomic next-generation sequencing. Significant changes to taxonomic and functional community assembly occurred at a concentration of 1500 mg U kg-1 sediment and above. At uranium concentrations of ≥ 1500 mg U kg-1 , genes associated with methanogenic consortia and processes increased in relative abundance, while numerous significant changes were also seen in the relative abundances of genes involved in nitrogen cycling. Such alterations in carbon and nitrogen cycling pathways suggest that taxonomic and functional changes to microbial communities may result in changes in ecosystem processes and resilience.

Direct and indirect effects of copper-contaminated sediments on the functions of model freshwater ecosystems.

Copper is acutely toxic to, and directly affects, primary producers and decomposers, which are key players in essential processes such as the nutrient cycle in freshwater ecosystems. Even though the indirect effects of metals (for example effects due to changes in species interactions) may be more common than direct effects, little is known about the indirect effects of copper on primary producers and decomposers. The effects of copper on phytoplankton, macrophytes, periphyton and organic matter decomposition in an outdoor lentic mesocosm facility were assessed, and links between the responses examined. Copper directly decreased macrophyte growth, subsurface organic matter decomposition, and the potential for high phytoplankton Chlorophyll a concentrations. However, periphyton cover and organic matter decomposition on the surface of the sediment were stimulated by the presence of copper. These latter responses were attributed to indirect effects, due to a reduction in grazing pressure from snails, particularly Physa acuta, in the higher copper-contaminated mesocosms. This permitted the growth of periphyton and other heterotrophs, ultimately increasing decomposition at the sediment surface. The present study demonstrates the pronounced influence indirect effects may have on ecological function, findings that may not be observed in traditional laboratory studies (which utilize single species or simplistic communities).

Multiple stressors unpredictably affect primary producers and decomposition in a model freshwater ecosystem.

Freshwater ecosystems are affected by various stressors, such as contamination and exotic species, making them amongst the most imperilled biological systems on the planet. In Australia and elsewhere, copper is one of the most common metal contaminants in freshwater systems and the European carp (Cyprinus carpio L.) is one of the most pervasive and widespread invasive fish species. Copper (Cu) and carp can both directly affect primary production and decomposition, which are critical and interrelated nutrient cycling processes and ecosystem services. The aim of this study was to explore the direct and indirect effects of Cu and carp individually, and together on periphyton cover, chlorophyll a concentration, growth of the macrophyte Vallisneria spiralis L., and the decomposition of leaf litter and cotton strips in a controlled, factorial experiment in outdoor experimental ponds. In isolation, Cu reduced macrophyte growth and organic matter decomposition, while chlorophyll a concentrations and periphyton cover remained unchanged, possibly due to the Low-Cu concentrations in the overlying water. Carp addition alone had a direct negative effect on the biomass of aquatic plants outside protective cages, but also increased plant biomass inside the cages, periphyton cover and chlorophyll a concentrations. Leaf litter was more decomposed in the carp only ponds compared to controls, while there was no significant effect on cotton strip decomposition. Aquatic plants were absent in the Cu + carp ponds caused by the combined effects of Cu toxicity, carp disturbance and the increase in turbidity due to carp bioturbation. Increases in periphyton cover in Low-Cu + carp, while absence in the High-Cu + carp ponds, and differences in the decomposition of surface and buried cotton strips were not as predicted, which highlights the need for such studies to understand the complex interactions among stressors for environmental risk assessment.

Response of Microbial Communities to Naturally Occurring Radioactive Material-Contaminated Sediments: A Microcosm-Based Study.

There is a growing need to understand the potential ecological impacts of contaminants in offshore oil and gas infrastructure, especially if that infrastructure is to be left in situ as a decommissioning option. Naturally occurring radioactive material (NORM) is one type of contaminant found in solid deposits on internal surfaces of infrastructure that poses potential ecological harm if released into the marine environment. Microbes are important components of marine sediment ecosystems because they provide ecosystem services, yet the impacts of NORM contamination to these communities are not well understood. The present study aimed to investigate the response of benthic microbial communities to NORM-contaminated scale, collected from an offshore oil and gas system, via controlled laboratory microcosm studies. Changes to microbial communities in natural sediment and sediments spiked with NORM at radium-226 activity concentrations ranging from 9.5 to 59.8 Bq/kg (in partial equilibria with progeny) over 7 and 28 days were investigated using high-throughput sequencing of environmental DNA extracted from experimental sediments. There were no significant differences in microbial community composition between control and scale-spiked sediments over 7 and 28 days. However, we observed a greater presence of Firmicutes in the scale-mixed treatment and Chloroflexi in the scale-surface treatments after 28 days. This could suggest selection for species with contaminant tolerance or potential resilience to radiation and metal toxicity. Further research is needed to explore microbial tolerance mechanisms and their potential as indicators of effects of radionuclide-contaminated sediments. The present study demonstrated that microcosm studies can provide valuable insights about the potential impacts of contamination from oil and gas infrastructure to sediment microbial communities. Environ Toxicol Chem 2024;43:1648-1661. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Saline mine water influences eukaryote life in shallow groundwater of a tropical sandy stream.

Eukaryotic communities in groundwater may be particularly sensitive to disturbance because they are adapted to stable environmental conditions and often have narrow spatial distributions. Traditional methods for characterising these communities, focussing on groundwater-inhabiting macro- and meiofauna (stygofauna), are challenging because of limited taxonomic knowledge and expertise (particularly in less-explored regions), and the time and expense of morphological identification. The primary objective of this study was to evaluate the vulnerability of eukaryote communities in shallow groundwater to mine water discharge containing elevated concentrations of magnesium (Mg) and sulfate (SO4). The study was undertaken in a shallow sand bed aquifer within a wet-dry tropical setting. The aquifer, featuring a saline mine water gradient primarily composed of elevated Mg and SO4, was sampled from piezometers in the creek channel upstream and downstream of the mine water influence during the dry season when only subsurface water flow was present. Groundwater communities were characterised using both morphological assessments of stygofauna from net samples and environmental DNA (eDNA) targeting the 18S rDNA and COI mtDNA genes. eDNA data revealed significant shifts in community composition in response to mine waters, contrasting with findings from traditional morphological composition data. Changes in communities determined using eDNA data were notably associated with concentrations of SO42-, Mg2+ and Na+, and water levels in the piezometers. This underscores the importance of incorporating molecular approaches in impact assessments, as relying solely on traditional stygofauna sampling methods in similar environments may lead to inaccurate conclusions about the responses of the assemblage to studied impacts.

Forest top canopy bacterial communities are influenced by elevation and host tree traits.

BACKGROUND: The phyllosphere microbiome is crucial for plant health and ecosystem functioning. While host species play a determining role in shaping the phyllosphere microbiome, host trees of the same species that are subjected to different environmental conditions can still exhibit large degrees of variation in their microbiome diversity and composition. Whether these intra-specific variations in phyllosphere microbiome diversity and composition can be observed over the broader expanse of forest landscapes remains unclear. In this study, we aim to assess the variation in the top canopy phyllosphere bacterial communities between and within host tree species in the temperate European forests, focusing on Fagus sylvatica (European beech) and Picea abies (Norway spruce). RESULTS: We profiled the bacterial diversity, composition, driving factors, and discriminant taxa in the top canopy phyllosphere of 211 trees in two temperate forests, Veluwe National Parks, the Netherlands and Bavarian Forest National Park, Germany. We found the bacterial communities were primarily shaped by host species, and large variation existed within beech and spruce. While we showed that there was a core microbiome in all tree species examined, community composition varied with elevation, tree diameter at breast height, and leaf-specific traits (e.g., chlorophyll and P content). These driving factors of bacterial community composition also correlated with the relative abundance of specific bacterial families. CONCLUSIONS: While our results underscored the importance of host species, we demonstrated a substantial range of variation in phyllosphere bacterial diversity and composition within a host species. Drivers of these variations have implications at both the individual host tree level, where the bacterial communities differed based on tree traits, and at the broader forest landscape level, where drivers like certain highly plastic leaf traits can potentially link forest canopy bacterial community variations to forest ecosystem processes. We eventually showed close associations between forest canopy phyllosphere bacterial communities and host trees exist, and the consistent patterns emerging from these associations are critical for host plant functioning.

Environmental DNA highlights the influence of salinity and agricultural run-off on coastal fish assemblages in the Great Barrier Reef region.

Agricultural run-off in Australia's Mackay-Whitsunday region is a major source of nutrient and pesticide pollution to coastal and inshore ecosystems of the Great Barrier Reef. While the effects of run-off are well documented for the region's coral and seagrass habitats, the ecological impacts on estuaries, the direct recipients of run-off, are less known. This is particularly true for fish communities, which are shaped by the physico-chemical properties of coastal waterways that vary greatly in tropical regions. To address this knowledge gap, we used environmental DNA (eDNA) metabarcoding to examine fish assemblages at four locations (three estuaries and a harbour) subjected to varying levels of agricultural run-off during a wet and dry season. Pesticide and nutrient concentrations were markedly elevated during the sampled wet season with the influx of freshwater and agricultural run-off. Fish taxa richness significantly decreased in all three estuaries (F = 164.73, P = <0.001), along with pronounced changes in community composition (F = 46.68, P = 0.001) associated with environmental variables (largely salinity: 27.48% contribution to total variance). In contrast, the nearby Mackay Harbour exhibited a far more stable community structure, with no marked changes in fish assemblages observed between the sampled seasons. Among the four sampled locations, variation in fish community composition was more pronounced within the wet season (F = 2.5, P = 0.001). Notably, variation in the wet season was significantly correlated with agricultural contaminants (phosphorus: 6.25%, pesticides: 5.22%) alongside environmental variables (salinity: 5.61%, DOC: 5.57%). Historically contaminated and relatively unimpacted estuaries each demonstrated distinct fish communities, reflecting their associated catchment use. Our findings emphasise that while seasonal effects play a key role in shaping the community structure of fish in this region, agricultural contaminants are also important contributors in estuarine systems.

Impacts of inundation and drought on eukaryote biodiversity in semi-arid floodplain soils.

Floodplain ecosystems are characterized by alternating wet and dry phases and periodic inundation defines their ecological character. Climate change, river regulation and the construction of levees have substantially altered natural flooding and drying regimes worldwide with uncertain effects on key biotic groups. In southern Australia, we hypothesized that soil eukaryotic communities in climate change affected areas of a semi-arid floodplain would transition towards comprising mainly dry-soil specialist species with increasing drought severity. Here, we used 18S rRNA amplicon pyrosequencing to measure the eukaryote community composition in soils that had been depleted of water to varying degrees to confirm that reproducible transitional changes occur in eukaryotic biodiversity on this floodplain. Interflood community structures (3 years post-flood) were dominated by persistent rather than either aquatic or dry-specialist organisms. Only 2% of taxa were unique to dry locations by 8 years post-flood, and 10% were restricted to wet locations (inundated a year to 2 weeks post-flood). Almost half (48%) of the total soil biota were detected in both these environments. The discovery of a large suite of organisms able to survive nearly a decade of drought, and up to a year submerged supports the concept of inherent resilience of Australian semi-arid floodplain soil communities under increasing pressure from climatic induced changes in water availability.

Assessing the ecological impacts of NORM-contaminated scale on marine infauna using sediment microcosms.

Naturally occurring radioactive materials (NORMs) can be found in decommissioned oil and gas infrastructure (e.g. pipelines), including scales. The effects of NORM contaminants from offshore infrastructure on benthic macroorganisms remain poorly understood. To test the potential ecological effects of NORM-contaminated scale, we exposed a marine amphipod, a clam and a polychaete to marine sediments spiked with low level concentrations of barium sulfate scale retrieved from a decommissioned subsea pipe. Only amphipods were included in further analysis due to treatment mortalities of the clam and polychaete. Barium (Ba) and copper (Cu) were elevated in the seawater overlying the spiked sediments, although no sediment metals exceeded guidelines. 210Po was the only NORM detected in the overlying waters while both 210Po and 226Ra were significantly elevated in the scale-contaminated sediments when compared with the control sediments. The whole-body burden of Ba and 226Ra were significantly higher in the scale-exposed amphipods. Using experiment- and scale-specific parameters in biota dose assessments suggested potential dose rates may elicit individual and population level effects. Future work is needed to assess the biological impacts and effects of NORM scale at elevated levels above background concentrations and the accumulation of NORM-associated contaminants by marine organisms.

Monitoring the Health of Coastal Environments in the Pacific Region-A Review.

Coastal areas provide important ecological services to populations accessing, for example, tourism services, fisheries, minerals and petroleum. Coastal zones worldwide are exposed to multiple stressors that threaten the sustainability of receiving environments. Assessing the health of these valuable ecosystems remains a top priority for environmental managers to ensure the key stressor sources are identified and their impacts minimized. The objective of this review was to provide an overview of current coastal environmental monitoring frameworks in the Asia-Pacific region. This large geographical area includes many countries with a range of climate types, population densities and land uses. Traditionally, environmental monitoring frameworks have been based on chemical criteria set against guideline threshold levels. However, regulatory organizations are increasingly promoting the incorporation of biological effects-based data in their decision-making processes. Using a range of examples drawn from across the region, we provide a synthesis of the major approaches currently being applied to examine coastal health in China, Japan, Australia and New Zealand. In addition, we discuss some of the challenges and investigate potential solutions for improving traditional lines of evidence, including the coordination of regional monitoring programs, the implementation of ecosystem-based management and the inclusion of indigenous knowledge and participatory processes in decision-making.

Short and informative DNA products to indirectly measure vascular plant biodiversity.

Anthropogenic activities are having a deleterious effect on biodiversity. To understand the magnitude of this issue, and ultimately temper its pace, requires reproducible biodiversity measurements at suitable spatio-temporal scales. Procuring such data solely by existing approaches is unachievable because of the costs, time and the taxonomic expertise required. High-throughput molecular biodiversity analysis shows great promise, increasing the breadth of biota sampled and accelerating the rate of data collection. In this issue of Molecular Ecology, Yoccoz et al. (2012) use short informative DNA product 'meta-barcodes' to provide an insight into above-ground vascular plant diversity from boreal, temperate and tropical environments. Interestingly, their molecular analysis was derived from the soils and not the plants themselves, with the molecular signatures of the soils not only strongly reflecting current diversity, but also traces of crops not sown for up to one hundred years. Importantly, the research examines the complexities associated with deriving biomass estimates from molecular data and the need to consider biomass turnover. The use of soil-derived meta-barcodes extends beyond estimating vascular plant diversity, with the approach being suited to the range of ecological applications, especially scenarios where DNA may be degraded.

Characterizing the spatial distributions of soil biota at a legacy base metal mine using environmental DNA.

Characterizing the distribution of biota in response to contaminants is a critical element of site risk assessments. In this study we investigated the spatial distributions of biota and soil chemistry data in surface soil from Sunny Corner, a legacy base metal sulfide mine, Australia. Our results showed that copper (Cu), zinc (Zn), arsenic (As) and lead (Pb) in the surface soil exceeded Australian national soil quality guidelines and posed risks to the environment. Environmental (e)DNA metabarcoding of prokaryote and eukaryote composition confirmed the suggestion of environmental risk posed by these elements collectively explaining 72.9 % and 60.5 % of the total variation in the composition of soil prokaryotes and eukaryotes, respectively. Prokaryotic taxa from the phyla Gemmatimonadetes, Verrucomicrobia and Deinococcus-Thermus showed similar spatial patterns to As and Pb, and were positively correlated. Eukaryotic taxa from the phylum Chlorophyta had similar positive correlations with As and Pb in the soil. In contrast, Amoebozoa and Cercozoa, were sensitive to metals and metalloids, having higher relative abundances in soils with lower concentrations of contaminants. Our study shows that metabarcoding is a promising ecological approach for rapid, large scale assessment of contaminated and potentially impacted sites.

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

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

Recolonisation of translocated metal-contaminated sediments by estuarine macrobenthic assemblages.

A recolonisation experiment was performed using sediments from three locations (Nords Wharf, Cockle Bay and Warners Bay) along a metal contamination gradient (Lake Macquarie, Australia). The study aimed to determine whether the source of the sediments would influence the recolonisation of benthic assemblages, providing additional information regarding the ecological risks associated with the more contaminated sediments. Sediments were translocated to two locations within the lake and retrieved after 22 weeks along with benthic samples from the surrounding sediments (ambient). Total abundance was greater in the reference treatment (Nords Wharf), with this difference being driven by polychaetes, especially capitellids. In general, univariate metrics were similar among the recolonised treatments, although evenness and diversity patterns were complex due to significant location-treatment interactions. PERMANOVA analysis demonstrated that the Nords Wharf treatments were significantly different from the more contaminated treatments (Cockle Bay and Warner's Bay) and the ambient assemblages, with no differences being detected among Cockle Bay and Warners Bay assemblages. Collectively, the findings showed that the source of the sediments influenced the composition of the recolonised assemblages, with the described approach being a powerful tool for examining the effects of location-specific sediments under environmentally relevant conditions.

Application of environmental DNA for assessment of contamination downstream of a legacy base metal mine.

Acid Rock Drainage (ARD) from legacy mines can negatively impact the biota in sediments and waters for tens of kilometers downstream. Here we used environmental (e)DNA metabarcoding to assess the impacts of metal contaminants on biota in sediment and water downstream of a legacy base metal sulfide mine in southeastern Australia, as exemplar of similar mines elsewhere. Concentrations of metals in water were below Australian water quality guideline values at 20 km downstream for copper (Cu), 40 km downstream for zinc (Zn) and 10 km downstream for lead (Pb). Sediment metal concentrations were below national guideline concentrations at 10 km downstream for Cu, 60 km downstream for Zn and 20 km downstream for Pb. In contrast, metabarcoding showed that biological communities from sediment samples at 10 km and 20 km downstream were similar to sites close to the mine and thus indicative of being impacted, despite metal concentrations being relatively low. As we illustrate, when combined with sediment and water chemistry, metabarcoding can provide more ecological robust perspective on the downstream effects of legacy mines, capturing the sensitivities of a diverse range of organisms.

The use of diversity indices for local assessment of marine sediment quality.

Diversity indices are commonly used to measure changes in marine benthic communities. However, the reliability (and therefore suitability) of these indices for detecting environmental change is often unclear because of small sample size and the inappropriate choice of communities for analysis. This study explored uncertainties in taxonomic density and two indices of community structure in our target region, Japan, and in two local areas within this region, and explored potential solutions. Our analysis of the Japanese regional dataset showed a decrease in family density and a dominance of a few species as sediment conditions become degraded. Local case studies showed that species density is affected by sediment degradation at sites where multiple communities coexist. However, two indices of community structure could become insensitive because of masking by community variability, and small sample size sometimes caused misleading or inaccurate estimates of these indices. We conclude that species density is a sensitive indicator of change in marine benthic communities, and emphasise that indices of community structure should only be used when the community structure of the target community is distinguishable from other coexisting communities and there is sufficient sample size.