An updated LSU database and pipeline for environmental DNA identification of arbuscular mycorrhizal fungi.

Recent work established a backbone reference tree and phylogenetic placement pipeline for identification of arbuscular mycorrhizal fungal (AMF) large subunit (LSU) rDNA environmental sequences. Our previously published pipeline allowed any environmental sequence to be identified as putative AMF or within one of the major families. Despite this contribution, difficulties in implementation of the pipeline remain. Here, we present an updated database and pipeline with (1) an expanded backbone tree to include four newly described genera and (2) several changes to improve ease and consistency of implementation. In particular, packages required for the pipeline are now installed as a single folder (conda environment) and the pipeline has been tested across three university computing clusters. This updated backbone tree and pipeline will enable broadened adoption by the community, advancing our understanding of these ubiquitous and ecologically important fungi.

Detection, movement and persistence of invertebrate eDNA in groundwater.

Sampling groundwater biodiversity is difficult because of limited access and issues with species identification. Environmental DNA (eDNA) provides a viable alternative to traditional sampling approaches, however limited knowledge of the abundance and fate of DNA in groundwater hinders the interpretation of data from these environments. Groundwater environments are dark and have lower oxygen concentrations and microbial activity than surface waters. Consequently, assumptions about DNA fate in surface ecosystems may not apply to groundwaters. Here, we test the longevity and transport of eDNA in groundwater within a static microcosm and a flow-through mesocosm. A variety of invertebrates were placed within a mesocosm and microcosm to enable DNA shedding, and then removed. DNA persisted for up to 5 weeks after their removal in the static experiment and was detected between 9 and 33 days in the flow-through experiment. Sediments and water both proved important for eDNA detection. Crustacean DNA was detected sporadically and unpredictably, whereas non-crustacean DNA was detected more frequently despite their lower densities. We suggest that detecting crustaceans poses a challenge to utilising eDNA approaches for stygofauna monitoring. This is confounded by the scarcity of sequences for stygofauna in reference databases. Further research is needed before eDNA alone can be routinely employed for stygofauna detection.

Effects of storage conditions on the stability of qPCR reagents: implications for environmental DNA detection.

OBJECTIVE: Environmental DNA (eDNA) detection is a transformative tool for ecological surveys which in many cases offers greater accuracy and cost-effectiveness for tracking low-density, cryptic species compared to conventional methods. For the use of targeted quantitative PCR (qPCR)-based eDNA detection, protocols typically require freshly prepared reagents for each sample, necessitating systematic evaluation of reagent stability within the functional context of eDNA standard curve preparation and environmental sample evaluation. Herein, we assessed the effects of long-term storage and freeze-thaw cycles on qPCR reagents for eDNA analysis across six assays. RESULTS: Results demonstrate qPCR plates (containing pre-made PCR mix, primer-probe, and DNA template) remain stable at 4 °C for three days before thermocycling without fidelity loss irrespective of qPCR assay used. Primer-probe mixes remain stable for five months of - 20 °C storage with monthly freeze-thaw cycles also irrespective of qPCR assay used. Synthetic DNA stocks maintain consistency in standard curves and sensitivity for three months under the same conditions. These findings enhance our comprehension of qPCR reagent stability, facilitating streamlined eDNA workflows by minimizing repetitive reagent preparations.

Spatiotemporal changes of pelagic food webs investigated by environmental DNA metabarcoding and connectivity analysis.

Environmental DNA metabarcoding (eDNA metaB) is fundamental for monitoring marine biodiversity and its spread in coastal ecosystems. We applied eDNA metaB to seawater samples to investigate the spatiotemporal variability of plankton and small pelagic fish, comparing sites with different environmental conditions across a coast-to-offshore gradient at river mouths along the Campania coast (Italy) over 2 years (2020-2021). We found a marked seasonality in the planktonic community at the regional scale, likely owing to the hydrodynamic connection among sampling sites, which was derived from numerical simulations. Nonetheless, spatial variability among plankton communities was detected during summer. Overall, slight changes in plankton and fish composition resulted in the potential reorganization of the pelagic food web at the local scale. This work supports the utility of eDNA metaB in combination with hydrodynamic modelling to study marine biodiversity in the water column of coastal systems. This article is part of the theme issue 'Connected interactions: enriching food web research by spatial and social interactions'.

Establishing Silphids in the invertebrate DNA toolbox: a proof of concept.

Environmental DNA (eDNA) analyses are an increasingly popular tool for assessing biodiversity. eDNA sampling that uses invertebrates, or invertebrate DNA (iDNA), has become a more common method in mammal biodiversity studies where biodiversity is assessed via diet analysis of different coprophagous or hematophagous invertebrates. The carrion feeding family of beetles (Silphidae: Coleoptera, Latreille (1807)), have not yet been established as a viable iDNA source in primary scientific literature, yet could be useful indicators for tracking biodiversity in forested ecosystems. Silphids find carcasses of varying size for both food and reproduction, with some species having host preference for small mammals; therefore, iDNA Silphid studies could potentially target small mammal communities. To establish the first valid use of iDNA methods to detect Silphid diets, we conducted a study with the objective of testing the validity of iDNA methods applied to Silphids using both Sanger sequencing and high throughput Illumina sequencing. Beetles were collected using inexpensive pitfall traps in Alberta, Michigan in 2019 and 2022. We successfully sequenced diet DNA and environmental DNA from externally swabbed Silphid samples and diet DNA from gut dissections, confirming their potential as an iDNA tool in mammalian studies. Our results demonstrate the usefulness of Silphids for iDNA research where we detected species from the genera Anaxyrus, Blarina, Procyon, Condylura, Peromyscus, Canis, and Bos. Our results highlight the potential for Silphid iDNA to be used in future wildlife surveys.

Streamlining large-scale oceanic biomonitoring using passive eDNA samplers integrated into vessel's continuous pump underway seawater systems.

Passive samplers are enabling the scaling of environmental DNA (eDNA) biomonitoring in our oceans, by circumventing the time-consuming process of water filtration. Designing a novel passive sampler that does not require extensive sample handling time and can be connected to ocean-going vessels without impeding normal underway activities has potential to rapidly upscale global biomonitoring efforts onboard the world's oceanic fleet. Here, we demonstrate the utility of an artificial sponge sampler connected to the continuous pump underway seawater system as a means to enable oceanic biomonitoring. We compared the performance of this passive sampling protocol with standard water filtration at six locations during a research voyage from New Zealand to Antarctica in early 2023. Eukaryote metabarcoding of the mitochondrial COI gene revealed no significant difference in phylogenetic α-diversity between sampling methods and both methods delineated a progressive reduction in number of Zero-Radius Operational Taxonomic Units (ZOTUs) with increased latitudes. While both sampling methods revealed comparable trends in geographical community compositions, distinct clusters were identified for passive samplers and water filtration at each location. Additionally, greater variability between replicates was observed for passive samplers, resulting in an increased estimated level of replication needed to recover 90 % of the biodiversity. Furthermore, traditional water filtration failed to detect three phyla observed by passive samplers and extrapolation analysis estimated passive samplers recover a larger number of ZOTUs compared to water filtration for all six locations. Our results demonstrate the potential of this passive eDNA sampler protocol and highlight areas where this emerging technology could be improved, thereby enabling large-scale offshore marine eDNA biomonitoring by leveraging the world's oceanic fleet without interfering with onboard activities.

Vertical and horizontal environmental DNA (eDNA) patterns of fish in a shallow and well-mixed North Sea area.

The integration of eDNA metabarcoding into monitoring programs provides valuable information about fish community structures. Despite the growing body of evidence supporting the method's effectiveness in distinguishing fine-scale eDNA signals, there is a limited understanding of eDNA distribution in shallow, well-mixed environments, especially related to sampling depth. We analyzed 167 samples collected from the surface and bottom water at 17 locations of the Belgian Part of the North Sea (BPNS), where the deepest sampling point was 31 m, and compared this to beam trawl catch data collected simultaneously at the same locations. eDNA metabarcoding identified an additional 22 species compared to beam trawl catch data. Diversity measures and patterns were very similar between surface and bottom samples and revealed community patterns that were previously described by long-term beam trawl catch data. Surface and bottom samples had 39 fish species in common, while six and eight rare species were uniquely detected, respectively. Our results demonstrate that eDNA metabarcoding effectively identifies spatial community patterns of fishes in the highly dynamic environment of the BPNS regardless of sampling depth. Out of the six most common species tested, eDNA metabarcoding read abundances correlated strongly with catch-based abundance data for one species, but moderately for two others, indicating that inferring fish abundance and biomass via eDNA metabarcoding remains challenging.

A sedimentary DNA record of the Atacama Trench reveals biodiversity changes in the most productive marine ecosystem.

The hadopelagic environment remains highly understudied due to the inherent difficulties in sampling at these depths. The use of sediment environmental DNA (eDNA) can overcome some of these restrictions as settled and preserved DNA represent an archive of the biological communities. We use sediment eDNA to assess changes in the community within one of the world's most productive open-ocean ecosystems: the Atacama Trench. The ecosystems around the Atacama Trench have been intensively fished and are affected by climate oscillations, but the understanding of potential impacts on the marine community is limited. We sampled five sites using sediment cores at water depths from 2400 to ~8000 m. The chronologies of the sedimentary record were determined using 210Pbex. Environmental DNA was extracted from core slices and metabarcoding was used to identify the eukaryote community using two separate primer pairs for different sections of the 18S rRNA gene (V9 and V7) effectively targeting pelagic taxa. The reconstructed communities were similar among markers and mainly composed of chordates and members of the Chromista kingdom. Alpha diversity was estimated for all sites in intervals of 15 years (from 1842 to 2018), showing a severe drop in biodiversity from 1970 to 1985 that aligns with one of the strongest known El Niño events and extensive fishing efforts during the time. We find a direct impact of sea surface temperature on the community composition over time. Fish and cnidarian read abundance was examined separately to determine whether fishing had a direct impact, but no direct relation was found. These results demonstrate that sediment eDNA can be a valuable emerging tool providing insight in historical perspectives on ecosystem developments. This study constitutes an important step toward an improved understanding of the importance of environmental and anthropogenic drivers in affecting open and deep ocean communities.

From eDNA to decisions using a multi-method approach to restoration planning in streams.

Reintroduction efforts are increasingly used to mitigate biodiversity losses, but are frequently challenged by inadequate planning and uncertainty. High quality information about population status and threats can be used to prioritize reintroduction and restoration efforts and can transform ad hoc approaches into opportunities for improving conservation outcomes at a landscape scale. We conducted comprehensive environmental DNA (eDNA) and visual encounter surveys to determine the distribution of native and non-native aquatic species in two high-priority watersheds to address key uncertainties-such as the distribution of threats and the status of existing populations-inherent in restoration planning. We then used these occurrence data to develop a menu of potential conservation actions and a decision framework to benefit an endangered vertebrate (foothill yellow-legged frog, Rana boylii) in dynamic stream systems. Our framework combines the strengths of multiple methods, allowing managers and conservation scientists to incorporate conservation science and site-specific knowledge into the planning process to increase the likelihood of achieving conservation goals.

Comparative analysis of bottom trawl and nanopore sequencing in fish biodiversity assessment: The sylt outer reef example.

The assessment of fish diversity is crucial for effective conservation and management strategies, especially in ecologically sensitive regions such as marine protected areas. This study contrasts the effectiveness of environmental DNA (eDNA) metabarcoding analysis employing Nanopore technology with compare beam trawl surveys at the Sylt Outer Reef, a Natura 2000 site in the North Sea, Germany. Out of the 17 fish species caught in a bottom trawl (using a 3m beam trawl), 14 were also identified through eDNA extracted from water samples. The three species not detected in the eDNA results were absent because they lacked representation in public DNA databases. The eDNA method detected twice as many fish species as the beam trawl, totalling 36 species, of which 14 were also detected by the trawl. Additionally, the selection of primers (Mifish) facilitated the identification of one marine mammal species, the harbour porpoise. In conclusion, the findings underscore the potential of eDNA coupled with MinION sequencing (Long read technology) as a robust tool for biodiversity assessment, surpassing traditional methods in detecting species richness.

Environmental DNA for the surveillance of biosecurity threats in Mediterranean lagoons.

Invasive species that outcompete endemic ones and toxic harmful algae that cause algal blooms threaten marine resources like fisheries, aquaculture, and even tourism. Environmental DNA (eDNA) metabarcoding can help as a method for early alert. In this study, we have analyzed communities inhabiting six lagoons within the Gulf of Lion (northwest Mediterranean Sea) with spatial protection as RAMSAR and Natura 2000 sites. Employing the COI gene as the only metabarcode, we found 15 genera that have caused recognized algal bloom outbreaks in the studied lagoons since 2000. In addition, seven alien invasive species that can pose risks to the rich marine resources of the zone and lagoons were also found. The results found from eDNA are consistent with events of toxic algae blooms before and after the sampling moment and with reported occurrences of the invasive species in nearby Mediterranean areas. Multivariate multiple analysis showed the importance of anthropic pressure in the abundance of these nuisance species. Mitigation actions and routine eDNA metabarcoding in zones of special interest like these fragile French Mediterranean lagoons are recommended for early alert of nuisance species in order to plan timely management actions.

Environmental DNA metabarcoding reveals diverse phytoplankton assemblages and potentially harmful algal distribution along the urban coasts of Türkiye.

Marine phytoplankton are widely used to monitor the state of the water column due to their rapid changes in response to environmental conditions. In this study, we aimed to investigate the coastal phytoplankton assemblages, including bloom-forming species using high-throughput sequencing of 18S rRNA genes targeting the V4 region and their relationship with environmental variables along the Istanbul coasts of the Sea of Marmara. A total of 118 genera belonging to six phyla were detected. Among them, Dinoflagellata (36) and Bacillariophyta (26) were represented with the highest number of genera. According to the relative abundance of DNA reads, the most abundant taxa were Dinoflagellata_phylum (18.1%), Emiliania (8.4%), Biecheleria (8.4), and Noctiluca (8.1%). The ANOSIM test showed that there was a significant temporal difference in the assemblages, while the driving environmental factors were pH, water temperature, and salinity. According to the TRIX index, the trophic state of the coasts was highly mesotrophic and eutrophic. In addition, 45 bloom-forming and HAB taxa were detected and two species of Noctiluca and Emiliania, which frequently cause blooms in the area, were recorded in high abundance. Our results provide insight into the phytoplankton assemblages along the urbanized coastlines by analysing the V4 region of 18S rRNA. This data can support future studies that use both traditional methods and metabarcoding, employing various primers and targeting different genes and regions.

Biofouling sponges as natural eDNA samplers for marine vertebrate biodiversity monitoring.

Environmental DNA (eDNA) analysis has now become a core approach in marine biodiversity research, which typically involves the collection of water or sediment samples. Yet, recently, filter-feeding organisms have received much attention for their potential role as natural eDNA samplers. While the indiscriminate use of living organisms as 'sampling tools' might in some cases raise conservation concerns, there are instances in which highly abundant sessile organisms may become a nuisance as biofouling on artificial marine structures. Here we demonstrate how a sea sponge species that colonizes the moorings of the world's largest curtain of hydroacoustic receivers can become a powerful natural collector of fish biodiversity information. By sequencing eDNA extracted from Vazella pourtalesii retrieved from moorings during routine biofouling maintenance, we detected 23 species of marine fish and mammals, compared to 19 and 15 species revealed by surface and bottom water eDNA respectively, and 28 species captured by groundfish survey in the surrounding area, which are more ecologically impactful and involve higher additional costs. Sponge-based species inventories proved at least as informative as those obtained by traditional survey methods, and are also able to detect seasonal differences in fish assemblages. We conclude that opportunistic sampling of marine sponge biofouling may become an efficient way to document and monitor biodiversity in our rapidly changing oceans.

Exploring uncharted territory: new frontiers in environmental DNA for tropical fisheries management.

Tropical ecosystems host a significant share of global fish diversity contributing substantially to the global fisheries sector. Yet their sustainable management is challenging due to their complexity, diverse life history traits of tropical fishes, and varied fishing techniques involved. Traditional monitoring techniques are often costly, labour-intensive, and/or difficult to apply in inaccessible sites. These limitations call for the adoption of innovative, sensitive, and cost-effective monitoring solutions, especially in a scenario of climate change. Environmental DNA (eDNA) emerges as a potential game changer for biodiversity monitoring and conservation, especially in aquatic ecosystems. However, its utility in tropical settings remains underexplored, primarily due to a series of challenges, including the need for a comprehensive barcode reference library, an understanding of eDNA behaviour in tropical aquatic environments, standardized procedures, and supportive biomonitoring policies. Despite these challenges, the potential of eDNA for sensitive species detection across varied habitats is evident, and its global use is accelerating in biodiversity conservation efforts. This review takes an in-depth look at the current state and prospects of eDNA-based monitoring in tropical fisheries management research. Additionally, a SWOT analysis is used to underscore the opportunities and threats, with the aim of bridging the knowledge gaps and guiding the more extensive and effective use of eDNA-based monitoring in tropical fisheries management. Although the discussion applies worldwide, some specific experiences and insights from Indian tropical fisheries are shared to illustrate the practical application and challenges of employing eDNA in a tropical context.

Marine eDNA sampling from submerged surfaces with paint rollers.

Environmental DNA (eDNA) analyses of species present in marine environments is the most effective biological diversity measurement tool currently available. eDNA sampling methods are an intrinsically important part of the eDNA biodiversity analysis process. Identification and development of eDNA sampling methods that are as rapid, affordable, versatile and practical as possible will improve rates of detection of marine species. Optimal outcomes of eDNA biodiversity surveys come from studies employing high levels of sampling replication, so any methods that make sampling faster and cheaper will improve scientific outcomes. eDNA sampling methods that can be applied more widely will also enable sampling from a greater range of marine surface micro-habitats, resulting in detection of a wider range of organisms. In this study, we compared diversity detection by several methods for sampling eDNA from submerged marine surfaces: polyurethane foam, nylon swabs, microfibre paint rollers, and sediment scoops. All of the methods produced a diverse range of species identifications, with >250 multicellular species represented by eDNA at the study site. We found that widely-available small paint rollers were an effective, readily available and affordable method for sampling eDNA from underwater marine surfaces. This approach enables the sampling of marine eDNA using extended poles, or potentially by remotely operated vehicles, where surface sampling by hand is impractical.

TREMATODE SPECIES DETECTION AND QUANTIFICATION BY ENVIRONMENTAL DNA-qPCR ASSAY IN LAKE CHANY, RUSSIA.

Environmental DNA (eDNA) surveys promise to be a sensitive and powerful tool for the detection of trematodes. This can contribute to the limited studies on trematode ecology, specifically in aquatic ecosystems. Here, we developed species-specific primer and probe sets for Moliniella anceps, Opisthioglyphe ranae, and Plagiorchis multiglandularis cercariae and applied a novel eDNA qPCR assay to detect larval trematodes quantitatively. We evaluated the effectiveness of the assays using filtered lake water samples collected from different sites of Lake Fadikha and Kargat River Estuary in Lake Chany, Russia, showing high species specificity and sensitivity in all 3 assays. Further, all 3 assays had high efficiencies ranging from 94.9 to 105.8%. Moliniella anceps, O. ranae, and P. multiglandularis were detected in the environmental water samples through real-time PCR. Thus, we anticipate that our approach will be beneficial for biomonitoring, measuring, and managing ecological systems.

An integrated spatio-temporal view of riverine biodiversity using environmental DNA metabarcoding.

Anthropogenically forced changes in global freshwater biodiversity demand more efficient monitoring approaches. Consequently, environmental DNA (eDNA) analysis is enabling ecosystem-scale biodiversity assessment, yet the appropriate spatio-temporal resolution of robust biodiversity assessment remains ambiguous. Here, using intensive, spatio-temporal eDNA sampling across space (five rivers in Europe and North America, with an upper range of 20-35 km between samples), time (19 timepoints between 2017 and 2018) and environmental conditions (river flow, pH, conductivity, temperature and rainfall), we characterise the resolution at which information on diversity across the animal kingdom can be gathered from rivers using eDNA. In space, beta diversity was mainly dictated by turnover, on a scale of tens of kilometres, highlighting that diversity measures are not confounded by eDNA from upstream. Fish communities showed nested assemblages along some rivers, coinciding with habitat use. Across time, seasonal life history events, including salmon and eel migration, were detected. Finally, effects of environmental conditions were taxon-specific, reflecting habitat filtering of communities rather than effects on DNA molecules. We conclude that riverine eDNA metabarcoding can measure biodiversity at spatio-temporal scales relevant to species and community ecology, demonstrating its utility in delivering insights into river community ecology during a time of environmental change.

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

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

Cost-effort analysis of Baited Remote Underwater Video (BRUV) and environmental DNA (eDNA) in monitoring marine ecological communities.

Monitoring the diversity and distribution of species in an ecosystem is essential to assess the success of restoration strategies. Implementing biomonitoring methods, which provide a comprehensive assessment of species diversity and mitigate biases in data collection, holds significant importance in biodiversity research. Additionally, ensuring that these methods are cost-efficient and require minimal effort is crucial for effective environmental monitoring. In this study we compare the efficiency of species detection, the cost and the effort of two non-destructive sampling techniques: Baited Remote Underwater Video (BRUV) and environmental DNA (eDNA) metabarcoding to survey marine vertebrate species. Comparisons were conducted along the Sussex coast upon the introduction of the Nearshore Trawling Byelaw. This Byelaw aims to boost the recovery of the dense kelp beds and the associated biodiversity that existed in the 1980s. We show that overall BRUV surveys are more affordable than eDNA, however, eDNA detects almost three times as many species as BRUV. eDNA and BRUV surveys are comparable in terms of effort required for each method, unless eDNA analysis is carried out externally, in which case eDNA requires less effort for the lead researchers. Furthermore, we show that increased eDNA replication yields more informative results on community structure. We found that using both methods in conjunction provides a more complete view of biodiversity, with BRUV data supplementing eDNA monitoring by recording species missed by eDNA and by providing additional environmental and life history metrics. The results from this study will serve as a baseline of the marine vertebrate community in Sussex Bay allowing future biodiversity monitoring research projects to understand community structure as the ecosystem recovers following the removal of trawling fishing pressure. Although this study was regional, the findings presented herein have relevance to marine biodiversity and conservation monitoring programs around the globe.

Combining environmental DNA and remote sensing for efficient, fine-scale mapping of arthropod biodiversity.

Arthropods contribute importantly to ecosystem functioning but remain understudied. This undermines the validity of conservation decisions. Modern methods are now making arthropods easier to study, since arthropods can be mass-trapped, mass-identified, and semi-mass-quantified into 'many-row (observation), many-column (species)' datasets, with homogeneous error, high resolution, and copious environmental-covariate information. These 'novel community datasets' let us efficiently generate information on arthropod species distributions, conservation values, uncertainty, and the magnitude and direction of human impacts. We use a DNA-based method (barcode mapping) to produce an arthropod-community dataset from 121 Malaise-trap samples, and combine it with 29 remote-imagery layers using a deep neural net in a joint species distribution model. With this approach, we generate distribution maps for 76 arthropod species across a 225 km2 temperate-zone forested landscape. We combine the maps to visualize the fine-scale spatial distributions of species richness, community composition, and site irreplaceability. Old-growth forests show distinct community composition and higher species richness, and stream courses have the highest site-irreplaceability values. With this 'sideways biodiversity modelling' method, we demonstrate the feasibility of biodiversity mapping at sufficient spatial resolution to inform local management choices, while also being efficient enough to scale up to thousands of square kilometres. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.