Environmental DNA Sequencing Research Articles

Co-occurrence of Bacillariophyceae-based- and Cryptophyceae-based planktonic food webs in a temperate estuarine ecosystem revealed via eDNA

We examined microbial food webs in Seomjin River and Gwangyang Bay in South Korea, using 18S rRNA amplicon sequencing of environmental DNA (eDNA) with a weighted gene correlation network analysis (WGCNA) – Cytoscape analysis, considering various environmental variables. WGCNA with weight >0.5 revealed two taxon-specific trophic interactions. One was composed of Chlorophyceae and Cryptophyceae-based primary producers and ciliates-based primary consumers. In another one, Bacillariophyceae was a main primary producer while rotifer and ciliates were primary consumers. The Bray-Curtis dissimilarity and correlation matrix of gene abundance with gene significance related to environmental variables showed that the Chlorophyceae and Cryptophyceae-based microbial food web was a function of dissolved inorganic nitrogen in the Seomjin River, whereas the Bacillariophyceae-based microbial food web was a function of dissolved inorganic phosphate in the Gwangyang Bay. Our study provides new insight into microbial food webs while considering the nutrient status of estuarine ecosystems.

High-Throughput Sequencing of Environmental DNA as a Tool for Monitoring Eukaryotic Communities and Potential Pathogens in a Coastal Upwelling Ecosystem.

The marine environment includes diverse microeukaryotic organisms that play important functional roles in the ecosystem. With molecular approaches, eukaryotic taxonomy has been improved, complementing classical analysis. In this study, DNA metabarcoding was performed to describe putative pathogenic eukaryotic microorganisms in sediment and marine water fractions collected in Galicia (NW Spain) from 2016 to 2018. The composition of eukaryotic communities was distinct between sediment and water fractions. Protists were the most diverse group, with the clade TSAR (Stramenopiles, Alveolata, Rhizaria, and Telonemida) as the primary representative organisms in the environment. Harmful algae and invasive species were frequently detected. Potential pathogens, invasive pathogenic organisms as well as the causative agents of harmful phytoplanktonic blooms were identified in this marine ecosystem. Most of the identified pathogens have a crucial impact on the aquacultural sector or affect to relevant species in the marine ecosystem, such as diatoms. Moreover, pathogens with medical and veterinary importance worldwide were also found, as well as pathogens that affect diatoms. The evaluation of the health of a marine ecosystem that directly affects the aquacultural sector with a zoonotic concern was performed with the metabarcoding assay.

Potential of high-throughput eDNA sequencing of soil fungi and bacteria for monitoring ecological restoration in ultramafic substrates: The case study of the New Caledonian biodiversity hotspot

Due to their central role in ecosystems functioning and their ability to rapidly respond to environmental changes, soil microorganisms could potentially be used for monitoring ecosystems recovery in the context of degraded land restoration. However, these belowground organisms have been, to date, largely neglected. Here, we investigated fungal and bacterial community diversity, composition, and structure from ultramafic soils in New Caledonia, an archipelago in the southwest Pacific recognized as a priority for conservation and restoration. The emerging approach of high-throughput amplicon sequencing of environmental DNA (eDNA) – metabarcoding of eDNA – was used to compare soil microbial communities from four different native vegetation types, representing different stages of a chronosequence and defined as reference ecosystems, to five distinct post-mining sites revegetated several years ago. Our results clearly revealed changes in soil microbial phyla and functional groups along the reference chronosequence and variable responses at the different revegetated sites, with two of the five sites showing a good trajectory of recovery. We thus propose three ratios as metrics for monitoring the restoration trajectory of soil microorganisms: the Ascomycota:Basidiomycota and Saprotrophic:Ectomycorrhizal ratios for fungi, and the Cyanobacteria:Chloroflexi ratios for bacteria. Our study, combined with recent works undertaken in other geographical areas, underpins the great promise that could represent soil microbial eDNA metabarcoding for monitoring restoration progress and success. With the emergence of these new cost-effective and scalable sequencing technologies, soil microbes could, in the near future, be included in guidelines for restoration operations in complement to more conventional approaches.

Soil protist function varies with elevation in the Swiss Alps.

SummaryProtists are abundant and play key trophic functions in soil. Documenting how their trophic contributions vary across large environmental gradients is essential to understand and predict how biogeochemical cycles will be impacted by global changes. Here, using amplicon sequencing of environmental DNA in open habitat soil from 161 locations spanning 2600 m of elevation in the Swiss Alps (from 400 to 3000 m), we found that, over the whole study area, soils are dominated by consumers, followed by parasites and phototrophs. In contrast, the proportion of these groups in local communities shows large variations in relation to elevation. While there is, on average, three times more consumers than parasites at low elevation (400–1000 m), this ratio increases to 12 at high elevation (2000–3000 m). This suggests that the decrease in protist host biomass and diversity toward mountains tops impact protist functional composition. Furthermore, the taxonomic composition of protists that infect animals was related to elevation while that of protists that infect plants or of protist consumers was related to soil pH. This study provides a first step to document and understand how soil protist functions vary along the elevational gradient.

Metagenomes, metatranscriptomes and microbiomes of naturally decomposing deadwood

Deadwood represents significant carbon (C) stock in a temperate forests. Its decomposition and C mobilization is accomplished by decomposer microorganisms – fungi and bacteria – who also supply the foodweb of commensalist microbes. Due to the ecosystem-level importance of deadwood habitat as a C and nutrient stock with significant nitrogen fixation, the deadwood microbiome composition and function are critical to understanding the microbial processes related to its decomposition. We present a comprehensive suite of data packages obtained through environmental DNA and RNA sequencing from natural deadwood. Data provide a complex picture of the composition and function of microbiome on decomposing trunks of European beech (Fagus sylvatica L.) in a natural forest. Packages include deadwood metagenomes, metatranscriptomes, sequences of total RNA, bacterial genomes resolved from metagenomic data and the 16S rRNA gene and ITS2 metabarcoding markers to characterize the bacterial and fungal communities. This project will be of use to microbiologists, environmental biologists and biogeochemists interested in the microbial processes associated with the transformation of recalcitrant plant biomass.

Consistent declines in aquatic biodiversity across diverse domains of life in rivers impacted by surface coal mining.

The rivers of Appalachia (United States) are among the most biologically diverse freshwater ecosystems in the temperate zone and are home to numerous endemic aquatic organisms. Throughout the Central Appalachian ecoregion, extensive surface coal mines generate alkaline mine drainage that raises the pH, salinity, and trace element concentrations in downstream waters. Previous regional assessments have found significant declines in stream macroinvertebrate and fish communities after draining these mined areas. Here, we expand these assessments with a more comprehensive evaluation across a broad range of organisms (bacteria, algae, macroinvertebrates, all eukaryotes, and fish) using high-throughput amplicon sequencing of environmental DNA (eDNA). We collected water samples from 93 streams in Central Appalachia (West Virginia, United States) spanning a gradient of mountaintop coal mining intensity and legacy to assess how this land use alters downstream water chemistry and affects aquatic biodiversity. For each group of organisms, we identified the sensitive and tolerant taxa along the gradient and calculated stream specific conductivity thresholds in which large synchronous declines in diversity were observed. Streams below mining operations had steep declines in diversity (-18 to -41%) and substantial shifts in community composition that were consistent across multiple taxonomic groups. Overall, large synchronous declines in bacterial, algal, and macroinvertebrate communities occurred even at low levels of mining impact at stream specific conductivity thresholds of 150-200 µS/cm that are substantially below the current U.S. Environmental Protection Agency aquatic life benchmark of 300 µS/cm for Central Appalachian streams. We show that extensive coal surface mining activities led to the extirpation of 40% of biodiversity from impacted rivers throughout the region and that current water quality criteria are likely not protective for many groups of aquatic organisms.

Exploring the plant environmental DNA diversity in soil from two sites on Deception Island (Antarctica, South Shetland Islands) using metabarcoding

AbstractThe few Antarctic studies to date to have applied metabarcoding in Antarctica have primarily focused on microorganisms. In this study, for the first time, we apply high-throughput sequencing of environmental DNA to investigate the diversity of Embryophyta (Viridiplantae) DNA present in soil samples from two contrasting locations on Deception Island. The first was a relatively undisturbed site within an Antarctic Specially Protected Area at Crater Lake, and the second was a heavily human-impacted site in Whalers Bay. In samples obtained at Crater Lake, 84% of DNA reads represented fungi, 14% represented Chlorophyta and 2% represented Streptophyta, while at Whalers Bay, 79% of reads represented fungi, 20% represented Chlorophyta and < 1% represented Streptophyta, with ~1% of reads being unassigned. Among the Embryophyta we found 16 plant operational taxonomic units from three Divisions, including one Marchantiophyta, eight Bryophyta and seven Magnoliophyta. Sequences of six taxa were detected at both sampling sites, eight only at Whalers Bay and two only at Crater Lake. All of the Magnoliophyta sequences (flowering plants) represent species that are exotic to Antarctica, with most being plausibly linked to human food sources originating from local national research operator and tourism facilities.

The role of internal transcribed spacer 2 secondary structures in classifying mycoparasitic Ampelomyces

Many fungi require specific growth conditions before they can be identified. Direct environmental DNA sequencing is advantageous, although for some taxa, specific primers need to be used for successful amplification of molecular markers. The internal transcribed spacer region is the preferred DNA barcode for fungi. However, inter- and intra-specific distances in ITS sequences highly vary among some fungal groups; consequently, it is not a solely reliable tool for species delineation. Ampelomyces, mycoparasites of the fungal phytopathogen order Erysiphales, can have ITS genetic differences up to 15%; this may lead to misidentification with other closely related unknown fungi. Indeed, Ampelomyces were initially misidentified as other pycnidial mycoparasites, but subsequent research showed that they differ in pycnidia morphology and culture characteristics. We investigated whether the ITS2 nucleotide content and secondary structure was different between Ampelomyces ITS2 sequences and those unrelated to this genus. To this end, we retrieved all ITS sequences referred to as Ampelomyces from the GenBank database. This analysis revealed that fungal ITS environmental DNA sequences are still being deposited in the database under the name Ampelomyces, but they do not belong to this genus. We also detected variations in the conserved hybridization model of the ITS2 proximal 5.8S and 28S stem from two Ampelomyces strains. Moreover, we suggested for the first time that pseudogenes form in the ITS region of this mycoparasite. A phylogenetic analysis based on ITS2 sequences-structures grouped the environmental sequences of putative Ampelomyces into a different clade from the Ampelomyces-containing clades. Indeed, when conducting ITS2 analysis, resolution of genetic distances between Ampelomyces and those putative Ampelomyces improved. Each clade represented a distinct consensus ITS2 S2, which suggested that different pre-ribosomal RNA (pre-rRNA) processes occur across different lineages. This study recommends the use of ITS2 S2s as an important tool to analyse environmental sequencing and unveiling the underlying evolutionary processes.

Assessing the in situ bacterial diversity and composition at anthropogenically active sites using the environmental DNA (eDNA)

In this study, we identified the in situ bacterial groups and their community structure in coastal waters influenced by anthropogenic inputs. The use of environmental DNA (eDNA) and high throughput sequencing (HTS) were employed to derive accurate and reliable information on bacterial abundance. The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified and the sequences were clustered into operational taxonomic units to analyze the site-specific variations in community composition. The percentage composition within the bacterial orders varied significantly among nearshore anthropogenic hotspots and offshore (5 km) samples. The microbial network constructed taking the bacterial abundance as nodes displayed strong positive and negative correlations within the bacterial families. Overall, the use of eDNA coupled with HTS is an incredible means for monitoring and assessing the abundance of bacterial communities and also serves as a biomonitoring tool to understand the degree of anthropogenic contamination in coastal waters.

Changes in protist communities in drainages across the Pearl River Delta under anthropogenic influence

Drainages in the Pearl River Delta urban agglomeration (PRDUA) host vital aquatic ecosystems and face enormous pressures from human activities in one of the largest urban agglomerations in the world. Despite being crucial components of aquatic ecosystems, the interactions and assembly processes of the protistan community are rarely explored in areas with serious anthropogenic disturbance. To elucidate the mechanisms of these processes, we used environmental DNA sequencing of 18S rDNA to investigate the influence of environmental factors and species interactions on the protistan community and its assembly in drainages of the PRDUA during summer. The protistan community showed a high level of diversity and a marked spatial pattern in this region. Community assembly was driven primarily by stochastic processes based on the Sloan neutral community model, explaining 74.28%, 75.82%, 73.67%, 74.40% and 51.24% of community variations in the BJ (Beijiang), XJ (Xijiang), PRD (Pearl River Delta), PRE (Pearl River Estuary) areas and in total, respectively. Meanwhile, environmental variables including temperature, pH, dissolved oxygen, transparency, nutrients and land use were strongly correlated with the composition and assembly of the protistan community, explaining 40.40% of variation in the protistan community. Furthermore, the bacterial community was simultaneously analysed by the 16S rDNA sequencing. Co-occurrence network analysis revealed that species interactions within bacteria (81.41% positive) or protists (82.80% positive), and those between bacteria and protists (50% positive and 50% negative) impacted the protistan community assembly. In summary, stochastic processes dominated, whereas species interactions and environmental factors also played important roles in shaping the protistan communities in drainages across the PRDUA. This study provides insights into the ecological patterns, assembly processes and species interactions underlying protistan dynamics in urban aquatic ecosystems experiencing serious anthropogenic disturbance.

Fieldable Environmental DNA Sequencing to Assess Jellyfish Biodiversity in Nearshore Waters of the Florida Keys, United States

Recent advances in molecular sequencing technology and the increased availability of fieldable laboratory equipment have provided researchers with the opportunity to conduct real-time or near real-time gene-based biodiversity assessments of aquatic ecosystems. In this study, we developed a workflow and portable kit for fieldable environmental DNA sequencing (FeDS) and tested its efficacy by characterizing the breadth of jellyfish (Medusozoa) taxa in the coastal waters of the Upper and Lower Florida Keys. Environmental DNA was isolated from seawater collection events at eight sites and samples were subjected to medusozoan 16S rRNA gene and metazoan mitochondrial cytochrome oxidase 1 gene profiling via metabarcoding onsite. In total, FeDS yielded 175,326 processed sequence reads providing evidence for 53 medusozoan taxa. Our most salient findings revealed eDNA from: (1) two venomous box jellyfish (Cubozoa) species, including taxa whose stings cause the notorious Irukandji envenomation syndrome; (2) two species of potentially introduced stalked jellyfish (Staurozoa); and (3) a likely cryptic species of upside-down jellyfish (Scyphozoa). Taken together, the results of this study highlight the merits of FeDS in conducting biodiversity surveys of endemic and introduced species, and as a potential tool for assessing envenomation and/or conservation-related threats.

Rappemonads are haptophyte phytoplankton

Rapidly accumulating genetic data from environmental sequencing approaches have revealed an extraordinary level of unsuspected diversity within marine phytoplankton,1-11 which is responsible for around 50% of global net primary production.12,13 However, the phenotypic identity of many of the organisms distinguished by environmental DNA sequences remains unclear. The rappemonads represent a plastid-bearing protistan lineage that to date has only been identified by environmental plastid 16S rRNA sequences.14-17 The phenotypic identity of this group, which does not confidently cluster in any known algal clades in 16S rRNA phylogenetic reconstructions,15 has remained unknown since the first report of environmental sequences over two decades ago. We show that rappemonads are closely related to a haptophyte microalga, Pavlomulina ranunculiformis gen. nov. et sp. nov., and belong to a new haptophyte class, the Rappephyceae. Organellar phylogenomic analyses provide strong evidence for the inclusion of this lineage within the Haptophyta as a sister group to the Prymnesiophyceae. Members of this new class have a cosmopolitan distribution in coastal and oceanic regions. The relative read abundance of Rappephyceae in a large environmental barcoding dataset was comparable to, or greater than, those of major haptophyte species, such as the bloom-forming Gephyrocapsa huxleyi and Prymnesium parvum, and this result indicates that they likely have a significant impact as primary producers. Detailed characterization of Pavlomulina allowed for reconstruction of the ancient evolutionary history of the Haptophyta, a group that is one of the most important components of extant marine phytoplankton communities.

EDNAFlow, an automated, reproducible and scalable workflow for analysis of environmental DNA sequences exploiting Nextflow and Singularity

Metabarcoding of environmental DNA (eDNA) when coupled with high throughput sequencing is revolutionising the way biodiversity can be monitored across a wide range of applications. However, the large number of tools deployed in downstream bioinformatic analyses often places a challenge in configuration and maintenance of a workflow, and consequently limits the research reproducibility. Furthermore, scalability needs to be considered to handle the growing amount of data due to increase in sequence output and the scale of project. Here, we describe eDNAFlow, a fully automated workflow that employs a number of state-of-the-art applications to process eDNA data from raw sequences (single-end or paired-end) to generation of curated and noncurated zero-radius operational taxonomic units (ZOTUs) and their abundance tables. This pipeline is based on Nextflow and Singularity which enable a scalable, portable and reproducible workflow using software containers on a local computer, clouds and high-performance computing (HPC) clusters. Finally, we present an in-house Python script to assign taxonomy to ZOTUs based on user specified thresholds for assigning lowest common ancestor (LCA). We demonstrate the utility and efficiency of the pipeline using an example of a published coral diversity biomonitoring study. Our results were congruent with the aforementioned study. The scalability of the pipeline is also demonstrated through analysis of a large data set containing 154 samples. To our knowledge, this is the first automated bioinformatic pipeline for eDNA analysis using two powerful tools: Nextflow and Singularity. This pipeline addresses two major challenges in the analysis of eDNA data; scalability and reproducibility.

Metabarcoding with MinION: Speeding up the detection of invasive aquatic species using environmental DNA and nanopore sequencing

Traditional detection of aquatic invasive species, via morphological identification is often time-consuming and can require a high level of taxonomic expertise, leading to delayed mitigation responses. Environmental DNA (eDNA) detection approaches of multiple species using Illumina-based sequencing technology have been used to overcome these hindrances, but sample processing is often lengthy. More recently, portable nanopore sequencing technology has become available, which has the potential to make molecular detection of invasive species more widely accessible and to substantially decrease sample turnaround times. However, nanopore-sequenced reads have a much higher error rate than those produced by Illumina platforms, which has so far hindered the adoption of this technology. We provide a detailed laboratory protocol and bioinformatic tools to increase the reliability of nanopore sequencing to detect invasive species, and we test its application using invasive bivalves. We sampled water from sites with pre-existing bivalve occurrence and abundance data, and contrasting bivalve communities, in Italy and Portugal. We extracted, amplified and sequenced eDNA with a turnaround of 3.5 days. The majority of processed reads were ≥ 99 % identical to reference sequences. There were no taxa detected other than those known to occur. The lack of detections of some species at some sites could be explained by their known low abundances. The approach is now being tested on other target taxa such as fish and other vertebrates.

Using the predictive functional profiling of microbial communities to assess the ecological quality of reclaimed waters

The use of proxies for ecosystem function in biomonitoring is desirable to move towards more holistic strategies. Next-generation sequencing of environmental DNA can clearly contribute to these advances. This research is part of a project aimed to use constructed wetlands (CWs), managed by the company Global Omnium (https://www.globalomnium.com/Group/Home/) and having the University of Valencia in charge of the scientific research, in order to improve the ecological quality of already treated wastewaters before being poured into natural environments. The rationale is that, although wastewaters treated by classical methods can meet the standards required by law, transitional constructed ecosystems, as the CWs, can promote ancillary benefits that contribute to the maintenance of the ecological health of receiving natural ecosystems. We propose that some functional traits of the microbial community can be used to outline the process of water renaturation. To assess this, the prokaryotic communities of different types of CWs were profiled by the MiSeq sequencing of the V4 region of the 16S rRNA. Using the sequences obtained, a prediction of the functional capabilities of these communities was made with the bioinformatic package PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States; https://github.com/picrust/picrust2), assuming that phylogeny and function are effectively related attributes. Predictions on the occurrence of functional marker genes, particularly those involved in the biogeochemical cycling of main nutrients (C, N, S, P), were then made. The PICRUSt2 predictions were based in the annotated genes catalog of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/). In general, results indicated a decline in the CWs effluents of microbial functions typical from wastewater in favor of those more suited for the receiving environment. An enhancement of aerobic metabolism was predicted to occur based on the increase of the gene encoding the cytochrome-c oxidase compared to fermentation pathways. Accordingly, both the denitrification and the dissimilatory reduction of sulfate, that are predominant in the anaerobic environments, also decreased in the effluents of the CWs to lower levels more similar to those observed in natural environments, showing a shift towards reactions at higher redox potentials. On the other hand, a development of metabolic skills for degrading plant materials (e.g., xylose, vanillin, syringate, protocatechuate) was also predicted, which was likely related to a parallel transformation of the organic matter pool in the CWs (more unreactive and natural). Additionally, increases observed in pathways for the synthesis of some cell structural compounds (e.g., mycolic acids) and coenzymes (e.g., F420 cofactor, NAD+) can be related with an enhancement of colonization and competitive potentials of the natural microbial community, as well as with changes in the actual availability of nutrients in the environment. Based on these findings, we raise the possibility of considering these functional surveys made on marker genes as a complementary strategy on the biomonitoring procedures, not only for CWs, but for the general study of freshwater ecosystem potentially affected by wastewater pollution.

Land-use types shape soil microbial compositions under rapid urbanization in the Xiong'an New Area, China.

For urban planning and infrastructural projects, considerable attention has been paid to the relationship between soil biota, especially protists, and edaphic conditions in various land-use types having different plant species in the Xiong'an New Area of China. To elucidate this relationship, we assessed edaphic variables and soil biota compositions and compared them among 5 habitat types: human-made forests, crop cultivations, arid rivers, Baiyangdian (BYD) Lake, and around oil wells. In all, 12 experimental plots from terrestrial and aquatic ecosystems were assessed using high-throughput sequencing of environmental DNA, targeting the V3-V4 region of the 16S rRNA gene, internal transcribed spacer 1, and V4 region of the 18S rRNA gene for bacteria, fungi, and protists, respectively. The abundance of bacterial and protist communities was higher than fungi, possibly because fungi prefer acidic soil conditions and likely have greater susceptibility to anthropogenic activities. Across all experimental plots, land-use types contributed the most to the β-diversity of soil biota, followed by soil moisture. Diversity and richness were significantly higher at aquatic habitats than at terrestrial habitats. Predictive metagenomic analysis of trophic groups predicted relatively high frequency of functional genes from bacterial metabolism pathways (carbohydrate and amino acid); contrary to expectation, phototrophic protists, but not fungal symbionts and protistan consumers, were the dominant group at the BYD Lake. Geographical coordinates showed significant (P<0.05) relationships with all microbiome taxa (nodes at network) from all land-use types. Moreover, soil-microbiome relationships were more complex and more intense at crop habitats. Links between protist and fungal taxa were the highest at the petroleum-contaminated sampling sites, indicating the importance of these two soil microbiomes in polluted soil. Thus, our findings suggest that human manipulation and land-use types are crucial factors for soil biota structure and composition across our sampling sites.

Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

The combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

Carbon fixation and rhodopsin systems in microbial mats from hypersaline lakes Brava and Tebenquiche, Salar de Atacama, Chile.

In this work, molecular diversity of two hypersaline microbial mats was compared by Whole Genome Shotgun (WGS) sequencing of environmental DNA from the mats. Brava and Tebenquiche are lakes in the Salar de Atacama, Chile, where microbial communities are growing in extreme conditions, including high salinity, high solar irradiance, and high levels of toxic metals and metaloids. Evaporation creates hypersaline conditions in these lakes and mineral precipitation is a characteristic geomicrobiological feature of these benthic ecosystems. The mat from Brava was more rich and diverse, with a higher number of different taxa and with species more evenly distributed. At the phylum level, Proteobacteria, Cyanobacteria, Chloroflexi, Bacteroidetes and Firmicutes were the most abundant, including ~75% of total sequences. At the genus level, the most abundant sequences were affilitated to anoxygenic phototropic and cyanobacterial genera. In Tebenquiche mats, Proteobacteria and Bacteroidetes covered ~70% of the sequences, and 13% of the sequences were affiliated to Salinibacter genus, thus addressing the lower diversity. Regardless of the differences at the taxonomic level, functionally the two mats were similar. Thus, similar roles could be fulfilled by different organisms. Carbon fixation through the Wood-Ljungdahl pathway was well represented in these datasets, and also in other mats from Andean lakes. In spite of presenting less taxonomic diversity, Tebenquiche mats showed increased abundance and variety of rhodopsin genes. Comparison with other metagenomes allowed identifying xantorhodopsins as hallmark genes not only from Brava and Tebenquiche mats, but also for other mats developing at high altitudes in similar environmental conditions.

A SYSTEMATIC REVIEW AND NARRATIVE SYNTHESIS OF THE USE OF ENVIRONMENTAL SAMPLES FOR THE SURVEILLANCE OF AVIAN INFLUENZA VIRUSES IN WILD WATERBIRDS.

Wild waterbirds are reservoir hosts for avian influenza viruses (AIV), which can cause devastating outbreaks in multiple species, making them a focus for surveillance efforts. Traditional AIV surveillance involves direct sampling of live or dead birds, but environmental substrates present an alternative sample for surveillance. Environmental sampling analyzes AIV excreted by waterbirds into the environment and complements direct bird sampling by minimizing financial, logistic, permitting, and spatial-temporal constraints associated with traditional surveillance. Our objectives were to synthesize the literature on environmental AIV surveillance, to compare and contrast the different sample types, and to identify key themes and recommendations to aid in the implementation of AIV surveillance using environmental samples. The four main environmental substrates for AIV surveillance are feces, feathers, water, and sediment or soil. Feces were the most common environmental substrate collected. The laboratory analysis of water and sediment provided challenges, such as low AIV concentration, heterogenous AIV distribution, or presence of PCR inhibitors. There are a number of abiotic and biotic environmental factors, including temperature, pH, salinity, or presence of filter feeders, that can influence the presence and persistence of AIV in environmental substrates; however, the nature of this influence is poorly understood in field settings, and field data from southern, coastal, and tropical ecosystems are underrepresented. Similarly, there are few studies comparing the performance of environmental samples to each other and to samples collected in wild waterbirds, and environmental surveillance workflows have yet to be validated or optimized. Environmental samples, particularly when used in combination with new technology such as environmental DNA and next generation sequencing, provided information on trends in AIV detection rates and circulating subtypes that complemented traditional, direct waterbird sampling. The use of environmental samples for AIV surveillance also shows significant promise for programs whose goal is early warning of high-risk subtypes.

Genomic Monitoring of the Critically Endangered Kākāpō by Real-Time Targeted Nanopore Sequencing of Environmental DNA

Genomic Monitoring of the Critically Endangered Kākāpō by Real-Time Targeted Nanopore Sequencing of Environmental DNA