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

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

Natrix: a Snakemake-based workflow for processing, clustering, and taxonomically assigning amplicon sequencing reads.

BACKGROUND: Sequencing of marker genes amplified from environmental samples, known as amplicon sequencing, allows us to resolve some of the hidden diversity and elucidate evolutionary relationships and ecological processes among complex microbial communities. The analysis of large numbers of samples at high sequencing depths generated by high throughput sequencing technologies requires efficient, flexible, and reproducible bioinformatics pipelines. Only a few existing workflows can be run in a user-friendly, scalable, and reproducible manner on different computing devices using an efficient workflow management system. RESULTS: We present Natrix, an open-source bioinformatics workflow for preprocessing raw amplicon sequencing data. The workflow contains all analysis steps from quality assessment, read assembly, dereplication, chimera detection, split-sample merging, sequence representative assignment (OTUs or ASVs) to the taxonomic assignment of sequence representatives. The workflow is written using Snakemake, a workflow management engine for developing data analysis workflows. In addition, Conda is used for version control. Thus, Snakemake ensures reproducibility and Conda offers version control of the utilized programs. The encapsulation of rules and their dependencies support hassle-free sharing of rules between workflows and easy adaptation and extension of existing workflows. Natrix is freely available on GitHub ( https://github.com/MW55/Natrix ) or as a Docker container on DockerHub ( https://hub.docker.com/r/mw55/natrix ). CONCLUSION: Natrix is a user-friendly and highly extensible workflow for processing Illumina amplicon data.

Monitoring freshwater fish communities in large rivers using environmental DNA metabarcoding and a long-term electrofishing survey.

Monitoring freshwater fish communities in a large human-impacted river is a challenging task. The structure of fish assemblages has been monitored yearly in the Marne and the Seine Rivers, across the Paris conurbation, France, using traditional electrofishing (EF) surveys since 1990, in accordance with the European Water Framework Directive. In addition, metabarcoding of DNA extracted from environmental samples (eDNA) was concomitantly conducted in nine sampling sites in 2017 and in 2018 to compare the estimates of species richness and relative abundance among three methods: annual, long-term EF monitoring and eDNA. The present study confirms better detection of fish species using eDNA compared to annual EF. eDNA metabarcoding was also more efficient for species detection than a 3-6-year EF survey but was similar or less efficient than a long-term EF survey of 14 years of monitoring. In addition, the numbers of reads per species relative to the total number of reads significantly increased with (a) increasing relative abundance (relative percentage of individuals caught per species) and (b) increasing number of years that a fish species was detected during the 2000-2018 period. These results suggest that eDNA could reflect local population persistence.

Predicting the fate of eDNA in the environment and implications for studying biodiversity.

Environmental DNA (eDNA) applications are transforming the standard of characterizing aquatic biodiversity via the presence, location and abundance of DNA collected from environmental samples. As eDNA studies use DNA fragments as a proxy for the presence of organisms, the ecological properties of the complex and dynamic environments from which eDNA is sampled need to be considered for accurate biological interpretation. In this review, we discuss the role that differing environments play on the major processes that eDNA undergoes between organism and collection, including shedding, decay and transport. We focus on a mechanistic understanding of these processes and highlight how decay and transport models are being developed towards more accurate and robust predictions of the fate of eDNA. We conclude with five recommendations for eDNA researchers and practitioners, to advance current best practices, as well as to support a future model of eDNA spatio-temporal persistence.

Environmental DNA Isolation, Validation, and Preservation Methods.

Environmental DNA (eDNA) workflows contain many familiar molecular-lab techniques, but also employ several unique methodologies. When working with eDNA, it is essential to avoid contamination from the point of collection through preservation and select a meaningful negative control. As eDNA can be obtained from a variety of samples and habitats (e.g., soil, water, air, or tissue), protocols will vary depending on usage. Samples may require additional steps to dilute, block, or remove inhibitors or physically break up samples or filters. Thereafter, standard DNA isolation techniques (kit-based or phenol:chloroform:isoamyl [PCI]) are employed. Once DNA is extracted, it is typically quantified using a fluorometer. Yields vary greatly, but are important to know prior to amplification of the gene(s) of interest. Long-term storage of both the sampled material and the extracted DNA is encouraged, as it provides a backup for spilled/contaminated samples, lost data, reanalysis, and future studies using newer technology. Storage in a freezer is often ideal; however, some storage buffers (e.g., Longmires) require that filters or swabs are kept at room temperature to prevent precipitation of buffer-related solutes. These baseline methods for eDNA isolation, validation, and preservation are detailed in this protocol chapter. In addition, we outline a cost-effective, homebrew extraction protocol optimized to extract eDNA.

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.

The update and optimization of an eDNA assay to detect the invasive rusty crayfish (Faxonius rusticus).

Environmental DNA (eDNA) is nuclear or mitochondrial DNA shed into the environment, and amplifying this DNA can serve as a reliable, noninvasive way to monitor aquatic systems for the presence of an invasive species. Assays based on the collection of eDNA are becoming increasingly popular, and, when optimized, can aid in effectively and efficiently tracking invasion fronts. We set out to update an eDNA assay to detect the invasive rusty crayfish, Faxonius rusticus. We tested for species specificity compared to other stream crayfish and field tested the assay at sites with known presence (N = 3) and absence (N = 4) in the Juniata River watershed in central Pennsylvania, USA. To maximize sensitivity, we field tested different storage buffers (Longmire's buffer and ethanol), DNA extraction methods (Qiagen's DNEasy and PowerWater kits), and quantitative polymerase chain reaction (qPCR) chemistries (TaqMan and SYBR green). Our assay confirmed the presence data and performed optimally when filter samples were stored in Longmire's buffer, DNA was extracted with DNeasy Blood and Tissue Kit, and TaqMan qPCR chemistry was utilized. With proper sample processing, our assay allows for accurate, noninvasive detection of F. rusticus in streams.

Environmental DNA gives comparable results to morphology-based indices of macroinvertebrates in a large-scale ecological assessment.

Anthropogenic activities are changing the state of ecosystems worldwide, affecting community composition and often resulting in loss of biodiversity. Rivers are among the most impacted ecosystems. Recording their current state with regular biomonitoring is important to assess the future trajectory of biodiversity. Traditional monitoring methods for ecological assessments are costly and time-intensive. Here, we compared monitoring of macroinvertebrates based on environmental DNA (eDNA) sampling with monitoring based on traditional kick-net sampling to assess biodiversity patterns at 92 river sites covering all major Swiss river catchments. From the kick-net community data, a biotic index (IBCH) based on 145 indicator taxa had been established. The index was matched by the taxonomically annotated eDNA data by using a machine learning approach. Our comparison of diversity patterns only uses the zero-radius Operational Taxonomic Units assigned to the indicator taxa. Overall, we found a strong congruence between both methods for the assessment of the total indicator community composition (gamma diversity). However, when assessing biodiversity at the site level (alpha diversity), the methods were less consistent and gave complementary data on composition. Specifically, environmental DNA retrieved significantly fewer indicator taxa per site than the kick-net approach. Importantly, however, the subsequent ecological classification of rivers based on the detected indicators resulted in similar biotic index scores for the kick-net and the eDNA data that was classified using a random forest approach. The majority of the predictions (72%) from the random forest classification resulted in the same river status categories as the kick-net approach. Thus, environmental DNA validly detected indicator communities and, combined with machine learning, provided reliable classifications of the ecological state of rivers. Overall, while environmental DNA gives complementary data on the macroinvertebrate community composition compared to the kick-net approach, the subsequently calculated indices for the ecological classification of river sites are nevertheless directly comparable and consistent.

Intraspecific genetic variability and diurnal activity affect environmental DNA detection in Japanese eel.

Environmental DNA (eDNA) analysis with species-specific primer/probe sets is promising as a tool to quantify fish abundance and distribution. Nevertheless, several factors could reduce the accuracy of this method. Here, we aimed to analyze whether intraspecific variability and diel activity rhythm affect eDNA detection in Japanese eels (Anguilla japonica). For this purpose, we performed tank experiments focusing on two points. First, we assessed the effects of base pair sequences with probe region polymorphism on eDNA detection. Next, we evaluated the influences of diel rhythm, activity, and individual differences in eDNA release rate on eDNA concentration. We examined the base pair sequences of the probe regions of 20 individuals and found genetic mismatches in two of them. The eDNA concentration was estimated to be much lower in these variants than it was in the other individuals. We conducted a rearing experiment on four non-variant individuals to explore the influences of diel activity and inter-individual differences in eDNA detection. Nocturnal eel activity was reflected in the eDNA detection but the inter-individual differences remained large. The observed weak positive correlations between eDNA concentration and activity suggest that eDNA emission is highly dependent on basal metabolism. The present study suggests that consideration of polymorphic sites at the probe region and diel activity rhythms should improve the accuracy and precision of abundance estimation through eDNA. Such fine-tuning is applicable not only for eels but also for other fishes to be targeted by eDNA technology.

Environmental DNA detection of an invasive ant species (Linepithema humile) from soil samples.

Alien ant species (Formicidae, Hymenoptera) cause serious damage worldwide. Early detection of invasion and rapid management are significant for controlling these species. However, these attempts are sometimes hindered by the need for direct detection techniques, such as capture, visual observation, or morphological identification. In this study, we demonstrated that environmental DNA (eDNA) analysis can be used as a monitoring tool for alien ants using Linepithema humile (Argentine ant), one of the most invasive ants, as a model species. We designed a new real-time PCR assay specific to L. humile and successfully detected eDNA from the surface soil. The reliability of eDNA analysis was substantiated by comparing eDNA detection results with traditional survey results. Additionally, we examined the relationship between eDNA concentration and distance from nests and trails. Our results support the effectiveness of eDNA for alien ant monitoring and suggest that this new method could improve our ability to detect invasive ant species.

Persistence and accumulation of environmental DNA from an endangered dragonfly.

Detection of environmental DNA (eDNA) has become a commonly used surveillance method for threatened or invasive vertebrates in both aquatic and terrestrial environments. However, most studies in this field favor vertebrate target species. Environmental DNA protocols can be especially useful for endangered invertebrates such as the Hine's emerald dragonfly (Somatochlora hineana) where conservation efforts have been greatly hindered by training, time, overall costs, and environmental impacts associated with conducting surveys in the calcareous fens occupied by this species. An essential step in developing such a protocol is to evaluate the dynamics of eDNA concentration under controlled conditions. We used the quantitative polymerase chain reaction (qPCR) to examine seasonal shifts in the persistence and net-accumulation of eDNA from captive S. hineana larvae in experimental mesocosms at temperatures corresponding with their overwintering (5.0 °C) and active (16.0 °C) seasons. Environmental DNA persisted longer at 5.0 °C but accumulated more readily at 16.0 °C. Differences in the accumulation and persistence of eDNA reflect differences in the longevity of eDNA at different temperatures and seasonal differences in larval S. hineana behavior. This study highlights the importance of considering how seasonal changes in temperature influence not only the speed of eDNA degradation but also the target species' eDNA shedding rates.

Molecular detection of giant snakeheads, Channa micropeltes (Cuvier, 1831), one of the most troublesome fish species.

A lack of reliable tools for determining the presence and distribution of fish species can impede understanding of predator-prey interactions and fishery management. Conventional fish survey methods are invasive, and can be size or species selective. Combining netting and electrofishing is a current method used to monitor fish species in Phayao Lake (Kwan Phayao), Thailand. However, the methods are inefficient and time-consuming. Recently, locals who rely on inland fisheries in Kwan Phayao expressed their deep concerns about the giant snakehead, Channa micropeltes (Cuvier, 1831) destroying other fish there. The giant snakehead prey on many commercially important fish species, as the prey species is reduced, negative effects on both biodiversity and the fishery sector could follow. Here, an eDNA-based survey was developed to detect the presence of the giant snakehead. Water samples were collected from six sites within Kwan Phayao and 17 sites in Ing River where water flowed into and out of Kwan Payao. The eDNA of the giant snakehead was detected in water samples from all collection sites using the developed qPCR assay with various concentrations. The eDNA was shown here to be a sensitive and reliable tool for fish surveillance so there will be a better chance for developing an effective management strategy.

A workflow for the relative quantification of multiple fish species from oceanic water samples using environmental DNA (eDNA) to support large-scale fishery surveys.

While the number of published marine studies using environmental DNA (eDNA) has increased substantially in recent years, marine fish surveys are still scarce. To examine the potential for eDNA to support marine fisheries monitoring surveys, we optimized an eDNA isolation method, developed a multispecies assay and tested it on eDNA samples collected along the Pacific coast of the United States. Four commercial DNA extraction kits that exploit the capability of the nucleic acids binding a solid phase (two using a silica matrix and two magnetic beads) as well an organic separation method were tested. A species-specific multiplex qPCR assay was developed and tested to simultaneously target Pacific hake (Merluccius productus), Pacific lamprey (Entosphenus tridentatus) and eulachon (Thaleichthys pacificus). The specificity of the assay was tested in silico, in vitro and in natura. Environmental DNA isolation using phenol:chloroform:isoamyl purification with a phase lock was optimized and yielded the highest amount of total and target DNA and was used to extract 46 marine water samples for the detection of the three species of interest. The multiplex qPCR assay used in the quantification process was also optimized to provide convenience and accuracy. Pacific hake was present in 44% of the eDNA samples while the other two species were absent. Here, we present a complete workflow for the simultaneous detection and quantification of multiple marine fish species using eDNA. This workflow supports large-scale at-sea sampling efforts with preservation at ambient temperatures and has demonstrated DNA extraction efficiency and reliability. The multiplex qPCR assay is shown to be sensitive and specific for the purposes of simultaneously monitoring the relative abundance of multiple targeted fish species.

Optimising sampling and analysis protocols in environmental DNA studies.

Ecological surveys risk incurring false negative and false positive detections of the target species. With indirect survey methods, such as environmental DNA, such error can occur at two stages: sample collection and laboratory analysis. Here we analyse a large qPCR based eDNA data set using two occupancy models, one of which accounts for false positive error by Griffin et al. (J R Stat Soc Ser C Appl Stat 69: 377-392, 2020), and a second that assumes no false positive error by Stratton et al. (Methods Ecol Evol 11: 1113-1120, 2020). Additionally, we apply the Griffin et al. (2020) model to simulated data to determine optimal levels of replication at both sampling stages. The Stratton et al. (2020) model, which assumes no false positive results, consistently overestimated both overall and individual site occupancy compared to both the Griffin et al. (2020) model and to previous estimates of pond occupancy for the target species. The inclusion of replication at both stages of eDNA analysis (sample collection and in the laboratory) reduces both bias and credible interval width in estimates of both occupancy and detectability. Even the collection of > 1 sample from a site can improve parameter estimates more than having a high number of replicates only within the laboratory analysis.

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.

Passive eDNA collection enhances aquatic biodiversity analysis.

Environmental DNA (eDNA) metabarcoding is a sensitive and widely used approach for species detection and biodiversity assessment. The most common eDNA collection method in aquatic systems is actively filtering water through a membrane, which is time consuming and requires specialized equipment. Ecological studies investigating species abundance or distribution often require more samples than can be practically collected with current filtration methods. Here we demonstrate how eDNA can be passively collected in both tropical and temperate marine systems by directly submerging filter membranes (positively charged nylon and non-charged cellulose ester) in the water column. Using a universal fish metabarcoding assay, we show that passive eDNA collection can detect fish as effectively as active eDNA filtration methods in temperate systems and can also provide similar estimates of total fish biodiversity. Furthermore, passive eDNA collection enables greater levels of biological sampling, which increases the range of ecological questions that eDNA metabarcoding can address.

Preliminary evidences of the presence of extracellular DNA single stranded forms in soil.

The relevance of extracellular DNA (eDNA) in the soil ecosystem is becoming more and more evident to the scientific community by the progressive discovery of functions accompanying to natural gene transformation. However, despite the increased number of published articles dedicated to eDNA in soil, so far only few are focused on its single stranded form (eDNAss). The present paper is the first to investigate the quantitative relevance of eDNAss in the total soil eDNA pool, discriminating between its linear (eDNAssl) and circular (eDNAssc) forms and the respective weakly (wa) and tightly (ta) adsorbed fractions. The results showed the prevalence of eDNAss and its linear form in both the total soil eDNA pool and its wa and ta fractions. Both of the eDNAss fractions (linear and circular) were characterized by small fragments.

A practical guide to sample preservation and pre-PCR processing of aquatic environmental DNA.

Environmental DNA (eDNA) is rapidly growing in popularity as a tool for community assessments and species detection. While eDNA approaches are now widely applied, there is not yet agreement on best practices for sample collection and processing. Investigators looking to integrate eDNA approaches into their research programme are required to examine a growing collection of disparate studies to make an often uncertain decision about which protocols best fit their needs. To promote the application of eDNA approaches and to encourage the generation of high-quality data, here we review the most common techniques for the collection, preservation and extraction of metazoan eDNA from water samples. Specifically, we focus on experimental studies that compare various methods and outline the numerous challenges associated with eDNA. While the diverse applications of eDNA do not lend themselves to a one-size-fits-all recommendation, in most cases, capture/concentration of eDNA on cellulose nitrate filters (with pore size determined by water turbidity), followed by storage of filters in Longmire's buffer and extraction with a DNeasy Blood & Tissue Kit (or similar) has been shown to provide sufficient, high-quality DNA. However, we also emphasize the importance of testing and optimizing protocols for the system of interest.

Assessment of fishery resources using environmental DNA: Small yellow croaker (Larimichthys polyactis) in East China Sea.

Species distribution monitoring and biomass assessment are crucial for fishery management and resource conservation. However, traditional methods such as motor trawling are costly and less effective than the novel environmental DNA (eDNA) approach. This study employs eDNA approach to investigate horizontal and vertical distributions of small yellow croaker (Larimichthys polyactis), an economically important species, in the East China Sea. The analysis of 171 eDNA samples collected from 44 stations using the species-specific primers and Taqman probe suggests a presence of small yellow croaker at 28 sampling layers in 44 stations. Significant differences in croaker eDNA concentrations were revealed among sampling stations and layers, consistent with previous findings through motor-trawl capture offshore and nearshore ichthyoplakton surveys, indicating small yellow croaker exhibits strong regional distribution and layer preference. In addition, we found a high eDNA concentration of small yellow croaker in the surface waters beyond the motor-trawl prohibition line, which confirms spawning grounds have been expanded from nearshore to offshore areas. Such expansion of spawning grounds could be a response by small yellow croaker to stressors such as overfishing, climate change, and nearshore environment contamination. To identify environmental variables potentially associated with small yellow croaker presence and absence, we conducted a correlation analysis between eDNA concentration and environmental variables, and the results provide a guideline for further investigation of fishery resources in the future. In conclusion, this study demonstrates the power of the eDNA approach in monitoring small yellow croaker at extensive geographic scales. The developed protocols and the findings are expected to assist in long-term monitoring and protection programs and benefit sustainable fishery in small yellow croaker.

Comparing the efficiency of open and enclosed filtration systems in environmental DNA quantification for fish and jellyfish.

Water sampling and filtration of environmental DNA (eDNA) analysis have been performed by several different methods, and each method may yield a different species composition or eDNA concentration. Here, we investigated the eDNA of seawater samples directly collected by SCUBA to compare two widely used filtration methods: open filtration with a glass filter (GF/F) and enclosed filtration (Sterivex). We referred to biomass based on visual observation data collected simultaneously to clarify the difference between organism groups. Water samples were collected at two points in the Sea of Japan in May, September and December 2018. The respective samples were filtered through GF/F and Sterivex for eDNA extraction. We quantified the eDNA concentration of five fish and two cnidarian species by quantitative polymerase chain reaction (qPCR) using species-specific primers/probe sets. A strong correlation of eDNA concentration was obtained between GF/F and Sterivex; the intercepts and slopes of the linear regression lines were slightly different in fish and jellyfish. The amount of eDNA detected using the GF/F filtration method was higher than that detected using Sterivex when the eDNA concentration was high; the opposite trend was observed when the eDNA concentration was relatively low. The concentration of eDNA correlated with visually estimated biomass; eDNA concentration per biomass in jellyfish was approximately 700 times greater than that in fish. We conclude that GF/F provides an advantage in collecting a large amount of eDNA, whereas Sterivex offers superior eDNA sensitivity. Both filtration methods are effective in estimating the spatiotemporal biomass size of target marine species.