Benthic invertebrates are the most commonly used organisms used to assess ecological status as required by the EU Water Framework Directive (WFD). For WFD-compliant assessments, benthic invertebrate ...communities are sampled, identified and counted. Taxa × abundance matrices are used to calculate indices and the resulting scores are compared to reference values to determine the ecological status class. DNA-based tools, such as DNA metabarcoding, provide a new and precise method for species identification but cannot deliver robust abundance data. To evaluate the applicability of DNA-based tools to ecological status assessment, we evaluated whether the results derived from presence/absence data are comparable to those derived from abundance data. We analysed benthic invertebrate community data obtained from 13,312 WFD assessments of German streams. Broken down to 30 official stream types, we compared assessment results based on abundance and presence/absence data for the assessment modules "organic pollution" (i.e., the saprobic index) and "general degradation" (a multimetric index) as well as their underlying metrics. In 76.6% of cases, the ecological status class did not change after transforming abundance data to presence/absence data. In 12% of cases, the status class was reduced by one (e.g., from good to moderate), and in 11.2% of cases, the class increased by one. In only 0.2% of cases, the status shifted by two classes. Systematic stream type-specific deviations were found and differences between abundance and presence/absence data were most prominent for stream types where abundance information contributed directly to one or several metrics of the general degradation module. For a single stream type, these deviations led to a systematic shift in status from 'good' to 'moderate' (n = 201; with only n = 3 increasing). The systematic decrease in scores was observed, even when considering simulated confidence intervals for abundance data. Our analysis suggests that presence/absence data can yield similar assessment results to those for abundance-based data, despite type-specific deviations. For most metrics, it should be possible to intercalibrate the two data types without substantial efforts. Thus, benthic invertebrate taxon lists generated by standardised DNA-based methods should be further considered as a complementary approach.
Stream ecosystems are impacted by multiple stressors worldwide. Recent studies have shown that the effects of multiple stressors are often complex and difficult to predict based on the effects of ...single stressors. More research is needed to understand stressor impacts on stream communities and to design appropriate counteractions. We carried out an outdoor mesocosm experiment to assess single and interactive multiple-stressor effects on stream macroinvertebrates in a setup with controlled application of three globally important stressors, namely, reduced stream flow velocity, deposition of fine sediment and increased chloride concentration in a full-factorial design. Each mesocosm comprised three compartments (channel substratum, leaf litter bag and drift net) that were individually analyzed and also compared. We identified 102,501 specimens in total (mainly to family level), 36.5% of which were found in the substratum, 60.6% in litter bags and 2.9% in the drift. Added fine sediment and reduced flow velocity had strong negative single-stressor effects on the abundances of EPT taxa, i.e. Ephemeroptera (mayflies), Plecoptera (stoneflies) and Trichoptera (caddisflies), and a positive effect on chironomid abundances in the substratum. Increased salt concentration reduced abundances of Ephemeroptera. Chironomids migrated from litter bag to channel substratum when water velocity was reduced and Leptophlebiidae in the opposite direction when sediment was added. All three stressors caused higher drift propensities, especially added fine sediment. Both additive and complex multiple-stressor effects were common. A complex three-way interaction affected EPT richness in the substratum, demonstrating the need to evaluate higher-order interactions for more than two stressors. Our results add further evidence that multiple-stressor interactions, notably increased salinity with other stressors, affect a variety of invertebrate taxa across different habitats of stream communities. The results have direct implications for water management as they highlight the need to re-evaluate defined salinity thresholds in the context of multiple-stressor interactions.
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•Multiple stressor effects on stream invertebrate communities are poorly understood.•We tested effects of three globally important stressors in a field experiment.•Increased salinity, added sediment and reduced flow affected many invertebrates.•Stressor responses can vary across microhabitats.•Multiple stressors may interact, often in unpredictable ways.
Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive ...coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.
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•DNA barcode representation in public databases of 28,000 aquatic species is analysed.•Gaps in barcode reference libraries are largest for diatoms and invertebrates.•Sequence coverage varies considerably among invertebrate groups.•Species monitored by one or few countries more frequently lack reference barcodes.•Strategies should be implemented to maintain quality of barcode reference libraries.
DNA metabarcoding is increasingly used as a tool to assess biodiversity in research and environmental management. Powerful analysis software exists to process raw data. However, the translation of ...sequence read data into biological information and downstream analyses may be difficult for end users with limited expertise in bioinformatics. Thus, there is a growing need for easy‐to‐use, graphical user interface (GUI) software to analyse and visualise DNA metabarcoding data. Here, we present TaxonTableTools (TTT), a new platform‐independent GUI that aims to fill this gap by providing simple, reproducible analysis and visualisation workflows. At its base, TTT uses a "TaXon table", which is a data format that can be generated easily within TTT from two input files: a read table and a taxonomy table obtained using various published metabarcoding pipelines. TTT analysis and visualisation modules include Venn diagrams to compare taxon overlap among replicates, samples, or analysis methods. TTT analyses and visualises basic statistics, such as read proportion per taxon, as well as more sophisticated visualisations, such as interactive Krona charts for taxonomic data exploration. Various ecological analyses can be produced directly, including alpha or beta diversity estimates, and rarefaction analysis ordination plots. Metabarcoding data can be converted into formats required for traditional, taxonomy‐based analyses performed by regulatory bioassessment programs. In addition, TTT is able to produce html‐based interactive graphics that can be analysed in any web browser. The software comes with a manual and tutorial, is free and publicly available through GitHub (https://github.com/TillMacher/TaxonTableTools) or the Python package index (https://pypi.org/project/taxontabletools/).
Freshwaters are exposed to multiple anthropogenic stressors, leading to habitat degradation and biodiversity decline. In particular, agricultural stressors are known to result in decreased abundances ...and community shifts towards more tolerant taxa. However, the combined effects of stressors are difficult to predict as they can interact in complex ways, leading to enhanced (synergistic) or decreased (antagonistic) response patterns. Furthermore, stress responses may remain undetected if only the abundance changes in ecological experiments are considered, as organisms may have physiological protective pathways to counteract stressor effects. Therefore, we here used transcriptome-wide sequencing data to quantify single and combined effects of elevated fine sediment deposition, increased salinity and reduced flow velocity on the gene expression of the amphipod Gammarus fossarum in a mesocosm field experiment.
Stressor exposure resulted in a strong transcriptional suppression of genes involved in metabolic and energy consuming cellular processes, indicating that G. fossarum responds to stressor exposure by directing energy to vitally essential processes. Treatments involving increased salinity induced by far the strongest transcriptional response, contrasting the observed abundance patterns where no effect was detected. Specifically, increased salinity induced the expression of detoxification enzymes and ion transporter genes, which control the membrane permeability of sodium, potassium or chloride. Stressor interactions at the physiological level were mainly antagonistic, such as the combined effect of increased fine sediment and reduced flow velocity. The compensation of the fine sediment induced effect by reduced flow velocity is in line with observations based on specimen abundance data.
Our findings show that gene expression data provide new mechanistic insights in responses of freshwater organisms to multiple anthropogenic stressors. The assessment of stressor effects at the transcriptomic level and its integration with stressor effects at the level of specimen abundances significantly contribute to our understanding of multiple stressor effects in freshwater ecosystems.
Land‐use changes have degraded ecosystems worldwide. A particular concern for freshwater biodiversity and ecosystem function are stressors introduced by intensified agriculture. Typically several ...stressors affect freshwater ecosystems simultaneously. However, the combined effects of these multiple stressors on streams and rivers are still poorly understood, yet of critical importance to improve freshwater management. We investigated responses of benthic macroinvertebrates to three globally important agricultural stressors affecting streams (nutrient enrichment, fine sediment deposition and reduced current velocity), using 64 stream mesocosms (full‐factorial 2 × 2 × 2 design, eight replicates of each treatment combination) established on the banks of the Breitenbach Stream (Hesse, Germany). The experiment ran for 1 month (16 days of colonisation, 14 days of manipulations), and all invertebrates in the mesocosms were collected at the end of this period. Fourteen of the 17 studied invertebrate response variables were affected by one or more stressors each. Negative effects on richness or abundance of pollution‐sensitive Ephemeroptera, Plecoptera and Trichoptera (EPT) taxa were particularly common. Overall, both sediment addition and stream flow velocity reduction had pervasive and strong effects. Responses to sediment addition were mostly negative, whereas decreased current velocity reduced several EPT metrics but increased the abundances of some of the other common taxa. Nutrient enrichment had few effects, but these were consistently negative. Combined stressor effects were mainly additive, with only two interactions found in total, both between reduced velocity and nutrients (on the crustacean Gammarus spp. and ceratopogonid midges). This finding implies that multiple‐stressor responses may be predicted from knowledge of single‐stressor effects in this stream community (unlike the often synergistic or antagonistic responses observed elsewhere). However, further taxon‐specific responses and interactions among stressors may have been obscured by limited taxonomic resolution, especially for the numerically dominant Chironomidae. Genetic approaches are required to address this limitation in the future.
Background
Chironomids, or non-biting midges, often dominate stream invertebrate communities in terms of biomass, abundance, and species richness and play an important role in riverine food webs. ...Despite these clear facts, the insect family Chironomidae is often treated as a single family in ecological studies or bioassessments given the difficulty to determine specimens further. We investigated stressor responses of single chironomid operational taxonomic units (OTUs) to three globally important stressors (increased salinity, fine sediment and reduced water flow velocity) in a highly replicated mesocosm experiment using a full-factorial design (eight treatment combinations with eight replicates each).
Results
In total, 183 chironomid OTUs (97% similarity) were obtained by applying a quantitative DNA metabarcoding approach. Whereas on the typically applied family level, chironomids responded positively to added fine sediment and reduced water velocity in the streambed and negatively to reduced velocity in the leaf litter, an OTU-level analysis revealed a total of 15 different response patterns among the 35 most common OTUs only. The response patterns ranged from (a) insensitivity to any experimental manipulation over (b) highly specific sensitivities to only one stressor to (c) additive multiple-stressor effects and even (d) complex interactions.
Conclusion
Even though most OTUs (> 85%) could not be assigned to a formally described species due to a lack of accurate reference data bases at present, the results indicate increased explanatory power with higher taxonomic resolution. Thus, our results highlight the potential of DNA-based approaches when studying environmental impacts, especially for this ecologically important taxon and in the context of multiple stressors.
DNA metabarcoding is a powerful tool to assess arthropod diversity in environmental bulk samples such as Malaise trap, pitfall trap, or hand net samples. While comparative performance tests for ...different extraction protocols, primers, and Taq polymerases have been made, the effect of different PCR volumes on bulk sample metabarcoding performance is less explored. Although using small PCR volumes reduces overall costs, they may lead to decreased taxon recovery or higher replicate variability due to increased pipetting imprecision, PCR stochasticity (PCR drift), or inhibition when using high amounts of template community DNA. We here performed a simple DNA metabarcoding experiment to test if species detection and the consistency of technical replicates decrease with decreasing PCR volume in standard reaction tubes. We used a mock community sample consisting of different amounts of DNA from 35 arthropod species, and a Malaise trap sample composed of many thousand insect specimens. PCR volumes tested were 5, 10, 15, 20, 25, and 50 µl. Both samples were replicated 14 times in the first PCR step with two technical replicates each in the second PCR step. Our data show that small PCR volumes did neither have systematically lower species detection or richness values, nor lower consistency between PCR replicates. We therefore recommend low volumes primarily depending on handling constraints. Further, we emphasize the importance of sequencing depth for taxon recovery.
In this study, we tested the influence of PCR volume on bulk sample metabarcoding outcomes. Our results show that neither species richness nor replicate consistency is systematically influenced by PCR volume. This opens up possibilities of saving costs or increasing the number of biological replicates.
Environmental DNA (eDNA) extracted from water is routinely used in river biodiversity research, and via metabarcoding eDNA can provide comprehensive taxa lists with little effort and cost. However, ...eDNA‐based species detection in streams and rivers may be influenced by sampling season and other key factors such as water temperature and discharge. Research linking these factors and also informing on the potential of eDNA metabarcoding to detect shifts in ecological signatures, such as species phenology and functional feeding groups across seasons, is missing. To address this gap, we collected water samples every 2 weeks for 15 months at a long‐term ecological research (LTER) site and at three different positions in the river's cross section, specifically the water surface, riverbed, and riverbank. For these 102 samples, we analyzed macroinvertebrate species and molecular operational taxonomic unit (OTU) richness and temporal community turnover across seasons based on cytochrome c oxidase subunit I (COI) metabarcoding data. Using Generalized Additive Models, we found a significant influence of sampling season on species richness. Community turnover followed a cyclic pattern, reflecting the continuous change of the macroinvertebrate community throughout the year (“seasonal clock”). Although water temperature had no influence on the inferred species richness, higher discharge reduced the number of Annelida and Ephemeroptera species detectable with eDNA. Most macroinvertebrate taxa showed the highest species richness in spring, in particular merolimnic species with univoltine life cycles. Further, we detected an increase in the proportion of shredders in winter and parasites in summer. Our results show the usefulness of highly resolved eDNA metabarcoding time series data for ecological research and biodiversity monitoring in streams and rivers.
Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality ...and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well-curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost-efficient and reliable DNA-based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two-thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution.
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