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100Effective identification of species using short DNA fragments (DNA barcoding and DNA 101 metabarcoding) requires reliable sequence reference libraries of known taxa. Both 102 taxonomically comprehensive coverage and content quality are important for sufficient 103 accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are 104 particularly important if molecular identification tools are to be implemented in biomonitoring 105 and reports in the context of the EU Water Framework Directive (WFD) and the Marine 106Strategy Framework Directive (MSFD). We analysed gaps in the two most important 107 reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a 108 focus on the taxa most frequently used in WFD and MSFD. Our analyses show that 109 coverage varies strongly among taxonomic groups, and among geographic regions. In 110 general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, 111 caddisflies and vascular plants) are well represented in the barcode libraries, while others 112 have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also 113 found that species monitored in several countries often are represented by barcodes in 114 reference libraries, while species monitored in a single country frequently lack sequence 115 records. A large proportion of species (up to 50%) in several taxonomic groups are only 116represented by private data in BOLD. Our results have implications for the future strategy to 117 fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the 118 monitoring of European aquatic biota under the WFD and MSFD. For example, missing 119 species relevant to monitoring in multiple countries should be prioritized. We also discuss 120 why a strategy for quality control and quality assurance of barcode reference libraries is 121 needed and recommend future steps to ensure full utilization of metabarcoding in aquatic 122 biomonitoring. 123 124

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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.

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.

1. 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. 2. 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 9 2 9 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. 3. 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. 4. 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.

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