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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

The caddisfly subfamily Drusinae BANKS comprises roughly 100 species inhabiting mountain ranges in Europe, Asia Minor and the Caucasus. A 3-gene phylogeny of the subfamily previously identified three major clades that were corroborated by larval morphology and feeding ecologies: scraping grazers, omnivorous shredders and filtering carnivores. Larvae of filtering carnivores exhibit unique head capsule complexities, unknown from other caddisfly larvae. Here we assess the species-level relationships within filtering carnivores, hypothesizing that head capsule complexity is derived from simple shapes observed in the other feeding groups. We summarize the current systematics and taxonomy of the group, clarify the systematic position of Cryptothrix nebulicola, and present a larval key to filtering carnivorous Drusinae. We infer relationships of all known filtering carnivorous Drusinae and 34 additional Drusinae species using Bayesian species tree analysis and concatenated Bayesian phylogenetic analysis of 3805bp of sequence data from six gene regions (mtCOI5-P, mtCOI3-P, 16S mrDNA, CADH, WG, 28S nrDNA), morphological cladistics from 308 characters, and a total evidence analysis. All analyses support monophyly of the three feeding ecology groups but fail to fully resolve internal relationships. Within filtering carnivores, variation in head setation and frontoclypeus structure may be associated with progressive niche adaptation, with less complex species recovered at a basal position. We propose that diversification of complex setation and frontoclypeus shape represents a recent evolutionary development, hypothetically enforcing speciation and niche specificity within filtering carnivorous Drusinae.

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BackgroundTaxonomy offers precise species identification and delimitation and thus provides basic information for biological research, e.g. through assessment of species richness. The importance of molecular taxonomy, i.e., the identification and delimitation of taxa based on molecular markers, has increased in the past decade. Recently developed exploratory tools now allow estimating species-level diversity in multi-locus molecular datasets.ResultsHere we use molecular species delimitation tools that either quantify differences in intra- and interspecific variability of loci, or divergence times within and between species, or perform coalescent species tree inference to estimate species-level entities in molecular genetic datasets. We benchmark results from these methods against 14 morphologically readily differentiable species of a well-defined subgroup of the diverse Drusinae subfamily (Trichoptera, Limnephilidae). Using a 3798 bp (6 loci) molecular data set we aim to corroborate a geographically isolated new species by integrating comparative morphological studies and molecular taxonomy.ConclusionsOur results indicate that only multi-locus species delimitation provides taxonomically relevant information. The data further corroborate the new species Drusus zivici sp. nov. We provide differential diagnostic characters and describe the male, female and larva of this new species and discuss diversity patterns of Drusinae in the Balkans. We further discuss potential and significance of molecular species delimitation. Finally we argue that enhancing collaborative integrative taxonomy will accelerate assessment of global diversity and completion of reference libraries for applied fields, e.g., conservation and biomonitoring.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-0972-5) contains supplementary material, which is available to authorized users.