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.

Taxonomy 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. Here 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. Our 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.

Adults and larvae of two new cryptic, endemic caddisflies, Ecclisopteryx keroveci sp.n. and Ecclisopteryx ivkae sp.n., are described and illustrated from the Western Balkans. Phylogenetic analysis (Bayesian MCMCMC) and association of different life history stages in both cryptic species were achieved through comparison of morphological characters and mitochondrial (mtCOI and mtLSU) and nuclear (nuWG) gene sequence data. The new species form a sister clade to the widely distributed E. dalecarlica and E. guttulata, with which they were formerly misidentified. Adults differ from each other and other species in the genus by the uniquely shaped inferior appendages in males and segment X in females. The larvae differ from each other and their congeners in the shape of the pronotum, and presence and constitution of additional spines on the parietalia. Larvae of both species are grazers and prefer stony substrate. Ecclisopteryx keroveci sp.n. has a wide distribution in the Western Balkans, while E. ivkae sp.n. is endemic to Dalmatia. Our findings demonstrate the significance of the Western Balkans as a freshwater biodiversity hotspot, and accentuate the importance of research focused on freshwater biodiversity and biogeography in southern Europe.

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.

In a recent 3-gene phylogeny of the trichopteran subfamily Drusinae Banks 1916, molecular data clearly correlated with the morphology and feeding ecology of larvae. The largest of three main groups, the Drusinae grazer clade, exhibits an unusual larval feeding ecology for Limnephilidae, and is the most diverse group. In this paper we describe four previously unknown Drusinae larvae included in this clade: Drusus balcanicus Kumanski, 1973 (micro-endemic to Eastern Balkans), Drusus botosaneanui Kumanski, 1968 (Dinaric Western Balkans, Hellenic and Eastern Balkan, Asia Minor), Drusus serbicus Marinkovi?-Gospodneti?, 1971 (micro-endemic to Dinaric Western Balkans), and Drusus tenellus (Klap?lek, 1898) (Carpathians, Dinaric Eastern Balkans). Characteristically, the larvae of these species have toothless mandibles typical of the Drusinae grazer clade. Larvae and adults were unambiguously associated using a phylogenetic analysis based on two mitochondrial [mtCOI, mtLSU (=16S) rDNA] and two nuclear genes (nuWG, nuCAD). In addition, information on the morphology of the larvae is given and the diagnostic features necessary for identification are illustrated.

In this paper we describe Drusus sharrensis sp. n., from the Sharr Mountains in Kosovo. Males of the new species are morphologically most similar to Drusus krusniki Malicky, 1981, D. kerek Oláh, 2011 and D. juliae Oláh, 2011 but differ mainly in exhibiting (1) a differently shaped spinose area on tergite VIII; (2) intermediate appendages anteriorly curved in lateral view with broad tips in dorsal view; (3) inferior appendages with a distinct dorsal protrusion in the proximal half. Females of the new species are morphologically most similar to D. krusniki, D. kerek, D. juliae, and D. plicatus Radovanovic, 1942 but mainly differ in (1) segment X that is longer than the supragenital plate with distinctly pointed tips; (2) supragenital plate quadrangular with a distinct round dorsal protrusion; (3) a vulvar scale with a small median lobe. Results of phylogenetic species delimitation support monophyly of Drusus sharrensis sp. n. and recover it as sister to a clade comprising (D. pelasgus Oláh, 2010 + D. juliae + D. arbanios Oláh, 2010 + D. plicatus + (D. dacothracus Oláh, 2010 + D. illyricus Oláh, 2010)). The new species is a micro-endemic of the Sharr Mountains, a main biodiversity hotspot in the Balkan Peninsula. Main threats to the aquatic ecosystems of this part of the Balkan Peninsula are discussed.