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.

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data.

The essential key to routine molecular species identification (DNA barcoding/metabarcoding) is the existence of an error-free DNA barcode reference library providing full coverage of all species. Published studies generally state the need to produce more barcodes, and control their quality, but unfortunately, the number of barcoded species is still low. However, to initiate real progress, we need to know where the gaps lie, how big they are and why they persist. Our aims were to draw and understand the current state of knowledge regarding species diversity, distribution, and barcode coverage, and offer solutions for improvement. In this study, we used two groups of aquatic insects, beetles and true bugs. We have compiled and critically evaluated an essentially complete and up-to-date European list, containing 1527 species. The list served as a basis for the barcode gap analyses in the Barcode-of-Life-Data-System (BOLD) conducted in three subsequent years (2020-2022). The overall barcode coverage of the pan-European fauna was around 50 % in both groups. The lowest coverage was in the Mediterranean, the Balkans and South-eastern Europe. The coverage in each country depended significantly on the local diversity, the number of rare, endemic species and the similarity of its fauna to that of the most active barcoding European countries. Gap analyses showed a very small increase in species coverage (<1 % in European aquatic beetles) despite an ~25 % increase in the number of barcodes. Hence, it is clear that future barcoding campaigns must prioritise quality over quantity. To visibly improve reference libraries, we need to increase the involvement of taxonomic experts and focus on targeted studies and underexplored but biodiversity-rich areas.

The riffle beetle genus Hedyselmis Hinton, 1976 includes two species from the Malay Peninsula, with adults with a highly deviating morphology. Its phylogenetic relationships are unclear, although it has been hypothesized to be related to Graphelmis Delève, 1968, a large genus widely distributed in the Oriental and East Palaearctic regions. In this paper the larva of H. opis Hinton, 1976 is described based on material collected in the Cameron Highlands (Malaysia) and the conspecificity with co-existing adults tested using sequences of one nuclear (5' end of 18S rRNA) and three mitochondrial gene fragments (5' end of the large ribosomal unit + tRNAleu + 5' end of the NADH dehydrogenase subunit 1; 5' end of cytochrome c oxidase subunit I; and a fragment of cytochrome b) with a total of ca. 2,600 bp. This is the first example of the use of molecular data to match different life stages within the family Elmidae. The larva of H. opis has a subcylindrical body typical of many other elmid genera; abdominal segments 1-7 with preserved pleura; and ninth segment with oval operculum. The last instar larvae have clearly visible prominent spiracles on mesothorax and abdominal segments 1-8. The phylogenetic position of Hedyselmis in relation to Graphelmis was investigated using molecular data for three species of Graphelmis plus a selection of other Elmidae genera. Hedyselmis opis is nested within Graphelmis, confirming their close relationship and suggesting that their status requires taxonomic revision.

The riffle beetle genus Hexanchorus Sharp, 1882 is distributed from Mexico to Argentina, forming an important component of the freshwater invertebrate fauna of Latin America. With 21 described species, Hexanchorus represents one of the most speciose Larainae genera, but its real diversity is likely much higher. We analysed material from a relatively small area in Ecuador, resulting in the first record of H.cordillierae for Ecuador and discovery of four new species and one subspecies: Hexanchorusvirilissp. n., Hexanchorusrostratussp. n., Hexanchorusshepardisp. n., Hexanchorusonoreisp. n. and Hexanchorusonoreisagittatusssp. n. For delimiting and characterizing species, both morphological and molecular (mtCOI DNA barcodes) data were used. A distribution map of Hexanchorus species is provided based on published records.