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.

Building reference libraries of DNA barcodes is relatively straightforward when specifically designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group (e.g. single class or single order). DNA barcoding marine communities have been comparatively harder to accomplish due to the broad taxonomic diversity and lack of consistently efficient primers. Although some of the so-called “universal” primers have been relatively successful, they still fail to amplify COI-5P of many marine animal groups, while displaying random success even among species within each group. Here we propose a new pair of primers designed to enhance amplification of the COI-5P region in a wide range of marine organisms.

The accuracy of specimen identification through DNA barcoding and metabarcoding relies on reference libraries containing records with reliable taxonomy and sequence quality. The considerable growth in barcode data requires stringent data curation, especially in taxonomically difficult groups such as marine invertebrates. A major effort in curating marine barcode data in the Barcode of Life Data Systems (BOLD) was undertaken during the 8^th International Barcode of Life Conference (Trondheim, Norway, 2019). Major taxonomic groups (crustaceans, echinoderms, molluscs, and polychaetes) were reviewed to identify those which had disagreement between Linnaean names and Barcode Index Numbers (BINs). The records with disagreement were annotated with four tags: a) MIS-ID (misidentified, mislabeled, or contaminated records), b) AMBIG (ambiguous records unresolved with the existing data), c) COMPLEX (species names occurring in multiple BINs), and d) SHARE (barcodes shared between species). A total of 83,712 specimen records corresponding to 7,576 species were reviewed and 39% of the species were tagged (7% MIS-ID, 17% AMBIG, 14% COMPLEX, and 1% SHARE). High percentages (>50%) of AMBIG tags were recorded in gastropods, whereas COMPLEX tags dominated in crustaceans and polychaetes. The high proportion of tagged species reflects either flaws in the barcoding workflow (e.g., misidentification, cross-contamination) or taxonomic difficulties (e.g., synonyms, undescribed species). Although data curation is essential for barcode applications, such manual attempts to examine large datasets are unsustainable and automated solutions are extremely desirable.

Past molecular studies using mtDNA sequences and alloenzymes signalled the existence of at least two cryptic species within the Hediste diversicolor morphotype, in European coasts. However, to this day, no new species descriptions have been made. In this study, we identified five completely sorted lineages using a multi-locus approach, including the mitochondrial DNA cytochrome oxidase I gene (COI-5P) and the nuclear markers ITS2 rRNA and 28S rRNA. The molecular data were complemented with morphometric measurements examined through multivariate statistical analysis and the incorporation of statistical dissimilarities. Apart from the Baltic Sea, where three of the lineages occur in sympatry, Hediste diversicolor comprises four deeply divergent allopatric lineages in the rest of Europe. They group populations from the NE Atlantic and part of the Western Mediterranean Sea; from the Tyrrhenian Sea; from the Adriatic and Ionian Sea; and, lastly, from the Caspian, Black and the northern Aegean Seas. The lineage from the Ionian Sea revealed low genetic distances compared with the one from the Adriatic Sea and lacked enough specimens for the morphometric analysis, preventing further conclusions about its independent status. Three independent morphometric clusters were identified mainly based on worm size, the distance between the anterior and posterior eyes, parapodia proportions and the length of several prostomial appendages. Two sympatric lineages present in the Baltic Sea, showed evidence of possible hybridization and lacked significant PCA morphometric variation between them. The two remaining lineages were formally described as new species, namely Hediste pontii sp. nov. (Adriatic Sea) and Hediste astae sp. nov. (northern Aegean, Caspian and Black Seas). These new species can now be formally recognized and used in biomonitoring or other relevant ecological studies. Finally, a neotype is defined for H. diversicolor, whose usage is restricted to the NE Atlantic lineage. http://www.zoobank.org/urn:lsid:zoobank.org:pub:948C73FC-B07F-40A3-B8FA-03B60DE2089D