Detailed DNA barcoding of mayflies in a small European country proved how far we are from having comprehensive barcode reference libraries.

Mayflies (Ephemeroptera) are among the crucial water and habitat quality bioindicators. However, despite their intensive long-term use in various studies, more reliable mayfly DNA barcode data have been produced in a negligible number of countries, and only ~40% of European species had been barcoded with less than 50% of families covered. Despite being carried out in a small area, our study presents the second-most species-rich DNA reference library of mayflies from Europe and the first comprehensive view from an important biodiversity hotspot such as the Western Carpathians. Within 1153 sequences, 76 morphologically determined species were recorded and added to the Barcode of Life Data System (BOLD) database. All obtained sequences were assigned to 97 BINs, 11 of which were unique and three represented species never barcoded before. Sequences of 16 species with high intraspecific variability were divided into 40 BINs, confirming the presence of cryptic lineages. Due to the low interspecific divergence and the non-existing barcoding gap, sequences of six species were assigned to three shared BINs. Delimitation analyses resulted in 79 and 107 putative species respectively. Bayesian and maximum-likelihood phylogenies confirmed the monophyly of almost all species and complexes of cryptic taxa and proved that DNA barcoding distinguishes almost all studied mayfly species. We have shown that it is still sufficient to thoroughly investigate the fauna of a small but geographically important area to enrich global databases greatly. In particular, the insights gained here transcend the local context and may have broader implications for advancing barcoding efforts.

Description of the larva of Hypsilara (Coleoptera: Elmidae).

The mature larva of the Larainae riffle beetle genus Hypsilara Maier & Spangler, 2011 is described here for the first time, based on larvae of Hypsilara autanai Lassová, Ciampor Jr, Ciamporová-Zatovicová, 2014. The larvae were collected with adults in the stream near the tepui Cerro Autana and Cerro Cuao (southwestern Venezuela) and associated together using DNA barcoding. Larvae of Hypsilara strongly resemble those of Phanoceroides, here we present important morphological diagnostic characters. This description complements the descriptions of the Larainae larvae in the Neotropics, as the larva of Hypsilara was the last one of the group missing so far.

50%, not great, not terrible: Pan-European gap-analysis shows the real status of the DNA barcode reference libraries in two aquatic invertebrate groups and points the way ahead.

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.

Fuzzy species borders of glacial survivalists in the Carpathian biodiversity hotspot revealed using a multimarker approach.

The Carpathians are one of the key biodiversity hotspots in Europe. The mountain chain uplifted during Alpine orogenesis and is characterised by a complex geological history. Its current biodiversity was highly influenced by Pleistocene glaciations. The goal of the current study was to examine the phylogenetic and demographic history of Gammarus balcanicus species complex in the Carpathians using multiple markers as well as to delimit, using an integrative approach, and describe new species hidden so far under the name G. balcanicus. Results showed that divergence of the studied lineages reaches back to the Miocene, which supports the hypothesis of their survival in multiple micro refugia. Moreover, the increase of their diversification rate in the Pleistocene suggests that glaciation was the driving force of their speciation. The climatic changes during and after the Pleistocene also played a major role in the demography of the local Carpathian lineages. Comparison of diversity patterns and phylogenetic relationships of both, the mitochondrial and nuclear markers, provide evidence of putative hybridisation and retention of ancient polymorphism (i.e., incomplete lineage sorting). The morphological examination supported the existence of two morphological types; one we describe as a G. stasiuki sp. nov. and another we redescribe as a G. tatrensis (S. Karaman, 1931).

Disersus otongachi/ a new species of Larainae riffle beetles from Ecuador (Coleoptera: Elmidae).

We describe here a new species in the genus Disersus Sharp, 1882 from the Otongachi Reserve in Ecuador. Disersus otongachi sp.nov. is externally similar to other representatives of the genus, however, this species can be clearly distinguished for significantly longer antennae and the unique shape of the male genitalia.

Ancyronyx clisteri, a new spider riffle beetle species from Borneo, redescription of A. sarawacensis Jäch including a description of the larva and new distribution data for A. procerus Jäch using DNA barcodes (Coleoptera, Elmidae).

Ancyronyx clisteri sp. nov. (Coleoptera, Elmidae) a new spider riffle beetle discovered from northern Borneo (Brunei; Sabah and Sarawak, Malaysia) and the larva of Ancyronyx sarawacensis Jäch are described. Illustrations of the habitus and diagnostic characters of the new species and the similar and highly variable A. sarawacensis are presented. Differences to closely related species, based on DNA barcodes and morphological characters, are discussed. Association of the larva and the imago of A. sarawacensis, and the occurrence of Ancyronyx procerus Jäch in Peninsular Malaysia and Sabah are confirmed by using COI mtDNA sequences.

Ancyronyx lianlabangorum sp. nov., a new spider riffle beetle from Sarawak, and new distribution records for A. pulcherrimus Kodada, Jäch & Ciampor based on DNA barcodes (Coleoptera, Elmidae).

Ancyronyx lianlabangorum sp. nov. (Coleoptera, Elmidae), a new spider riffle beetle from the Kelabit Highlands (Sarawak, northern Borneo), is described. Illustrations of the habitus and diagnostic characters of the new species and the similar, polymorphic A. pulcherrimus Kodada et al. are presented. Differences to closely related species, based on COI nucleotide sequences and morphological characters, are discussed. Ancyronyx pulcherrimus is here recorded from Sarawak for the first time, based on DNA barcoding.

Ictelmis, a new riffle beetle genus from Ecuador (Coleoptera: Elmidae).

A new minute riffle beetle genus Ictelmis gen. nov. and the type species Ictelmis martae sp. nov. are described from Ecuador. The description is supported by characteristic morphological features and DNA barcoding data, drawings and habitus photographs are provided. Based on molecular data and morphology it is suggested that Ictelmis is closely related with Onychelmis Hinton, and Notelmis Hinton.

Four new species of Hexanchorus Sharp from Ecuador (Coleoptera, Elmidae) with DNA barcoding and notes on the distribution of the genus.

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: Hexanchorusvirilis sp. n., Hexanchorusrostratus sp. n., Hexanchorusshepardi sp. n., Hexanchorusonorei sp. n. and Hexanchorusonoreisagittatus ssp. 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.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

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.

Phanoceroides Hinton, 1939: description of new species, morphology of larvae, and revised taxonomic position of the genus (Coleoptera: Elmidae) based on molecular evidence.

The monotypic genus Phanoceroides was initially classified within the subfamily Elminae, based solely on the distribution and type of pubescence on the body surface. Although a close relationship with Larainae was suggested (Hinton 1939, Maier 2013), its taxonomic position and phylogeny remained unresolved. Based on newly collected material, we describe here P. fernandesi sp. n. and document morphological characters and differences of adults and larvae of P. aquaticus Hinton, 1939 and P. fernandesi sp. n. To clarify the taxonomic position of Phanoceroides within Elmidae, we used molecular data (cox1 and mtDNA). The results support position of the genus within the subfamily Larainae with Hypsilara Maier & Spangler, 2011 as a close relative.

Catchment land use as a predictor of the macroinvertebrate community changes between inlet and outlet of small water dams.

Changes in land use practices have affected the integrity and the quality of stream water worldwide. Effective catchment management, in terms of land use alteration, depends on our ability to quantify ecologically significant changes and to discriminate among varying levels of impact. In this study, we assessed the structural and functional changes upstream and downstream of eight small water reservoirs in western Slovakia and the relationship between these changes and shifts in physicochemical parameters as a consequence of stream damming and catchment land use. Dams were categorized into two groups, the first with both inlet and outlet situated in deforested and agricultural landscape and the second with inlet flowing through natural and forested landscape, while outlet is situated in deforested and urban region. Using a "between-groups" principal components analysis and a "between-groups" fuzzy principal components analysis, we found significant differences in structural and functional composition of macroinvertebrate communities between inlets with forested catchment and outlets flowing through agricultural, urban landscape. The structural dissimilarity is best explained by the physicochemical and biological characteristics of the aquatic environment, while functional variation of communities is best explained by land use of an area surrounding the stream. The distance in structural and functional community composition between inlet and outlet was linked with difference in environmental conditions between these habitats. The change of structural community composition significantly reflected shift in the water temperature and phosphorus concentration, while the change of functional community composition was determined by change of nutrient concentrations (e.g., PO4, NO3, NH4) and pH.

Two new Larainae species from Guayana region, Venezuela (Coleoptera: Elmidae).

Two new species of the subfamily Larainae (Insecta: Coleoptera: Elmidae), Hexanchorus angeli n. sp. and Hypsilara autanai n. sp., are described from Guyana region in Venezuela. We provide habitus photographs, detail drawings of both male and female genitalia, and description of morphological features important for discrimination of the new species. Molecular differences within genera were measured using 816bp fragment of mtDNA gene for cytochrome oxidase c subunit I. Sequence divergences among species are discussed.

Hypsilara breweri n.sp. from Venezuela: description of new species with notes on the morphology and phylogenetic relationships of the genus (Coleoptera: Elmidae: Larainae).

A new species of the genus Hypsilara Maier & Spangler, 2011, collected in the Eastern Venezuela, is described and illustrated. We provide habitus photographs, detail drawings of both male and female genitalia, and description of some morphological features, which were omitted in the description of the genus. Included are also remarks on the distribution and genetic distance of the Hypsilara species and possible relations to other South American Larainae.