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Accurate identification of invaders, and especially their juveniles and eggs, is a difficult task if several morphologically similar species co-occur. The aim of the study was to develop and test a rapid and cost-effective procedure for identification of five species of invasive gobies occurring in the middle Danube basin, namely round goby Neogobius melanostomus, bighead goby Ponticola kessleri, monkey goby Neogobius fluviatilis, racer goby Babka gymnotrachelus and tubenose goby Proterorhinus semilunaris. First, a 708 bp fragment of the cytochrome oxidase I gene was amplified and sequenced for representative samples of these five species. Appropriate sequences of the five species available in public databases were used for in silico analysis. A digestion of the amplified fragment with the BfaI enzyme was found to be suitable for the species identification, as it showed unique restriction patterns for each species. The technique was also successfully applied for fish remains from burbot Lota lota stomachs. Thus the technique could be a useful tool in monitoring biological invasions, especially by identifying specimens that could not be determined on the basis of morphological features. The results demonstrate that the PCR- RFLP method may in some cases be more reliable for species identification than a standard DNA sequencing. [ABSTRACT FROM AUTHOR]

The Danube River was originally inhabited by six native sturgeon species, but currently, the sterlet (Acipenser ruthenus L.) is the only native sturgeon species still occupying the Slovak–Hungarian stretch of the Middle Danube. All sturgeon species are facing extinction, suffering from overfishing, water pollution, illegal fishing, poaching or other negative impacts. Urgent and proper actions are needed to prevent their extinction, and evaluating its genetic diversity is one of the essential tools of conservation programs. Since the management actions are primarily local in nature, we first focused on comparing and analysing local sources of fish for population recovery and natural (wild) population in the adjacent stretch of the Danube River. We used 2 fragments of mitochondrial DNA and 12 microsatellites to analyse the genotype of the three groups of sterlets, i.e. wild, broodstock and stocked individuals from Slovak part of the Danube. Mitochondrial markers of all groups were diversified similarly to populations from other parts of the Danube. This confirmed that broodstock and stocked fish belong to the original Danube population. Microsatellites revealed very similar patterns among groups compared, but we detected possible negative trends reflected in losing polymorphism in a few loci in broodstock and stocked individuals.

Anther smuts are fungal diseases that have detrimental effects on the biology of their host plants. We investigated the transmission of Anther smut Antherospora vindobonensis to its host, Scilla vindobonensis under natural and laboratory conditions. Plants flowering early in spring were less vulnerable to disease (∼ 20-30% of plants which appeared early in the season were diseased) than flowers that bloom at the end of the season (∼ 60% of plants which appeared late in the season were diseased) which contradicts the patterns observed for other anther smuts species. Diseased plants showed thicker stems, lower nectar production and smaller flower radius than healthy plants. The disease prevalence in S. vindobonensis was not influenced by plant density. The production of spores in diseased flowers was 125 times higher than the production of pollen in healthy flowers. The number of pollen grains attached to pollinator bodies from healthy plants under controlled conditions was similar to the number of spores attached from diseased plants. Experienced honeybees in the field showed almost identical preferences for healthy plants (65%) over diseased ones, as did naïve bumblebees under controlled laboratory conditions (69%). eDNA metabarcoding revealed that healthy flowers attracted a significantly higher number of arthropod species (M = 6.23, SE = 1.48) than diseased flowers (M = 2.32, SE = 0.31). Strong seasonal differences in the risk of infection suggest that S. vindobonensis solves the trade-off between low pollinator availability and low risk of infection at the beginning of the season and high risk of infection and high pollinator availability at the end of season. By reproducing early in the season, plants can reduce the risk of disease., (© 2024. The Author(s).)

This study confirms the extended distribution of two invasive species of the genus Ameiurus in Ukraine. Specifically, A. melas is recorded for the first time in the Southern Buh basin and A. nebulosus has expanded further eastward within the Dnipro basin. Material collected in 2019 and 2022 was identified by morphological features and confirmed by molecular genetic analysis. The most reliable morphological characters for distinguishing these two species include anal-fin membrane pigmentation (light or black), gill raker count (fewer or more than 16), and serrations on the pectoral-fin spine (well-developed along the full length or small, absent near the tip). The analysis of the cytochrome oxidase subunit I barcoding marker identified all samples from the Dnipro Basin (Tnia and Velykyi Luh localities) as A. nebulosus, while all specimens from the Vinnytsia region within the Southern Buh basin (Sotskoho and Vyshenske lakes) were attributed to A. melas. The maximum-likelihood analysis revealed clearly separated clades with high bootstrap support (>75%), strongly supporting the presence of the two separate species. This study suggests the potential for further eastward expansion of both species within Ukraine: A. nebulosus in the northern direction and A. melas in the southern direction., (© 2024 Fisheries Society of the British Isles.)

Assessments of biodiversity and ecosystem status can benefit from DNA metabarcoding as a means to streamline sample processing and specimen identification. Moreover, processing the fixation medium instead of the precious material introduces straightforward protocols that allow subsequent focus on certain organisms detected among the preserved specimens. In this study, we present a proof of concept via the analysis of freshwater invertebrate samples from the Tatra Mountain lakes (Slovakia). Besides highlighting a match between the lake-specific environmental conditions and the results of our fixative DNA metabarcoding, we observed an option to fine-tune the fixation time: to prefer two weeks over a day or a month. This effect emerged from the presence/absence of individual taxa rather than from coarse per-sample records of taxonomic richness, demonstrating that metabarcoding studies-and efforts to optimize their protocols-can use the robust metrics to explore even subtle trends. We also provide evidence that fixative DNA might better capture large freshwater species than terrestrial or meiofauna., (© 2024. The Author(s).)

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., (© 2024 John Wiley & Sons Ltd.)

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 Laššová, Čiampor Jr, Čiamporová-Zaťovičová, 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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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)., (© 2021. The Author(s).)

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 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., (Ján Kodada, Manfred A. Jäch, Hendrik Freitag, Zuzana Čiamporová-Zaťovičová, Katarína Goffová, Dávid Selnekovič, Fedor Čiampor Jr.)

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., (Ján Kodada, Manfred A. Jäch, Hendrik Freitag, Zuzana Čiamporová-Zaťovičová, Katarína Goffová, Dávid Selnekovič, Fedor Čiampor Jr.)

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.

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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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.

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.