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.

Checklist, distribution, diversity, and rarity of mayflies (Ephemeroptera) in Slovakia.

Despite the essential role of mayflies (Ephemeroptera) in freshwater ecosystems and their long-term use in research and routine biomonitoring in the Carpathian and Pannonian ecoregions, their distribution data are fragmentary and outdated. All published and unpublished data on mayflies from Slovakia was gathered and a database of > 15,000 species records from 2206 localities built with the aims (i) to critically revise available data and assess the completeness of the species inventory, (ii) to identify hotspots of species diversity, and (iii) to provide a benchmark for assessment of species rarity and conservation status in the region. After the critical revision of the data covering more than 100 years, the occurrence of 109 mayfly species in Slovakia was confirmed. The species inventory appears to be nearly complete, as evidenced by the rarefaction curve and a nonparametric species richness estimator. The highest mayfly gamma diversity was recorded below 500 m a.s.l. and in streams of the fifth order, which can be considered hotspots of mayfly diversity in the region. Six species were last recorded before 1990 and thus can be considered extinct in Slovakia. Twenty-nine species could be classified as very rare, with their occurrence frequency decreasing with increasing altitude and most of them being restricted to large lowland rivers and stagnant water habitats in their floodplains. In conclusion, our study provides comprehensive data on key freshwater bioindicators and suggests increasing conservation priorities, especially in lowland river floodplains occupied by several very rare mayfly species.

An unwanted companion reaches the country: the first record of the alien mosquito Aedes japonicus japonicus (Theobald, 1901) in Slovakia.

BACKGROUND: Invasive mosquitoes of the genus Aedes are quickly spreading around the world. The presence of these alien species is concerning for both their impact on the native biodiversity and their high vector competence. The surveillance of Aedes invasive mosquito (AIM) species is one of the most important steps in vector-borne disease control and prevention. METHODS: In 2020, the monitoring of AIM species was conducted in five areas (Bratislava, Zvolen, Banská Bystrica, Presov, Kosice) of Slovakia. The sites were located at points of entry (border crossings with Austria and Hungary) and in the urban and rural zones of cities and their surroundings. Ovitraps were used at the majority of sites as a standard method of monitoring. The collected specimens were identified morphologically, with subsequent molecular identification by conventional PCR (cox1) and Sanger sequencing. The phylogenetic relatedness of the obtained sequences was inferred by the maximum likelihood (ML) method. The nucleotide heterogeneity of the Slovak sequences was analysed by the index of disparity. RESULTS: A bush mosquito, Aedes japonicus japonicus, was found and confirmed by molecular methods in three geographically distant areas of Slovakia-Bratislava, Zvolen and Presov. The presence of AIM species is also likely in Kosice; however, the material was not subjected to molecular identification. The nucleotide sequences of some Slovak strains confirm their significant heterogeneity. They were placed in several clusters on the ML phylogenetic tree. Moreover, Ae. j. japonicus was discovered in regions of Slovakia that are not close to a point of entry, where the mosquitoes could find favourable habitats in dendrothelms in city parks or forests. CONCLUSION: Despite being a first record of the Ae. j. japonicus in Slovakia, our study indicates that the established populations already exist across the country, underlining the urgent need for intensified surveillance of AIM species as well as mosquito-borne pathogens.

A rare new species of Leptohyphes Eaton (Ephemeroptera: Leptohyphidae) from the Lost World.

A new species of Leptohyphes Eaton (Ephemerotera: Leptohyphidae) is described, diagnosed and illustrated from nymphs collected in the Pantepui region in Southeastern Venezuela. Leptohyphes kukenan sp. nov. is similar to Leptohyphes populus Allen because they share a unique character for the genus: male compound eyes are divided. Other unique characters for the new species include a plesiomorphic gill structure, with imbricated ventral lamellae, and paired subapical denticles on tarsal claw. The new species show a unique combination of characters that distinguish it from L. populus and all other species of Leptohyphes: fore and hind margin of femora with spines on strong elevated sockets, part of body and legs covered by small pale spots, tarsal claws with 4-5 marginal denticles and a subapical pair of submarginal denticles, pronotum with large rounded lateral projection, hind wing pads absent in female, gill formula 8/7/7/4/1, operculate gill with imbricated ventral lobes, and gill V without ventral extension on dorsal lamella.

Enderleina preclara Jewett, 1960 (Plecoptera, Perlidae), from the Venezuelan Guayana-A description of a putative female, nymph and egg.

The putative female, nymph and egg of Enderleina preclara are described from specimens collected in the Kukenán-tepui foothills and Acopán-tepui foothills and plateau of Venezuela. Morphological and molecular approaches were employed to characterize these stages. Diagnostic keys for males and females of the described Enderleina species are also presented. The nymph is also morphologically compared with those of other Enderleina species. Comments on the ecology of this species and erroneous information regarding the putative type locality of E. preclara are provided.

A new species of Cryptonympha Lugo-Ortiz McCafferty (Ephemeroptera, Baetidae) from Cerro Duida (Venezuela).

The genus Cryptonympha includes three species: C. copiosa Lugo-Ortiz McCafferty, 1998; C. dasilvai Salles Francischetti, 2004 and C. genevievae Thomas, Manchon Glémet, 2013, known only from the nymphal stage. A description of the nymph of a fourth representative, Cryptonympha tracheata sp. n. is provided here, based on material collected in a blackwater stream on the slopes of the Cerro Duida mountain in south-western Venezuela. The nymph of the new species can be easily distinguished from the other species of the genus by very long tracheal gills II-VII, 2.5 times the length of each tergum, gill I small, nearly half length of gill IV and by posterior margin of terga with rounded spines. A complete description of the new species and a key for nymphs of all known Cryptonympha species are provided.

Local environment rather than past climate determines community composition of mountain stream macroinvertebrates across Europe.

Community assembly is determined by a combination of historical events and contemporary processes that are difficult to disentangle, but eco-evolutionary mechanisms may be uncovered by the joint analysis of species and genetic diversity across multiple sites. Mountain streams across Europe harbour highly diverse macroinvertebrate communities whose composition and turnover (replacement of taxa) among sites and regions remain poorly known. We studied whole-community biodiversity within and among six mountain regions along a latitudinal transect from Morocco to Scandinavia at three levels of taxonomic hierarchy: genus, species and haplotypes. Using DNA barcoding of four insect families (>3100 individuals, 118 species) across 62 streams, we found that measures of local and regional diversity and intraregional turnover generally declined slightly towards northern latitudes. However, at all hierarchical levels we found complete (haplotype) or high (species, genus) turnover among regions (and even among sites within regions), which counters the expectations of Pleistocene postglacial northward expansion from southern refugia. Species distributions were mostly correlated with environmental conditions, suggesting a strong role of lineage- or species-specific traits in determining local and latitudinal community composition, lineage diversification and phylogenetic community structure (e.g., loss of Coleoptera, but not Ephemeroptera, at northern sites). High intraspecific genetic structure within regions, even in northernmost sites, reflects species-specific dispersal and demographic histories and indicates postglacial migration from geographically scattered refugia, rather than from only southern areas. Overall, patterns were not strongly concordant across hierarchical levels, but consistent with the overriding influence of environmental factors determining community composition at the species and genus levels.

Four new species and new records of Atopsyche Banks (Trichoptera: Hydrobiosidae) from Pantepui biogeographical region (Venezuela).

Four new species of the genus Atopsyche Banks (Hydrobiosidae) from Pantepui biogeographical region (Venezuela) are described and illustrated: Atopsyche (Atopsaura) inmae n. sp., Atopsyche (Atopsaura) cristinae n. sp., Atopsyche (Atopsaura) svitoki n. sp., and Atopsyche (Atopsaura) carmenae n. sp. Molecular analyses (cox1 sequences) allowed larva-adult associations and the morphological description of larvae of two of the new species (A. inmae and A. cristinae). A phylogenetic tree is performed to assess the molecular validity of the species, establish evolutionary relation among them and interpret historical biogeography of tepuis. Finally, two new distributional records for A. (Atopsaura) atahuallpa are included, which up to now was known only from the type locality.

Hydrolutos piaroa sp.n. (Orthoptera: Anostostomatidae), a new semiaquatic Lutosini species from south-western Venezuelan lowland streams.

Hydrolutos piaroa, a new species of Lutosini (Orthoptera: Anostostomatidae) from Tobogán de la Selva (Puerto Ayacucho region, SW Venezuela) is described and figured. Inhabiting aquatic environment it represents an unusual orthopteran with sternal and pleural area covered by fine microtrichia, forming a plastron. This is the first known Hydrolutos species sampled in lowland streams of Venezuelan Guayana.

A new species of Parakari (Ephemeroptera: Baetidae) from Guiana Highlands.

The genus Parakari was described from Guiana Highlands in southeastern Venezuela by Nieto & Derka in 2011 for two species inhabiting streams draining isolated, flat-topped table mountains called tepuis. A description of a third representative, Parakari roraimensis sp. n., is given here based on material collected from a coldwater stream at the foothills of Roraima-tepui (SE Venezuela). Detailed morphological descriptions of mature nymph and female adult are given. A differential diagnosis and a key to nymphs of the three Parakari species are provided.

Enderleina khazeni n. sp. (Plecoptera, Perlidae), a new stonefly from Venezuelan Guayana.

A new species of Enderleina (Plecoptera), E. khazeni n. sp., is described from two males collected in Bolivar Province, Southeast Venezuela. It appears to be more closely related to E. preclara Jewett that to other Enderleina species, but can be clearly distinguished from that species by shape of the penial sclerites. With the description of this new taxon, six species of the genus are known, of these males of four of them have been described.

Hydrolutos gransabanensis sp.n. (Orthoptera: Anostostomatidae), a new semi-aquatic Lutosini species from Gran Sabana (Venezuela).

Hydrolutos gransabanensis, a new species of Lutosini (Orthoptera: Anostostomatidae) from the cave Cueva El Tigre (Santa Elena de Uairén, SE Venezuela) is described and figured for both sexes. This is the first record of Hydrolutos species out of tepui systems. The genus is recently known by 6 apterous species from Venezuelan Guayana region.