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and routine biomonitoring in the Carpathian and Pannonian ecoregions, their distribution data are fragmentary and outdated. All published and unpublished data on mayflies from Slovakia was gathered and a database of 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.