Essential for implementation of molecular tools in bioassessments are comprehensive databases with high-quality reference DNA sequence data. A recent survey arising from the DNAqua-Net EU COST Action WG1 has revealed significant gaps in the barcode reference data of aquatic biota both on a taxonomic and a geographical level (Weigand 2019). Although reference barcode libraries of all main freshwater groups are continuously improved through a number of national DNA-barcoding campaigns, still little data is available from Central and Eastern Europe. In Slovakia, the barcode coverage of the aquatic biota was very low when DNAqua-Net started. At the end of 2016 only 165 public records (60 OTUs) were available in BOLD (< 3 % of aquatic macroinvertebrates reported from Slovakia), and most of them were mined from GenBank. This led us to launch the campaign “Barcoding aquatic biota of Slovakia” (www.AquaBOL.SK), aiming to improve this unpleasant state. Since many common species in Slovakia are covered by barcodes from other countries, initially we focused on least explored habitat types within Europe - alpine glacial lakes in Tatra Mts and karstic springs. Both are located in Western and Slovak part of Eastern Carpathians, which are considered one of the most valuable biodiversity centers in Europe. More than 2700 sequences of COI-5P (337 BINs), originating in Slovakia, are currently in our dataset. Mostly they represent aquatic insects (86 %) and crustaceans (12 %), followed by minor taxonomic groups. From insects, the best covered orders are Coleoptera (43 % of sequences, 21% of species), followed by Trichoptera (22 % of sequences, 24 % of species) and Ephemeroptera (18 % of sequences, 33 % of species). And even if other groups (Hemiptera, Plecoptera) represent only around 5 % of all sequences each, their species coverage is already 21 % or 28 %, respectively. Relatively high number of unique BINs (35 out of 337) from our area, and separate intraspecific lineages within BINs of many widespread species, supports the premise that the Western Carpathians belong to the most important biodiversity hotspots. Our local reference database, continuously updated, is useful for the analysis and interpretation of metabarcoding data - a prerequisite for effective and nondestructive monitoring of aquatic biotopes including vulnerable, threatened, and protected habitats. It is also beneficial for a number of other purposes such as taxonomic revisions and phylogenetic analyses. This contribution was partially supported by VEGA2/0030/17, VEGA2/0084/2021 and COST CA15219.
Mayflies (Ephemeroptera) represent a small but diverse order of amphibiotic insects, whose larvae contribute to several essential processes in freshwater habitats, such as bioturbation and bioirrigation, decomposition, nutrient cycling, and also serve as a primary source of nutrients for numerous organisms. Due to their cosmopolitan distribution and high-quality water requirements, they are also important indicators of ecosystem health and an integral part of biomonitoring protocols. Although the Slovak mayfly fauna is well researched, studies on genetic diversity, including DNA barcoding, are still lacking. The absence of the comprehensive DNA barcode reference libraries from various biogeographical regions and the presence of so-called cryptic lineages may prevent further efficient use and application of new approaches to aquatic ecosystem biomonitoring (Biomonitoring 2.0) based on eDNA analyses. Therefore, in the initial stage of our research, we bring the first insight into the genetic diversity of mayflies (based on mtDNA COI-5P barcoding fragment) from 47 localities of Slovakia mostly situated in the biogeographically significant Western Carpathians' territory. A total of 403 sequences of 42 morphologically determined species were added to the BOLD (Barcode of Life Data System) database, representing more than 1/3 of the mayfly fauna of Slovakia and covering 10 of 16 families. Sequences of these species were finally assigned to 62 BINs (Barcode Index Numbers) in BOLD (Fig. 1), whereby sequences of 12 species were divided into more than one BIN, indicating the presence of cryptic lineages. The largest number of BINs was represented by widely distributed species such as Baetis rhodani Pictet, 1843-1845 (6 BINs), Habroleptoides confusa Sartori & Jacob, 1986 (4 BINs) and Ecdyonurus venosus (Fabricius, 1775) (3 BINs). The sequences of the remaining nine species were split into two BINs. Maximum intraspecific variability (calculated by K2P) of some representatives was surprisingly high [e.g., E. venosus – 27.1 %; Baetis muticus (Linneaus, 1758) – 23.6 %; Caenis luctuosa (Burmeister, 1839) – 23.34 %, Baetis rhodani – 18.66 % and B. vernus Curtis, 1834 – 15.25 %] and far exceeded the level of intraspecific variability of the COI fragment based on the BOLD standards. The sequences of 23 individuals determined as Habroleptoides confusa, Baetis rhodani, B. buceratus Eaton, 1870, Caenis beskidensis Sowa, 1973 and Torleya major (Klapálek, 1905) created seven unique BINs, which represent the distant phylogenetic lineages of already existing BINs, that are currently unique to Slovakia. The coexistence of Baetis rhodani individuals of two different BINs was confirmed at five localities. Our study indicates clear importance of more detailed sampling and DNA barcoding due to the presence of unexpected intraspecific genetic diversity of mayflies captured in a relatively small area of the Western Carpathians.
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