Background: Dragonflies and damselflies (Odonata) are important components in biomonitoring due to their amphibiotic lifecycle and specific habitat requirements. They are charismatic and popular insects, but can be challenging to identify despite large size and often distinct coloration, especially the immature stages. DNA-based assessment tools rely on validated DNA barcode reference libraries evaluated in a supraregional context to minimize taxonomic incongruence and identification mismatches.Methods: This study reports on findings from the analysis of the most comprehensive DNA barcode dataset for Central European Odonata to date, with 103 out of 145 recorded European species included and publicly deposited in the Barcode of Life Data System (BOLD). The complete dataset includes 697 specimens (548 adults, 108 larvae) from 274 localities in 16 countries with a geographic emphasis on Central Europe. We used BOLD to generate sequence divergence metrics and to examine the taxonomic composition of the DNA barcode clusters within the dataset and in comparison with all data on BOLD.Results: Over 88% of the species included can be readily identified using their DNA barcodes and the reference dataset provided. Considering the complete European dataset, unambiguous identification is hampered in 12 species due to weak mitochondrial differentiation and partial haplotype sharing. However, considering the known species distributions only two groups of five species possibly co-occur, leading to an unambiguous identification of more than 95% of the analysed Odonata via DNA barcoding in real applications. The cases of small interspecific genetic distances and the observed deep intraspecific variation inCordulia aenea(Linnaeus, 1758) are discussed in detail and the corresponding taxa in the public reference database are highlighted. They should be considered in future applications of DNA barcoding and metabarcoding and represent interesting evolutionary biological questions, which call for in depth analyses of the involved taxa throughout their distribution ranges.

Lake Skadar with its surrounding springs, wetlands and larger affluents is among the most diverse freshwater ecosystems in the Mediterranean region and a key biodiversity/endemism hotspot in Europe. It is also highly endangered due to climate change and rapid tourism development in the area. Being abundant, diverse and mostly predatory, true aquatic bugs play an important role in the functioning of freshwater ecosystems and are used as indicators of aquatic habitat quality. Nevertheless, this taxonomic group has been scarcely studied in the area. Our survey provides the first comprehensive DNA barcode library for 24 out of 25 species of aquatic Heteroptera collected in the Skadar Lake basin and adjacent regions. By this, we extend the list of species known from the area by 60%. In the case of three species, Notonecta maculata, Hydrometra stagnorum and Nepa cinerea, we detected multiple highly divergent, and also new BINs indicating possible taxonomic inconsistencies, the potential for (pseudo)cryptic diversity and intricate phylogeographic patterns. We show that presumably well-known hotspots, such as Lake Skadar region, are heavily understudied regarding even the prominent insect taxa and, thus, particularly vulnerable to undocumented biodiversity loss. Finally, we underline the value of simple DNA-barcoding-based surveys for providing reference barcode libraries for effective biomonitoring and signalling taxonomic and biogeographic issues.

Molecular traces are increasingly being applied to assess the presence of species and communities. Studies on environmental DNA (eDNA) have, to a large extent, become common practice in species detection, but less studies have compared biodiversity estimations with the more temporary environmental RNA (eRNA). This study compares metabarcoding results from pond water obtained from both molecule types by sequencing the V4 region in the 18S rRNA marker. Water was collected from two depths, 20 and 80 cm, and filtered sequentially through two filter porosities, 0.45 and 0.22 μm. Each filter was cut in half before fixation in either 96% ETOH or RNAlater. The results showed no differences between the fixatives for either molecule. Overall, biodiversity estimates from eDNA significantly overperformed eRNA, likely due to higher concentrations of eDNA from terrestrial sources. Comparisons of the two depths showed variation for eDNA only, with increasing levels of biodiversity found at the upper water layer. Both filter pore sizes captured distinctive compositions of taxa, where about 30% of the diversity was uniquely identified from the second, finer filter. Taken together, these findings imply that the choice of molecular marker, depth and filter pore size affects the obtained biodiversity estimations in a pond.

DNA barcoding has become a cornerstone for species identification and biodiversity monitoring, enabling applications from ecological research to conservation and environmental policy. The International Barcode of Life (iBOL) provides global coordination, but national nodes are essential for implementing barcoding at scale, building local capacity and translating scientific advances into practice. This paper synthesises experiences from 20 countries (17 in Europe), drawing on a survey and a workshop conducted under the Horizon Europe Biodiversity Genomics Europe project. We examine how national nodes are initiated, governed and sustained and identify common challenges, such as defining scope, securing funding, harmonising methods and engaging stakeholders. Most nodes were initiated by research communities and operate as informal networks with heterogeneous governance and staffing models. Key priorities include constructing comprehensive DNA barcode reference libraries, aligning activities with biomonitoring needs and promoting FAIR and CARE data principles. We highlight strategies for capacity building, methodological standardisation and stakeholder engagement, alongside approaches for diversifying funding and strengthening communication. Based on these insights, we present ten practical recommendations to guide the establishment and long-term success of national DNA barcoding nodes. Strengthening these infrastructures will enhance Europe’s ability to deliver robust DNA-based biodiversity monitoring, underpin metabarcoding and metagenomic studies and contribute to global efforts in species discovery, conservation and environmental management.