The diversity of non-biting midges (Chironomidae, Diptera) remains an unresolved topic, with estimates of species numbers ranging from 6000 to 15,000 according to various authors. To assess Chironomidae diversity in Lithuania, we evaluate the effectiveness of COI gene-based species delimitation methods for providing rapid diversity estimates. Nevertheless, differences between tree-based and distance-based approaches can result in varying group classifications, which may cause species numbers to be overestimated or underestimated. For our study, we analyzed a dataset of 109 specimens sampled from six Lithuanian streams. By applying multiple methods, such as Assemble Species by Automatic Partitioning (ASAP), Automatic Barcode Gap Discovery (ABGD), the generalized mixed Yule-coalescent (GMYC) model, and the Bayesian implementation of the Poisson Tree Processes (bPTP) model, we found that species estimates ranged from 28 to 58. Among these methods, ASAP proved to be the most effective for our dataset, identifying 58 putative species. These results reinforce our assumption that the current understanding of Chironomidae species diversity is incomplete.

Chironomidae, commonly known as non-biting midges, are key indicators of the health and biodiversity of freshwater ecosystems. They are also one of the most abundant and diverse groups of aquatic invertebrates. Although Chironomidae are ecologically important, abundant and diverse, there has been limited focused research on this group in Lithuania. Our study addresses this gap by analysing the diversity of Chironomidae in six Lithuanian streams. In this study, we present a comprehensive list of Chironomidae collected from six streams with similar hydrological characteristics: three dammed and three undammed. Lithuania is home to 158 species of known species of Chironomidae, comprised of 22 species in the subfamily Tanypodinae, 87 in the Chironominae, 42 in the Orthocladiinae, four in the Diamesinae and three in the Prodiamesinae. Throughout our research, we collected 11,296 chironomid specimens using a D-shaped aquatic net. Amongst these specimens, we identified 89 species representing 65 genera and five subfamilies, including 37 species and 12 genera, were recorded for the first time in Lithuania. The subfamily Chironominae, with 28 genera and 6,816 specimens, has exhibited the highest abundance of non-biting midges both in terms of genera and individuals. Amongst the streams investigated, the Luknelė was the richest in diversity, harbouring 37 genera and 2,657 individuals, accounting for about 55% of the Chironomidae genera found during the research. Our findings significantly enhance the understanding of the Lithuanian Chironomidae fauna, marking the first comprehensive study of such a kind, as previous knowledge of this fauna has been derived only from related studies.