Strong mito-nuclear discordance and sharing of mitochondrial DNA among species of the Simulium variegatum group (Diptera, Simuliidae) in Europe

Abstract Simuliidae are an important component of freshwater ecosystems and act as bloodsucking pests. The accurate identification of black fly species is crucial for effective pest management and lotic ecosystem biodiversity assessment. Nowadays, molecular markers are widely used for species identification. We analysed two mitochondrial and two nuclear molecular markers—COI, COII, ITS1, and ECP1—to assess their suitability for species assignment and delimitation within the well-defined morphospecies of the Simulium variegatum group ( Simulium argyreatum , S. maximum , S. monticola , S. monticoloides , S. sicanum , and S. variegatum ). The molecular analyses revealed strong discordance between mitochondrial and nuclear data, as well as sharing of mtDNA among several morphospecies. Four species— S. argyreatum , S. monticola , S. sicanum , and S. variegatum —shared common haplotypes and could not be effectively determined based on the mitochondrial markers. Nuclear markers have been proven to be more effective in species delimitation, especially the ECP1 gene, which correctly determined the studied species, following their morphological identification. Using only the mitochondrial COI gene barcodes for species identification in black flies may not be sufficient. A possible explanation for the mito-nuclear discordance is multiple mitochondrial introgression events among several species.