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Abstract Background Prompt and precise identification of black flies (Simuliidae) is crucial, given their biting behaviour and significant impact on human and animal health. To address the challenges presented by morphology and chromosomes in black fly taxonomy, along with the limited availability of molecular data pertaining to the black fly fauna in Vietnam, this study employed DNA-based approaches. Specifically, we used mitochondrial and nuclear-encoded genes to distinguish nominal species of black flies in Vietnam. Methods In this study, 135 mitochondrial cytochrome c oxidase subunit I (COI) sequences were established for 45 species in the genus Simulium in Vietnam, encompassing three subgenera (Gomphostilbia, Nevermannia, and Simulium), with 64 paratypes of 27 species and 16 topotypes of six species. Of these COI sequences, 71, representing 27 species, are reported for the first time. Results Combined with GenBank sequences of specimens from Malaysia, Myanmar, Thailand, and Vietnam, a total of 234 DNA barcodes of 53 nominal species resulted in a 71% success rate for species identification. Species from the non-monophyletic Simulium asakoae, S. feuerborni, S. multistriatum, S. striatum, S. tuberosum, and S. variegatum species groups were associated with ambiguous or incorrect identifications. Pairwise distances, phylogenetics, and species delimitation analyses revealed a high level of cryptic diversity, with discovery of 15 cryptic taxa. The current study also revealed the limited utility of a fast-evolving nuclear gene, big zinc finger (BZF), in discriminating closely related, morphologically similar nominal species of the S. asakoae species group. Conclusion This study represents the first comprehensive molecular genetic analysis of the black fly fauna in Vietnam to our knowledge, providing a foundation for future research. DNA barcoding exhibits varying levels of differentiating efficiency across species groups but is valuable in the discovery of cryptic diversity. Graphical abstract |
