Systematic evaluation of rDNA ITS2 as a DNA barcode for molecular identification of cyclorrhapha flies in China

Morphological identification of medically important flies is often hindered by incomplete or damaged specimens, cryptic species, and morphological conservatism among closely related species. In this study, common fly species were collected from 15 cities across 10 provinces in China to evaluate rDNA internal transcribed spacer 2 (ITS2) as a potential DNA barcode for molecular identification. A total of 1252 fly specimens were collected, of which 933 were morphologically classified into 19 species, 15 genera, 9 families, and 3 superfamilies; 319 remained unidentified due to the loss of key morphological characters. Seventeen mitochondrial and ribosomal candidates were evaluated using sequences retrieved from GenBank across Cyclorrhapha. ITS2 was selected based on its relatively low intraspecific divergence, higher interspecific divergence, and conserved flanking regions suitable for universal primer design. The designed ITS2 primers successfully amplified and sequenced all 144 tested specimens, including morphologically damaged samples. Integrating morphological and molecular data, 27 fly species were identified. Barcode gap analyses indicated clear interspecific discrimination in the genera of Muscidae, Calliphoridae, and Sarcophagidae. Phylogenetic analyses further supported species-level identification and revealed ITS2 sequence diversity within Musca domestica specimens. These results demonstrate that ITS2 is a promising candidate DNA barcode for Cyclorrhapha flies, particularly for specimens with ambiguous or damaged morphology.