Abstract
Diptera comprises more than 154,000 described species, representing approximately 10-12% of insects. Members have successfully colonized all continents and a wide range of habitats. However, higher-level phylogenetic relationships within Diptera have remained ambiguous. Mitochondrial genomes (mitogenomes) have been used as valuable molecular markers for resolving phylogenetic issues. To explore the effect of such markers in solving the higher-level phylogenetic relationship of Diptera, we sequenced and annotated the mitogenomes of 25 species, combined with 180 mitogenomes from 33 superfamilies of dipteran insects to conduct a phylogenetic analysis based on the PCGsrRNA and PCGs12rRNA datasets using IQ-TREE under the partition model. The phylogenetic analysis failed to recover the monophyly of the two suborders Nematocera and Brachycera. Two of six infraorders within the Nematocera-Tipulomorpha and Ptychopteromorpha-were monophyletic. The ancestral Deuterophlebiidae were a strongly supported sister group of all remaining Diptera, but Anisopodidae, as the closest relative of Brachycera, received only weak support. Three of four infraorders within Branchycera-Tabanomorpha, Xylophagomorpha, and Stratiomyomorpha-were, respectively, supported as a monophyletic clade, except Muscomorpha due to the strong long-branch attraction between Cecidomyiidae and Nycteribiidae. The inferred infraordinal relationships followed the topology Tabanomorpha + (Xylophagomorpha + (Stratiomyomorpha + Muscomorpha)). However, the proposed topology lacks strong statistical support, suggesting alternative relationships remain plausible. Based on mitogenome data alone, we infer that Diptera originated earlier than the Late Triassic at 223.43 Mya (95% highest posterior density [HPD] 166.60-272.02 Mya) and the earliest brachyeran Diptera originated in the mid-Jurassic (171.61 Mya).