LAUSR

Simple Summary Flesh flies (Diptera: Sarcophagidae) make up the second-largest family of Oestroidea. They are well known for their veterinary, forensic, and medical importance due to their extremely diverse feeding habits, making them a hotspot in dipterology research. Therefore, efforts have been devoted to accumulating the mitochondrial genomes of Sarcophagidae. However, the mitogenome of flesh flies has mostly been sequenced for the genus Sarcophaga of the subfamily Sarcophaginae, and the other two subfamilies, Miltogramminae and Paramacronychiinae, have barely been touched. Here, we sequenced the mitochondrial genomes of five sarcophagid species from four genera representing all three subfamilies and investigated the phylogeny and evolution of flesh flies from the perspective of the mitogenome via comprehensive comparative analyses employing all mitogenomic data. This study will broaden our knowledge of flesh flies and make contributions to forensics, veterinary science, entomology, and ecology. Abstract Flesh flies (Diptera: Sarcophagidae) represent a rapid radiation belonging to the Calyptratae. With more than 3000 known species, they are extraordinarily diverse in terms of their breeding habits and are therefore of particular importance in human and veterinary medicine, forensics, and ecology. To better comprehend the phylogenetic relationships and evolutionary characteristics of the Sarcophagidae, we sequenced the complete mitochondrial genomes of five species of flesh flies and performed mitogenomic comparisons amongst the three subfamilies. The mitochondrial genomes match the hypothetical condition of the insect ancestor in terms of gene content and gene arrangement. The evolutionary rates of the subfamilies of Sarcophagidae differ significantly, with Miltogramminae exhibiting a higher rate than the other two subfamilies. The monophyly of the Sarcophagidae and each subfamily is strongly supported by phylogenetic analysis, with the subfamily-level relationship inferred as (Sarcophaginae, (Miltogramminae, Paramacronychiinae)). This study suggests that phylogenetic analysis based on mitochondrial genomes may not be appropriate for rapidly evolving groups such as Miltogramminae and that the third-codon positions could play a considerable role in reconstructing the phylogeny of Sarcophagidae. The protein-coding genes ND2 and ND6 have the potential to be employed as DNA markers for species identification and delimitation in flesh flies.