Sepsid flies (Diptera: Sepsidae) are important model insects for sexual selection research. In order to develop mitochondrial (mt) genome data for this significant group, we sequenced the first complete mt genome of the sepsid fly Nemopoda mamaevi Ozerov, 1997. The circular 15,878 bp mt genome is typical of Diptera, containing all 37 genes usually present in bilaterian animals. We discovered inaccurate annotations of fly mt genomes previously deposited on GenBank and thus re-annotated all published mt genomes of Cyclorrhapha. These re-annotations were based on comparative analysis of homologous genes, and provide a statistical analysis of start and stop codon positions. We further detected two 18 bp of conserved intergenic sequences from tRNAGlu-tRNAPhe and ND1-tRNASer(UCN) across Cyclorrhapha, which are the mtTERM binding site motifs. Additionally, we compared automated annotation software MITOS with hand annotation method. Phylogenetic trees based on the mt genome data from Cyclorrhapha were inferred by Maximum-likelihood and Bayesian methods, strongly supported a close relationship between Sepsidae and the Tephritoidea.

A traditionally controversial taxon, the Tipulomorpha has been frequently discussed with respect to both its familial composition and relationships with other Nematocera. The interpretation of internal relationships within the Tipuloidea, which include the Tipulidae sensu stricto, Cylindrotomidae, Pediciidae and Limoniidae, is also problematic. We sequenced the first complete mitochondrial (mt) genome of Symplecta hybrida (Meigen, 1804), which belongs to the subfamily Chioneinae of family Limoniidae, and another five nearly complete mt genomes from the Tipuloidea. We did a comparative analysis of these mt genomics and used them, along with some other representatives of the Nematocera to construct phylogenetic trees. Trees inferred by Bayesian methods strongly support a sister-group relationship between Trichoceridae and Tipuloidea. Tipulomorpha are not supported as the earliest branch of the Diptera. Furthermore, phylogenetic trees indicate that the family Limoniidae is a paraphyletic group.

In the present paper, the first complete mitochondrial genome of the family Dixidae is reported. The complete mitochondrial genome of Dixella sp. is a circular molecule of 15,574 bp in length, containing all 37 genes including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (srRNA and lrRNA), and a long control region. Its gene arrangement is conserved with the ancestral gene order of Drosophila yakuba, which is considered to exhibit the ground pattern of Hexapoda mitochondrial genome. Most PCGs start with standard ATN codons, while COI uses CCG, ND1 uses TTG and ND5 uses GTG as start codons. All PCGs terminate in the common stop codons TAA, except for COII and ND5 which end with a single thymine stop codon. There is a 703 bp of the control region, located between srRNA and tRNA(lle)-tRNA(Gln)-tRNA(Met) (IQM) cluster, without conserved blocks or long tandem repeats.

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Culicomorpha is a monophyletic group containing most bloodsucking lower dipterans, including many important vectors of pathogens. However, the higher-level phylogenetic relationships within Culicomorpha are largely unresolved, with multiple competing hypotheses based on molecular sequence data. Here we sequenced four nearly complete mitochondrial (mt) genomes representing four culicomorph families, and combined these new data with published mt genomes to reconstruct the phylogenetic relationships of all eight extant culicomorph families. We estimated phylogenies using four datasets and three methods. We also used four-cluster likelihood mapping to study potential incongruent topologies supported by the different datasets and phylogenetic questions generated by the previous studies. The results showed that a clade containing Ceratopogonidae, Thaumaleidae and Simuliidae was the sister group to all other Culicomorpha; in another clade, the Dixidae was basal to the remaining four families; Chaoboridae, Corethrellidae and Culicidae formed a monophyletic group and the Chironomidae was the sister group to this clade; Culicidae and Corethrellidae were sister groups in all trees. Our study provides novel mt genome data in Culicomorpha for three new family representatives, and the resulting mt phylogenomic analysis helps to resolve the phylogeny and taxonomy of Culicomorpha.