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.

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.

Tipulomorpha has long been a problematic taxon in terms of familial composition, phylogenetic relationships among families and position relative to other ‘lower’ Diptera. Whole-transcriptome shotgun sequencing provides a powerful basis for phylogenetic studies. We performed de novo transcriptome sequencing to produce the first transcriptome datasets representing the families Pediciidae, Limoniidae and Cylindrotomidae using high-throughput sequencing technologies. We assembled cDNA libraries for Pedicia vetusta (Alexander) (Pediciidae), Rhipidia sejuga Zhang, Li and Yang (Limoniidae) and Liogma simplicicornis Alexander (Cylindrotomidae). Using the Illumina RNA-Seq method, we obtained 28,252, 44,152 and 44,281 unigenes, from the three respective species. Based on sequence similarity searches, 12,475 (44.16%), 20,334 (46.05%) and 17,478 (39.47%) genes were identified. Analysis of genes highly conserved at the amino acid sequence level revealed there were 1,709 single-copy orthologs genes across the analyzed species. Phylogenetic trees constructed using maximum likelihood (ML) based on the 1,709 single-copy orthologs genes indicated that the relationship between the four major infraorders of lower Diptera was: Culicomorpha + (Tipulomorpha + (Psychodomorpha + (Bibionomorpha + Brachycera))). Trichoceridae belongs within Tipulomorpha as the sister-group of Tipuloidea. Highly supported relationships within the Tipuloidea are Pediciidae + (Limoniidae + (Cylindrotomidae + Tipulidae)). Four-cluster likelihood mapping was used to study potential incongruent signals supporting other topologies, however, results were congruent with the ML tree.

The genus Tipula Linnaeus, 1758 Linnaeus C . 1758. Systema naturae per regna tria naturae, secundum classes, ordines, genera, species, cum caracteribus, differentiis, synonymis, locis. 10th ed. Salvii, Holmiae [= Stockholm]: Lipsiae; Vol. 1; p. i–iv, 1–824. [Google Scholar] is a large group of crane flies with more than 2400 known species from 41 subgenera. In this study, we report the first complete mitochondrial (mt) genome sequence of the subgenus Tipula (Yamatotipula), which is a circular molecule of 15,668 bp with an AT content of 77.2%. The mt genome contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a long non-coding region. Three conserved overlapping regions, 8 bp between tRNA^Trp and tRNA^Cys, 7 bp between ATP8 and ATP6, and 7 bp between ND4 and ND4L, are found. Phylogenetic analysis reveals that the Tipulomorpha includes the family Trichoceridae and the Trichoceridae is sister-group to the remaining Tipulomorpha.

The genus Tanyptera Latreille, 1804 is recorded from Shandong Province, China for the first time with T. (T.) hebeiensis Yang et Yang, 1988 found in Mount Kunyu, Shandong. In this study, we report the complete mitochondrial genome sequence of T. (T.) hebeiensis, representing the first mitochondrial genome of the subfamily Ctenophorinae (Diptera: Tipulidae), which is a circular molecule of 15,888 bp with an AT content of 77.6%. The mitochondrial genome contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a non-coding region. Gene overlaps are found at nine gene junctions, ranging from 1 to 8 bp in length. The canonical mitochondrial start codons for invertebrate mitochondrial genomes are found in 12 PCGs, except for COI which uses the uncanonical start codons TCG. Stop codons of 10 PCGs are invariably complete TAA and TAG, while COII, ND4, and ND5 end with a single thymine stop codon. Phylogenetic analysis reveals that the Pediciidae is a sister group to the remaining Tipuloidea, the Cylindrotomidae has a sister-group relationship with the Tipulidae, and the Limoniidae is not a monophyletic clade.

Limoniidae, the most speciose family in the superfamily Tipuloidea, consists of four subfamilies and more than 11,000 species. However, mitochondrial (mt) genome sequences, which have been widely used for phylogenetic study, are available for only 11 species across three subfamilies. Thus, a larger variety of mt genome sequences in Limoniidae are required to improve our understanding of tipuloid phylogeny and genomic evolution. Here we present mt genomes of Elephantomyia (Elephantomyia) inulta Alexander, 1938 and Helius (Helius) pluto Alexander, 1932, representing the first mt genomes of the tribe Elephantomyiini (Limoniidae). The two mt genomes are typical circular DNA molecules and show similar gene order, nucleotide composition and codon usage. Standard ATN start and TAR stop codons are present in most protein-coding genes. All transfer RNA (tRNA) genes exhibited the cloverleaf secondary structure typical for metazoans except in tRNASer(AGN), which lacks the dihydrouridine arm. Phylogenetic analyses were performed based on four nucleotide matrixes for the currently sequenced species of Tipuloidea using Bayesian inference and maximum likelihood methods. Four-cluster likelihood mapping was used to study incongruent signals between different topologies. Pediciidae is supported as the earliest lineage in Tipuloidea, and the sister-group relationship between Cylindrotomidae and Tipulidae is also supported, but the monophyly of Limoniidae is not supported. Our study also supports the monophyly of Elephantomyiini (Elephantomyia + Helius), as one of origins of flower-visiting in Limoniidae. Although Elephantomyiini is sister to Limoniinae + Epiphragma (Limnophilinae) in our study, a more precise understanding of its phylogenetic position in Tipuloidea will require additional studies that include a broader species sample.