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The superfamily Lauxanioidea is a significant dipteran clade including over 2500 known species in three families: Lauxaniidae, Celyphidae and Chamaemyiidae. We sequenced the first five (three complete and two partial) lauxanioid mitochondrial (mt) genomes, and used them to reconstruct the phylogeny of this group. The lauxanioid mt genomes are typical of the Diptera, containing all 37 genes usually present in bilaterian animals. A total of three conserved intergenic sequences have been reported across the Cyclorrhapha. The inferred secondary structure of 22 tRNAs suggested five substitution patterns among the Cyclorrhapha. The control region in the Lauxanioidea has apparently evolved very fast, but four conserved structural elements were detected in all three complete mt genome sequences. Phylogenetic relationships based on the mt genome data were inferred by Maximum Likelihood and Bayesian methods. The traditional relationships between families within the Lauxanioidea, (Chamaemyiidae + (Lauxaniidae + Celyphidae)), were corroborated; however, the higher-level relationships between cyclorrhaphan superfamilies are mostly poorly supported.

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.

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.

Muscoidea is a significant dipteran clade that includes house flies (Family Muscidae), latrine flies (F. Fannidae), dung flies (F. Scathophagidae) and root maggot flies (F. Anthomyiidae). It is comprised of approximately 7000 described species. The monophyly of the Muscoidea and the precise relationships of muscoids to the closest superfamily the Oestroidea (blow flies, flesh flies etc) are both unresolved. Until now mitochondrial (mt) genomes were available for only two of the four muscoid families precluding a thorough test of phylogenetic relationships using this data source. Here we present the first two mt genomes for the families Fanniidae (Euryomma sp.) (family Fanniidae) and Anthomyiidae (Delia platura (Meigen, 1826)). We also conducted phylogenetic analyses containing of these newly sequenced mt genomes plus 15 other species representative of dipteran diversity to address the internal relationship of Muscoidea and its systematic position. Both maximum-likelihood and Bayesian analyses suggested that Muscoidea was not a monophyletic group with the relationship: (Fanniidae + Muscidae) + ((Anthomyiidae + Scathophagidae) + (Calliphoridae + Sarcophagidae)), supported by the majority of analysed datasets. This also infers that Oestroidea was paraphyletic in the majority of analyses. Divergence time estimation suggested that the earliest split within the Calyptratae, separating (Tachinidae + Oestridae) from the remaining families, occurred in the Early Eocene. The main divergence within the paraphyletic muscoidea grade was between Fanniidae + Muscidae and the lineage ((Anthomyiidae + Scathophagidae) + (Calliphoridae + Sarcophagidae)) which occurred in the Late Eocene.

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.

Phylogenetic relationships within the Tabanidae are largely unknown, despite their considerable medical and ecological importance. The first robust phylogenetic hypothesis for the horse fly tribe Scionini is provided, completing the systematic placement of all tribes in the subfamily Pangoniinae. The Scionini consists of seven mostly southern hemisphere genera distributed in Australia, New Guinea, New Zealand and South America. A 5757 bp alignment of 6 genes, including mitochondrial (COI and COII), ribosomal (28S) and nuclear (AATS and CAD regions 1, 3 and 4) genes, was analysed for 176 taxa using both Bayesian and maximum likelihood approaches. Results indicate the Scionini are strongly monophyletic, with the exclusion of the only northern hemisphere genus Goniops. The South American genera Fidena, Pityocera and Scione were strongly monophyletic, corresponding to current morphology-based classification schemes. The most widespread genus Scaptia was paraphyletic and formed nine strongly supported monophyletic clades, each corresponding to either the current subgenera or several previously synonymised genera that should be formally resurrected. Molecular results also reveal a newly recognised genus endemic to New Zealand, formerly placed within Scaptia. Divergence time estimation was employed to assess the global biogeographical patterns in the Pangoniinae. These analyses demonstrated that the Scionini are a typical Gondwanan group whose diversification was influenced by the fragmentation of that ancient land mass. Furthermore, results indicate that the Scionini most likely originated in Australia and subsequently radiated to New Zealand and South American by both long distance dispersal and vicariance. The phylogenetic framework of the Scionini provided herein will be valuable for taxonomic revisions of the Tabanidae., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Members of the Calliphoridae (blowflies) are significant for medical and veterinary management, due to the ability of some species to consume living flesh as larvae, and for forensic investigations due to the ability of others to develop in corpses. Due to the difficulty of accurately identifying larval blowflies to species there is a need for DNA-based diagnostics for this family, however the widely used DNA-barcoding marker, cox1, has been shown to fail for several groups within this family. Additionally, many phylogenetic relationships within the Calliphoridae are still unresolved, particularly deeper level relationships. Sequencing whole mt genomes has been demonstrated both as an effective method for identifying the most informative diagnostic markers and for resolving phylogenetic relationships. Twenty-seven complete, or nearly so, mt genomes were sequenced representing 13 species, seven genera and four calliphorid subfamilies and a member of the related family Tachinidae. PCR and sequencing primers developed for sequencing one calliphorid species could be reused to sequence related species within the same superfamily with success rates ranging from 61% to 100%, demonstrating the speed and efficiency with which an mt genome dataset can be assembled. Comparison of molecular divergences for each of the 13 protein-coding genes and 2 ribosomal RNA genes, at a range of taxonomic scales identified novel targets for developing as diagnostic markers which were 117-200% more variable than the markers which have been used previously in calliphorids. Phylogenetic analysis of whole mt genome sequences resulted in much stronger support for family and subfamily-level relationships. The Calliphoridae are polyphyletic, with the Polleninae more closely related to the Tachinidae, and the Sarcophagidae are the sister group of the remaining calliphorids. Within the Calliphoridae, there was strong support for the monophyly of the Chrysomyinae and Luciliinae and for the sister-grouping of Luciliinae with Calliphorinae. Relationships within Chrysomya were not well resolved. Whole mt genome data, supported the previously demonstrated paraphyly of Lucilia cuprina with respect to L. sericata and allowed us to conclude that it is due to hybrid introgression prior to the last common ancestor of modern sericata populations, rather than due to recent hybridisation, nuclear pseudogenes or incomplete lineage sorting., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The monogeneric family Fergusoninidae consists of gall-forming flies that, together with Fergusobia (Tylenchida: Neotylenchidae) nematodes, form the only known mutualistic association between insects and nematodes. In this study, the entire 16,000 bp mitochondrial genome of Fergusonina taylori Nelson and Yeates was sequenced. The circular genome contains one encoding region including 27 genes and one non-coding A+T-rich region. The arrangement of the protein-coding, ribosomal RNA (rRNA) and transfer RNA (tRNA) genes was the same as that found in the ancestral insect. Nucleotide composition is highly A+T biased. All of the protein initiation codons are ATN, except for nad1 which begins with TTT. All 22 tRNA anticodons of F. taylori match those observed in Drosophila yakuba, and all form the typical cloverleaf structure except for tRNA-Ser((AGN)) which lacks a dihydrouridine (DHU) arm. Secondary structural features of the rRNA genes of Fergusonina are similar to those proposed for other insects, with minor modifications. The mitochondrial genome of Fergusonina presented here may prove valuable for resolving the sister group to the Fergusoninidae, and expands the available mtDNA data sources for acalyptrates overall.

Glow-worms are bioluminescent fly larvae (Order Diptera, genus Arachnocampa) found only in Australia and New Zealand. Their core habitat is rainforest gullies and wet caves. Eight species are present in Australia; five of them have been recently described. The geographic distribution of species in Australia encompasses the montane regions of the eastern Australian coastline from the Wet Tropics region of northern Queensland to the cool temperate and montane rainforests of southern Australia and Tasmania. Phylogenetic trees based upon partial sequences of the mitochondrial genes cytochrome oxidase II and 16S mtDNA show that populations tend to be clustered into allopatric geographic groups showing overall concordance with the known species distributions. The deepest division is between the cool-adapted southern subgenus, Lucifera, and the more widespread subgenus, Campara. Lucifera comprises the sister groups, A. tasmaniensis, from Tasmania and the newly described species, A. buffaloensis, found in a high-altitude cave at Mt Buffalo in the Australian Alps in Victoria. The remaining Australian glow-worms in subgenus Campara are distributed in a swathe of geographic clusters that extend from the Wet Tropics in northern Queensland to the temperate forests of southern Victoria. Samples from caves and rainforests within any one geographic location tended to cluster together within a clade. We suggest that the morphological differences between hypogean (cave) and epigean (surface) glow-worm larvae are facultative adaptations to local microclimatic conditions rather than due to the presence of cryptic species in caves.