Phylogeny and Evolutionary Timescale of Muscidae (Diptera: Calyptratae) Inferred from Mitochondrial Genomes.

House flies (Muscidae) comprise the most species-rich family of the muscoid grade with over 5000 described species worldwide, and they are abundant in various terrestrial and aquatic ecosystems. The high number of species, varied appearances, complex feeding habits, and wide distributions have hindered researchers from understanding their phylogeny and evolutionary history. Here, we newly sequenced fifteen mitochondrial genomes and reconstructed the phylogenetic relationships and divergence time among eight subfamilies of Muscidae (Diptera). The best phylogenetic tree, which was inferred by IQ-Tree, recovered the monophyly for seven out of eight subfamilies (except for Mydaeinae). Based on phylogenetic analyses and morphological characteristics, we prefer the subfamily status of Azeliinae and Reinwardtiinae, and separate Stomoxyinae from Muscinae. Genus Helina Robineau-Desvoidy, 1830 was synonymized with Phaonia Robineau-Desvoidy, 1830. The divergence time estimation indicated Muscidae originated at 51.59 Ma (early Eocene). Most subfamilies had originated around 41 Ma. We provided a mtgenomic viewpoint on the phylogenetic relationships and divergence time estimation of Muscidae.

Complete mitochondrial genome of Coenomyia ferruginea (Scopoli). (Diptera, Coenomyiidae).

The dipteran family Coenomyiidae was firstly separated from the Xylophagidae by Akrira Nagatomi in 1975. We sequenced and annotated the mitochondrial genome of Coenomyia ferruginea, the first representative of genera Coenomyia with complete mitochondrial data. This mitogenome is 17,283 bp totally, which consists of 22 transfer RNAs, 13 protein-coding genes, 2 ribosomal RNAs, and 1 non-coding control region. All genes have the conservational arrangement like other published species of brachyceran flies. The nucleotide composition biases toward A and T, the overall A + T% was up to 75.4% of the entire mitogenome. Both Bayesian inference and ML analysis strongly supported the sister relationship between Coenomyiidae and Xylophagidae. Our results also suggested that Xylophagomorpha is the sister group to Stratiomyomorpha.

The phylogeny and evolutionary timescale of stoneflies (Insecta: Plecoptera) inferred from mitochondrial genomes.

Phylogenetic analysis based on mitochondrial genomic data from 25 stonefly species recovered a well-supported tree resolving higher-level relationships within Plecoptera (stoneflies). The monophyly of both currently recognized suborders was strongly supported, concordant with previous molecular analyses of Plecoptera. The southern hemisphere suborder Antarctoperlaria formed two clades: Eustheniidae + Diamphipnoidae and Austroperlidae + Gripopterygidae; consistent with relationships proposed based on morphology. The largely northern hemisphere suborder Arctoperlaria also divided into two groups, Euholognatha and Systellognatha, each composed of the five families traditionally assigned to each infraorder (the placement Scopuridae by mt genome data remains untested at this time). Within Euholognatha, strong support for the clade Nemouridae + Notonemouridae confirmed the northern origin of the currently southern hemisphere restricted Notonemouridae. Other family level relationships within the Arctoperlaria differ from those recovered by previous morphology and molecular based analyses. A fossil-calibrated divergence estimation suggests the formation of two suborders dates back to the Jurassic (181 Ma), with subsequent diversification of most stonefly families during the Cretaceous. This result confirms the hypothesis that initial divergence between the suborders was driven by the breakup of the supercontinent Pangaea into Laurasia and Gondwanaland (commencing 200 Ma and complete by 150 Ma).

Mitochondrial Genomes Provide Insights into the Phylogeny of Culicomorpha (Insecta: Diptera).

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.

The mitochondrial genome of Drino Sp. (Diptera, Tachinidae).

The tachinid fly Drino sp. belongs to the subfamily Exoristinae of Tachinidae. We sequenced and annotated the mitogenome of Drino sp. which makes this species first representative of the tribe Eryciini (Tachinidae: Exoristinae) with nearly complete mitochondrial data. This mitogenome is 15437 bp in total, which consists of 22 transfer RNAs, 13 protein-coding genes, 2 ribosomal RNAs and non-coding control region. All genes have the conservational arrangement with other published species of Tachinidae. The nucleotide composition biases toward A and T, the overall A + T% was up to 80.4% of the entire mitogenome. Bayesian inference analysis strongly supported the monophyly of Tachinidae and Exoristinae. Our results also suggested that Exoristinae is the sister group to Phasiinae, and Dexiinae is the sister group to the clade of Phasiinae + Exoristinae.

Mitochondrial Genomes Provide Insights into the Phylogeny of Lauxanioidea (Diptera: Cyclorrhapha).

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.

Transcriptomes of three species of Tipuloidea (Diptera, Tipulomorpha) and implications for phylogeny of Tipulomorpha.

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.

Complete mitochondrial genome of Allognosta vagans (Diptera, Stratiomyidae).

The complete mitochondrial genome (mitogenome) of Allognosta vagans (Loew, 1873) has been reported in this study. This is the first sequenced mitogenome of the subfamily Beridinae. The genome is 15,982 bp in length, including 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a partial sequence of the AT-rich region, and the AT-rich region contains several characteristic repeated sequences. In addition, the nucleotide composition of the coding region was 38.8% of A, 38.7% of T, 9.4% of C, 13.1% of G, 77.5% of A + T content. So far, five complete mitochondrial genome data of related species are available in our lab, all of them are used in Maximum Likelihood and Neighbour-Join analyses. The result supported that Xylomyidae and Stratiomyidae are sister group.

Complete mitochondrial genome of Hydrellia griseola (Diptera, Ephydridae).

The complete mitochondrial genome (mitogenome) of Hydrellia griseola has been reported in this study. This is the first sequenced complete mitogenome of the family Ephydridae. The complete mitogenome is 16,159 bp in length, including 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and a partial sequence of the AT-rich region, and the AT-rich region contains several characteristic repeated sequences. In addition, the nucleotide composition of the coding region was 38.7% of A, 37.0% of T, 14.2% of C, 10.1% of G, 75.7% of A + T content. Four complete mitochondrial genome data of related species are download from GenBank, and all of them are used in Neighbor-Join analyses. The result consistently supports the monophyly of Ephydroidea.

Mitochondrial genome analysis of Ectophasia roundiventris (Diptera, Tachinidae).

The mitochondrial genome of Ectophasia roundiventris (Loew, 1858), the first representative of subfamily Phasiinae, was sequenced and annotated. So far, there are four Tachinidae mitochondrial genomes, here, all of them are used in Neighbour-Join and Maximum Likelihood analyses. The nucleotide composition of Ectophasia roundiventris mitochondrial genome was 40.4% of A, 39.0% of T, 11.8% of C, 8.8% of G, 79.4% of A + T content. The codon ATG was the most popular start codon. The conservative stop codon was TAA, COX2, and ND5 terminated with an incomplete stop codon T, while the gene ND4 was ended with stop codon TA.

The complete mitochondrial genome analysis of Eristalis tenax (Diptera, Syrphidae).

The complete mitochondrial genome of Eristalis tenax (Linnaeus, 1758) (Diptera, Syrphidae) is reported here. This is the first sequenced mitogenome from the subfamily Milesiinae. The whole mitochondrial genome is 16,091 bp in length and contains 37 canonical genes, which include 22 transfer RNA genes, 13 protein-coding genes and two ribosomal RNA genes, the control region is 1125 bp in length. Most PCGs start with standard ATN codons, while CO1 and ND1 use TTG, CO3 uses TGG as start codons. All PCGs terminate in the common stop codons TAA. In addition, the nucleotide composition of the coding region was 40.0% of A, 40.1% of T, 11.2% of C, 8.7% of G and 80.1% of A + T content. The phylogenetic tree shows that Syrphidae is the sister group of Pipunculidae.

The complete mitochondrial genome of the Atylotus miser (Diptera: Tabanomorpha: Tabanidae), with mitochondrial genome phylogeny of lower Brachycera (Orthorrhapha).

Brachycera is a clade with over 80,000 described species and originated from the Mesozoic, and its larvae employ comprehensive feeding strategies. The phylogeny of the lower Brachycera has been studied intensively over the past decades. In order to supplement the lack of genetic data in this important group, we sequenced the complete mitochondrial (mt) genome of Atylotus miser as well as the nearly complete mt genomes of another 11 orthorrhaphous flies. The mt genome of A. miser is 15,858bp, which is typical of Diptera, with 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a 993bp control region. The rest of the orthorrhaphous mt genomes in our study have the similar structure with A. miser. Additionally, we conducted a phylogenetic analysis of 20 mt genomes using Maximum-likelihood and Bayesian methods in order to reconstruct the evolutionary relationship of Orthorrhapha. The results show that all infraorders of Brachycera are monophyletic, and a relationship of Tabanomorpha+((Xylophagomorpha+Stratiomyomorpha)+Muscomorpha) has been proposed. Within Xylophagomorpha, Nemestrinoidae forms the sister group of Xylophagidae.

The complete mitochondrial genome of a stonefly species, Kamimuria chungnanshana Wu, 1948 (Plecoptera: Perlidae).

This study determined the complete mitochondrial (mt) genome of the stonefly, Kamimuria chungnanshana Wu, 1948. The mt genome is 15, 943 bp in size and contains 37 canonical genes which include 22 transfer RNA genes, 13 protein-coding genes, and two ribosomal RNA genes, the control region is 1062 bp in length. The phylogenetic tree shows that Kamimuria chungnanshana is sister group of Kamimuria wangi.

The Phylogeny and Evolutionary Timescale of Muscoidea (Diptera: Brachycera: Calyptratae) Inferred from Mitochondrial Genomes.

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.

The first mitochondrial genome of the sepsid fly Nemopoda mamaevi Ozerov, 1997 (Diptera: Sciomyzoidea: Sepsidae), with mitochondrial genome phylogeny of cyclorrhapha.

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.