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Muscidae are a megadiverse dipteran family that exhibits extraordinary diversity in morphology and life history as both immatures and adults. The classification of Muscidae has been long debated, and most higher-level relationships remain unknown. In this study, we used multilocus Sanger sequencing (mS-seq), anchored hybrid enrichment (AHE) and restriction-site associated DNA sequencing (RAD-seq) approaches to examine relationships within Muscidae. The results from AHE and RAD-seq largely correspond to those obtained from mS-seq in terms of overall topology, yet phylogenomic approaches received much higher nodal support. The results from all molecular approaches contradict the traditional classification based predominantly on adult morphology, but provide an opportunity to re-interpret the morphology of immature stages. Rearrangements in Muscidae classification are proposed as follows: (i) Mesembrina Meigen and Polietes Rondani are transferred from Muscinae to Azeliinae; (ii) Reinwardtiinae stat. rev. is resurrected as a subfamily distinct from Azeliinae; (iii) Eginia Robineau-Desvoidy, Neohelina Malloch, Syngamoptera Schnabl and Xenotachina Malloch are transferred to Reinwardtiinae stat. rev.

Sarcophagidae is one of the most species-rich families within the superfamily Oestroidea. This diversity is usually represented by three lineages: Miltogramminae, Paramacronychiinae and Sarcophaginae. Historically, the phylogenetic relationships among these lineages have been elusive, due to poorly supported hypotheses or small taxon sets, or both. This study provides a dramatic increase in molecular data, more balanced sampling of all three lineages from all biogeographical regions and a reassessment of morphological characters using scanning electron microscopy in the most comprehensive assessment of subfamily-level phylogeny in Sarcophagidae to date. This analysis of the largest molecular dataset ever produced for a phylogenetic analysis of a fly lineage, with 950 loci from anchored hybrid enrichment comprising 435 930 bp from 101 species, revealed Paramacronychiinae as sister to Miltogramminae, not to Sarcophaginae, as suggested by adult morphology. Maximum likelihood analysis produced a well-supported topology, with 91% of the nodes receiving strong bootstrap proportions (> 97%). In contrast to the molecular data, three out of nine morphological characters studied point to a sister-group relationship of (Sarcophaginae + Paramacronychiinae) and the remaining six characters are either silent on subfamily relationships or in need of further study. Re-examination of morphological structures provides new insights into the evolution of male genitalic traits within Sarcophagidae and highlights their convergence producing conflicting phylogenetic signal. Our phylogeny reconciles older and widely used systems of classification with tree-based thinking and sets up a classification of flesh flies that is more aligned with their evolutionary history.

The therevoid clade represents a group of four families (Apsilocephalidae, Evocoidae, Scenopinidae and Therevidae) of lower brachyceran Diptera in the superfamily Asiloidea. The largest of these families is that of the stiletto flies (Therevidae). A large-scale (i.e. supermatrix) phylogeny of Therevidae is presented based on DNA sequence data from seven genetic loci (16S, 18S and 28S ribosomal DNA and four protein-encoding genes: elongation factor 1-alpha, triose phosphate isomerase, short-wavelength rhodopsin and the CPSase region of carbamoyl-phosphate synthase-aspartate transcarbamoylase-dihydroorotase). Results are presented from Bayesian phylogenetic analyses of approximately 8.7 kb of sequence data for 204 taxa representing all subfamilies and genus groups of Therevidae. Our results strongly support the sister-group relationship between Therevidae and Scenopinidae, with Apsilocephalidae as sister to Evocoidae. Previous estimates of stiletto fly phylogeny based on morphology or DNA sequence data, or supertree analysis, have failed to find significant support for relationships among subfamilies. We report for the first time strong support for the placement of the subfamily Phycinae as sister to the remaining Therevidae, originating during the Mid Cretaceous. As in previous studies, the sister-group relationship between the species-rich subfamilies Agapophytinae and Therevinae is strongly supported. Agapophytinae are recovered as monophyletic, inclusive of the Taenogera group. Therevinae comprise the bulk of the species richness in the family and appear to be a relatively recent and rapid radiation originating in the southern hemisphere (Australia + Antarctica + South America) during the Late Cretaceous. Genus groups are defined for all subfamilies based on these results.

Horse flies, family Tabanidae, are the most diverse family-level clade of bloodsucking insects, but their phylogeny has never been thoroughly explored using molecular data. Most adult female Tabanidae feed on nectar and on the blood of various mammals. Traditional horse fly classification tends towards large heterogeneous taxa, which impede much-needed taxonomic work. To guide renewed efforts in the systematics of horse flies and their relatives, we assembled a dataset of 110 exemplar species using nucleotide data from four genes—mitochondrial CO1, and nuclear 28S, CAD and AATS. All commonly recognized tribes in Tabanidae are represented, along with outgroups in Tabanomorpha. The phylogeny is reconstructed using Bayesian inference, and divergence times are estimated using Bayesian relaxed clock methods with time constraints from tabanid fossils. Our results show Athericidae strongly supported as the lineage most closely related to Tabanidae, and Pangoniinae and Tabaninae as monophyletic lineages. However, Chrysopsinae is nonmonophyletic, with strong support for both a nonmonophyletic Bouvieromyiini and for Rhinomyzini as sister to Tabaninae. Only the tribes Philolichini, Chrysopsini, Rhinomyzini and Haematopotini are recovered as monophyletic, although Scionini is monophyletic with exclusion of the peculiar genus Goniops Aldrich. Mycteromyia Philippi and Adersia Austen, two enigmatic genera sometimes placed in separate family-level groups, are recovered inside Pangoniini and Chrysopsini, respectively. Several species-rich genera are not recovered as monophyletic, including Esenbeckia Rondani, Silvius Meigen, Dasybasis Macquart and Tabanus L. Tabanidae likely originated in the Cretaceous, and all major extant groups were present by the early Palaeogene. This newly revised phylogenetic framework for Tabanidae forms the basis for a new assessment of tabanid diversification and provides context for understanding the evolution of trophic specialization in horse flies.

Members of the megadiverse insect order Diptera (flies) have successfully colonized all continents and nearly all habitats. There are more than 154 000 described fly species, representing 10-12% of animal species. Elucidating the phylogenetic relationships of such a large component of global biodiversity is challenging, but significant advances have been made in the last few decades. Since Hennig first discussed the monophyly of major groupings, Diptera has attracted much study, but most researchers have used non-numerical qualitative methods to assess morphological data. More recently, quantitative phylogenetic methods have been used on both morphological and molecular data. All previous quantitative morphological studies addressed narrower phylogenetic problems, often below the suborder or infraorder level. Here we present the first numerical analysis of phylogenetic relationships of the entire order using a comprehensive morphological character matrix. We scored 371 external and internal morphological characters from larvae, pupae and adults for 42 species, representing all infraorders selected from 42 families. Almost all characters were obtained from previous studies but required revision for this ordinal- level study, with homology assessed beyond their original formulation and across all infraorders. We found significant support for many major clades (including the Diptera, Culicomorpha, Bibionomorpha, Brachycera, Eremoneura, Cyclorrhapha, Schizophora, Calyptratae and Oestroidea) and we summarize the character evidence for these groups. We found low levels of support for relationships between the infraorders of lower Diptera, lower Brachycera and major lineages of lower Cyclorrhapha, and this is consistent with findings from molecular studies. These poorly supported areas of the tree may be due to periods of rapid radiation that left few synapomorphies in surviving lineages.

The dipteran clade Calyptratae is comprised of approximately 18 000 described species (12% of the known dipteran diversity) and includes well-known taxa such as houseflies, tsetse flies, blowflies and botflies, which have a close association with humans. However, the phylogenetic relationships within this insect radiation are very poorly understood and controversial. Here we propose a higher-level phylogenetic hypothesis for the Calyptratae based on an extensive DNA sequence dataset for 11 noncalyptrate outgroups and 247 calyptrate species representing all commonly accepted families in the Oestroidea and Hippoboscoidea, as well as those of the muscoid grade. DNA sequences for genes in the mitochondrial (12S, 16S, cytochrome c oxidase subunit I and cytochrome b) and nuclear genome [18S, 28S, the carbamoyl phosphate synthetase region of CAD (rudimentary), Elongation factor one alpha] were used to reconstruct the relationships. We discuss problems relating to the alignment and analysis of large datasets and emphasize the advantages of utilizing a guide tree-based approach for the alignment of the DNA sequences and using the leaf stability index to identify ‘wildcard’ taxa whose excessive instability obscures the phylogenetic signal. Our analyses support the monophyly of the Calyptratae and demonstrate that the superfamily Oestroidea is nested within the muscoid grade. We confirm that the monotypic family Mystacinobiidae is an oestroid and further revise the composition of the Oestroidea by demonstrating that the previously unplaced and still undescribed ‘McAlpine’s fly’ is nested within this superfamily as a probable sister group to Mystacinobiidae. Within the Oestroidea we confirm with molecular data that the Calliphoridae are a paraphyletic grade of lineages. The families Sarcophagidae and Rhiniidae are monophyletic, but support for the monophyly of Tachinidae and Rhinophoridae depends on analytical technique (e.g. parsimony or maximum likelihood). The superfamilies Hippoboscoidea and Oestroidea are consistently found to be monophyletic, and the paraphyly of the muscoid grade is confirmed. In the overall relationships for the calyptrates, the Hippoboscoidea are sister group to the remaining Calyptratae, and the Fanniidae are sister group to the nonhippoboscoid calyptrates, whose relationships can be summarized as (Muscidae (Oestroidea (Scathophagidae, Anthomyiidae))).

Tipuloidea, the crane flies, are a diverse lineage of true flies (Insecta: Diptera) whose phylogenetic classification and taxonomy remain a challenge. Here we present the results of a quantitative phylogenetic analysis of Tipuloidea based on combined morphological characters (adult, larvae and pupae) and nuclear gene sequence data (28S rDNA and CAD). Forty-five species, from 44 genera and subgenera, were sampled, representing the four putative families of Tipuloidea (Cylindrotomidae, Limoniidae, Pediciidae and Tipulidae sensu stricto). Analyses of individual datasets, although differing in overall topology, support the monophyly of several major lineages within Tipuloidea. Parsimony and Bayesian analyses using individual morphological and molecular datasets resulted in incongruent topologies. Increased resolution and tree support was obtained when both datasets (morphology and genes) were combined, in both combined evidence parsimony and Bayesian analyses, than when analysed separately. The recovered consensus phylogeny was not consistent with any previously proposed Tipuloidea classification, with previous importance assigned to character states shown here to represent losses and reversals seen as a major factor influencing erroneous classification. The results provided here, together with evidence from previous analyses, were used to append the Tipuloidea classification to supported evolutionary lineages. Tipuloidea is presented as two families: Pediciidae and Tipulidae. Pediciidae is recovered as the sister group to all remaining Tipuloidea. Our current phylogenetic hypothesis is not consistent with the existing subfamilial classification of the ‘Limoniidae’, which is paraphyletic with respect to a well-supported Tipulinae + Cylindrotominae clade, whereas the three ‘limoniid’ subfamilies are para- or polyphyletic. The recognition of ‘Limoniidae’ as a valid monophyletic family is discouraged and the subfamilies of ‘Limoniidae’ are amended and placed within Tipulidae. A revised phylogenetic classification is proposed for the crane flies based on a synthesis of evidence from multiple genes and morphology.

Neuropterida comprise the holometabolan orders Neuroptera (lacewings, antlions and relatives), Megaloptera (alderflies, dobsonflies) and Raphidioptera (snakeflies) as a monophyletic group sister to Coleoptera (beetles). The higher-level phylogenetic relationships among these groups, as well as the family-level hierarchy of Neuroptera, have to date proved difficult to reconstruct. We used morphological data and multi-locus DNA sequence data to infer Neuropterida relationships. Nucleotide sequences were obtained for fragments of two nuclear genes (CAD, 18S rDNA) and two mitochondrial genes (COI, 16S rDNA) for 69 exemplars representing all recently recognized families of Neuropterida as well as outgroup exemplars from Coleoptera. The joint posterior probability of phylogeny and divergence times was estimated using a Bayesian relaxed-clock inference method to establish a temporal sequence of cladogenesis for the group over geological time. Megaloptera were found to be paraphyletic with respect to the rest of Neuropterida, calling into question the validity of the ordinal status for Megaloptera as presently defined. Ordinal relationships were weakly supported, and monophyly of Megaloptera was not recovered in any total-evidence analysis; Corydalidae were frequently recovered as sister to Raphidioptera. Only in relaxed-clock inferences were Raphidioptera and a paraphyletic Megaloptera recovered with strong support as a monophyletic group sister to Neuroptera. A monophyletic Neuroptera diverged from a common Raphidioptera + ‘Megaloptera’ ancestor during the Late Carboniferous. Contrary to some previous hypotheses, Coniopterygidae, not Nevrorthidae, were recovered as sister to the rest of Neuroptera, with Nevrorthidae recovered with Osmylidae and Sisyridae. The monophyly of the universally recognized Myrmeleontiformia was confirmed, with an origin in the mid-Triassic, but a monophyletic Hemerobiiformia was not recovered in any analysis. Dilaridae were not closely related to the clade comprising Mantispidae and Berothidae, and diverged earlier than proposed previously. The phylogenetic status and taxonomic composition of Polystoechotidae and Ithonidae are in need of re-evaluation, as Oliarces Carpenter (presently Ithonidae) was placed well within the present circumscription of Polystoechotidae

Relationships among families of the lower Diptera (formerly suborder 'Nematocera') have been exceptionally difficult to resolve. Multiple hypotheses based on morphology have been proposed to identify the earliest lineages of flies and place the phylogenetic origin of the higher flies (Brachycera), but convincing support is limited. Here we resolve relationships among the major groups of lower Diptera using sequence data from four nuclear markers, including both ribosomal (28S rDNA) and protein-coding (CAD, TPI and PGD) genes. Our results support both novel and traditional arrangements. Most unexpectedly, the small, highly-specialized family Deuterophlebiidae appears to be sister to all remaining Diptera. Other results include the resolution of the traditional infra-orders Culicomorpha (including a novel superfamily Simulioidea ¼ Thaumaleidae þ Simuliidae), Tipulomorpha (Tipulidae sensu lato þ Trichoceridae) and Bibionomorpha sensu lato. We find support for a limited Psychodomorpha (Blephariceridae, Tanyderidae and Psycho- didae) and Ptychopteromorpha (Ptychopteridae), whereas the placement of several enigmatic families (Nymphomyiidae, Axymyiidae and Perissommatidae) remains ambiguous. According to genetic data, the infra-order Bibionomorpha is sister to the Brachycera. Much of the phylogenetic signal for major lineages was found in the 28S rDNA gene, whereas protein-coding genes performed variably at different levels. In addition to elucidating relationships, we also estimate the age of major lower dipteran clades, based on molecular divergence time estimates using relaxed-clock Bayesian methods and fossil calibration points.

Recent independent phylogenetic analyses of membracid relationships based on molecular and morphological data have identified monophyletic lineages within the family. However, the results of these studies have not fully resolved treehopper phylogeny, and relationships among some higher membracid lineages remain in doubt. Portions of three datasets (958 aligned nucleotides from elongation factor-1α, 2363 aligned nucleotides from 28S ribosomal DNA, and eighty-three morphological features of adults and nymphs) introduced in recent studies were reanalysed separately and in combination with two new molecular datasets (321 aligned nucleotides from wingless and 1829 aligned nucleotides from 18S ribosomal DNA). The results of the combined data analyses, contrary to previous analyses of morphological data alone, grouped membracids into two well-supported lineages, one comprising Stegaspidinae and Centrotinae, the other comprising Membracinae, Darninae and Smiliinae. The analyses recovered Centrotinae, Membracinae and Darninae as monophyletic groups, but Stegaspidinae was paraphyletic with respect to Centrotinae, and Smiliinae was polyphyletic with Micrutalini placed as a sister group to the clade comprising Membracinae, Darninae and Smiliinae. These results are consistent with the following hypotheses, proposed previously based on an analysis of morphological data: (1) the posterior pronotal process was derived and lost multiple times during the evolution of Membracidae; (2) Membracidae originated in the New World and reached the Old World subsequently via dispersal; (3) maternal care evolved independently multiple times and may or may not have been preceded by the acquisition of ant mutualism.

The muscid genus Thricops Rondani comprises forty-four species and two subspecies restricted to the northern hemisphere. A species-level phylogenetic analysis of Thricops was conducted using forty-four morphological characters, 426 bp of the nuclear gene white and 523 bp spanning the 5′ end of the cytochrome c oxidase subunit I (COI), the tRNA leucine gene (L2 region) and the 3′ end of the cytochrome c oxidase subunit II (COII). Thirty-nine species and two subspecies of Thricops were included in the analysis. Two species of Azelia Robineau-Desvoidy and one species of Hydrotaea Robineau-Desvoidy were used as outgroups. Morphological characters were coded for all included species, the mitochondrial gene fragment (COI + II) was sequenced for a subset of seventeen species of Thricops and three outgroup species, and white for twelve of those seventeen Thricops species and two outgroup species. Six separate maximum parsimony analyses were performed on three taxon sets of different sizes (n = 14, n = 20, n = 44). Results from the partition homogeneity test indicated no significant incongruence between data partitions, and four combined maximum parsimony analyses were conducted (DNA + morphology for n = 14; COI + II + morphology for n = 20; DNA + morphology for n = 20; DNA + morphology for n = 44). The relative contribution of each data partition to individual nodes was assessed using partitioned Bremer support. Strict consensus trees resulting from the unweighted analyses of each dataset are presented. Combination of datasets increased resolution for the small taxon set (n = 14), but not for the larger ones (n = 20, n = 44), most probably due to increasing amounts of missing data in the larger taxon sets. Results from both individual and combined analyses of the smaller taxon sets (n = 14, n = 20) provided support for the monophyly of Thricops and a complete division of the genus into two monophyletic subgroups. The strict consensus cladograms resulting from the analysis of the morphological data alone and the combined data for the large taxa set (n = 44) both supported the monophyly of the genus, but placed the species Thricops foveolatus (Zetterstedt) and Thricops bukowskii (Ringdahl) at the base of the ingroup, in a polytomy with a relatively well-resolved branch comprising all remaining species of the genus. The basal position of these two species, included in the morphological taxon set but absent in the others, illustrates the potential pitfalls of taxon sampling and missing data in phylogenetic analyses. The synonymy of Alloeostylus with Thricops as proposed by previous authors was supported by our results. Relative contributions of different data partitions is discussed, with the mitochondrial sequence generally providing finer resolution and better branch support than white.

The monotypic new family Ocoidae is described to accommodate Ocoa gen.n., small, elongate, delicate, asiloid flies known from west-central Chile, South America. Both sexes of adult O. chilensis sp.n. are described and illustrated; immature stages are unknown. Diagnostic morphological features of adults include the antennal postpedicel comprising a single, undivided structure consisting of a bulbous base and a long, threadlike terminal element; vein M3 lacking, anal vein complete; acropod heterodactylus; spurs lacking on all tibiae; and anterior surface of hind coxa with strong, knoblike, bulbous projection; and abdominal tergite 2 lacking sensory patches of hairs. The epandrium of the male terminalia is divided along the midline into two sicklelike sections; gonostyli articulate in a horizontal plane. The female terminalia has well-developed acanthophorite spines; posterior margin of hypoproct with several ventrally projecting, elongate, needlelike setae; two large, poorly sclerotized spermathecae; and a spermathecal sac that is smaller than each spermatheca. The relationships of this enigmatic family are discussed in relation to recent findings on the phylogeny and classification of the Asiloidea. Ocoidae are similar to Therevidae and close relatives Scenopinidae and Apsilocephalidae, comprising the therevoid group of families; however, they lack defining synapomorphies of those families. Morphological evidence supports a sister-group relationship between Ocoidae and Scenopinidae. Molecular evidence from 28S rDNA provides further support for the monophyly of the therevoid group of families, and suggests that Ocoidae belongs within this clade, in agreement with the morphological data. The nucleotide data place Ocoidae as sister to the Scenopinidae + Therevidae, but the morphology-based hypothesis of relationships (Ocoidae + Scenopinidae) is only two additional steps (0.2%) on the optimal trees from the molecular data.

Agapophytinae subf.n. is a highly diverse lineage of Australasian Therevidae, comprising eight described and two new genera: Agapophytus Guerin-Meneville, Acupalpa Krober, Acraspisa Krober, Belonalys Krober, Bonjeania Irwin & Lyneborg, Parapsilocephala Krober, Acatopygia Krober, Laxotela Winterton & Irwin, Pipinnipons gen.n. and Patanothrix gen.n. A genus-level cladistic analysis of the subfamily was undertaken using sixty-eight adult morphological characters and c. 1000 base pairs of the elongation factor-1 alpha (EF-1 alpha) protein coding gene. The morphological data partition produced three most parsimonious cladograms, whereas the molecular data partition gave a single most parsimonious cladogram, which did not match any of the cladograms found in the morphological analysis. The level of congruence between the data partitions was determined using the partition homogeneity test (HTF) and Wilcoxon signed ranks rest. Despite being significantly incongruent in at least one of the incongruence tests, the partitions were combined in a simultaneous analysis. The combined data yielded a single cladogram that was better supported than that of the individual partitions analysed separately. The relative contributions of the data partitions to support for individual nodes on the combined cladogram were investigated using Partitioned Bremer Support. The level of support for many nodes on the combined cladogram was non-additive and often greater than the sum of support for the respective nodes on individual partitions. This synergistic interaction between incongruent data partitions indicates a common phylogenetic signal in both partitions. It also suggests that criteria for partition combination based solely on incongruence may be misleading. The phylogenetic relationships of the genera are discussed using the combined data. A key to genera of Agapophytinae is presented, with genera diagnosed and figured. Two new genera are described: Patanothrix with a new species (Pat. skevingtoni) and Pat. wilsoni (Mann) transferred from Parapsilocephala, and Pipinnipons with a new species (Pip. kroeberi). Pipinnipons fascipennis (Krober) is transferred from Squamopygin Krober and Pip. imitans (Mann) is transferred from Agapophytus. Agapophytus bicolor (Krober) is transferred from Parapsilocephala. Agapophytus varipennis Mann is synonymised with Aga, queenslandi Krober and Aga. flavicornis Mann is synonymised with Aga. pallidicornis (Krober).