Flies are one of four superradiations of insects (along with beetles, wasps, and moths) that account for the majority of animal life on Earth. Diptera includes species known for their ubiquity (Musca domestica house fly), their role as pests (Anopheles gambiae malaria mosquito), and their value as model organisms across the biological sciences (Drosophila melanogaster). A resolved phylogeny for flies provides a framework for genomic, developmental, and evolutionary studies by facilitating comparisons across model organisms, yet recent research has suggested that fly relationships have been obscured by multiple episodes of rapid diversification. We provide a phylogenomic estimate of fly relationships based on molecules and morphology from 149 of 157 families, including 30 kb from 14 nuclear loci and complete mitochondrial genomes combined with 371 morphological characters. Multiple analyses show support for traditional groups (Brachycera, Cyclorrhapha, and Schizophora) and corroborate contentious findings, such as the anomalous Deuterophlebiidae as the sister group to all remaining Diptera. Our findings reveal that the closest relatives of the Drosophilidae are highly modified parasites (including the wingless Braulidae) of bees and other insects. Furthermore, we use micro-RNAs to resolve a node with implications for the evolution of embryonic development in Diptera. We demonstrate that flies experienced three episodes of rapid radiation-lower Diptera (220 Ma), lower Brachycera (180 Ma), and Schizophora (65 Ma)-and a number of life history transitions to hematophagy, phytophagy, and parasitism in the history of fly evolution over 260 million y.T he history of life is often portrayed as an ongoing series of evolutionary bursts, with each representing the origin and diversification of unique life forms with different and ecologically significant adaptations. Although the radiations of some groups, such as cichlid fishes of the lakes of East Africa or Darwin's finches, are well documented (1), the big radiations that account for most of the diversity of life on Earth have been more challenging to explore. To understand these radiations, we must resolve the relationships among major taxa, date the origin of these lineages (many of them ancient), and then explicitly consider whether the diversification events are really pulse-like adaptive radiations or, more simply, the result of nonadaptive, or even random, neutral processes.Although the paradigm of adaptive radiation has been applied to every level of biological classification, the large-scale macroevolutionary pattern expected from ancient repeated episodes of adaptive radiation is unclear. It has been predicted that at this scale, ecologically driven diversification may result in (i) significant variation in clade size, uncorrelated to the age of the clade (2), and (ii) shifts in average diversification rate coincident with major shifts in morphology, life history, or ecology (3). Another macroevolutionary prediction of repeated adaptive radiation is the widespre...

Abstract. 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 treebased 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))).

Background: More than 80% of all animal species remain unknown to science. Most of these species live in the tropics and belong to animal taxa that combine small body size with high specimen abundance and large species richness. For such clades, using morphology for species discovery is slow because large numbers of specimens must be sorted based on detailed microscopic investigations. Fortunately, species discovery could be greatly accelerated if DNA sequences could be used for sorting specimens to species. Morphological verification of such "molecular operational taxonomic units" (mOTUs) could then be based on dissection of a small subset of specimens. However, this approach requires cost-effective and low-tech DNA barcoding techniques because wellequipped, well-funded molecular laboratories are not readily available in many biodiverse countries. Results: We here document how MinION sequencing can be used for large-scale species discovery in a specimenand species-rich taxon like the hyperdiverse fly family Phoridae (Diptera). We sequenced 7059 specimens collected in a single Malaise trap in Kibale National Park, Uganda, over the short period of 8 weeks. We discovered > 650 species which exceeds the number of phorid species currently described for the entire Afrotropical region. The barcodes were obtained using an improved low-cost MinION pipeline that increased the barcoding capacity sevenfold from 500 to 3500 barcodes per flowcell. This was achieved by adopting 1D sequencing, resequencing weak amplicons on a used flowcell, and improving demultiplexing. Comparison with Illumina data revealed that the MinION barcodes were very accurate (99.99% accuracy, 0.46% Ns) and thus yielded very similar species units (match ratio 0.991). Morphological examination of 100 mOTUs also confirmed good congruence with morphology (93% of mOTUs; > 99% of specimens) and revealed that 90% of the putative species belong to the neglected, megadiverse genus Megaselia. We demonstrate for one Megaselia species how the molecular data can guide the description of a new species (Megaselia sepsioides sp. nov.). Conclusions: We document that one field site in Africa can be home to an estimated 1000 species of phorids and speculate that the Afrotropical diversity could exceed 200,000 species. We furthermore conclude that low-cost MinION sequencers are very suitable for reliable, rapid, and large-scale species discovery in hyperdiverse taxa. MinION sequencing could quickly reveal the extent of the unknown diversity and is especially suitable for biodiverse countries with limited access to capital-intensive sequencing facilities.

In spite of the growth of molecular ecology, systematics and next-generation sequencing, the discovery and analysis of diversity is not currently integrated with building the tree-of-life. Tropical arthropod ecologists are well placed to accelerate this process if all specimens obtained through mass-trapping, many of which will be new species, could be incorporated routinely into phylogeny reconstruction. Here we test a shotgun sequencing approach, whereby mitochondrial genomes are assembled from complex ecological mixtures through mitochondrial metagenomics, and demonstrate how the approach overcomes many of the taxonomic impediments to the study of biodiversity. DNA from approximately 500 beetle specimens, originating from a single rainforest canopy fogging sample from Borneo, was pooled and shotgun sequenced, followed by de novo assembly of complete and partial mitogenomes for 175 species. The phylogenetic tree obtained from this local sample was highly similar to that from existing mitogenomes selected for global coverage of major lineages of Coleoptera. When all sequences were combined only minor topological changes were induced against this reference set, indicating an increasingly stable estimate of coleopteran phylogeny, while the ecological sample expanded the tip-level representation of several lineages. Robust trees generated from ecological samples now enable an evolutionary framework for ecology. Meanwhile, the inclusion of uncharacterized samples in the tree-of-life rapidly expands taxon and biogeographic representation of lineages without morphological identification. Mitogenomes from shotgun sequencing of unsorted environmental samples and their associated metadata, placed robustly into the phylogenetic tree, constitute novel DNA “superbarcodes” for testing hypotheses regarding global patterns of diversity.

Background DNA barcodes are a useful tool for discovering, understanding, and monitoring biodiversity which are critical tasks at a time of rapid biodiversity loss. However, widespread adoption of barcodes requires cost-effective and simple barcoding methods. We here present a workflow that satisfies these conditions. It was developed via “innovation through subtraction” and thus requires minimal lab equipment, can be learned within days, reduces the barcode sequencing cost to < 10 cents, and allows fast turnaround from specimen to sequence by using the portable MinION sequencer. Results We describe how tagged amplicons can be obtained and sequenced with the real-time MinION sequencer in many settings (field stations, biodiversity labs, citizen science labs, schools). We also provide amplicon coverage recommendations that are based on several runs of the latest generation of MinION flow cells (“R10.3”) which suggest that each run can generate barcodes for > 10,000 specimens. Next, we present a novel software, ONTbarcoder, which overcomes the bioinformatics challenges posed by MinION reads. The software is compatible with Windows 10, Macintosh, and Linux, has a graphical user interface (GUI), and can generate thousands of barcodes on a standard laptop within hours based on only two input files (FASTQ, demultiplexing file). We document that MinION barcodes are virtually identical to Sanger and Illumina barcodes for the same specimens (> 99.99%) and provide evidence that MinION flow cells and reads have improved rapidly since 2018. Conclusions We propose that barcoding with MinION is the way forward for government agencies, universities, museums, and schools because it combines low consumable and capital cost with scalability. Small projects can use the flow cell dongle (“Flongle”) while large projects can rely on MinION flow cells that can be stopped and re-used after collecting sufficient data for a given project.