Convergence of dominance and neglect in flying insect diversity.

Most of arthropod biodiversity is unknown to science. Consequently, it has been unclear whether insect communities around the world are dominated by the same or different taxa. This question can be answered through standardized sampling of biodiversity followed by estimation of species diversity and community composition with DNA barcodes. Here this approach is applied to flying insects sampled by 39 Malaise traps placed in five biogeographic regions, eight countries and numerous habitats (>225,000 specimens belonging to >25,000 species in 458 families). We find that 20 insect families (10 belonging to Diptera) account for >50% of local species diversity regardless of clade age, continent, climatic region and habitat type. Consistent differences in family-level dominance explain two-thirds of variation in community composition despite massive levels of species turnover, with most species (>97%) in the top 20 families encountered at a single site only. Alarmingly, the same families that dominate insect diversity are 'dark taxa' in that they suffer from extreme taxonomic neglect, with little signs of increasing activities in recent years. Taxonomic neglect tends to increase with diversity and decrease with body size. Identifying and tackling the diversity of 'dark taxa' with scalable techniques emerge as urgent priorities in biodiversity science.

Network analysis with either Illumina or MinION reveals that detecting vertebrate species requires metabarcoding of iDNA from a diverse fly community.

DNA obtained from invertebrates (iDNA) can be metabarcoded in order to survey vertebrate communities. However, little attention has been paid to the interaction between the invertebrate and vertebrate species. Here, we tested for specialization by sampling the dung and carrion fly community of a swamp forest remnant along a disturbance gradient (10 sites: 80-310 m from a road). Approximately, 60% of the baited 407 flies yielded 294 vertebrate identifications based on two COI fragments and 16S. A bipartite network analysis found no statistically significant specialization in the interactions between fly and vertebrate species, but uncommon fly species can carry the signal for vertebrate species that are otherwise difficult to detect with iDNA. A spatial analysis revealed that most of the 20 vertebrate species reported in this study could be detected within 150 m of the road (18 spp.) and that the fly community sourced for iDNA was unexpectedly rich (24 species, 3 families). They carried DNA for rare and common species inhabiting different layers of the forest (ground-dwelling: wild boar, Sunda pangolin, skinks, rats; arboreal: long-tailed macaque, Raffles' banded langur; flying: pin-striped tit-babbler, olive-winged bulbul). All our results were obtained with a new, greatly simplified iDNA protocol that eliminates DNA extraction by obtaining template directly through dissolving fly faeces and regurgitates with water. Lastly, we show that MinION- and Illumina-based metabarcoding yield similar results. We conclude by urging more studies that use different baits and involve experiments that are capable of revealing the dispersal capabilities of the flies carrying the iDNA.

Monophyletic blowflies revealed by phylogenomics.

BACKGROUND: Blowflies are ubiquitous insects, often shiny and metallic, and the larvae of many species provide important ecosystem services (e.g., recycling carrion) and are used in forensics and debridement therapy. Yet, the taxon has repeatedly been recovered to be para- or polyphyletic, and the lack of a well-corroborated phylogeny has prevented a robust classification. RESULTS: We here resolve the relationships between the different blowfly subclades by including all recognized subfamilies in a phylogenomic analysis using 2221 single-copy nuclear protein-coding genes of Diptera. Maximum likelihood (ML), maximum parsimony (MP), and coalescent-based phylogeny reconstructions all support the same relationships for the full data set. Based on this backbone phylogeny, blowflies are redefined as the most inclusive monophylum within the superfamily Oestroidea not containing Mesembrinellidae, Mystacinobiidae, Oestridae, Polleniidae, Sarcophagidae, Tachinidae, and Ulurumyiidae. The constituent subfamilies are re-classified as Ameniinae (including the Helicoboscinae, syn. nov.), Bengaliinae, Calliphorinae (including Aphyssurinae, syn. nov., Melanomyinae, syn. nov., and Toxotarsinae, syn. nov.), Chrysomyinae, Luciliinae, Phumosiinae, Rhiniinae stat. rev., and Rhinophorinae stat. rev. Metallic coloration in the adult is shown to be widespread but does not emerge as the most likely ground plan feature. CONCLUSIONS: Our study provides the first phylogeny of oestroid calyptrates including all blowfly subfamilies. This allows settling a long-lasting controversy in Diptera by redefining blowflies as a well-supported monophylum, and blowfly classification is adjusted accordingly. The archetypical blowfly trait of carrion-feeding maggots most likely evolved twice, and the metallic color may not belong to the blowfly ground plan.

ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone.

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.

Beyond Drosophila: resolving the rapid radiation of schizophoran flies with phylotranscriptomics.

BACKGROUND: The most species-rich radiation of animal life in the 66 million years following the Cretaceous extinction event is that of schizophoran flies: a third of fly diversity including Drosophila fruit fly model organisms, house flies, forensic blow flies, agricultural pest flies, and many other well and poorly known true flies. Rapid diversification has hindered previous attempts to elucidate the phylogenetic relationships among major schizophoran clades. A robust phylogenetic hypothesis for the major lineages containing these 55,000 described species would be critical to understand the processes that contributed to the diversity of these flies. We use protein encoding sequence data from transcriptomes, including 3145 genes from 70 species, representing all superfamilies, to improve the resolution of this previously intractable phylogenetic challenge. RESULTS: Our results support a paraphyletic acalyptrate grade including a monophyletic Calyptratae and the monophyly of half of the acalyptrate superfamilies. The primary branching framework of Schizophora is well supported for the first time, revealing the primarily parasitic Pipunculidae and Sciomyzoidea stat. rev. as successive sister groups to the remaining Schizophora. Ephydroidea, Drosophila's superfamily, is the sister group of Calyptratae. Sphaeroceroidea has modest support as the sister to all non-sciomyzoid Schizophora. We define two novel lineages corroborated by morphological traits, the 'Modified Oviscapt Clade' containing Tephritoidea, Nerioidea, and other families, and the 'Cleft Pedicel Clade' containing Calyptratae, Ephydroidea, and other families. Support values remain low among a challenging subset of lineages, including Diopsidae. The placement of these families remained uncertain in both concatenated maximum likelihood and multispecies coalescent approaches. Rogue taxon removal was effective in increasing support values compared with strategies that maximise gene coverage or minimise missing data. CONCLUSIONS: Dividing most acalyptrate fly groups into four major lineages is supported consistently across analyses. Understanding the fundamental branching patterns of schizophoran flies provides a foundation for future comparative research on the genetics, ecology, and biocontrol.

Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing.

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 well-equipped, 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 specimen- and 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.

Soup to Tree: The Phylogeny of Beetles Inferred by Mitochondrial Metagenomics of a Bornean Rainforest Sample.

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.

Complete tribal sampling reveals basal split in Muscidae (Diptera), confirms saprophagy as ancestral feeding mode, and reveals an evolutionary correlation between instar numbers and carnivory.

With about 5000 species in ca. 180 genera, the Muscidae is the most species-rich family in the muscoid grade of Calyptratae (Diptera: Cyclorrhapha), the others being the Fanniidae, Scathophagidae and Anthomyiidae. Muscidae is remarkable for its young age, high species diversity in all biogeographic regions, and an unusually diverse range of feeding habits at the larval stage (e.g., saprophagy, phytophagy, carnivory, endoparasitism, haematophagy). We here review muscid classification and biology and present a molecular phylogeny based on four mitochondrial genes (12S, 16S, COI, CYTB) and three nuclear genes (28S, Ef1a, and CAD) for 84 species from 40 genera. Our analysis is the first to include species from all biogeographic regions and all currently recognised muscid subfamilies and tribes. We provide strong support for the monophyly of the Muscidae, and for the first time also for the first split within this family. The ancestral larval feeding habit is reconstructed to be saprophagy with more specialised coprophagous saprophagy, phytophagy, and carnivory evolving multiple times from saprophagous ancestors. The origins of carnivory in larvae are significantly correlated with a reduction of the number of larval instars from three (ancestral) to two and one. The genus Achanthiptera which was previously in its own subfamily is shown to be closely related to Azeliini. However, it appears that Azeliinae is paraphyletic because Muscinae is sister-group to the Azeliini while the azeliine Reinwardtiini are polyphyletic. Coenosiinae and Muscinae are monophyletic, but Muscini is paraphyletic with regard to Stomoxyini. Because many subfamilies are apparently para- or even polyphyletic, we review the history of muscid classification in order to reveal how the currently used classification originated.

A phylogenetic analysis of Sciomyzidae (Diptera) and some related genera.

Sciomyzidae is a family of acalyptrate flies with 546 species in 61 genera that is among the most extensively studied groups of higher Diptera. Most of the known larvae are obligate enemies of Gastropoda. Hundreds of studies published over the past 50 years have resulted in detailed information concerning morphology of adults and immature stages, biology, development, behaviour, phenology and distribution. However, studies of phylogenetic relationships are based almost exclusively on morphological characters of adults, and no comprehensive molecular analysis across the family has been published. Here we fill this void by generating and analysing molecular data for 54 species of Sciomyzidae (22 genera), including Phaeomyiidae (one genus), and seven representative species of five other families of Sciomyzoidea (Coelopidae, Dryomyzidae, Helcomyzidae, Heteromyzidae and Huttoninidae) as outgroups. The reconstruction is based on morphological characters as well as nucleotide sequences for genes from the mitochondrial (12S, 16S, COI, COII, Cytb) and nuclear genome (28S, EF1α). The results are compared with recent morphological analyses. Our analyses support the monophyly of Sciomyzidae + Phaeomyiidae, and place Phaeomyiinae as a unique lineage within Sciomyzidae. A modified classification comprising three subfamilies is proposed. The major subfamily, Sciomyzinae, consists of two monophyletic and well separated groups, the tribes Sciomyzini and Tetanocerini.

Episodic radiations in the fly tree of life.

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.

The Muscoidea (Diptera: Calyptratae) are paraphyletic: Evidence from four mitochondrial and four nuclear genes.

Approximately 5% of the known species-level diversity of Diptera belongs to the Muscoidea with its approximately 7000 described species. Despite including some of the most abundant and well known flies, the phylogenetic relationships within this superfamily are poorly understood. Previous attempts at reconstructing the relationships based on morphology and relatively small molecular data sets were only moderately successful. Here, we use molecular data for 127 exemplar species of the Muscoidea, two species from the Hippoboscoidea, ten species representing the Oestroidea and seven outgroup species from four acalyptrate superfamilies. Four mitochondrial genes 12S, 16S, COI, and Cytb, and four nuclear genes 18S, 28S, Ef1a, and CAD are used to reconstruct the relationships within the Muscoidea. The length-variable genes were aligned using a guide tree that was based on the protein-encoding genes and the indel-free sections of the ribosomal genes. We found that, based on topological considerations, this guide tree was a significant improvement over the default guide trees generated by ClustalX. The data matrix was analyzed using maximum parsimony (MP) and maximum likelihood (ML) and yielded very similar tree topologies. The Calyptratae are monophyletic and the Hippoboscoidea are the sister group to the remaining calyptrates (MP). The Muscoidea are paraphyletic with a monophyletic Oestroidea nested within the Muscoidea as sister group to Anthomyiidae+Scathophagidae. The monophyly of three of the four recognized families in the Muscoidea is confirmed: the Fanniidae, Muscidae, and Scathophagidae. However, the Anthomyiidae are possibly paraphyletic. Within the Oestroidea, the Sarcophagidae and Tachinidae are sister groups and the Calliphoridae are paraphyletic.

The phylogeny and evolution of host choice in the Hippoboscoidea (Diptera) as reconstructed using four molecular markers.

Hippoboscoidea is a superfamily of Diptera that contains the Glossinidae or tsetse flies, the Hippoboscidae or louse flies, and two families of bat flies, the Streblidae and the Nycteribiidae. We reconstruct the phylogenetic relationships within Hippoboscoidea using maximum parsimony and Bayesian methods based on nucleotide sequences from fragments of four genes: nuclear 28S ribosomal DNA and the CPSase domain of CAD, and mitochondrial 16S rDNA and cytochrome oxidase I. We recover monophyly for most of the presently recognized groups within Hippoboscoidea including the superfamily as a whole, the Hippoboscidae, the Nycteribiidae, the bat flies, and the Pupipara (=Hippoboscidae+Nycteribiidae+Streblidae), as well as several subfamilies within the constituent families. Streblidae appear to be paraphyletic. Our phylogenetic hypothesis is well supported and decisive in that most competing topological hypotheses for the Hippoboscoidea require significantly longer trees. We confirm a single shift from a free-living fly to a blood-feeding ectoparasite of vertebrates and demonstrate that at least two host shifts from mammals to birds have occurred. Wings have been repeatedly lost, but never regained. The hippoboscoid ancestor also evolved adenotrophic viviparity and our cladogram is consistent with a gradual reduction in the motility of the deposited final instar larvae from active burrowing in the soil to true pupiparity where adult females glue the puparium within the confines of bat roosts.

Sensitivity analysis, molecular systematics and natural history evolution of Scathophagidae (Diptera: Cyclorrhapha: Calyptratae).

The 60 000 described species of Cyclorrhapha are characterized by an unusual diversity in larval life-history traits, which range from saprophagy over phytophagy to parasitism and predation. However, the direction of evolutionary change between the different modes remains unclear. Here, we use the Scathophagidae (Diptera) for reconstructing the direction of change in this relatively small family (≈ 250 spp.) whose larval habits mirror the diversity in natural history found in Cyclorrhapha. We subjected a molecular data set for 63 species (22 genera) and DNA sequences from seven genes (12S, 16S, Cytb, COI, 28S, Ef1-alfa, Pol II) to an extensive sensitivity analysis and compare the performance of three different alignment strategies (manual, Clustal, POY). We find that the default Clustal alignment performs worst as judged by character incongruence, topological congruence and branch support. For this alignment, scoring indels as a fifth character state worsens character incongruence and topological congruence. However, manual alignment and direct optimization perform similarly well and yield near-identical trees, although branch support is lower for the direct-optimization trees. All three alignment techniques favor the upweighting of transversion. We furthermore confirm the independence of the concepts "node support" and "node stability" by documenting several cases of poorly supported nodes being very stable and cases of well supported nodes being unstable. We confirm the monophyly of the Scathophagidae, its two constituent subfamilies, and most genera. We demonstrate that phytophagy in the form of leaf mining is the ancestral larval feeding habit for Scathophagidae. From phytophagy, two shifts to saprophagy and one shift to predation has occurred while a second origin of predation is from a saprophagous ancestor.