Fossil insect eyes shed light on trilobite optics and the arthropod pigment screen.

Fossilized eyes permit inferences of the visual capacity of extinct arthropods1-3. However, structural and/or chemical modifications as a result of taphonomic and diagenetic processes can alter the original features, thereby necessitating comparisons with modern species. Here we report the detailed molecular composition and microanatomy of the eyes of 54-million-year-old crane-flies, which together provide a proxy for the interpretation of optical systems in some other ancient arthropods. These well-preserved visual organs comprise calcified corneal lenses that are separated by intervening spaces containing eumelanin pigment. We also show that eumelanin is present in the facet walls of living crane-flies, in which it forms the outermost ommatidial pigment shield in compound eyes incorporating a chitinous cornea. To our knowledge, this is the first record of melanic screening pigments in arthropods, and reveals a fossilization mode in insect eyes that involves a decay-resistant biochrome coupled with early diagenetic mineralization of the ommatidial lenses. The demonstrable secondary calcification of lens cuticle that was initially chitinous has implications for the proposed calcitic corneas of trilobites, which we posit are artefacts of preservation rather than a product of in vivo biomineralization4-7. Although trilobite eyes might have been partly mineralized for mechanical strength, a (more likely) organic composition would have enhanced function via gradient-index optics and increased control of lens shape.

Discovery of a novel ejaculation mechanism in danceflies (Diptera: Empididae), with implications for genital elongation.

Genitalia are known to evolve rapidly and are among the most variable structures in insect morphology, making them a target of active research. However, function and evolutionary significance of internal genital structures remain less well understood. Here, we report the morphology and mechanism of a novel ejaculatory system that has evolved in the dancefly genus Rhamphomyia (Insecta: Diptera: Empididae). Using synchrotron µCT technology, we examined the male genitalia of five dancefly species and identified an ejaculatory system resembling a leverage hydraulic jack, which is thought to have derived from a plunger-like pumping system. This jacking system amplifies the applied muscle power by up to 4.2 times, allowing the system to produce the same pumping power with much smaller muscles. However, the volume of the pumping muscle in the jacking system is comparable to that of the plunger system, indicating a significant increase in ejaculation power in this genus. We hypothesize that the greater pumping power evolved through sexual selection favoring strong ejaculation to rapidly pass semen through a thin and elongated phallus and spermathecal duct.

Carrion insects living within the bones of large mammals: insect conservation and forensic entomology implications.

Succession patterns of carrion insects on large mammal's carrion has been widely studied, notably to estimate the post-mortem interval in forensic investigations as accurately as possible. However, little attention has been paid to the carrion insects living inside these bones once a carcass is skeletonized. One very recent study documented flies emerging from pig carcasses, and only scarce authors reported the presence of other carrion insects taking advantage of the bone marrow. We, thus, aimed to (1) estimate the frequency of inner-bone space colonization by carrion insects, with particular attention to bone-skipper flies; (2) identify the insects living inside the carrion bones; and (3) determine whether or not carrion insects found within the bones can successfully exit the bones and complete their development. We extensively sampled 185 large mammals' bones collected from twelve vulture feeding stations and four isolated carcasses in southwest France and northern Spain. Sampled bones were opened, and the insects found inside were identified. For two bones, foramen, i.e., the holes providing a natural entrance and exit to the bone's inner cavity, was monitored with a camera to assess the insect's putative exit. We describe the entomofauna, i.e., the set of insect species, living within the bones, and illustrate insects' ability to exit the bones for their subsequent development and maturity. These results are discussed in the framework of carrion insect conservation and forensic entomology perspectives.

Revision of the Brazilian species of Ptilodexia Brauer & Bergenstamm, 1889 (Diptera: Tachinidae: Dexiinae), with the description of a new species.

The Brazilian species of the New World genus Ptilodexia Brauer & Bergenstamm, 1889 are revised. Before this study, only one species of Ptilodexia was recorded from Brazil, viz. P. lateralis (Walker, 1836). Herein we record, for the first time, two new records of known Ptilodexia species in Brazil, viz. P. striata (Wulp, 1891) and P. rubricornis (Wulp, 1891). In addition, a new species is described, Ptilodexia matogrossensis sp. nov. from Mato Grosso and Mato Grosso do Sul states in Brazil; hence four species of this genus are currently recorded from Brazil. The species Neomyostoma ptilodexioides Townsend, 1935, from Brazil, placed in the monotypic genus Neomyostoma Townsend, 1935, is proposed as junior synonym of P. lateralis. Illustrations and detailed descriptions are presented for P. lateralis, P. matogrossensis sp. nov., P. striata and P. rubricornis and the male terminalia is described for P. lateralis and P. striata. The female terminalia and the first larval instar are described for the first time for the genus, based on the descriptions of P. lateralis and P. striata. A key to the identification of all recognized Brazilian species of Ptilodexia is presented. Finally, an updated distributional record is given for all studied species.

Phylogenomics resolves long-standing questions about the affinities of an endangered Corsican endemic fly.

Recent studies on oestroidean Diptera (Brachycera) are providing a comprehensive and nuanced understanding of the evolutionary history of this remarkably diverse clade of holometabolous insects. The Oestroidea, which includes formidable pests such as various blowflies, botflies, and flesh flies that infest livestock, pets and humans, are mostly composed of beneficial species that act as scavengers or parasitoids on various pest insects. In our research, we used genomic methods to elucidate the phylogenetic position of Nesodexia corsicana Villeneuve, 1911 (Diptera: Calliphoridae), a mysterious oestroid species endemic to Corsica and characterized by distinctive morphological features that have puzzled taxonomists for years. Contrary to initial hypotheses, our results place Nesodexia Villeneuve, 1911 within the Calliphoridae subfamily Rhinophorinae, a small lineage of terrestrial isopod parasitoids. Through detailed morphological analysis of adults of both sexes and eggs, we uncovered significant insights consistent with our phylogenomic reconstruction. The unique morphological features of the species, coupled with its restricted and fragmented habitat, highlight its potential conservation importance. We delineated the area of occupancy for N. corsicana and assessed its "threatened" category using specific IUCN Red List criteria. In addition, we mapped the available habitat within its range and determined potential key biodiversity areas (KBA) triggered by N. corsicana. New potential KBAs are only partially covered by the Corsican Regional Park. Finally, we mapped the distribution of habitats on the island to assess the potential distribution of the species beyond its currently known geographic range.

A window on remarkable cryptic diversity of the Merodon planifacies subgroup (Diptera: Syrphidae) in the Afrotropical Region.

The genus Merodon Meigen (Diptera: Syrphidae) is one of the most species-rich hoverfly genera distributed across the Palaearctic and Afrotropical regions. In the Palaearctic, the genus Merodon boasts 195 described species, while its Afrotropical region pales in comparison, with a mere 17 species documented thus far. As a result of 8 years of fieldwork conducted in the Republic of South Africa, in this paper, we present the description of 11 new species for science with a description of immature stages for 2 species, which increases the diversity of this genus in the Afrotropical region by remarkable 39%. These revelations are based on integrating morphology, molecular analysis (COI gene and 28S rRNA) and geometric morphometry. All described species belong to the Merodon planifacies subgroup, the Merodon desuturinus lineage and, within that, to the Afrotropical Merodon melanocerus group. Additionally, we provide an illustrated key to 15 species belonging to the subgroup, a detailed discussion on relevant taxonomic characters, a morphological diagnosis, a distribution map and clarification of the association between M. capi complex and host plants from the genus Merwilla.

Size rather than complexity of sexual ornaments prolongs male metamorphosis and explains sexual size dimorphism in sepsid flies.

Male sexual ornaments often evolve rapidly and are thought to be costly, thus contributing to sexual size dimorphism. However, little is known about their developmental costs, and even less about costs associated with structural complexity. Here, we quantified the size and complexity of three morphologically elaborate sexually dimorphic male ornaments that starkly differ across sepsid fly species (Diptera: Sepsidae): (i) male forelegs range from being unmodified, like in most females, to being adorned with spines and large cuticular protrusions; (ii) the fourth abdominal sternites are either unmodified or are converted into complex de novo appendages; and (iii) male genital claspers range from small and simple to large and complex (e.g. bifurcated). We tracked the development of 18 sepsid species from egg to adult to determine larval feeding and pupal metamorphosis times of both sexes. We then statistically explored whether pupal and adult body size, ornament size and/or ornament complexity are correlated with sex-specific development times. Larval growth and foraging periods of male and female larvae did not differ, but the time spent in the pupal stage was ca 5% longer for sepsid males despite emerging 9% smaller than females on average. Surprisingly, we found no evidence that sexual trait complexity prolongs pupal development beyond some effects of trait size. Evolving more complex traits thus does not incur developmental costs at least in this system.

The Acquisition and Retention of Lumpy Skin Disease Virus by Blood-Feeding Insects Is Influenced by the Source of Virus, the Insect Body Part, and the Time since Feeding.

Lumpy skin disease virus (LSDV) is a poxvirus that causes severe systemic disease in cattle and is spread by mechanical arthropod-borne transmission. This study quantified the acquisition and retention of LSDV by four species of Diptera (Stomoxys calcitrans, Aedes aegypti, Culex quinquefasciatus, and Culicoides nubeculosus) from cutaneous lesions, normal skin, and blood from a clinically affected animal. The acquisition and retention of LSDV by Ae. aegypti from an artificial membrane feeding system was also examined. Mathematical models of the data were generated to identify the parameters which influence insect acquisition and retention of LSDV. For all four insect species, the probability of acquiring LSDV was substantially greater when feeding on a lesion compared with feeding on normal skin or blood from a clinically affected animal. After feeding on a skin lesion LSDV was retained on the proboscis for a similar length of time (around 9 days) for all four species and for a shorter time in the rest of the body, ranging from 2.2 to 6.4 days. Acquisition and retention of LSDV by Ae. aegypti after feeding on an artificial membrane feeding system that contained a high titer of LSDV was comparable to feeding on a skin lesion on a clinically affected animal, supporting the use of this laboratory model as a replacement for some animal studies. This work reveals that the cutaneous lesions of LSD provide the high-titer source required for acquisition of the virus by insects, thereby enabling the mechanical vector-borne transmission. IMPORTANCE Lumpy skin disease virus (LSDV) is a high consequence pathogen of cattle that is rapidly expanding its geographical boundaries into new regions such as Europe and Asia. This expansion is promoted by the mechanical transmission of the virus via hematogenous arthropods. This study quantifies the acquisition and retention of LSDV by four species of blood-feeding insects and reveals that the cutaneous lesions of LSD provide the high titer virus source necessary for virus acquisition by the insects. An artificial membrane feeding system containing a high titer of LSDV was shown to be comparable to a skin lesion on a clinically affected animal when used as a virus source. This promotes the use of these laboratory-based systems as replacements for some animal studies. Overall, this work advances our understanding of the mechanical vector-borne transmission of LSDV and provides evidence to support the design of more effective disease control programmes.

Mutation predicts 40 million years of fly wing evolution.

Mutation enables evolution, but the idea that adaptation is also shaped by mutational variation is controversial. Simple evolutionary hypotheses predict such a relationship if the supply of mutations constrains evolution, but it is not clear that constraints exist, and, even if they do, they may be overcome by long-term natural selection. Quantification of the relationship between mutation and phenotypic divergence among species will help to resolve these issues. Here we use precise data on over 50,000 Drosophilid fly wings to demonstrate unexpectedly strong positive relationships between variation produced by mutation, standing genetic variation, and the rate of evolution over the last 40 million years. Our results are inconsistent with simple constraint hypotheses because the rate of evolution is very low relative to what both mutational and standing variation could allow. In principle, the constraint hypothesis could be rescued if the vast majority of mutations are so deleterious that they cannot contribute to evolution, but this also requires the implausible assumption that deleterious mutations have the same pattern of effects as potentially advantageous ones. Our evidence for a strong relationship between mutation and divergence in a slowly evolving structure challenges the existing models of mutation in evolution.

Morphological and molecular characterization of puparia of Piophilidae species of forensic relevance.

Piophilidae are a small family of Diptera with a worldwide distribution and which are historically associated with human activities. In addition to their economic importance, piophilid larvae can also be of medical and legal relevance. Within a medicolegal context, piophilids are frequently associated with cadavers in advanced stages of decomposition, thus being potentially useful forensic indicators and they have been reported also from archaeo-funerary contexts. An accurate species identification is therefore an essential prerequisite to ensure the reliable analysis of insect material in medical, forensic and archaeological investigations. Identification of the adult piophilid flies is possible because of the availability of identification keys, in contrast immature insects, especially puparia, have been poorly investigated and described. In this paper, puparia of 11 species of forensic interest (Piophila casei, Piophila megastigmata, Parapiophila atrifrons, Parapiophila flavipes, Parapiophila vulgaris, Protopiophila litigata, Liopiophila varipes, Prochyliza nigrimana, Prochyliza xanthosoma and Stearibia nigriceps in subtribe Piophilina and Centrophlebomyia furcata in subtribe Thyreophorina) are described and a molecular analysis, based on the COI sequencing, is presented to show the potential of the molecular approach in their identification.

Larval anatomy of the digestive and excretory systems of the pharyngeal bot fly, Pharyngomyia picta (Diptera: Oestridae).

Oestrid flies (Diptera: Oestridae) are obligate parasites of mammals during their larval stage and show anatomical adaptations for the infestation of host tissues. Unlike the species that parasitize domestic mammals, those oestrid species that infest wild mammal hosts remain poorly known. With the use of x-ray micro-computed tomography, we describe for the first time the anatomy of the digestive and excretory systems of the second and third larval instars of Pharyngomyia picta (Meigen), a parasite of cervids that, like other species within the subfamily Oestrinae, causes nasopharyngeal myiasis. Both larval instars of P. picta show a pair of remarkably large salivary glands arranged in a characteristic 'glandular band', a convoluted and thickly uniform midgut and a greatly enlarged distal region of the anterior pair of Malpighian tubules. These anatomical features also have been described in other species within the subfamily Oestrinae, whereas they differ from the observations in other oestrid subfamilies. We discuss the potential functional significance of the anatomy of the digestive and excretory systems of Oestrinae larvae as specific adaptations to parasitize the nasopharyngeal cavities of mammal hosts.

Sperm competition in yellow dung flies: No consistent effect of sperm size.

The male competition for fertilization that results from female multiple mating promotes the evolution of increased sperm numbers and can impact sperm morphology, with theory predicting that longer sperm can at times be advantageous during sperm competition. If so, males with longer sperm should sire more offspring than competitors with shorter sperm. Few studies have directly tested this prediction, and findings are inconsistent. Here we assessed whether longer sperm provide a competitive advantage in the yellow dung fly (Scathophaga stercoraria; Diptera: Scathophagidae). Initially, we let brothers with different temperature-mediated mean sperm lengths compete - thus minimizing confounding effects of genetic background - and found no clear advantage of longer sperm. We then used flies from lines subjected to bidirectional selection on phenoloxidase activity that had shown correlated evolutionary responses in sperm and female spermathecal duct lengths. This experiment also yielded no main effect of sperm size on siring success. Instead, there was a trend for a shorter-sperm advantage, but only when competing in females with longer spermathecal ducts. Our data corroborated many previously reported findings (last-male precedence, effects of copula duration and body size), suggesting our failure to find sperm size effects is not inherently due to our experimental protocols. We conclude that longer sperm are not competitively superior in yellow dung flies under most circumstances, and that, consistent with previous work, in this species competitive fertilization success is primarily determined by the relative numbers of sperm competing.

Effects of genetic vs. environmental quality on condition-dependent morphological and life history traits in a neriid fly.

Condition is assumed to reflect both genes and environment, enabling condition-dependent signals to reveal genetic quality. However, because the phenotypic effects of variation in genetic quality could be masked by environmental heterogeneity, the contribution of genetic quality to phenotypic variation in fitness-related traits and condition-dependent signals remains unclear. We compared effects of ecologically relevant manipulations of environmental quality (nutrient dilution in the larval diet) and genetic quality (one generation of inbreeding) on male and female morphology, life history and reproductive performance in the neriid fly Telostylinus angusticollis. We found that larval diet quality had strong, positive effects on male and female body size, male secondary sexual traits, and aspects of male and female reproductive performance. By contrast, inbreeding had weak effects on most traits, and no trait showed clear and consistent effects of both environmental and genetic quality. Indeed, inbreeding effects on body size and male competitive performance were of opposite sign in rich vs. poor larval diet treatment groups. Our results suggest that environmental quality strongly affects condition, but the effects of genetic quality are subtle and environment-dependent in this species. These findings raise questions about the genetic architecture of condition and the potential for condition-dependent traits to function as signals of genetic quality.

Meiotic drive does not cause condition-dependent reduction of the sexual ornament in stalk-eyed flies.

Meiotic drive systems are associated with low-frequency chromosomal inversions. These are expected to accumulate deleterious mutations due to reduced recombination and low effective population size. We test this prediction using the 'sex-ratio' (SR) meiotic drive system of the Malaysian stalk-eyed fly Teleopsis dalmanni. SR is associated with a large inversion (or inversions) on the X chromosome. In particular, we study eyespan in males carrying the SR chromosome, as this trait is a highly exaggerated, sexually dimorphic trait, known to have heightened condition-dependent expression. Larvae were raised in low and high larval food stress environments. SR males showed reduced eyespan under the low and high stress treatments, but there was no evidence of a condition-dependent decrease in eyespan under high stress. Similar but more complex patterns were observed for female eyespan, with evidence of additivity under low stress and heterosis under high stress. These results do not support the hypothesis that reduced sexual ornament size in meiotic drive males is due to a condition-dependent response to the putative increase in mutation load. Instead, reduced eyespan likely reflects compensatory resource allocation to different traits in response to drive-mediated destruction of sperm.

Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

Evolution of multivariate wing allometry in schizophoran flies (Diptera: Schizophora).

The proximate and ultimate mechanisms underlying scaling relationships as well as their evolutionary consequences remain an enigmatic issue in evolutionary biology. Here, I investigate the evolution of wing allometries in the Schizophora, a group of higher Diptera that radiated about 65 million years ago, by studying static allometries in five species using multivariate approaches. Despite the vast ecological diversity observed in contemporary members of the Schizophora and independent evolutionary histories throughout most of the Cenozoic, size-related changes represent a major contributor to overall variation in wing shape, both within and among species. Static allometries differ between species and sexes, yet multivariate allometries are correlated across species, suggesting a shared developmental programme underlying size-dependent phenotypic plasticity. Static allometries within species also correlate with evolutionary divergence across 33 different families (belonging to 11 of 13 superfamilies) of the Schizophora. This again points towards a general developmental, genetic or evolutionary mechanism that canalizes or maintains the covariation between shape and size in spite of rapid ecological and morphological diversification during the Cenozoic. I discuss the putative roles of developmental constraints and natural selection in the evolution of wing allometry in the Schizophora.

Intraspecific mating system evolution and its effect on complex male secondary sexual traits: Does male-male competition increase selection on size or shape?

Sexual selection is generally held responsible for the exceptional diversity in secondary sexual traits in animals. Mating system evolution is therefore expected to profoundly affect the covariation between secondary sexual traits and mating success. Whereas there is such evidence at the interspecific level, data within species remain scarce. We here investigate sexual selection acting on the exaggerated male fore femur and the male wing in the common and widespread dung flies Sepsis punctum and S. neocynipsea (Diptera: Sepsidae). Both species exhibit intraspecific differences in mating systems and variation in sexual size dimorphism (SSD) across continents that correlates with the extent of male-male competition. We predicted that populations subject to increased male-male competition will experience stronger directional selection on the sexually dimorphic male foreleg. Our results suggest that fore femur size, width and shape were indeed positively associated with mating success in populations with male-biased SSD in both species, which was not evident in conspecific populations with female-biased SSD. However, this was also the case for wing size and shape, a trait often assumed to be primarily under natural selection. After correcting for selection on overall body size by accounting for allometric scaling, we found little evidence for independent selection on any of these size or shape traits in legs or wings, irrespective of the mating system. Sexual dimorphism and (foreleg) trait exaggeration is therefore unlikely to be driven by direct precopulatory sexual selection, but more so by selection on overall size or possibly selection on allometric scaling.

The implication of morphometrics and growth rate of dipteran flies in forensic entomotoxicology research: a review.

Forensic entomotoxicology integrates toxicology into forensic entomology to estimate minimum postmortem interval (PMImin) and circumstances of death where toxicants and poisonous substances are the suspected cause of death. Forensic entomotoxicology not only confirms the presence of toxicants in insects feeding off a cadaver but also studies its effect on the bio-morphometry and growth rate of insects. This review article highlights the effects of various toxicants on forensically important species of dipteran flies. It also discusses the parameters that may affect accuracy in estimation of time since death. The bio-physical effects of toxicants (excluding the analytical approach for qualitative detection) would help understand the trends in forensic entomotoxicological research worldwide.

Evolutionary adaptations in four hippoboscid fly species belonging to three different subfamilies.

Lipoptena cervi (Linnaeus, 1758), Lipoptena fortisetosa Maa, 1965, Hippobosca equina Linnaeus, 1758, and Pseudolynchia canariensis (Macquart, 1840) (Diptera: Hippoboscidae) are haematophagous ectoparasites that infest different mammal and bird species and occasionally attack humans. They are known for the health implications they have as vectors of pathogens to humans and animals, and for the injuries they inflict on their host's skin. This study focused on the morphological structures evolved by parasites in terms of their biology and the different environment types that they inhabit. To this aim, we examined four hippoboscid species, as well as their hosts' fur (ungulate and horse), and feather (pigeon) through light and Scanning Electron Microscopy (SEM) observations in order to highlight the main morphological features that evolved differently in these flies and to explain the effect of hosts' fur/feather microhabitats on the morphological specializations observed in the investigated ectoparasites. The studied species showed main convergent characters in mouthparts while remarkable differences have been detected on the antennal sensillar pattern as well as on the leg acropod that displayed divergent characters evolved in relation to the host.

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.