The genus Megaselia Rondani (Diptera: Phoridae) is one of the largest in the animal kingdom, with nearly 1700 described species and many remaining to be discovered. Work on this group is notoriously challenging due to the extreme species diversity, poor knowledge of higher‐level relationships and lack of molecular data. In this paper, we present the largest study to date of Megaselia relationships based on molecular data from one nuclear (28S rDNA) and three mitochondrial (ND1, COI and 16S) markers for 175 Nordic specimens representing 145 species of Megaselia, plus outgroups. Based on phylogenetic analyses of these data, we propose 22 informal Megaselia species groups, all of which match well‐supported terminal clades. Relationships among these groups, and between them and several isolated species, remain largely uncertain. Of the 22, 20 species groups fall into a moderately well‐supported monophyletic clade of ‘core Megaselia’. Two species groups, the spinigera and ruficornis groups, fall outside of core Megaselia, as does the single representative of Myriophora, a genus that is included in Megaselia by some specialists. Here, we explore the morphology of these molecular species groups to aid future studies, and we discuss the implications of our findings for the generic circumscription of Megaselia. Hopefully, our results can aid further characterization of subgroups within the enormous Megaselia radiation and among its closest relatives, thus facilitating future work on this challenging but fascinating group of small flies.

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.