1D MinION sequencing for large-scale species discovery: 7000 scuttle flies (Diptera: Phoridae) from one site in Kibale National Park (Uganda) revealed to belong to >650 species

Abstract

BackgroundMore 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 using 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.\n\nResultsWe here document how MinION sequencing can be used for large-scale species discovery in a specimen- and species-rich taxon like the hyper-diverse fly family Phoridae (Diptera). We sequenced 7,059 specimens collected in a single Malaise trap in Kibale National Park, Uganda over the short period of eight weeks. We discovered >650 species which exceeded 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 3,500 barcodes per flowcell. This was achieved by adopting 1D sequencing, re-sequencing 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 a 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.).\n\nConclusionsWe 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 100,000 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.