ABSTRACT
Background Most animal species await discovery. Most of these undiscovered species share two features: they live in tropical regions and they belong to animal clades that combine high specimen abundance with immense species richness. Using morphology for species discovery in such taxa is slow because large numbers of specimens must be sorted to species based on detailed microscopic work. Fortunately, species discovery can be greatly accelerated by using molecular methods for sorting specimens into putative species before validating the species with morphology by examining a subset of specimens. This requires cost-effective and low-tech DNA barcoding techniques given that well equipped molecular laboratories are not readily available in many biodiverse countries.
Results We here document how MinION can be used to reveal the extent of the undiscovered biodiversity in specimen-rich taxa. We sequenced 7,062 specimens belonging to Phoridae, a hyper-diverse family of flies (Diptera). All specimens were collected in a single Malaise trap in Kibale National Park, Uganda over the period of eight weeks. We discover >650 species which exceeds the number of phorid species currently described for the entire Afrotropical region. We were able to obtain these barcodes at low cost using MinION sequencing by increasing the barcoding capacity per flowcell to 3500 (i.e., 700% increase over existing protocols). This is achieved by adopting 1D sequencing, re-sequencing weak amplicons on a used flowcell, and increasing the bioinformatics speed via parallelization and improved demultiplexing. Comparison with Illumina data reveals that the MinION barcodes yield very similar putative species (match ratio: 0.989) which is due to the high accuracy of the MinION barcodes (99.99% accuracy, 0.4% Ns). Morphological examination of 100 mOTUs confirms good congruence with molecular clusters (93% of mOTUs; >99% of specimens) and reveals that 90% of the putative species belong to the neglected megadiverse genus Megaselia. Lastly, we demonstrate for one species how the molecular data guides the description of new species (Megaselia sepsioides sp. nov.).
Conclusions We conclude that MinION is very suitable for reliable, rapid, and large-scale species discovery in hyperdiverse taxa, especially in biodiverse countries with limited access to capital-intensive sequencing facilities.
Footnotes
clarifications with regard to methods results of additional analyses text changes
List of abbreviations
- mOTU
- molecular Operational Taxonomic Units
- NGS
- Next Generation Sequencing
- NuMTs
- Nuclear mitochondrial DNA sequences
- BIN
- Barcode Index Number