ABSTRACT
Background More than 80% of all animal species remain unknown to science. Most of these species tend to live in the tropics and belong to animal taxa that combine small 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 species-level sorting. Morphological verification of “molecular taxonomic operational units” (mOTUs) delineated with DNA sequences could then be based on inspecting 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 to reveal the extent of the undiscovered biodiversity in specimen-rich taxa such as Phoridae, a hyper-diverse family of flies (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 exceeds the number of phorid species currently described for the entire Afrotropical region. The barcodes were obtained using a low-cost MinION pipeline that increases the barcoding capacity per flowcell from 500 to 3,500 barcodes. This was achieved by adopting 1D sequencing, re-sequencing weak amplicons on a used flowcell, improving demultiplexing, and introducing parallelization. Comparison with Illumina data revealed that the MinION barcodes are very accurate (99.99% accuracy, 0.46% Ns) and thus yield very similar putative species (match ratio: 0.991). Morphological examination of 100 mOTUs also confirmed good congruence with molecular clusters (93% of mOTUs; >99% of specimens) and revealed that 90% of the putative species belong to a neglected megadiverse genus, i.e., Megaselia. We demonstrate for one species how the molecular data can guide the description of a new species (Megaselia sepsioides sp. nov.).
Conclusions We conclude that low-cost MinION sequencers are very suitable for reliable, rapid, and large-scale species discovery in hyperdiverse taxa. MinION sequencing can reveal the extent of the unknown diversity quickly and is especially suitable for 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
- PTP
- Poisson Tree Processes
- MSA
- Multiple Sequence Alignment