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Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing
Stockholm University, Faculty of Science, Department of Zoology. Station Linné, Sweden.
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2019 (English)In: BMC Biology, E-ISSN 1741-7007, Vol. 17, article id 96Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2019. Vol. 17, article id 96
Keywords [en]
NGS barcoding, DNA barcoding, Nanopore sequencing, MinION, Large-scale species discovery
National Category
Zoology
Identifiers
URN: urn:nbn:se:su:diva-192270DOI: 10.1186/s12915-019-0706-9ISI: 000501016500001OAI: oai:DiVA.org:su-192270DiVA, id: diva2:1544787
Available from: 2021-04-16 Created: 2021-04-16 Last updated: 2024-01-17Bibliographically approved
In thesis
1. A multi-faceted approach to a "dark taxon": The hyperdiverse and poorly known scuttle flies (Diptera: Phoridae)
Open this publication in new window or tab >>A multi-faceted approach to a "dark taxon": The hyperdiverse and poorly known scuttle flies (Diptera: Phoridae)
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Most of the unknown animal biodiversity on earth is in groups of invertebrates that are hyperdiverse and abundant, yet poorly known (“dark taxa”). The study of these organisms requires a multi-faceted approach and methodologies designed to tackle large numbers of species and specimens. The scuttle flies (Diptera: Phoridae) are a classic example of a dark taxon and the focus of this thesis. Paper I is a molecular phylogeny of the phorid genus Megaselia based on one nuclear (28S rDNA) and three mitochondrial (ND1, COI and 16S) markers from 145 species of Nordic Megaselia. Molecular data was analysed with Bayesian analysis, maximum likelihood, and parsimony methods. Based on these results, and supporting morphological data, we divide Megaselia into 22 informal species groups, 20 of which fall into a monophyletic “core Megaselia”. We discuss implications for the future circumscription of Megaselia and associated genera. Paper II presents a pipeline for rapid and cost-effective species discovery using the Oxford Nanopore mobile sequencing technology MinION. This paper reveals the presence of ca. 650 species of Phoridae from a single Malaise trap placed in Kibale National Park, Uganda. Based on our data, we estimate that the phorid fauna of the Afrotropical region could be as high as 100 000 species: this figure dwarfs previous diversity estimates. The implications for species discovery and description are discussed, and a new species (Megaselia sepsioides sp. nov.) is described. Paper III outlines a large-scale integrative approach to species discovery and delimitation in hyperdiverse groups, exemplified using a dataset of 18 000 phorid flies from Sweden. COI minibarcodes (313 bp) were obtained for all specimens and classified into putative species using different clustering methods (objective clustering, Poisson tree process, automatic barcode gap discovery and refined single linkage). No clustering method was accurate enough to use for species delimitation without confirmation from additional data. We found that the stability of a cluster to change across genetic-distance thresholds and the genetic variation within a cluster both accurately predict clusters where morphology is likely to be incongruent with barcode data. With molecular clustering integrated with morphological validation, we found that we could examine less than 5% of specimens and still delimit all species fully and accurately. Paper IV addresses questions about the scuttle fly fauna of Sweden with data from 32 000 scuttle flies from 37 sites and 4 time periods. We estimate that the total Swedish fauna contains 652-713 (based on Chao 1 or CNE estimates, respectively) species of scuttle flies, 1.5 times the 372 species currently documented from Sweden. Ordination techniques show that scuttle fly communities are organized in a gradient across Sweden, which is well correlated with plant hardiness zones defined by the Swedish Horticultural Society. Hierarchical modelling of species communities (HMSC) reveals that phorid community composition is largely determined by climatic and temporal variables, but much of the variance remains unexplained by the models we explored. Comparison of our phorid data with that of species more commonly utilised for biodiversity assessments revealed that phorids may allow more fine-scaled analysis as they may exist in smaller ranges, and that they additionally may give unique patterns of distribution that are unlike those seen in other taxa.

Place, publisher, year, edition, pages
Stockholm: Department of Zoology, Stockholm University, 2021. p. 43
Keywords
biodiversity, taxonomy, dark taxa
National Category
Zoology
Research subject
Systematic Zoology
Identifiers
urn:nbn:se:su:diva-192276 (URN)978-91-7911-472-5 (ISBN)978-91-7911-473-2 (ISBN)
Public defence
2021-06-08, online via Zoom, public link is available at the department website, 13:00 (English)
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Available from: 2021-05-12 Created: 2021-04-16 Last updated: 2022-02-25Bibliographically approved

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