Abstract
DNA barcoding using the nuclear internal transcribed spacer (ITS) has become prevalent in surveys of fungal diversity. This approach is, however, associated with numerous caveats, including the desire for speed, rather than accuracy, through the use of automated analytical pipelines, and the shortcomings of reference sequence repositories. Here we use the case of a specimen of the bracket fungus Trametes s.lat. (which includes the common and widespread turkey tail, T. versicolor) to illustrate these problems. The material was collected in Vietnam as part of a biodiversity inventory including DNA barcoding approaches for arthropods, plants and fungi. The ITS barcoding sequence of the query taxon was compared against reference sequences in GenBank and the curated fungal ITS database UNITE, using BLASTn and MegaBLAST, and was subsequently analysed in a multiple alignment-based phylogenetic context through a maximum likelihood tree including related sequences. Our results initially indicated issues with BLAST searches, including the use of pairwise local alignments and sorting through Total score and E value, rather than Percentage identity, as major shortcomings of the DNA barcoding approach. However, after thorough analysis of the results, we concluded that the single most important problem of this approach was incorrect sequence labelling, calling for the implementation of third-party annotations or analogous approaches in primary sequence repositories. In addition, this particular example revealed problems of improper fungal nomenclature, which required reinstatement of the genus name Cubamyces (= Leiotrametes), with three new combinations: C. flavidus, C. lactineus and C. menziesii. The latter was revealed as the correct identification of the query taxon, although the name did not appear among the best BLAST hits. While the best BLAST hits did correspond to the target taxon in terms of sequence data, their label names were misleading or unresolved, including [Fungal endophyte], [Uncultured fungus], Basidiomycota, Trametes cf. cubensis, Lenzites elegans and Geotrichum candidum (an unrelated ascomycetous contaminant). Our study demonstrates that accurate identification of fungi through molecular barcoding is currently not a fast-track approach that can be achieved through automated pipelines.Citation: Lucking R., Truong B. V., Huong D. T. T., Le N. H., Nguyen Q. D., Nguyen V. D., Raab-Straube E. von, Bollendorff S., Govers K. & Di Vincenzo V. 2020: Caveats of fungal barcoding: a case study in Trametes s.lat. (Basidiomycota: Polyporales) in Vietnam reveals multiple issues with mislabelled reference sequences and calls for third-party annotations. – Willdenowia 50: 383–403. doi: https://doi.org/10.3372/wi.50.50302Version of record first published online on 15 September 2020 ahead of inclusion in December 2020 issue.
Highlights
The true fungi (Fungi) represent the third largest kingdom in terms of known species and the second largest with respect to estimated richness, with between 2.2 and 3.8 million species (Hawksworth & Lücking 2018)
Our study demonstrates that accurate identification of fungi through molecular barcoding is currently not a fast-track approach that can be achieved through automated pipelines
MegaBLAST returned as best matches for the query sequence numerous unidentified or incompletely identified reference sequences, including [Fungal endophyte], Ba sidiomycota sp., [Uncultured fungus], Polyporales sp., Agaricales sp. and Trametes sp
Summary
The true fungi (Fungi) represent the third largest kingdom in terms of known species and the second largest with respect to estimated richness, with between 2.2 and 3.8 million species (Hawksworth & Lücking 2018). Due to their simple body plan, fungi have few diagnostic characters compared to plants and animals Molecular barcoding has become an important tool in the identification of fungi, as well as other organisms, and is even being implemented in citizen science projects Phenomena such as hybridization, introgression or gene duplication complicate the use of the ITS barcoding marker (Lindner & Banki 2013; Li & al. 2013, 2017; Lücking & al. 2020)
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