Testing DNA barcoding in Usnea (Parmeliaceae) in Colombia using the internal transcribed spacer (ITS)

Abstract

We tested the functionality of ITS-based DNA barcoding in lichen fungi using Colombian samples of the genus Usnea as an example. New ITS sequences were generated for 15 samples from five localities in two different ecoregions, representing varying morphologies and medullary chemistries. We employed five strategies to identify the samples: (1) BLASTn on the NCBI BLAST site with the original identifications of the best matching reference sequences; (2) as previous, but with revised identifications of the reference sequences based on a separately published revision of ITS sequences published for the genus; (3) local BLASTn in BioEdit using a separately published, revised and curated set of ITS reference sequences for the genus; (4) multiple alignment based phylogenetic analysis within the framework of all available ITS sequences for Usnea s.str.; and (5) integrative taxonomy, combining molecular phylogeny and comparative analysis of phenotype and chemical data. Using the latter approach as reference, we found that NCBI BLASTn with original identifications performed poorly, resulting in an identification success rate of only 7% (a single sample). NCBI BLASTn with revised identifications more than tripled identification success (23%), but was still unsatisfactory. Local BLASTn in BioEdit using the revised, curated reference data further doubled identification success (47%), but remained inadequate. Multiple alignment-based phylogenetic analysis achieved an identification success rate of 80% compared to the result from integrative taxonomy. Based on these results, we conclude that ITS-based DNA barcoding of the genus Usnea under the current circumstances performs poorly, but can be substantially improved using three strategies: (1) update identifications of reference sequences in primary repositories such as GenBank or alternatively use a curated reference data set; (2) perform local BLAST with a curated reference data set focusing on the target genus only, combined with multiple alignment-based phylogenetic analysis as a verification step; and (3) close substantial geographic and taxonomic gaps in the existing reference data. Our analyses suggest that if a near-complete reference data set with correct identifications existed for the genus, then standard BLAST approaches could achieve high levels of identification success close to 100%. As part of our DNA barcoding exercise, which generated the first 15 ITS sequences for Colombian samples of the genus Usnea, we confirm the presence of U. aranea and U. wasmuthii in Colombia and we report for the first time U. tenuicorticata for the country.

Species boundaries in the messy middle—A genome‐scale validation of species delimitation in a recently diverged lineage of coastal fog desert lichen fungi

“…The standard DNA barcoding marker—the ITS—has been a powerful tool for understanding species diversity in lichen‐forming fungi (Lücking et al, 2020 ; Schoch et al, 2012 ), although issues with using this marker in some clades have been demonstrated (Pino‐Bodas et al, 2013 ). In taxonomically challenging groups, the ITS can provide an important first pass for grouping closely related species (Leavitt et al, 2013 ; Lücking et al, 2014 ; Moncada et al, 2020 ). While the taxonomic implications of DNA barcoding approaches remain unsettled (see Meier et al, 2021 ), our results show that species partitions inferred from the ITS marker coincide with clades comprised of closely related species‐level lineages in Niebla inferred from phylogenomic data (Figure 3 ).…”

How to publish a new fungal species, or name, version 3.0

“…Sequences intended to be submitted as DNA barcodes should only include the generally used region for that marker in order to allow BLAST searches to work optimally. For example, submissions for the ITS region (Schoch et al 2012) should be submitted with only short SSU and LSU flanking sequences (Moncada et al 2020). b.…”

A molecular re-evaluation of Parmelia encryptata with notes on its distribution

“…Such databases facilitate quick determination of taxa based on DNA sequences, but are of limited use since the identification using a simple blast search of a single marker may bias the recognition of genetically similar species (e.g. Lücking et al 2020b;Moncada et al 2020). Furthermore, numerous records are mislabelled in GenBank, as recently reported by Hofstetter et al (2019).…”

“…Although BLAST searches facilitate quick determination of taxa based on DNA sequences, the recognition of genetically similar species may be challenging (e.g. Lücking et al 2020a, b;Moncada et al 2020).…”