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

Bibiana Moncada,Robert Lücking,Harrie Sipman

doi:10.35535/pfsyst-2020-0026

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.

Highlights

DNA barcoding has become an important tool in mycology to provide species identifications or verify determinations based on phenotype characters (Begerow et al 2010; Kelly et al 2011; Schoch et al 2012; Xu 2016; Truong et al 2017; Hofstetter et al 2019; Lücking et al 2020a)
Moncada et al.: Testing DNA barcoding in Usnea in Colombia in sequence data pertaining to Usnea: first and foremost taxonomic and geographic gaps, and substantial issues with sequence identifications and voucher information
NCBI BLASTn resulted in a total of different potential identifications for the query sequences, taking into account the three best hits plus up to three addition hits with a percentage identity of 98.5% or higher (Table 2)

Summary

Introduction

DNA barcoding has become an important tool in mycology to provide species identifications or verify determinations based on phenotype characters (Begerow et al 2010; Kelly et al 2011; Schoch et al 2012; Xu 2016; Truong et al 2017; Hofstetter et al 2019; Lücking et al 2020a). Among the original NCBI BLASTn results, we noticed an unusually high number of appearances of the same reference sequence as hit, namely Usnea subfusca (MG262534), which was found among the best hits for no less than eight query sequences (Table 2).

Results

Conclusion