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

Q2 Agricultural and Biological Sciences
B. Moncada, H. Sipman, R. Lücking
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引用次数: 8

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.
利用内部转录间隔段(ITS)检测哥伦比亚香豆属植物的DNA条形码
我们以哥伦比亚的Usnea属样品为例,测试了基于ITS的DNA条形码在地衣真菌中的功能。来自两个不同生态区的五个位置的15个样本产生了新的ITS序列,代表了不同的形态和髓质化学。我们采用了五种策略来鉴定样品:(1)在NCBI BLAST位点上进行BLASTn与原始鉴定的最佳匹配参考序列;(2) 如前所述,但具有基于针对属发表的ITS序列的单独发表的修订版的参考序列的修订鉴定;(3) BioEdit中的局部BLASTn,使用该属的一组单独发表、修订和策划的ITS参考序列;(4) 在Usnea s.str.所有可用ITS序列的框架内进行基于多重比对的系统发育分析。;(5)综合分类学,结合分子系统发育和表型与化学数据的比较分析。使用后一种方法作为参考,我们发现具有原始鉴定的NCBI BLASTn表现不佳,导致鉴定成功率仅为7%(单个样本)。改进鉴定的NCBI BLASTn鉴定成功率增加了两倍多(23%),但仍不令人满意。BioEdit中使用修订的、精心策划的参考数据的局部BLASTn进一步使鉴定成功率翻了一番(47%),但仍然不足。与综合分类学的结果相比,基于多重比对的系统发育分析获得了80%的鉴定成功率。基于这些结果,我们得出结论,在目前的情况下,基于ITS的Usnea属DNA条形码表现不佳,但可以使用三种策略进行实质性改进:(1)更新原始库(如GenBank)中参考序列的鉴定,或者使用精心策划的参考数据集;(2) 使用仅关注目标属的精心策划的参考数据集进行局部BLAST,结合基于多重比对的系统发育分析作为验证步骤;以及(3)填补现有参考数据在地理和分类学上的巨大空白。我们的分析表明,如果该属存在具有正确识别的接近完整的参考数据集,那么标准BLAST方法可以实现接近100%的高水平识别成功。作为我们DNA条形码工作的一部分,我们为Usnea属的哥伦比亚样本生成了前15个ITS序列,我们确认了哥伦比亚存在U.aranea和U.wasmuthii,并首次为该国报告了U.tenuicorticata。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Plant and Fungal Systematics
Plant and Fungal Systematics Agricultural and Biological Sciences-Plant Science
CiteScore
2.40
自引率
0.00%
发文量
2
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