Ceballos-Escalera Angelina, Richards John, Arias Maria Belen, Inward Daegan J G, Vogler Alfried P
Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK Natural History Museum London United Kingdom.
Department of Life Sciences, Imperial College London, Silwood Park Campus, Ascot SL5 7PY, UK Imperial College London Ascot United Kingdom.
MycoKeys. 2022 Mar 8;88:1-33. doi: 10.3897/mycokeys.88.77106. eCollection 2022.
Full taxonomic characterisation of fungal communities is necessary for establishing ecological associations and early detection of pathogens and invasive species. Complex communities of fungi are regularly characterised by metabarcoding using the Internal Transcribed Spacer (ITS) and the Large-Subunit (LSU) gene of the rRNA locus, but reliance on a single short sequence fragment limits the confidence of identification. Here we link metabarcoding from the ITS2 and LSU D1-D2 regions to characterise fungal communities associated with bark beetles (Scolytinae), the likely vectors of several tree pathogens. Both markers revealed similar patterns of overall species richness and response to key variables (beetle species, forest type), but identification against the respective reference databases using various taxonomic classifiers revealed poor resolution towards lower taxonomic levels, especially the species level. Thus, Operational Taxonomic Units (OTUs) could not be linked via taxonomic classifiers across ITS and LSU fragments. However, using phylogenetic trees (focused on the epidemiologically important Sordariomycetes) we placed OTUs obtained with either marker relative to reference sequences of the entire rRNA cistron that includes both loci and demonstrated the largely similar phylogenetic distribution of ITS and LSU-derived OTUs. Sensitivity analysis of congruence in both markers suggested the biologically most defensible threshold values for OTU delimitation in Sordariomycetes to be 98% for ITS2 and 99% for LSU D1-D2. Studies of fungal communities using the canonical ITS barcode require corroboration across additional loci. Phylogenetic analysis of OTU sequences aligned to the full rRNA cistron shows higher success rate and greater accuracy of species identification compared to probabilistic taxonomic classifiers.
对真菌群落进行全面的分类学表征对于建立生态关联以及早期检测病原体和入侵物种至关重要。真菌的复杂群落通常通过对核糖体RNA基因座的内部转录间隔区(ITS)和大亚基(LSU)基因进行代谢条形码分析来表征,但依赖单个短序列片段会限制鉴定的可信度。在这里,我们将来自ITS2和LSU D1-D2区域的代谢条形码分析联系起来,以表征与树皮甲虫(小蠹科)相关的真菌群落,树皮甲虫可能是几种树木病原体的传播媒介。两种标记物都揭示了总体物种丰富度和对关键变量(甲虫种类、森林类型)的响应的相似模式,但使用各种分类学分类器针对各自的参考数据库进行鉴定时,对较低分类水平,尤其是物种水平的分辨率较差。因此,无法通过分类学分类器将操作分类单元(OTU)跨ITS和LSU片段进行关联。然而,使用系统发育树(聚焦于具有流行病学重要性的粪壳菌纲),我们将通过任何一种标记物获得的OTU相对于包括这两个基因座的整个rRNA顺反子的参考序列进行定位,并证明了ITS和LSU衍生的OTU在系统发育分布上基本相似。两种标记物一致性的敏感性分析表明,粪壳菌纲中OTU划分在生物学上最合理的阈值对于ITS2为98%,对于LSU D1-D2为99%。使用经典ITS条形码对真菌群落进行的研究需要通过其他基因座进行确证。与概率分类学分类器相比,将OTU序列与完整rRNA顺反子比对后的系统发育分析显示出更高的成功率和物种鉴定的更高准确性。