Diversity and phylogenetic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and environmental PCR
Diversity and phylogenetic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and environmental PCR
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DOI：
10.3852/mycologia.99.2.185
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发表时间：
2007-03-01
期刊：
影响因子：
2.8
通讯作者：
Vilgalys, Rvtas
中科院分区：
文献类型：
--
作者：
Arnold, A. Elizabeth;Henk, Daniel A.;Vilgalys, Rvtas
我们研究了火炬松无症状叶片中的内生真菌（Pinus taeda）的系统发育学研究，其目标有四：（i）基于序列相似性评价形态分类单元、BLAST匹配和组作为功能分类单元;（ii）探索最大化环境数据集系统发育信号的方法，环境数据集通常包含许多分类单元但很少特征;（iii）比较培养与无培养方法（表面灭菌叶的环境PCR）用于估计内生菌多样性和物种组成;（iv）探讨传统生态指标之间的关系（例如Shannon指数）和系统发育多样性（PD）在估计内生菌多样性和空间异质性方面的应用。从90片火炬松叶片中的87片中回收内生菌，得到439个分离株，代表24种形态分类群。150个分离株的核核糖体内转录间隔区（ITS）序列数据显示，59个不同的ITS基因型，分别代表24和37个独特的组，基于90%和95%的序列相似性。通过重新编码模糊对齐的区域提取系统发育信号，并实施一个保守的系统发育骨干约束，我们恢复了良好的支持基于ca的系统发育。对72个子囊菌门和担子菌门、145个培养的内生菌和33个环境样品的核核糖体大亚基（LSUrDNA）进行了PCR扩增。与LSUrDNA界定的物种的比较表明，形态分类充分估计总物种丰富度，但很少对应的生物学意义的群体。ITS BLAST结果在其效用方面是可变的。但ITS基因型组的90%序列相似性与LSUrDNA界定的物种一致。环境PCR产生了更多的基因型每采样努力，并恢复了几个不同的分支相对于培养，但一些常见的培养分支从未发现（Sordariomycetes）或罕见的相对于他们的高频率之间的文化（Leotiomycetes）。与传统指标相比，PD显示了火炬松树木和研究地块内生菌组合的空间异质性。我们的研究结果强调，需要谨慎指定分类单位的基础上毛文化形态或ITS BLAST匹配，系统发育工具的实用性，从环境样品中提取强大的peptigenies，培养和环境PCR的互补性，PD相对于传统的生态指标的实用性，以及在这个简化的温带生态系统中的叶面真菌内生菌的多样性非常高。
We examined endophytic fungi in asymptomatic foliage of loblolly pine (Pinus taeda) in North Carolina, USA, with four goals: (i) to evaluate morphotaxa, BLAST matches and groups based on sequence similarity as functional taxonomic units; (ii) to explore methods to maximize phylogenetic signal for environmental datasets, which typically contain many taxa but few characters; (iii) to compare culturing vs. culture-free methods (environmental PCR of surface sterilized foliage) for estimating endophyte diversity and species composition; and (iv) to investigate the relationships between traditional ecological indices (e.g. Shannon index) and phylogenetic diversity (PD) in estimating endophyte diversity and spatial heterogeneity. Endophytes were recovered in culture from 87 of 90 P. taeda leaves sampled, yielding 439 isolates that represented 24 morphotaxa. Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) for 150 isolates revealed 59 distinct ITS genotypes that represented 24 and 37 unique groups based on 90% and 95% sequence similarity, respectively. By recoding ambiguously aligned regions to extract phylogenetic signal and implementing a conservative phylogenetic backbone constraint, we recovered well supported phylogenies based on ca. 600 by of the nuclear ribosomal large subunit (LSUrDNA) for 72 Ascomycota and Basidiomycota, 145 cultured endophytes and 33 environmental PCR samples. Comparisons with LSUrDNA-delimited species showed that morphotaxa adequately estimated total species richness but rarely corresponded to biologically meaningful groups. ITS BLAST results were variable in their utility. but ITS genotype groups based on 90% sequence similarity were concordant with LSUrDNA-delimited species. Environmental PCR yielded more genotypes per sampling effort and recovered several distinct clades relative to culturing, but some commonly cultured clades were never found (Sordariomycetes) or were rare relative to their high frequency among cultures (Leotiomycetes). In contrast to traditional indices, PD demonstrated spatial heterogeneity in endophyte assemblages among P. taeda trees and study plots. Our results highlight the need for caution in designating taxonomic units based on gross cultural morphology or ITS BLAST matches, the utility of phylogenetic tools for extracting robust phylogenies from environmental samples, the complementarity of culturing and environmental PCR, the utility of PD relative to traditional ecological indices, and the remarkably high diversity of foliar fungal endophytes in this simplified temperate ecosystem.