Understudied, underrepresented, and unknown: Methodological biases that limit detection of early diverging fungi from environmental samples
Understudied, underrepresented, and unknown: Methodological biases that limit detection of early diverging fungi from environmental samples
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研究不足、代表性不足和未知：方法学偏差限制了环境样本中早期分化真菌的检测
DOI：
10.1111/1755-0998.13540
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发表时间：
2022
影响因子：
7.7
通讯作者：
Smith, Matthew E.
中科院分区：
文献类型：
--
作者：
Reynolds, Nicole K.;Jusino, Michelle A.;Stajich, Jason E.;Smith, Matthew E.
元条形码是了解真菌群落的重要工具。内部转录间隔（ITS） rDNA是公认的真菌条形码，但存在已知的问题。大亚基rDNA （large subunit， LSU）也被用于真菌群落的研究，但现有的LSU元编码引物大多针对子囊菌门（Dikarya）和担子菌门（Basidiomycota），很少关注早期分化真菌（EDF）。然而，来自多项研究的证据表明，EDF构成了群落抽样中未知多样性的很大一部分。在这里，我们研究了DNA标记选择和方法偏差如何影响环境样本中EDF的恢复。我们以一个EDF谱系Zoopagomycota为例。我们利用Illumina MiSeq对3组引物（ITS1F/ITS2, LROR/LR3， LR3与新引物LR22F配对）进行了扩增，并对Zoopagomycota模拟群落和146个环境样本进行了测序。我们比较了两种分类法分配方法，并创建了与AMPtk软件兼容的LSU参考数据库。两种分类分配方法恢复了明显不同的真菌和EDF群落。靶片段长度变化加剧了PCR扩增偏差，影响了下游的分类定位，但EDF的影响大于Dikarya。为了提高LSU扩增子的鉴定，我们进行了系统发育重建，并说明了这一关键工具在研究已识别和未识别序列方面的优势。我们的研究结果表明，许多EDF群落可能被“标准”元条形码方法遗漏或错误识别，需要改进技术来了解这些分类群在更广泛的生态背景下的作用。
Metabarcoding is an important tool for understanding fungal communities. The internal transcribed spacer (ITS) rDNA is the accepted fungal barcode but has known problems. The large subunit (LSU) rDNA has also been used to investigate fungal communities but available LSU metabarcoding primers were mostly designed to target Dikarya (Ascomycota + Basidiomycota) with little attention to early diverging fungi (EDF). However, evidence from multiple studies suggests that EDF comprise a large portion of unknown diversity in community sampling. Here, we investigate how DNA marker choice and methodological biases impact recovery of EDF from environmental samples. We focused on one EDF lineage, Zoopagomycota, as an example. We evaluated three primer sets (ITS1F/ITS2, LROR/LR3, and LR3 paired with new primer LR22F) to amplify and sequence a Zoopagomycota mock community and a set of 146 environmental samples with Illumina MiSeq. We compared two taxonomy assignment methods and created an LSU reference database compatible with AMPtk software. The two taxonomy assignment methods recovered strikingly different communities of fungi and EDF. Target fragment length variation exacerbated PCR amplification biases and influenced downstream taxonomic assignments, but this effect was greater for EDF than Dikarya. To improve identification of LSU amplicons we performed phylogenetic reconstruction and illustrate the advantages of this critical tool for investigating identified and unidentified sequences. Our results suggest much of the EDF community may be missed or misidentified with “standard” metabarcoding approaches and modified techniques are needed to understand the role of these taxa in a broader ecological context.
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影响因子：
2.7
作者：
Palmer JM;Jusino MA;Banik MT;Lindner DL
通讯作者：
Lindner DL
新的Zoopagaceae捕获并消耗土壤阿米巴原虫
DOI：
--
发表时间：
1938
期刊：
影响因子：
--
作者：
C. Drechsler
通讯作者：
C. Drechsler
影响因子：
--
作者：
FEIBELMAN, T;BAYMAN, P;CIBULA, WG
通讯作者：
CIBULA, WG
影响因子：
2.9
作者：
Picard, Kathryn T.
通讯作者：
Picard, Kathryn T.
影响因子：
7.7
作者：
Furneaux, Brendan;Bahram, Mohammad;Ryberg, Martin
通讯作者：
Ryberg, Martin