Diversity patterns of uncultured Haptophytes unravelled by pyrosequencing in Naples Bay
Diversity patterns of uncultured Haptophytes unravelled by pyrosequencing in Naples Bay
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DOI：
10.1111/mec.12108
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发表时间：
2013-01-01
影响因子：
4.9
通讯作者：
De Vargas, Colomban
中科院分区：
文献类型：
--
作者：
Bittner, Lucie;Gobet, Angelique;De Vargas, Colomban
触媒生物是海洋原生生物的一个重要门，目前已知的形态种有300种，形态属有80种。我们利用454焦磷酸测序技术对大亚基核糖体DNA （LSU rDNA）片段进行测序，以评估在那不勒斯湾收集的浮游生物样本的多样性。一组针对LSU rDNA D1/D2区域的群体特异性引物被设计用于扩增两个大小部分（0.83或320 μ m）和两个采样深度[地下，1 m或深层叶绿素最大值（DCM） 23 m]的核酸提取物（总DNA或RNA）的Haptophyte序列。使用覆盖当前测序的整个附生植物多样性的数据库鉴定了454个reads。我们的数据集揭示了数百个Haptophyte集群。然而，这些聚类中的大多数不能与分类上已知的序列相关联：考虑到我们全球数据集上的OTUs97%（序列一致性水平为97%的聚类），不到1%的reads与来自培养的序列聚类，不到12%的reads与之前从克隆和Sanger测序中获得的参考序列聚类。因此，我们强调了大量未表征的环境遗传多样性，这清楚地表明，目前栽培的物种很难反映自然环境中存在的实际多样性。随着深度的增加，植物群落呈现出明显的结构性。DCM样品的多样性和均匀性最高，在DCM采集的大粒径馏分（320 μ m）样品的共有OTUs97%的比例低于其他样品。从Chrysoculter romboideus物种的读数在DCM中被发现，而它们可以在地下被检测到。在最小粒径段（0.83 mu m）的DCM处收集到的完全未知OTUs97%的比例最高。总的来说，这项研究强调了探索单细胞真核生物的大量和大部分未知的多样性所固有的几个技术和理论障碍。
Haptophytes are a key phylum of marine protists, including 300 described morphospecies and 80 morphogenera. We used 454 pyrosequencing on large subunit ribosomal DNA (LSU rDNA) fragments to assess the diversity from size-fractioned plankton samples collected in the Bay of Naples. One group-specific primer set targeting the LSU rDNA D1/D2 region was designed to amplify Haptophyte sequences from nucleic acid extracts (total DNA or RNA) of two size fractions (0.83 or 320 mu m) and two sampling depths [subsurface, at 1 m, or deep chlorophyll maximum (DCM) at 23 m]. 454 reads were identified using a database covering the entire Haptophyta diversity currently sequenced. Our data set revealed several hundreds of Haptophyte clusters. However, most of these clusters could not be linked to taxonomically known sequences: considering OTUs97% (clusters build at a sequence identity level of 97%) on our global data set, less than 1% of the reads clustered with sequences from cultures, and less than 12% clustered with reference sequences obtained previously from cloning and Sanger sequencing of environmental samples. Thus, we highlighted a large uncharacterized environmental genetic diversity, which clearly shows that currently cultivated species poorly reflect the actual diversity present in the natural environment. Haptophyte community appeared to be significantly structured according to the depth. The highest diversity and evenness were obtained in samples from the DCM, and samples from the large size fraction (320 mu m) taken at the DCM shared a lower proportion of common OTUs97% with the other samples. Reads from the species Chrysoculter romboideus were notably found at the DCM, while they could be detected at the subsurface. The highest proportion of totally unknown OTUs97% was collected at the DCM in the smallest size fraction (0.83 mu m). Overall, this study emphasized several technical and theoretical barriers inherent to the exploration of the large and largely unknown diversity of unicellular eukaryotes.