CNRS, UMR7144 & Université Pierre et Marie Curie, Team EPPO, Station biologique de Roscoff, Place Georges Tessier, Roscoff, France.
Mol Ecol. 2013 Jan;22(1):87-101. doi: 10.1111/mec.12108. Epub 2012 Nov 19.
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 (0.8-3 or 3-20 μm) 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 OTUs(97%) (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 (3-20 μm) taken at the DCM shared a lower proportion of common OTUs(97%) 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 OTUs(97%) was collected at the DCM in the smallest size fraction (0.8-3 μm). Overall, this study emphasized several technical and theoretical barriers inherent to the exploration of the large and largely unknown diversity of unicellular eukaryotes.
甲藻是海洋原生生物的一个重要门,包括约 300 种已描述的形态种和 80 种形态属。我们使用 454 焦磷酸测序技术对从那不勒斯湾大小分级浮游生物样本中提取的大核糖体亚基 DNA(LSU rDNA)片段进行分析,以评估其多样性。我们设计了一组针对 LSU rDNA D1/D2 区的组特异性引物,用于从两个大小分级(0.8-3 或 3-20 μm)和两个采样深度(表层、1 m 或 23 m 深的叶绿素最大层)的核酸提取物(总 DNA 或 RNA)中扩增甲藻序列。我们使用涵盖当前测序的整个甲藻多样性的数据库来识别 454 读取。我们的数据集中揭示了数百个甲藻群。然而,大多数这些群无法与分类学上已知的序列相关联:考虑到我们的全局数据集上的 OTUs(97%)(在序列同一性水平为 97%的聚类),不到 1%的读取与培养物的序列聚类,不到 12%的读取与之前从环境样本的克隆和 Sanger 测序中获得的参考序列聚类。因此,我们强调了大量未被描述的环境遗传多样性,这清楚地表明,目前培养的物种不能很好地反映自然环境中存在的实际多样性。甲藻群落似乎根据深度而显著分层。在 DCM 处获得了最高的多样性和均匀度,并且在 DCM 处从大尺寸分级(3-20 μm)采集的样本与其他样本共享较低比例的共同 OTUs(97%)。在 DCM 处发现了 Chrysoculter romboideus 物种的大量读段,而在表层也可以检测到。在最小尺寸分级(0.8-3 μm)中,在 DCM 处采集的完全未知 OTUs(97%)的比例最高。总体而言,这项研究强调了在探索单细胞真核生物的大量且大部分未知多样性时存在的几个技术和理论障碍。