Data from: Diversity patterns of uncultured Haptophytes unravelled by pyrosequencing in Naples Bay

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 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 (3–20 μm) 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 (0.8–3 μm). Overall, this study emphasized several technical and theoretical barriers inherent to the exploration of the large and largely unknown diversity of unicellular eukaryotes.定鞭藻门（Haptophyta）是一类重要的海洋原生生物门类，包含约300个已描述的形态种以及80个形态属。本研究针对那不勒斯湾采集的分级粒径浮游生物样本，利用454焦磷酸测序（454 pyrosequencing）技术对大亚基核糖体DNA（large subunit ribosomal DNA, LSU rDNA）片段进行测序，以评估定鞭藻的物种多样性。研究团队设计了一组靶向LSU rDNA D1/D2区域的类群特异性引物，用于从两个粒径分级（0.8–3 μm或3–20 μm）以及两个采样深度（表层1米处，或23米处的深层叶绿素最大值层（deep chlorophyll maximum, DCM））的核酸提取物（总DNA或总RNA）中扩增定鞭藻序列。本研究采用覆盖当前已测序全部定鞭藻门多样性的数据库，对454测序读段进行物种鉴定。本数据集共揭示了数百个定鞭藻聚类簇，但其中绝大多数聚类簇无法与已分类的已知序列相匹配：在基于97%序列同一性阈值构建的全局数据集的操作分类单元（Operational Taxonomic Unit, OTU）97%中，仅有不到1%的读段与实验室培养物种的序列聚类，不到12%的读段与此前通过环境样本克隆及桑格测序（Sanger sequencing）获得的参考序列聚类。由此可见，当前已培养的定鞭藻物种远未能反映自然环境中实际存在的物种多样性，本研究凸显了大量未被表征的环境遗传多样性。定鞭藻群落结构呈现出显著的深度依赖性分布特征：在深层叶绿素最大值层采集的样本中，物种多样性与均匀度均达到最高；且该层采集的大粒径分级（3–20 μm）样本与其余样本共有的OTU97%占比更低。菱形金囊藻（Chrysoculter romboideus）的测序读段在深层叶绿素最大值层中检出量显著更高，而在表层1米处的样本中也可被检测到。在最小粒径分级（0.8–3 μm）的深层叶绿素最大值层样本中，完全未知的OTU97%占比最高。综上，本研究凸显了探索单细胞真核生物庞大且尚未被充分认知的多样性时，所固有的多项技术与理论障碍。