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（Haptophytes）是海洋原生生物的关键门类，包含约300个已描述的形态种与80个形态属。本研究针对那不勒斯湾采集的粒径分级浮游生物样本，采用454焦磷酸测序技术对大亚基核糖体DNA（large subunit ribosomal DNA, LSU rDNA）片段进行测序，以评估其物种多样性。研究设计了一组靶向LSU rDNA D1/D2区域的类群特异性引物组合，用于扩增两个粒径分级（0.8–3 μm或3–20 μm）以及两个采样深度（表层1米处、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RNA）中的定鞭藻序列。本研究采用覆盖当前已完成测序的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。本数据集共揭示数百个定鞭藻聚类簇，但其中绝大多数无法与分类学上已确证的序列建立对应关系：针对全局数据集以97%序列同一性阈值构建的97%同一性操作分类单元（OTU97%）中，仅有不到1%的读段与培养物来源的序列聚类，不足12%的读段与此前通过环境样本克隆及桑格（Sanger）测序获得的参考序列聚类。由此可见，当前已培养物种难以反映自然环境中实际存在的生物多样性，本研究凸显了大量未被表征的环境遗传多样性。定鞭藻群落结构显著随采样深度呈现分化：深层叶绿素最大值层样本的物种多样性与均匀度最高，且该层中取自大粒径分级（3–20 μm）的样本与其余样本共享的OTU97%共有比例较低。菱形金定鞭藻（Chrysoculter romboideus）的测序读段在深层叶绿素最大值层中显著检出，同时在表层样本中亦可被检测到。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。总体而言，本研究凸显了探索单细胞真核生物庞大且绝大多数尚未被认知的多样性时，固有的多项技术与理论壁垒。