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）是海洋原生生物的关键门类，目前已报道约300个已描述形态种及80个形态属。本研究针对那不勒斯湾采集的分级过滤浮游生物样本，通过454焦磷酸测序（454 pyrosequencing）分析大亚基核糖体DNA（LSU rDNA）片段的多样性。我们设计了一套靶向LSU rDNA D1/D2区域的群体特异性引物组，用于扩增两种粒径分级（0.8–3 μm或3–20 μm）、两种采样深度[表层下1米处，或23米处的深层叶绿素最大值层（DCM）]的核酸提取物（总DNA或总RNA）中的定鞭藻序列。我们采用覆盖当前已测序全定鞭藻门多样性的数据库，对454测序读段进行物种注释。本数据集共鉴定出数百个定鞭藻聚类簇，但其中绝大多数无法与已分类的已知序列匹配：对全局数据集进行97%序列相似度阈值下的操作分类单元（OTUs97%）分析后发现，仅有不足1%的读段与培养物来源序列聚类，不足12%的读段与此前通过环境样本克隆及Sanger测序获得的参考序列聚类。由此可见，当前已培养物种远不足以反映自然环境中的真实多样性，本研究揭示了大量未被鉴定的环境遗传多样性。定鞭藻群落结构随深度呈现显著分化：深层叶绿素最大值层样本的多样性与均匀度最高，且该层中大粒径分级（3–20 μm）样本与其余样本的共有97%相似度OTUs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显著检出，同时亦可在表层下样本中检测到。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97%相似度OTUs占比最高。综上，本研究凸显了在探索单细胞真核生物庞大且绝大多数尚未探明的多样性过程中固有的多项技术与理论瓶颈。