Marine heterotrophic alveolates: a genomic and morphometric single cell approach.

微生物真核生物（原生生物）显然占大多数的真核生物物种，但很少有分类单位已从形态和分子标准进行了分类学研究，因为只有有限的序列数据。对于异养生物，已知和未知微生物真核生物之间的差距甚至更大，因为大多数描述和基于核酸的研究依赖于实验室培养，并且异养生物往往比自养生物更难培养。新开发的单细胞方法，如单细胞PCR，使分类无需培养，但大多数基于核酸的研究仍然几乎完全依赖于已公布的序列，使未知物种不可见的不良或完全未分配的序列。此外，关于异养生物的数据几乎完全缺乏从metatranscriptomics的新领域。因此，显然需要对新序列进行广泛的注释，并对相应的异养物种进行正确的分类。在这项研究中，我们将重点放在海洋异养原生生物，即肺泡，因为有时他们代表的浮游生物量的主要组成部分，将生物物质转移到更高的营养水平，并可能在海洋生态系统中发挥关键的功能作用。我们将在格陵兰岛进行冬季和夏季实地研究，并在Spitzbergen周围进行巡航，以研究异养群落的演替过程及其生物多样性在年度变化中的差异。通过开发一个工具箱，将显微镜、单细胞多重聚合酶链反应、单细胞转录组学和对所收集的环境样本的元分析结合起来，将实现为完成数据库收集全面数据的目标。通过单细胞PCR和核糖体DNA（SSU，LSU和ITS）测序，我们将获得用于物种分类或鉴定的序列，并将鉴定隐蔽物种。通过ITS 2 rRNA二级结构建模和补偿性碱基变化（CBC）将实现更高的序列分辨率。光学显微镜分析将有助于找到与序列数据一致的已建立的和新的形态分类特征。此外，将对微生物拍照和/或拍摄并固定存档。对于选定的未充分研究的物种，单细胞转录组学将用于开发新的分类标记，并填补元转录组学数据库中的主要空白。对非生物和生物环境数据的元分析将完善物种数据集，以提供更多的地理信息。现有的数据库将提供全面的数据收集后，彻底修订和命名分析。新的工具箱将有助于今后的识别，所获得的数据将大大丰富现有的数据库，从而更全面地了解微生物真核生物的多样性及其在海洋生态系统中的功能作用。

Microbial eukaryotes (protists) apparently account for the majority of eukaryotic species, but comparably few taxa have been taxonomically investigated from both morphological and molecular criteria, as there are only limited sequence data. The gap between known and unknown microbial eukaryotes is even higher for heterotrophs, because most descriptions and nucleic acid based investigations depend upon laboratory cultures and heterotrophs tend to be more difficult to cultivate than autotrophs. Newly developed single cell methods, such as single cell PCR, enable classification without cultivation, but most nucleic acid based studies still rely almost entirely on published sequences, rendering unknown species invisible as poorly or completely unassigned sequences. Furthermore, data about heterotrophs is almost completely lacking from the new field of metatranscriptomics. The need for an extensive annotation of new sequences and correct taxonomic classification of the respective heterotrophic species is therefore evident. In this study, we will focus on marine heterotrophic protists, i.e. alveolates, since at times they represent the major component of planktonic biomass, transferring biological matter to higher trophic levels, and may play key functional roles in marine ecosystems. We will conduct a winter and summer field study in Greenland and a cruise around Spitzbergen to study the successional processes of heterotrophic communities and the differences in their biodiversity during annual shifts. The goal of collecting comprehensive data for completing databases will be reached with the development of a toolbox integrating microscopy, single cell multiplex PCR, single cell transcriptomics and meta-analysis of the collected environmental samples. With single cell PCR and sequencing of ribosomal DNA (SSU, LSU and ITS), we will obtain sequences for classification or identification of species and will identify cryptic species. Higher sequence resolution will be achieved with ITS2 rRNA secondary structure modeling and compensatory base change (CBC). Light microscopic analysis will serve to find established and new morphological classification features congruent with sequence data. Additionally, the organisms will be photographed and/or filmed and fixed for archiving. For selected understudied species, single cell transcriptomics will be utilized to develop new classification markers and to fill major gaps in metatranscriptomic databases. Meta-analyses of abiotic and biotic environmental data will complete the dataset of the species to offer additional biogeographical information. Existing databases will be supplied with the collected comprehensive data after thorough revision and nomenclatural analysis. The new toolbox will facilitate future identification and the data obtained will substantially enrich existing databases, thereby yielding a more comprehensive view of diversity of microbial eukaryotes and their functional role in marine ecosystems.