Dimensions: Collaborative Research: Functional Diversity of Marine Eukaryotic Phytoplankton and Their Contributions to the C and N Cycling

知识价值：浮游植物构成了海洋食物网的基础，因此是生物泵中的关键元素，通过生物泵，大气中的二氧化碳被隔离在深海中。几十年来，生物海洋学一直专注于真核浮游植物。20世纪70-80年代，人们发现海洋中的单细胞微囊藻在数量上占主导地位，并负责海洋光合作用的很大一部分。随之而来的是一种新的范式的增长，在这种范式中，苦味细菌主导了上层海洋生物学和生物地球化学。事实上，新的数据支持了经典的观点，即真核浮游植物在N和C循环中都不成比例地重要，即使在非常小的细胞主导生物量的地区，以及真核生物本身处于“微微”大小的部分。在营养稀少的环境中，非常小的细胞占主导地位，原位回收似乎提供了初级生产所需的大部分氮(即铵)。然而，新的硝酸盐供应必须平衡系统中的氮损失，新的数据表明，这种硝酸盐是浮游植物氮的重要贡献，即使在海洋沙漠中也是如此。目前尚不清楚这些系统中的硝酸盐供应是由整个群落使用，还是主要由较大的浮游植物(基本上完全是真核生物)使用。根据仍然相当有限的分子调查，我们现在认识到，大型和小型真核浮游植物的多样性比之前认为的要大，最丰富和最广泛的真核生物可能不在培养中，也可能与已知的栽培生物没有密切关系。这个项目将调查北大西洋两个地点(亚北极和亚热带)在两个季节的真核浮游植物的分类、遗传和功能多样性。PIS将使用诊断微阵列进行基于功能基因(DNA和RNA)的群落分析和下一代测序(即使用454技术的转录组学)，以确定参与者的群落组成和活动，并探索自然组合的功能多样性。为了确定哪些基团在碳氮同化中是活跃的，以及哪些氮源被不同大小和不同功能基团所利用，将使用过滤分离和流式细胞仪分选的样品来1)通过同位素示踪剂孵育来测量13C初级产物和15N同化，2)测量不同分类基团的自然稳定的N同位素特征，3)将分子多样性与C和N转化中的功能多样性联系起来。利用与质谱学相结合的流式细胞术，这些研究人员发现了出人意料的强烈分化，以被原核生物和真核生物同化的N的形式存在，其中真核生物更具活力。整合：该项目将调查真核浮游植物的分类、遗传和功能多样性，并将这种多样性和组装组成与表层海洋的碳和氮生物地球化学联系起来。将通过使用分子、化学和显微镜方法确定浮游植物组合的组成来调查分类多样性。将在几个层面上探索遗传多样性，包括对关键功能基因克隆文库的直接测序，以及对大小分级/分类的浮游植物组合的后转录测序和微阵列分析。利用自然丰度和示踪稳定同位素方法，遗传和分类多样性将与大小分离和分类分类的种群中碳和氮同化的功能多样性联系在一起。更广泛的影响：该项目的更广泛影响包括对基础研究和教育的贡献：1)继续开发环境样品中先进的新方法，在生物地球化学和生物多样性方面的应用日益广泛；2)在气候和环境科学的基础课程中进行本科教学，以招收理科专业的一年级和二年级学生；3)通过实习和高级论文研究(普林斯顿大学的要求)获得本科研究经验；4)通过研究生一级的课堂和研究经验，培训下一代微生物生态学/生物地球化学研究人员。此外，还将为普林斯顿中学教师的外展计划创建一个新的模块(海洋的森林和沙漠)(探索、提问是有效的科学教学的基础)。

Intellectual Merit: Phytoplankton form the basis of the marine food web and thus are a crucial element in the biological pump whereby CO2 from the atmosphere is sequestered in the deep ocean. For decades, biological oceanography focused on the eukaryotic phytoplankton. In the 1970-80s, it was discovered that the single celled picocyanobacteria are numerically dominant in the oceans and are responsible for a large fraction of ocean photosynthesis. What ensued was the growth of a new paradigm in which the picocyanobacteria dominate upper ocean biology and biogeochemistry. In fact, new data support the classic view that the eukaryotic phytoplankton are disproportionately important in both N and C cycling, even in regions where very small cells dominate the biomass and where the eukaryotes themselves are in the "pico" size fraction. In oligotrophic environments, where very small cells dominate, in situ recycling appears to supply most of the nitrogen (i.e., ammonium) required for primary production. New nitrate supply must balance N loss from the system, however, and new data suggest that this nitrate is an important contribution to phytoplankton N, even in the oceanic deserts. It is not known whether the nitrate supply in these systems is used by the entire assemblage or predominantly by larger phytoplankton (essentially entirely eukaryotic). On the basis of still quite limited molecular surveys, we now recognize that the diversity of both large and small eukaryotic phytoplankton is greater than previously thought and that the most abundant and widespread eukaryotes are probably not in culture and may not be closely related to known cultivated organisms. This project will investigate the taxonomic, genetic and functional diversity of eukaryotic phytoplankton at two North Atlantic sites (subarctic and subtropical) in two seasons.The PIs will use diagnostic microarrays for community analysis based on functional genes (both DNA and RNA) and next generation sequencing (i.e., transcriptomics using 454 technology) to identify the players, both in terms of community composition and activity, and to explore the functional diversity of the natural assemblage. In order to identify which groups are active in C and N assimilation and which N source is being utilized by the different size and functional groups, both filter-separated and flow cytometry-sorted samples will be used to 1) measure 13C primary production and 15N assimilation by incubations with isotope tracers, 2) measure the natural stable N isotope signatures of different taxonomic groups and 3) link the molecular diversity to the functional diversity in C and N transformations. Using flow cytometry linked to mass spectrometry, these investigators have found an unexpectedly strong differentiation in the form of N assimilated by prokaryotes and eukaryotes, with eukaryotes being more dynamic.Integration: This project will investigate the taxonomic, genetic and functional diversity of eukaryotic phytoplankton and to link this diversity and assemblage composition to the carbon and nitrogen biogeochemistry of the surface ocean. Taxonomic diversity will be investigated by identifying the components of the phytoplankton assemblages using molecular, chemical and microscope methods. Genetic diversity will be explored at several levels, including direct sequencing of clone libraries of key functional genes and metatranscriptomic sequencing and microarray analysis of size fractionated/sorted phytoplankton assemblages. Using natural abundance and tracer stable isotope methods, genetic and taxonomic diversity will be linked to functional diversity in C and N assimilation in size- fractionated and taxon-sorted populations.Broader Impacts: The broader impacts of this project include contributions to fundamental research and education: 1) continued development of new advanced methods of isotope analysis in environmental samples, with increasing breadth of applications in biogeochemistry and biodiversity; 2) undergraduate teaching in foundation courses on climate and environmental science to recruit freshmen and sophomore students into the science majors; 3) undergraduate research experience through internships and senior thesis research (a requirement at Princeton) for upper level undergraduates; 4) training the next generation of microbial ecology/ biogeochemistry researchers through classroom and research experience at the graduate level. In addition, a new module will be created (The Forests and Deserts of the Ocean) for the Princeton outreach program for middle school teachers (QUEST, Questioning Underlies Effective Science Teaching).