EAGER: Novel genome-based method to measure taxon-specific phytoplankton growth rates in natural communities

浮游植物是海洋中主要的初级生产者，即，海洋食物链的基础 它们贡献了全球氧气产量的一半，从而维持了地球上的所有生命。因此，浮游植物生长和产生生物量的速度是决定海洋肥力（鱼类生产的分布和规模）和浮游植物在海洋地球化学循环中的作用（例如，氧气生产、营养消耗）。 浮游植物生长速率的重要性在已经提出并应用于该问题的大量方法中是显而易见的。许多现有的方法在实验室中对浮游植物培养效果很好，但没有一种方法能够测量单个物种在其自然环境中的生长速度，作为一个非常复杂多样的自然群落的成员。该项目的研究人员将开发一种新的方法，该方法没有与现有方法相关的许多工件。 它将有助于确定作为自然群落组成部分的个别物种的增长率。 这种方法将使研究浮游植物对环境条件的反应-变化的温度或营养物质的可用性-独立于草食性浮游动物放牧造成的细胞损失。改进的生长速率信息将在浮游植物实地研究的各个方面以及将实地研究转化为模型和理论方面都是有用的。 作为该项目的一部分，两个全球重要的浮游植物属将在实验室中生长，生长率实验将纳入普林斯顿大学海洋学入门课程的教学实验室。因此，学生的研究将有助于浮游植物生理特征的基础知识，产生的数据是至关重要的，以了解是什么决定了自然组合的增长率。非培养方法的发展增加了我们对浮游植物多样性和代谢潜力的了解。然而，一般不可能将生物地球化学活动，如营养同化和生长，与自然组合中的单个分类群联系起来。也很难区分浮游植物瞬时生长率与损失的重要性，例如，在控制生物量积累率方面，放牧压力是一个重要因素，因为即使是最好的细胞损失量化方法也非常耗时，而且存在很大的不确定性。 这种区别是至关重要的，因为生物量积累是大多数生长率测定的基础。 所有这些方法都需要孵化和/或不能解释放牧，并且往往具有有限的分类分辨率，这限制了对生态系统功能的理解。该项目将开发一种基于基因组序列分析的新方法，称为iRep，用于测量特定分类群的生长速率。 该方法不需要孵育或时间序列采样，并已被其他人应用于评估复杂的细菌群落，如人类肠道微生物组。 将使用聚球藻（在亚热带海洋的广阔区域中占主导地位的生物量和生产的蓝细菌）培养物来测试和验证该方法。 此外，将iRep应用于真核浮游植物的第一步将通过表征叶绿体DNA（cpDNA）的周转和确定角毛藻（硅藻，往往占主导地位的生物量的上升流区域）的cpDNA复制的起源。这个EAGER建议集中在两组实验，以建立该方法的潜力：1。iRep方法在聚球藻培养和人工混合聚集体中的应用。2.叶绿体DNA更新与硅藻比生长速率关系的测定。关于真核藻类中的cpDNA复制模式知之甚少，高等植物中的cpDNA复制问题仍然存在争议。分类特异性生长速率测定是海洋学长期探索的目标。因此，这个探索性的建议是一个很好的适合EAGER计划。

Phytoplankton are the main primary producers in the ocean, i.e., the basis of the marine food chain. They contribute about half of the global oxygen production, and thereby sustain all life on earth. Thus, the rate at which phytoplankton grow and produce biomass is a key variable in determining the fertility of the seas (the distribution and magnitude of fish production) and the role of phytoplankton in biogeochemical cycles (e.g., oxygen production, nutrient consumption). The importance of phytoplankton growth rate is evident in the plethora of methods that have been suggested and applied to the problem. Many of the existing methods work well for phytoplankton cultures in the lab, but none of them are capable of measuring the growth rate of an individual species in its natural environment, as a member of a very complex diverse natural community. The investigators on this project will develop a new method, which is free of many of the artifacts associated with existing methods. It will allow the determination of growth rates of individual species as components of natural assemblages. This method will enable the study of phytoplankton response to environmental conditions -- changing temperature or nutrient availability -- independently of cell loss from grazing by herbivorous zooplankton. Improved growth rate information will be useful in just about every aspect of phytoplankton field research and in translating that field research into models and theory. As part of the project, two globally important phytoplankton genera will be grown in the laboratory, and growth rate experiments will be incorporated into the teaching laboratory for the introductory Oceanography course at Princeton University. Student research will thus contribute fundamental knowledge on phytoplankton physiological traits, producing data that are crucial to understanding what determines growth rate in natural assemblages.The ability to obtain DNA sequence information from natural seawater samples, i.e., the development of culture-independent methods, has increased our understanding of the diversity and metabolic potential of phytoplankton. It is generally not possible, however, to link biogeochemical activities, such as nutrient assimilation and growth, to individual taxa in natural assemblages. It is also difficult to distinguish the importance of phytoplankton instantaneous growth rate vs. losses to e.g., grazing pressure, in controlling biomass accumulation rates because even the best methods for quantifying cell loss are very time-consuming and subject to large uncertainties. This distinction is crucial because biomass accumulation is the basis of most assays for growth rate. All these methods require incubations and or cannot account for grazing, and often have limited taxonomic resolution, which limits understanding of ecosystem functioning. This project will develop a novel method for taxon-specific growth rate measurement, called iRep, that is based on genome sequence analysis. The method does not require incubations or time series sampling, and has been applied by others to evaluate complex bacterial communities such as the human gut microbiome. It will be tested and validated using cultures of Synechococcus (cyanobacteria that dominate the biomass and production in vast expanses of the subtropical ocean). In addition, the first steps towards applying iRep to eukaryotic phytoplankton will be undertaken by characterizing turnover of chloroplast DNA (cpDNA) and determining the origins of cpDNA replication in Chaetoceros (diatoms that often dominate the biomass of upwelling regions). This EAGER proposal focuses on two sets of experiments to establish the potential of the method:1. Application of the iRep method to Synechococcus in culture and artificial mixed assemblages. 2. Determination of the relationship between cpDNA turnover and diatom specific growth rate. Very little is known about the mode of cpDNA replication in eukaryotic algae, and controversy still exists over the question of cpDNA replication in higher plants. The goal of taxon-specific determination of growth rate has been a long term quest in oceanography. Thus this exploratory proposal is a good fit for the EAGER program.