Award: OCE-1153588

INTELLECTUAL MERIT Ocean element cycles (e.g. carbon, nitrogen, and phosphorus) are largely driven by microorganisms and the metabolisms encoded by their genes. These properties are distributed across a diverse community of microbial cells which act in concert to move energy and matter through the ecosystem. But how are these systems assembled and what mechanisms are responsible for the ?division of labor? within and between different sub-species or ?ecotypes?? Studies of Prochlorococcus, the smallest and most abundant oxygen evolving photosynthetic cell in the oceans, have shed light on this question. The 3 billion billion billion Prochlorococcus cells in the global ocean encompasses an immense level of diversity. Our ability to map this diversity onto environmental gradients provides a powerful lens into key selective pressures that shape the functional capacity of Prochlorococcus populations. In our project, we explored a single ecologically important trait in Prochlorococcus: the ability of cells to use nitrate as a nitrogen source. Nitrogen is often an important limiting factor for the growth of phytoplankton which in turn consume carbon dioxide and supply fixed carbon to higher levels of the food web. Nitrate is an abundant inorganic nitrogen source in the oceans and can fuel the growth of photosynthetic organisms in the sunlit surface layer when nitrate is supplied from deeper nutrient rich water. After the first isolates of Prochlorococcus were obtained in the early 1990s, it was observed that no Prochlorococcus could use nitrate for growth. This suggested, surprisingly, that there was a large standing stock of photosynthetic organisms that were unresponsive to stimulation by nitrate. Later, we discovered that some fraction of Prochlorococcus could indeed use nitrate, isolated these cells, and through the support of this grant examined the first genome sequences of cultured Prochlorococcus capable of assimilating nitrate. We designed protocols to enumerate Prochlorococcus harboring the trait for nitrate assimilation in the wild and followed seasonal changes in their abundance in the North Pacific and North Atlantic subtropical gyres. Up to 50% of high-light adapted Prochlorococcus, typically the most abundant Prochlorococcus subgroup in surface waters, had the genes required for nitrate assimilation. Highest abundances were observed when overall nitrogen concentrations were low and likely limiting production. Thus, Prochlorococcus with access to a wide pool of potential nitrogen sources are at a selective advantage when nitrogen is a limiting resource. When understanding the role that nitrate has in fueling marine photosynthesis, we must now recognize Prochlorococcus as an important player and take into account how populations are assembled and how nitrogen assimilation potential varies within those populations. We have also uncovered distinct evolutionary processes that occur on different timescales that drive the observed diversity of nitrate assimilation in Prochlorococcus. Exploring the genomes of over 500 Prochlorococcus wild single cells and cultured isolates, it was observed that this trait was patchily distributed among a few of the recently evolved ecotypes and virtually absent from the others, likely reflecting the partitioning of ecotypes into different layers in the ocean water column. As new ecotypes appeared and expanded within the surface layers, others were pushed deeper into layers with elevated nitrogen concentrations. Random loss of the trait across ecotypes resulted in the relative retention of the trait in ecotypes in environments where nitrate assimilation can be advantageous. Near elimination of the trait is observed in other ecotypes when costs outweighed the benefits. These evolutionary trajectories have likely helped shape the biogeochemistry of the contemporary ocean by facilitating consumption of inorganic nitrogen at the surface. This work illustrates the power of combining genomic studies of a model organism with detailed knowledge of its ecology to better explore the evolutionary forces that shape marine ecosystems. BROADER IMPACTS The outcomes of our work have the potential to benefit society through a better understanding of marine microbial ecosystems. Prochlorococcus are expected to increase in abundance as the ocean warms over the coming century. Developing baseline information about the inherent diversity of these cells and how they respond to environmental perturbation will enhance our ability to predict the function of future marine ecosystems. This grant supported in part the development of a large genomic data set of Prochlorococcus, benefiting the extended research community. These data will facilitate studies on evolution, ecology, and the potential development of biofuels. Throughout the lifetime of the grant, the principle investigator has published a series of children?s books (The Sunlight Series) in order to instill an appreciation of photosynthesis in our younger generation. She has also presented this work to general audiences, most recently at TED2018, to promote a better understanding of the importance of marine ecosystems to human society. The project has further supported the training of 4 technicians who have used their experience on the project to move onto the next stages of their career; 3 are now in Ph.D. programs and one is in the biotechnology industry. Last Modified: 04/30/2018 Submitted by: Sallie W Chisholm