Humans are made up of many cells, but for billions of years, life on earth consisted of unicellular organisms whose bodies were a single cell. Why and how unicellular organisms evolved into multicellular individuals is a major unsolved question in biology. Answering this question will help scientists understand one of the more familiar features of life on earth, which is its hierarchical structure. Genes exist in chromosomes, chromosomes exist in cells, cells exist in multicellular organisms, and multicellular organisms exist in complex societies. Each of these levels in the hierarchy of life represents a level of organization on which natural selection may act. During evolution, each one of these levels has arisen from a group of lower-level units. How did groups of cells evolve into a new multicellular individual is the basic question addressed by this proposal. The researchers approach this question by testing a general theory of evolutionary transitions. The researchers present a novel hypothesis that stress responses are instrumental in the origin of cell groups and in the origin of specialized types of cells that arise in these groups. They will test this hypothesis in the volvocine green algae, a lineage of organisms that span hierarchical levels from single cells to multicellular individuals. Testing this hypothesis will help the scientists to better understand human disease. For example, the evolution of cancer in a human body is the reversal of the processes studied in the research. A cancerous cell has stopped cooperating with other cells in the multicellular group and has regained its capacity to evolve at the single cell level. In other words, cancer is a disease in which individuality changes from the level of the multicellular group back to the level of the cell. The project will result in training of undergraduate and graduate students and also will develop curricula to better teach concepts related to the evolution of complex organisms to middle school students. The proposed work will provide societal benefits through the development of methods of high-throughput phenotyping and imaging of cell groups that may help with medical diagnosis. Evolution by natural selection requires heritable variation in fitness at the individual level. During evolutionary transitions in individuality, such as the evolution of multicellular individuals, fitness must be remapped from the cell level to the new level of the multicellular individual. Previous work has shown that stress responses can be a major impetus for the reorganization of fitness during the transition to multicellular individuality. There are two basic criteria of multicellular individuality investigated in the work: group inseparability and somatic cell division of labor. The researchers have shown that these two individuality criteria respond to stress in species with intermediate complexity and levels of individuality. The researchers also will examine the phenotypic and underlying genetic responses to stress that affect group formation and the origin of new cell types. The application of phylogenetic methods to reconstruct the evolutionary history of plastic somatic cells will allow the researchers to determine whether the plastic development of somatic cells preceded or followed the origin of key genes necessary for cellular differentiation. Furthermore, they will use experimental evolution to test whether obligate cellular differentiation evolves in lab populations under the conditions predicted by their models. Finally , the researchers will identify the genetic basis for these stress responses via experiments utilizing gene expression analyses and gene knockouts to understand the role played by gene co-option in the origin of multicellularity. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.