Cells with nuclei—eukaryotes—existed exclusively as microorganisms 0.5 – 1.5 billion years before the evolution of plants, animals, and fungi. These microbial eukaryotes, or protists, are a diverse assemblage of organisms that encompass the phylogenetic breadth of eukaryotic evolution. Protists are characterized by numerous innovations in cell biology (e.g. multiple acquisitions of chloroplasts), play an essential role in ecosystems (e.g. carbon fixation in marine systems), and some are causative agents of prevalent infectious diseases (e.g. malaria) that impact the social and economic fortunes of entire countries.

Our collaborative, interdisciplinary approach will elucidate relationships among lineages of predominantly free-living microbial eukaryotes. To achieve this goal, DNA sequences of nine genes will be determined from at least 200 protist species. The sampling plan includes well-circumscribed taxonomic diversity of eukaryotes, but also extends to taxa of as yet unknown affinities. Phylogenetic analyses of these data will combine existing approaches with newly developed methods for partitioning multigene data. The resulting comprehensive phylogeny is essential for: 1) unifying the universal tree of life that includes both prokaryotes and eukaryotes, 2) understanding the multiple origins of multicellular eukaryotes, and 3) interpreting the origins of disease-causing protists.

The project is based in six laboratories at five institutions: the Burgess laboratory (ATCC) will focus on generating DNA and, in some cases, RNA; the Katz (Smith College), Bhattacharya, and Logsdon (Univ. Iowa) laboratories are responsible for acquisition and analysis of the molecular data; and the Patterson lab (MBL) leads the microscopy training and incorporates the findings of this project into the micro*scope web resource. As lead PI, Katz oversees coordination of the project, including DNA acquisition and distribution, and annual meetings. The Univ. of Iowa provides the central facility for phylogenetic analysis and database maintenance. Finally, the Huelsenbeck laboratory (UCSD) provides expertise in phylogenetic analyses and will further develop data partitioning methods.

Broader Impacts and Educational Component: The proposed study will invigorate protist research in the U.S. while answering fundamental questions about the eukaryotic tree of life.

• "Protist Diversity" workshop: We will offer a workshop on collection and identification (by light microscopy) of protists. Students will also be exposed to ultrastructural data. No such course exists in the U.S., and we expect fewer than half of participants will be from our labs.
• Graduate and post-doctoral training: Three postdoctoral fellows and three graduate students will be integrated into the proposed research. These participants will be trained in both molecular and microscopy techniques.
• Undergraduate training: PIs will seek REU supplements to train undergraduates, with a continued commitment to recruiting students from traditionally underrepresented groups. The PIs will also incorporate aspects of this proposal into courses they teach.
• Other outreach: This project will expand micro*scope, a web-based tool for exploring eukaryotic diversity (http://www.mbl.edu/microscope). We will develop micro*scope into a central resource for information and education on microbial diversity. Further, through its use of the Universal Biological Indexer and Organizer, micro*scope offers a mechanism for making taxon-specific links with other tree of life sites. Finally, our project will inform future decisions about expansion of the protists at the American Type Culture Collection.