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 plastids), play an essential role in ecosystems (e.g. carbon fixation in marine systems), and some are the causative agents of prevalent infectious diseases (e.g. malaria).

This proposal uses a collaborative, interdisciplinary approach to elucidate relationships among predominantly free-living microbial eukaryotes. To achieve this goal, DNA sequences of nine target genes (actin, a- and b- tubulin, cob , EF-1 a , Hsp70, Hsp90, RPB1, SSU rRNA) will be determined from at least 200 protist species. The sampling plan will capture the known taxonomic diversity of eukaryotes, while also positioning taxa of currently unknown affinities. Phylogenetic analyses of these data will combine existing approaches with newly developed methods for partitioning combined multigene data. The resulting comprehensive phylogeny will be critical to many issues, including : 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 will be 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 will be responsible for the acquisition and analysis of the molecular data; and the Patterson lab (MBL) will lead the microscopy training and incorporate the findings of this project into the micro*scope web resource. As lead PI, Katz will oversee coordination of the project, including DNA acquisition and distribution, and annual meetings. The University of Iowa will provide a central facility for phylogenetic analysis and database maintenance. Finally, the Huelsenbeck laboratory (UCSD) will provide 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. To achieve these goals, the proposal includes the following training and outreach components:

• 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 biology and microscopy techniques.
• Undergraduate training: PIs will seek REU supplements to train undergraduates, with a continued committed to recruiting students from traditionally underrepresented groups. The PIs will also incorporate aspects of this proposal into courses they teach.
• Community outreach: We will expand micro*scope, which is 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 educational resources on microbial diversity. Through use of the Universal Biological Indexer and Organizer that is being developed for all life forms, micro*scope will provide a mechanism for linking to other tree of life sites.