Zoosporic fungi (fungi that reproduce through motile spores) represent the earliest lineages in the fungal tree of life; unfortunately, they are also the most poorly understood. Limited sampling of these fungi, especially from marine and estuarine habitats, constrains attempts to reconstruct the early evolution of the kingdom Fungi. The primary hindrance to uncovering the full extent of zoosporic fungal diversity is the historical reliance on culture-dependent methods, resulting in the description of only the most abundant taxa, or those amenable to isolation in the laboratory. The recent use of culture-independent molecular methods to characterize fungal diversity and community composition in terrestrial, freshwater, and extreme environments has revealed novel early-diverging fungal lineages. Additionally, these studies have found that zoosporic fungal lineages are often the dominant fungal component of their respective ecosystems. As environmental cloning studies have high time and supply costs, next-generation sequencing has been promoted as a rapid, cost-effective method for characterizing communities of cryptic microorganisms in previously neglected habitats. Several metagenomic analysis pipelines and reference databases now exist to assist in the clustering and taxonomic identification of molecular sequences recovered from environmental samples. However, the available databases are populated with large numbers of sequences from previous environmental cloning studies outside a phylogenetic framework and without a corresponding taxonomic classification, hindering efforts to classify poorly known groups. To address these issues, the research proposed herein aims to: 1) use culture-independent methods to discover novel marine fungal taxa; 2) infer the phylogenetic relationships of these taxa with all known zoosporic counterparts, consisting primarily of terrestrial and freshwater fungi; and 3) use the phylogenetic dataset produced for objective 2 to date the loss(es) of the fungal flagellum and estimate the minimum age(s) of the adoption of a primarily terrestrial lifestyle in the evolution of the Fungi.The proposed research will provide training for a motivated undergraduate student, in addition to informing the development of child-focused interactive exhibits on fungi and their symbiotic interactions with plants and animals for two local science museums (North Carolina Museum of Life and Science, Durham, NC; North Carolina Museum of Natural Sciences, Raleigh, NC).