Fungi are essential to ecosystem functioning, occur virtually everywhere, and form complex relationships with diverse organisms including prokaryotes, algae, plants, invertebrates, vertebrates, and other fungi. Nonetheless, fungi are the most undersampled and poorly documented major lineage of eukaryotes, even though total species estimates range conservatively from 1.5–6.1 million species. This uncertainty reflects numerous gaps in knowledge about fungal distributions, especially regarding range and extent of unculturable fungi, microscopic fungi (microfungi), parasitic fungi, and tropical fungi. This project will document diversity of fungi in two groups that represent major gaps in our knowledge—the first are unculturable microfungi that associate with insects, called Labouls, and the other are unculturable microfungi that associate with plants, called Rust Fungi. Members of Labouls and Rust Fungi are expected to have profound effects on ecosystem health and functioning through both beneficial and harmful interactions with their animal and plant hosts. The research team will explore several global habitats in tropical and temperate regions that have not previously been surveyed for these fungi. It is expected that hundreds of new species will be discovered and described during this project. In addition to enhancing the documented biodiversity on the planet, this project will resolve the “tree of life” for both groups and use this information to resolve long-standing questions regarding the evolution of the Fungi. Broader impacts include graduate and undergraduate training, international workshops to enhance collaboration between US and local researchers, and outreach to the public through “Bat Night” and “Roach Hunt” field trips.<br/><br/>These fungal lineages are being studied for several reasons: 1) both represent groups poorly documented from tropical world regions; 2) both represent groups that are not detected by commonly applied culture-based and high throughput sequencing efforts; 3) neither group has been resolved, phylogenetically, by application of multi-locus analyses of known species; 4) both represent obligately parasitic lineages that are rarely studied but expected to have profound effects on ecosystems; and 5) both are or are related to heteroecious fungi (requiring two different hosts), a phenomenon that is extremely rare in Fungi. A complementary team of US and other global experts on these fungi, ranging from junior to senior scientists, have designed an experimental approach that includes standardized sampling strategies from tropical and extra-tropical regions with multi-locus phylogenetic, phylogenomic, comparative genomic, and statistical analyses to: 1) Determine whether accurate estimates from obligate microparasitic fungi, not included in any other estimates, will affect overall Fungi species estimates; 2) whether parasitic microfungi follow the reverse latitudinal gradient posited for other fungal groups; and 3) resolve long-standing unresolved phylogenetic nodes by the incorporation of “missing” lineages.<br/><br/>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.