Fungi are essential to ecosystem functioning, occur virtually everywhere including extreme habitats, and form complex mutualisms with diverse organisms including prokaryotes, algae, plants, invertebrates, vertebrates, and other fungi. Fungi are the most undersampled and poorly documented major lineage of eukaryotes, even though total estimates range conservatively from 1.5 to 6.1 million species. This uncertainty is a reflection of numerous gaps in knowledge about fungal distributions, especially in regard to range and extent of unculturable fungi, microfungi, parasitic fungi, and tropical fungi. In this project we aim to document diversity of fungi in two orders—Laboulbeniales, unculturable microfungal parasites of arthropods, and Pucciniales, unculturable microparasites of plants. These lineages were targeted 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, a phenomenon that is extremely rare in fungi. We have assembled a complimentary team of global experts on these fungi, ranging from junior to senior scientists, and 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) determine whether parasitic microfungi follow the reverse latitudinal gradient posited for other fungal groups; (3) resolve long-standing unresolved phylogenetic nodes by the incorporation of “missing” lineages; and (4) compare genome architecture for paired heteroecious and autoecious taxa to determine if genomes of heteroecious species do not conform to the genome streamlining of other obligate parasites.