Neotropical ants of the Attini tribe evolved the innate ability to farm fungi. Agaricomycetidae (mushroom forming fungi) contains two clades that are cultivated by ants: the lepiotaceous and pterulaceous cultivars. However, there are free-living relatives phylogenetically distributed throughout each cultivar clade. Comparison between the free-living relatives and attine system cultivars may identify the evolutionary differences caused by, or initially enabling, agricultural symbiosis. Our research compares free-living relatives and cultivars through genomic and metabolic analysis. We first performed DNA sequencing of the ITS barcode region and then whole genome sequencing of the cultivars. ITS sequencing allowed us to build phylogenetic trees to examine the relationships between the free-living fungi and cultivars. Whole genome sequencing allowed genomic cultivar to cultivar comparison. Attines undertake significant cultivar maintenance through the regulation of pathogenic contamination of their fungal "gardens" as well as the provision of specific growth substrates, including frass (insect feces). Noting the apparently specialized substrates cultivars receive from the ants, we conducted a pilot test for a growth media preference between a lepiotaceous cultivar, a pterulaceous cultivar, and a free-living pterulaceous relative. We placed the fungi on regular PDY media and PDY media infused with caterpillar frass and observed their growth. Cultivars either changed growth form or had improved growth on the frass-infused media. Following the pilot test, we will run metabolic assays on the cultivars and free-living relatives on different media types for further investigation that may indicate a media preference and give further insight to the relationship between the cultivars and free-living relatives. By looking at fungal metabolic assays and genomic data, we hope to gain insight into the fungi's evolutionary history and agricultural symbiosis.