Phylogenomic expansion within the genus Psilocybe and molecular evolution of the psilocybin-producing gene cluster

The mushroom genus Psilocybe s.l contains approximately 300 described species worldwide and is best known as the core group of psychedelic mushrooms. Psilocybe mushrooms have immense cultural value and have been used for centuries in indigenous Mesoamerica. In more recent years, Psilocybe spp. and their psychoactive compounds, psilocybin, and psilocin, have been targeted as a promising therapeutic for a growing mental health crisis. Nevertheless, a thorough understanding of the diversity, taxonomy and general biology of Psilocybe is very incomplete. To date, molecular diversity studies of the Psilocybe genus are severely lacking, and much of the known diversity has no evolutionary context. In this work, we set out to improve the predictive power to investigate questions about the evolution of Psilocybe spp. and their psychoactive chemistry by generating a more representative and highly supported phylogeny than has been available previously. We sequenced the metagenomes of 71 fungarium specimens vouchered as Psilocybe, including 23 types, expanding the diversity of Psilocybe genomic data nearly twentyfold. Further, using phylogenomic analysis of 2,983 single-copy genes, we produced a robust phylogeny with all branches receiving strong statistical support. Our results largely confirm two major clades, Clade I and Clade II, previously recognized and provide new data on species relationships not previously known. Utilizing ab initio gene predictions, our results also reveal two distinct gene orders within the psilocybin biosynthetic gene cluster that show both phylogenetic and ecological correlations. Using this new phylogenomic framework, we investigate the evolution of psilocybin-producing gene clusters within Psilocybe and other non-Psilocybe genera, creating a better understanding of the acquisition of psilocybin production outside of Psilocybe. This new information will be useful for a formal, molecular phylogenetic-based classification and future routes of the study of Psilocybe.