Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution of the psilocybin biosynthetic gene cluster.

Psychoactive mushrooms in the genus Psilocybe have immense cultural value and have been used for centuries in Mesoamerica. Despite the recent surge of interest in these mushrooms due to the psychotherapeutic potential of their natural alkaloid psilocybin, their phylogeny and taxonomy remain substantially incomplete. Moreover, the recent elucidation of the psilocybin biosynthetic gene cluster is known for only five of ~165 species of Psilocybe, four of which belong to only one of two major clades. We set out to improve the phylogeny of Psilocybe using shotgun sequencing of fungarium specimens, from which we obtained 71 metagenomes including from 23 types, and conducting phylogenomic analysis of 2,983 single-copy gene families to generate a fully supported phylogeny. Molecular clock analysis suggests the stem lineage of Psilocybe arose ~67 mya and diversified ~56 mya. We also show that psilocybin biosynthesis first arose in Psilocybe, with 4 to 5 possible horizontal transfers to other mushrooms between 40 and 9 mya. Moreover, predicted orthologs of the psilocybin biosynthetic genes revealed two distinct gene orders within the biosynthetic gene cluster that corresponds to a deep split within the genus, possibly a signature of two independent acquisitions of the cluster within Psilocybe.

DNA Authentication and Chemical Analysis of Psilocybe Mushrooms Reveal Widespread Misdeterminations in Fungaria and Inconsistencies in Metabolites.

The mushroom genus Psilocybe is best known as the core group of psychoactive mushrooms, yet basic information on their diversity, taxonomy, chemistry, and general biology is still largely lacking. In this study, we reexamined 94 Psilocybe fungarium specimens, representing 18 species, by DNA barcoding, evaluated the stability of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens across the most commonly vouchered species (Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe semilanceata), and explored the metabolome of cultivated P. cubensis. Our data show that, apart from a few well-known species, the taxonomic accuracy of specimen determinations is largely unreliable, even at the genus level. A substantial quantity of poor-quality and mislabeled sequence data in public repositories, as well as a paucity of sequences derived from types, further exacerbates the problem. Our data also support taxon- and time-dependent decay of psilocybin and psilocin, with some specimens having no detectable quantities of them. We also show that the P. cubensis metabolome possibly contains thousands of uncharacterized compounds, at least some of which may be bioactive. Taken together, our study undermines commonly held assumptions about the accuracy of names and presence of controlled substances in fungarium specimens identified as Psilocybe spp. and reveals that our understanding of the chemical diversity of these mushrooms is largely incomplete. These results have broader implications for regulatory policies pertaining to the storage and sharing of fungarium specimens as well as the use of psychoactive mushrooms for recreation and therapy. IMPORTANCE The therapeutic use of psilocybin, the active ingredient in "magic mushrooms," is revolutionizing mental health care for a number of conditions, including depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spotlighted the current state of knowledge of psilocybin, including the organisms that endogenously produce it. However, because of international regulation of psilocybin as a controlled substance (often included on the same list as cocaine and heroin), basic research has lagged far behind. Our study highlights how the poor state of knowledge of even the most fundamental scientific information can impact the use of psilocybin-containing mushrooms for recreational or therapeutic applications and undermines critical assumptions that underpin their regulation by legal authorities. Our study shows that currently available chemical studies are mainly inaccurate, irreproducible, and inconsistent, that there exists a high rate of misidentification in museum collections and public databases rendering even names unreliable, and that the concentration of psilocybin and its tryptamine derivatives in three of the most commonly collected Psilocybe species (P. cubensis, P. cyanescens, and P. semilanceata) is highly variable and unstable in museum specimens spanning multiple decades, and our study generates the first-ever insight into the highly complex and largely uncharacterized metabolomic profile for the most commonly cultivated magic mushroom, P. cubensis.

New Species of Bioluminescent Mycena Sect. Calodontes (Agaricales, Mycenaceae) from Mexico.

Mycena section Calodontes is macromorphologically distinguished by the collybioid or mycenoid basidiome, which is pink, purple, or violet, and, rarely, reddish-brown or yellowish. It is further characterized by the presence of oxalate crystals in the basal mycelium. The section comprises approximately 40 taxa, of which only five species and one variety exhibit bioluminescence. As part of an extensive study on Mycena sect. Calodontes in Mexico, specimens belonging to this section were collected and subjected to morphological analysis. Sequences from the nuclear internal transcribed spacer (ITS) of nuclear ribosomal DNA, RNA polymerase II large subunit Rpb1 (rpb1), and translation elongation factor-1α (Tef-1α) were generated to infer the relationships within Mycena sect. Calodontes using maximum likelihood and Bayesian inference. The phylogenetic evidence, along with the macro- and micromorphological features, supported the recognition of five new bioluminescent species within Mycena sect. Calodontes. Detailed macro- and micromorphological descriptions, line-drawing illustrations, and light and dark photographs of the new species are provided.

New species and records of bioluminescent Mycena from Mexico.

Seven species of bioluminescent fungi are recorded from the cloud forests in Mexico. Six represent new species belonging to the genus Mycena, whereas Mycena globulispora is a new distribution record for the country. Descriptions, illustrations, photographs, and an identification key to bioluminescent fungi species from Mexico are provided. Sequences of nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region were generated for barcoding purposes and comparisons with similar species.

A new stipitate species of Crepidotus from India and Thailand, with notes on other tropical species.

A new Asian species of Crepidotus (Basidiomycota, Agaricales), C. asiaticus, is presented based on morphological and nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) and large subunit (28S) sequence data. This new species, found in India and Thailand, is characterized by the centrally stipitate medium-sized basidiomata, orange to reddish brown pileus, white to brownish orange lamellae, and white stipe. Based on morphology, C. asiaticus is similar to the neotropical C. thermophilus. However, the microscopic characters, especially the size and shape of the basidiospores, can be used to distinguish these two taxa, as well as their geographic distributions. Further, the phylogenetic position of C. asiaticus is unique based on ITS and 28S nuc rDNA sequences. Melanomphalia argipoda, described by Singer from Ecuador, is also a stipitate Crepidotus based on an ITS sequence of the type specimen, so the new combination is proposed here. Phylogenetically, the three species form a monophyletic group with the Asiatic C. asiaticus forming the sister lineage to the neotropical C. argipodus and C. thermophilus.