Bryn T. M. Dentinger

The most famous chemicals produced by mushrooms are the psychedelic compound psilocybin from “magic mushrooms” and amatoxins from deadly poisonous mushrooms. These compounds are known to occur in multiple phylogenetically disjunct fungal lineages but have never been shown to co-occur within a single species. Here we show that the “dark magic mushroom” Galerina indica produces both psilocybin and amatoxins. Mass spectrometry revealed psilocybin and amatoxins in mushroom tissues, and genomic analyses identified corresponding biosynthetic genes. Phylogenetic analyses suggest that G. indica acquired psilocybin biosynthesis via horizontal gene transfer after amatoxin biosynthesis was already established, and that psilocybin biosynthesis was acquired twice independently within Galerina . Intriguingly, acquisition of psilocybin biosynthesis in G. indica may have coincided with reduced amatoxin potency. These findings reveal how horizontal gene transfer can combine powerful bioactive systems in a single species, potentially altering the ecological roles of both compound classes and the evolutionary fitness of the species.

Psilocybe is a well-known genus of fungi, commonly referred to as “magic mushrooms”, due to its production of psychoactive compounds. Research interest in this genus has experienced a resurgence due to its relevance as a potential therapy for a growing global mental health crisis. In this study, 62 specimens representing 10 species of Psilocybe from southern Brazil were examined using morphological data and rDNA ITS sequences in some of them. Here, we report new records of P. subaeruginascens and P. yungensis, providing new information about the occurrence and range of these species. In addition, a novel species from southern Brazil, Psilocybe araucariicola, is characterized and introduced using morphological characteristics and DNA sequencing. Phylogenetic analyses using rDNA ITS, rpb2, and tef1 sequences were performed to determine the relationship of P. araucariicola to other species within Psilocybe. Morphological description, illustrations, photographs, and phylogenetic analysis results of P. araucariicola are provided.

Nearly all species in the genus Psilocybe sensu stricto produce psilocybin, yet P. fuscofulva is a striking exception in which psilocybin has not been detected. To elucidate the evolutionary events leading to differential psilocybin production in Psilocybe, we produced genome assemblies for two P. fuscofulva strains, one P. polytrichoides strain, and one P. tampanensis strain, complemented by reannotated public genomes and metagenome-derived assemblies from fungarium specimens representing both major Psilocybe clades and related genera. Phylogenomic analysis based on 100 single-copy orthologs curated for high branch support strongly placed P. fuscofulva as the earliest-diverging lineage in Psilocybe Clade I. No psilocybin gene cluster (PGC) homologs, whether clustered or dispersed, were identified in P. fuscofulva, whereas a single intact PGC was present in all other examined Psilocybe genomes. The PGC resides in two distinct, clade-specific genomic loci: one conserved in Clade I and another in Clade II, each displaying characteristic gene orders and orientations consistent with rearrangement through circular intermediates. Time-calibrated phylogenies estimated the Psilocybe crown group at approximately 28 million years ago, with major clade divergences occurring in the Miocene. The absence of the PGC in P. fuscofulva, together with clade-specific structural conservation and the lack of remnant sequences at alternate loci, supports two independent origins of the PGC within Psilocybe: one in the ancestor of Clade II and a subsequent origin in Clade I following divergence from P. fuscofulva, most likely via horizontal gene transfer (HGT). Gene phylogenies provide weak support for transfer from Clade I to Clade II, although broader sampling is required for confirmation. These results constrain the timeframe of PGC emergence and dispersal to the Miocene, implying rapid HGT events possibly driven by ecological pressures in expanding grassland ecosystems. This study challenges the assumption of an ancestral psilocybin pathway in Psilocybe and its close relatives and underscores multiple recent acquisitions of an ecologically important metabolic trait.

The “magic mushroom” Psilocybe cubensis is cultivated worldwide for recreational and medicinal uses. Described initially from Cuba in 1904, there has been substantial debate about its origin and diversification. The prevailing view, first proposed by the Psilocybe expert Gastón Guzmán in 1983, is that P. cubensis was inadvertently introduced to the Americas when cattle were introduced to the continents from Africa and Europe (∼1500 CE), but that its progenitor was endemic to Africa. This hypothesis has never been tested. Here, we report the discovery of the closest wild relative of P. cubensis from sub-Saharan Africa, P. ochraceocentrata nom. prov. Using DNA sequences from type specimens of all known and accessable African species of Psilocybe , multi-locus phylogenetic and molecular clock analysis strongly support recognizing the African samples as a new species that last shared a common ancestor with P. cubensis ∼1.5 million years ago (∼710k - 2.55M years ago 95% HPD). Even at the latest estimated time of divergence, this long predates cattle domestication and the origin of modern humans. Both species are associated with herbivore dung, suggesting this habit likely predisposed P. cubensis to its present specialization on domesticated cattle dung. Ecological niche modeling using bioclimatic variables for global records of these species indicates historical presence across Africa, Asia, and the Americas over the last 3 million years. This discovery sheds light on the wild origins of domesticated P. cubensis and provides new genetic resources for research on psychedelic mushrooms.

Fungus-farming ants cultivate multiple lineages of fungi for food, but, because fungal cultivar relationships are largely unresolved, the history of fungus-ant coevolution remains poorly known. We designed probes targeting >2000 gene regions to generate a dated evolutionary tree for 475 fungi and combined it with a similarly generated tree for 276 ants. We found that fungus-ant agriculture originated ~66 million years ago when the end-of-Cretaceous asteroid impact temporarily interrupted photosynthesis, causing global mass extinctions but favoring the proliferation of fungi. Subsequently, ~27 million years ago, one ancestral fungal cultivar population became domesticated, i.e., obligately mutualistic, when seasonally dry habitats expanded in South America, likely isolating the cultivar population from its free-living, wet forest–dwelling conspecifics. By revealing these and other major transitions in fungus-ant coevolution, our results clarify the historical processes that shaped a model system for nonhuman agriculture.

Inbreeding, the mating of individuals that are related through common ancestry, is of central importance in evolutionary and conservation biology due to its impacts on individual fitness and population dynamics. However, while advanced genomic approaches have revolutionised the study of inbreeding in animals, genomic studies of inbreeding are rare in plants and lacking in fungi. We investigated global patterns of inbreeding in the prized edible porcini mushroom Boletus edulis using 225 whole genomes from seven lineages distributed across the northern hemisphere. Genomic inbreeding was quantified using runs of homozygosity (ROHs). We found appreciable variation both among and within lineages, with some individuals having over 20% of their genomes in ROHs. Much of this variation could be explained by a combination of elevation and latitude, and to a lesser extent by predicted habitat suitability during the last glacial maximum. In line with this, the majority of ROHs were short, reflecting ancient common ancestry dating back approximately 200–1700 generations ago, while longer ROHs indicative of recent common ancestry (less than approximately 50 generations ago) were infrequent. Our study reveals the inbreeding legacy of major climatic events in a widely distributed forest mutualist, aligning with prevailing theories and empirical studies of the impacts of historical glaciation events on the dominant forest tree species of the northern hemisphere.

The porcini mushroom family Boletaceae is a diverse, widespread group of ectomycorrhizal (ECM) mushroom-forming fungi that so far has eluded intrafamilial phylogenetic resolution based on morphology and multilocus data sets. In this study, we present a genome-wide molecular data set of 1764 single-copy gene families from a global sampling of 418 Boletaceae specimens. The resulting phylogenetic analysis has strong statistical support for most branches of the tree, including the first statistically robust backbone. The enigmatic Phylloboletellus chloephorus from non-ECM Argentinian subtropical forests was recovered as a new subfamily sister to the core Boletaceae. Time-calibrated branch lengths estimate that the family first arose in the early to mid-Cretaceous and underwent a rapid radiation in the Eocene, possibly when the ECM nutritional mode arose with the emergence and diversification of ECM angiosperms. Biogeographic reconstructions reveal a complex history of vicariance and episodic long-distance dispersal correlated with historical geologic events, including Gondwanan origins and inferred vicariance associated with its disarticulation. Together, this study represents the most comprehensively sampled, data-rich molecular phylogeny of the Boletaceae to date, establishing a foundation for future robust inferences of biogeography in the group.

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 .

The species-rich porcini mushroom family Boletaceae is a widespread and well-known group of ectomycorrhizal (ECM) mushroom-forming fungi that has eluded intrafamilial phylogenetic resolution despite many attempts using morphological traits and multi-locus molecular datasets. In this study, we present a genome-wide molecular dataset of 1764 single-copy gene families from a global sampling of 418 Boletaceae specimens. The resulting phylogenetic analysis has strong statistical support for most branches of the tree, including the first statistically robust backbone. The enigmatic Phylloboletellus chloephorus from non-ECM Argentinian subtropical forests was recovered as an early diverging lineage within the Boletaceae. Time-calibrated branch lengths estimate that the family first arose in the early- to mid-Cretaceous and underwent a rapid radiation in the Eocene, possibly when the ECM nutritional mode arose with the emergence and diversification of ECM angiosperms. Biogeographic reconstructions reveal a complex history of vicariance and episodic long-distance dispersal correlated with historical geologic events, including Gondwanan origins and cladogenesis patterns that parallel its fragmentation. Ancestral state reconstruction of sporocarp morphological traits predicts that the ancestor of the Boletaceae was lamellate with ornamented basidiospores, contrary to most contemporary “bolete” morphologies. Transition rates indicated that the lamellate hymenophore and sequestrate sporocarp are reversible traits. Together, this study represents the most comprehensively sampled, data-rich molecular phylogeny of the Boletaceae to date, enabling robust inferences of trait evolution and biogeography in the group.

Hybridization among diverged species is common across all domains of eukaryotic life, but appears to be particularly common in fungi. However, some fungi exhibit a greater tendency for hybridization than others and it is unclear what mechanisms facilitate or prevent hybridization. Here, we generated 253 whole genome sequences and 22 reference genomes within the globally distributed ectomycorrhizal species complex Boletus edulis Bull. and use a multi-faceted genomic approach to identify patterns of ongoing hybridization and determine whether hybridization is mediated by changes in genome structure, ecology, or both. We found that hybridization is common among species of B. edulis despite 2.1 million years of divergence. However, not all lineages that hybridize today exhibit patterns of introgression, indicating the presence of strong reproductive boundaries among some species. Using a pan-genomic approach we find that genome structural variation is abundant within B. edulis , but that the presence of ongoing introgression does not correlate with genome structural similarity or overall gene content. Instead, we find that the composition of ecologically relevant gene families more accurately predicts the presence of introgression among lineages and B. edulis as a group may specialize on chitin decomposition. Altogether, we show that ecological preferences are likely the primary driver of reproductive barriers in B. edulis .