Discussion
Psilocybin has breakthrough potential for treatment of mental health disorders, and as momentum builds in the clinical landscape, knowledge of diversity in magic mushrooms will impact development of natural medicines. Our results suggest that Psilocybe subaeruginosaoriginated in Australasia as evidenced by its widespread distribution in natural areas, high allelic diversity of mating genes, high genetic diversity at mitochondria and psilocybin loci, and high phenotypic diversity. Mushrooms in the P. subaeruginosa species complex were likely introduced to the northern hemisphere through movement of plants, soil, or wood chips, as they cluster among Australian populations in analyses of genetic diversity based on SNPs or genes, have low genetic diversity in their invaded areas (Gießler, 2018), and behave as weedy taxa, occurring in disturbed rather than natural areas (Borovička et al., 2012; Dennis & Wakefield, 1946).
Psilocybe subaeruginosa colonises wood chips and leaf litter, and mushrooms from one vicinity often fruit from the same mycelial genotype, based on sibling relationships supported by the AJK statistic and genetic distance within populations defined by DAPC. We studied 86 haplotypes across eastern Australia, although the overall effective sample size is reduced as many haplotypes were siblings, and greater genetic diversity is expected with wider sampling, potentially including kin genotypes of P. azurescens and P. cyanescens . Mushrooms collected from geographically different areas that were recovered as siblings support that P. subaeruginosa spreads as a saprotrophic invader of garden beds. Genotypes of P. subaeruginosa likely persist perennially, and anecdotal evidence from citizen scientists in the present study shows that fruiting sites are re-visited to collect mushrooms, likely with the same genotype, year after year. This contrasts with P. cubensis , in which genotypes are ephemeral, with mycelia disappearing after manure is degraded, akin to annual plants.
Our findings indicate that P. subaeruginosa is one taxon rather than a complex of species, supported by evidence from population analyses, phylogenetic analyses, gene flow measured by FST, and mating compatibility. The alternative hypothesis of cryptic species diversity is rejected by evidence of gene flow among sexually compatible populations and shared mating type alleles. Low phenotypic diversity or a fixed phenotype in populations from the northern hemisphere is likely caused by underlying low genetic diversity from an invasion event, as P. azurescens and P. cyanescens share close ancestry with P. subaeruginosa in all analyses. Additional species described in this taxonomic complex are likely phenotypic/geographic variants and are con-specific with P. subaeruginosa . Taxonomic synonyms may be useful to describe invasive populations, such as P. cyanescens in Europe, however, these taxa have an origin from Australia and could be considered subpopulations ofP. subaeruginosa . The ITS region is intraspecifically variable in the Australian population, and this genetic diversity is expected in the centre of origin. Dabao Sun et al. (2023) found that differentiated lineages in a fungal taxon at a global scale had complicated species boundaries because sympatric inter-sterile populations could theoretically exchange genetic material by crossing through other compatible populations. More crosses in the case of P. subaeruginosa will be needed to determine if any reproductive isolation exists, however, most populations show some degree of mating compatibility.
Mushrooms produce copious spores that are wind dispersed (Dam, 2013), and allopatric speciation of mushrooms has occurred at the scale of continental geographic boundaries (Geml, Tulloss, Laursen, Sazanova, & Taylor, 2008; James, Moncalvo, Li, & Vilgalys, 2001; Li, Han, Liu, Zhao, & Yang, 2020; M.-Z. Zhang et al., 2023). Our study, which found isolated populations of P. subaeruginosa on mountain ranges in Australia, may add to evidence that mushrooms limited by their available habitat and spore dispersal, by nature, have more opportunities for allopatric speciation than panmictic organisms that migrate. For example, Amend et al. (2010) found montane populations ofTricholoma matsutake were isolated based on topography, with mountain ranges a barrier to gene flow. Another study found that the ectomycorrhizal species Suillus brevipes was structured into subpopulations within North America due to isolation by and on mountain ranges (Branco et al., 2017). The mean level of population differentiation we report here from haploid genomes, FST=0.36, may be high compared to other taxa, yet, these values vary considerably across species of mushroom (Carriconde, Gryta, Jargeat, Mouhamadou, & Gardes, 2008; Mi et al., 2016; J. Zhang et al., 2022). This level of differentiation suggests that spatial populations of P. subaeruginosa have had sufficient time to show the effects of isolation within their centre of origin. Why some species show strong intracontinental population substructure while others do not is uncertain and highlights how little we understand fungal niche breadth, gene flow, distribution, and the temporal and geographic scale of the centre of origin.
Isolation and infrequent gene flow lead to divergence; in fungi with dominant asexual stages, isolation generates near clones, in which clonal reproduction is interspersed with infrequent sexual reproduction that maintains species cohesion (Taylor, Hann-Soden, Branco, Sylvain, & Ellison, 2015). In obligate outcrossing fungi, mating compatibility loci maintain species connectivity because allelic diversity benefits compatibility, and MAT genes diverge but maintain key amino acids at functional sites (Peris et al., 2022; van Diepen et al., 2013). Populations of P. subaeruginosa were sexually compatible, and slight differences at mating compatibility loci among populations may be caused by genetic drift and isolation, or alternatively, we under-sampled potentially shared alleles. Allopatric species boundaries may be interrupted given that humans move soils and their accompanying microorganisms, and as shown with magic mushrooms here, species connectivity through mate compatibility persists even in disparate populations with small evidence of gene flow.
Psilocybin loci were genetically different within and among populations of P. subaeruginosa in Australia, whether from allelic diversity, or potential differences in presence or absence of functional homologs of psiH . Some haplotypes contained two putatively functional paralogs of the psiH gene family (psiH1 and psiH2 ), whereas others contained one (psiH1 ). One isolate, BRIP75275 has a putative functional psiH in the psiH3 position, but a pseudogene in the psiH2 position. That sequence groups at the base of the psiH2 clade with several pseudogenes that are also in the psiH3 position. Analyses of FST and Tajima’s D indicated that differentiation of the psilocybin locus among populations may be a result of genetic drift, such as from a founder effect in isolated populations, and populations maintain allelic diversity through balancing selection. McTaggart et al. (2023) found the psilocybin locus was homozygous in five siblings of P. subaeruginosa , however, with increased sampling, we show heterozygosity in dikaryons at the psilocybin locus.
Allelic differences of genes in the psilocybin pathway may increase/decrease metabolism of tryptamines, and the ratios of psilocybin and its analogues may differ among genotypes. Humans have at least 14 types of serotonin receptors; 5-HT2A has the highest affinity for psilocin and is linked to hallucinogenic effects of magic mushrooms (Glennon, Titeler, & McKenney, 1984; Lee & Roth, 2012; Madsen et al., 2019). The suite of tryptamines produced by magic mushrooms in the psilocybin pathway may have different affinities for types of serotonin receptors beyond 5-HT2A (Glatfelter et al., 2022). We put forward the hypothesis that alternate allelic combinations at paralogs ofpsiH may cause wood lover’s paralysis by producing a derivative of tryptamine that agonises peripheral serotonin receptors, such as those linked to Parkinson’s disease (Ohno, Shimizu, & Tokudome, 2013).
High genetic diversity of all examined alleles/loci in the centre of origin of P. subaeruginosa contrasts with low diversity in naturalised and cultivated populations of P. cubensis . Our findings put perspective on what may be expected in terms of genetic diversity in the centre of origin of P. cubensis . Mitochondrial diversity and allelic diversity at mating loci was variable between and among all examined populations, as should be expected in the centre of origin of P. cubensis .
Our study shows that P. subaeruginosa is a widespread and invasive mushroom with a centre of origin in Australia. Geographically limited populations are sexually compatible, although there is little evidence of contemporary gene flow, with mitochondria, mating genes, and alleles at psilocybin loci differentiated among populations.Psilocybe subaeruginosa produces high concentrations of psilocybin and is a commercially attractive species if the cause of wood lover’s paralysis can be determined and excluded for safe clinical use.