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.