Diversity of alleles at psilocybin loci
We examined variation at the psilocybin gene cluster in P.
subaeruginosa with two approaches, namely comparing individual genes
annotated by FunAnnotate, and comparing aligned, concatenated coding
sequences across the entire locus. We used a phylogenetic hypothesis to
analyse the putatively functional and non-functional copies of thepsiH gene family (Fig. S3). There were 178 amino acid differences
among haplotypes across the translated alignment, (number of amino acid
differences psiD =10, psiK =24, psiM =15,psiR =14, psiT1 =35, psiT2 =23, psiH1 =15, andpsiH2 =42).
A SplitsTree network of 11,768 nucleotides from concatenated coding
sequences of psiD, psiK, psiM, psiT1, psiT2, psiR and two
paralogs of psiH (psiH1 and psiH2 ) show that
genotypes of psilocybin loci cluster by geographic location (Fig. 5A).
The locus is heterozygous in some dikaryotic parents, as siblings from
the same parental genotype had different alleles (e.g., the Bunya
population in Fig. 5A), and there is possible evidence of recombination
within the locus with siblings from Bunya and Shelley sharing three
genotypes.
We calculated FST, nucleotide diversity (pi), and
Tajima’s D index across coding sequence of the psilocybin locus as
measures of differentiation between populations, diversity in different
populations, and selection. FST did not vary in
comparisons of populations, which may indicate that allelic differences
in the psilocybin pathway have occurred by genetic drift in
differentiated populations (Fig. 5B). There was high nucleotide
diversity (pi) in genes of the psilocybin pathway within and among
populations (Fig. 5C), expected under balancing selection. Analyses of
Tajima’s D index that recovered mostly positive values indicate that
some genes of the psilocybin pathway may be under balancing selection,
specifically psiT2 , psiT1 , and psiH2 , suggesting
that there is some advantage to maintaining multiple alleles (Fig. 5D).psiH2 had the highest values of the Tajima’s D index, which may
reflect differential functionality at the locus.
We examined gene diversity of the psiH family, annotated with
exonerate, with a phylogenetic analysis, which delineated clades closely
approximating the psiH1 , psiH2 , and psiH3 positions
(Fig. S3A). psiH 1 formed a strongly supported clade with short
branch lengths. psiH2 appears paraphyletic in respect topsiH1 , and psiH3 appears to have originated frompsiH 2. Alternate topologies in psiH2 and psiH3could reflect recombination among populations or ambiguity in the
alignment and splice sites of pseudogenes. All but one ortholog ofpsiH3 were considered pseudogenes, and only 29 of 76 isolates
annotated for psiH2 had a putatively functional allele. We
plotted gene synteny and identity using Clinker and noted structural
variation and differential sequence conservation at psiH2 andpsiH3 positions.