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.