Figure captions
Fig. 1. Phenotypic diversity of Psilocybe subaeruginosa from populations in Australia, including pilei that are conical, papillate, or sinuate, and fruiting from diverse substrates, including grass, leaf litter, moss, and wood. A. Ellendale, Tasmania. B. Dover, Tasmania. C. Tasmania (image courtesy of Karen Keats). D. Ellendale, Tasmania. E. Victoria (image courtesy of Tannar Coolhaas). F. Tasmania (image courtesy of Karen Keats). G. kunanyi, Tasmania. H. Ellendale, Tasmania. I. Western Australia (image courtesy of Otto). J. Victoria (image courtesy of Tannar Coolhaas). K. Victoria. L. Bunya Mountains, Queensland.
Fig. 2. Analyses of Australian populations of Psilocybe subaeruginosa (n=85), and P. azurescens (n=2) and P. cyanescens (n=1) from the northern hemisphere. A. Provenance map of Australian collections used in the study. B. 2-dimensional plot of Discriminant Analysis of Principle Components (DAPC) based on 6,757 SNPs with indels and sites under LD removed from the dataset, and individuals coloured based on K=7 in the barplot of 2C. C. DAPC exploring K-values 2–8 based on the same dataset in 2B. Facets reflect geographic sampling from local populations. D. SplitsTree network based on 1,555,848 LD-corrected SNPs, including indels, with all genomes treated as haploids. Individuals are coloured by populations defined in DAPC analysis at K=7. Sibling haplotypes sampled from the same pileus are labelled with the same letter. Edge length reflects genetic difference and reticulation may indicate recombination. Figures B and C produced by R packages adegenet, vcfR, and ggplot2.
Fig. 3. Phylograms from maximum likelihood searches based on translated, aligned, concatenated genes at mating-compatibility loci. Individuals are coloured based on structured populations in Fig. 2C. ConcatenatedSTE3 genes at the pheromone/receptor (PR) locus, with compatible (black) and incompatible (red) crosses among connected haplotypes. B. Relationships among concatenated HD1 and HD2 genes at the homeodomain (HD) locus. Alleles at mating compatibility loci are mostly private to the sampled populations, which indicates high allelic diversity in Australia.
Fig. 4. Measures of differentiation, nucleotide diversity and selection across contigs containing mate compatibility genes among populations ofP. subaeruginosa . A–C homeodomain (HD) locus (826,289 base pairs). D–F pheromone/receptor (PR) locus (122,582 base pairs). A. FST plotted in 10,000 base pair windows of the HD locus as a measure of differentiation among populations. Line colour indicates which population has been removed from the comparison of all populations. FST values approach 0 in admixed, recombinant populations, and high FST values may indicate divergence or a lack of recombination. FSTdecreases when the South Australian, Tasmanian, and Victorian population is removed from the comparison, which indicates genetic differentiation from populations east of the Great Dividing Range. B. Nucleotide diversity (pi) plotted across 10,000 base pair windows of the HD locus as a measure of diversity within defined populations. Diversity is low within all populations (pi < 0.2). C. Tajima’s D index plotted in 10,000 base pair windows across the HD locus as a measure of selection across all populations. Positive values may be a signature of balancing selection, in particular negative frequency dependent selection, in which multiple alleles are maintained in populations and no allele becomes dominant. D. FST plotted in 3,500 base pair windows of the PR locus, with similar levels of differentiation across the locus in all populations. E. Nucleotide diversity (pi) plotted across 3,500 base pair windows of the PR locus, with divergence among populations driven by diversity in the South Australian, Tasmanian, and Victorian population. F. Tajima’s D index plotted in 3,500 base pair windows across the PR locus, with support for balancing selection based on positive values comparable to the HD locus.
Fig. 5. Analyses of genetic diversity at the psilocybin locus. A. SplitsTree network of aligned coding sequences of psiD, psiK, psiM, psiT1, psiT2, psiR and two paralogs of psiH (psiH1and psiH2 ). Individuals are coloured based on structured populations in Fig. 1A. Sibling isolates sampled from the same pileus are linked by dashed lines. Siblings that have different alleles at psilocybin loci indicate heterozygosity in the parental genotype. Siblings with more than two genotypes (in populations from Bunya and Shelley) may reflect recombination within the psilocybin locus. B. FST plotted across individual genes as a measure of differentiation among populations at the psilocybin locus. FST is comparable among populations. C. Nucleotide diversity (pi) plotted across individual genes as a measure of genetic diversity at the psilocybin locus. The Khancoban population is the most genetically diverse at the psilocybin locus relative to other populations. D. Tajima’s D index plotted across individual genes as a measure of selection at the psilocybin locus. Most genes have neutral to positive values of Tajima’s D, which indicates balancing selection or maintenance of diversity at the psilocybin locus. Measures of genetic diversity were plotted in 50 base pair windows of all genes exceptpsiM , which used a 30-base pair window.
Fig. 6. Phylogram from a maximum likelihood search of an alignment of 26 ITS types of Psilocybe subaeruginosa and related taxa, showing a sister relationship to P. cubensis . Sequences were obtained from genomes in the present study, and from GenBank for sequences of P. allenii, P. azurescens, P. cyanescens, P. makarorae , and P. weraroa . Sequence abundance for each ITS type is shown in the adjacent haplotype networks for P. subaeruginosa and P. cubensis , with dashes indicating the number of parsimony informative sites between ITS types. The ITS region is intraspecifically variable in P. subaeruginosa , which is paraphyletic with respect to other related taxa.
Fig. S1. SplitsTree network based on 76,076 amino acids aligned from 194 single copy orthologs in Psilocybe subaeruginosa and relatives. Branch length is informative for genetic distance between points, reticulation is indicative of recombination, homoplasy, or incomplete lineage sorting.
Fig. S2. Heatmap of sibling relationships based on pairwise comparisons of the AJK statistic. Several of the relationships are known siblings, and based on likelihood values of the AJK statistic, pairwise relationships ≥0.91 are an indication that isolates are siblings.
Fig. S3. Evolution of psiH paralogs in the psilocybin locus inPsilocybe subaeruginosa .
A. Maximum likelihood tree of positional orthologs of the threepsiH paralogs extracted from cluster loci using exonerate. Bold alleles are annotated as pseudogenes, and all sequences in thepsiH3 clade were pseudogenes. Isolate BRIP75275 has a predicted functional paralog in the psiH3 position and a predicted pseudogene in the psiH2 position. B. Clinker plot of synteny and relatedness of genes in the psilocybin metabolic pathway. Darker connections between plots indicate higher percent nucleotide identity.psiH2 is likely a duplicated copy of psiH1 , andpsiH3 is likely a duplication of psiH2 .
Fig. S4. Haplotype network of SNP diversity in the mitochondrial genome based on 1,334 SNPs. Sibling populations shared the same mitochondrial genotype (e.g., populations from Bunya, Ravensbourne, and Clifton Hill and Geelong).