Diversity of alleles at mitochondria
We called 1,334 SNPs in mitochondrial contigs using a k-mer approach (k=31) with kSNP and plotted mitochondrial genotypes in a haplotype network (Fig. S4). There were 17 mitochondrial genotypes across the 9 populations defined by DAPC analyses and 14 geographic locations (including P. azurescens and P. cyanescens ), indicating high mitochondrial diversity. Psilocybe cyanescens had a near-identical mitochondrial genotype to populations of P. subaeruginosa in Australia, and P. azurescens clustered among Australian genotypes differing by as few as two parsimony informative characters.
High intraspecific diversity of the ITS region in P. subaeruginosa
We examined whether the ITS region was informative at species rank inP. subaeruginosa , as phylogenetic relationships based on the ITS region are used as a proxy for species identification. Phylogenetic relationships were compared across 26 different ITS types, reflective of ITS sequence diversity within and among populations of P. subaeruginosa and related taxa identified on GenBank (Fig. 6). We used a haplotype network to visualise the proportions of individuals that shared a particular ITS type. Psilocybe subaeruginosa was paraphyletic in the ITS region with respect to P. allenii ,P. azurescens , P. cyanescens , P. makarorae , andP. weraroa . Based on parsimony informative characters, ITS sequences of P. allenii , P. azurescens , and P. cyanescens clustered with samples of P. subaeruginosa from Australia, whereas P. makarorae and P. weraroa had unique ITS sequences not sampled from Australia. Psilocybe cubensis , sampled from eight ITS-types in 166 individuals, is a sister taxon toP. subaeruginosa (Bradshaw et al., 2022) and most sequences cluster with cultivars (exemplified by the AlbinoAplus sequence, Fig. 6). These sequences of P. cubensis are representative of naturalised and cultivated populations rather than diversity in its centre of origin.