Allelic diversity at psilocybin loci and mitochondria
tBLASTn was used to identify contigs that contained the psilocybin gene cluster and BLASTp was used to search annotated assemblies based on genes in the psilocybin pathway identified in wood-loving species ofPsilocybe (Reynolds et al., 2018). Amino acid sequences of psilocybin genes annotated by FunAnnotate were aligned with MAFFT and their homology was confirmed with a search for the most likely tree using IQTree. The coding sequences of genes in the psilocybin pathway, including psiD, psiM, psiT2, psiH (paralog 1), psiT1, psiH(paralog 2), psiK, psiR , were aligned with MAFFT, concatenated with FASconCAT-G, and visualised with a neighbour net in SplitsTree. FST, nucleotide diversity, and Tajima’s D were calculated across coding sequences of the psilocybin locus using vcfools and plotted with ggplot2 in R.
The psiH gene family, including pseudogenes, was annotated with exonerate (Slater & Birney, 2005). We aligned all alleles of thepsiH family with MAFFT, and searched for a maximum likelihood tree with IQTree v.2. We used Clinker (Gilchrist & Chooi, 2021) to align representative genotypes of the entire psilocybin locus.
Mitochondrial contigs of P. subaeruginosa were identified using a BLASTn search against the mitochondrial genome (NW_025952838) of theP. cubensis reference assembly (K. McKernan et al., 2021). SNPs were called from all mitochondrial contigs using kSNP (k=31, min frac=1), and relationships were visualised with a haplotype network using PopART (Leigh & Bryant, 2015) with sites masked that had more than 5% missing data.