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.