DOI: 10.5061/dryad.tmpg4f52s
title: Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution of the psilocybin biosynthetic gene cluster

description: Psychoactive mushrooms in the genus Psilocybe have immense cultural value
 and have been used for centuries in Mesoamerica. Despite the recent surge
 of interest in these mushrooms due to the psychotherapeutic potential of
 their natural alkaloid psilocybin, their phylogeny and taxonomy remain
 substantially incomplete. Moreover, the recent elucidation of the
 psilocybin biosynthetic gene cluster is known for only five of ~165
 species of Psilocybe, four of which belong to only one of two major
 clades. We set out to improve the phylogeny of Psilocybe using shotgun
 sequencing of fungarium specimens, from which we obtained 71 new
 metagenomes including from 23 types, and conducting phylogenomic analysis
 of 2,983 single-copy gene families to generate a fully supported
 phylogeny. Molecular clock analysis suggests the stem lineage of Psilocybe
 arose ~67 mya and diversified ~56 mya. We also show that psilocybin
 biosynthesis first arose in Psilocybe, with 4–5 possible horizontal
 transfers to other mushrooms between 40 and 9 mya. Moreover, predicted
 orthologs of the psilocybin biosynthetic genes revealed two distinct gene
 orders within the biosynthetic gene cluster that corresponds to a deep
 split within the genus, possibly a signature of two independent
 acquisitions of the cluster within Psilocybe. 
description: # Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution
 of the psilocybin biosynthetic gene cluster *** Data set contains:
 =====Assembly\_graphs.zip===== Assembly graph outputs from SPades genome
 assembly in .gfa format. These asseembly graphs were used as input for the
 DNA barcode extraction using PATHRacer (see methods for paramaters).
 =====Assembly\_scaffolds.zip===== Assembled genome assembly scaffolds from
 SPades. These draft metagenome assemblies where used for down stream
 phylogenetic analysis and protein preductions (see methods for
 paramaters).
 =====Protein\_predictions\_of\_Psilocybin\_producing\_taxa.zip=====
 Protein predictions in GFF format for pislocybin-producing taxa. Proteins
 predections were perfomred on bioinformatically reduced genomes (from
 diploid to haploid) using Redundands. Profiles were generated using the
 Program Augustus (see methods for paramaters).
 =====Raw\_phylogenetic\_trees.zip===== ==Mega11\_calibration\_tests== Time
 divergence trees and mega analysis environments(see methods for
 paramaters) =LSU tree= Divergence analysis for LSU species tree Multiple
 sequence alignment (mafft\_alignment.fasta), gene tree output from iqtree
 (.tre), Mega11 tree output (.nwk), and full Mega11 sesion file (.MTSX).
 =Psilocybe top100 tree= Divergence analysis for the Psilocybe species tree
 Multiple sequence alignment (mafft\_alignment.fasta), gene tree output
 from iqtree (.tre), Mega11 tree output (.nwk), and full Mega11 sesion file
 (.MTSX). ==Phylogenomic\_trees== Unaltered phylogenetic trees for each
 analysis, main and supplementary (see methods for paramaters) =Figure\_2=
 raw tree file output from iqtree for only the psilocybe species used in
 figure 2. =Figure\_3= mafft sequence alignment and raw tree file output
 from iqtree for samples used in PsiD analysis in figure 3. Also Includes
 PDF files with full PsiD contig infromation, and with clean labels that
 only include sample name. =constraint4= Raw tree output from iqtree for
 psilocybe that was used as the constraint tree for all public barcode data
 found in supplementary figures. ==Psilocybin\_cluster\_gene\_trees== Mafft
 alignments used for each individual Psi cluster gene tree (see methods for
 paramaters). Raw tree output from iqtree or each individual Psi cluster
 gene tree (see methods for paramaters). PDF file with tip labels only
 presenting sample names. ==Supplementary figure phylogenetic trees==
 =Alignments= Mafft alignments used for each public barcode gene tree (see
 methods for paramaters). =Trees= Raw tree output for all supplementary
 figures (see methods for paramaters). =====ASR.zip===== Input and output
 data used for ansectral state reconstruction (ASR) (see methods for
 paramaters). Includes the input tree and states, as well as the alter
 output file to remove outgroups from the phylogenetic tree. Also includes
 the ASR outputs altered to be compatible with Tracer.
 =====Supplementary\_tables.zip===== This directory contains supplementary
 tables 1-r Supplementary Table 1: Voucher table of Specimens Voucher table
 of all specimens sequenced and analyzed. Table include relevant voucher
 information, current name designation, and SRA and BioSample accession
 numbers. Supplementary Table 2: Genome assembly and BUSCO stats for each
 specimen Genome assembly statistics, and BUSCO complete gene recovery from
 the Agaricales\_db10 database. Tab 1: Psilocybe Includes Psilocybe s.s.
 taxa from this study Tab2: Non-Psilocybe Taxa found to not be Psilocybe
 s.s. from this study Supplementary Table 3: RBB gene numbers, contigs,
 clustering results, and RBB method comparison to Exonerate Gene and
 cluster recovery comparison of Reciprocal Best Blast and Exonerate
 methodology. Tab1: Augustus gene prediction Results for Psi cluster gene
 prediction using Augustus and RBB (reverse best blast) approach. Tab2:
 Exonerate protein alignment Results for Psi cluster gene prediction using
 Exonerates protein2genome function Tab3: Augustus-Exonerate comparison
 Comparison of Augustus and Exonerate output for gene prediction and contig
 placement. Supplementary Table 4: Toplogy constraint testing: Individual
 model results Individual model results model results and +/- symbols to
 indicate significance.
 =====Supplementary\_Information\_and\_Figures.zip=====
 ====Figures\_full\_resolution.zip==== High resolution copies of Figures
 1-3 in both PDF and PNG format. PDF file including all supplementary
 results and discussion along with supplementary table and figue legends.
 PDF file Including all supplementary figures. =====MSA\_stats.zip=====
 Multiple sequece alignment statistics generatned for the Psilocybe only
 data set used for Summary coalescent and concatened phylogenetic analysis.
 Stats files were generated using AMAS.py (see methods for paramaters).
 =====PsiD\_topology\_test.zip===== Toplogy constraint testing for HGT in
 Panaeolus and Pholiotina(see methods for paramaters).
 ==Constrained\_tree== Full IQtree directory output for PsiD Constrained
 tree analysis ==Unconstrained\_tree== Full IQtree directory output for
 PsiD unconstrained tree analysis ==Combined== Full IQtree directory output
 for combined PsiD unconstrained toplogy constraint testing analysis