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Convergent evolution of psilocybin biosynthesis by psychedelic mushrooms

Ali R. AwaniD, Jaclyn M. WinteriD, Dann TurneriD, William M. ShawiD, Laura M. SuziD, Alexander James BradshawiD, Tom EllisiD, Bryn T. M. DentingeriD

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Abstract

Psilocybin is a psychoactive compound with clinical applications produced by dozens of mushroom species1. There has been a longstanding interest in psilocybin research with regard to treatment for addiction2, depression3, and end-of-life suffering4. However, until recently very little was known about psilocybin biosynthesis and its ecological role. Here we confirm and refine recent findings5 about the genes underpinning psilocybin biosynthesis, discover that there is more than one psilocybin biosynthesis cluster in mushrooms, and we provide the first data directly addressing psilocybins ecological role. By analysing independent genome assemblies for the hallucinogenic mushrooms Psilocybe cyanescens and Pluteus salicinus we recapture the recently discovered psilocybin biosynthesis cluster5,6 and show that a transcription factor previously implicated in its regulation is actually not part of the cluster. Further, we show that the mushroom Inocybe corydalina produces psilocybin but does not contain the established biosynthetic cluster, and we present an alternative cluster. Finally, a meta-transcriptome analysis of wild-collected mushrooms provides evidence for intra-mushroom insect gene expression of flies whose larvae grow inside Psilocybe cyanescens. These larvae were successfully reared into adults. Our results show that psilocybin does not confer complete protection against insect mycophagy, and the hypothesis that it is produced as an adaptive defense compound may need to be reconsidered.

In-depth summary

Claude Opus 5.5 · xhigh effort · 2 Nov 2026 · from the full text of version 3 (16 Jan 2026)

Comparative mushroom genomics and fly-rearing experiments reveal an alternative psilocybin biosynthetic gene cluster in Inocybe corydalina, horizontal transfer of the canonical cluster, and incomplete defense against dipteran larvae.

The genetic basis and ecological function of psilocybin production across diverse mushroom lineages remain incompletely understood, with prior work proposing a single biosynthetic gene cluster and an insect-deterrent role. Comparative genome assemblies and larval rearing refine canonical cluster annotations, uncover an independently evolved pathway in Inocybe corydalina, and show that mycophagous fly larvae develop inside psilocybin-producing fruiting bodies.

Key results

  1. ShownInocybe corydalina produces psilocybin and baeocystin despite lacking the canonical psilocybin biosynthetic cluster, instead harboring an alternative clustered set of biosynthetic enzymes.

    Liquid chromatography-mass spectrometry confirmed psilocybin and baeocystin in a resequenced I. corydalina specimen (1193 contigs, N50 ~91,700 nt, higher CEGMA completeness than Pluteus salicinus), where homologs of the established cluster were unclustered and divergent, whereas a single candidate cluster encoded all four necessary enzyme types plus an MFS transporter. · Abstract; A novel psilocybin biosynthetic pathway; Pluteus salicinus and Inocybe corydalina hybrid…; Figure legends

  2. ShownHybrid genome and transcriptome analyses of Psilocybe cyanescens and comparative synteny show that the core canonical psilocybin cluster is highly expressed, excludes a previously implicated transcription factor, and was misassigned to P. cyanescens instead of Psilocybe serbica in a prior study.

    All five core cluster genes in P. cyanescens (645 contigs, N50 ~244,500 nt) ranked within the top 5% of RNA-seq expression; the flanking transcription factor occurs syntenically in non-producers Galerina marginata and Agaricus bisporus and at separate loci in Gymnopilus dilepis and Pluteus salicinus; ITS phylogeny placed the Fricke et al. P. cyanescens sequence with P. serbica (100% bootstrap support). · Abstract; Refining the annotation of the established…; Figure legends

  3. SuggestedPhylogenetic discordance and circular permutation of the five core cluster genes across ecological guilds indicate horizontal gene transfer (HGT), potentially mediated by a subtelomeric extra-chromosomal circular DNA intermediate.

    Core cluster gene trees group hardwood-dwelling Gymnopilus dilepis with Pluteus salicinus rather than the more closely related genus Psilocybe, and group dung-dwelling Psilocybe cubensis with Panaeolus cyanescens (1000 ultrafast bootstrap replicates across 209 single-copy gene families, 400,675 aligned nucleotide positions); core clusters (< 13kb) conserve intron numbers across three circular permutations. · Horizontal gene transfer; Construction of species phylogenetic tree for…; Construction of gene phylogenetic trees for…; Figure legends

  4. ShownWild Psilocybe cyanescens fruiting bodies harbor expressed dipteran genes and support complete larval development of Sciaridae fungus gnats, showing that psilocybin does not confer complete protection against insect mycophagy.

    Taxon-annotated GC plots and de novo metatranscriptome BLASTP (e-value < 1e-10) identified hundreds of dipteran contigs and proteins, including Exechia fusca; in rearing jars of washed wild P. cyanescens and co-occurring non-producer Stropharia aeruginosa, 4-5 larvae emerged per jar after 3-7 days and pupated into adult Sciaridae flies within 7-14 days. · Abstract; Interaction of diptera with psilocybin-producing…; Fly-rearing experiment; Figure legends

Methods, data and reproducibility

Hybrid genome assemblies combining Oxford Nanopore MinION long reads and Illumina short reads were generated for Psilocybe cyanescens, Pluteus salicinus, and Inocybe corydalina, with pileus and stipe RNA-seq for P. cyanescens. Candidate biosynthetic clusters were identified via InterProScan and custom Python colocalization scripts, compared phylogenetically with MAFFT and IQ-TREE, and paired with fly-rearing assays.

  • Whole-genome Nanopore and Illumina sequencing and pileus/stipe RNA-seq of fruiting bodies · Psilocybe cyanescens (with internal Diptera including Exechia fusca) · n = ~400,000 Nanopore, ~77M HiSeq, ~4.2M MiSeq reads; 3 fruiting bodies
  • Whole-genome Nanopore and Illumina HiSeq X sequencing of dried sporocarps · Pluteus salicinus · n = ~570,000 Nanopore reads and ~7.5 million Illumina HiSeq read pairs
  • Whole-genome Nanopore and Illumina HiSeq X sequencing and LCMS of dried sporocarps · Inocybe corydalina · n = 1 specimen; ~130,000 Nanopore reads and ~3.7 million Illumina HiSeq read pairs
  • Public comparative fungal genome assemblies and Panaeolus cyanescens scaffolds · Psilocybe, Panaeolus, Gymnopilus, Galerina, and Agaricus species · n = 6 comparative fungal species · NHTK01005322.1, NHTK01001377.1, NHTK01003280.1, UniProt P0DPB0
  • Wild-collected mushroom fruiting bodies for fly-rearing experiment · Psilocybe cyanescens, Stropharia aeruginosa, and Sciaridae flies · n = 2 jars (1 per mushroom species), 4-5 larvae and 1 adult fly emerged per jar
Methods & tools
MaSuRCA, BRAKER, Augustus, InterProScan, Trinity, Trimmomatic, RSEM, IQ-TREE, MAFFT, CEGMA, BLAST, blobology
Reagents & environment
Trinity v2.6.5; other assembly, alignment, and annotation tools were run largely with default settings.
Experimental conditions
RSEM transcript abundance estimation run with 70 threads (--thread_count 70); hardware and runtime not stated.
Reproducibility gaps
Custom Python scripts for cluster detection and taxonomic ORF aggregation are not shared in a public repository, and database accessions for the newly generated genome assemblies and RNA-seq reads are not provided.
Limitations (4)
  • Whether mycophagous animals display innate or learned attraction or avoidance toward psilocybin-containing mushrooms, or experience altered fitness from consuming them, remains untested. · stated by the authors
  • Direct experimental data demonstrating that psilocybin is bioactive in mycophagous invertebrates are currently lacking. · stated by the authors
  • The candidate alternative psilocybin biosynthetic gene cluster identified in Inocybe corydalina relies on genomic colocalization and functional domain annotations without biochemical validation of enzyme activity. · noted in this summary
  • The fly-rearing assay used a single jar per mushroom species from one wood-chip patch, yielding 4-5 larvae and one adult fly per jar without quantitative comparison of infestation rates. · noted in this summary

Read this if you study fungal secondary metabolite gene cluster evolution, horizontal gene transfer in basidiomycetes, or the chemical ecology of psilocybin.

Citations
27

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