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Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution of the psilocybin biosynthetic gene cluster

January 2024
Proceedings of the National Academy of Sciences 121(3):e2311245121

January 2024
121(3):e2311245121

DOI:10.1073/pnas.2311245121

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CC BY-NC-ND 4.0

Authors:

Alexander J. Bradshaw

University of Utah

Virginia Ramírez Cruz

University of Guadalajara

Giuliana Furci

Fundación Fungi

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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 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 to 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 .

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PNAS 2024 Vol. 121 No. 3 e2311245121 https://doi.org/10.1073/pnas.2311245121 1 of 9

RESEARCH ARTICLE

Signiﬁcance

Therapeutic use of psilocybin from

“magic mushrooms” is

revolutionizing mental health

treatment for many conditions,

including depression, PTSD, and

end- of- life care. However,

knowledge of Psilocybe diversity

and its evolutionary history is

substantially incomplete. Our

study presents the most extensive

phylogenomic dataset across

Psilocybe to date, with 23 samples

derived from type specimens.

Using ~3,000 single- copy gene

families, we recovered a robust

and well- supported phylogeny.

Mapping psilocybin biosynthetic

gene orthologs on the phylogeny

revealed two types of gene cluster

order corresponding to a deep

split in the genus. Molecular

dating suggests psilocybin

biosynthesis arose in Psilocybe ~67

mya, concurrent with the K- Pg

mass extinction event. A

signicant advancement in the

understanding of Psilocybe

evolution and psilocybin

biosynthesis is presented.

Author contributions: A.J.B., G.F., and B.T.M.D. designed

research; A.J.B., V.R.- C., and A.R.A. performed research;

V.R.- C., A.R.A., L.G.- D., and B.T.M.D. contributed new

reagents/analytic tools and fungal specimens; A.J.B.,

V.R.- C., and A.R.A. analyzed data; and A.J.B., V.R.- C.,

L.G.- D., and B.T.M.D. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

This article is distributed under Creative Commons

Attribution- NonCommercial- NoDerivatives License 4.0

(CC BY- NC- ND).

1A.J.B. and V.R.- C. contributed equally to this work.

2To whom correspondence may be addressed. Email:

alexander.j.bradshaw@gmail.com or bryn.dentinger@

gmail.com.

This article contains supporting information online at

https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.

2311245121/- /DCSupplemental.

Published January 9, 2024.

EVOLUTION

Phylogenomics of the psychoactive mushroom genus Psilocybe

and evolution of the psilocybin biosynthetic gene cluster

AlexanderJ.Bradshawa,b,1,2 , VirginiaRamírez- Cruzc,1 , AliR.Awand, GiulianaFurcie, LauraGuzmán- Dávalosf, and BrynT.M.Dentingera,b,2

Edited by Pamela Soltis, University of Florida, Gainesville, FL; received July 20, 2023; accepted November 28, 2023

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 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 to 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.

phylogenomics | psychoactive | mushroom | Psilocybe | evolution

Psilocybin, the psychoactive natural product found in some mushrooms and other Fungi,

is at the forefront of a wave of recent research showing the tremendous potential of psy-

chedelic drugs for a wide range of mental health therapies and understanding human

consciousness (1–3). Although species of Psilocybe (Fr.) P. Kumm. (rst described as

Agaricus semilanceatus Fr.) have been known to the scientic community since the early

19th century (4), Psilocybe mushrooms have been used across Mesoamerica for centuries,

particularly in spiritual ceremonies (5–13). In many of these indigenous cultures, their

use is considered sacramental or devoted to healing practices. It was for this reason that

with the arrival of European colonialism, in most cases, these cultures experienced religious

persecution for their use of these mushrooms (5). is renewed interest in psilocybin has

shined a spotlight on the organisms that produce them, most of which belong to a single

genus of mushroom, Psilocybe. Colloquially known as “magic mushrooms,” their psycho-

active properties were made widely known in 1957 when Robert Gordon Wasson pub-

lished his personal experiences with them in Life magazine (14). However, since Nixon’s

1971 “war on drugs” campaign in the United States, psilocybin and its analogs have been

listed on Schedule 1 of the Controlled Substances Act, making possession of the com-

pounds without a special license a federal crime. is legal status stied research on

psilocybin and the organisms that produce it for the last 50+ years (11).

New research in the last six years has yielded surprising insights into the biosynthesis

and evolution of psilocybin. In most Fungi, psilocybin is synthesized by four core enzymes

[PsiD (tryptophan decarboxylase), PsiK (kinase), PsiM (methyltransferase), and PsiH

(P450 monooxegynase)] that convert the amino acid tryptophan into psilocybin and whose

genes occur in a biosynthetic gene cluster (BGC) (15). BGCs constitute close proximity

clustering of genes related to the biosynthesis of secondary metabolites, which are com-

monly used for accessory metabolic functions such as chemical defense or metabolism of

variable carbon sources (16). e close proximity allows for ecient and coordinated

expression of the genes in the cluster, which often serve complementary functions in the

production of a specic secondary metabolite (17). In Fungi, secondary metabolite pro-

duction through BGCs is common in the biosynthesis of many important compounds,

such as melanin and penicillin (18). However, BGCs are not strictly limited to Fungi and

can be found across a wide variety of prokaryotes and eukaryotes. For example, the

well- studied lactose operon (“lac operon”) encodes enzymes directly related to the metab-

olism of lactose as a carbon source and is analogous to a BGC in that genes of the operon

are tightly linked but regulated by a single promoter. Similarly, “pathogenicity islands,”

such as SPI- II in Salmonella spp., contain a multitude of genes that are expressed in a

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2 of 9 https://doi.org/10.1073/pnas.2311245121 pnas.org

coordinated and systematic manner, akin to BGC expression, and

directly confer the ability to become pathogenic in numerous hosts

(19, 20). In plants, BGCs are involved in chemical defenses against

insect herbivory, such as in the biosynthesis of morphine and

nepetalactone (21, 22). BGCs have also been found in animals,

such as ancient plant- like terpene BGCs in corals that generate

antifeedant chemicals (23). While secondary metabolites are not

necessarily directly related to primary growth and development,

they provide a system in which an organism can favorably alter

interactions within its environment, making them essential to the

evolutionary ecology of all life (16).

e canonical psilocybin BGC is highly conserved across the

handful of species investigated (24–27) and has been reportedly

acquired by mushrooms outside of Psilocybe through horizontal

gene transfer (HGT) (25) and convergent evolution (24).

However, only ve of the ~165 species of Psilocybe (11) have been

investigated at a genomic- level and only 24 have been included

in multi- locus molecular phylogenetic datasets (10, 28). us, the

tempo, mode, and timing of the evolution of the psilocybin BGC

remains largely unknown, impeding our ability to construct a

robust predictive framework for elucidating evolutionary patterns

and the discovery of novel therapeutics.

In this study, we generated genomic- level data from 71 Psilocybe

taxa using fungarium specimens, including 23 types. Type specimens

are the ultimate authority for applying names to other collections

and are therefore essential references for accurately naming and

describing diversity (29). In recent years, generating DNA sequences

from historical specimens (“museomics”) has beneted from advances

in DNA extraction methods, and the increasing accessibility and

cost reduction of amplicon- independent high- throughput sequenc-

ing (30–33). Historical collections represent hundreds of years of

the collective eort and serve as a signicant concentration of rare

and unique samples (34–36). Outside of a handful of common spe-

cies, Psilocybe spp. are not commonly collected, and many specimens

are only represented by a single collection, making research with

them tremendously dicult (11).

Using the metagenomic data derived from our voucher specimens,

we set out to construct a robust, time- calibrated phylogeny. We then

bioinformatically mined the primary psilocybin BGC and mapped

the core genes on the phylogeny to investigate the tempo, mode, and

patterns of its evolution. We also investigated the timing of the origin

and purported horizontal acquisition events of the psilocybin BGC

by combining publicly available data from all known mushrooms

with the psilocybin BGC to gain insight into the evolutionary forces

that gave rise to psilocybin biosynthesis.

Results

Sequencing and Genome Assembly. Metagenomic shotgun

sequencing was performed on 74 samples, including 71 identied

as Psilocybe and three from other taxa known to produce psilocybin.

ese specimens were all derived from museum vouchers ranging

in age from 3 to 74 y, with one sample missing a collection date

(SIAppendix, TableS1). Total reads for samples ranged from 3,909,488

reads (Psilocybe_mexicana_IBUG- 13593, ~11.7x coverage) to

225,748,454 reads (Psilocybe_baeocystis_WTU- F- 011245, ~ 677.2x

coverage). Across all of our samples, we achieved an average of

94,490,694 reads (~283.5x coverage) and a median of 99,070,482

reads (~297.2x coverage). Genome assembly completeness as

measured by the N50 statistic ranged from N50 = 554 (Psilocybe_

tuberosa_WTU- F- 011378) to N50 = 60,042 (Psilocybe_stuntzii_

WTU- F- 011520). e number of contigs ranged from 7,004

(Psilocybe_stuntzii_WTU- F- 011520) to 784,732 (Psilocybe_

caerulescens_var_mazatecorum_SFSU- F- 029971). BUSCO scores

ranged from 30.7% (Psilocybe_fuliginosa_NY- 1901148) to 95.4%

(Psilocybe_stuntzii_WTU- F- 011520). It should be noted that

these assemblies are likely to be highly discontiguous due to low

endogenous DNA content from museum specimens and should be

treated as metagenome assemblies, but that are nonetheless useful

for phylogenomics (37). A complete summary of sequencing results

and genome assembly statistics is available in SIAppendix, TableS2.

Psilocybe Phylogenomic Analysis and Divergence Time of Major

Clades. Multiple sequence alignment statistics for the Psilocybe-

only dataset revealed 87% (2,615) single- copy genes were recovered

for all samples, 11% (339) recovered between 60 and 61 samples,

and less than 1% (29) recovered fewer than 60 (SI Appendix).

Phylogenetic analysis revealed 52/71 were true Psilocybe (Psilocybe

sensu stricto) (incl. 20 types) (Fig.1) and the other 19 were found

to belong to other genera: 14 specimens correspond to Deconica

(W.G. Sm.) P. Karst. (three types, combined in SI Appendix,

Results and Discussion), three form a monophyletic group of un-

certain placement within Strophariaceae, one corresponded to

Kuehneromyces (Psilocybe_laticystis_UBC- F16759, sister to all of

Deconica), and one (Psilocybe_washingtonensis_WTU- F- 055019)

was unable to be accurately assigned to an existing genus and was

therefore removed from further analysis (SIAppendix, Fig.S1 and

TableS1).

Phylogenetic analysis of the concatenated supermatrix and sum-

mary coalescent analysis of individual gene trees yield topologically

identical phylogenies with strong support for most nodes except

for two nodes internal to species clades with multiple representa-

tives (SI Appendix, Figs. S1 and S2). An ancient divergence within

Psilocybe was recovered, corresponding to Clades I & II in

Ramírez- Cruz et al. (10). e phylogenetic relationships of 39

Psilocybe species are reported (Fig. 2, Left).

e stem age of Psilocybe was estimated at 67.61 mya with the

crown age estimated at 56.43 mya (Fig. 2, Left; see also supple-

mentary data on Dryad). e LTT plot does not deviate from the

null hypothesis of linear growth (Fig. 2, Left). If a pattern of expo-

nential growth were present instead, it would suggest that Psilocybe

has undergone an evolutionary radiation or contraction, which is

not supported by our data.

Incorporating Publicly Available Sequences. Across all publicly

available sequences, 40 species were represented, ~24% of the

approximately 165 currently accepted Psilocybe s.s. (11). However,

sequence number and taxonomic representation of molecular

markers varied widely. No species retrieved from the public

databases had all gene regions representing a single voucher

specimen. We retrieved 26 ITS, 32 Ef1a, 30 RPB1, and 32 RPB2

sequences from public databases representing 11, six, nine, and

three species not represented in the genomic dataset, respectively.

In total, the combined datasets were represented by 67 unique

taxa, 17 of which were only available from public databases

(SIAppendix, Figs.S3–S6).

Misidentication is a common issue in fungaria and Psilocybe

specimens in particular (11, 29). In our study, we identied four

specimens that were consistently incongruent with the other

taxa they clustered with: “Psilocybe acutissima” (GAM00011063),

“Psilocybe baeocystis” (WTU- F011245), “Psilocybe quebecensis”

(NY1901130), and “Psilocybe silvatica” (VPI- F0003693). ese

specimens were reexamined microscopically and redetermined as

Psilocybe hoogshagenii R. Heim (GAM00011063), Psilocybe cya-

nescens Wakef. (WTU- F01124), and Psilocybe caerulipes (Peck) Sacc.

(NY1901130, VPI- F0003693) (SI Appendix, Table S1).

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PNAS 2024 Vol. 121 No. 3 e2311245121 https://doi.org/10.1073/pnas.2311245121 3 of 9

Identication of Psilocybin BGC Genes in Psilocybe. In most

cases, the gene predictions from exonerate matched those from

Augustus/RBB, with disagreements still having correct gene hits.

In a few cases, we found that exonerate identied genes more

accurately and with less ambiguity compared to our RBB method

(SIAppendix, TableS3).

We found variability in the order of the four- core psilocybin-

producing cluster genes within our expanded representation of

Psilocybe species, identifying two distinct patterns. e rst pattern

followed a gene order of PsiD > PsiM > PsiH > PsiK, the canonical

gene order originally reported from Psilocybe cubensis and Psilocybe

serbica M. M. Moser & E. Horak, which was represented entirely

Fig.1. Diversity of Psilocybe sensu stricto. (A) Psilocybe azurescens (image credit, Paul Stamets), (B) Psilocybe cyanescens (image credit Bryn T.M. Dentinger), (C) Psilocybe

semilanceata (image credit Giuliana Furci), (D) Psilocybe zapotecorum (image credit Bryn T.M. Dentinger), (E) Psilocybe cubensis (image credit Oscar Castro- Jauregui),

(F) Psilocybe bohemica (image credit Jan Borovička), (G) Psilocybe yungensis (image credit Virginia Ramírez- Cruz), and (H) Psilocybe mexicana (image credit Oscar Castro- Jauregui).

PsiD PsiKPsiHPsiM

PsiD PsiM PsiK

PsiH PsiH

Psilocybe pleurocystidiosa Isotype(NY-761619)

Psilocybe yungensis(SFSU-F-29944)

Psilocybe heimii Isotype (NY-761610)

Psilocybesemilanceata(SFSU-F-029972)

Psilocybefagicola var. mesocystidiata (NY-761608)

Psilocybe tampanensis(UBC-F10177)

Psilocybecubensisv1.0

Psilocybe weilii Isotype(WTU-F-063525)

Psilocybecolumbiana Isotype (NY-761607)

Psilocybe galindii Isotype (NY-761609)

Psilocybe liniformansvar. americana (NY-1797145)

Psilocybe muliercula(GAM-00011071)

Psilocybezapotecorum(FFCL-689)

Psilocybe pelliculosa (WTU-F-012331)

Psilocybe caerulipes (PUL-00030154)

Psilocybe caerulipes*(VPI-F-0003693)

Psilocybe cyanescens(WTU-F-011306)

Psilocybesamuiensis(WTU-F-055014)

Psilocybe singeri Isotype(NY-761622)

Psilocybesubyungensis Isotype (NY-1197500)

Psilocybe guilartensis Paratype(CFMR-PR-5680)

Psilocybe cyanofibrillosa Isotype(NY-761605)

Psilocybe callosa(NY-1595861)

Psilocybe congolensis Isotype (NY-1652567)

Psilocybeportoricensis(CFMR-PR-4572)

Psilocybe subfimetaria (SFSU-F-029945)

Psilocybe azurescens (UT-M0001775)

Psilocybe hoogshageniivar.convexa Isotype(NY-761612)

Psilocybeaztecorum (SFSU-F-029933)

Psilocybe hoogshagenii* (GAM-00011063)

Psilocybeovoideocystidiata Isotype (XAL-51B)

Psilocybecaerulescens (NY-1920304)

Psilocybeaztecorum var.bonetii(NY-1595856)

Psilocybe cyanescens (NHYD01003446.1)

Psilocybe xalapensis Isotype(NY-761630)

Psilocybe mexicana (IBUG-13593)

Psilocybe bohemica(SFSU-F-029930)

Psilocybe hoogshagenii(SFSU-F-029980)

Psilocybesubcubensis (SFSU-29974)

Psilocybe fimetaria (UBC-F-30923)

Psilocybezapotecorum(GAM-00011076)

Psilocybeangustipleurocystidiata Isotype(NY-761597)

Psilocybesubhoogshagenii(NY-915004)

Psilocybe serbica v1.0

Psilocybe stuntzii (WTU-F-011520)

Psilocybe caerulescens var.mazatecorum (SFSU-F-029971)

Psilocybe cubensis (IBUG-4367)

Psilocybeargentipes(NY-1595850)

Psilocybe cyanescens (KM-189047)

Psilocybewrightii Isotype(NY-761629)

Psilocybearcana Isotype(SFSU-F-000737)

Psilocybe cyanescens*(WTU-F-011245)

PsilocybeazurescensHolotype(WTU-F19095)

Psilocybe hopii Isotype (XAL)

Psilocybemoravica Isotype(SFSU-F-000732)

Psilocybe caerulipes* (NY-1901130)

“C”

“cubensae”

“mexicanae”

“zapotecorum”

“cordisporae”

PsiD

PsiH

Psilocybe angustipleurocystidiata Isotype (NY-761597)|Psi M

Psilocybe xalapensis Isotype (NY-761630)|Psi M

Psilocybe wrightii Isotype (NY-761629)|Psi M

Psilocybe zapatoecorum (GAM00011076)|Psi M

Psilocybe pelliculosa (WTU-F-012331)|Psi M

Psilocybe subcubensis (SFSU-29974)|Psi M

Psilocybe arcana Isotype (SFSU-F-000737)|Psi M

Psilocybe fimetaria (UBC-F30923)|Psi M

Psilocybe columbiana Isotype (NY-761607)|Psi M

Psilocybe weilii Isotype (WTU-F-063525)|Psi M

Psilocybe cyanofibrillosa Isotype (NY-761605)|Psi M

Psilocybe samuiensis (WTU-F-055014)|Psi M

Psilocybe pleurocystidiosa Isotype (NY-761619)|Psi M

Psilocybe argentipes (NY-1595850)|Psi M

Psilocybe aztecorum (SFSU-F-029933)|Psi M

Psilocybe hopii Isotype (XAL) |Psi M

Psilocybe tampanensis (UBC-F10177)|Psi M

Psilocybe

cyanescens

(WTU-F-011245)|Psi M*

Psilocybe semilanceata (SFSU-F-029972)|Psi M

Psilocybe azurescens (UT-M0001775)|Psi M

Psilocybe subhoogshagenii (NY-915004)|Psi M

Psilocybe aztecorum var. bonetii (NY-1595856)|Psi M

Psilocybe callosa (NY-1595861)|Psi M

Psilocybe moravica Isotype (SFSU-F-000732)|Psi M

Psilocybe heimii Isotype (NY-761610)|Psi M

Psilocybe cubensis (IBUG-4367)|Psi M

Psilocybe muliercula (GAM00011071)|Psi M

Psilocybe bohemica (SFSU-F-029930)|Psi M

Psilocybe azurescens Holotype (WTU-F19095)|Psi M

Psilocybe hoogshagenii (SFSU-F-029980)|Psi M

Psilocybe portoricensis (CFMR-PR-4572)|Psi M

Psilocybe fagicola var. mesocystidiata (NY-761608)|Psi M

Psilocybe mexicana (IBUG-13593)|Psi M

Psilocybe subyungensis Isotype NY-1197500|Psi M

Psilocybe caerulipes(VPI-F-0003693)|Psi M*

Psilocybe liniformans var. americana (NY-1797145)|Psi M

Psilocybe yungensis (SFSU-29944)|Psi M

Psilocybe cyanescens (WTU-F-011306)|Psi M

Psilocybe singeri Isotype (NY-761622)|Psi M

Psilocybe caerulipes(NY-1901130)|Psi M*

Psilocybe caerulescens var. mazatecorum (SFSU-F-029971)|Psi M

Psilocybe caerulescens (NY-1920304)|Psi M

Psilocybe subfimetaria (SFSU-F-029945)|Psi M

Psilocybe guilartensis Paratype (CFMR-PR-5680)|Psi M

Psilocybe caerulipes (PUL00030154)|Psi M

Psilocybe galindii Isotype (NY-761609)|Psi M

Psilocybe stuntzii (WTU-F-011520)|Psi M

Psilocybe zapotecorum (FFCL689)|Psi M

Psilocybe congolensis Isotype (NY-1652567)|Psi M

Psilocybe hoogshagenii var. convexa Isotype (NY-761612)|Psi M

Psilocybe ovoideocystidiata Isotype (XAL-51B)|Psi M

Psilocybe hoogshagenii (GAM00011063)|Psi M*

100

PsiM

Psilocybe muliercula (GAM00011071)|Psi D

Psilocybe portoricensis (CFMR-PR-4572)|Psi D

Psilocybe hoogshageni (SFSU-F-029980)|Psi D

Psilocybe caerulipes(VPI-F-0003693)|Psi D*

Psilocybe cyanofibrillosa Isotype (NY-761605)|Psi D

Psilocybe azurescens Holotype (WTU-F19095)|Psi D

Psilocybe congolensis Isotype(NY-1652567)|Psi D

Psilocybe heimii Isotype (NY-761610)|Psi D

Psilocybe weilii Isotype (WTU-F-063525)|Psi D

Psilocybe arcana Isotype (SFSU-F-000737)|Psi D

Psilocybe cyanescens (WTU-F-011245)|Psi D*

Psilocybe hoogshagenii (GAM00011063)|Psi D*

Psilocybe aztecorum (SFSU-F-029933)|Psi D

Psilocybe guilartensis Paratype (CFMR-PR-5680)|Psi D

Psilocybe galindii Isotype (NY-761609)|Psi D

Psilocybe stuntzii (WTU-F-011520)|Psi D

Psilocybe azurescens (UT-M0001775)|Psi D

Psilocybe liniformans var. americana (NY-1797145)|Psi D

Psilocybe caerulipes (PUL00030154)|Psi D

Psilocybe angustipleurocystidiata Isotype (NY-761597)|Psi D

Psilocybe caerulipes(NY-1901130)|Psi D*

Psilocybe cubensis (IBUG-4367)|Psi D

Psilocybe cyanescens (WTU-F-011306)|Psi D

Psilocybe ovoideocystidiata Isotype (XAL-51B)|Psi D

Psilocybe mexicana (IBUG-13593)|Psi D

Psilocybe moravica Isotype (SFSU-F-000732)|Psi D

Psilocybe wrightii Isotype (NY-761629)|Psi D

Psilocybe subyungensis Isotype (NY-1197500)|Psi D

Psilocybe hopii Isotype (XAL) |Psi D

Psilocybe fagicola var. mesocystidiata (NY-761608)|Psi D

Psilocybe subcubensis (SFSU-29974)|Psi D

Psilocybe pleurocystidiosa Isotype (NY-761619)|Psi D

Psilocybe yungensis (SFSU-29944) |Psi D

Psilocybe subhoogshagenii (NY-915004)|Psi D

Psilocybe tampanensis UBC-F10177|Psi D

Psilocybe zapotecorum (GAM00011076)|Psi D

Psilocybe hoogshagenii var. convexa Isotype (NY-761612)|Psi D

Psilocybe xalapensis Isotype (NY-761630)|Psi D

Psilocybe aztecorum var. bonetii (NY-1595856)|Psi D

Psilocybe columbiana Isotype (NY-761607)|Psi D

Psilocybe caerulescens var. mazatecorum (SFSU-F-029971)|Psi D

Psilocybe argentipes (NY-1595850)|Psi D

Psilocybe bohemica (SFSU-F-029930)|Psi D

Psilocybe callosa (NY-1595861)|Psi D

Psilocybe fimetaria (UBC-F30923)|Psi D

Psilocybe subfimetaria (SFSU-F-029945)|Psi D

Psilocybe zapotecorum (FFCL689)|Psi D

Psilocybe pelliculosa (WTU-F-012331)|Psi D

Psilocybe singeri Isotype (NY-761622)|Psi D

Psilocybe caerulescens (NY-1920304)|Psi D

Psilocybe samuiensis (WTU-F-055014|Psi D

Psilocybe semilanceata (SFSU-F-029972)|Psi D

100

Psilocybemuliercula(GAM00011071)|Psi H

Psilocybezapotecorum(FFCL689)|Psi H

Psilocybeaztecorumvar. bonetii (NY-1595856)|Psi H

Psilocybe pleurocystidiosa Isotype(NY-761619)|Psi H

Psilocybeaztecorum(SFSU-F-029933)|Psi H

a(IBUG-13593)|Psi H

Psilocybeargentipes(NY-1595850)|Psi H

Psilocybecaerulescens(NY-1920304)|Psi H

Psilocybe

cyanescens

(WTU-F-011245)|Psi H*

Psilocybeangustipleurocystidiata Isotype(NY-761597)|Psi H

10177)|Psi H

is(UBC-F10177)|Psi H

Psilocybe pelliculosa(WTU-F-012331)|Psi H

Psilocybesubhoogshagenii(NY-915004)|Psi H

Psilocybe guilartensis Paratype(CFMR-PR-5680)|Psi H

Psilocybecaerulescensvar.mazatecorum(SFSU-F-029971)|Psi H

Psilocybe heimii Isotype(NY-761610)|Psi H

PsilocybeazurescensHolotype(WTU-F19095)|Psi H

Psilocybeyungensis(SFSU-29944)|Psi H

Psilocybe weilii Isotype(WTU-F-063525)|Psi H

m(GAM00011076)|Psi H

Psilocybestuntzii(WTU-F-011520)|Psi H

Psilocybe liniformansvar.americana(NY-1797145)|Psi H

Psilocybecallosa(NY-1595861)|Psi H

Psilocybecyanescens(WTU-F-011306)|Psi H

Psilocybe ovoideocystidiata Isotype(XAL-51B)|Psi H

Psilocybe hopii Isotype(XAL) |Psi H

Psilocybecongolensis Isotype(NY-1652567)|Psi H

Psilocybe zapotecorum(GAM00011076)|Psi H

scye sage AM00011063)|Psi H*

Psilocybe wrightii Isotype(NY-761629)|Psi H

Psilocybe hoogshagenii(SFSU-F-029980)|Psi H

Psilocybecaerulipes (PUL00030154)|Psi H

Psilocybesemilanceata (SFSU-F-029972)|Psi H

Psilocybecaerulipes(VPI-F-0003693)|Psi H*

13593)|Psi H

Psilocybecolumbiana Isotype(NY-761607)|Psi H

Psilocybexalapensis Isotype(NY-761630)|Psi H

Psilocybe portoricensis(CFMR-PR-4572)|Psi H

-055014)|Psi H

Psilocybeazurescens (UT-M0001775)|Psi H

Psilocybesubfimetaria(SFSU-F-029945)|Psi H

Psilocybecubensis (IBUG-4367)|Psi H

Psilocybesubyungensis Isotype(NY-1197500)|Psi H

Psilocybecyanofibrillosa Isotype (NY-761605)|Psi H

Psilocybefimetaria(UBC-F30923)|Psi H

Psilocybecaerulipes(NY-1901130)|Psi H*

Psilocybecyanescens(WTU-F-011306)|Psi H

Psilocybesubcubensis(SFSU-29974)|Psi H

Psilocybesingeri Isotype(NY-761622)|Psi H

Psilocybe bohemica(SFSU-F-029930)|Psi H

var.convexa Isotype (NY-761612)|Psi H

m(FFCL689)|Psi H

Psilocybeazurescens(UT-M0001775)|Psi H

Psilocybemoravica Isotype(SFSU-F-000732)|Psi H

Psilocybefagicolavar.mesocystidiata(NY-761608)|Psi H

Psilocybea

rcana Isotype(SFSU-F-000737)|Psi H

sotype(NY-71609)|Psi H

Psilocybe caerulescens (NY-1920304)|Psi K

Psilocybe hoogshageni (SFSU-F-029980)|Psi K

Psilocybe singeri Isotype (NY-761622)|Psi K

Psilocybe cyanofibrillosa Isotype (NY-761605)|Psi K

Psilocybe argentipes (NY-1595850)|Psi K

Psilocybe bohemica (SFSU-F-029930)|Psi K

Psilocybe aztecorum var. bonetii (NY-1595856)|Psi K

Psilocybe weilii Isotype (WTU-F-063525)|Psi K

Psilocybe zapotecorum (FFCL689|)Psi K

Psilocybe callosa (NY-1595861)|Psi K

Psilocybe hoogshagenii (GAM00011063)|Psi K*

Psilocybe caerulescens var. mazatecorum (SFSU-F-029971)|Psi K

Psilocybe pelliculosa (WTU-F-012331)|Psi K

Psilocybe samuiensis (WTU-F-055014)|Psi K

Psilocybe portoricensis (CFMR-PR-4572)|Psi K

Psilocybe subyungensis Isotype (NY-1197500)|Psi K

Psilocybe moravica Isotype (SFSU-F-000732)|Psi K

Psilocybe aztecorum (SFSU-F-029933)|Psi K

Psilocybe hoogshagenii var. convexa Isotype (NY-761612)|Psi K

Psilocybe fagicola var. mesocystidiata (NY-761608)|Psi K

Psilocybe hopii Isotype (XAL) |Psi K

Psilocybe fimetaria (UBC-F30923)|Psi K

Psilocybe caerulipes(NY-1901130)|Psi K*

Psilocybe guilartensis Paratype (CFMR-PR-5680)|Psi K

Psilocybe subhoogshagenii (NY-915004)|Psi K

Psilocybe stuntzii (WTU-F-011520)|Psi K

Psilocybe mexicana (IBUG-13593)|Psi K

Psilocybe azurescens Holotype (WTU-F19095)|Psi K

Psilocybe caerulipes(VPI-F-0003693)|Psi K*

Psilocybe arcana Isotype (SFSU-F-000737)|Psi K

Psilocybe cyanescens (WTU-F-011306)|Psi K

Psilocybe congolensis Isotype (NY-1652567)|Psi K

Psilocybe yungensis (SFSU-29944)|Psi K

Psilocybe muliercula (GAM00011071)|Psi K

Psilocybe subfimetaria (SFSU-F-029945)|Psi K

Psilocybe columbiana Isotype (NY-761607)|Psi K

Psilocybe semilanceata (SFSU-F-029972)|Psi K

Psilocybe ovoideocystidiata Isotype (XAL-51B)|Psi K

Psilocybe galindii Isotype (NY-761609)|Psi K

Psilocybe heimii Isotype (NY-761610)|Psi K

Psilocybe angustipleurocystidiata Isotype (NY-761597)|Psi K

Psilocybe subcubensis (SFSU-29974)|Psi K

Psilocybe cyanescens

(WTU-F-011245)|Psi K*

Psilocybe caerulipes (PUL00030154)|Psi K

Psilocybe wrightii Isotype (NY-761629)|Psi K

Psilocybe liniformans var. americana (NY-1797145)|Psi K

Psilocybe tampanensis (UBC-F10177|)Psi K

Psilocybe pleurocystidiosa Isotype (NY-761619)|Psi K

Psilocybe zapotecorum (GAM00011076)|Psi K

Psilocybe xalapensis Isotype NY-761630|Psi K

Psilocybe cubensis (IBUG-4367)|Psi K

Psilocybe azurescens (UT-M0001775)|Psi K

100

PsiK

Agrocybe pediades

Galerina marginata

Gymnopilus chrysopellus

Gymnopilus junonius

outgroup

Loremipsum

010203040506070

(

-13593

)

177

)

(

)

10177

)

3593

)

055014)

ilo

resce

(

9095

)

ilo

esce

(

U-

011306

)

ilo

resce

M0001775)

ilo

anescens

(

WTU-

-011245

)

resce

-M0001775)|

sce

(

011306

)

(

)

ilo

(

)

(

AM00011076

)

(

AM00011076)

var

vex

(

689

)

Fig.2. Psilocybe phylogenomic tree and evolution of the psilocybin BCG. Left: Phylogenomic tree of Psilocybe estimated from a gene- partitioned concatenated

supermatrix of 2,983 single- copy gene families estimated in IQ- TREE. Tree is rooted with an outgroup consisting of Hebeloma cylindrosporum and Pholiota alnicola. All

nodes received 100% ultrafast bootstrap support in the concatenated analysis except where indicated. Topology is identical to summary coalescence of individual

gene trees except for the terminal relationships within the P. cyanescens species cluster. Time- transformed branch lengths and divergence dates were estimated

from loci with 100 clock- like, well- supported gene trees in RelTime using two internal calibrations based on the ages for Strophariaceae/Hymenogastraceae and

Gymnopilus, reported by Varga etal. (38) and Ruiz- Dueñas etal. (39). Divergence time 95% HPD condence bars are depicted in gold. Branch labels are percent

nonparametric bootstraps implemented using the within- partition ultrafast bootstrapping option in IQ- TREE. The lineage through time plot is represented by

a purple dashed line. Sequences derived from type specimens are labeled in red. The psilocybin BGC is depicted by arrows to the right of the terminal labels.

Arrows are oriented according to the coding direction for each gene as determined through reciprocal best BLAST hits and color- coded by gene (PsiD = red, PsiK

= green, PsiH = purple, PsiM = gold). Absence of genes indicates that placement in context of the whole cluster could not be reliably determined bioinformatically.

Right: Maximum- likelihood phylograms for each of the four core genes from the psilocybin- producing gene cluster, PsiD (red), PsiK (green), PsiM (gold), and PsiH

(purple). For enhanced readability, trees are midpoint rooted or rooted at the node leading to Clade II (PsiH). Duplicated PsiH genes are highlighted in yellow (full

length) or blue (truncated). The two major clades from the organismal phylogeny are labeled with roman numerals and with shaded boxes in the background.

Numbers on branches are percent nonparametric rapid bootstraps implemented using the within- partition ultrafast bootstrapping option in IQ- TREE.

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4 of 9 https://doi.org/10.1073/pnas.2311245121 pnas.org

by Clade II of our dataset. e second was PsiD > PsiK > PsiH >

PsiM, found in Clade I of our organismal phylogeny (Fig. 2,

Right). Across our phylogeny, the original pattern was represented

by 17 Psilocybe specimens, while 35 of our Psilocybe specimens

exhibited the non- canonical pattern.

In addition to gene order and orientation, we investigated

the evolution of each gene separately. Phylogenetic analysis was

performed without using our species constraint tree for each of

the core psilocybin- producing genes, PsiD, PsiK, PsiM, and

PsiH (Fig. 2, Right). We found that PsiD, PsiK, and PsiM were

topologically congruent with the organismal phylogeny, sug-

gesting a vertical inheritance pattern for these genes in Psilocybe.

In contrast, PsiH was largely topologically consistent with the

organismal phylogeny, but multiple taxa in both Clade I and

Clade II were found to have duplicated copies of PsiH that were

recovered as paraphyletic (Fig. 2, Right, and SI Appendix,

Table S3). In all cases, both copies of PsiH are found in tandem

on the same contig (SI Appendix, Table S3). One copy of PsiH

in each of P. zapotecorum, P. mexicana, and P. tampanensis is

truncated, indicating they are not functional. e phylogenetic

topology of the Clade I duplications is consistent with an

ancient duplication followed by gene loss in most taxa, with

relict, incompletely degenerated copies persisting in only a few

lineages.

Psilocybin Homologs Genes Present Outside of Psilocybe and

Ecological Niche Patterns. We also investigated the known

HGT events from Panaeolus (Fr.) Quél., Gymnopilus P. Karst.,

and Pluteus Fr., using publicly available data and specimens

sequenced in this study vouchered as Gymnopilus luteofolius

(Peck) Singer, Pluteus albostipitatus (Dennis) Singer, and Pluteus

salicinus (Pers.) P. Kumm (SI Appendix, TableS1 and Fig.3,

Left). Gene prediction and RBB analysis yielded representation

of the four- core psilocybin- producing genes from the published

genomes of Panaeolus cyanescens and Gymnopilus dilepis (Berk.

& Broome) Singer, and our Gymnopilus luteofolius and Pluteus

salicinus data (SIAppendix, TableS1).

Phylogenetic analysis of the PsiD gene, including those from

our Psilocybe genomic samples, and publicly available sequences,

revealed three branching patterns for our non- Psilocybe taxa. e

rst branch contained Conocybe smithii Watling (= Pholiotina

smithii (Watling) Enderle) as a monophyletic branch sister to our

Psilocybe caerulipes (Peck) Sacc. samples (BS 100%). e second

branch placed Panaeolus cyanescens as a sister group to Psilocybe

cubensis (BS 100%), and the nal contained species of both

Gymnopilus and Pluteus sharing a most recent common ancestor

with all of Psilocybe (BS 100%) (Fig. 3, Left).

After expanding Psilocybe diversity and rening the placement

of known HGT events, we sought to identify whether phyloge-

netic clusters could be attributed to specic ecological niches.

Ancestral state reconstruction was performed on the organismal

phylogeny, showing a wood decay ecology as the ancestral condi-

tion in the most recent common ancestor (MRCA) of Clades I

and II, with two independent specializations to coprophilic life-

styles in each (SI Appendix, Fig. S7). Interestingly, our two primary

phylogenetic clades correspond to distinct ecological lifestyles,

with a few notable exceptions (Fig. 3, Left). Clade I, identied

through our PsiD analysis, corresponds almost entirely to the

soil- dwelling saprotrophic lifestyle, except for the Psilocybe caer-

ulipes cluster, which exhibits wood decay ecology (i.e., rotting

wood rather than soil enriched with woody debris). Psilocybe Clade

II, Gymnopilus, and Pluteus were associated primarily with wood

decay, and in the case of Psilocybe cubensis and Panaeolus cyanescens,

a coprophilous lifestyle (Fig. 3, Left).

Evolutionary Origins and Timing of the Psilocybin BGC in

Mushrooms. Divergence dating is consistent with the earliest

possible origin of the psilocybin BGC in Psilocybe approximately

~67 mya with subsequent acquisitions by four other genera

between ~40 (Panaeolus) and ~9 (Gymnopilus) mya (Fig.3, Right

and SIAppendix). e crown age of Psilocybe was estimated at ~57

mya in the phylogenomic dataset, which predates the estimated

origin of psilocybin in the next possible oldest lineage, Panaeolus.

However, the relative ages of the psilocybin- producing Panaeolus

stem and the crown age of Psilocybe in the LSU dataset suggest

the earliest possible origin could be in Panaeolus. Additionally,

two species of Panaeolus and two species of Gymnopilus known

to contain the psilocybin BGC were paraphyletic in the LSU

phylogeny. e most parsimonious interpretation is that the

psilocybin BGC was gained twice independently in each genus.

e rst acquisition in Panaeolus (Pa.) is in Pa. subalteatus ~40

mya with a second acquisition ~16 mya in Pa. cyanescens. e

rst acquisition in Gymnopilus is in G. aeruginosus ~22 mya with

a second in G. junonius ~9 mya. e psilocybin BGC was gained

in Pluteus ~22 mya and in Pholiotina ~11 mya (SIAppendix). To

further test the notion that these acquisitions were due to HGT,

we performed a topological constraint test. e likelihoods of

the best ML trees for PsiD from topologically constrained and

unconstrained searches were −8780.501249 and −8699.391458,

respectively. Tree topology test statistics universally rejected the

constrained topology, corroborating the results of Reynolds etal.

(25) and consistent with expectations from HGT (SIAppendix,

TableS4).

Discussion

Phylogenomics of Psilocybe. Our phylogenomic analysis resulted

in a single, unambiguous and statistically well- supported

phylogeny for 52 specimens of Psilocybe s.s., resolving key nodes

that were previously unsupported (Fig.2, Left). Total topological

congruence inferred from a partitioned supermatrix analysis and

a summary coalescence of individual gene trees demonstrates that

the inferred phylogeny is robust to the methodological approach

and indicates low levels of gene discordance within Psilocybe.

e ancient divergence within Psilocybe rst reported by

Ramírez- Cruz et al. (10) was also recovered here. is early split

coincides with the genic arrangement of the psilocybin BGC

(Fig. 2, Left) but precedes a shift in ecology. Our results suggest

that Psilocybe arose as primarily a wood- decomposing group that

transitioned to soil after the split, with two independent special-

izations on herbivore dung (SI Appendix, Fig. S7). However, addi-

tional critical species, such as the African dung- dwelling

P. natalensis Gartz, D.A. Reid, M.T. Sm. & Eicker, will need to

be included to reconstruct evolution of ecologies more condently

in Psilocybe (further discussed in SI Appendix).

Origin of the Psilocybin BGC.

Reynolds etal. (25) speculated that

the psilocybin BGC may have originated in Fibularhizoctonia

G.C. Adams & Kropp, a genus of anamorphic (asexually

reproducing) Athelia Pers. that has multiple copies of Psi

homologs, albeit not contained in a cluster (40). e Atheliales

are close relatives of the Agaricales with a broad range of ecologies,

including termite symbionts (41), plant pathogens, mycorrhizal

mutualists, and saprobes. Reynolds etal. (25) speculated that

this insect association may have provided the selective force for

the evolution of psilocybin as a modulator of the symbiosis.

Interestingly, Athelia arachnoidea (Berk.) Jülich is mycoparasitic

on lichens common on tree bark in north temperate regions

(42, 43), whereas its anamorph Fibularhizoctonia carotae (Rader)

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PNAS 2024 Vol. 121 No. 3 e2311245121 https://doi.org/10.1073/pnas.2311245121 5 of 9

G.C. Adams & Kropp is pathogenic on carrot roots (44). Our

results suggest wood decomposition as the ancestral ecology

of Psilocybe. Both Gymnopilus and Pluteus are also wood

decomposers. e mycoparasitic ecology of the teleomorphic

(sexually reproducing) Athelia arachnoidea on bark is intriguing

because the habitat is shared with wood- dwelling mushrooms

and physical interaction may be one way the psilocybin BGC

could be transferred horizontally. However, the true vector of

these HGTs is unknown.

Tempo, Mode, and Timing of the Evolution of the Psilocybin BGC.

Previous studies have shown that the psilocybin BGC has been

transferred horizontally between several genera of mushrooms

within the order Agaricales (24, 25). Our analysis combined these

data with a much greater representation of Psilocybe, recovering the

PsiD genes in Gymnopilus and Pluteus outside of those in Psilocybe.

In contrast, the copies in Pholiotina and Panaeolus are nested

within the Psilocybe PsiD genes. is topology is consistent with

ancient divergence between the Psilocybe and Gymnopilus/Pluteus

psilocybin BGCs and indicates more recent HGT events between

Psilocybe and both Panaeolus and Pholiotina. Panaeolus cyanescens

appears to have a HGT event with the ancestor of Psilocybe

cubensis/subcubensis. However, the LSU tree indicates that Pa.

subalteatus is relatively older than Pa. cyanescens, and therefore the

possibility remains that an earlier HGT event occurred between

Panaeolus and Psilocybe. Interestingly, Pholiotina smithii appears

to have an HGT event with an ancestor of Clade I, which was

recovered with the highest likelihood of a dung ecology in our

ancestral state reconstruction. Pholiotina smithii and its potentially

conspecic relative Ph. cyanopus both have terrestrial ecologies

in water- saturated and Sphagnum moss- dominated soils and

grasslands, respectively. However, critical taxa are still missing

from the Psilocybe phylogeny, such as the Sphagnum- associated

and non- psilocybin- producing P. fuscofulva that has previously

been shown to occupy an early- diverging position (45), which

may substantially alter the ancestral state reconstructions.

HGT seems to be the most likely explanation for the polyphy-

letic distribution of the psilocybin BGC. Topological constraint

tests of the PsiD phylogeny was performed, and results rejected

the alternative hypothesis that recovered psilocybin- producing

Panaeolus and Pholiotina outside of Psilocybe, consistent with the

hypothesis that the gene cluster was acquired by these two genera

~67 mya

~9 mya

Panaeouls cyanescens

Panaeouls subbalteatus

Panaeolus

Pholiotina/ Conocybe

Pholiotina cyanopus

Psilocybe

Psilocybe atrobrunnea

Gymnopilus

Gymnopilus junonius

Gymnopilus aeruginosus

Pluteus

Pluteus salicinus

~40 mya

~11-22 mya

0.2

Psilocybe portoricensis (CFMR-PR-4572)|Psi D

Psilocybe zapotecorum (GAM00011076)|Psi D

Psilocybe heimii Isotype (NY-761610)|Psi D

Psilocybe arcana Isotype (SFSU-F-000737)|Psi D

Psilocybe caerulescens (NY-1920304)|Psi D

Psilocybe mexicana (IBUG-13593)|Psi D

Psilocybe cyanofibrillosa Isotype (NY-761605)|Psi D

Psilocybe hoogshagenii (SFSU-F-029980)|Psi D

Psilocybe guilartensis Paratype (CFMR-PR-5680)|Psi D

Psilocybe callosa (NY-1595861)|Psi D

Fibsp1 938295

Psilocybe galindii Isotype (NY-761609)|Psi D

Psilocybe zapotecorum (FFCL689)|Psi D

Psilocybe xalapensis Isotype (NY-761630)|Psi D

Psilocybe pleurocystidiosa Isotype (NY-761619)|Psi D

Psilocybe bohemica (SFSU-F-029930)|Psi D

Psilocybe ovoideocystidiata Isotype (XAL-51B)|Psi D

Psilocybe muliercula (GAM00011071)|Psi D

Gymnopilus dilepis (ASM293838v1)|PsiD

Psilocybe congolensis Isotype (NY-1652567)|Psi D

Gymjun1|Psi D

Psilocybe tampanensis (UBC-F10177)|Psi D

Psilocybe hopii Isotype (XAL) |Psi D

Psilocybe subhoogshagenii (NY-915004)|Psi D

Psilocybe hoogshangenii* (GAM00011063)|Psi D

Psicy01 2263

Psilocybe yungensis (SFSU-29944)|Psi D

Psilocybe azurescens (UT-M0001775)|Psi D

Psilocybe cyanescens (WTU-F-011306)|Psi D

Pluteus albostipitatus (KM-54312)|Psi D

Psilocybe subfimetaria (SFSU-F-029945)|Psi D

Pholiotina smithii 2634DeC

Pluteus salicinus (KM-265162)|Psi D

Psilocybe singeri Isotype (NY-761622)|Psi D

Psilocybe cyanescens (KY984104-1)*|Psi D

Psilocybe caerulipes* (NY-1901130)|Psi D

Psilocybe caerulipes (PUL00030154)|Psi D

Psilocybe columbiana Isotype (NY-761607)|Psi D

Psilocybe liniformans var. americana (NY-1797145)|Psi D

Psilocybe azurescens Holotype (WTU-F19095)|Psi D

Psilocybe cubensis

Pancy01 5060

Psubaeruginosa

Gymlu01 2525

Psilocybe wrightii Isotype (NY-761629)|Psi D

Psilocybe angustipleurocystidiata Isotype (NY-761597)|Psi D

ocy

e caeru

escens var. mazatecorum

(SFSU

029971)|P

Psilocybe cyanescens* (WTU-F-011245)|Psi D

Psilocybe weilii Isotype (WTU-F-063525)|Psi D

Psilocybe cubensis (KY984101-1)|Psi D

Psilocybe pelliculosa (WTU-F-012331)|Psi D

Psilocybe aztecorum var. bonetii (NY-1595856)|Psi D

Psilocybe samuiensis (WTU-F-055014)|Psi D

Psilocybe stuntzii (WTU-F-011520)|Psi D

Gymnopilus luteofolius (KM-27062)|Psi D

Psilocybe caerulipes* (VPI-F-0003693)|Psi D

Psilocybe cubensis (IBUG-4367)|Psi D

Psilocybe argentipes (NY-1595850)|Psi D

Fibsp1 949465

Psilocybe hoogshagenii var. convexa Isotype (NY-761612)|Psi D

Pholiotina smithii

Psilocybe subyungensis Isotype (NY-1197500)|Psi D

Psilocybe fagicola var. mesocystidiata (NY-761608)|Psi D

Gymch1 1578740

Psilocybe semilanceata (SFSU-F-029972)|Psi D

Psilocybe moravica Isotype (SFSU-F-000732)|Psi D

Psilocybe aztecorum (SFSU-F-029933)|Psi D

Psilocybe subcubensis (SFSU-29974)|Psi D

Panaeolus cyanescens (ASM293835v1)|Psi D

Psilocybe fimetaria (UBC-F30923)|Psi D

Pazurescens

100

Soil with

Wood

Soil

wood debris

Dung

uteu

s s

ali

inu

Fig.3. PsiD gene tree and LSU gene tree of psilocybin- producing mushrooms and their inactive relatives. Left: Maximum- likelihood phylogram of Agaricomycetes

PsiD homologs. Tree is rooted with Phallomycetidae following Reynolds etal. (25). Numbers on branches are percent nonparametric rapid bootstraps implemented

using the within- partition ultrafast bootstrapping option in IQ- TREE. Icons depicting ecological niche are indicated to the Right of the terminal labels and legend is

depicted in the Upper left. Major clades are denoted with roman numerals and background shading. Right: Maximum- likelihood time tree of all publicly available LSU

sequences for species with the homologous canonical psilocybin BGC and their non- active relatives. Tree is rooted with Calocybe gambosa following the Agaricales

topology in Dentinger etal. (37). Species with the psilocybin BGC are indicated by blue branches. Genera are color- coded in each tree with lines and terminal

labels to corresponding psilocybin- producing taxa outside of Psilocybe in the PsiD tree. Nodes where the earliest possible acquisition of the psilocybin BGC likely

occurred are indicated by blue dots. Time scale is at the bottom with the inferred dates of psilocybin BGC acquisition for each lineage indicated by hashmarks.

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6 of 9 https://doi.org/10.1073/pnas.2311245121 pnas.org

through HGT (25) (SI Appendix, Table S4). Additionally, our

phylogenetic analyses are consistent with these prior reports and

show that the psilocybin BGC has been acquired (likely by

HGT) in mushrooms with increasing frequency over the past

~67 mya. In contrast, our molecular dating results provide evi-

dence that the psilocybin BGC has been present in Psilocybe

between ~56 (MRCA of the genus Psilocybe) and ~67 mya

(MRCA of Psilocybe and Panaeolus) (Fig. 3, Right). While our

LSU dataset presented a more recent divergence estimate for the

MRCA of Psilocybe, we only included sequences from genera

containing psilocybin- producing species so these estimates are

likely to be unreliable due to missing taxa. In comparison, diver-

gence dating of our phylogenomic dataset placed the MRCA of

all of Psilocybe at ~57 mya and the MRCA of Psilocybe and

Agrocybe at ~67 mya (SI Appendix).

Our molecular dating analyses relied entirely on secondary

calibrations, which are vulnerable to inaccuracies (46). Although

these secondary calibrations were derived from studies that used

fossil calibrations and we applied uniform rather than normal

prior distributions, our absolute dates should still be considered

tentative estimates until primary calibrations are available or cor-

roborating evidence is provided. Although a handful of fossil

Agaricales exist, the closest fossils to Psilocybe are Nidulariaceae,

estimated to be between 45 and 90 mya, but this family is still

very distantly related to Psilocybe (38, 47). An expanded dataset

to enable a single calibration point would be compromised by

additional phylogenetic uncertainty. Moreover, many of the avail-

able fossils cannot be placed phylogenetically with high condence

due to the dearth of reliable morphological characters. However,

regardless of the absolute timing, the presence of the gene cluster

in all Psilocybe s.s. specimens from this study suggests that the

psilocybin BGC is primarily inherited vertically within the genus,

although some patterns suggest that this may not be the only

mechanism.

e evolution and organization of the psilocybin BGC in

Psilocybe exhibit some patterns that are consistent with multiple

gains or losses. Two dierent gene orders within the BGC correlate

with the earliest divergence within Psilocybe (Fig. 2, Left), indicat-

ing an ancient reconstruction of genes within the genus.

Interestingly, the dierence in gene order is consistent with a cir-

cular transmission model (48), in which the core cluster genes

undergo HGT via a circularized intermediate, which linearizes

upon insertion into a new genome, proposed for Pluteus salicinus

in Awan et al. (24). is nding also indicates that transcriptional

regulation of the genes may vary among taxa, resulting in

taxon- specic metabolic proles (49, 50). Due to a high number

of discontiguous assemblies in our dataset, future studies should

place considerable eort on obtaining highly contiguous genomes

from modern collections to better rene the psilocybin BGC

within Psilocybe.

Duplication of PsiH. Upon deeper investigation of PsiH, we

found that it had considerably more sequence variation than any

of the other core genes and has undergone multiple duplications

or multiple losses (Fig.2 and SIAppendix, TableS3). When we

included second- best hit identications to the PsiH phylogeny, we

found that the splitting of clade II became less severe, suggesting

multiple paralogs are present among many species of Psilocybe.

PsiH encodes an indole- 4- monooxygenase that catalyzes the

oxidation of tryptamine at the 4- carbon position of the indole

ring (15), a feature that is critical to the pharmacological properties

of psilocybin/psilocin and that is both rare in nature and dicult

to achieve with traditional synthetic chemistry. is innovation in

the psilocybin BGC could be considered the most important to

the functioning of the pathway due to its specic activity and is

critical for invivo synthesis of psilocybin in heterologous systems

(51, 52). us, our study provides additional PsiH sequences that

may be useful for optimizing production of psilocybin or other

analogs using heterologous expression.

Ecological Role of Psilocybin. Despite its prominence in the

scientic and public imagination, experimental evidence for the

ecological role of psilocybin is largely lacking and speculation reigns

supreme, as thoroughly investigated and reviewed in Meyer and Slot

(2023) (53). Psilocybin is a prodrug that is rapidly converted to

the dephosphorylated psilocin, which mimics serotonin and binds

tightly to serotonin receptors, especially 5- HT

, a pharmacological

action common to psychedelics (54). Serotonin receptors are also

found in high concentration in the human gastrointestinal tract,

where serotonin signaling is involved in a wide variety of functions,

including pain and vomiting (55–57). High- anity binding to

these receptors in mammals, and homologs in distantly related

organisms such as insects and arachnids, produces unnatural and

altered behaviors (58–61). is disorientation may be a direct

deterrent or could render the subject more vulnerable to predation

(or, alternatively, may be a reward). However, in humans, the onset

of symptoms is delayed by 30 min or more, eectively decoupling

the eects from their source. Because of the potentially large eect

of psilocybin on GI processes through interactions with serotonin

receptors in the gut, it is also possible that psilocybin functions as an

emetic or laxative to promote the dispersal of spores before they are

rendered inviable by digestion. In fact, nausea is commonly reported

in human clinical trials (62). However, other than humans, there

are few documented cases of vertebrates consuming psilocybin-

containing mushrooms (consisting entirely of domesticated dogs)

(63). Many Psilocybe mushrooms are uncommon or scarce, and it

is dicult to imagine how animals could learn to recognize them

with infrequent encounters and delayed pharmacological activity,

limiting the selective potential of psilocybin as a psychoactive

defense.

Fungal–insect interactions are ancient and widespread and pro-

vide a more logical hypothesis for development of psilocybin as a

chemical defense in mushrooms (25, 64, 65). To date, this has

been the most commonly asserted hypothesis for the ecological

role of psilocybin, but empirical studies are still lacking.

Furthermore, anecdotes and personal observations conrm that

psilocybin- containing mushrooms regularly have living insect

larvae in them and they can be reared to adults (24). While not

substantive, these anecdotes and initial studies indicate that psil-

ocybin may be ineective against insects or that some insects may

have evolved detoxifying capabilities in response. Moreover, stud-

ies demonstrating that psilocybin alters behavior are absent in

many non- model insects.

An alternative hypothesis is that psilocybin functions indirectly

in an inducible chemical defense system. Lenz et al. (64) eluci-

dated the chemical basis of the blue pigment that forms when

psilocybin- containing mushrooms are damaged as oligo/polymers

of psilocin. e conversion of psilocybin to psilocin and the link-

ing of them into chains is enzymatically controlled. Lenz et al.

(64, 65) pointed out that the psilocin oligo/polymers have chem-

ical properties similar to plant avonoids and polyphenolic tan-

nins, which produce reactive oxygen molecules that can damage

gut tissue. ey proposed the “polymer hypothesis,” where the

psilocin oligo/polymers may be an inducible defense against fun-

givory and psilocybin is simply the artillery kept in reserve for the

true chemical weaponry (64, 65). Intriguingly, the formation of

the blue psilocin oligo/polymers is invariably connected to psilo-

cybin biosynthesis throughout the multiple independent

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PNAS 2024 Vol. 121 No. 3 e2311245121 https://doi.org/10.1073/pnas.2311245121 7 of 9

inheritances (24, 25) and convergent evolution (24). e main-

tenance of the enzymatic capacity to induce psilocybin conversion

lends further support to the hypothesis that it is the blue oligo/

polymer that has an ecological function rather than the possibly

accidental pharmacological eects of psilocybin itself.

Materials and Methods

DNA Extraction and Genomic Sequencing. Hymenophore fragments (5 to

15 mg) from dried fungarium samples, derived from a large collection of insti-

tutional loans made to the Natural History Museum of Utah (UMNH) (11) were

homogenized by placing them in 2.0 mL screw- cap tubes containing a single

3.0- mm and 8 × 1.5- mm stainless steel beads and shaking them in a BeadBug™

microtube homogenizer (Sigma- Aldrich, #Z763713) for 120 s at a speed setting

of 3,500 rpm. For samples collected after 1950, DNA extraction was performed

with Monarch® Genomic DNA Puriﬁcation Kit (NEB, #T3010S) following the man-

ufacturer’s protocol for Tissue Lysis with a 1 h incubation at 56 °C except with

double the volume of lysis buffer and increasing the amount of wash buffer

to 550 µL during each of the wash steps. For samples collected before 1950,

including many of the type specimens (see SIAppendix, TableS1 for holding

institution and collection information), DNA extraction was performed using a

phenol- chloroform DNA extraction protocol. In short, after physical homogeni-

zation, lysis was performed as above, after which total lysate was placed in Phase

Lock Gel™ Light tubes (QuantaBio, #2302820) along with an equal volume of

OmniPur® Phenol:Chloroform:Isoamyl Alcohol (25:24:1, TE- saturated, pH 8.0)

solution (MilliporeSigma, Calibiochem #D05686) and then mixed by gentle

inversion for 15 min using a ﬁxed speed tube rotator. After mixing, tubes were

centrifuged at maximum speed (14,000×g) for 10 min; then, the aqueous (top)

layer was transferred to a new phase- lock gel tube and the process repeated. DNA

precipitation of the aqueous phase was performed by adding 5 M NaCl to a ﬁnal

concentration of 0.3 M and two volumes of room temperature absolute ethanol,

inverting the tubes 20× for thorough mixing followed by an overnight incubation

at −20 °C. The next day, DNA was pelleted by centrifugation at 14,000×g for

5 min. The DNA pellet was washed twice with freshly prepared, ice- cold 70%

ethanol, air- dried for 15 min at room temperature, and then resuspended in 150

µL of Elution Buffer from the Monarch® Genomic DNA kit. DNAs were submitted to

the High Throughput Genomics Core at the University of Utah, where sequencing

libraries were prepared using the Nextera™ DNA Flex Library Prep (Illumina®,

#20018704) and sequenced on a full lane of Illumina® NovaSeq6000 PE 2 ×

150 bp using an S4 ﬂow cell.

Sequence Processing and Genome Assembly. Sequencing run statistics and

quality metric were visualized for each sample using FastQC version 0.11.9 and

then compared to each other using MultiQC version 1.10 (66). Library complex-

ity was estimated using the “EstimateLibraryComplexity” function of the Picard

toolkit (http://broadinstitute.github.io/picard/). Raw sequencing reads were

trimmed and quality ﬁltered using fastP version 0.20.1 (67) and then assembled

using the paired- end assembly in SPAdes version 3.15.2 (68) with kmer values

alternating every other digit between 21 and 127, inclusive. Genome assembly

stats were quantiﬁed using a custom Perl script (https://github.com/hobrien/Perl/

blob/master/ContigStats.pl).

Homolog Extraction and Phylogenetic Analysis. Single- copy gene families

were identiﬁed from the Psilocybe serbica genome (Psiser1) Markov Cluster

(MCL) proﬁle (clustering.2497, https://mycocosm.jgi.doe.gov/clm/run/Psiser1-

comparative- qc.2497;SvdlKh?organism=Psiser1) generated by the Joint

Genome Institute’s (JGI) Mycocosm genome portal (69, 70). This proﬁle included

two species of Psilocybe (P. cubensis (Earle) Singer and P. serbica) (15) and six

closely related taxonomic outgroups: Agrocybe pediades (Fr.) Fayod (Agrped1)

(39), Galerina marginata (Batsch) Kühner (Galma1) (71), Gymnopilus chrysopellus

(Berk. & M.A. Curtis) Murrill (Gymch1) (39), G. junonius (Fr.) P.D. Orton (Gymjun1)

(39), Hebeloma cylindrosporum Romagn. (Hebcy2) (72), and Pholiota alnicola

(Fr.) Singer [=Flammula alnicola (Fr.) P. Kumm.)] (Phoaln1) (39). Amino acid

sequences of the resulting 2,983 genes in the cluster from Galma1:v1 (Galerina

marginata) (71) were used as queries for homolog identiﬁcation in the new

assemblies using the “protein2genome” model in exonerate version 2.4.0 (73).

The protein2genome model allows for amino acid sequences to be aligned to a

genome allowing introns, frameshifts, and exon phase changes in the alignment

to better identify orthologous genes, even when distantly related. The output-

speciﬁc ﬂag “- - ryo “%tcs\n”” was also used to extract the sequence information

that occurs in the target genome coding sequence of the protein2genome align-

ment, which was used for further downstream phylogenetic analysis. Nucleotide

sequence output from exonerate for each amino acid query was combined and

aligned using the multiple sequence alignment program MAFFT version 7.475

(74) with the parameters - - maxiterate 1000 - - localpair - - reorder. Concatenation

and multiple sequence alignment summary statistics were performed using the

package AMAS (75). Individual gene trees were estimated with IQ- TREE multi-

core version 2.2.0.3 (76) using ModelFinder (77) and 1,000 ultrafast bootstrap

replicates optimized using the - - bnni ﬂag (78). A summary coalescent tree was

constructed from the individual gene trees using ASTRAL- III (79) after removing

branches with low support (BS < 10%). A phylogenomic tree was also constructed

from a concatenated supermatrix of all genes using a partitioned analysis in IQ-

TREE allowing model selection and model parameter estimation for each gene

partition separately with branch lengths shared among all partitions (option - p),

and branch supports estimated as above.

Divergence Dating. The fast- dating method of the relative rate framework (RRF),

implemented in RelTime and incorporated into the MEGA 11 software package

(80–82), was used to estimate divergence times using the ML topology and branch

lengths estimated from the concatenated supermatrix with Psilocybe s.s. as the in-

group and six selected outgroup taxa (SIAppendix, Fig.S1). We chose this method

as it has been shown to have comparable accuracy to Bayesian- based approaches

but requires far less computational resources (83). However, with 2,983 loci, the

computational burden was excessive even with RelTime. Therefore, to further reduce

computational burden, we selected a subset of 100 loci from the 2,983 genes whose

gene trees were the most clock- like (had the least amount of root- to- tip variation)

and had maximal average bipartition support using the package SortaDate (84) to

use in RelTime. To calibrate the divergence time estimations, the node representing

the most recent common ancestor (MRCA) of Psilocybe plus Agrocybe pediades,

Galerina marginata, and Gymnopilus spp. [=Strophariaceae sensu Varga etal. (38)

and Hymenogastraceae sensu Ruiz- Dueñas et al. (39)] was constrained using a

uniform prior with a minimum of 57 mya [well- supported core shift stem age of

Strophariaceae s.s.; Varga etal. (38)] and maximum of 71 mya [stem age of the

Hymenogastraceae; Ruiz- Dueñas etal. (39)], and ﬁxing the stem age of Gymnopilus

to 54 mya [Varga etal. (38)]. The number of lineages- through- time (LTT) was esti-

mated with the “ltt” function in the R package ape (85) using log- transformed

y- values to account for progressively fewer extinction events toward the present.

Incorporating Publicly Available Sequences. Four traditional phylogenetic

loci for Fungi (ITS, Ef1a, RPB1, and RPB2) were bioinformatically extracted from the

genome assemblies using PathRacer (86). PathRacer uses hidden Markov models

(HMMs) to query de Bruijn assembly graphs for compatible paths, independent

of assembled contiguous sequences, minimizing the limitations of discontiguous

assemblies for sequence mining. HMMs were built using nucleotide sequences

from 10,859 complete ITS sequences obtained from the NCBI RefSeq Targeted

Loci Project (87), and 487 partial EF1a exons, 639 partial RPB1 exons, and 937

partial RPB2 exons generated by AFTOL (88) and downloaded from GenBank,

and aligned automatically with the L- INS- i algorithm in MAFFT version 7.475

(74). HMM proﬁles for each gene were generated with hmmer version 3.1b2

(hmmer.org) using the multiple sequence alignments as inputs. ITS sequences

were manually trimmed from edge sequence outputs. Sequences of EF1a,

RPB1, and RPB2 were extracted from edge sequence outputs with exonerate

using the ﬂag - protein2genome using query protein sequences from Psilocybe

cyanescens Wakef. in GenBank (GenBank accessions: Ef1a=ADI71893.1;

RPB1=AHB18799.1; RPB2=AHH34099.1). All Psilocybe sequences correspond-

ing to the same loci (ITS, EF1a, RPB1, and RPB2) were retrieved from NCBI as

well as all Psilocybe species hypotheses (SH) ITS sequences from the curated

fungal database UNITE (89). Each dataset from NCBI was checked for overlap

of different genes for a single vouchered specimen using fungarium accession

numbers or collections IDs in the GenBank records. The loci extracted from the

genomes generated in this study were combined with the publicly available data

in single locus matrices and then automatically aligned with the L- INS- i algorithm

in MAFFT. Phylogenetic trees for each gene as well as a concatenated supermatrix

of the four traditional phylogenetic loci were constructed using IQ- TREE with the

same parameters as above, including gene partitions in the concatenated matrix

to allow for separate models and model parameters to be estimated per gene,

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8 of 9 https://doi.org/10.1073/pnas.2311245121 pnas.org

and with the addition of a backbone constraint using the rooted phylogenomic

topology implemented with the - g ﬂag.

Identication of Psilocybin BGC Genes in Psilocybe.

We used gene prediction

and reciprocal- best BLAST (RBB) to identify homologs of gene from the psilocy-

bin BGC in our genome assemblies. Due to the diploid nature of our genomes,

we ﬁrst used the software package Redundans version 0.14a (90) to phase our

assemblies before gene prediction. Psilocybin gene cluster detection was per-

formed using the Augustus version 3.4.0 gene prediction software (91) with the

ﬂags –singlestrand=true to predict genes independently on each strand with the

coprinus_cinereus training set. Following gene prediction, the most likely psilo-

cybin BGC for each species was chosen as follows: Using the protein sequences

for PsiD (tryptophan decarboxylase), PsiK (kinase), PsiM (methyltransferase), and

PsiH (P450) based on the publicly available chromosomal level assembly for

P. cubensis (92), we used BLASTP with default parameters to search for homologs

in the predicted translated proteins for each species. The top three hits for each

gene were then used together to ﬁnd putative clusters by examining the gene

numbers assigned by augustus. Putative “clusters” were identiﬁed as any subset

of the 21 genes where the predicted gene numbers of any gene within the subset

was at most two genes away from one other gene in the subset. This set of com-

putationally generated, putative psilocybin BGCs was then manually curated by

querying the top three hits against the Psi gene sequences from the chromosomal

assembly of P. cubensis using BLASTn to measure their similarities. The largest

subset with high similarities to the Psi genes was chosen as the cluster candidate

for each Psilocybe species (Fig.2). To corroborate our gene predictions, we utilized

exonerate with the same Psi protein queries. Identiﬁcations were compared to

gene prediction output to determine the order and orientation of each gene in the

cluster based on the strand position (+/- ) of the start codon for each gene (Fig.2).

Individual gene multiple sequence alignments and maximum likelihood trees

were estimated for each psi gene using MAFFT and IQ- TREE as above.

Global PsiD Phylogenetic Analysis, Topological Constraint Testing, and

Ancestral State Reconstruction. PsiD nucleotide sequences extracted from

all primary psilocybin BGC- containing mushrooms with exonerate were tran-

scribed utilizing codon- aware multiple sequence alignment through HYPHY

version 2.5.36 (93) following the tutorial outlined at https://github.com/veg/

hyphy- analyses/blob/master/codon- msa/README.md. Extracted PsiD amino acid

sequences were combined with the PsiD amino acid sequences published in

Reynolds etal. (25), undergoing multiple sequence alignment and phylogenetic

analysis under the same parameters used for individual gene trees. Ecological

lifestyle was determined from original species descriptions and monographs

when available, or directly from collection notes (SIAppendix, TableS1).

To test the hypothesis that psilocybin- producing Panaeolus (Fr.) Quél. and

Pholiotina Fayod PsiD are derived within Psilocybe PsiD, consistent with HGT, topo-

logical constraint testing was performed by comparing the best ML tree constrained

to recover a monophyletic Psilocybe in IQ- TREE version 1.6.12 with the uncon-

strained PsiD tree using bootstrap proportion (BP), weighted Kishino–Hasegawa

test (94), weighted Shimodaira–Hasegawa test (95), expected likelihood weights

(96) with 1,000 RELL replicates, and the approximately unbiased (AU) test (97). An

unrooted topological constraint tree was rendered in Mesquite version 3.2 (98).

Ancestral state reconstruction was performed on our concatenated supermatrix

phylogenetic tree using RASP 4 (4.3 build 20220517 x64) (99). Each sample was

coded for their respective ecological niches (soil, wood, dung, and soil with wood

debris) and run under default settings using the MCMC Bayesian option of the

Multistate Reconstruction in BayesTraits model (100) (SIAppendix). MCMC output

was analyzed using Tracer version 1.7.2 (101), which displayed a unimodal distribution

supporting convergence with an effective samples size (ESS) of 450 (SIAppendix).

Tempo, Mode, and Timing of the Primary Psilocybin BGC.

In order to assess

the relative timing of psilocybin biosynthesis across mushrooms, a dataset was

constructed of all publicly available sequences (194 individuals across 147 spe-

cies) of the ribosomal large subunit DNA region (28S; downloaded from NCBI

on 14 August 2022) for all mushroom genera with species containing the pri-

mary psilocybin BGC: Conocybe Fayod /Pholiotina, Gymnopilus, Panaeolus Fr.,

Pluteus, and Psilocybe. A sequence of Calocybe gambosa (Fr.) Donk was used as

an outgroup. Due to the poor overlap between taxa for other gene regions, the

28S region had the best species representation that could be aligned across all

genera. Multiple sequence alignment was performed using the L- INS- i algorithm

in MAFFT. Phylogenetic analysis was performed under maximum likelihood using

IQ- TREE with automatic model selection and up to 1,000 nonparametric rapid

bootstraps. Divergence dating of the LSU gene tree was performed with RelTime

in the same manner as above but with ﬁxed node ages for Gymnopilus p.p. (54

mya), Panaeolus (60 mya), and Conocybe (45 mya) based on Varga etal. (38).

Data, Materials, and Software Availability. Raw short- read sequences

for this project have been deposited in the Sequence Read Archive (SRA) and

assigned the bioproject number PRJNA904752 and SRA and Biosample acces-

sion numbers are reported in SIAppendix, TableS1. Raw tree ﬁles, assemblies,

gene prediction ﬁles, multiple sequence alignments, and Psilocybe constraint

tree are available through Dryad (https://datadryad.org/stash/landing/show?id=

doi%3A10.5061%2Fdryad.tmpg4f52s) (102). Any code or speciﬁc script requests

should be sent to the corresponding author.

ACKNOWLEDGMENTS. We acknowledge the Natural History Museum of Utah for

its commitment to collaborative Science and the Genomics Core Facility, a part of the

Health Sciences Cores at The University of Utah, for their input and high- quality work.

Additionally, we acknowledge the numerous institutions that provided specimens

for destructive sampling, many of which were rare and irreplaceable. Further, we wish

to recognize the dedicated and hard work performed by Isabelle Galland and Talia

A. Backman in their help processing many of these samples for DNA sequencing.

Further, we wish to thank David Scott Flocken for his insight and thought- provoking

conversations on the work generated here. Additionally, we would like to thank

Jan Borovička and Oscar Castro- Jauregui for providing high- quality images and

graciously allowing us to use them for publication. We would also like to thank Paul

Stamets who provided images and feedback on this publication, and Fungi Perfecti

LLC who provided funding which helped facilitate this work. Additionally, we would

like to thank Dr. Jason Slot for providing data from previous publications as well as

intriguing conversions about Psilocybin evolution. V.R.- C. and L.G.- D. would like

to thank the University of Guadalajara and CONAHCYT for their support of their

research. This work was supported by a grant from NSF (DEB #2114785) and partially

from a generous donation to the NHMU from Fungi Perfecti LLC.

Author aliations: aSchool of Biological Sciences, University of Utah, Salt Lake City, UT 84112;

bNatural History Museum of Utah, Collections and Research, University of Utah, Salt Lake

City, UT 84108; cConsejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT),

Departamento de Botánica y Zoología, Universidad de Guadalajara, Zapopan 45147,

Mexico; dGenomics Innovation Unit, Guy’s and St.Thomas’ NHS Foundation Trust, St Thomas’

Hospital, London SE1 7EH, United Kingdom; eFungi Foundation, Brooklyn, NY 11216; and

fDepartamento de Botánica y Zoología, Universidad de Guadalajara, Zapopan 45147, Mexico

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Los hongos alucinógenos (Psilocybe, Hymenogastraceae) en el desarrollo evolutivo de la consciencia del hombre

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Full-text available

May 2024

Esta revisión bibliográfica explora el papel de los hongos alucinógenos, específicamente el género Psilocybe de la familia Hymenogastraceae, en el desarrollo evolutivo de la conciencia humana. A través de un enfoque multidisciplinario que abarca biología, etnobotánica y neurociencia, se examina la influencia de compuestos como la psilocibina y la psilocina en los consumidores, revelando su potencial para desencadenar efectos neurológicos y psicológicos significativos. Se explora el impacto en la conciencia humana, señalando cambios perceptuales, cognitivos y emocionales. Se analiza el desarrollo evolutivo y antropológico de los seres humanos, explorando cómo la disponibilidad de alimentos, el uso de la psilocibina y las adaptaciones fisiológicas han podido influir en el éxito reproductivo y la supervivencia a lo largo del tiempo.

Synthetic biology enables mushrooms to meet emerging sustainable challenges

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Feb 2024

With the increasing sustainability challenges, synthetic biology is offering new possibilities for addressing the emerging problems through the cultivation and fermentation of mushrooms. In this perspective, we aim to provide an overview on the research and applications mushroom synthetic biology, emphasizing the need for increased attention and inclusion of this rapidly advancing field in future mushroom technology over China and other countries. By leveraging synthetic biology, mushrooms are expected to play a more versatile role in various area, including traditional fields like circular economy, human wellness and pharmaceutics, as well as emerging fields like vegan meat, mushroom-based materials and pollution abatement. We are confident that these efforts using synthetic biology strategies have the potential to strengthen our capacity to effectively address sustainable challenges, leading to the development of a more sustainable social economy and ecology.

Cultivation, chemistry, and genome of Psilocybe zapotecorum

Preprint

Nov 2023

Psilocybe zapotecorum is a strongly blue-bruising psilocybin mushroom used by indigenous groups in southeastern Mexico and beyond. While this species has a rich history of ceremonial use, research into its chemistry and genetics have been limited. Herein, we detail mushroom morphology and report on cultivation parameters, chemical profile, and the full genome sequence of P. zapotecorum . First, growth and cloning methods are detailed that are simple, and reproducible. In combination with high resolution microscopic analysis, the strain was barcoded, confirming species-level identification. Full genome sequencing reveals the architecture of the psilocybin gene cluster in P. zapotecorum, and can serve as a reference genome for Psilocybe Clade I. Characterization of the tryptamine profile revealed a psilocybin concentration of 17.9±1.7 mg/g, with a range of 10.6-25.7 mg/g (n=7), and similar tryptamines (psilocin, baeocystin, norbaeocystin, norpsilocin, aeruginascin, 4-HO-tryptamine, and tryptamine) in lesser concentrations for a combined tryptamine concentration of 22.5±3.2 mg/g. These results show P. zapotecorum to be a potent – and variable – Psilocybe mushroom. Chemical profiling, genetic analysis, and cultivation assist in demystifying these mushrooms. As clinical studies with psilocybin gain traction, understanding the diversity of psilocybin mushrooms will assure that psilocybin therapy does not become synonymous with psilocybin mushrooms.

Los hongos sagrados del género Psilocybe en Jalisco

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Sep 2022

Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution of the psilocybin biosynthetic gene cluster

Preprint

Full-text available

Dec 2022

Psychoactive mushrooms in the genus Psilocybe have immense cultural value and have been used for centuries in Mesoamerica. Despite a recent surge in interest in these mushrooms due to emerging evidence that psilocybin, the main psychoactive compound, is a promising therapeutic for a variety of mental illnesses, their phylogeny and taxonomy remain substantially incomplete. Moreover, the recent elucidation of the psilocybin biosynthetic gene cluster is known for only five species of Psilocybe, four of which belong to only one of two major clades. We set out to improve the phylogeny for Psilocybe using shotgun sequencing of 71 fungarium specimens, including 23 types, and conducting phylogenomic analysis using 2,983 single-copy gene families to generate a fully supported phylogeny. Molecular clock analysis suggests the stem lineage arose ~66 mya and diversified ~53 mya. We also show that psilocybin biosynthesis first arose in Psilocybe, with 4-5 possible horizontal transfers to other mushrooms between 40 and 22 mya. Moreover, predicted orthologs of the psilocybin biosynthetic genes revealed two distinct gene orders within the cluster that corresponds to a deep split within the genus, possibly consistent with the independent acquisition of the cluster. This novel insight may predict differences in chemistry between the two major clades of the genus, providing further resources for the development of novel therapeutics.

Assessing the relative performance of fast molecular dating methods for phylogenomic data

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Dec 2022
BMC GENOMICS

Advances in genome sequencing techniques produced a significant growth of phylogenomic datasets. This massive amount of data represents a computational challenge for molecular dating with Bayesian approaches. Rapid molecular dating methods have been proposed over the last few decades to overcome these issues. However, a comparative evaluation of their relative performance on empirical data sets is lacking. We analyzed 23 empirical phylogenomic datasets to investigate the performance of two commonly employed fast dating methodologies: penalized likelihood (PL), implemented in treePL, and the relative rate framework (RRF), implemented in RelTime. They were compared to Bayesian analyses using the closest possible substitution models and calibration settings. We found that RRF was computationally faster and generally provided node age estimates statistically equivalent to Bayesian divergence times. PL time estimates consistently exhibited low levels of uncertainty. Overall, to approximate Bayesian approaches, RelTime is an efficient method with significantly lower computational demand, being more than 100 times faster than treePL. Thus, to alleviate the computational burden of Bayesian divergence time inference in the era of massive genomic data, molecular dating can be facilitated using the RRF, allowing evolutionary hypotheses to be tested more quickly and efficiently.

DNA Authentication and Chemical Analysis of Psilocybe Mushrooms Reveal Widespread Misdeterminations in Fungaria and Inconsistencies in Metabolites

Article

Full-text available

Nov 2022
APPL ENVIRON MICROB

The mushroom genus Psilocybe is best known as the core group of psychoactive mushrooms, yet basic information on their diversity, taxonomy, chemistry, and general biology is still largely lacking. In this study, we reexamined 94 Psilocybe fungarium specimens, representing 18 species, by DNA barcoding, evaluated the stability of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens across the most commonly vouchered species (Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe semilanceata), and explored the metabolome of cultivated P. cubensis. Our data show that, apart from a few well-known species, the taxonomic accuracy of specimen determinations is largely unreliable, even at the genus level. A substantial quantity of poor-quality and mislabeled sequence data in public repositories, as well as a paucity of sequences derived from types, further exacerbates the problem. Our data also support taxon- and time-dependent decay of psilocybin and psilocin, with some specimens having no detectable quantities of them. We also show that the P. cubensis metabolome possibly contains thousands of uncharacterized compounds, at least some of which may be bioactive. Taken together, our study undermines commonly held assumptions about the accuracy of names and presence of controlled substances in fungarium specimens identified as Psilocybe spp. and reveals that our understanding of the chemical diversity of these mushrooms is largely incomplete. These results have broader implications for regulatory policies pertaining to the storage and sharing of fungarium specimens as well as the use of psychoactive mushrooms for recreation and therapy. IMPORTANCE The therapeutic use of psilocybin, the active ingredient in "magic mushrooms," is revolutionizing mental health care for a number of conditions, including depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spotlighted the current state of knowledge of psilocybin, including the organisms that endogenously produce it. However, because of international regulation of psilocybin as a controlled substance (often included on the same list as cocaine and heroin), basic research has lagged far behind. Our study highlights how the poor state of knowledge of even the most fundamental scientific information can impact the use of psilocybin-containing mushrooms for recreational or therapeutic applications and undermines critical assumptions that underpin their regulation by legal authorities. Our study shows that currently available chemical studies are mainly inaccurate, irreproducible, and inconsistent, that there exists a high rate of misidentification in museum collections and public databases rendering even names unreliable, and that the concentration of psilocybin and its tryptamine derivatives in three of the most commonly collected Psilocybe species (P. cubensis, P. cyanescens, and P. semilanceata) is highly variable and unstable in museum specimens spanning multiple decades, and our study generates the first-ever insight into the highly complex and largely uncharacterized metabolomic profile for the most commonly cultivated magic mushroom, P. cubensis.

Ancient plant-like terpene biosynthesis in corals

Article

Full-text available

Jun 2022
NAT CHEM BIOL

Octocorals are major contributors of terpenoid chemical diversity in the ocean. Natural products from other sessile marine animals are primarily biosynthesized by symbiotic microbes rather than by the host. Here, we challenge this long-standing paradigm by describing a monophyletic lineage of animal-encoded terpene cyclases (TCs) ubiquitous in octocorals. We characterized 15 TC enzymes from nine genera, several of which produce precursors of iconic coral-specific terpenoids, such as pseudopterosin, lophotoxin and eleutherobin. X-ray crystallography revealed that coral TCs share conserved active site residues and structural features with bacterial TCs. The identification of coral TCs enabled the targeted identification of the enzyme that constructs the coral-exclusive capnellane scaffold. Several TC genes are colocalized with genes that encode enzymes known to modify terpenes. This work presents an example of biosynthetic capacity in the kingdom Animalia that rivals the chemical complexity generated by plants, unlocking the biotechnological potential of octocorals for biomedical applications. Marine natural products are often made by symbiotic microbes, but octocorals encode their own terpene cyclases that share features with bacterial versions and produce terpenoid precursors, including the coral-exclusive capnellane scaffold.

Genetic Survey of Psilocybe Natural Products

Article

Full-text available

Jun 2022
CHEMBIOCHEM

Psilocybe magic mushrooms are best known for their main natural product, psilocybin, and its dephosphorylated congener, the psychedelic metabolite psilocin. Beyond tryptamines, the secondary metabolome of these fungi is poorly understood. The genomes of five species (P. azurescens, P. cubensis, P. cyanescens, P. mexicana, and P. serbica) were browsed to understand more profoundly common and species‐specific metabolic capacities. The genomic analyses revealed a much greater and yet unexplored metabolic diversity than evident from parallel chemical analyses. P. cyanescens and P. mexicana were identified as aeruginascin producers. Lumichrome and verpacamide A were also detected as Psilocybe metabolites. The observations concerning the potential secondary metabolome of this fungal genus support pharmacological and toxicological efforts to find a rational basis for yet elusive phenomena, such as paralytic effects, attributed to consumption of some magic mushrooms.

Diversity, biology, and history of psilocybin-containing fungi: Suggestions for research and technological development

Article

Full-text available

Feb 2022

Therapeutic use of psilocybin has become a focus of recent international research, with preliminary data showing promise to address a range of treatment-resistant mental health conditions. However, use of psilocybin as a healing entheogen has a long history through traditional consumption of mushrooms from the genus Psilocybe. The forthcoming adoption of new psilocybin-assisted therapeutic practices necessitates identification of preferred sources of psilocybin; consequently, comprehensive understanding of psilocybin-containing fungi is fundamental to consumer safety. Here we examine psilocybin producing fungi, discuss their biology, diversity, and ethnomycological uses. We also review recent work focused on elucidation of psilocybin biosynthetic production pathways, especially those from the genus Psilocybe, and their evolutionary history. Current research on psilocybin therapies is discussed, and recommendations for necessary future mycological research are outlined.

The Evolution and Ecology of Psilocybin in Nature

Article

May 2023
FUNGAL GENET BIOL

Fungi produce diverse metabolites that can have antimicrobial, antifungal, antifeedant, or psychoactive properties. Among these metabolites are the tryptamine-derived compounds psilocybin, its precursors, and natural derivatives (collectively referred to as psiloids), which have played significant roles in human society and culture. The high allocation of nitrogen to psiloids in mushrooms, along with evidence of convergent evolution and horizontal transfer of psilocybin genes, suggest they provide a selective benefit to some fungi. However, no precise ecological roles of psilocybin have been experimentally determined. The structural and functional similarities of psiloids to serotonin, an essential neurotransmitter in animals, suggest that they may enhance the fitness of fungi through interference with serotonergic processes. However, other ecological mechanisms of psiloids have been proposed. Here, we review the literature pertinent to psilocybin ecology and propose potential adaptive advantages psiloids may confer to fungi.

Ancestral State Reconstruction Using BayesTraits

Chapter

Sep 2022

The fossil record is the best evidence of the characteristics of extinct species, but only a narrow range of traits fossilize or survive the fossilization process. Lacking fossil or other evidence about the past, ancestral states can be reconstructed. Three pieces of information are combined when reconstructing ancestral states: extant or known trait values (data); the evolutionary history, linking the species of interest (phylogeny); and the evolutionary model of trait change. These reconstructed ancestral states can be interpreted as our best guess as to the route evolution took, given the distribution of the trait across species, the relationships among them, and our model of evolution. Because the information we use to reconstruct the past is often not known without error, uncertainty about their true values should be accounted for when reconstructing ancestral states. In this chapter we describe how ancestral states can be reconstructed using a Bayesian framework implemented in the software BayesTraits to account for uncertainty in the phylogenetic tree and the model of evolution.

A whole genome atlas of 81 Psilocybe genomes as a resource for psilocybin production.

Article

Sep 2021

The Psilocybe genus is well known for the synthesis of valuable psychoactive compounds such as Psilocybin, Psilocin, Baeocystin and Aeruginascin. The ubiquity of Psilocybin synthesis in Psilocybe has been attributed to a horizontal gene transfer mechanism of a ~20Kb gene cassette. A recently published highly contiguous reference genome derived from long read single molecule sequencing has underscored interesting variation in this Psilocybin synthesis gene cassette. This reference genome has also enabled the shotgun sequencing of spores from many Psilocybe strains to better catalog the genomic diversity in the Psilocybin synthesis pathway. Here we present the de novo assembly of genomes of 81 Psilocybe genomes compared to the P.envy reference genome. Surprisingly, the genomes of Psilocybe galindoi , Psilocybe tampanensis and Psilocybe azurescens lack sequence coverage over the previously described Psilocybin synthesis pathway but do demonstrate amino acid sequence homology to an alternative pathway and may illuminate previously proposed convergent evolution of Psilocybin synthesis.

Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution of the psilocybin biosynthetic gene cluster

Abstract

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