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

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DNA Authentication and Chemical Analysis of Psilocybe
Mushrooms Reveal Widespread Misdeterminations in Fungaria
and Inconsistencies in Metabolites
Alexander J. Bradshaw,a,b Talia A. Backman,aVirginia Ramírez-Cruz,dDale L. Forrister,aJaclyn M. Winter,cLaura Guzmán-Dávalos,e
Giuliana Furci,fPaul Stamets,gBryn T. M. Dentingera,b
a
School of Biological Sciences, University of Utah, Salt Lake City, Utah, USA
b
Natural History Museum of Utah, University of Utah, Salt Lake City, Utah, USA
c
Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, USA
d
CONACYT-UdeG, Departamento de Botánica y Zoología, Universidad de Guadalajara, Zapopan, Jalisco, Mexico
e
Departamento de Botánica y Zoología, Universidad de Guadalajara, Zapopan, Jalisco, Mexico
f
Fungi Foundation, Brooklyn, New York, USA
g
Fungi Perfecti LLC Laboratories, Shelton, Washington, USA
ABSTRACT The mushroom genus Psilocybe is best known as the core group of psy-
choactive 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 sta-
bility of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens
across the most commonly vouchered species (Psilocybe cubensis,Psilocybe cyanes-
cens, 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 reposito-
ries, 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 uncharacter-
ized 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 mush-
rooms,”is revolutionizing mental health care for a number of conditions, including
depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spot-
lighted the current state of knowledge of psilocybin, including the organisms that endo-
genously produce it. However, because of international regulation of psilocybin as a con-
trolled 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 assump-
tions 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
Editor Irina S. Druzhinina, Royal Botanic
Gardens
Copyright © 2022 Bradshaw et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Alexander J.
Bradshaw, alexander.j.bradshaw@gmail.com, or
Bryn T. M. Dentinger,
bdentinger@nhmu.utah.edu.
The authors declare a conflict of interest. Paul
Stamets is the current CEO of Fungi Perfecti
LLC, Co-founder of MycoMedica Life Sciences,
PBC and has patents pending and approved
using psilocybin. All other authors report no
conflicting interests in the body or intent of
this work.
Received 31 August 2022
Accepted 6 November 2022
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GENETICS AND MOLECULAR BIOLOGY
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rendering even names unreliable, and that the concentration of psilocybin and its trypt-
amine derivatives in three of the most commonly collected Psilocybe species (P. cubensis,
P. cyanescens,andP. 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 culti-
vated magic mushroom, P. cubensis.
KEYWORDS mycology, Psilocybe, fungaria, metabolomics
Mushrooms belonging to the genus Psilocybe (Fr.) P. Kumm. have become a major
scientific and public curiosity since 1957 when R. Gordon Wasson published a
popular account of his first-hand experience with psychedelic mushrooms in Life mag-
azine (1). Although species of Psilocybe (the first of which was originally described as
Agaricus semilanceatus Fr.) have been known to the scientific community since the
early 19th century (2), Wasson’s article was the first to make their psychoactive proper-
ties widely known. However, the ceremonial use of the psychoactive mushrooms has
been practiced by the indigenous peoples of Mesoamerica for centuries.
The use of psychoactive mushrooms is reportedly depicted in petroglyphs and murals
found in Siberia, Africa, and Spain, demonstrating a long-held interest in these organisms
across the history of humankind (3). Further, in the 16th century, the Spanish Franciscan
Friar Bernardino de Sahagún was the first European to document the ceremonial use of
psychoactive mushrooms in Mexico (4–7).Sahagún,havingspent50yearsthere,began
studying the language and culture of the Aztec, and much of this work was published in
his Historia general de las Cosas de la Nueva España, in which he refers to psychoactive
mushrooms as “teonanácatl”(later updated to “teotlaquilnanácatl”as the correct Nahuatl
word), meaning “God’sflesh,”the sacred mushrooms of Mesoamerica. While being first
recorded by Europeans and Western scientists in the Nahuatl population, ritual usage of
psychoactive mushrooms has occurred in indigenous Mexican cultures including the
Chatins, Chinantecs, Matlazincs, Mazatecs, Mixes, Purepechs, Totonacs, and Zapotecs, with
names for these mushrooms specifictoeachculture(7,8).
Despite psychoactive mushrooms being known and used in Mesoamerica for centu-
ries, the publicity from Wasson’s article spurred widespread use of so-called “magic
mushrooms”as recreational drugs during the psychedelic era of the 1960s and 1970s,
culminating in the criminalization of psilocybin and its analogs (and by extension the
organisms that produce them) as part of the “war on drugs”promulgated by Richard
Nixon in 1971 (9). Based on assumptions about the universal presence of these chemi-
cals in all Psilocybe spp., it soon became illegal to possess putative psilocybin-contain-
ing mushrooms in numerous countries, hobbling research on their diversity, biology,
and unique psychoactive properties (10).
Recently, a renewed interest in psilocybin and Psilocybe spp. has emerged due to
increasing evidence that psilocybin is highly effective in treating a variety of mental
health problems, including anxiety and depression (11–17). This has led to a resur-
gence in clinical studies, many of which have demonstrated the therapeutic efficacy of
psilocybin (18), as well as basic research that led to the identification of the enzymes
and underlying genes responsible for psilocybin and psilocin biosynthesis (19, 20).
Despite these recent advances, fundamental knowledge of Psilocybe, including its di-
versity, distribution, taxonomy, ecology, and chemistry, remains lacking or in dire need
of update. In fact, even the functional role of psilocybin and psilocin under natural con-
ditions remains a mystery (21, 22).
The genus Psilocybe sensu lato has been estimated to contain between 277 and 326
known species (23–25), of which 144 exhibit a characteristic inducible “bluing”reaction
when damaged. This blue pigment is caused by the enzymatic oligomerization of psi-
locin and indicates the presence of the psychoactive compounds psilocybin and psilo-
cin (22–24). It is now accepted that most of the non-bluing species likely belong in the
genus Deconica (W.G. Sm.) P. Karst., and none of them are known to synthesize psilocy-
bin or psilocin (26, 27), and yet the transfer of these species from Psilocybe is largely
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incomplete (28–32) (see Supplementary Information 1). In addition to the ongoing
refinement of Psilocybe taxonomy, many species are considered rare, with many having
been collected only once, leading to poor specimen representation in fungaria (equal
to herbaria for Fungi; Supplementary Information 1) (33–35). This makes identification
difficult without highly detailed and complete taxonomic keys (30, 36, 37) or impossi-
ble because of the dearth of specimens for authentication and poor representation
in DNA sequence databases. This situation is not uncommon in mycology, where
characters useful for field identification are often insufficient, resulting in rampant
misidentification that cannot be resolved without molecular data (38, 39). On top of
this, ambiguities about legal requirements in several countries for their storage, care,
and sharing exacerbate the problem by further suppressing research, creating a chal-
lenging situation for researchers and legal authorities alike.
Although Psilocybe specimens may contain controlled substances, no evidence
exists on their consistency, and psilocybin has been empirically identified in only ;45
accepted Psilocybe spp., which are only 32% of the ;140 putative bluing species (26,
35, 40, 41). Moreover, even for the species examined, the accuracy and reliability of
these reports are dubious due to a lack of standards for taxonomic identification
(including deposition of vouchers to allow for reexamination), lack of peer review for
some studies, and inconsistency of methods used for chemical analysis (see Table S1 in
the supplemental material) (40, 42). In addition, no data on the preservation of psilocy-
bin/psilocin in fungarium specimens exist, further obfuscating if and to what degree
current regulation of controlled substances applies to them, especially if these com-
pounds actively degrade or have become unmeasurable during storage. This absence
of a common set of methods, the almost complete lack of voucher-based studies, and
the deficiency of information on the resilience of the controlled substances under
standard fungarium storage conditions render existing information unreliable and
highly suspect, with the result that there is actually little authoritative information on
the chemistry of most Psilocybe spp.
Psilocybe mushrooms may produce a diversity of metabolites in addition to psilocybin
and psilocin, including compounds such as bioactive
b
-carbolines in Psilocybe cubensis
(Earle) Singer and Psilocybe mexicana R. Heim that may provide monoamine oxidase-inhibi-
ting roles and contribute synergistically to the activity of psilocybin (43). Yet, much of the
metabolome for most mushroom species remains uncharacterized. The metabolomes of a
few Psilocybe species have been investigated recently, and genetic evidence for the biosyn-
thesis of additional bioactive compounds was reported (44). Thus, the potential for other
bioactive compounds within the metabolome of Psilocybe spp. to have synergistic effects
when the mushrooms are consumed as whole organisms is very real, and this information
will be important for potential future implementation of whole-organism-based therapies,
such as those being developed in Oregon, USA (14, 45). A more comprehensive view of
the metabolome of Psilocybe spp. is therefore urgently needed to mitigate any potential
unanticipated bioactivity or adverse side effects of whole-organism consumption, as well
as to provide a baseline for understanding the possible benefits of a whole-organism
approach over psilocybin as a sole therapeutic.
In this study, we aimed to establish a baseline of current knowledge of Psilocybe
identity and chemistry in public repositories, including vouchered specimens, DNA
sequence databases, and the published literature. We then set out to provide an
update to this knowledge with new insights on the chemical stability of psilocybin and
its tryptamine intermediates in collections over time and to acquire a bird’s-eye view
of the metabolome of the most widely cultivated psychedelic mushroom, P. cubensis.
RESULTS
Fungarium specimens and DNA extraction. We curated a database of Psilocybe
museum voucher records utilizing MycoPortal, the most inclusive online aggregator of
fungal voucher specimen records (see online supplemental data) (46). After filtering
out observations (which are mostly not linked to a physical voucher; n= 7,462) and
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non-Psilocybe species under older names, as well as reconciling synonyms, the final
database consisted of 3,707 records of physical vouchers across 72 institutions found
on MycoPortal. Many institutions either could not or refused to provide loan material
due to the legal ambiguity of Psilocybe specimens. Despite the large number of vouch-
ers present across these institutions, approximately one-third were identified only to
genus level (1,151/3,707). Most of the specimens identified to species level were domi-
nated by a few well-known taxa, i.e., P. cubensis (319/3,707), Psilocybe cyanescens
Wakef. (100/3,707), and Psilocybe semilanceata (Fr.) P. Kumm. (138/3,707). Of the
;3,700 specimens requested, ;1,200 were received from 35 institutions.
A total of 18 species names across 94 specimens were used for DNA barcoding.
Across all samples, DNA extractions yielded 5.5 to 166.1 ng/
m
L with 260:230 values
between 0.22 and 6.52 and with 260:280 values ranging from 1.29 to 2.04 (see Table S2
in the supplemental material). Our best extraction results usually came from larger ma-
terial inputs between 10 and 15 mg.
Phylogenetic analysis. In our analysis of all publicly available sequences identified as
Psilocybe spp. from the National Center for Biotechnology Information (NCBI), we found a
large number of likely incorrect or inconsistent names and sequences that of were very
poor quality or that are highly divergent from mushroom-forming fungi. Four hundred
twenty-three internal transcribed spacer (ITS) sequences in NCBI were labeled as Psilocybe
or Deconica and other labels (when Psilocybe was included as a name in the sequence re-
cord), including one sequence labeled Stropharia rugosoannulata Farl. ex Murrill and one
sequence labeled “Uncultured fungus.”Thirty-two of 39 sequences (82%) labeled as
Deconica had specific epithets (9 species names), and six did not. Three hundred sixteen of
382 sequences (83%) labeled as Psilocybe had specific epithets (69 species names), and 66
did not. The most abundant Psilocybe species labels were P. cubensis (31 sequences; 10%),
P. cyanescens (25 sequences; 8%), and P. semilanceata (20 sequences; 6%). Fifty-seven
sequences labeled as Psilocybe were attributable to Deconica (Fig. S1).
A number of erroneous sequences were encountered in the NCBI database that will
require either removal due to poor sequence quality or artifacts, or updates to the sequence
identity. Some of these (MN510675 to MN510688 and MG969992)hadmanyambiguous
bases and potential errors that introduced excessive numbers of autapomorphies, leading to
exceedingly long branches in the phylogenetic tree. Two sequences labeled as Psilocybe
cubensis (ON415277 and ON415278)areinsteadFusarium spp. (pathogenic fungi distantly
relatedtomushrooms)basedonBLASTresults.Onesequence(
DQ900972)fromanenviron-
mental barcoding study (47) appears to be chimeric, with some portions of the sequence
potentially being random and other parts matching Amoebozoa rather than Fungi based on
BLAST. These sequences contribute nothing to species identification and are likely only to add
more confusion, and we recommend removing them from the public records. One sequence
labeled Psilocybe montana (Pers.) Kumm. (which should be updated to the current name
Deconica montana (Pers.) P. D. Orton; AY129360) clustered with P. cubensis. Sequences labeled
as P. cubensis were recovered in six clades. Some of these were other species of Psilocybe (e.g.,
P. mexicana and P. chuxiongensis T. Ma & K.D. Hyde) whereas others belong to Deconica
(AY129351)orGalerina (AY281023). A specimen from the Universidad de Guadalajara (IBUG)
identified as Psilocybe cubensis that was newly sequenced clustered with sequences labeled as
Stropharia rugosoannulata. Similar to sequences labeled as P. cubensis, sequences labeled as P.
cyanescens were recovered in three clades, two of which correspond to Panaeolus (EU029946)
and Deconica (MF467896). Fifty-five sequences labeled as Psilocybe (including labels with spe-
cific epithets, no epithets, and uncultured) clustered with sequences labeled as Deconica.
Some phylogenetic relationships between Psilocybe species are recovered for the first
time. Sequences of P. cubensis were most closely related to a sequence labeled as
Psilocybe natalensis Gartz, D.A. Reid, M.T. Sm. & Eicker (89% rapid bootstrap support [BS]),
together sharing a most recent common ancestor (MRCA) with P. chuxiongensis (100%
BS) (Fig. S1). However, the sequence labeled as P. natalensis in GenBank (OK491080)is
not derived from the type and is of uncertain provenance. The GenBank record refers to
an observation on an online forum (https://mushroomobserver.org/472561) and lists
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the sequence isolation source as “Cultivated from a spore print”and the country of
origin as “South Africa,”but no voucher specimen is indicated, and it is unclear if the
data are reproducible or the source can be reexamined. Overall, the inclusion of NCBI
sequences resulted in a large amount of noise in our analysis, which intensified con-
fusion and contributed to a severe lack of clarity in sequence-based identification of
Psilocybe spp.
Due to the large number of misidentifications and the noise in our Psilocybe-only
analysis, we performed a second phylogenetic analysis utilizing species hypothesis
(SH) sequences from the family Strophariaceae and the closely related genera
Gymnopilus P. Kumm. and Agrocybe Fayod from UNITE (48). Of the 94 new ITS sequen-
ces generated, five have no comparable SH in the UNITE fungal ITS database (Table
S2). Only 48 unique SH sequences were found in UNITE: 13 lacked species-level identifi-
cation, and two are incorrectly assigned (Fig. 1). Like the NCBI records, the UNITE data-
base also includes outdated taxonomic names such as Psilocybe coprophila (Bull.) P.
Kumm. and Psilocybe subviscida (Peck) Kauffman, both of which belong to the nonpsy-
choactive genus Deconica.
Our phylogenetic analysis revealed Psilocybe sensu stricto to be a monophyletic
group (98% BS) sharing a most recent common ancestor with species of the genus
FIG 1 Phylogenetic tree of Strophariaceae and Psilocybe. (Right) Phylogenetic tree of museum specimen ITS sequences along with all species hypothesis
(SH) sequences for the family Strophariaceae and the genera Agrocybe and Galerina from the UNITE fungal sequence database. Correct identifications
within the genus Psilocybe are highlighted in blue, misidentifications at the genus level are highlighted in red, and misidentifications at the species level
are highlighted in orange. (Left) Visual representation of the three main species used for this study, Psilocybe cubensis (photo provided by F. Landeros) (A),
Psilocybe cyanescens (photo by B. Dentinger) (B), and Psilocybe semilanceata (photo by P. Stamets) (C).
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Agrocybe (Fig. 1), in contrast with previous work that reported other members of
Strophariaceae as related taxa (49). Approximately 31 moderately to highly supported
clades (i.e., species) of Psilocybe are currently represented by ITS sequences. Thirteen of
the 94 specimens newly sequenced and two SH sequences from UNITE that had been
identified as Psilocybe clustered outside the Psilocybe sensu stricto clade. These speci-
mens were phylogenetically placed within clades corresponding to the genera
Agrocybe,Deconica,Galerina Earle, Stropharia (Fr.) Quél., and Tubaria (W.G. Sm.) Gillet.
Additionally, multiple groupings within Psilocybe had incongruent taxonomic names,
which suggests either misidentification or possible synonyms (Fig. 1).
Although taxonomic inconsistencies occurred in every clade, some clades contained more
divergent taxonomic names than others. Groupings within the sister clade to Tubaria included
eight different generic or family-level-only identifications [Pachylepyrium Singer, Phaeomarasmius
Scherff., Pholiota (Fr.) P. Kumm., Pleuroflammula Singer, Psilocybe,Mythicomyces Redhead & A.H.
Sm., Strophariaceae,andTubaria]. Additionally, the branch belonging to Deconica included two
SH sequences attributed to Psilocybe, six of our vouchered specimens, and eight SH sequences
labeled Strophariaceae species SH sequences (Fig. 1). Agrocybe and Galerina were recovered as
paraphyletic, with a single branch (63% BS) that also included two of our vouchered specimens
labeled as Psilocybe and SH sequences labeled as Pholiota spp.
Tryptamine metabolite profiling in voucher specimens. We chose specimens of
the three most commonly vouchered species (P. cubensis,P. cyanescens,andP. semilan-
ceata) for chemical analysis and taxonomically verified each using full-length ITS barcoding
that corresponded to the SH for their respective voucher identification. A single specimen
of P. cubensis was misidentified (IBUG-10258, equals Stropharia rugosoannulata), and one
specimen of P. cyanescens was misidentified (SFSU-F-029946, equals Psilocybe allenii Borov.,
Rockefeller & P.G. Werner) (Table 1 and Table S2). The quantities of all metabolites analyzed
varied substantially between species (Fig. 2). Very few fungarium specimens of P. cubensis
showed detectable levels of psilocybin or psilocin compared to most dried specimens of
TABLE 1 Specimen voucher table of Psilocybe used for chemical analysis
Institution code
a
Accession no. Voucher taxonomic designation Collection yr
WTU WTU-F-054871 Psilocybe cubensis 1970
WTU WTU-F-011258 Psilocybe cubensis 1974
WTU WTU-F-054869 Psilocybe cubensis 1978
IBUG 2924 Psilocybe cubensis 1986
IBUG 13422 Psilocybe cubensis 1990
IBUG 14027 Psilocybe cubensis 1995
IBUG 11267 Psilocybe cubensis 2000
NY 1901145 Psilocybe cubensis 2003
WTU WTU-F-011538 Psilocybe cyanescens 1972
WTU WTU-F-011519 Psilocybe cyanescens 1978
WTU WTU-F-011079 Psilocybe cyanescens 1982
SFSU SFSU-F-029949 Psilocybe cyanescens 1993
WTU WTU-F-011523 Psilocybe cyanescens 1999
SFSU SFSU-F-029967 Psilocybe cyanescens 2007
UBC F24013 Psilocybe cyanescens 2011
UBC F30955 Psilocybe cyanescens 2012
UBC F32228 Psilocybe cyanescens 2015
SFSU SFSU-F-029972 Psilocybe semilanceata 1973
WTU WTU-F-055015 Psilocybe semilanceata 1979
UBC F15293 Psilocybe semilanceata 1999
UBC F17025 Psilocybe semilanceata 2007
UBC F22104 Psilocybe semilanceata 2010
UCSC UCSC-F-00856 Psilocybe semilanceata 2011
UBC F32307 Psilocybe semilanceata 2013
FLAS FLAS-F-63134 Psilocybe semilanceata 2015
a
WTU, University of Washington Herbarium; IBUG, Universidad de Guadalajara; NY, New York Botanical Garden;
SFSU, Harry D. Thiers Herbarium, San Francisco State University; UBC, University of British Columbia Herbarium;
UCSC, University of California, Santa Cruz; FLAS, University of Florida Herbarium.
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P. cyanescens, which had detectable levels of both compounds, albeit reduced, even after
nearly50yearsofstorage(Fig.2).
Concentrations of psilocybin and psilocin, when detectable at all, varied in P. cuben-
sis voucher specimens from 0.55 to 1.9
m
g/mg (dry weight), with only a single sample
having detectable amounts of psilocin at 0.44
m
g/mg (dry weight). Psilocybe cyanescens
specimens contained relatively more and consistently higher alkaloid concentrations,
with psilocybin content ranging from 3.0 to 15.6
m
g/mg (dry weight) and psilocin con-
tent ranging from 0.22 to 5.22
m
g/mg (dry weight). Psilocybe semilanceata specimens
contained psilocybin content ranging from 0.33 to 15.77
m
g/mg (dry weight) and psilo-
cin content ranging from 0.1 to 3.88
m
g/mg (dry weight) (Fig. 2). Specimen age was
poorly correlated with quantity of alkaloids. Alkaloids were low or nearly undetectable
in some specimens of P. cubensis and P. cyanescens ,20 years old but also detectable
at multiple-microgram-per-milligram concentrations in nearly 50-year-old specimens
of P. cyanescens. Older specimens (>20 years) of P. semilanceata had barely detectable
levels of the indole alkaloids.
Psilocybin and psilocin metabolite content in cultivated P. cubensis.Psilocybin
was the major alkaloid recovered from mycelium (6.44
m
g/mg), caps (10.5 to 20
m
g/mg),
and stipes (15.44 to 18.44
m
g/mg) (Table 2). Psilocin was a minor component in all sam-
ples (0.44 to 2.0
m
g/mg). In comparison, psilocybin-to-psilocin ratios ranged from ;5:1
to 47:1 in the caps and ;7.5:1 to 37:1 in the stipes (Table 2). Performing an unpaired t
test, we found no significant difference in psilocybin or psilocin between whole individ-
ual sporocarps (Psi 5 and Psi 6) from the same reproductive event in a single growth bin
(psilocybin percentage point [pp] = 0.1349 and psilocin pp = 0.4756). Unpaired ttests
also showed no significant difference in concentrations between the same anatomical
tissues (caps and stipes) or between the two sporocarps Psi 5 and Psi 6 (psilocybin
pp = 0.5081 and psilocin pp = 0.4563) or measured across Psi 4, Psi 5, and Psi 6 (psilocy-
bin pp = 0.8182 and psilocin pp = 0.6898). However, at the full sporocarp level, quantities
Psilocin Psilocybin Tryptophan Baeocystin Norbaeocystin
0
5
10
15
20
50
40302010
Psilocybe cubensis
Compound Concentration (ug/mg)
Specimen Age
0
5
10
15
20
Psilocybe cyanescens
Compound Concentration (ug/mg)
Specimen Age
0
5
10
15
20
Psilocybe semilanceata
Compound Concentration (ug/mg)
Specimen Age
50
40302010 50
40302010
FIG 2 Content of tryptophan, psilocybin, and related alkaloids in fungarium specimens of three species of Psilocybe over 5 decades. Species are Psilocybe
cubensis (left), Psilocybe cyanescens (center), and Psilocybe semilanceata (right). Specimen age (years) is on the xaxis, and concentration of metabolites is on
the yaxis in micrograms per milligrams (dry weight). Points indicate concentrations of individual compounds: tryptophan (green), baeocystin (blue),
norbaeocystin (purple), psilocybin (yellow-green), and psilocin (red).
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of psilocybin and psilocin were statistically different between growth bins (psilocybin
pp = 0.0560 and psilocin pp = 0.0389).
Metabolomic profiling and tissue comparison of cultivated material. We identi-
fied 1,465 distinct spectral features based on their unique mass-to-charge (m/z) ratio and
retention time. Using hierarchical clustering to compare the intensity profiles of each of
our P. cubensis samples across the two growing environments, we found that metabolic
profiles of caps and stipes were most like one another, with the mycelium being most dif-
ferent from all other samples, regardless of which container they were cultivated in (Fig. 3).
However, in the case of both caps and stipes, Psi 5 and Psi 6 were shown to be more alike,
while Psi 4 formed an outgroup for each tissue type (Fig. 3). We investigated this further by
performing a principal-component analysis (PCA) on the intensity of each feature and par-
tial least-squares discriminant analysis (PLS-DA) for each sample (Fig. S3 and S4). PCA
showed largely overlapping spectral intensities, but the same pattern as that in the hier-
archical clustering was present, with dimension 1 accounting for 15.6% of the variability
and dimension 2 accounting for 63.3% of the variability (Fig. S3). PLS-DA on the full profiles
for each sample, rather than the individual spectral features, again showed a pattern of
caps and stipes being most similar but distinct separation between samples Psi 4 (growth
bin 1) and Psi 5 and Psi 6 (growth bin 2), even though all samples were cultivated and har-
vested in the same manner.
In addition to looking at broad-scale patterns of metabolomic profiles, we also wished to
investigate if any of the identified spectral features could be annotated to known compounds.
We collected tandem mass spectrometry (MS/MS) data for sample Psi-6-Cap for comparison to
the Global Natural Products Social Molecular Networking database (GNPS; https://gnps.ucsd
.edu/ProteoSAFe/static/gnps-splash.jsp); (50). Using the GNPS spectral library matching the com-
pound database provides an annotation along with a cosine score (MQscore) that indicates
spectral similarity to the annotated compound. An MQscore of 1 suggests an identical match of
a spectral feature to the annotated compound in the database, with decreasing scores suggest-
ing compounds of similar structure as the annotation. Spectral library matching for Psi-6-Cap
revealed 30 features that could be annotated to known compounds (0.02% of the identified
1,465 features) (Table S5). Many of these annotations belonged to compounds involved in cell
membrane formations, sugars, and cholesterol (sn-glycero-3-phosphocholine MQscore = 0.97,
sucrose MQscore = 0.93, and epicoprostanol MQscore = 0.92), which are likely to be found in
high quantities because these profiles were derived from freshly preserved specimens. However,
three annotated spectral features were of particular interest as they had structural similarity to
serotonin (MQscore = 0.9292), cocamidopropyl betaine (CAPB) (MQscore = 0.9191), and traza-
done hydrochloride (MQscore = 0.73).
DISCUSSION
Our study shows that fungarium specimens of Psilocybe spp. are regularly misidenti-
fied, little has been done to investigate the stability of the known psychoactive com-
pounds under standard storage conditions, and the chemical complexity of these
organisms has been overlooked by an almost exclusive focus on the principal psycho-
active agents. These deficiencies cast a shadow on what little information is available
TABLE 2 Psilocybin and psilocin concentrations in cultivated sporocarps of Psilocybe cubensis
Sample
identifier
UT-M catalog
no.
Amt (mg) sampled of
voucher specimen
Psilocybin concn
(mg/mg)
Psilocin concn
(mg/mg) Source (origin of sample)
Psi-mycelium UT-M0001771 7 6.3 0.01 Stock mycelium culture
Psi-4-Cap UT-M0001772 7 10.5 2.11 Single sporocarp from one growth bin
Psi-4-Stipe UT-M0001772 8 15.44 1.9 Single sporocarp from one growth bin
Psi-5-Cap UT-M0001773 7 17.3 0.5 One of two sporocarps from one growth bin
Psi-5-Stipe UT-M0001773 9 16.33 0.4 One of two sporocarps from one growth bin
Psi-6-Cap UT-M0001774 9 19.9 0.44 Two of two sporocarps from one growth bin
Psi-6-Stipe UT-M0001774 7 18.3 1.5 Two of two sporocarps from one growth bin
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on Psilocybe and underscore the increasingly urgent need for comprehensive basic
research on them.
Authentication and diversity of Psilocybe fungarium specimens. Fungi often ex-
hibit cryptic morphology and can be difficult to identify, even for highly trained experts.
Cryptic traits, lack of extensive training, and even clerical errors in databasing can all con-
tribute to misidentification(38,51).Inthissense,itislikelythatspecimensinfungariaof
nearly all taxa suffer from some level of taxonomic uncertainty, but the extent to which
misidentification is a problem has been rarely investigated. A dearth of authentic DNA
barcodes for Psilocybe and other closely related groups further exacerbates the problem.
Without DNA barcodes from type or other authentic material, names can be applied incon-
sistently, leading to different applications of species names to modern collections. This also
makes it difficult to know when a specimen represents a new species, resulting in further
misapplication of named species to novel taxa. We found that close to 13% of the speci-
mens we studied that were cataloged as Psilocybe belonged to other genera, none of
which are known to contain controlled substances. This result challenges the use of names
in collections for meta-analysis or evidence-based decisions on their custody and care.
Many publicly available sequences are incorrectly labeled or are of very poor quality, fur-
ther aggravating the problem for identifying specimens or other samples using a DNA bar-
coding approach. Even some of the most recognizable and frequently collected species (P.
cubensis and P. cyanescens) had sequences that belonged to other related genera and in
two cases to fungi from a different phylum (Ascomycota). Some of these are likely misiden-
tification of the source material, including cultures, highlighting the need for an authorita-
tive, expert-curated reference database of DNA sequences derived from types or other
authentic specimens.
While misidentifications in museum collections and public DNA repositories are not
uncommon (38, 39), the ambiguous legal status of Psilocybe specimens makes misidentifi-
cations detrimental not only to scientific research but also to properly applying regulatory
policies. Misidentification of putative psilocybin-producing mushroom specimens may lead
to an unnecessary effort and poor use of resources, or even miscarriage of justice in legal
cases.
A review of the currently known species of Psilocybe suggests that 165 belong to
FIG 3 Comparison of chemical profiles across cultivated Psilocybe cubensis samples. Three sporocarps
and one mycelium were used for chemical profiling. Sporocarps were harvested simultaneously from
the same growth bin (Psi 5 and 6) and from a second growth bin (Psi 4) grown from a single
mycelial culture. Hierarchical clustering of samples (yaxis, left) and spectra (xaxis, top) based on
Euclidean distance similarities of total chemical profile and spectral feature similarity, respectively.
Heatmap shows chemical factor contribution using the intensity values for each identified factor
represented by a color gradient from blue (low contribution) to red (high contribution).
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Psilocybe sensu stricto while 98 belong to Deconica or other genera (see Supplementary
Information 1 in the supplemental material). Fifty-two species that likely belong to
Deconica are currently still classified in Psilocybe. However, these likely include synonyms
and cryptic species that are presently unknown and do not account for undocumented
species that likely exist. Only five species of Psilocybe sensu stricto are represented by ITS
barcodes derived from types. Critically, sequences from types of the best-known species,
such as Psilocybe cubensis, do not exist. This fundamental information is critical for accu-
rate application of names and should be considered an urgent priority to aid in the iden-
tification of this medically and societally important group.
While our data set had limited representation of the known species of Psilocybe,new
phylogenetic patterns were uncovered. While Ramírez-Cruz et al. (34) reported that the
American species P. cubensis shared a most recent common ancestor (MRCA) with the
southeast Asian species Psilocybe thaiaerugineomaculans Guzmán,Karun.&Ram.-Guill.,our
inclusion of the South African P. natalensis and the more recently described Asian species P.
chuxiongensis revealed that P. cubensis likely shares an MRCA with P. natalensis and not the
Asian species. This is consistent with the African-origin hypothesis for P. cubensis and/or its
progenitor proposed by Guzmán et al. (52) and Froese et al. (53) rather than the Asian-origin
hypothesis suggested by the relationships to Asian species in the work of Ramírez-Cruz
et al. (34) and Ma et al. (54). However, it should be noted that the sequences of P. natalensis
and P. thaiaerugineomaculans are not derived from type specimens (which is also true for P.
cubensis), and so while the geographic implications remain, the taxonomic ones are uncer-
tain. To gain a better understanding of the geographic origins of Psilocybe spp., especially
the enigmatic P. cubensis,newfieldwork to expand documentation of Psilocybe in Africa
andAsiashouldbeapriority.
Psilocybin, psilocin, and related metabolites in Psilocybe spp. Decades of incon-
sistency in the identification of Psilocybe and methods for measuring its metabolites render
most previous studies unreliable and unreproducible. In our review of the published litera-
ture on alkaloid chemistry in Psilocybe, there was an almost complete lack of thorough sam-
ple identification, few studies designated vouchers, and literature citations frequently con-
tained incorrect information or opposing statements (see Table S1 in the supplemental
material). In a few instances, reports for some species, such as Psilocybe ovoideocystidiata
Guzmán & Gaines and Psilocybe kumaenorum R. Heim, lacked peer review or were not acces-
sible. Although our focus was specifically on literature pertaining to Psilocybe,psilocybinhas
been reported from other species such as Agrocybe praecox (Pers.) Fayod (55, 56), although
there is good reason to believe this may have been a misidentification of Psilocybe subcu-
bensis Guzmán (57). Unfortunately, because no vouchers were deposited, it is impossible to
reexamine the material to determine the true identity. Nonetheless, this dubious report has
persisted through repetition without critical review or scrutiny of the data. A much more rig-
orous set of standards is needed to establish a reliable baseline for information on the
chemistry of psilocybin and related alkaloids in mushrooms.
We chose to quantify psilocybin and related metabolites in fungarium specimens of
Psilocybe to determine their presence and stability over time. One recent study showed
that the method of drying and extent of light exposure can affect psilocybin/psilocin
stability in P. cubensis (58), but this examined storage over only a single year. Our speci-
mens from three species spanned multiple decades, and we found that metabolite
content and stability varied widely and metabolite content was undetectable in most
samples stored for more than 40 years (Fig. 2). However, the sample size for any given
specimen age was small, and information on confounding factors, such as the original
preservation method, was often not available. In one instance, we were able to mea-
sure the chemical content of a specimen of P. cubensis (WTU-F-054869) deposited
around the same time as specimens used for previous chemical analysis (26, 59, 60).
Although there is a strong possibility that this specimen was the same one used in the
original study or was harvested from the same cultivated strain around the same time
(M. Beug, personal communication), no voucher information was available from the
original study and so it was not possible to confirm its identity. Bigwood and Beug (60)
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noted that multiple flushes of P. cubensis contained over 5.0
m
g/mg (dry weight) of psi-
locybin. However, when we measured the WTU-F-054869 voucher, now 42 years old,
we detected less than half of their reported psilocybin content (1.9
m
g/mg) and no psi-
locin (Fig. 2). This provides further evidence that these chemicals are highly unstable in
preserved specimens of P. cubensis, degrading either in a time-dependent manner or
possibly during or soon after the initial preservation. A study designed to specifically
test time-dependent degradation with replicates and controls would be needed to
determine the exact causes of the metabolite instability we observed.
Our results also reveal taxon-dependent patterns of metabolite instability. For example,
psilocybin and psilocin in P. cyanescens had a comparably lower rate of degradation over
time than in P. cubensis, with the youngest specimen having 15.6
m
g/mg psilocybin and
5.8
m
g/mg psilocin and the oldest (40-year) specimen having 6.11
m
g/mg psilocybin and
3.66
m
g/mg psilocin. Similarly, in P. semilanceata the youngest sample contained 11.2
m
g/
mg of psilocybin and 3.88
m
g/mg psilocin, while the oldest contained 0.2
m
g/mg psilocybin
and no detectable psilocin. One possible explanation for the generally better chemical pres-
ervation in P. cyanescens and P. semilanceata is that these species have sporocarps that are
comparatively lower in biomass than those of P. cubensis.Thismayresultinamorerapid
desiccation during heat-assisted drying, minimizing the opportunity for spontaneous and/or
enzymatic degradation of psilocybin and psilocin. Another possible explanation may be
from differences in the expression or catalytic activity of the enzymes involved in conversion
of psilocybin and psilocin (22). However, the overall pattern of degradation for the other
metabolites we measured mirrored that of psilocybin and psilocin (Fig. 2), suggesting spon-
taneous rather than enzyme-assisted degradation. Nonetheless, in any given specimen, deg-
radation patterns are likely to differ based on a complex set of factors such as age, preserva-
tion method, and species. Taken together, our results illustrate that the presence and
quantities of psilocybin and psilocin in fungarium specimens are highly unpredictable.
To investigate if the initial method of specimen preservation has a large effect on psilocy-
bin and psilocin degradation (58), we compared psilocybin and psilocin in freshly harvested
specimens that were flashfrozeninliquidN
2
and lyophilized. This preservation method is
thought to optimally preserve naturally occurring concentrations of psilocybin and psilocin
by protecting psilocybin against hydrolytic dephosphorylation (61). Our lyophilized sporo-
carp samples had 10.55 to 19.9
m
g/mg psilocybin and 0.44 to 2.0
m
g/mg psilocin, which is
consistent with previous reports, including the high ratio of psilocybin to psilocin (Table S1).
These quantities are much higher than anything we detected in heat-dried fungarium speci-
mens of P. cubensis, indicating lyophilization may be the best method for preserving psycho-
active alkaloids in this species.
Psilocybin and psilocin concentrations have been shown to vary among mycelium
and parts of the mushroom (21, 62). In an effort to document this variation in our culti-
vated P. cubensis strain, we independently analyzed psilocybin and psilocin concentra-
tions in different samples (mycelium, stipe, and cap) from multiple sporocarps within
the same flush (Psi 5 and Psi 6) and from sporocarps between two growth containers
(Psi 5/Psi 6 versus Psi 4). Mycelium showed one-third the content of psilocybin and five
to 20 times less psilocin than did the stipe and cap, respectively (Table 2). Although we
found comparatively more alkaloids in the caps than in the stipes, the concentrations
of psilocybin and psilocin at the full sporocarp level were statistically different between
growth bins despite using the same mycelial stock and growth conditions. This sug-
gests that using consistent strain and growth conditions would yield predictably simi-
lar concentrations of both psilocin and psilocybin within a growing environment and
between caps and stipes but that inconspicuous microenvironmental conditions could
be relevant to the metabolomic phenotype (Fig. 2 and Table 2). These results have im-
portant implications for the use of whole sporocarps for therapeutic or recreational
applications, and better replicated studies are needed to elucidate the nuances of this
variation.
Untargeted metabolomics reveals a large diversity of uncharacterized metabo-
lites in P. cubensis.Although the vast majority of chemical studies of Psilocybe have
focused on the principal psychoactive alkaloids and their intermediates, Psilocybe spp.
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almost certainly produce a diversity of other metabolites, at least some of which may
be bioactive (43). Our total metabolomic profile of cultivated P. cubensis detected
1,465 unique spectral features that varied among tissue types and growth replicates
(Fig. 3 and Fig. S3 and S4). Only 0.2% of these could be assigned to known chemicals
using the GNPS reference database. While becoming more common, studies of untar-
geted metabolomics in fungi are relatively new and often focus on Ascomycota rather
than Basidiomycota because of their historical use for antimicrobial and natural prod-
uct discovery (63). The general lack of these studies makes it difficult to judge if our
metabolic profile is typical of Basidiomycota, as some species, such as Trametes versi-
color (L.) Lloyd, have been shown to produce different profiles based on the presence
of other fungi (64). Developmental variation is also likely. For example, Ganoderma
sichuanense J.D. Zhao & X.Q. Zhang (equals Ganoderma lingzhi Sheng H. Wu, Y. Cao &
Y.C. Dai) can have as many as 9,000 spectral features that differ depending on the life
cycle stage in which they are investigated (65). While undoubtedly highly variable, the
metabolomic profile of our sporocarps represents a snapshot of mature P. cubensis
mushrooms grown in a conventional medium, which may be useful for future compari-
son and chemical exploration of cultivated material.
Spectral features with similarity to both serotonin and trazodone hydrochloride were
detected in P. cubensis. Serotonin is a neurotransmitter that modulates many neurological
processes (66), while trazodone hydrochloride is a synthetic triazolopyridine derivative with
serotonin-selective reuptake inhibition (SSRI) activity commonly prescribed as an antide-
pressant (67). Serotonin has been reported from Panaeolus (Fr.) Quél., some species of
which also produce psilocybin (68, 69), and many other species of Basidiomycota (70). The
synthetic trazodone hydrochloride is far less likely to be present in the mushrooms since it
is not a natural product. However, motifs and chemical structures with similarity to syn-
thetic compounds have been found to be naturally occurring in other fungi (71), so the
possibility that some fungal natural products could mimic the known function of these syn-
thetic compounds is not unreasonable. Even if these two compounds were incorrectly
identified, these results clearly demonstrate that additional chemicals in the metabolome
of P. cubensis have real potential to contribute to physiological and neurological activity if
consumed.
In addition to endogenous metabolites, the accumulation of chemical compounds
from the growth substrate is a potential source of other physiologically active metabolites.
For example, we detected cocamidopropyl betaine (CAPB), a chemical component of co-
conut (72). CAPB is a common human irritant and allergen (72–74),anditspresenceinthe
substrate when cultivating P. cubensis sporocarp may be a cause for concern for some peo-
ple.Whilewecannotabsolutelyconfirm that this spectral feature is CAPB, neither can we
rule out that this compound is endogenously produced by P. cubensis,asoursporocarps
were produced using a casing of coconut coir fiber, a likely source of this compound. This
would suggest that the accumulation of chemicals and the overall chemical composition
of cultivated sporocarps may be a combination of both endogenous and exogenous sour-
ces. Indeed, mushrooms are known to sequester chemicals from the environment, where
they have been used to remove toxins (75, 76). This is of particular importance for the
potential therapeutic or recreational use of whole mushrooms and requires empirical data
to make informed decisions on policies around their production and regulation (77).
Despite a surge in interest in Psilocybe due to accumulating evidence of the therapeutic
potential of psilocybin, the current state of knowledge of Psilocybe and the psychoactive
compoundsitproducesisstillinitsinfancy.Ourstudyshowsthatmuchmoreresearchis
needed to achieve a more thorough, accurate, and reliable understanding of this enigmatic
and socially important group to develop meaningful policies around its use and care, and
improve its application for human wellbeing.
MATERIALS AND METHODS
Fungarium voucher authentication with DNA barcodes and phylogenetics. (i) Fungarium speci-
mens. We created a curated database of voucher information for all collections identified as the genus
Psilocybe using MycoPortal (46) accessed on 1 June 2020. Records without physical vouchers (i.e.,
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observations only) were removed, and the remaining names were corrected by comparing them to the
most up-to-date list of currently accepted names in Species Fungorum (78). We subsampled specimens
specifically for vouchers that were identified to species level and attempted to sample a minimum of
three specimens with the same taxonomic name, totaling 94 specimens, to maximize the diversity of
our data set for accurate species comparison (see Table S1 in the supplemental material).
(ii) DNA extraction and barcode sequencing. Total DNA was extracted from the hymenophore in an
amount ranging from 5 to 15 mg. These fragments were ground to a fine powder by placing them in 2.0-mL
screw-cap tubes containing a single 3.0-mm bead and 8- by 1.5-mm stainless steel beads and shaking them
in a BeadBug microtube homogenizer (catalog no. Z763713; Sigma) for 120 s at speed setting 350. The
groundsamplewasusedasinputtotheMonarchgenomicDNApurification kit (New England Biolabs [NEB]
catalog no. T3010S) following the manufacturer’s instructions except using twice the volume of lysis buffer
and increasing the amount of wash buffer to 550
m
L during both of the washing steps. These samples were
amplified using the PCR primers ITS-8F (59-AGTCGTAACAAGGTTTCCGTAGGTG-39)andITS-6R(59-TTCCCG
CTTCACTCGCAGT-39), which were specifically designed for Agaricomycetes (79), and submitted for Sanger
sequencing on both strands through the Genewiz Corporation as well as the DNA sequencing core at the
University of Utah.
Complementary sequencing chromatograms were then processed by creating a consensus sequence
using Sequencher 5.4 (http://www.genecodes.com) and trimming the 59and 39ends to conserved motifs 59-
CATTA- and -GACCT-39following the method in reference 79 for downstream analysis.
(iii) Phylogenetic analysis. To validate the identities of the voucher specimens, we subjected the ITS
sequences to phylogenetic analysis using two data sets. The first consisted of all currently available ITS sequen-
ces from the National Center for Biotechnology Information (NCBI) identified as Psilocybe spp. using the query
‘Psilocybe [ORGN] AND “internal transcribed spacer”’ (n= 384). The second included all sequences belonging to
the Species Hypothesis (SH) (generated at the 1.5% similarity threshold) of family Strophariaceae Taxon
Hypothesis (80) TH006803 (244 SH, 26 sequences assigned to Psilocybe) found in the UNITE database (UNITE
classifies Psilocybe in Strophariaceae; general release, 10.05.2021) (81). Outgroup SH sequences for the genera
Agrocybe (TH012107) and Gymnopilus (TH012136) were included based on phylogenetic relationships provided
by JGI MycoCosm (https://mycocosm.jgi.doe.gov/mycocosm/species-tree/tree;Y-nUEB?organism=agaricales).
The motivation for using the UNITE SH sequences independent of NCBI sequences is that the UNITE database is
curated to minimize noise by reducing minor sequence variation using representative sequences chosen man-
ually by expert curators or automatically using a standard dynamic similarity threshold. All SHs are assigned a
unique digital object identifier (DOI) to allow stable, unambiguous reference across studies, even in the com-
plete absence of meaningful taxonomic names (82).
For Psilocybe, expert verification has been completed for 9/48 SHs, for which a representative
sequence has been manually chosen, whereas the remaining SHs are represented by a sequence auto-
matically chosen by the SH algorithm.
ITS barcode sequences generated in this study were combined with data set 1 and data set 2 for
phylogenetic analysis.
For all data sets, sequences were automatically aligned using the multiple sequence alignment soft-
ware MAFFT v7.475 (83) with the L-INS-i algorithm followed by phylogenetic inference under maximum
likelihood (ML) using IQ-TREE v22.0.3 (84) with the automatic ModelFinder setting (85) and 1,000 ultra-
fast bootstrap replicates (86). The best ML trees were rendered in FigTree v1.4.4 (http://tree.bio.ed.ac.uk/
software/figtree/).
Tryptamine metabolite profiling in voucher specimens. (i) Specimen preparation and chemical
extraction. Eight Psilocybe cubensis vouchers (including specimens deposited in the University of
Washington Herbarium [WTU] around the same time as those analyzed in reference 26), six cultivated P.
cubensis samples (three sporocarps split into caps [=pileus] and stipes), nine P. cyanescens samples, and
eight P. semilanceata samples were used for chemical analysis across multiyear time points (P. cubensis =
17 to 50 years, P. cyanescens = 5 to 48 years, and P. semilanceata = 5 to 47 years). Samples were ground
to a fine powder, as described above, prior to solvent extraction as described in the work of Fricke et al.
(20). Briefly, homogenized samples were resuspended in a 1:20 ratio of sample (milligrams) to methanol
(microliters) and then sonicated in a Branson 1510 sonicator for 30 min. The sonicated solution was then
passed through a 0.22-
m
mfilter, and the filtered solution was diluted to 100
m
g/mL with a methanol-
water (50:50) solution and injected on the ultrahigh-performance liquid chromatograph/mass spectrom-
eter (UHPLC-MS).
(ii) UHPLC-MS method to identify and quantify psilocybin and tryptamine alkaloid intermedi-
ates. Hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry was performed using an
Acquity Arc UHPLC-MS (Waters, Milford, MA, USA) in both positive and negative mode with a Waters
Acquity UPLC ethylene-bridged hybrid (BEH) amide column (1.7
m
m, 2.1 by 100 mm) set to 40°C. The
l
range was set at 200 to 500 nm with a resolution of 1.2 nm and a sampling rate of 10 points/s. The mass
range in positive mode was set at 100 to 800 Da with a gain of 1, probe at 600°C, a capillary voltage of
0.8 kV, and collection at 10 points/s. A flow rate of 0.5 mL/min and a linear solvent gradient of 99.9% sol-
vent A (10 mM ammonium acetate, 95% acetonitrile, 0.014% ammonium hydroxide, pH 9.0) to 30% sol-
vent B (10 mM ammonium acetate with 0.014% ammonium hydroxide, pH 9.0) over 7 min were used.
All samples were resuspended to a concentration of 100
m
g/mL in methanol-water (50%:50%).
(iii) Chemical standard curve creation and calculation. First, we calculated control curves by
measuring the content of six chemical standards for baeocystin, norbaeocystin, norpsilocin, psilocin, psi-
locybin, and tryptophan at three different concentrations in triplicate (Fig. S2 and Table S3). This allowed
us to identify our compounds in spectral data based on retention times and calculate the concentration
of each compound in each sample based on extracted m/z intensities (Table S4). Standard curves were
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measured by running standards at 1.0-, 1.2-, and 1.5-
m
g injections in triplicate and in three replicates.
The baseline integrations were recorded for each, and the standard curves were generated using the
line of best fit from the standard measurements.
Cultivation of P. cubensis.Specimen cultivation and processing were carried out as follows. Specimens
of P. cubensis were cultivated from spore syringes acquired from Spore Works (sporeworks.com; Golden
Teacher spore syringe microscopy kit, SKU PSMICsy-090) inoculated on 2% malt agar plates with 50
m
g/mL
ampicillin antibiotic. Isolated sections of contiguous mycelium from initial spore plates were subcultured by
removing ;0.2cmoftissuefromtheedgeofthegrowingmyceliumwithaflame-sterilized scalpel to
another plate of 2% malt agar and ampicillin with a thin sheet of sterilized cellophane plastic placed on the
surface to facilitate the removal of mycelium for chemical processing. A single culture was used for chemical
analysis as well as cultivation of sporocarps. Agar plugs from this culture were placed into spawn growth me-
dium and incubated at room temperature without a consistent light cycle for ;2to3weeksuntilmedium
was entirely colonized by mycelium based on visual inspection. Spawn growth was achieved using 1-L wide-
mouth Mason jars (Amazon, ASIN: B07HGG3DD1) containing one bag of Ben’s Original Ready Rice whole-
grain brown rice (Amazon, ASIN: B00G9U46DW), topped with custom lids that included a 0.22-
m
mfilter for
sterile airflow (Amazon, ASIN: B08M5XPFTP), with autoclaving for 40 min on a liquid cycle. To induce sporo-
carps, fully colonized spawn was mixed aseptically with one brick of coconut coir (Amazon, ASIN:
B01M8FUUSJ), 300 g of vermiculite (Amazon, ASIN: B001693Y3Y), and enough tap water (anecdotally thought
to be better than deionized [DI] H
2
O) to fill half a container with a moist but not muddy consistency within
an 18-qt plastic bin (Amazon, ASIN: B01C3BGTAS).
After 2 to 3 weeks, the lid was left ajar for increased airflow to promote sporocarp production (;1week).
Mycelium and sporocarps underwent lyophilization prior to chemical analysis. Viable culture plugs were
retained for long-term storage at room temperature in 50 mL of distilled water (87), and lyophilized voucher
samples used for chemistry are deposited in the Garret Herbarium at the Natural History Museum of Utah
(UT-M) under the accession numbers listed in Table 2.
UPLC-MS of cultivated Psilocybe cubensis samples. We applied a UPLC-MS-based untargeted
metabolomics pipeline to identify shared and unique metabolites across three cultivated P. cubensis
sporocarps and vegetative mycelium. Tissue samples were lyophilized, ground, and extracted in 80:20
(vol/vol) methanol-water, producing 2 mL of retained supernatant from 100 mg (62.5 mg) of sample for
spectroscopic analysis. Small molecules (detector range of 50 to 2,000 Da) from the extraction were ana-
lyzed using UHPLC (Waters Acquity I-Class, 2.1- by 100-mm BEH amide columns) and mass spectrometry
(Waters Xevo G2 quadrupole time of flight [QToF]) (UPLC-MS) in positive and negative ionization mode.
Additionally, MS/MS spectra were acquired for sample Psi-6-Cap by running data-dependent acquisition
mode (DDA), whereby MS/MS data were collected for all metabolites that were ionized above a set
threshold (total ion current [TIC] of 5,000).
Data processing and spectral feature analysis. Raw data from the UPLC-MS analysis were processed
using the R package XCMS (88) for peak detection, peak alignment, and peak filtering. We used the following
parameters: peak detection method ‘centWave’[ppm = 15, peakwidth = c(0.2, 5), snthresh = 5, prefilter =
c(1,500)]; peak grouping method ‘density’(bw = 3); and retention time correction method “peakGroups,”in
which retention time standards containing eight known compounds as a reference sample and integrate-
areas-of-missing-peaks method “FillChromPeaksParam”were used. We then used the R package Camera (89)
to assign the various coeluting features (unique m/z-retention time combinations) derived from one com-
pound into pc groups. The parameters used were peak grouping after retention time ‘groupFWHM’
(perfwhm, 0.7; sigma = 6), verify grouping ‘groupCorr’,annotateisotopes‘findIsotopes’, and annotate adducts
‘findAdducts’(polarity = ‘positive’).
Chemical profile comparison was performed by using the intensity values for each identified factor across
all cultivated samples. Intensity values were processed and normalized by the total using the decostand com-
mand from the R package Vegan (90). Normalized values were then used for PCA and PLS-DA, which were
calculated and visualized using the R package mixOmics (91). To investigate the similarity of the chemical
profiles, Euclidean distances were calculated using the base R function dist. These calculations were then
assigned hierarchical clustering using the base R function hclust. These two measurements were then visual-
ized together using the aheatmap function of the R package NMF (92).
GNPS annotation to known compounds. The identification of compounds from metabolic studies is
often one of the most difficult parts of chemical profiling,asthousandsofcompoundsandtheirderivatives
are often produced but most lack standard references. In an attempt to make annotation and exploration of
metabolomic data sets more accessible, GNPS was created as an initiative to make a standardized and acces-
sible platform to compare spectral data across numerous organisms and chemical databases, as well as pro-
viding a platform that would allow older data to be reanalyzed as databases become better over time (50,
93). GNPS utilizes a molecular networking approach that groups sets of spectra from related molecules to-
gether to allow for comparison to known compounds even if the specific spectrum being compared has
never been identified. In this regard, annotations with high MQscores may have structure and function highly
similar to those of the known compounds, while those with lower scores may be similar in structure but
have novel or altered function compared to the known compound.
A molecular network was created using the online workflow (https://ccms-ucsd.github.io/GNPSDocumentation/)
on the GNPS website (http://gnps.ucsd.edu). The data were filtered by removing all MS/MS fragment ions
within 617 Da of the precursor m/z.MS/MSspectrawerewindowfiltered by choosing only the top 6 frag-
ment ions in the 650-Da window throughout the spectrum. The precursor ion mass tolerance was set to
2.0 Da, and the MS/MS fragment ion tolerance was set to 0.5 Da. A network was then created where edges
were filteredtohaveacosinescoreabove0.7andmorethan6matchedpeaks.Further,edgesbetweentwo
nodes were kept in the network only if each of the nodes appeared in the other’s respective top 10 most
DNA and Chemical Analysis of Psilocybe Mushrooms Applied and Environmental Microbiology
Month YYYY Volume XX Issue XX 10.1128/aem.01498-22 14
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similar nodes. Finally, the maximum size of a molecular family was set to 100, and the lowest-scoring edges
were removed from them until the molecular family size was below this threshold. The spectra in the net-
work were then searched against the GNPS spectral libraries. The library spectra were filtered in the same
manner as the input data. All matches kept between network spectra and library spectra were required to
have a score above 0.7 and at least 6 matched peaks.
Data availability. All edited ITS sequence data for all specimens were submitted to the National
Center for Biotechnology Information with NCBI accession numbers reported in Table S2. The collection
database, multiple sequence alignments, and trees are available in Figshare project number 129332. The
whole metabolomic spectra have been submitted as a MASSIVE data set available at https://massive
.ucsd.edu/ProteoSAFe/private-dataset.jsp?task=1b798991ec6b40d48b49b74e971e2ecf.
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 0.5 MB.
SUPPLEMENTAL FILE 2, XLSX file, 0.02 MB.
SUPPLEMENTAL FILE 3, XLSX file, 0.02 MB.
SUPPLEMENTAL FILE 4, XLSX file, 0.04 MB.
SUPPLEMENTAL FILE 5, XLSX file, 0.01 MB.
SUPPLEMENTAL FILE 6, XLSX file, 0.02 MB.
ACKNOWLEDGMENTS
We acknowledge the Natural History Museum of Utah for their commitment to
collaborative science as well as the DNA Sequencing Core Facility, the University of Utah,
where DNA barcode sequencing was performed. In addition, we thank the institutions who
provided samples for this study and specifically thank the University of Washington
Herbarium (WTU), Universidad de Guadalajara (IBUG), the New York Botanical Garden (NY),
the Harry D. Thiers Herbarium, San Francisco State University (SFSU), the University of British
Columbia Herbarium (UBC), and the University of Florida Herbarium (FLAS), who allowed us
to perform destructive sampling for chemistry in addition to DNA barcoding. We also thank
the Fungi Foundation for supporting this work in its genesis and with Giuliana Furci’stime.
Further, we acknowledge the dedicated and hard work performed by Isabelle Galland, who
helped to process many of these samples for DNA sequencing. Finally, we acknowledge the
Drug Enforcement Agency (DEA), for providing the permission to perform this work under
license RW0535508.
Paul Stamets is the current CEO of Fungi Perfecti LLC, Co-founder of MycoMedica Life
Sciences, PBC and has patents pending and approved using psilocybin. All other authors
report no conflicting interests in the body or intent of this work.
Alexander J. Bradshaw developed the conceptual framework and performed initial
experimental details and planning and outreach to collection institutions, as well as databasing
and curation of samples, DNA extraction and sequencing, phylogenetic analysis of samples,
initial chemical work, cultivation of specimens, data analysis, and preparation and editing of
the manuscript. Talia A. Backman performed DNA extraction of samples, the chemistry analysis
of voucher samples, the creation of chemical standard curves, and the preparation and editing
of the manuscript. Virginia Ramírez-Cruz provided expertise and advice on Psilocybe taxonomy,
a comprehensive review of the current status of rare specimens in collection institutions, and a
compilation of all previous chemical work done in Psilocybe and aided in the preparation and
editing of the manuscript. Jaclyn M. Winter (DEA license RW0535508) provided the mass
spectrometry equipment and expertise in developing the HILIC-MS methodology for analyzing
and quantifying the tryptamine alkaloids and generating chemical standard curves, as well as
the preparation and editing of the manuscript. Dale L. Forrister performed the untargeted
metabolomic analysis of samples as well as the preparation and editing of the manuscript.
Laura Guzmán-Dávalos contributed with outreach to collection institutions and comments,
additions, and editing of the manuscript. Bryn T. M. Dentinger provided expertise to oversee all
DNA sequencing and phylogenetic work as well as contributing to the original experimental
design of this work in addition to the preparation and editing of the manuscript. Giuliana Furci
contributed with outreach to collection institutions and comments and additions to the
manuscript. Paul Stamets contributed comments and additions to the manuscript, as well as
high-resolution images for specimens.
DNA and Chemical Analysis of Psilocybe Mushrooms Applied and Environmental Microbiology
Month YYYY Volume XX Issue XX 10.1128/aem.01498-22 15
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This work was generously supported by a donation to the NHMU from Fungi Perfecti
LLC.
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