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Discovery of the closest free-living relative of the domesticated “magic mushroom”
Psilocybe cubensis
in
1
Africa
2
3
Alexander J Bradshaw
1,5
, Cathy Sharp
2
, Breyten Van Der Merwe
3
, Keaton Tremble
4
, Bryn T.M. Dentinger
5
4
5
1
Biology Department, Clark University, Worcester, MA 01610
6
2
Natural History Museum of Zimbabwe, Centenary Park, cnr Park Road &, Leopold Takawira Ave,
7
Bulawayo, Zimbabwe
8
3
Department of Microbiology, Stellenbosch University, Private Bag X1, Stellenbosch, 7600, South Africa
9
4
Department of Biology, Duke University, Durham, NC, USA
10
5
Natural History Museum of Utah and School of Biological Sciences, University of Utah, 301 Wakara
11
Way, Salt Lake City UT 84108
12
13
14
Emails:
15
Abradshaw@clarku.edu
16
mycofreedom@gmail.com,
17
breytenvdmerwe@gmail.com
18
keaton.tremble@duke.edu
19
bryn.dentinger@gmail.com
20
Corresponding Authors:
Alexander J Bradshaw, Bryn T.M. Dentinger
21
22
ORCID:
23
Alexander J Bradshaw: https://orcid.org/0000-0002-6261-621X
24
Cathy Sharp:
https://orcid.org/0009-0003-4985-1543
25
Breyten Van Der Merwe: http://orcid.org/0000-0003-0546-5619
26
Keaton Tremble: https://orcid.org/0000-0002-0788-2830
27
Bryn T.M. Dentinger: https://orcid.org/0000-0001-7965-4389
28
29
Keywords: psychedelic mushroom, psilocybin, diversity, Zimbabwe, Southern Africa
30
31
32
33
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Abstract:
34
The “magic mushroom”
Psilocybe cubensis
is cultivated worldwide for recreational and medicinal uses.
35
Described initially from Cuba in 1904, there has been substantial debate about its origin and
36
diversification. The prevailing view, first proposed by the
Psilocybe
expert Gastón Guzmán in 1983, is
37
that
P. cubensis
was inadvertently introduced to the Americas when cattle were introduced to the
38
continents from Africa and Europe (~1500 CE), but that its progenitor was endemic to Africa. This
39
hypothesis has never been tested. Here, we report the discovery of the closest wild relative of
P.
40
cubensis
from sub-Saharan Africa,
P. ochraceocentrata
nom. prov. Using DNA sequences from type
41
specimens of all known and accessable African species of
Psilocybe
, multi-locus phylogenetic and
42
molecular clock analysis strongly support recognizing the African samples as a new species that last
43
shared a common ancestor with
P. cubensis
~1.5 million years ago (~710k - 2.55M years ago 95% HPD).
44
Even at the latest estimated time of divergence, this long predates cattle domestication and the origin of
45
modern humans. Both species are a ssociated with herbivore dung, suggesting this habit likely
46
predisposed
P. cubensis
to its present specialization on domesticated cattle dung. Ecological niche
47
modeling using bioclimatic variables for global records of these species indicates historical presence
48
across Africa, Asia, and the Americas over the last 3 million years. This discovery sheds light on the wild
49
origins of domesticated
P. cubensis
and provides new genetic resources for research on psychedelic
50
mushrooms.
51
INTRODUCTION:
52
Psilocybe
cubensis
(Earle) Singer
is the most widely known, collected, and cultivated "magic
53
mushroom" in the world (A. J. Bradshaw et al., 2022).
P.
cubensis
was first described as
Stropharia
54
cubensis
Earle from a cattle-grazed field in Cuba in 1904 and today is globally distributed where it is
55
common in association with domesticated cattle across subtropical and tropical regions of America,
56
Asia, and Australia (T. Froese et al., 2016; Guzman, 2005; McTaggart et al., 2023; Thomas et al., 2002).
P.
57
cubensis
is one of the core species of psychoactive mushrooms used traditionally and
58
contemporaneously for cultural and spiritual ceremonies across Mexico, and has been domesticated
59
with many strains developed by an active social subculture due to its ease of cultivation (Castro Jauregui
60
et al., 2022; Guzmán, 2008; Van Court et al., 2022).
P. cubensis
is also the target of ongoing biochemical
61
and biomedical studies for drug discovery and whole organism therapies for a wide range of psychiatric
62
illnesses (Blei et al., 2020; Brownstien et al., 2024; Fricke et al., 2017; Lerer et al., 2024; Matsushima et
63
al., 2009; Shahar et al., 2024; Zhuk et al., 2015). Yet, despite the cultural, scientific and medical
64
importance of
P. cubensis
, we know little of its specific ecology or evolutionary origins.
65
The prevailing hypothesis, first proposed by Guzmán (1983), is that
P. cubensis
originated in
66
Africa and was transported to the Americas by Spanish colonizers during the 15th and 16th centuries.
67
This hypothesis was largely predicated on the speculation that other species closely related to it remain
68
to be discovered in Africa, a prediction rooted in the observation that the African continent is historically
69
undersampled for
Psilocybe
(Piepenbring et al., 2020; Tsakem et al., 2024)
.
The closest relatives of
P.
70
cubensis
were recently shown to be from both Asia and Africa, but sampling from these and other
71
regions remains vastly incomplete and definitive statements about origins and biogeography have
72
remained untenable (A. J. Bradshaw et al., 2024). Interestingly, despite substantial popular knowledge
73
and conspicuousness of
P. cubensis
in cattl e-grazed pastu res, it has not been officially confirmed from
74
subtropical or tropical Africa, although casual reports of a
P. cubensis
lookalikes have appeared in peer-
75
reviewed literature (Froese et al. 2016) and online databases such as iNaturalist and MycoPortal.
P.
76
cubensis
does occur in Asia, recorded for Thailand (Ma et al. 2014) and India based on an ITS sequence
77
matching
P. cubensis
in GenBank (accession OK165610.1), and multiple records of it occur across South
78
and Southeast Asia in iNaturalist and MycoPortal.
79
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One obstacle to determining the existence of named species in a given place at a given time i
s
80
the availability of type specimens as references. Molecular identification is particularly sensitive to th
e
81
lack of type reference sequences because sequences in public databases can carry names that have no
t
82
been validated through comparison with them. In the absence of type reference sequences, this ca
n
83
result in a broad misapplication of names resulting in a collective misunderstanding of species
’
84
identities, ecologies, and distributions. The recent study by Bradshaw et al. (2024) explicitly targete
d
85
type specimens for whole genome sequencing in an effort to remedy this long-standing problem i
n
86
Psilocybe
. While many type specimens were included in the study, it was not exhaustive, and critica
87
types such as
S. cubensis
and species known only from Africa were not included.
88
Recent fieldwork across sub-Saharan Africa between 2013 and 2022 resulted in multipl
e
89
specimens of an unknown
Psilocybe
sp.
that is superficially similar to
P. cubensis
in habit, habitat, an
d
90
general appearance. Upon further comparison of microscopic and molecular characters with the type o
f
91
S. cubensis
and other
P. cubensis
specimens from around the world, the recognition of the Africa
n
92
specimens as a distinct species is warranted, provisionally named
Psilocybe ochraceocentrata
. A multi
-
93
locus molecular phylogenetic analysis was used to confirm its close relationship to
P. cubensis
, an
d
94
divergence dating, ecological niche modeling, and species distribution modeling were used to predic
t
95
when, in time, these two lineages diverged and where they were likely to be found over the last
3
96
million years.
97
98
MATERIALS AND METHODS:
99
Collections and Sampling
100
Six collections of mushrooms resembling
P. cubensis
were collected across Zimbabwe and South Africa,
101
occurring on or near decomposing herbivore dung. All collections were made on public or private land
102
with the owner's permission. Sporcarp collections were air-dried over low heat and preserved from
103
insect damage using naphthalene. All other voucher details and deposition are reported in Table 1.
104
105
Table 1: Sample and voucher information
106
107
108
Genomic sequencing, assembly, and barcode extraction from Type specimens.
109
s
e
t
n
’
d
n
l
e
d
f
n
-
d
t
3
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Type specimens of the Cubensae complex (Table 1) were extracted and sequenced in the same manner
110
as (A. J. Bradshaw et al., 2024). Hymenophore fragments (5 to 15 mg) from dried fungarium samples
111
were homogenized by placing them in 2.0 mL screw-cap tubes containing a single 3.0-mm and 8 × 1.5-
112
mm stainless steel beads and shaking them in a BeadBug™ microtube homogenizer (Sigma-Aldrich,
113
#Z763713) for 120 s at a speed setting of 3,500 rpm. DNA extraction of mechanically homogenized
114
samples was performed using a Phenol-chloroform DNA extraction protocol. Lysis was performed with
115
Monarch® Genomic DNA Purification Kit (NEB, #T3010S) following the manufacturer’s protocol for
116
Tissue Lysis with an overnight incubation at 56 °C , using a volume of 500 ul lysis buffer, 10ul of
117
Proteinase K and increasing the amount of wash buffer to 550 μL during each of the wash steps.
118
Overnight incubation was then followed by a 2-hour incubation with 4ul of RNase A, after which total
119
lysate was placed in homemade Phase Lock gel tubes made using Dow Corning™ High Vacuum Grease
120
(ASIN:
B001UHMNW0)
along with an equal volume of OmniPur® Phenol:Chloroform:Isoamyl Alcohol
121
(25:24:1, TE-saturated, pH 8.0) solution (MilliporeSigma, Calibiochem #D05686) and then mixed by
122
gentle inversion for 15 min using a fixed speed tube rotator. After mixing, tubes were centrifuged at
123
maximum speed (14,000×g) for 10 min; then, the aqueous (top) layer was transferred to a new phase-
124
lock gel tube and the process repeated. DNA precipitation of the aqueous phase was performed by
125
adding 5 M NaCl to a final concentration of 0.3 M and two volumes of room temperature absolute
126
ethanol, inverting the tubes 20× for thorough mixing followed by an overnight incubation at −20 °C. The
127
next day, DNA was pelleted by centrifugation at 14,000×g for 5 min. The DNA pellet was washed twice
128
with freshly prepared, ice-cold 70% ethanol, air-dried for 15 min at room temperature, and then
129
resuspended in 150 μL of Elution Buffer from the Monarch® Genomic DNA kit.
130
131
DNAs were then cleaned using the Zymo Research™ DNA Clean & Concentrator-5 (#D4003) kit, with the
132
5:1 binding buffer protocol to account for the short fragments common of older herbarium samples.
133
Cleaned gDNAs were submitted to the High Throughput Genomics Core at the University of Utah, where
134
sequencing libraries were prepared using the Nextera™ DNA Flex Library Prep (Illumina®, #20018704)
135
and sequenced on a full lane of Illumina® NovaSeq 6000 PE 2 × 150 bp using an S4 flow cell. SRA
136
accession and biosample numbers are provided in Table 1. Raw reads were trimmed using fastP v0.23.4
137
(Chen et al., 2018), assembled using MetaSPades v3.15.5 (Nurk et al., 2017; Prjibelski et al., 2020), and
138
barcodes were extracted using Pathracer v3.16.0.dev (Shlemov & Korobeynikov, 2019) with custom
139
hidden Markov models (A. Bradshaw et al., 2023). Genome assembly stats and resulting BUSCO (Simão
140
et al., 2015) scores are reported in Supplementary Table 2.
141
142
DNA barcoding and sequencing of additional Specimens
143
The Internal transcribed spacer (ITS) DNA barcodes, commonly used for species delineation (Schoch et
144
al., 2012), of specimens “T1,” “T2,” and “Harding” were amplified using the primers set ITS1 and ITS4
145
(White et al., 1990). All ITS sequences were sequenced using Sanger sequencing and processed in the
146
same manner as Van Der Merwe et al. (2024). gDNA from dried basidiomata was extracted using the
147
Zymo Research™ Quick-DNA Fungal/BacterialMiniprep kits ( #D6005). Approximately 10 mg of fungal
148
tissue was used for extraction, which was carried out according to the manufacturer’s instructions.
149
Successful DNA extraction was visualized on a 1% agarose gel with ethidium bromide. Amplified
150
barcodes were sequenced at the Central Analytical Facility, Stellenbosch University, using an ABI 3730xl
151
DNA analyzer (Thermo Fisher Scientific™, Waltham, Massachusetts) with a 50 cm capillary array and
152
POP-7. All ITS sequences were deposited into NCBI GenBank (Sayers et al., 2019) with accession
153
numbers reported in Table 1.
154
155
Phylogenetic Inference
156
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Four independent loci were selected for phylogenetic analysis: the nuclear ribosomal internal
157
transcribed spacers (ITS), translation elongation factor 1-alpha (Ef1a), the largest subunit of RNA
158
polymerase II (RPB1), and the second largest subunit of RNA polymerase II (RPB2). In addition to newly
159
generated sequences, selected sequences from NCBI GenBank (Sayers et al., 2019) and the UNITE
160
database sh_general_release_dynamic_04.04.2024 (Abarenkov et al., 2010), including sequences from
161
recently named species from Africa and elsewhere (Canan et al., 2024; Ostunii et al., 2024; Van Der
162
Merwe et al., 2024) and sequences from type specimens (A. Bradshaw et al., 2023; A. J. Bradshaw et al.,
163
2024); Supplementary data, Table 1), were used for phylogenetic analysis. ITS sequences were trimmed
164
at the 5’ and 3’ end conserved motifs 5’- CATTA- and -GACCT-3’ for downstream analysis (Dentinger et
165
al., 2010). Only the coding sequence of the protein-coding genes were used. ITS barcodes were
166
partitioned into ITS1, 5.8S, and ITS2, using ITSx v1.1.3 (Bengtsson-Palme et al., 2013), aligned separately
167
using MAFFT v7.490 with the flags --maxiterate 1000 --localpair for slow but accurate analysis (Katoh,
168
2002). Concatenation of multiple sequence alignments of the three ITS partitions was achieved using
169
SEGUL 0.22.1 (Handika & Esselstyn, 2024). Phylogenetic analyses were performed using IQTREE2 2.3.6
170
with the flags -m MFP+MERGE -bnni -bb 1000, to enable automatic model finder (Kalyaanamoorthy et
171
al., 2017) and 1000 ultrafast bootstrap replicates (Minh et al., 2013). Evolutionary models were
172
automatically determined for ITS1, 5.8S, and ITS2 partitions separately, and for each codon position for
173
EF1a, RPB1, and RPB2. Edges were linked across all partitions. Phylogenetic trees were rendered in
174
FigTree (http://tree.bio.ed.ac.uk/software/figtree/).
175
176
Molecular dating
177
A reduced dataset was constructed using sequences of the three protein-coding loci for all specimens
178
represented by two or more loci. A concatenated matrix was constructed using Segul (Handika &
179
Esselstyn, 2022), and each codon position was given its own partition. A root calibration normally
180
distributed around a mean of 67.6 Ma (SD +- 6) following Bradshaw et al. (2024), and a calibrated Yule
181
speciation model were used to estimate divergence times. Bayesian inference was conducted using
182
BEAST2 v 2.7.7 (Bouckaert et al., 2019) with Markov Chain Monte Carlo (MCMC) sampling to estimate
183
posterior distributions of phylogenetic parameters. The best fitting models of evolution determined with
184
automatic model finder in IQTREE (Nguyen et al., 2015) were used for each partition. A chain length of 1
185
x 10
8
steps was used, sampling every 5000 steps to ensure a thorough parameter space exploration.
186
Convergence and effective sample sizes (ESS) of parameter estimates were assessed using Tracer v1.7.2
187
(Rambaut et al., 2018), with ESS values above 200 considered indicative of sufficient mixing and
188
convergence. The resulting posterior tree distribution was downsampled to every 2000 trees using
189
LogCombiner due to computational limitations and then summarized using TreeAnnotator v1.10
190
(Drummond & Rambaut, 2007) to produce a maximum clade credibility tree, with node heights
191
representing median posterior estimates. The annotated tree was visualized using FigTree as above,
192
with posterior probabilities indicated on nodes to reflect the robustness of inferred relationships and
193
95% highest posterior density (HPD) of estimated divergence times indicated with bars.
194
195
Species Distribution Modeling
196
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To predict the ranges of
P. cubensis
and
P. ochraceocentrata
, we conducted species distribution
197
modeling (SDM) using GPS coordinates of known collections and the contemporary 19 bioclimatic
198
variables at 2.5M resolution (Fick & Hijmans, 2017) as environmental predictors. In addition, we sought
199
to predict their historical ranges by conducting SDM of
P. cubensis
using four 19 bioclimatic datasets
200
modeled to have occurred during the Current age, Anthropocene (1979 – 2013),
last interglacial (LIG)
201
~130KYA , Pleistocene, MIS19 (~787 KYA) and the Pliocene ~3.3Mya (Brown et al., 2018; Dolan et al.,
202
2015; Hill, 2015; Karger et al., 2017) at 2.5M resolution accessed from paleoclim.com (Brown et al.,
203
2018). All collection GPS coordinates used for SDM were pulled from MycoPortal (Miller & Bates, 2017)
204
entries, including data from Mushroom Observer (https://mushroomobserver.org/) and iNaturalist
205
(https://www.inaturalist.org). All data points were filtered to remove non-wild collections, including the
206
removal of all entries with specific mentions of samples being cultivated, confiscated by police, or those
207
labeled as known cultivated strains of
P. cubensis
. The locations of all collections and observations were
208
plotted using GPS coordinates over a global map with R packages ggmap v. 4.0.0 (Kahle & Wickham,
209
2013) and sf v1.0-16 (Pebesma, 2018), and then visualized using ggplot2 v3.5.1 (Wickham, 2016).
210
To account for lumping of
P. ochraceocentrata
in prior records of
P. cubensis
, SDM for all
211
environmental datasets was conducted both with and without African collections of
P .cubensis
, using
212
the SDM R package (v1.2-46). We tested six of the most common SDM models ("bioclim",
213
"domain.dismo", "glm", "gam", "rf", and "svm"), using 1000 random points as “absence” points to
214
validate the model (Supplement Data). The best-performing model, according to AUC, COR Deviance,
215
TSS, MCC, and F1 score, was chosen for each environmental dataset.
216
217
Microscopy and Morphological analysis
218
Microscopic analysis on specimens CS-3006, CS-5783, and CS-3309 was performed using a Leitz Wetzlar
219
Orthoplan microscope with a Leitz Wetzlar drawing tube. All spore measurements were taken from
220
thirty spores whose side-view was clearly visible. Spore prints were studied using Melzer’s Reagent, and
221
sections of dried lamellae were studied in Congo Red. Color descriptions were derived from (Rayner,
222
1970), and codes are recorded parenthetically for each specimen. Microscopic analysis of additional
223
specimens T1, T3, and Harding was performed using Methods outlined in Van Der Merwe et al. (2024).
224
The length and width were measured, and the length/width quotient (Q) was calculated and reported in
225
Supplementary Table 1 for all samples.
226
227
RESULTS
228
Molecular systematics
229
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Independent phylogenetic analysis of ITS, EF1a, and RPB2 all recovered African specimens originally
230
identified as
P. cubensis
,
P.
cf.
cubensis
, and
P.
cf.
natalensis
as a monophyletic group sister to a clade
231
containing sequences from the holotype of
Stropharia cubensis
Earle (=
Psilocybe cubensis
) (Fig. 1) with
232
strong bootstrap support (ITS=97%, EF1a=99%, RPB2=100%). These same African sequences were not
233
reciprocally monophyletic with respect to sequences of
P. cubensis
using RPB1. Sequences of the types
234
of the other known species of
Psilocybe
endemic to Africa were consistently recovered in other clades.
235
Sequences from the holotype of
Stropharia aquamarina
Pegler were placed sister to
Psilocybe
236
wayanadensis
K.A. Thomas, Manim. & Guzmán
and other species from Asia and Australia (BS; ITS=100,
237
EF1a=92, RPB1=88, RPB2=97, Concat=100). Sequences from the holotype of
Psilocybe natalensis
Gartz,
238
D.A. Reid, M.T. Sm. & Eicker
did not match any publicly available sequences and was placed sister to
239
Psilocybe chuxiongensis
T. Ma & K.D. Hyde (typified from Yunnan, China) and
Psilocybe maluti
B. Van der
240
Merwe, Rockefeller & K. Jacobs (typified from South Africa) ( BS; ITS=99, Concat=99). Sequences of the
241
holotype of
Psilocybe jaliscana
Guzmán and the sequences of
Psilocybe subcubensis
Guzmán were
242
nested within a clade of sequences from
P. cubensis
across all loci. Sequences from the holotype of
243
Psilocybe keralensis
K.A. Thomas, Manim. & Guzmán was recovered in Clade I, and its ITS sequence was
244
identical to the ITS from the type specimen of
Psilocybe ingeli
B. Van der Merwe, Rockefeller & K.
245
Jacobs.
246
247
Molecular dating and geological timeline
248
Due to the close relationship between
P. ochraceocentrata
and
P. cubensis
, we performed molecular
249
dating using Bayesian inference with BEAST of a concatenated dataset of EF1a, RPB1, and RPB2 (Figure
250
1). Phylogenetic reconstruction generated a tree with highly supported nodes (posterior probability
251
>95%). Only a minority of nodes received less than a 0.95 posterior probability and most of these were
252
near the tips among closely related species and species complexes. Molecular dating places the MRCA
253
of
P. ochraceocentrata
and
P. cubensis
at ~1.56 million years ago (MYA)(0.71-2.55 95% HPD). This
254
estimated divergence date corresponds to the Pleistocene epoch (2.5 MYA - 11.7 KYA) following the
255
mass emergence of grass biomes in warm climates with the evolution of the C
4
photosynthetic pathway
256
(8 to 3 MYA) (Edwards et al., 2010).
257
258
259
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The copyright holder for this preprintthis version posted December 7, 2024. ; https://doi.org/10.1101/2024.12.03.626483doi: bioRxiv preprint
260
Figure 1.
Phylogeny, photo documentation, and geographical distribution of
Psilocybe ochraceocentrata
.
261
LEFT: Maximum clade credibility chronogram of
Psilocybe
spp. resulting from Bayesian divergence
262
analysis of three loci (EF1a, RPB1, RPB2). All nodes received >95% posterior probability except for
263
internal nodes with posterior probabilities indicated. Shaded bars at nodes represent 95%HPD. Shaded
264
bars have been removed from nodes where descendants represent multiple individuals of a species for
265
improved readability. The mean divergence time for the common ancestor of
P. cubensis
and
P.
266
ochraceocentrata
is indicated by a red arrow. Scale bar at the bottom represents the estimated time
267
using a root calibration normally distributed around a mean of 67.6 Ma (SD +- 6) following Bradshaw et
268
al. (2024). Geologic epoch time scale is approximate. Colored vertical bars represent noteworthy global
269
events that illustrate correlation of major divergences within
Psilocybe
: the K-Pg event ~65 MYA, the
270
Eocene Epoch Climate Optimum (EECO), the Terminal Eocene Event (TEE), the Mid-Miocene Climate
271
Optimum (MMCO), and the emergence of C4 grass biomes (C4). Right: in situ photos of
Psilocybe
272
cubensis
(BD1406, top),
P. ochraceocentrata
(CS3006, middle), and records of
P. cubensis
(gold squares)
273
from Mycoportal, iNatrualist, and Mushroom Observer with African records reinterpreted as
P.
274
ochraceocentratta
(Purple squares).
275
276
Ecological niche modeling and species distribution of
P. cubensis
277
.
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The copyright holder for this preprintthis version posted December 7, 2024. ; https://doi.org/10.1101/2024.12.03.626483doi: bioRxiv preprint
Due to the close relatedness of
P. ochraceocentrata
and
P. cubensis
, we chose to investigate the
278
theoretical species distribution of
P. cubensis
through time using ecological niche modeling (ENM) and
279
species distribution modeling (SDM). To do so, we used publicly available data from MycoPortal, which
280
includes data from iNaturalist and Mushroom Observer. Occurrence data was filtered for only those
281
with georeferences and removed any entries that suggested occurrences were acquired from non-
282
natural sources, such as cultivation. After filtering, we found 1001 occurrences, 12 of which were from
283
the African continent (Supplementary data). Due to the inability to authenticate specimens from Africa
284
as
P. cubensis
, and sparse literature reports of its distribution across Africa and India (Guzmán, 2014;
285
Thomas & Manimohan, 2003) , we performed ENM and SNM in two ways. The first analysis included all
286
geopoints (minus confirmed
P. ochraceocentrata
specimens), assuming that
P. cubensis
specimens from
287
Africa were correctly identified (Figure 2). The second analysis removed African specimens, assuming
288
that these specimens are
P. ochraceocentrata
(Supplementary Figure 5)
.
ENM and SDM were done using
289
multiple geological datasets, ranging from the Pliocene (~3MYA) to the Modern day (Figure 2,
290
Supplementary data).
291
Both data sets exhibited high similarity with ENM and SDM across time, predicting similar
292
species distributions (Figure 2, Supplementary Figure 5). The most extensive predicted ranges occurred
293
across the Southern United States, Central America, and South America (ENM >0.8, SDM =1), with a
294
presence across Southern Africa, Southeast Asia, and Australasia, albeit as a more restricted range
295
(ENM<0.5, SNM=1) (Figure 2). Across time, ENM and SDM indicated reduced distribution across Africa,
296
while the Americas, South East Asia, Australasia, and Europe exhibited an increased presence and
297
distribution up to the Last Interglacial (LIG) ~130 thousand years ago (KYA). Post LIG into contemporary
298
times, ENM and SDM predict distributions more in line with the Pleistocene, MIS19 (~787 KYA) and the
299
Pliocene (~3MYA), but with a slightly increased predicted presence in Africa and distribution across
300
Southeast Asia and Australasia remaining similar to the LIG (Figure 2).
301
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302
Figure 2.
Ecological niche modeling (ENM) and Species distribution modeling (SDM) of
Psilocybe cubensi
s
303
and
P. ochraceocentrata
through time. All occurrence data for
P. cubensis
from public sources (likely
304
including
P. ochraceocentrata
misidentified as
P. cubensis
in Africa) across three time scales since the
305
mid-Pleistocene.
Top Row:
Contemporary (Modern day),
Middle Row:
Pleistocene, MIS19 (~787 KYA) ,
306
Bottom Row:
Pliocene (~3MYA).
Left:
ENM with distribution likelihood indicated as a heat gradient from
307
purple (0%) to yellow (100%).
Right:
SDM with predicted species presence as present (1, yellow) or
308
absent (0, purple).
309
310
Taxonomy
311
Psilocybe ochraceocentrata
C. Sharp, A. Bradshaw, B. Dentinger & B. van der Merwe
nov. sp.
312
Index Fungorum Registration #: IF 902894
313
Holotype Deposition: Natural History Museum of Zimbabwe, BUL8013
314
GenBank Accession: PQ315824 (ITS), PQ317397( EF1a), PQ317404 (RPB1), PQ317412(RPB2)
315
SRA Accession:
316
317
s
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Etymology:
318
Pileus with a yellow-ochre center.
319
320
Diagnosis:
321
Typus: Zimbabwe, Matabeleland South Province, The Farmhouse, Kezi Road, Matobo Hills. QDS 2028A4.
322
In thick leaf litter in mixed deciduous woodland on granitic sand. 14 Jan 2013. Collectors C. Sharp & R.
323
Aldridge. Holotype: CS3006 (Field ID, Private Fungarium of C. Sharp), Holotype split: Natural History
324
Museum of Zimbabwe, BUL 8013.
325
326
General field description:
327
Fruiting body
medium-sized, up to 105 mm tall and growing in tight clusters; a pale fungus that bruise
s
328
blue-green.
Pileus
65-75 mm diam.; first cream-colored, then vinaceous-grey (115) with center ochreou
s
329
(44) to fulvous (43); pale yellow towards margin; convex then planate, often undulating; surface finel
y
330
and radially silky with a dull sheen.
Flesh
bright cream-coloured, firm to pithy.
Margin
first down-curved
;
331
edge smooth often with radial cracking.
Lamellae
adnate; first pale then greyish-sepia (106) to brown
-
332
vinaceous (84) to very dark sepia and almost black near margin; face of gill speckled as spores ripen; t
o
333
12 mm deep, thin, papery and fragile; edge thin, smooth or finely scalloped, pale; sparsely t
o
334
moderately crowded with lamellulae, 6-8/cm.
Stipe
central; up to 95 mm long x 9-10 mm; cream
-
335
coloured, bluing where handled; cylindrical, often twisted; apex minutely tufted (x10), streake
d
336
longitudinally, dull sheen, base often with white silky hairs.
Flesh
fibrous in walls and center hollow.
Rin
g
337
median to higher; white first then with dark spores; membranous, striate, very fragile and soon clingin
g
338
to stipe and disintegrating.
Mycelium
is white to cream-coloured, forming a thick, compact mat amongs
t
339
leaf litter.
Bruising
instantly to blue-green (94) where handled or damaged.
Odour
of typical mushroom
340
Spore-print colour
dark vinaceous-grey (116) to purplish-grey (128).
341
342
343
Figure 3:
344
s
s
y
;
-
o
o
-
d
g
g
t
.
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(A)
Psilocybe ochraceocentrata
. Holotype collection (CS-3006). Photo credit: C.Sharp,(B) Older
345
fruiting body of
Psilocybe ochraceocentrata
; color change possibly due to water logging,
346
(C)
Psilocybe ochraceocentrata
found on dung; (D) Cluster of
Psilocybe ochraceocentrata
(South
347
Africa). Photo credit: Talan Moult, (E) Young sporocarp of
Psilocybe ochraceocentrata
(Harding).
348
Photo credit: Talan Moult.
349
350
Habit and habitat:
351
Miombo woodland, mixed deciduous woodland, all on granitic sand; CS3309 was found growing on old,
352
decomposed herbivore dung of unknown origin.
353
354
Macroscopic Description:
355
Fruiting body
in a tight cluster, to 105 mm tall.
Pileus
to 65 mm diameter; first cream-colored to pale
356
vinaceous-grey (115) with ochreous (44) or fulvous (43) center; first convex then planate; surface finely
357
streaked radially with dull sheen.
Flesh
cream-coloured, firm.
Margin
straw-yellow (40), down-curved,
358
smooth; some radial cracking.
Lamellae
greyish-sepia (106) to brown vinaceous (84), face speckled with
359
ripening spores; adnate; thin, papery, fragile; edge finely scalloped and pale; with lamellulae.
Stipe
to 95
360
mm long x 9 (apex) – 10 (mid) – 10 (base) mm; cylindrical, twisted or not; apex minutely tufted (x10
361
lens), longitudinally streaked to silky-hairy at the base.
Flesh
hollow with fibrous walls.
Ring
just above
362
median, white, striate, membranous to clinging, and very fragile.
Bruising
immediately to glaucous sky-
363
blue (93) to glaucous blue-green (94).
Odour
typical mushroom.
Spore-print
dark vinaceous-grey (116)
364
to purplish-grey (128).
Chemical reactions:
not recorded
365
366
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Microscopic description:
367
Figure 4:
368
369
Microscopic illustrations and microscopic feature morphology of
Psilocybe ochraceocentrata
: (A)Basidi
a
370
from Holotype specimen CS-3006; (B): Basidiospores from Holotype specimen CS-3006; (C
371
Cheilocystidia from specimen CS-5783. Microscopic images are derived from specimen
P
372
ochraceocentrata
"Harding.”
373
Spores derived from Holotype CS-3006,:
Ranges are given parenthetically with mean values underlined
;
374
ellipsoid, a few lenticulars; thick-walled, smooth-walled with germ-pore; (11)11.8(12.5) x (6.5)7.6(8.0
375
µm; Q = (1.44)1.55(1.71).
Basidia
elongate or clavate and variable in size, 22-38 x 8-12.5 µm; fou
r
376
sterigmata, 4-5µm long with rounded or acute apex.
377
Notes from additionally examined samples:
378
[1]
Spores
can be highly variable in size; note CS3309 has much smaller spores with a few in the extrem
e
379
range (Supplementary Table 1)
380
a
)
.
;
)
r
e
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[2]
Spores
shape ellipsoid, some collections having more lens-shaped spores than others; thick-walled,
381
smooth-walled with apical germ-pore.
Basidia
elongate or clavate; 22-30 x 8-11 µm; four sterigmata, 4-
382
5µm long with rounded or acute apex.
Cystidia
no pleurocystidia were observed; despite numerous
383
sections, no cheilocystidia were observed in the Type collection but very clear in CS3309 and CS5783;
384
capitate in shape, thin-walled, 21-26 x 9-13µm across the widest part.
Hyphae
clamp connections
385
observed in the Type.
386
[3] CS-2680. Mashonaland Central Province, Mukuvisi Woodlands, Harare. QDS 1731C3. 18 Jan 2012.
387
Collector Daniel Nyamajiwa. The spore range is a good match even if a bit smaller, e.g.
388
(10)10.8(11.5)[13] x [6.5](7)7.4(8) µm but shape slightly different, Q = (1.33)1.4 5 (1.57) (Supplementary
389
Table 1).
390
[4] Even though the ochre-yellow center is diagnostic, one noticeable morphological difference is that
391
the green-blue pigments turn black and remain dark while the specimen is fresh. This may be due to the
392
older fruiting bodies or because they contain more water in a very wet season. All signs of green or black
393
coloration are absent in the dried samples (Figure 2, B).
394
Synonomizations
395
396
Psilocybe cubensis
(Earle) Singer, Sydowia 2(1-6): 37 (1948)
397
398
Basionym:
399
Stropharia cubensis
Earle, Inf. an. Estac. Cent. agr. Cuba 1: 240 (1906)
400
401
Synonyms:
402
=
Naematoloma caerulescens
Pat., Bull. Soc. mycol. Fr. 23(2): 78 (1907)
403
c
Hypholoma caerulescens
(Pat.) Sacc. & Trotter, Syll. fung. (Abellini) 21: 212 (1912)
404
c
Psilocybe cubensis
var.
caerulescens
(Pat.) Singer & A.H. Sm., Mycologia 50(2): 269 (1958)
405
=
Stropharia cyanescens
Murrill, Mycologia 33(3): 279 (1941)
406
c
cubensis
var.
cyanescens
(Murrill) Singer & A.H. Sm., Mycologia 50(2): 269 (1958)
407
=
Psilocybe jaliscana
Guzmán, Docums Mycol. 29(no. 116): 46 (2000)
408
409
DISCUSSION:
410
Psilocybe
has become a world-renowned genus of mushrooms, primarily due to their
411
psychoactivity. Currently,
Psilocybe
mushrooms have been targeted as a source of natural products for
412
use in a global mental health crisis (Carhart-Harris et al., 2017; Daniel & Haberman, 2017; Gandy et al.,
413
2020; Johnson & Griffiths, 2017). However, due to the issue of legality in their collection,
414
characterization of their diversity and studies of their biology have been severely stifled. Despite the
415
~160 species of true
Psilocybe
described around the world, most were described in the Americas
416
(Borovička et al., 2011; Guzmán, 2014; Guzman et al., 2013; Johnston & Buchanan, 1995; Ma et al.,
417
2014; Picker & Rickards, 1970). In contrast, only seven species of
Psilocybe
(including
P.
418
ochraceocentrata)
have been typified from Africa, ranging from the cedar forests of Northern Africa
419
(
Psilocybe mairei)
to the grassland of South Africa (
Psilocybe maluti
) (Guzmán, 2014; Van Der Merwe et
420
al., 2024); many more still undocumented species are predicted.
421
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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The subject of psychoactive mushrooms, in particular
Psilocybe
, and their proposed medical
422
benefits is rapidly gaining interest globally, and no less so in Zimbabwe. Recreational use in Zimbabwe is
423
generally isolated and often dependent on the availability of imported products, likely the frequently
424
cultivated
P. cubensis
(Musshoff et al., 2000). However, historical indigenous knowledge of these fungi is
425
lacking outside of Mesoamerica, making traditional-use claims challenging to validate. However, there
426
have been suggestions that access to the information has been restricted due to a sense of protected
427
information among traditional healers (T. Froese et al., 2016; Guzmán, 2014; Van Der Merwe et al.,
428
2024). This issue is likely a consequence of the historical lack of mycological studies across Africa, which
429
remains one of the most understudied geographic locals for fungal diversity (Antonelli et al., 2024; Crous
430
et al., 2006). Consequently, the people of Zimbabwe's use of
Psilocybe
for ceremonial or medicinal
431
purposes is unknown.
432
Psilocybe
has been previously grouped into four major sections, including the “Cordisporae”,
433
“Mexicanae,” “Zapotecorum,” and “Cubensae” sections. Of these sections, the Cubensae has had little
434
documentation of novel species. The Cubensae section includes species found across Central America,
435
Southeast Asia, India, and Africa, includ ing the most iconic species
P. cubensis
(A. J. Bradshaw et al.,
436
2024; Guzmán, 1983, 1995; Ramírez-Cruz et al., 2013). However, the abundance of diversity within the
437
section remains uncertain, primarily due to a limited representation of type specimens. Type specimens
438
serve as the authoritative description of a species, and their representation is essential to characterize
439
new species definitively. In particular, some specimens of the Cubensae complex, such as
Psilocybe
440
jaliscana
(typified from Mexico) and
Psilocybe aquamarina
(typified from Kenya), were thought to be
441
synonymous with
P. cubensis
and
P. subcubensis
, respectively (Guzman 2014, and personal
442
Communication, Virginia Ramírez-Cruz and Alonso Cortés-Pérez). While the case for
Psilocybe jaliscana
443
being synonymous with
P. cubensis
is true, the same is not true for
P. aquamarina
, which is reported
444
here as genuinely novel.
445
Further, a new issue arises when publicly deposited data with type specimens is validated. The
446
commercially sold “Natal Super Strength (NSS)” (OK491080.1) strain of
P. natalensis
(typified from
447
KwaZulu-Natal) does not ma tch the type specimen of
P. natalensis
. Instead, four of the five publicly
448
deposited sequences cluster with
P. ochraceocentrata
, indicating misidentification. This could lead to
449
future regulatory issues and confusion over the species identity of commercially sold
Psilocybe
strains.
450
Misidentifications at these levels illustrate the difficulty of identifying many species of
Psilocybe
, which
451
often exhibit plastic morphology, necessitating type specimen validation for taxonomic accuracy and
452
stability. This phenomenon is not unique to the Cubensae complex, as it has also been shown to occur
453
across the genus multiple times in commonly collected species (Awan et al., 2018; A. J. Bradshaw et al.,
454
2022, 2024). Without the ability to accurately describe what species are being consumed medically or
455
being commercialized, we jeopardize future work in understanding the species-specific secondary
456
metabolism of
Psilocybe.
With this work, we explicitly targeted type specimens, allowing for direct and
457
accurate comparison of future specimens
of
Psilocybe
from around the world. Notably, we provide the
458
molecular data from the holotype of
Stropharia cubensis
Earle, confirming the identity of the majority of
459
commercially available strains of “magic mushrooms” as
P. cubensis
for the first time
.
460
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted December 7, 2024. ; https://doi.org/10.1101/2024.12.03.626483doi: bioRxiv preprint
Psilocybe cubensis
is a globally distributed species whose origin is debated. Our ecological
461
modeling suggests that
P. cubensis
could have been found historically but discontinuosly across the
462
Americas, Southeast Asia, Southern and Central Africa, and Australasia between 0.71 and 2.55MYA
463
(Figure 2, Supplementary data). While there is an extensive predicted range, it has been shown that
P.
464
cubensis
from Australia has relatively low genetic diversity, suggesting recent arrival with domesticated
465
cattle to the continent (McTaggart et al., 2023). There are also no authenticated specimens of
P.
466
cubensis
from Africa and only two sequences of it from India (iNat144581118, OK165610.1). While it is
467
possible that
P. cubensis
may occur naturally in Africa,
P. ochraceocentrata
has a strikingly
468
morphological and genetic similarity and shares its almost exclusively ruminant coprophilic habit, which
469
is likely to lead to misidentification. Our divergence analysis suggests that their MRCA likely originated
470
alongside the large herbivores, possibly during the expansion of the C4 grasslands in East Africa 1.8-1.2
471
MYA (Cerling, 1992; Cerling et al., 1988). Coincidentally, this is also the period when
Homo erectus
472
became the dominant hominin in East Africa and the first to spread from Africa through Eurasia via the
473
Levantine corridor alongside large herbivores, including bovids (Antón & Swisher, Iii, 2004; Belmaker,
474
2010; Dennell & Roebroeks, 2005; Zhu et al., 2018). These major migration events present a possible
475
avenue for dispersal of the MRCA of
P. cubensis
and
P. ochraceocentrata
from Africa and their
476
subsequent divergence in Asia and Africa after aridification lead to loss of habitat in the intervening
477
region.
478
P. cubensis
was typified from Cuba in 1904 and is regularly found across the Americas, where it
479
is associated with herbivore dung. However, cattle did not reach the Americas until 1493 (Sluyter, 2023)
480
during the European colonization of Mesoamerica. While the origins and modern domestication of
481
cattle are still debated, there is no question that their evolutionary origins reside in the Old World as
482
opposed to the New World. Domestication of large grazing cattle (
Bos taurus
) and Asian zebu (
Bos
483
indicus
) occurred between ~8-10KYA. The MRCA of both of these domesticated animals, aurochs (
Bos
484
primigenius
), occupied a range from northern Africa to both coasts of Eurasia (Hanotte et al., 2002; Pitt
485
et al., 2019; Zeuner, 1963), overlapping with estimated historic species distribution of
P. cubensis.
486
Precolonial presence of
P. cubensis
in the Americas is not known, but its estimated divergence
487
from
P. ochraceocentrata
and ecological niche modeling suggest it may have existed there before the
488
arrival of Europeans. Bison (
Bison
spp.) are bovids that dominated grasslands in the Americas
489
throughout the Pleistocene and may have facilitated the precolonial arrival and spread of
P. cubensis
.
490
Bison are thought to have arrived from Asia to the Americas in multiple waves, with the first occurring
491
~195–135KYA during the LIG and then the secondc45–21KYA (D. Froese et al., 2017). After their arrival,
492
bison became one of the dominant herbivores in the Americas and were widely distributed across North
493
America and Central America. Bison are known ecological engineers, expanding and maintaining
494
grassland ecosystems wherever they roam (Gates et al., 2010; Ripple et al., 2015), making them
495
important vectors for coprophilic fungi, potentially including
P. cubensis
. The predicted ranges that we
496
see in the ENM and SDM results overlap with reported range of bison across the Americas, including
497
their co-occurrence across the coastlines of Alaska and the Pacific Northwest, and the peninsula of
498
Florida during the LIG (Supplementary data). The occurrence of
P. cubensis
in the Antilles archipelago is
499
most likely a recent development since no large herbivore existed that could have supported it except
500
for giant ground sloths (
Megalocnus
spp.), although this seems unlikely given these were probably
501
browsers with grasses making up a limited portion of their diets (Dantas et al., 2023). While we cannot
502
confirm the Americas are an origin of
P. cubensis
, we also cannot rule it out with the presently available
503
data. However, the inferred divergence of
P. cubensis
and
P. ochraceocentrata
millions of years ago
504
rather than hundreds of years makes the hypothesis that
P. cubensis
was brought to the Americas from
505
its ancestral home in Africa, where it then would have to have gone extinct, less parsimonious.
506
.CC-BY-NC 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted December 7, 2024. ; https://doi.org/10.1101/2024.12.03.626483doi: bioRxiv preprint
The recent characteriz ation of novel species of
Psilocybe
from Africa (Van Der Merwe et al.,
507
2024) indicates that more diversity remains to be described there. The research landscape of
Psilocybe
508
has been heavily stigmatized due to the near-global government restrictions on possession of the
509
psychoactive compounds psilocybin and psilocin. As a consequence of this long-standing impediment,
510
research on
Psilocybe
has a rich history of cooperation between citizens and professional scientists. The
511
study presented here was made possible by the collections and observations of numerous citizen
512
scientists, analysis with modern phylogenetic analysis and ecological modeling, and validation against
513
type specimens maintained in museum collections. Together, these resources provided a robust,
514
collaborative framework that enabled the discovery of
P. ochraceocentrata
and further refined our
515
understanding of the possible geographic origins of one of the world’s most infamous mushrooms,
P.
516
cubensis
. More investment and less regulation on the collecting of fungi, including species and
517
specimens predicted but not proven to contain controlled substances, is the only way to accelerate
518
scientific discovery to gain a more complete understanding of biodiversity and its importance to human
519
well-being before it is lost.
520
521
ACKNOWLEDGEMENTS:
522
The Authors would like to acknowledge Virginia Ramírez-Cruz and Laura Guzmán-Dávalos for assistance
523
in identifying difficult-to-find specimens. Sariah VanderVeur and Toma Ipsen for assistance in sample
524
preparation and help with technical lab duties. We would also like to thank the holding institutions who
525
provided material, much of which is rare and irreplaceable: Instituto de Ecología (INECOL, XAL),
526
University of Leipzig (LZ), Universidad de Guadalajara (IBUG), and Royal Botanic Gardens KEW (K). We
527
would also like to thank Talan Moult for documenting and collecting the South African specimens of
528
Psilocybe ochraceocentrata
used in this study
.
Dr. David Minter is thanked for his thoughts and
529
information on correct Latin designation.
530
531
Data availability:
532
All extracted DNA barcodes have been deposited on NCBI GenBank with corresponding accession
533
numbers reported in Table 1. All raw genomic sequencing data has been deposited in the Short Read
534
Archive (SRA) under Bioproject number PRJNA1159811; Biosample accession numbers are reported in
535
Table 1. All Type specimens derived molecular data has also been provided for RefSeq designation and
536
curation. Any code or specific script requests should be sent to the corresponding author.
537
Supplementary data, including microscopic features, genome assembly statistics, sequence alignments,
538
raw phylogenetic trees, and ecological and species distribution modeling outputs, can be downloaded
539
from the DRYAD data repository at https://doi.org/10.5061/dryad.5x69p8df2.
540
541
Conflict of interest:
542
The authors declare that there is no conflict of interest.
543
544
List of Supplementary Figures:
545
SF1 ML tree of ITS
546
SF2 ML tree of EF1a
547
SF3 ML tree of RPB1
548
SF4 ML tree of RPB2
549
SF5 ENM and SDM without African occurrence data
550
551
List of Supplementary Tables:
552
ST1 Microscopic characteristics and measurements
553
ST2 Genome assembly and BUSCO statistics
554
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted December 7, 2024. ; https://doi.org/10.1101/2024.12.03.626483doi: bioRxiv preprint
555
Supplementary data:
556
All occurrence data pulled from MycoPortal, including filtered sets used for ENM and SDM
557
558
References:
559
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