Extensive Collection of Psychotropic Mushrooms with Determination of Their Tryptamine Alkaloids

In this section, non-Psilocybe species of the genera Gymnopilus, Inocybe, Panaeolina, Panaeolus, Pholiotina, and Pluteus were analyzed: 12 species, 23 collections, and 54 individual fruiting bodies (Table 1).

Among the genus Gymnopilus, relatively low concentrations of PS, PSB, and BA have been reported for G. purpuratus and G. validipes, with the highest level of 3.3 mg/g PSB [34,41,53]. We analyzed two fruiting bodies of our G. dilepis collection from Malaysia for which tryptamine data have never been published. When harvested, the mushrooms strongly turned blue-green but their tryptamine contents were lower than those formerly reported for Gymnopilus species, with the highest content of only 0.131 mg/g PSB and without detection of AE, NA, and NB.

Leaving aside Boletus manicus, which may contain hitherto unknown psychotropic indolic substances [54], Inocybe is the only ectomycorrhizal fungal genus reported to produce PSB and related tryptamine alkaloids. We analyzed three Inocybe species previously reported or suspected to be psychotropic, specifically I. aeruginascens, I. calamistrata, and I. corydalina. These mushrooms are often difficult to find, and we only had the opportunity to analyze the fungarium specimens. Analysis of such collections may be problematic due to the poor stability of tryptamine alkaloids [55].

In two fruiting bodies of I. aeruginascens, we quantified all of the monitored tryptamines except NB. Both samples contained similar concentrations of AE, PS, and PSB (0.280–0.283 mg/g AE, 0.005 mg/g PS, and 0.124–0.128 mg/g PSB), and the BA concentration was lower (0.038–0.039 mg/g). These results are comparable with Stijve et al. [56], but Gartz [50] reported circa 10× higher values for AE and PSB, and 100× higher values for BA. Such discrepancies may be attributed not only to analytical problems, but more likely to the age of the material analyzed in our laboratory.

In I. corydalina, we analyzed four fruiting bodies of two individual fungarium collections (Figure 2). Curiously, the concentrations we found were even higher than those reported by Stijve and Kuyper [52], especially BA (0.499–0.975 mg/g) and 0.016–0.365 mg/g AE, 0.036–0.097 mg/g NB, less than or equal to 0.006 mg/g PS, and 0.076–0.282 mg/g PSB. It is interesting to compare the concentrations in PS-33 and PS-67, the latter analyzed nearly nine years after harvest. PS could not be detected in PS-67, but the concentrations of AE, BA, NB, and PSB were not remarkably lower than those in PS-33.

On the other hand, I. calamistrata used to be suspected of being psychotropic because of the blue-green tinge present at the stipe base (see, e.g., [57]), but PSB has not been found in the fruiting bodies [15,52,58]. Gartz [49] reported the occurrence of PSB, PS, and BA, but these data can be considered false-positive [59]. This study confirms the negative results of the previous studies.

Panaeolina and Panaeolus species (including taxa formerly classified as Copelandia or Anellaria) are commonly listed within the literature of psychotropic fungi ([29] and many others), but only a few species have been confirmed to contain psychotropic tryptamines. Despite the fact that a world monograph of Panaeolus exists [60], no molecular studies have been published to date on this genus and a critical revision relying on genetic markers is needed. Therefore, we have documented some of our collections by fungarium specimens and/or ITS rDNA sequences so that their determination can be corrected by future research.

Panaeolus cinctulus (referred to as Panaeolus subbalteatus in many previous studies) is known to contain psychotropic tryptamines. We analyzed 12 fruiting bodies from three different collections. Unfortunately, AE was not measured because these collections were processed before AE analysis was introduced into the method. These were quantified as 0.118–1.525 mg/g BA, 0.045–0.477 mg/g NB, 0.007–0.257 mg/g PS, and 0.114–1.578 mg/g PSB in P. cinctulus, which is shown in Figure 2. As pointed out by Stamets [61], tryptamine concentrations reported from this species are highly variable. Interestingly, Stijve and Kuyper [52] and Stijve and de Meier [62] found no PS, which was present in relatively low amounts in our samples.

In three additional taxa—Panaeolus olivaceus, P. papilionaceus, and Panaeolina foenisecii—none of the observed tryptamines were detected. In the latter two species, negative data were published earlier, but several positive detections of PS/PSB have also been published for P. foenisecii, which was reviewed by Allen and Merlin [63]. According to these authors, the positive results may be due to the misidentification of P. foenisecii with P. cinctulus.

A very interesting and rare species in our dataset is Pholiotina cyanopus, for which we analyzed three fruiting bodies from a single fungarium collection. P. cyanopus contained 0.821–1.360 mg/g BA, 0.247–0.565 mg/g NB, less than or equal to 0.062 mg/g PS, and less than or equal to 0.859 mg/g PSB. Our results were very similar to the previous study by Halama et al. [13], who also reported a trace concentration of AE.

Several Pluteus species from the section Pluteus are also known to contain psychotropic compounds [11]. We report the data for P. salicinus, which has already been analyzed for BA, PS, and PSB, and for P. americanus and P. glaucotinctus, which are supposed to be psychotropic due to their bluing reaction. Our data on BA, PS, and PSB in P. salicinus match those in previous studies [43,52], and concentrations of AE and NB (not yet reported) were generally low. An elevated NB content was observed in the single sample of P. glaucotinctus. In both P. glaucotinctus and P. americanus (and also P. salicinus), PSB was the major compound with concentrations exceeding 1 mg/g in most samples.

The genus Psilocybe is the largest and the best-known group of psychotropic mushrooms. We analyzed 87 fruiting bodies from 31 individual collections of 14 Psilocybe species. The phylogenetic tree (ITS rDNA) of the genus is presented in Figure 3, where the maximum PSB concentrations are also indicated; the phylogenetic position of all analyzed species is indicated in the tree. Here, we comment on the results for the Psilocybe species from the top to the bottom of the tree, but not on the species complex of P. serbica, which will be discussed separately.

P. cubensis is the most frequently cultivated psychotropic mushroom in the world, and it produces large fruiting bodies. We analyzed nine fruiting bodies from four individual collections, where we quantified 0.026–0.053 mg/g AE, 0.139–0.881 mg/g BA, 0.044–0.161 mg/g NB, 0.208–5.344 mg/g PS, and 0.651–3.509 mg/g PSB. Concentrations of BA, PS, and PSB corresponded to the tryptamine ranges from previous studies [3,27,41], and similar NB and AG concentrations were reported in our previous study [55]. Psilocybe ovoideocystidiata is rather closely related to P. cubensis (Figure 3). The tryptamine concentration ranges determined in eight fruiting bodies from three collections were similar to P. cubensis (Table 1, Figure 4) and higher than those reported by Allen et al. [40].

The taxa belonging to the P. cyanescens complex only showed subtle differences in the ITS rDNA marker, as can be seen in the tree (Figure 3). We only analyzed two species of this complex: P. cyanescens and P. subaeruginosa. Psilocybe cyanescens is known as the “Wavy cap” on the Pacific Coast of the USA and is probably the best-known wood-rotting species growing in the USA. This mushroom is not indigenous to Europe where it was introduced in the 20th century [31]. P. cyanescens is known for its high BA, PS, and PSB concentrations [3,41,45,52]. Our results for four European collections analyzed in this study confirmed that P. cyanescens is one of the most “potent” species: 0.011–0.039 mg/g AE, 0.216–2.852 mg/g BA, 0.102–0.978 mg/g NB, 0.409–10.018 mg/g PS, and 2.340–13.808 mg/g PSB. Literature data for PS and PSB in P. subaeruginosa are based on methods using older detection techniques, namely, the UV detector and fluorescence detector [64] and chemiluminescence [65], where the reported PSB range was 0.0–2.1 mg/g and the PS content was only 0.038 mg/g [65]. Our results for all tryptamines in P. subaeruginosa were very low (the highest of 0.33 mg/g PS) and did not correspond much to those found in other species of the P. cyanescens complex. We speculate that this species may produce much higher tryptamine concentrations and additional analyses are therefore desired.

Tryptamines were not detected in Psilocybe fuscofulva (P. atrobrunnea auct., P. turficola nom. inval.). This species is present in an isolated lineage in the tree (Figure 3) and does not turn blue/green when bruised. Our negative results for this species confirm those of the previous studies [66,67] and correspond with the molecular evidence provided by Reynolds et al. [68]. P. fuscofulva is the only non-psychotropic member of the genus Psilocybe in the modern concept [69]. However, lower in the tree, another tryptamine-negative species can be seen: Psilocybe fimetaria.

Although one of the P. fimetaria collections was analyzed only a few months after sampling (PS-65), we were unable to detect any of the analyzed tryptamines. This collection (PRM 951396) was found in the Czech Republic and was primarily determined according to the morphological characters. The phylogenetic analysis shows its affinity to P. pelliculosa (Figure 3), a psychotropic species known from the USA [29]. One of the ITS rDNA sequences of P. pelliculosa was even 100% similar to that of P. fimetaria, but their EF1-a gene sequences are apparently distinct (the sequences HF912341↗ and LR760712↗ show a match of only 94%). This would suggest that the ITS rDNA molecular marker is not always a reliable character for species delimitation in Psilocybe (which is also indicated in the Psilocybe cyanescens complex). According to the literature [70,71], Psilocybe fimetaria turns blue when bruised, however, the bluing reaction was not observed in the Czech collection. Because no other sequences of P. fimetaria are available in public databases, we were unable to discuss the taxonomic identity of our collection. The absence of tryptamine alkaloids in our samples could be explained, for example, by (i) low initial concentration and following degradation before analysis, and (ii) an existence of two Psilocybe species corresponding to the morphological concept of P. fimetaria, one psychotropic and one non-psychotropic, as not only the bluing reaction but also PSB was reported in P. fimetaria [33]. Therefore, a thorough analysis and multi-gene sequencing of additional collections of P. fimetaria are needed.

Psilocybe medullosa is a rare European species that does not turn blue when bruised but the chemical tests indicated an occurrence of PSB and PS at low levels [67]. We analyzed 11 fruiting bodies from three different collections and measured no AE, 0.007–0.406 mg/g BA, 0.000–0.215 mg/g NB, 0.000–0.051 mg/g PS, and 0.143–1.003 mg/g PSB. The tryptamine concentrations were rather low, but certainly not negligible.

Psilocybe caerulipes is widespread but not common in the USA, with no available analytical data on tryptamines [29]. Our results for the two collections showed relatively high concentrations: 0.018–0.028 mg/g AE, 0.63–0.141 mg/g BA, 0.019–0.052 mg/g NB, 0.501–2.770 mg/g PS, and 2.234–5.674 mg/g PSB.

Furthermore, we had the opportunity to analyze three well-known Mexican Psilocybe species: P. caerulescens, P. mexicana, and P. zapotecorum (for current taxonomic concepts of these species see [72]). Due to the lack of material (two fruiting bodies per collection), no fungarium material was deposited, but all collections were documented by ITS rDNA sequencing. The lowest tryptamine content was found in P. caerulescens with the highest concentration of only 0.413 mg/g PS. Higher concentrations were found in P. mexicana, from which PS and PSB were isolated in 1958 by Albert Hofmann [73]. We quantified traces of AE, 0.254–0.324 mg/g BA, 0.159–0.203 mg/g NB, 1.944–1.974 mg/g PS, and 3.286–3.934 mg/g PSB in these mushrooms which is consistent with the historical findings by Hofmann et al. [74]. When compared to P. mexicana, in P. zapotecorum, we found a similar NB content, but lower PS (0.293–0.367 mg/g) and considerably higher PSB (9.022–9.653 mg/g). To the best of our knowledge, these are the first analytical data for tryptamines in P. zapotecorum.

One undetermined Psilocybe species donated from Australia appeared to have the same ITS rDNA sequence as one labeled as P. tasmaniana (GenBank MH289756↗), but the spore size of our collection did not match that reported for this species [70]; we therefore considered this collection Psilocybe cf. tasmaniana. Tryptamine measurements revealed relatively low concentrations, which are published for the first time for this species: 0.282–0.965 mg/g BA, 0.031–0.144 mg/g NB, 1.489–2.051 mg/g PS, and 0.512–1.892 mg/g PSB; AE was not analyzed because the method was not available.

Last but not least, grassland inhabiting P. semilanceata (Liberty Cap) was analyzed as one of the most well-known and widespread temperate species. There is rich analytical data on tryptamines in this species [3,41,42,43,45,46,52], but our quantification of AE and NB is novel. Relatively high BA, high PSB, and low PS concentrations are typical for this species, which is consistent with our measurements (Figure 4).

The Psilocybe serbica complex contains several taxa formerly classified at the species level [31]. However, phylogenetic analysis based on three molecular markers did not prove a significant difference between P. serbica, P. bohemica, P. arcana, P. moravica, and P. moravica var. sternberkiana. Therefore, all taxa are now considered conspecific with P. serbica or may be treated as varieties [67,71,75]. The phylogenetic analysis (Figure 3) also revealed that the invalidly described “Psilocybe germanica” (Gartz & Wiedemann 2015, Drug Testing and Analysis 7: 853–857) did not differ from the validly described taxa of the Psilocybe serbica complex (however, no material was available for tryptamine analysis).

Psilocybe serbica and its varieties are very common in certain regions of the Czech Republic, which enabled us to analyze a rich sample population, particularly of var. arcana and var. bohemica; one collection could not be attributed to a specific variety and is indicated as P. serbica in the dataset. Unfortunately, the tryptamine concentration data presented here (Table 1, Figure 5) cannot be accurately compared with earlier data published in the literature because the particular varieties were not well-described or distinguished in the past.

The most abundant taxon we studied from this complex was var. arcana, where we analyzed 54 fruiting bodies from 18 individual collections. Our dataset is the most extensive out of all published studies. We measured 0.000–0.024 mg/g AE, 0.000–2.237 mg/g BA, 0.000–0.794 mg/g NB, 0.412–7.922 mg/g PS, and 0.002–8.878 mg/g PSB. The concentrations are highly variable, which was also observed in var. bohemica. Curiously enough, the concentrations of PS (0.027–2.485 mg/g) and PSB (1.553–15.543 mg/g) determined in var. bohemica (20 fruiting bodies, eight collections) were significantly higher (t-test, p < 0.05) than those in var. arcana; the concentrations of AE, BA, and NB were roughly similar. The concentration of 15.543 mg/g PSB in var. bohemica was the highest determined in the whole dataset and P. serbica var. bohemica is therefore comparable with the highly potent P. azurescens with PSB concentrations reported of up to 17.8 mg/g PSB [58].

Previous studies [3,52,53] referring to var. bohemica have reported similar or slightly lower tryptamine levels—in light of our findings, this could be caused by including var. arcana in the dataset. On the other hand, Stříbrný et al. [45], who distinguished between var. bohemica and var. arcana, did not observe such a difference in the PS/PSB content in these varieties and PS was significantly higher than PSB in var. bohemica. This discrepancy could be attributed to numerous factors, especially a low sample population (only one collection per variety was analyzed). Psilocybe serbica var. moravica is rare and only six fruiting bodies of one collection could be analyzed. The tryptamine concentrations were similar to those of var. bohemica, where low PS (0.061–0.386 mg/g) and high PSB (5.655–14.158 mg/g) concentrations were observed.

Despite the fact that the morphological differences between var. arcana and var. bohemica have not been confirmed by molecular studies, there is a significant difference in their PS and PSB composition. However, this result should be taken with care, and a more detailed study focused on this difference should carefully verify this observation. Because of the insufficient sample population, we were not able to compare the PS and PSB concentrations in var. moravica; the available data suggest a similarity to var. bohemica. We also cannot explain the striking variation in the tryptamine concentrations observed in samples probably originating from the same mycelium, and possible factors that influence this phenomenon, both environmental and biological, should be investigated in the future.

The large variability in the concentrations of tryptamines in fruiting bodies observed in this study deserves more attention. According to our data, some Psilocybe species possibly contain higher concentrations of tryptamines than others (species-dependent). The differences between species might correspond to the activity of enzymes (PsiD, PsiH, PsiK, PsiM) that catalyze the biosynthesis of NB, BA, PSB, and PS [4,76].

However, the development stages of fruit-bodies and the climatic conditions may also represent important factors that regulate the production of psychotropic tryptamines [77]. These are also hydrophilic substances [78], so we did not exclude the possibility that they could be leached from the fruiting bodies into the surrounding environment by rainwater. Furthermore, the concentration of tryptamines may depend on the substrate composition: the content and bioavailability of nitrogen, phosphate, and the amino acid tryptophan, which serves as a precursor for the biosynthesis of PSB and similar tryptamines [77,79]. Tryptophan can be enriched in the substrate by fertilizing the soil with decomposed plants or animal dung [80], so fruiting bodies harvested, e.g., from woodchip beds might contain higher tryptamine concentrations than those from natural sites. However, the actual influence of such factors has yet to be investigated. Last but not least, the concentrations may be artificially affected by sample processing and storage before analysis [55].

According to our experience during this study, there is great scientific and public interest in the concentrations of tryptamine alkaloids in psychotropic mushrooms. However, their illegal status greatly complicates their taxonomic and chemical investigation. Donating PS/PSB containing mushroom samples for scientific study is associated with the risk of criminal prosecution, but without such assistance, our study would be limited to the mushroom species growing in the Czech Republic, where the work of the analytical team was within the legal boundaries.

Furthermore, the poor stability of tryptamines complicates the evaluation of the data [55], especially when the mushrooms were harvested by volunteers with limited laboratory equipment and experience—it is essentially not possible to ensure completely identical processing of the samples prior to analysis. The delay between collecting and analysis also affects the analytical results to some extent, but the relatively high tryptamine concentrations in old fungarium samples (e.g., Inocybe corydalina PS-67) suggest that the degradation of tryptamines in dried fruit-bodies is not as rapid as in the pulverized samples investigated by Gotvaldová et al. [55].

In addition, some psychotropic mushrooms are difficult to find in the field (or difficult to find in sufficient quantities), so only old fungarium collections were available for analysis. In any case, we tried to ensure and present the most interesting composition of psychotropic mushrooms available with the quality corresponding the analytical results.

Methanol LC-MS CHROMASOL™ and eluent additives for UHPLC-MS/MS—acetic acid, formic acid, and ammonium formate—were obtained from Honeywell, Fluka™, Charlotte, NC, USA. Ultrapure water, 18.2 MΩ·cm, was produced by the Smart2Pure 12 UV system (Thermo Fisher Scientific—Barnstead, Germany). The analytical standards including PSB and PS were purchased from Cayman Chemical, Ann Arbor, MI, USA. The reference standards of AE, BA, and NB were synthesized at the Forensic Laboratory of Biologically Active Substances at UCT Prague according to our previous work [55].

A major part of the mushroom samples was obtained by the authors of this study and their co-workers. These were collected in their natural habitats in the Czech Republic and Malaysia. Furthermore, mushroom collections were provided in response to requests on social media by anonymous donors from Australia, Bosnia and Herzegovina, France, Germany, Italy, Mexico, Sweden, and the USA. Some mushroom samples (mainly Inocybe spp.) were donated for analysis from the fungarium of the Mycological Department of the National Museum, Prague (PRM) and private fungaria.

The collectors were instructed to dry the mushrooms gently in the air (near a heater or in an air dryer at no more than about 40 °C) and promptly sent them to the Forensic Laboratory of Biologically Active Substances in Prague, which is authorized to handle illegal psychotropic substances. Dried fungal samples were stored in the dark at 20 °C until analysis. The time between mushroom collection and analysis is shown in Supplementary Table S1. Mushroom species were identified by Jan Borovička according to the current taxonomy [32,67,71,75,81,82] using micromorphological and/or molecular characters (DNA sequencing, see below). When possible, representative parts of the mushroom collections were deposited in the fungarium of the Mycological Department, National Museum, Prague, Czech Republic (PRM). Wild Stropharia aeruginosa and cultivated Agaricus bisporus (sold on the market) were used as mushroom negative controls in the tryptamine measurements. For the list of analyzed mushroom species and further details on the collections (collection date, geographic origin, etc.) see Supplementary Table S1. For the list of complete scientific mycological names of mushroom species including the authors, see Supplementary Table S2.

The dried fruiting bodies were homogenized by a ceramic mortar and pestle to a fine powder. To extract the sample, fungal powder (10.0 ± 0.5 mg) was mixed with 1.0 mL of 0.5% acetic acid (v/v) in methanol. The mixtures were shaken by a vortex at 13× g at 20 °C for one hour. In the next step, the testing tubes were centrifuged for 10 min at 857× g at 20 °C. These suspensions were filtered using Eppendorf tubes with a 0.2 µm PTFE microfilter (Ciro, Hudson, WI, USA) in a centrifuge (15,256× g, 5 min, 20 °C). Then, re-extractions of the remaining pellets were carried out with 1 mL methanol and vortexed and ultrafiltrated using the same conditions as described above. To prevent degradation of the analyzed alkaloids, the extraction solvents were purged with a stream of nitrogen, and the sets of testing tubes were covered with aluminum foil.

Mixtures of 100 µL of the final supernatant and 900 µL solvent of 10 mM ammonium formate with 0.1% formic acid in methanol/water (1/9, v/v) stored in brown glass vials (Assistent-Glaswarenfabrik Karl Hecht, Sondheim vor der Rhön, Germany) were used for analysis using UHPLC-MS/MS. Consequently, these samples were further diluted (10×, 100×, and 1000×) due to different concentrations of analytes.

The separation and quantification of the five analyzed tryptamines AE, BA, NB, PS, and PSB in mushroom biomass was carried using the UHPLC 1290 Infinity assembly (Agilent Technologies, Santa Clara, CA, USA) with a Zorbax Eclipse Plus C18 column, 100 × 2.1 mm, 1.8 µm and with a Zorbax Eclipse Plus C18, 5 × 2.1 mm, 1.8 µm pre-column (Agilent Technologies, Santa Clara, CA, USA) coupled with a triple quadrupole 6460 spectrometer (Agilent Technologies, Santa Clara, CA, USA). This method was used based on a previous publication (Gotvaldová et al., 2021 [55]). Column separation was performed at 40 °C. In the method, mobile phase A—10 mM ammonium formate with 0.1% (v/v) formic acid—and mobile phase B—10 mM ammonium formate with 0.1% (v/v) formic acid in methanol—were used. The gradient elution was used, the flow rate was 0.25 mL/min, and the injection volume of the sample was 3 μL.

In the MS system, an electrospray ionization in positive mode (ESI⁺) was used for all analytes. Optimal MS operating conditions were set as follows: drying gas temperature (nitrogen) of 340 °C, drying gas flow of 10 L/min, nebulizer (nitrogen) of 25 psig, sheath gas temperature (nitrogen) of 400 °C, sheath gas flow of 12 L/min, and capillary voltage of 2300 V. The dynamic multiple reaction monitoring (dMRM) acquisition mode was utilized for analysis. For all analytes except PS, the two most intensive transitions were used, whereas for PS, three transitions were used. The detection limit (LOD) was 1.0 ng/mL for NB and 0.1 ng/mL for other analytes. The quantification limit (LOQ) was 5.0 ng/mL for NB and 0.5 ng/mL for the other analytes. The total analysis time was 7 min.

In order to determine or document some of the studied mushroom collections, automated DNA sequencing of the ITS rDNA, LSU, and EF1-α molecular markers was performed at Macrogen Europe. Genomic DNA was extracted from dried fruiting bodies using either the NucleoSpin Plant II DNA Isolation Kit (Macherey-Nagel) or the NaOH extraction protocol [83]. For details on the DNA extraction, PCR conditions, primers, PCR product purification, and sequencing, see [75]. The sequences were submitted to the GenBank/EMBL-Bank databases under the accession numbers MN901948-MN901955↗, MN900720↗, MW352021-MW352022↗, MT028350↗, and LR760712↗. For the list of sequenced collections, see Supplementary Table S1.

For the phylogenetic analysis (ITS rDNA), reliable Psilocybe sequences were retrieved from the GenBank database, mostly based on taxonomic papers on this genus [67,75,82,84,85,86,87,88,89], but also unpublished sequences that the authors believed to be correctly identified (direct submissions to GenBank, with no available reference). Sequences of insufficient length were omitted. The sequences were aligned with MAFFT online version 7 [90] using the E-INS-i strategy with default settings. The poorly aligned positions in the alignment were removed with Gblocks v. 0.91b [91] with options for a less stringent selection. The evolutionary history was inferred by MEGA X using the maximum likelihood (ML) method based on the Kimura 2-parameter model [92], which was the best-fitting model according to the model test analysis in MEGA X. The tree with the highest log-likelihood (−2513.31) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches (maximum likelihood bootstrap values). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 52 nucleotide sequences and a total of 588 positions were in the final dataset, of which 147 were variable and 35 were singletons. Stropharia luteonitens (MT028350↗) was used as the outgroup.