Psilocybe azurescens taxonomy paper

Paul Stamets and Jochen Gartz
A new caerulescent Psilocybe from the Pacific Coast of Northwestern America

Abstract: A new taxon from the Pacific Northwest of North American is described. This species can be placed into Stirps Cyanescens of the Sections Caerulescents Singer (Singer, 1948; Singer & Smith, 1958a) or into the more recently constructed Section Cyanescens as amended by Guzman (1983). This new species is autumnal and lignicolous, living in soils enriched with deciduous wood- debris, characterestic of riparian woodlands of the coastal regions of the Pacific Northwest of North America. Strongly bruising bluish to indigo-black, this Psilocybe species features a hazelnut to caramel-colored pileus and a stipe whose base radiates clusters of white rhizomorphs. This novel species is delineated through a combination of morphological and microscopic feautures and possesses unusally high concentrations of psilocybin, psilocin and baeocystin.

Psilocybe azurescens Stamets & Gartz sp. nova

Pileo ochreato-brunneo, hygrophano, viscido, pellicula separabili intructo, conico dein convexo, plano 30-100mm lato, umbonato. Lamellis sinuato-adnatis, pallidis vel brunneo. Stipite albo, stricto, elongato, 90-200mm, fibrillis cum strigositate basis caerulescentibus. Carne caerulescente. Sporis 12-13,5 x 6,5-8.0 um. Cystidiis fusoid-ventricosis. Cheilocystidiis 23-28 x 6.5-8.0 um; pleurocystidiis 23-35 x 9-10 um.

Macroscopic Features: Pileus 30-100mm in diameter, conic to convex, expanding to broadly convex and eventually flattening with age with a pronounced, persistent broad umbo; surface smooth, viscous when moist, covered by a separable gelatinous pellicle; chestnut to ochraceous brown to caramel in color often becoming pitted with dark blue or bluish black zones, hygrophanous, fading to light straw color in drying, strongly bruising blue when damaged; margin even, sometimes irregular and eroded at maturity, slightly incurved at first, soon decurved, flattening with maturity, translucent striate and often leaving a fibrillose annular zone in the upper regions of the stem. Lamellae ascending, sinuate to adnate, brown, often stained info-black where injured, close, with two tiers of lamellulae, mottled, edges withish. Spore-print dark purplish brown to purplish black in mass. Stipe 90-200mm long by 3-6mm thick, silky white, dingy brown from the base or in age, hollow at maturity. Composed of twisted, cartilaginous tissue. Base of stem thickening downwards, often curved, and characterized by coarse white aerial tufts of mycelium, often with azure tones. Mycelium surrounding stipe base densely rhizomorphic, silky white, tenaciously holding the wood-chips together, strongly bruising bluish upon disturbance. Odor none to slightly farinaceous. Taste extremely bitter.

Microscopic Features: Clamp connections abundant. Ixocutis gelatinous, hyaline hyphae, 1.5 - 5.5um in diameter. Sub-pellis a brownish band, more highly pigmented than pileal trama. Lamellar trama regular, composed of hyphae 5 - 15 um in diameter, slightly encrusted with brown pigments; subhumenium a subcellular compact layer, 10um thick. Pileal trama 5 - 15um thick. Pleurocystidia abundant, fusoid-ventricose, tapering to a narrow but short neck, bluntly papillate, 23-35 x 9-10 um. Cheilocystidia forming a nongelatinized sterile band, nearly identical to pleurocystidia measuring 23-28 x 6.5-8.0 um. Basidia 4-spored, measuring 27-30.5 x 6.3-7.2 um. Spores 12-13.5 x 6.5-8.0 um, rich reddish brown in KOH and light purplish vinaceous in aqeous ammoniacal solutions. Wall thickness less than 1 um. Caulocystidia abundant above the annular zone and similar to pleurocystidia but thicker walled and more irregular, measuring 43um long with undulated necks. Cortial hyphae on stipe slightly thickened, almost subgelatinized walls, 3 - 5 um in diameter with clamps and brown intra-perital pigment. Caulocystidia absent below annular zone. Tissue notably awash with bluish tones.

Habit & Habitat: Cespitose to gregarious on deciduous wood-chips and/or in sandy soils rich in lignicolous debris. Aspect collyboid, generating an extensive, dense and tenacious mycelial mat, Psilocybe azurescens causes the whitening of wood. Fruitings begin in late September and continue until harsh frost, usually mid-November.

Distribution: Specimens were first collected on an alluvial plain along the Columbia river network near Astoria, Oregon in 1979. Fruitings of this species are known from Oregon and Washington. Holotype: A dried collection of fruitbodies cultivated on alder (Alnus rubra) wood-chips using the methods described by Stamets (1993) outdoors, harvested on 11/21/93 and deposited at WTU. Original clone used for propagation was from Astoria, Oregon on 10/30/79. Additional collections from Tillamook and Astoria, Oregon in October 1990 were collected by one of the authors (Jochen Gartz) and deposited in LZ.

Taxonomic Considerations: Psilocybe azurescens generally resembles Psilocybe bohemica Sebek, Psilocybe cyanofibrillosa Guzman & Stamets, Psilocybe cyanescens Wakefield, Psilocybe eucalypta Guzman & Walting, Psilocybe mairei Singer, Psilocybe serbica Moser & Horak and Psilocybe collybioides Singer & Smith. Complete reproductive barriers have been found be one of the authors (Jochen Gartz) between Psilocybe azurescens and Psilocybe bohemica as well as between Psilocybe azurescens and Pacific Northwest European collections of Psilocybe cyanescens.

In it's natural habitat, the general aspect of Psilocybe azurescens is most similar to Psilocybe cyanofibrillosa Stamets & Guzman but differs in several significant macroscopic features. Psilocybe azurescens has pleurocystidia whereas Psilocybe cyanofibrillosa has long necked, lageniform cheilocystidia, often forked, while Psilocybe azurescens has singly formed, fusoid ventricose cheilocystidia with short necks. Macroscopically Psilocybe azurescens is much larger in form and quickly bruises bluish to indigo-black upon handling. The bruising reaction in P. cyanofibrillosa is less intense and comparatively slow in appearing, which directly reflects it's low psilocybin content (Bocks, 1968; Stamets et al. 1980). Both species are characterized by non- undulating pileal margins.

Psilocybe azurescens also closely resembles a variety of Pacific Northwest Psilocybe widely reported as Psilocybe cyanescens Wakefield, a species originally discovered in the British Isles. This variety of Psilocybe cyanescens gained considerable notoriety in the mid-1970's (Weil, 1975, 1977; Pollock, 1975; Ott, 1975; Guzman & Ott, 1976; Guzman et al., 1976). Yet, this mushroom has probably been confused with other taxa. The mushroom portrayed in many popular field guides and identified as Psilocybe cyanescens (Arora, 1979 & 1991; Lincoff & Mitchel, 1977; Lincoff, 1981; Menser, 1977; Ott & Bigwood, 1978; Stamets, 1978) differs from the type in the relative number of surface cystidia.

In the Pacific Northwest, Psilocybe azurescens can be macroscopically distinguished from Psilocybe cyanescens by the following combination of features. Psilocybe azurescens has a cap margin characteristically even, not undulating and has a persitent, pronounced umbo at the disc when the pileus fully expands. The variety of P. cyanescens from the Pacific Northwest is characterized by distinctive, exaggerated undulating margin, resembling a sine-wave at maturity and is notably non-umbonate. In general, Psilocybe azurescens, as it is presently understood, is substantially larger than most collections of Psilocybe cyanescens. Microscopically, the pleurocystidia in Psilocybe azurescens are mucronate whereas the Pacific Northwest form Psilocybe cyanescens can become distinctly capitate at maturity. Otherwise, the microscopic features of Psilocybe azurescens are largely coincident within the range reported for the Pacific Northwest Psilocybe cyanescens.

Krieglsteiner (1984, 1986) extensively studies collections of Psilocybe from Europe, some of which were determined to be Psilocybe cyanescens. He proposed that Psilocybe mairei Singer, Psilocybe serbica Moser & Horak and Psilocybe bohemica could be conspecific with Psilocybe cyanescens Wakefield because these taxa could not be delineated microscopically. However, one significant feature which characterizes Psilocybe bohemica and separates this species from these aforementioned taxa and from Psilocybe azurescens is that the pilei of Psilocybe bohemica become white upon drying. Furthermore, one author (Jochen Gartz) has found complete reproductive barriers between 80 random pairings of monokaryons from Psilocybe azurescens, Psilocybe cyanescens and Psilocybe bohemica. Since monokaryons from single spore isolates from each of these species have proved to be incompatible, these taxa appear to be auto- nomous. Former research (Gartz, 1993) has also shown that complete repro- ductive barriers exist between Pacific Northwest strains of Psilocybe cyanescens and Czechoslovakian collections of Psilocybe bohemica. Mating studies paired single spore isolations and clamp connections failed to form, an indication of incompatibility. Furthermore, monokaryons from a collection of Psilocybe cyanescens (non-pleurocystidiate form) from Austria in October of 1992 also failed to form dikaryotic mycelia when paired with strains of Pacific Northwest Psilocybe cyanescens (pleurocystidiate form), Psilocybe azurescens and Psilocybe bohemica, respectively.

A closely related species is Psilocybe eucalypta Guzman & Watling. Psilocybe eucalypta has smaller and narrower cheilocystidia, only 15-25 x 4.4-6.6 um in comparison to Psilocybe azurescens cheilocystidia which measure 23-28 x 6.5-8.0 um. Furthermore, Psilocybe azurescens produces a more massive fruitbody, with a pileal diameter of 30 to 100 mm whereas P. eucalypta is smaller, falling within a range of 15-38 mm. P. eucalypta has thus far only been reported from the region centering around eastern Australia. Lastly, Psilocybe serbica Moser & Horak (1968), a temperate species, can be easily separated from Psilocybe azurescens by it's lack of pleurocystidia and it's non-umbonate form. Another related species, Psilocybe collybioides Singer & Smith, known at present from Argentina, shares many features common to Psilocybe azurescens save for it's exceptionally small spores, measuring only 5.5-10 x 3.5-6.5 um. These com- binations of features separate Psilocybe azurescens from the aforementioned taxa. Psilocybe azurescens is being named for the soft blue tones present on the mushroom, before handling or damage, especially along the cap margin and in the basal mycelium. Additionally the name also honors the son of one of the authors (Paul Stamets).

Chemical Analyses: In comparison to other species of Psilocybe, the fruitbodies of Psilocybe azurescens contain unusually high concentrations of psilocybin, psilocin and baeocystin - accumulating to more than 2% of the dry biomass of the mushrooms (Beug & Bigwood, 1982; Gartz, 1989, 1992-1994; Wurst et al., 1984). Thin Layer Chromatography (TLC) reveals a nearly identical profile of extracts from P. semilanceata and Psilocybe azurescens (psilocybin, baeocystin and six minor alkaloids), differing only in psilocin content (Gartz, 1985). Baeocystin is present in high concentrations in Psilocybe azurescens and P. semilanceata. Gartz (1993) has determined that baeocystin is also a hallucinogenic compound.

Research by Gartz (1989) showed alkaloid synthesis in Psilocybe cubensis (Earle) Singer is suppressed when the mycelium is grown using agar media supplemented with more than 10% mal sugar. Psilocybe azurescens reacts similarly. Research has also shown that alkaloid content is generally low in the mycelia compared to the fruitbodies. With Psilocybe cubensis, the main alkaloid synthesis occurs during the differentiation of the mycelia to the fruitbodies (Gartz & Muller, 1989). Further, younger fruitbodies frequently have higher alkaloid levels than more mature ones (Gartz, 1992/1993). As Table III shows, specimens grown outdoors in Germany did not vary significantly from those grown in the United States, after dehydratation, even when the supporting substrates were dissimilar.

Table I Indole alkaloid content of collection (LZ) of wild fruitbodies of Psilocybe azurescens from Tillamook, Oregon, USA, October, 1989.

Sample 1: 50mg dry weight, 1.71% Psilocybin, 0.34% Psilocin, 0.41% Baeocyst.
Sample 2: 101mg dry weight, 1.68% Psilocybin, 0.28% Psilocin, 0.38% Baeocyst.
Sample 3: 167mg dry weight, 1.56% Psilocybin, 0.30% Psilocin, 0.32% Baeocyst.
Sample 4: 213mg dry weight, 1.51% Psilocybin, 0.31% Psilocin, 0.28% Baeocyst.
Sample 5: 270mg dry weight, 1.40% Psilocybin, 0.28% Psilocin, 0.19% Baeocyst.
Sample 6: 317mg dry weight, 1.29% Psilocybin, 0.26% Psilocin, 0.27% Baeocyst.
Sample 7: 450mg dry weight, 1.20% Psilocybin, 0.25% Psilocin, 0.31% Baeocyst.

Table II Indole alkaloid content from naturalized, outdoor cultivated specimens of Psilocybe azurescens, from Astoria, Oregon, USA, October, 1990.

Sample 1: 062mg dry weight, 1.78% Psilocybin, 0.38% Psilocin, 0.35% Baeocyst.
Sample 2: 123mg dry weight, 1.75% Psilocybin, 0.39% Psilocin, 0.36% Baeocyst.
Sample 3: 170mg dry weight, 1.58% Psilocybin, 0.34% Psilocin, 0.37% Baeocyst.
Sample 4: 224mg dry weight, 1.43% Psilocybin, 0.28% Psilocin, 0.31% Baeocyst.
Sample 5: 331mg dry weight, 1.18% Psilocybin, 0.19% Psilocin, 0.25% Baeocyst.
Sample 6: 472mg dry weight, 1.20% Psilocybin, 0.20% Psilocin, 0.21% Baeocyst.

Table III Indole alkaloid content from dried, outdoor-cultivated, naturalized specimens of Psilocybe azurescens, from Germany (A) and USA (B).

Sample 1: A - 156mg dry weight, 1.62% Psilocybin, 0.42% Psilocin, 0.38% Baeocystin B - 165mg dry weight, 1.72% Psilocybin, 0.38% Psilocin, 0.39% Baeocystin
Sample 2: A - 213mg dry weight, 1.56% Psilocybin, 0,32% Psilocin, 0.28% Baeocystin B - 233mg dry weight, 1.62% Psilocybin, 0.25% Psilocin, 0.24% Baeocystin
Sample 3: A - 312mg dry weight, 1.43% Psilocybin, 0.26% Psilocin, 0.31% Baeocystin B - 341mg dry weight, 1.32% Psilocybin, 0.25% Psilocin, 0.35% Baeocystin
Sample 4: A - 412mg dry weight, 1.17% Psilocybin, 0.31% Psilocin, 0.28% Baeocystin B - 403mg dry weight, 1.21% Psilocybin, 0.38% Psilocin, 0.19% Baeocystin
Sample 5: A - 450mg dry weight, 1.19% Psilocybin, 0.36% Psilocin, 0.24% Baeocystin B - 465mg dry weight, 1.24% Psilocybin, 0.24% Psilocin, 0.30% Baeocystin

Table IV Variation of the amounts of alkaloids in the mycelium of Psilocybe azurescens depending on the concentration of malt extract in solidified agar (1,5%) after 3 weeks of colonization.

1% Malt Extract: 0.31% Psilocybin dry weight, 0.12% Psilocin, 0.12% Baeocyst.
2% Malt Extract: 0.25% Psilocybin dry weight, 0.09% Psilocin, 0.08% Baeocyst.
3% Malt Extract: 0.28% Psilocybin dry weight, 0.08% Psilocin, 0.05% Baeocyst.
4% Malt Extract: 0.27% Psilocybin dry weight, 0.04% Psilocin, 0.03% Baeocyst.
5% Malt Extract: 0.25% Psilocybin dry weight, 0.02% Psilocybin, 0% Baeocystin
6% Malt Extract: 0.18% Psilocybin dry weight, 0% Psilocin, 0% Baeocystin
8% Malt Extract: 0.05% Psilocybin dry weight, 0% Psilocin, 0% Baeocystin
10% Malt Extract: At and above 10% malt extract, the mycelium is non-blueing.

Acknowledgments: The authors would like to thank Scott Redhead and Roy Watling for reviewing the manuscript. The senior author is grateful to Michael Beug and The Evergreen State College for their continued support and to Azureus Stamets for his assistance in the field. The authors also express gratitude to G. K. Mueller from the University of Leipzig Herbarium (LZ) and Marta Semerdzieva of Prague. Paxton Hoag and Mark Herke are credited for first collecting this mushroom. John Allen and Eric Iseman helped in field collections.

Notes: (1) A mushroom reportedly responsible for the death of a child in Kelso, Washington in 1962 was identified by Alexander Smith as Psilocybe baeocystis Singer & Smith (see McCawley et al., 1962; Singer & Smith, 1958a) From the Kelso site, cultures were isolated and mushrooms were grown from mycelial spawn. The cultured specimens have an aspect atypical of Psilocybe baeocystis Singer & Smith and strikingly typical of the Pacific Northwest form of Psilocybe cyanescens. These two species can be easily delineated from one another macroscopically. Furthermore, these authors know of no successes in cultivating P. baeocystis, despite many attempts. In contrast, Psilocybe cyanescens can easily be cultivated on wood-chip substrates. When one of the authors (Paul Stamets) noted these discrepancies to Alexander Smith (1982), he responded that the identification was tentative, made from poorly preserved spore material retrieved from stomach washings and was not without uncertainity.

(2) Psilocybe cyanescens Wakefield was originally described from a collection at the Kew Gardens, Surrey, England. (See Dennis & Wakefield, 1946; Singer & Smith, 1958a; Guzman, 1983). The variety of Psilocybe cyanescens from the Pacific Northwest has abundant and conspicuous pleurocystidia, often with distinctive swollen apices, evenly dispersed over the surface plane of the lamellae. The type collection of Psilocybe cyanescens by Wakefield has so few surface cystidia that they could easily be overlooked (Wakefield, 1946; Singer & Smith, 1958b; Krieglsteiner, 1984). The discrepancies seen between these varieties may be significant at the species level. Further study of these taxa is warranted.

(3) Chang & Mills (1992) propose that P. eucalypta is actually P. subaeruginosa. Guzman et al. (1993) strongly disagree with this synonymy. Krieglsteiner (1984, 1986) believes that P. eucalypta is conspecific with Psilocybe cyanescens.