Species of Psilocybe (Hymenogastraceae) from Yunnan, Southwest China

Home , Agaricales, Hymenogastraceae, Psilocybe, Secotioid, Strophariaceae, Zhaoyang District

http://dx.doi.org/10.11646/phytotaxa.284.3.3

Species of Psilocybe (Hymenogastraceae) from Yunnan, southwest China

TAO MA1, XIAO-FEI LING1 & KEVIN D. HYDE1, 2* 1 Research Institute of Resource Insects, Chinese Academy of Forestry, the Key Laboratory of Cultivating and Utilization of Resource Insects of State Forestry Administration, Kunming, Yunnan 650224, China 2 Center of Excellence in Fungal Research, Mae Fah Luang University 333 Moo l, Tasud, Muang, Chiang Rai 57100, Thailand Corresponding author: [email protected]

Abstract

Species of Psilocybe collected in Yunnan Province in southwest China were identified using both morphological characteristics and phylogenetic analyses of ITS and nrLSU sequence data. Six phylogenetic species were recognized among the studied collections. In this paper, Psilocybe ruliensis is introduced as a new species, while Psilocybe keralensis is new to China, P. aff. fasciata is new to Yunnan and P. chuxiongensis is recollected in Yunnan Province. The remaining two phylogenetic species are not described or detailed due to the paucity of the material. The four species appear to be endemic to Asia.

Key words: basidiomycota, hallucinogenic, taxonomy

Introduction

The genus Psilocybe sensu lato (sensu Guzmán 1983, 1995) includes the genus Deconica. The name Psilocybe was recently conserved with P. semilanceata (Fr.) P. Kumm. (1871:71) as the conserved type (Redhead et al. 2007), and Psilocybe is now applied to the clade of psychotropic species: Psilocybe sensu stricto (Ramírez-Cruz et al. 2013). However, Psilocybe fuscofulva Peck (1887:7) [formerly known as P. atrobrunnea (Lasch) Gillet (1878:586)] probably does not produce psychotropic alkaloids (Borovička et al. 2015). Eleven species of Psilocybe have been reported from China (Ma et al. 2014, Li et al. 2014, Wang & Tzean 2015) and among these species, P. chuxiongensis T. Ma & K.D. Hyde (2014:213), P. cinnamomea J.F. Liang, Yang K. Li & Ye Yuan (2014:216) and P. angulospora Wang & Tzean (2015:165) were originally described from China. Psilocybe taiwanensis Zhu L. Yang & Guzmán (2010: 185) was described from Taiwan (Guzmán & Yang 2010), but Guzmán et al. (2013) considered it with P. argentipes K. Yokoy (1976: 349) and P. thaizapoteca Guzmán, Karun. & Ram.-Guill. (2012:77) to be a synonym of P. subcaerulipes Hongo (1958:44). Psilocybe chuxiongensis was described from Yunnan Province in southwest China. In this study five phylogenetic species besides P. chuxiongensis were recognized among the studied collections. One of them is described as Psilocybe ruliensis sp. nov. based on evidences from morphology and ITS and LSU sequence data analyses. Psilocybe keralensis K.A. Thomas, Manim. & Guzmán (2002:196) is new to China and P. aff. fasciata Hongo (1957:144) is new to Yunnan Province. The other two phylogenetic species are not described due to inadequate materials and are not detailed further.

Materials and Methods

Sampling and morphological study Macro-morphological features are described based on detailed field notes made from fresh material and documented by photographs. Materials were dried using an electric food drier and specimens were deposited in the herbarium of the International Fungal Research and Development Centre (IFRDC), Research Institute of Resource Insects, Chinese Academy of Forestry. Facesoffungi and Index Fungorum numbers are registered (Jayasiri et al. 2015, Index Fungorum 2016). Colour designations (e.g. 4A5) are from Kornerup and Wanscher (1981). Microscopic features are described

Accepted by Samantha Karunarathna: 25 Oct. 2016; published: 16 Nov. 2016 181 from dried material mounted in 5% KOH, Congo red, or water, measured and illustrated under a compound microscope (Nikon 80i). For terminology and methodology for descriptions see Ma et al. (2014).

DNA extraction, PCR, Sequencing and phylogenetic analyses DNA extraction, PCR, sequencing and sequence alignment were followed Ma et al. (2014) and referenced therein. Phylogenetic analyses were conducted based on two nuclear genes (nrLSU and ITS). Sequences newly generated were aligned with selected reference sequences from GenBank. Seven reference taxa from GenBank lacked nrLSU and three lacked ITS sequence data, but were also included in this dataset and omissions were treated as missing data. Most of the reference sequences used are from the studies of Borovička et al. (2011, 2015) and Ramírez-Cruz et al. (2013). Species from the genera Stropharia were used as outgroup taxa, based on former studies (Moncalvo et al. 2002, Matheny et al. 2006). The combined nuclear dataset (LSU and ITS) was analysed using RAxML 7.2.6 (Stamatakis 2006) and MrBayes v3.1.2 (Huelsenbeck & Ronquist 2005), respectively. ML trees were inferred by running 1000 replicates combined with a ML+Rapid bootstrap. Bayesian inference (BI) best-fit models were determined by Akaike Information Criterion (AIC) in Modeltest Version 2.3 (Posada & Crandall 1998), and GTR+I+G model was suggested as the substitution model for each of the two nuclear regions and the combined dataset. BI analyses using six chains were run for 1 000 000 generations and trees sampled every 100th generation. The first 25% (2500) of samples were discarded as burn-in. Node support was estimated as posterior probabilities (pp) from the remaining trees. Clades receiving ML Bootstrap values (BS)>70% and BI pp>0.95 were considered to be significantly supported. Tree visualization and drawing were carried out with TreeView version 1.5.2 (Page 1996).

Results

Molecular studies Twenty-four new sequences (12 for ITS and LSU) were generated from twelve specimens of the seven species (Table 1) and are deposited in GenBank. The combined LSU+ITS dataset consisted of 69 taxa representing 44 species and 1375 nucleotide sites were included in the final alignment matrix. For this dataset, RAxML and BI analysis yielded nearly identical tree topologies and only the tree inferred from BI analysis is shown for the datasets. Phylogenetic trees generated showed that the samples collected from Yunnan grouped into six lineages which including P. chuxiongensis. Two lineages corresponded to P. fasciata and P. keralensis. Psilocybe aff. fasciata from Yunnan and P. fasciata from Japan clustered together with 83% RAxML BS support, but with a relatively low BI pp value. Four collections of P. keralensis from three sites in Yunnan clustered together although with relatively low support. Psilocybe ruiliensis, a novel species described in the present paper, with three samples from the same site, formed an independent clade with strong support (RAxML BS=100%, PP=1.00), and had a sister relationship with P. samuiensis (RAxML BS=79%, PP=0.80). The remaining species had limited material and are tentatively named Psilocybe spp. 1 and 2. Psilocybe sp. 1 formed a monophyletic clade, but with little support, and is related with P. keralensis, P. antioquiensis, P. zapotecorum, P. thaizapoteca and P. argentipes. Psilocybe sp. 2 and P. stuntzii formed a group with 78% RAxML BS and 0.92 Bayesian PP support (Fig. 1).

TABLE 1. New sequences generated in this study GenBank accession Species name Voucher Coll. No. Locality ITS nrLSU Psilocybe aff. fasciata IFRD415224 MS007 Mangshi, Yunnan, ChinaKX357868 KX357869 P. keralensis IFRD414040 CX063 Chuxiong, Yunnan, China KX357870 KX357871 P. keralensis IFRD415173 ZY039 Zhaotong, Yunnan, China KX357872 KX357873 P. keralensis IFRD415174 ZY040 Zhaotong, Yunnan, China KX357874 KX357875 P. keralensis IFRD414213 ZD040 Shangri-La, Yunnan, China KX357876 KX357877 P. ruiliensis IFRD415240 RLC004 Ruili, Yunnan, China KX357878 KX357879 P. ruiliensis IFRD415241 holotype RLC005 Ruili, Yunnan, China KX357880 KX357881 P. ruiliensis IFRD415243 RLC007 Ruili, Yunnan, China KX357882 KX357883 Psilocybe sp.1 IFRD415205 SB143 Shuangbai county, Yunnan, China KX357884 KX357885 Psilocybe sp.2 IFRD414050 CX025 Chuxiong, Yunnan, China KX357886 KX357887 P. thaiduplicatocystidiata MFLU10-0850 NTS-120 Chiang Mai, Thailand KX357888 KX357889 P. thaiaerugineomaculans MFLU10-0851 NTS-121 Chiang Mai, Thailand KX357890 KX357891

182 • Phytotaxa 284 (3) © 2016 Magnolia Press MA ET AL. FIGURE 1. Phylogram inferred from analyses of a combined LSU and ITS dataset using MrBayes. Values above and below branches are posterior probabilities (pp≥0.80) and RAxML bootstrap (BS≥50%) values. For node with little difference between RAxML and BI analyses, only the pp value is displayed and BS value is not shown but indicated by ‘/’. The terminal taxa represent individual specimens with GenBank accession numbers. nrLSU and ITS numbers are indicated as LSU/ITS and the sequences that are lacking are not shown, and only one number is represented the sequence that is including nrLSU and ITS data. Species from Yunnan province and new sequences generated in this study are in bold.

Psilocybe ruiliensis T. Ma, X.F. Ling & K.D. Hyde, sp. nov. (Figs. 2, 3) Index Fungorum number: IF552166, Facesoffungi number: FoF 02246

Etymology:—the species epithet ‘ruiliensis’ refers to the location Rili where the type collections were found. Holotype:—CHINA, Yunnan Province: Dehong Dai-Jingpo Autonomous Prefecture, Ruili, Longchuan County, on grassland near a reservoir, E097°48´03.6”, N24°15´48.9”, 940m, 24 September 2011, Tao Ma, Xiao-Fei Ling RLC005 (IFRD 415241). Diagnosis:―the new species is characteristic by growing on grassland in the southern subtropical region, basidiomata are somewhat bluish when touched or bruised, having bright reddish orange-brown to yellowish brown and hemisphaeric or subhemisphaeric pileus, the pileus with or without umbo, ellipsoid to subhexagonal basidiospores of medium size 9–11 × (5.5–) 6–7.5 × 5.5–7 μm, slender to stout and fusiform to clavate cheilocystidia, and the form of pleurocystidia similar to the stout pleurocystidia. Original description:―Pileus 10–20 mm diameter, at first irregularly hemisphaeric, conic to subcampanulate, bright reddish orange-brown (5B8), then hemisphaeric, hemisphaeric-conic, hemispheric-convex to almost plane, with or without umbo or small acutely papillate at the disk, yellowish orange-brown (5B6) or dirty yellowish brown when moist, and often with reddish tinge, straw yellow or brownish when dry, frequently whitish at edge; surface smooth, hygrophanous and more or less translucently striate when moist; margin often decorated with somewhat fugacious white to bluing veil remnants, and with cortinate white veil and sometimes small scales in young stages; context yellowish (1A2), sometimes turning bluish when bruised in matured stages, watery brown when wet. Lamellae adnate to subsinuate or adnexed, 1.7–4.2 mm high, subdistant to close, yellowish or beige (4A2) when young, then becoming brownish buff, earth yellow, yellowish brown to chocolate brown (6E7) with gray-purple or purple tinge; edges serrulate, slightly wavy, and remaining whitish and sometimes slightly blackish. Stipe 27–62 mm × 1.5–3.5 mm, central, occasionally slightly eccentric, equal to slightly bulbous at the base, straight to flexuous, yellow-white to brownish, somewhat bluish when touched or bruised; surface covered with appressed whitish pruinose-fibrils; base of stipe with white mycelium; context hollow, fibrous or soft, whitish to brownish, bluish when bruised; annulus absent. Odour null or with somewhat fresh grassy smell. Microscopic characteristics:―Pileipellis an ixocutis, 15–60 μm thick, made up of creeping, hyaline and colourless, subregular to interwoven, 2–5 μm wide filamentous to slender tubular hyphae, sometimes including dark yellow and occasionally blue intracellular pigments, wall smooth or with finely incrusted pigments; subpileipellis yellowish brown to brown in KOH, composed of colourless to dark yellowish or brownish, tubular to inflated, 5–17 μm wide hyphae, and the wall often rough for incrusted pigments. Subhymenium subcellular, hyaline, composed of irregular vesiculose to polygonal or subglobose cells. Hymenophoral trama dark yellowish or brownish yellow, regular, with cylindrical hyphae 3–19 μm diameter, hyaline, colourless to yellowish, thin-walled to slightly thick-walled (≤1.0 μm), and the wall smooth to with somewhat dark incrusted pigments. Basidia (18.5–) 21–29.5 (–32) × (7–) 8.5–11 (–12) μm, hyaline and colourless, nearly cylindric to clavate, often narrowed in lower half and constricted in the middle, 4-spored, sometimes 2-spored; sterigmata (2–) 3–5.5 (–7) μm long. Basidiospores (345/10/5) (7.5–) 9–11 (–12.5) × (5.5–) 6–7.5 (–8) × 5.5–7 (–7.5) μm, ellipsoid, ovoid, subrhomboid to subhexagonal in face view, Q = (1.2–) 1.3–1.6 (–1.8), Q = 1.49±0.10; ellipsoid or subellipsoid in side view, Q = (1.4–) 1.5–1.9 (–2), Q = 1.64±0.12, sometimes containing 1–2 oil drops, brown with purple tinge in water, dark yellow in KOH, purplish brown in deposit; wall smooth, slightly thick (0.5–1 μm), complex, with distinct 1–1.5 μm wide apical germ pore. Cheilocystidia (14–) 16–26 (–30) × 3.5–8.5 (–9.5) μm, slender to stout, hyaline, fusiform, fusiform-lageniform to fusiform-clavate or clavate, sometimes sublageniform or broadly lageniform, and the stout similar to pleurocystidia, but bigger, often with a 1.5–9.5 × 1.5–2.5 μm rostrum or neck, occasionally forked, the top or apex frequently seems wall thickened or contain some matter. Pleurocystidia scattered, 13.5–22 (–24) × 5.5–9 μm, thin-walled, hyaline, fusiform to fusiform-clavate or clavate, sometimes sublageniform or ventricose, apex often protruded or extended into a 0.5–4.5 (–6) × 1.5–3.5 μm rostrum or sometimes neck, the top or apex frequently with a thickened wall or containing some matter. Caulocystidia relatively rare, scarce in young individuals, (16–) 22–70 (–90) × (4.5–) 5.5–15.5 (–24.5) μm, often clustered at the upper part of the stipe, thin-walled, hyaline, elongated lageniform, fusiform-lageniform, ventricose, clavate, obclavate or nearly cylindric, occasionally ping-pong racket shape, often with an elongated (2–) 5–26.5 (–44.5) × 1.5–3.5 μm neck, the top or apex frequently with a thickened wall or containing some matter. Clamp connections common in all parts of the basidioma.

184 • Phytotaxa 284 (3) © 2016 Magnolia Press MA ET AL. FIGURE 2. Basidiomata of Psilocybe species. A–G: Psilocybe keralensis, A, E: IFRD415173, B: IFRD415174, C–D: IFRD414040, F–G: IFRD414213; H, L: Psilocybe aff. fasciata, IFRD415224; I–K, M–N: Psilocybe ruiliensis, I–J: IFRD415243, K, N: holotype, IFRD415241, M: IFRD415240.

Distribution:—Rili, Yunnan Province, southwest China. Habitat:—Growing solitary to scattered and gregarious on grassland near a reservoir where cattle and horses have grazed in early autumn. Material examined:—CHINA, Yunnan Province: Dehong Dai-Jingpo Autonomous Prefecture, Ruili, Longchuan County, on grassland near a reservoir, E097°48´03.6”, N24°15´48.9”, 940m, 24 September 2011, Tao Ma, Xiao-Fei Ling RLC005 (IFRD 415241, holotype), RLC004 (IFRD 415240, paratype), RLC006 (IFRD 415242, paratype), RLC007 (IFRD 415243, paratype) and RLC008 (IFRD 415244, paratype). Note:—Psilocybe ruiliensis was only found growing on grassland in the southern subtropical region (close to tropical) of western Yunnan at a relatively low altitude. It can be distinguished by its bright reddish orange-brown to yellowish brown and hemisphaeric or subhemisphaeric pileus, ellipsoid to subhexagonal basidiospores with medium size in face view, slender to stout cheilocystidia, and the presence of pleurocystidia. Phylogenetically, P. ruiliensis forms a monophyletic clade with high BS support (100%) and pp value (1.00) and groups with P. mexicana, P. tampanensis and P. samuiensis (sect. Mexicana) and P. caerulescens (sect. Cordispora) sensu Guzmán (1995). In this group, P. ruiliensis and P. samuiensis Guzmán, Bandala & J.W.Allen cluster together. These taxa are similar in some features of their basidiomata, habitat and basidiospores, but P. samuiensis differs in having a smaller and convex to conic-convex to campanulate pileus and relatively slender stem. Microscopically, the slightly larger hexagonal and subrhomboid basidiospores (9.6–12.0 × 6.4–8.4 μm), the ventricose-lageniform cheilocystidia and pleurocystidia, and the relatively slender pleurocystidia and basidia can easily separate P. samuiensis from P. ruiliensis (Guzmán et al. 1993, Ramírez-Cruz et al. 2013).

PSILOCYBE (HYMENOGASTRACEAE) Phytotaxa 284 (3) © 2016 Magnolia Press • 185 FIGURE 3. Psilocybe ruiliensis A–C, E–G:(holotype, IFRD415241), D: (IFRD415240), A: Pileipellis, B: Caulocystidia, C–D: Cheilocystidia, E: Pleurocystidia, F: Basidia, G: Basidiospores. Scal bar=10 μm.

Morphologically, P. ruiliensis is related to stirps Mexicana according to Singer (1986) because of the medium sized spores, mycenoid habit and growing in open places on soil. It is also related to Sect. Mexicana sensu Guzmán because of the size of spores, the mycenoid habit and the subtropical habitat, although the shape of its basidiospores is partly subrhomboid. In sect. Mexicana sensu Guzmán, besides P. samuiensis mentioned above, P. ruiliensis bears

186 • Phytotaxa 284 (3) © 2016 Magnolia Press MA ET AL. some superficial resemblance to P. mexicana. Both species grow on subtropical grasslands, but the different shape of fruit body, the absence of pleurocystidia, and the subrhomboid spores of P. mexicana easily distinguish them (Singer and Smith 1958, Guzmán 1983). In many aspects, P. ruiliensis is also similar to P. gallaeciae Guzmán & M.L. Castro and P. galindii Guzmán, but the latter two species have pseudorhiza. In addition, P. gallaeciae has a different coloured pileus, slightly broader and thicker-walled spores, and lageniform pleurocystidia and cheilocystidia. Psilocybe galindii has relatively slender basidiomata, smaller basidia, and slightly larger and subrhomboid thicker-walled basidiospores, as well as grows at 1700–1800 m elevation (Guzmán 1983, Guzmán & Castro 2003, Ramírez-Cruz et al. 2013).

Psilocybe keralensis K.A. Thomas, Manim. & Guzmán, Mycotaxon 83: 196, 2002 (Figs. 2, 4)

Pileus 15–20 mm diameter, hemisphearic, subconic or campanulate, not umbonate or papillate, hygrophanous, brown (6C4 or 4B8) to orange brown (5B6-5C8), fading to pale earth yellow (5A4) to straw yellow, surface glabrous, lucidus when dry and often somewhat bluish when touched or on drying, especially at the edge, not viscid and the margin finely translucent striate when moist; context cream white or yellowish white (2A3), bruising ink blue. Lamellae adnate to slightly sinuate, close, beige (5A4) to orange-yellow (5A6) to greyish purple (9C2) or purple-brown (6C2 to 9E5), often with ink blue tinge, edges serrulate and remaining whitish. Stipe 45–75 × 2–4 mm, equal, sometimes flattened and becoming tapered toward the base, nearly concolorous with the pileus, darker below, often with ink blue to blackish tinge when touched or when dry, and shiny when dry; surface longitudinally striate and covered with appressed whitish fibrils or flocculose, sometimes uneven and with scrobicula and grooves; base with white mycelium and often radicating; annulus absent; context fragile, yellowish and yellowish brown towards the surface and base, staining somewhat ink blue when bruised. Odour null or with fresh grassy or slightly mushroomy smell. Pileipellis an ixocutis, 15–80 μm thick, made up of creeping, interwoven, 2–6 μm wide filiform to slender tubular hyphae, hyaline and colourless, wall-smoothed and thin; subpileipellis more pigmented to dark yellow in KOH. Subhymenium subcellular, hyaline, composed of irregular vesiculose to polygonal or subglobose cells. Hymenium contained rich granular and extracellular dark blue pigment. Hymenophoral trama regular, with filamentous to cylindrical smooth hyphae 2–18 μm diameter, hyaline, colourless to dark yellowish, thin-walled to slightly thick-walled (≤1 μm). Basidia 18.5–28.5 × 5.5–8 μm, hyaline and colourless, long subcylindric to clavate, often constricted in middle and narrowed in lower half, 4-spored and 2-spored, rarely 1-spored; sterigmata (1.5–) 3–5 (–6) μm. Basidiospores (136/6/4) 6–9 (–10, –12) ×5–6.5(–7.5) × 4–6 μm, often subrhomboid or ovoid, sometimes ellipsoid, occasionally inconspicuous subhexagonal in face view, Q = 1.1–1.6 (–1.7, –1.9), Q = 1.35±0.13, ellipsoid or subovoid, sometimes nearly oblong in side view, Q = (1.2–) 1.3–1.8, Q = 1.55±0.15; yellowish brown with purple tinge in water, dark yellow to yellowish brown in KOH, dark purplish brown in deposit; wall smooth, slightly thick (0.5–1 μm), complex, with 0.8–1.5 μm wide apical germ pore. Cheilocystidia abundant, 14–27 (–34.5) × 4–8 μm, hyaline and colourless, ventricose to sublageniform, sometime fusoid or clavate, occasionally nearly cylindric-clavate or obclavate, irregularly branched or not branched, mostly with a 1.5–9.5 (–13.5) × 1–3 μm rostrum or neck, sometimes with an acuminate apex, the top or apex often seems wall thickened or contain some matter. Pleurocystidia relatively rare and scattered, 13–22 × 4–6.5 μm, hyaline, similar to cheilocystidia, but smaller, often with a 1–7.5×1–2 μm neck or rostrum, not branched, the top or apex often seems wall thickened or contain some matter. Caulocystidia abundant, (14.5–) 21–45.5 (–49) × 3.5–8 (–11) μm, scattered, gregarious to clustered at the upper part of the stipe, hyaline, similar to cheilocystidia, but more variable in size, and the neck occasionally with a septum, the top or apex often with a thickened wall or containing some matter. Clamp connections common in all parts of the basidioma. Distribution:—India (Thomas et al. 2002) and Southwestern China (Chuxiong, Shangri-La and Zhaotong, Yunnan Province) (new record to China). Habitat:—Growing solitary to scattered on dung or soil of meadows, or grassland in open area of the forest where cattle have grazed in summer, at 2400–3400m altitude. Material examined:—CHINA, Yunnan Province: Diqing Tibetan Autonomous Prefecture, Shangri-La County, Namucuo Ecological Park, on dung in grassland, E99°50′32.9, N27°47′30.2ʺ, 3379m, 8 August 2009, Tao Ma, Hui Yang ZD040 (IFRD414213); Chuxiong, Zixi Mountain, on soil of grassland in forest, E101°24′06.7ʺ, N25°01′03.7ʺ, 2465m, 4 August 2010, Tao Ma, Xiao-Fei Ling CX063 (IFRD414040); Zhaotong, Zhaoyang district, Dashanbao, on soil of grassland, E103°22′33.4ʺ, N27°27′01.1ʺ, 2909m, 3 August 2011, Tao Ma, Xiao-Fei Ling ZY039 (IFRD415173), ZY040 (IFRD415174). Note:—Psilocybe keralensis was found on grassland or dung in subtropical to high temperate mountains at an altitude of 2400–3400 m in Yunnan Province. It is characterized by bluing of the basidiomata and hymenium, the subrhomboid or ovoid basidiospores in face view, with medium size 6–9 (–10) × 5–6.5 (–7.5) × 4–6 μm, and often has pseudorhiza at the stem base.

PSILOCYBE (HYMENOGASTRACEAE) Phytotaxa 284 (3) © 2016 Magnolia Press • 187 FIGURE 4. Psilocybe keralensis (IFRD415173), A: Basidia, B: Basidiospores, C: Cheilocystidia, D: Pleurocystidia, E: Caulocystidia. Scal bar=10 μm.

Psilocybe keralensis belongs to the Psilocybe fagicola-complex in Sect. Cordispora Guzmán because of its subrhomboid on face-view and relatively small to medium spores, the characteristic bluing reaction, and the presence of pseudorhiza (Guzmán et al. 2005). It is closely related to P. columbiana Guzmán of this complex, because of the similar basidiomata and basidiospores. The latter species also grows in meadows in high mountains (altitude 3300–3500m). Psilocybe columbiana differs significantly in its absence of pleurocystidia, as well as having relatively slender cheilocystidia (22–30 × 3.3–6.5 μm) and slightly stout spores [(6.6–) 7.1–8 (–8.8) × (4.9–) 6–6.6 (–7.1) × 4.4–5.5 μm] (Guzmán 1978, 1983). Psilocybe keralensis is only known from Kerala, India (Thomas et al. 2002). The Yunnan material shows some variation compared with the India material, such as the pileus without umbo or papilla, and somewhat diversity in colour of the pileus, gills and stems of basidiomata. Phylogenetically, P. keralensis does not group with species of Sect. Cordispora Guzmán, but nested within

188 • Phytotaxa 284 (3) © 2016 Magnolia Press MA ET AL. the “Zapotecorum”clade (Ramírez-Cruz et al. 2013), together with P. antioquiensis, P. argentipes, P. thaizapoteca, P. zapotecoantillarum, P. zapotecorum and Psilocybe sp. 1. In this clade, most members have pseudorhiza and pleurocystidia. Pseudorhiza were not mentioned in the original description of P. zapotecoantillarum Guzmán, T.J. Baroni & Lodge and were not recorded in Psilocybe sp. 1 in the field (Guzmán et al. 2003). In Guzmán’s sense, P. antioquiensis Guzmán, Saldarr., Pineda, G. García & L.-F. Velázquez belongs to sect. Mexicana as it has relatively larger subrhomboid thick-walled basidiospores compared with that of sect. Cordispora, while the latter four species including Psilocybe sp. 1 belong to sect. Zaptecorum Guzmán due to their subellipsoid, thin-walled basidiospores. Morphologically, P. antioquensis is quite similar to P. keralensis, but P. antioquiensis differs significantly in having relatively slender basidiomata and larger basidiospores [(6–) 8–10 (–11) × (5–)6–6.5 (–7) × 5–6 μm] (Guzmán 1994, Guzmán et al. 2006). Guzmán et al. (2013) considered P. argentipes, P. thaizapoteca as synonyms of P. subcaerulipes, which are closely related to P. keralensis in their similar basidiomata, as well as most microscopic features. The relatively larger basidiomata, smaller 4-spored basidia (16–25 × 5–6 μm), smaller subellipsoid spores [(5–) 6–7 (–8) × (3–) 4–4.5 × (3–) 3.5–4 μm] and gregarious or caespitose habitat can differentiate P. subcaerulipes from P. keralensis. Psilocybe keralensis is obviously different with P. zapotecorum R. Heim and P. zapotecoantillarum in both macro- and microscopic features (Guzmán 1983, Guzmán et al. 2003).

Psilocybe aff. fasciata Hongo, J. Jap. Bot. 32: 144, 1957 (Figs. 2, 5)

Pileus 1.5–2 mm diameter, about 10 mm high, conic to hemispheric-campanulate, with a small acute papilla or umbo, hygrophanous, dark yellowish brown to ochre-brown/chestnut brown (5C6-5D8) and whitish at margin, with somewhat olive tinge when young, mixed with blackish blue when touching and drying, fading to pale brown, sometimes with reddish brown tone; surface smooth, translucently striate when moist; margin decorated with somewhat fugacious, whitish to blackish blue, minute fibrillose veil remnants. Lamellae adnate to sinuate, subclose to subdistant, earth yellow (5A5) to greyish purple (11E3) with irregularly dark and pale mottles, blackish blue tinge during drying or when handled; edges serrulate and remaining whitish. Stipe 57–65 mm × 2–2.5 mm, equal, subbulbous at base, slightly flexuous, yellowish to yellowish brown or brown, somewhat with blackish blue tinge when touched or on drying; surface longitudinally striate and covered with appressed whitish pruinose-fibrils, shiny when dry; annulus absent; context hollow and fragile. Odour with somewhat fresh grassy smell. Pileipellis an ixocutis, 15–90 μm thick, made up of creeping, interwoven, 2–6 μm wide filamentous to slender tubular hyphae, hyaline and colourless, sometimes with loops; subpileipellis yellowish brown to brown in KOH, composed of tubular to inflated, 4–22 μm wide hyphae. Subhymenium subcellular, hyaline, composed of irregular vesiculose to polygonal or subglobose cells. Hymenophoral trama regular, with cylindrical hyphae 3–10 μm diameter, hyaline, colourless to dark yellowish, sometimes with dark blue pigmentation in mature specimens. Basidia 20–24 × 6.5–9 μm, hyaline and colourless, nearly cylindric to clavate, 4-spored; sterigmata 2–4 μm long. Basidiospores (101/3/1) 9–11 (–14) × 5–6.5 (–7.5) × 5–6.5 (–7) μm, ellipsoid to elongate-ellipsoid, hexagonal-ellipsoid, sometimes subrhomboid/subovoid in face view, Q = 1.6–1.9 (–2.1), Q = 1.78±0.12; ellipsoid to elongate-ellipsoid in side view, Q = (1.5–) 1.6–1.9 (–2), Q = 1.77±0.10, yellowish brown with a purple tinge in water, dark yellowish to yellowish brown in KOH, dark purplish brown in deposit; wall-smoothed, slightly thick (0.5–1 μm), with 0.8–1.5 μm wide apical germ pore. Cheilocystidia 19–31.5 × 4.5–8 μm, hyaline, sometimes with dark blue pigmentation, lageniform to fusiform, with a 1–2 μm wide neck, sometimes forked, occasionally with a septa, apex acute or obtuse and often with a thickened wall or containing some matter. Pleurocystidia absent. Caulocystidia 21–30 (–38) × 6.5–9 (–10) μm, solitary or clustered at the upper part of the stipe, similar to cheilocystidia, but relatively broader, with 1–2 μm wide neck, apex often with a thickened wall or containing some matter. Clamp connections common in all parts of the basidioma. Distribution:—Known only from Japan (Hongo, 1957) and China (Guangxi, Hongkong, and Yunnan (Mao 1998, 2000, Bau & Sarentoya 2009). This is a new record for Yunnan. Habitat:—Growing scattered to gregarious on soil in grasslands near dung (at the edge of forest). Material examined:—CHINA, Yunnan Province: Dehong Dai-Jingpo Autonomous Prefecture, Mangshi, Heihelaopo Natural Scenic District, E98°35´58.1”, N24°13´34.4”, 2638m, 22 September 2011, Tao Ma, Xiao-Fei Ling MS007 (IFRD415224).

PSILOCYBE (HYMENOGASTRACEAE) Phytotaxa 284 (3) © 2016 Magnolia Press • 189 FIGURE 5. Psilocybe aff. fasciata (IFRD415224), A: Pileipellis, B: Cheilocystidia, C: Basidia, D: Basidiospores, E: Caulocystidia. Scale bar = 10 μm.

Note:—Psilocybe aff. fasciata from Yunnan is characterized by its yellowish brown to ochre-brown and olive brown pileus, the ellipsoid to subovoid basidiospores of medium size (9–11 × 5–6.5 μm in face view), the lageniform

190 • Phytotaxa 284 (3) © 2016 Magnolia Press MA ET AL. to fusiform cheilocystidia with a slender neck, and lack of pleurocystidia. Psilocybe fasciata was described from Japan by Hongo (1957) who described it with a ‘olivaceo-brunneo (“oliue-brown” vel “clove brown”)’pileus, 9.5–11 × 5–6 μm, ellipsoid to slightly ovoid spores, ventricose cheilocystidia and with a slender neck. The specimen from Yunnan mostly conforms with the characters of P. fasciata, and the sequence clustered together in the phylogenetic tree with significant BS support, but low pp value. The loop structure of hyphae in the pileipellis, the blue pigmentation of cheilocystidia and hymenophoral trama, and the small acute papilla of the pileus in the Yunnan specimen, was not recorded in the original description of P. fasciata. Phylogenetically, P. fasciata is closely related with P. stuntzii, P. semilanceata. and P. hispanica. In appearance, it is somewhat similar to P. stuntzii Guzmán & J. Ott in the colour and form of the basidiocarp, as well as some microscopic features, but the annulus, the form of the spores and the habitat separate these two species (Guzmán 1983, Noordeloos 2011). Psilocybe fasciata is easily differentiated from P. semilanceata and P. hispanica Guzmán by its macro- and microscopic features (Guzmán 1983, 2000, Noordeloos 2011). Hongo (1957) related P. fasciata to Psilocybe caerulipes (Peck) Sacc., but the latter has smaller basidiospores (7–) 8.2–9.9 (–11) × 3.8–5.5 (–6) × 3.8–5 μm, and grows on rotten wood or debris in hardwood forests (Guzmán 1983, Singer and Smith 1958). Psilocybe fasciata is somewhat similar to P. fimetaria (P.D. Orton) Watling and P. subfimetaria Guzmán & A.H. Sm. because of the form of the basidiocarp, but it can easily be distinguished by its smaller basidia and basidiospores, and absent of annulus, as well as the growing on dung habitat of the latter two species (Guzmán 1983, Noordeloos 2011). Psilocybe fasciata was considered a synonym of Psilocybe venenata (S. Imai) Imazeki & Hongo by Guzmán (1983), but the latter has an annulus, so we treat them as two species (Hongo 1957, Mao 2000, Bau & Sarentoya 2009).

Psilocybe chuxiongensis T. Ma & K.D. Hyde, Phytotaxa 156(4): 213, 2014

This species is found in subtropical habitats of central Yunnan Province, and characterized by hemisphaeric to hemisphaeric-convex pilei, the absence of annulus, large ellipsoid, elongate-ellipsoid to subhexagonal basidiospores (13–16 × 8–10.5 × 7.5–10 μm) and a coprophilous or subcoprophilous habitat. It is closely related to the wide-spread subtropical species P. cubensis (Earle) Singer and the pantropical species P. subcubensis Guzmán because of the similar basidiomata and microscopic characteristics (basidiospores, pleurocystidia and cheilocystidia), as well as having a similar habitat (Guzmán 1983, 1995).

Discussion

The distribution centre of the genus Psilocybe is Latin America (Guzmán 2005, 2009). In four years of collecting, in 30 cities and counties almost throughout Yunnan Province at 150–4300 m altitude, we only collected 14 specimens from six sites which belonged to six phylogenetic species. Four described species showed high specificy and diversity in geographical distribution. Psilocybe keralensis only grows in subtropical to temperate high mountains at an altitude of 2400–3400m, P. chuxiongensis and P. aff. fasciata were found in subtropic mountains at 2200–2600m, and P. ruiliensis was only found growing on southern subtropical grassland at 900m. They are all endemic in Asia.

Acknowledgments

Financial support was provided by the Grant for Essential Scientific Research of Chinese National Nonprofit Institute (no. riricaf2011003z and Riri200707M). The authors thank Xiaoming Chen, Ying Feng and Hang Chen for their valuable help. Special thanks go to Prof. Zhu-Liang Yang for his guidance in taxonomic study. Many thanks to the late Prof. Gastón Guzmán for providing his monograph and some important papers. We would like to thank Wen-Feng Zhang for her assistant of the tracing of line drawings, and Hui Yang and Yan-Ming Zhang for their assistant in field explorations. We are also grateful to Samantha C. Karunarathna for providing the specimens of two species included in molecular study.

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