Crepidotus yuanchui sp. nov. and C. caspari found in subalpine areas of China

Abstract

Only a few Crepidotus species have been previously reported from subalpine areas of China. Members of Crepidotus possessing an orange-yellow pileus are rare in China as well. Here, we describe Crepidotus yuanchui, a new species characterized by an orange-yellow pileus, ovoid basidiospores, and abundant cylindric to narrowly utriform cheilocystidia. This species is widely distributed above 2000 m asl in Yunnan Province. In addition, C. caspari, which is a distinct species based on its combined characteristics of smooth basidiospores and clamp connections, is newly recorded and detailed from subalpine China. The results of phylogenetic analyses of ITS + nLSU sequences based on maximum likelihood and Bayesian inference methods support the recognition of these two species. Photographs, descriptions, line drawings, and comparisons of related species are provided.

1. Introduction

Crepidotus (Fr.) Staude, a typically pleurotoid genus with dark-colored basidiospores and a rudimentary stem, includes more than 200 documented species (Senn-Irlet, 1995; Consiglio & Setti, 2008; Kirk, Cannon, Minter, & Stalpers, 2008). Originally described as Agaricus L. tribe Crepidotus Fr., Crepidotus was later elevated to generic rank by Staude (Fries, 1821; Staude, 1857; Donk, 1949; Consiglio & Setti, 2008). Tribe Crepidotus initially contained eight species, most of which have subsequently been combined into Entoloma P. Kumm. and Tapinella E.-J. Gilbert, with only three species remaining in the genus Crepidotus (Consiglio & Setti, 2008). Numerous Crepidotus species have been discovered in Europe and America over the past 200 y, and much progress has been made in regard to the species diversity and taxonomy of the genus (Hesler & Smith, 1965; Senn-Irlet, 1995; Senn-Irlet & de Meijer, 1998; Aime, Vilgalys, & Miller, 2005; Ripkova, Aime, & Lizon, 2005; Bandala, Esteve-Raventós, & Montoya, 2008; Bandala, Montoya, & Mata, 2008; Capelari, 2011; Kasuya & Kobayashi, 2011; Kasuya, Uno, & Hosaka, 2014; Kumar, Aime, Vrinda, & Pradeep, 2020). Several new species have recently been identified in Asia and South America. Many of these species are stipitate, which is atypical of the pleurotoid morphology thought to be characteristic of this genus (Guzmán-Dávalos, Pradeep, Vrinda, Kumar, & Aime, 2017; Capelari, 2011; Kumar et al., 2020).

Two basidiospore types are generally observed in Crepidotus: ornamented and smooth. According to the classification of Consiglio and Setti (2008), ornamented globose, amygdaliform to lachrymiform basidiospores is primarily associated with presence of clamp connections on hyphae; one cited example is subgenus Dochmiopus (Pat.) Pilát, although some species of this subgenus, such as C. autochthonus J.E. Lange, have smooth to almost smooth basidiospores. In contrast, smooth basidiospores are mostly related to the clampless subgenus Crepidotus (Fr.) Staude, which also includes some species with ornamented basidiospores, such as C. cinnabarinus Peck and C. versutus (Peck) Sacc. (Senn-Irlet, 1995; Consiglio & Setti, 2008). In Crepidotus, the presence of smooth spores along with clamp connections is an unusual combination of characteristics. During our on-going work on the taxonomy of Crepidotaceae Singer and Inocybaceae Jülich, we found a species possessing smooth basidiospores and clampless hyphae in subalpine localities of Yunnan Province, China; this was the second smooth-spored Crepidotus species with clamp connections collected in China (Ge & Bau, 2020).

Although we conducted extensive field work on Crepidotaceae and Inocybaceae in Yunnan Province, most specimens were collected from no more than 2000 m asl and were initially identified as subalpine Crepidotaceae or Inocybaceae taxa (Yu, Chang, Qin, Zeng, & Fan, 2020; Ge & Bau, 2020). During subsequent field work at subalpine elevations, however, two unique species were identified in Yunnan Province: C. yuanchui, a new species found in various areas from 2200 to 2400 m asl, and C. caspari Velen. located at more than 3400 m asl in Pudacuo National Forest Park. On the basis of previous documentation, our records thus correspond to the highest known elevations of collected Crepidotus species (Senn-Irlet & de Meijer, 1998; Aime, Baroni, & Miller, 2002).

2. Materials and methods

2.1. Morphology

All specimens were photographed using an Olympus E-M1 Mark III camera (Olympus, Tokyo) equipped with a M. Zuiko Digital Ed 12-40 mm or 60 mm lens (Olympus, Tokyo). The white balance was calibrated before image acquisition. The longitude, latitude, and elevation of collection localities were measured with a portable GPS, and the surrounding habitat and any identifiable tree species were recorded. Odor and taste were determined from fresh basidiocarps, and the materials were then packaged in aluminum paper. After recording characters, fresh specimens were dried in a drying apparatus (Stöckli, Netstal) at 40 °C.

Basidiocarp characters were determined from fresh materials, with a magnifying lens used when necessary. Color terms and notation followed Kornerup and Wanscher (1978). Specimen pieces were mounted in 5% KOH (w/v) and observed under a Lab A1 microscope (Carl Zeiss AG, Jena). Congo Red was applied if necessary. Microscopic observations were aided by the use of ZEN 2.3 (blue edition) software (Carl Zeiss Microscopy GmbH, 2016), with calibrations conducted before measurements. Basidiospore data were processed according to Aime et al. (2002), with at least 20 spores selected from each basidiocarp, and with the total number of basidiospores recorded. In our descriptions, extreme values (top and bottom 5% of values) are indicated in brackets; the average length and width is shown as L^m and W^m, respectively, and Q^m represents the average Q ± standard deviation. For scanning electron microscopy (SEM), individual lamellae were mounted on a stub, coated with gold, and examined under a Hitachi SU8010 scanning electron microscope (Hitachi, Tokyo) at an accelerating voltage of 2 kV.

2.2. Molecular phylogeny

2.2.1. DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted using a Plant Genomic DNA kit (CoWin Biosciences, Beijing). The nuclear large subunit (nLSU) region of ribosomal DNA was amplified using primers LR0R/LR7 following methods detailed in Aime et al. (2005) and Guzmán-Dávalos et al. (2017). The internal transcribed spacer (ITS) region was amplified with the primer pair ITS1 and ITS4 (Kasuya et al., 2014). The following touchdown program was used to amplify the DNA of some specimens: 95 °C for 5 min, followed by 15 cycles of 95 °C for 1 min (decreasing by 1 °C per cycle), 65 °C for 30 s, and 72 °C for 1 min and then 20 cycles of 95 °C for 1 min, 50 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min (Don, Cox, Wainwright, Baker, & Mattick, 1991). All PCR products were sequenced by the Beijing Genomics Institute. For specimens yielding sequences of low quality, high-quality sequencing data were obtained by applying plasmid vectors using VELUTE Gel Mini Purification and pBLUE-T kits (Beijing Zoman Biotechnology Co., Beijing).

2.2.2 Sequence alignment and phylogenetic analysis

Sequencing results were checked with Chromas 2.6.5 (https://chocolatey.org/packages/chromas/2.6.5). Eight sequences were generated from the collected specimens and submitted to the GenBank database, and additional sequences were downloaded from GenBank. A total of 103 sequences from 57 taxa were used for the phylogenetic analysis. Species of Neopaxillus Singer, the genus most closely related to Crepidotus (Watling & Aime, 2013), were used as outgroups. A combined dataset of ITS + nLSU sequences was constructed and aligned using the MAFFT v7 online tool with automatic settings (FFT-NS-1, FFT-NS-2, FFT-NS-I, or L-INS-i) (Katoh, Rozewicki, & Yamada, 2019), followed by manual adjustment in BioEdit 7.2.5 (Hall, 1999). The final aligned dataset used for the phylogenetic analysis comprised 1638 nucleotide sites, including gaps (Table 1).

Table 1. Sequences used in phylogenetic analysis.

Taxa	Voucher	ITS accession no.	LSU accession no.	Locality
Crepidotus aff. subverrucisporus	G0716	―	MK277891	Hungary
C. affinis	PDD:72848	KY827291	―	New Zealand
C. alabamensis	MCA778	―	AF367960	the United States
C. albolanatus	PDD:72865	KY827292	―	New Zealand
C. amygdalosporus	OKM26740	―	AF205678	the United States
C. antillarum	OKM26827	―	AF205680	the United States
C. applanatus	TM03_415	―	EU522804	Canada
C. applanatus	SLO1492	MF621029	MF621023	the United States
C. applanatus var. applanatus	MCA170	―	AF205694	the United States
C. asiaticus	TJB9995	MF077337	MF077336	India
C. asiaticus	TJB10018	MF077339	MF077338	India
C. aureus	OKM27300	―	AF205685	the United States
C. betulae	MCA384	―	AF205679	the United States
C. brunnescens	MCA864	―	AF367936	the United States
C. calolepis	ECV4050	KY777396	MF797668	the United States
C. caspari	FFAAS0343	MZ401360	MW581520	China
C. caspari	FFAAS0342	MZ401361	MW581521	China
C. cesatii	OKM26976	―	AF205681	the United States
C. cesatii	HBAU15522	MZ145080	―	China
C. cesatii	HBAU15551	MZ145081	―	China
C. cf. albissimus	MCA697	―	AF367959	the United States
C. cf. subaffinis	MCA604	―	AF205703	the United States
C. cinnabarinus	MCA387	―	AF205686	the United States
C. croceitinctus	MCA580	―	AF367932	the United States
C. croceotinctus	TBGT17271	―	MK878547	India
C. crocophyllus	OKM26173	―	AY029707	the United States
C. crocophyllus	RGT930924/02	―	AF139946	the United States
C. epibryus	G0515	―	MK277884	Hungary
C. eucalyptorum	G1749	―	MK277885	Hungary
C. exilis	TBGT17157	―	MK878548	India
C. fragilis	MCA904	―	AF367931	the United States
C. fraxinicola	OKM26739.5	―	AF205699	the United States
C. fraxinicola	OKM26748.2	―	AF205701	the United States
C. globisporus	TBGT17341	―	MK878540	India
C. indicus	TBGT17161	―	MG735357	India
C. inhonestus	420526MF0778	―	MG696605	China
C. kauffmanii	G1956	―	MK277887	Hungary
C. lanuginosus	OKM27331	―	AF367940	the United States
C. lateralipes	PDD:72572	KY827297	―	New Zealand
C. lateralipes	PDD:98274	KY827298	―	New Zealand
C. lundellii	G0516	―	MK277888	Hungary
C. lundellii	SJ6	―	AF367942	the United States
C. luteolus	16834	JF907963	―	Italy
C. lutescens	HMJAU 37002	NR_158400	―	China
C. macedonicus	G0394	―	MK277889	Hungary
C. macedonicus	PV773	MH780921	―	Hungary
C. macedonicus	DB3859	MH780922	―	Hungary
C. malachioides	SLO1250	NR_132047	KF154018	Czech Republic
C. malachius	SLO1497	MF621032	MF621026	the United States
C. malachius	SLO479	MF621033	MF621027	the United States
C. martinii	MCA640	―	AF367944	the United States
C. mollis	OKM26279	―	AF205677	the United States
C. mollis	PBM1036	―	DQ986293	the United States
C. novae-zealandiae	PDD:95850	HQ533046	―	New Zealand
C. nyssicola	TJB8699	―	AF205690	the United States
C. nyssicola	S.D. Russell MycoMap # 7399	MN906236	―	India
C. nyssicola	S.D. Russell MycoMap # 7426	MN906237	―	India
C. praecipuus	PDD:72624	KY827312	―	New Zealand
C. roseus	TBGT15507	MK567976	MK567977	India
C. rufofloccosus	PDD:72601	KY827296	―	New Zealand
C. sinuosus	OKM26290	―	AF367945	the United States
C. sphaerosporus	OKM27013	―	AF205682	the United States
C. stenocystis	PRM911279	MF621030	MF621024	the United States
C. subverrucisporus	15720	JF907961	―	Italy
C. tigrensis	B2200	―	MK277892	Hungary
C. tobolensis	TCSS UB RAS9477	MK522392	―	Russia
C. tobolensis	LE287655	MK522393	NG068881	Hungary
C. trulliformis	PDD:72572	KY827297	―	New Zealand
C. trulliformis	PDD:98274	KY827298	―	New Zealand
C. uber	MCA1403	―	AF367961	the United States
C. variabilis	MCA633	―	AF367949	the United States
C. variabilis	SLO2019	MT055892	―	Slovakia
C. variabilis	SLO2023	MT055893	―	Slovakia
C. variabilis	SLO2024	MT055894	―	Slovakia
C. versutus	IBNR1997/0962	―	AF367958	the United States
C. versutus	PBM856	―	AY820890	the United States
C. volubilis	TBGT15648	―	MH310742	India
C. yuanchui	FFAAS0339a	MZ401362	MW581518	China
C. yuanchui	FFAAS0341	MZ401363	MW581519	China
Neopaxillus dominicanus	MCVE26928	JN033216	JN033217	Italy
N. dominicanus	MCVE25727	HQ452479	HQ452478	Italy
N. dominicanus	F1133966	JN033220	JN033221	Mexico
N. echinospermus	MA-Fungi49404	AJ419194	JN033222	Brazil
N. plumbeus	F1068564	JN033223	NG_060271	Puerto Rico

^a Ex-holotype.

Phylogenetic analyses based on Bayesian inference and maximum likelihood were respectively carried out on the aligned dataset using MRBAYES 3.2.6 and raxmlGUI 1.5b1 with default settings (Ronquist & Huelsenbeck, 2003; Stamatakis, Ludwig, & Meier, 2005). For the Bayesian analysis, GTR + G was selected as the best-fit model of nucleotide substitution based on the standard Akaike information criterion in MODELTEST 3.7 (Posada & Crandall, 1998). Fifteen million generations were run, with sampling performed every 10,000 generations and with 25% of trees removed as burn-in before generating final result. For the maximum likelihood analysis, 1000 bootstrap replicates were conducted using raxmlGUI 1.5b1 with default settings. The resulting trees were edited with FigTree 1.4.4 and Photoshop CS4 (Rambaut, 2016). The final alignment and the results of the phylogenetic analysis were uploaded to TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S28430).

3. Results

3.1. Taxonomy

Crepidotus caspari Velen. Figs. 1, 2, 3.

Fig. 1 - Basidiomes of Crepidotus caspari (FFAAS 0342). A: Lamellae brownish orange when old. B: Stipe short. C: Pileus orange when old. D, F: Lamellae grayish orange when matured. E, G, H: Pileus whitish to grayish orange at maturity. Bars: A-H 1 cm.

Fig. 2 - Crepidotus caspari (FFAAS 0342). A: Basidiocarps. B: Basidia. C: Basidiospores. D: Cheilocystidia. E: Pileipellis. F: Lamellae edge. Bars: A 1 cm; B, D-F 10 μm; C 5 μm.

Fig. 3 - Scanning electron micrographs of basidiospores. A: Crepidotus caspari (FFAAS 0342). B-D: Crepidotus yuanchui (FFAAS 0339).

Pileus 15-28 mm; spathuliform, petaloid, and slightly campanulate when young; plano-convex, shell-shaped, rounded flabelliform to semicircular, lobelike, occasionally irregularly circular when mature; plano-convex when old; initially white to orange white or pale orange (6A1-6A3), then whitish to gray (6C1, 6D1) at maturity, and grayish orange to orange (6B6, 6B7) when old, darker toward the margin, surface with pubescence, more fibrillose at maturity, wavy, uneven, with tiny squama under the lens, margin slightly hygrophanous. Lamellae L = 13-18, l = 3-5, moderately crowded, adnate to slightly sinuate, 2-3 mm wide, subventricose, gray (6B1) near the margin, darker toward the center, grayish orange (6B3, 6B4); reddish golden to brownish orange (6C7, 6C8), lighter [grayish orange to light orange (6A4, 6B4)] at the edge, and still intact when old; sometimes wavy at maturity. Stipe short, 2-4 × 1-2 mm wide, cylindric to clavate, grayish orange (6B3, 6B4), rudimentary, knob-like, or only observed as white hyphae when mature; context white to whitish, 1.0-1.5 mm. Taste and odor indistinct.

Basidiospores (6.3-) 6.5-7.6 (-7.8) × (4.3-) 4.6-5.2 (-5.7) μm (n/4 = 137, L^m = 7.1 ± 0.40, W^m = 4.88 ± 0.23), Q = (1.28-) 1.33-1.58 (-1.60), Q^m = 1.45 ± 0.07], broadly ellipsoid to ellipsoid, ovoid in dorso-ventral view, amygdaliform to slightly reniform in lateral view, sulfur yellow to straw yellow in KOH; apex obtuse, thin-walled (<0.5 μm thick), smooth under an optical microscope, very faintly ornamented under SEM. Basidia 19-25 × 6-8 μm, clavate, sometimes ventricose, 4-spored; sterigmata 2.4-3.6 (-5) μm long, hyaline, clamped. Pleurocystidia absent. Cheilocystidia (29-) 38-48 μm, abundant, mostly cylindric-clavate, sublageniform to ventricose; apex round to blunt, occasionally segmented, bifurcate in the upper part, conspicuously flexuous, hyaline. Pileipellis a cutis, with a parallel arrangement of cylindrical hyphae, 67-85 × 4.5-5.5 μm, thin- to thick-walled (0.5-0.8 μm thick), hyaline, with occasional intersecting, crooked hyphae; a few thicker hyphae, up to 7 μm, changing to light sulfur yellow, present mainly at the terminal and more numerous when old. Clamps present in all tissues.

Habitat: Solitary or scattered on fallen trunks and branches of Hippophae rhamnoides subsp. yunnanensis and Populus sp.

Known distribution: America (the United States), Asia (China), Europa (Bulgaria, Czech Republic, Estonia, France, Germany, Greece, Italy, Netherlands, Sweden, Switzerland).

Specimens examined: CHINA. Yunnan Province: Pudacuo National Forest Park, Diqing Tibetan Autonomous Prefecture, N 27°84¢90², E 99°91¢76², 3468 m asl, 29 Jul 2020, leg. Qin Na, Yupeng Ge, Junqing Yan, & Zewei Liu, FFAAS 0343 (collection number GN 0686). Pudacuo National Forest Park, Diqing Tibetan Autonomous Prefecture, N 27°85¢50², E 99°91¢43², 3473 m asl, 29 Jul 2020, leg. Qin Na, Yupeng Ge, Junqing Yan, & Zewei Liu, FFAAS 0342 (collection number GN 0684).

Note: The smooth basidiospores and clamped hyphae represent a relatively unique combination of characters for identifying C. caspari. Relatively few species with these characteristics have been previously recorded. According to Consiglio and Setti (2008), C. autochthonus is similar, but it possesses bigger basidiospores and a trichoderm-like pileipellis. Smooth-spored species with clamped hyphae were rarely recorded by Hesler and Smith (1965). Most clamped species with ellipsoid basidiospores belong to subgenus Dochmiopus; among them, species such as C. phaseoliformis Hesler & A.H. Sm. and C. amarus Murrill have the same range of basidiospore sizes but possess distinct ornamented basidiospores or a gelatinous pileipellis. Senn-Irlet (1995) and Senn-Irlet and de Meijer (1998) documented several species with smooth basidiospores, including C. antillarum (Pat.) Singer, C. albidus Ellis & Everh., C. acanthosyrinus Singer, and C. epibryus (Fr.) Quél. that have clamp connections. Several species have been documented from Asia, but none have the combined characteristics of smooth basidiospores and clamped hyphae (Han et al., 2004). Kasuya and Kobayashi (2011) described C. byssinus T. Kasuya & Takah. Kobay. from warm temperate areas of Japan. Although morphologically similar to C. caspari, C. byssinus possesses thickly rugulose-verruculose basidiospores, which is distinctly different from C. caspari (Kasuya & Kobayashi, 2011)

Under SEM, faint ornamentation can be observed on basidiospores of C. caspari collected in China, which is consistent with the results of Senn-Irlet (1995). Furthermore, this ornamentation can be classified as type I (very faintly ornamented), the weakest ornamentation type of Consiglio and Setti (2008). The weak ornamentation on the specimens collected in China matches the description of Senn-Irlet (1995), who reported very low ridges and warts.

Crepidotus yuanchui Q. Na, Z.W. Liu & Y.P. Ge, sp. nov. Figs. 4, 5

MycoBank no: MB838746.

Fig. 4 - Basidiomes of Crepidotus yuanchui (FFAAS 0339). A, H: Pileus ungulate when young. B, D: Lamellae salmon to Persian orange. C, I: Rounded flabelliform to petaloid pileus. E, F: Stipe only observed at primordial stages. Bars: A-I 1 cm.

Fig. 5 - Crepidotus yuanchui (FFAAS 0339). A: Basidiocarps. B: Basidia. C: Basidiospores. D: Cheilocystidia. E: Pileipellis. F: Lamellae edge. Bars: A 1 cm; B 5 μm; C-F 10 μm.

Diagnosis: Pileus medium-sized, rounded flabelliform to petaloid, orange-yellow; surface felty to villose. Basidiospores ovoid in dorso-ventral view; cheilocystidia cylindric to narrowly utriform, rarely forked. Pileipellis a trichoderm. Clamps present in all tissues.

Type: CHINA. Yunnan Province: Wulaoshan National Forest Park, Lincang City, N 23°54¢51², E 100°11¢06², 2463 m asl, 31 Jul 2020, leg. Qin Na, Yupeng Ge, Junqing Yan, & Zewei Liu (holotype, FFAAS 0339, collection number GN 0704).

Gene sequence ex-holotypes: MW581518 (nLSU), MZ401362 (ITS).

Etymology: from ‘yuanchui’, a mythical Chinese yellow-orange creature who attaches to a vigorous, talented young man, thereby referring to the yellow-orange basidiocarps.

Pileus (5-) 9-23 mm wide, ungulate to spathuliform at primordial stages, becoming convex, rounded flabelliform to semicircular, then rounded flabelliform to petaloid when mature, sometimes lobe-like, plano-convex; margin slightly incurved at maturity, wavy when old, not sulcate; pale orange to light orange (5A3, 5A4) at first, orange yellow to brownish yellow (5B8, 5C8) when mature; surface white to whitish, villose-strigose at the very young stage, becoming fibrillose, felty to villose when mature, dense toward the point of attachment, dorsally attached to the substratum, covered by white woolly hyphae. Lamellae adnate to nearly sinuate, 2-3.5 mm wide, initially pale yellow to pastel yellow (2A3, 2A4), with edge mostly darker [orange-yellow to brownish-yellow (5B8, 5C8)]; salmon to Persian orange (6A6, 6A7), with edge reddish orange to copper red (7B8, 7C8) at maturity, sometimes paler when old; subventricose, with dark brown spots observed on surface of lamellae at maturity. Stipe only observed at primordial stages, cylindrical, directly attached to the substratum, pale yellow, rudimentary when mature, knob-like, covered by white hyphae. Context thin, white to translucent. Taste and odor indistinct.

Basidiospores (4.8-) 5-6 (-6.2) × (4-) 4.2-5.2 (-5.5) μm, (n/4 = 90, L^m = 5.5 ± 0.32, W^m = 4.60 ± 0.30), Q = (1.04-) 1.08-1.30 (-1.40), Q^m = 1.20 ± 0.07] , HOLOTYPE n = 45, (4.8-) 5-6 (-6.2) × 4-4.9 (-5.5) μm, L^m = 5.4 ± 0.30, W^m = 4.5 ± 0.31), Q = 1.13-1.28 (-1.31), Q^m = 1.20 ± 0.05], buff yellow to light orange-yellow in KOH, subglobose to broadly ellipsoid, ovoid in dorso-ventral view, slightly amygdaliform to ovoid in lateral view, thin-walled (<0.5 μm thick), punctate to verrucose, warts up to 0.8 μm high, base broader than the height, slightly hemispherical under an optical microscope, warts irregular in shape, isolated or finely confluent under SEM. Basidia 24-26 × 6-8 (-9) μm, subcylindric to clavate, slightly subfusoid, attenuated thinner toward the base, hyaline, 4-spored; sterigmata 2-2.8 μm long. Cheilocystidia 36-44 × 5-9 μm, cylindric to narrowly utriform, sublageniform, mostly tapering toward the round apex, rarely forked, abundant, conspicuous and clumped, flexuous, occasionally septate, hyaline. Pileipellis a trichoderm composed of two layers; upper part up to 80 μm, composed of erect cylindric hyphae, dense, 3-4 μm wide, thin-walled (<0.5 μm thick), hyaline to slightly pale yellow; lower part consisting of cylindric to sausage-shaped or nearly fusiform hyphae, regularly arranged, parallel with the pileus surface, thin-walled (<0.5 μm thick), yellow to light buff yellow. Clamps present in all tissues.

Habitat: Solitary or scattered on fallen trunks and branches of hardwoods.

Known distribution: Yunnan Province, China

Additional specimens examined: CHINA. Yunnan Province: Mopanshan National Forest Park, Xinping Yi and Dai Autonomous County, Yuxi City, N 23°56¢40², E 101°57¢53², 2278 m asl, 25 Jul 2020, leg. Qin Na, Yupeng Ge, Junqing Yan, & Zewei Liu, FFAAS 0340 (collection number GN 0638). Wulaoshan National Forest Park, Lincang City, N 23°54¢52², E 100°10¢56², 2432 m asl, 31 Jul 2020, leg. Qin Na, Yupeng Ge, Junqing Yan, & Zewei Liu, FFAAS 0341 (collection number GN 0696).

Note: According to the classification proposed by Senn-Irlet (1995), C. yuanchui belongs to subsection Fibulatini Singer, section Crepidotellae Hesler & A.H. Sm., and subgenus Dochmiopus. Three species of subsection Fibulatini―C. lundellii Pilát, C. luteolus Sacc., and C. subverrucisporus Pilát―are documented in Senn-Irlet (1995). All three species possess substantially larger basidiospores than those of C. yuanchui. Of the 15 other species assigned to the two sections of subgenus Dochmiopus by Senn-Irlet (1995), only C. carpaticus Pilát possesses basidiospores similar to those of C. yuanchui; however, the cheilocystidia of C. carpaticus are rarely forked and are not cylindric to narrowly utriform as in C. yuanchui. Other species of subgenus Dochmiopus can be distinguished on the basis of basidiocarp color and the presence of clamp connections.

Crepidotus croceotinctus Peck, which was recently recorded in India, is the species of Crepidotus morphologically most similar to C. yuanchui (Kumar et al., 2020). Crepidotus croceotinctus was previously only known to be distributed on the North and South American continents; we thus did not notice, prior to its discovery in Asia, that the pileus color and basidiospores of C. croceotinctus are similar to those of C. yuanchui (Hesler & Smith, 1965; Krisai-Greilhuber, Senn-Irlet, & Voglmayr, 2002; Bandala & Montoya, 2000, 2004, 2008; Singer, 1973; Senn-Irlet & de Meijer, 1998). The major distinctions between these two species are that C. croceotinctus typically possesses vesiculose clavate cheilocystidia and larger basidiospores.

3.2. Molecular phylogeny

The Bayesian inference and maximum likelihood trees had similar topologies. In the maximum likelihood tree in Fig. 6, four species are grouped into clade 1: C. tobolensis Kapitonov, Biketova & Zmitr., C. macedonicus Pilát, C. croceotinctus, and the newly described C. yuanchui. The results of our phylogenetic analyses provide further evidence for the recognition of C. yuanchui, which is strongly supported in the tree as distinct from all other Crepidotus taxa. Crepidotus caspari forms a distinct clade (clade 2) with C. lundellii from Hungary and the United States. Based on the research of Pouzar (2005), C. lundellii has been incorporated into C. caspari. The identity and status of C. caspari has therefore been phylogenetically verified.

Fig. 6 - Maximum Likelihood tree based on the combined ITS + nLSU dataset (Maximum likelihood bootstrap ≥ 75%, Bayesian posterior probabilities≥ 0.95 are indicated). The tree is rooted with Neopaxillus species. The new species are marked by red.

4. Discussion

More Crepidotus species may occupy high altitudes than currently recognized, but the elevation was not indicated in most Crepidotus records we were able to access. In several reports that include elevational data, most specimens were collected at or below 500 m asl, with some found at 350 to 1700 m (Bandala, Montoya, & Horak, 2006). Only a few Crepidotus species have been documented above 2000 m asl (Senn-Irlet & de Meijer, 1998; Aime et al., 2002; Bandala et al., 2006). In addition, C. trichocraspedotus T. Bau & Y.P. Ge, which we recently published as a new taxon (Ge & Bau, 2020), is distributed at 2372 m asl. like C. caspari, C. trichocraspedotus has smooth basidiospores and clamped hyphae, features that are rare in Crepidotus. We thus predict that additional high-elevation Crepidotus species remain to be discovered and studied.

Yellow-orange members of Crepidotus exemplify the diversity of the genus. Consiglio and Setti (2008) described 25 Crepidotus taxa from Europe, most of which are white, whitish, or pinkish; the only orange-gray one, C. ehrendorferi Hauskn. & Krisai, differs from C. yuanchui in having typical globose basidiospores. Among species documented in North America, C. luteicolor Hesler & A.H. Sm. is the morphologically closest to C. yuanchui. Crepidotus yuanchui and C. luteicolor both possess a yellow pileus and ovoid, similar-sized basidiospores, but pleurocystidia, which are rare in the genus, are only present in C. luteicolor. Pleurocystidia presence/absence is therefore a useful character for distinguishing Crepidotus taxa. Most species with basidiospore sizes similar to C. yuanchui can be distinguished by their pileus characters. Only two species with the same pileus color as C. yuanchui have been previously recorded in China: C. lutescens T. Bau & Y.P. Ge and C. sulphurinus Imazeki & Toki (Ge, 2017; Ge, Yang, & Bau, 2017). In many respects, Crepidotus sulphurinus substantially differs from other yellow-orange Crepidotus species. Interestingly, C. lutescens has only been recorded from low-latitude and subalpine localities in China, and all specimens have been found during low temperatures (ca. 20 °C) (Ge et al., 2017). Moist, low-temperature environments may be suitable habitats for yellow-orange Crepidotus species. Over 5 d, we collected 12 yellow-orange Crepidotus specimens at 2000 to 3600 m asl (ca. 20 °C or lower) that represent at least two different unrecorded species. Low-temperature areas harboring yellow-orange Crepidotus may thus contribute to the species diversity of this genus.

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.

Acknowledgments

We are grateful to National Natural Science Foundation of China (No. 3190012), the Natural Science Foundation of Shandong Province (No: ZR2019PC028, No: ZR2020QC001), the Innovation Team of Shandong Agricultural Industry Technology System (2016 No.18), and the Major Scientific and Technological Innovation Projects of Key R & D Programs in Shandong Province (No.2019JZZY010717). We sincerely thank Dr. Weihuan Li, Dr. Rui Zhang, Xiaohong Wang, Kemeng Shi, Na Yu, Tian Wang (Ludong University) for the help in the fieldwork and researching. We thank Barbara Goodson and Mallory Eckstut from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

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