Species Diversity of Calocybe (Agaricales, Lyophyllaceae) from Shanxi Province of Northern China

Abstract

Many species of Calocybe are of great edible value. Nineteen species of Calocybe have been reported from China, which represents about 40% of the global population. However, in Shanxi Province, located in northern China, no Calocybe species had been reported before this study. Our present study showed there are at least eight Calocybe species distributed in this province, including seven known species and a new species. The known species are C. badiofloccosa, C. coacta, C. fulvipes, C. gambosa, C. gangraenosa, C. ionides, and C. pseudoflammula. The new species, Calocybe confusa sp. nov., is described and illustrated, and, morphologically, it is diagnosed by the combination of basidiomata turning black when exposed, cream grey, grey yellowish white to pale brown pileus, narrowly ellipsoid to subcylindrical basidiospores, and the absence of hymenial cystidia. The molecular data for known species are provided. The geography of the Calocybe species in Shanxi Province is discussed.

1. Introduction

The genus Calocybe Kühner ex Donk was officially erected by Donk [1], with Calocybe gambosa (Fr.) Donk as its type species. Many species of Calocybe are edible and some are cultivated, such as Calocybe indica [2]. This genus is dominantly distributed in the northern hemisphere, and is characterized by brightly coloured pilei, small basidiospores, and pileipelles of a cutis or cellular type [3]. In earlier studies, Calocybe had been placed in the genus Lyophyllum as a section [4] or a genus of Tricholomataceae [3]. Molecular studies revealed that Calocybe is a monophyletic genus [5,6,7,8,9] and that Rugosomyces Raithelh. should be merged into Calocybe [10].

Index Fungorum registers approximately 124 records, but about 46 species are accepted according to He et al. [11]. In China, 19 species of Calocybe have been documented, with the majority discovered in northern China. However, the knowledge of Calocybe in Shanxi Province is significantly insufficient because of the lack of records of this genus in this region before this study. During recent fieldwork in Shanxi Province, we gathered numerous Calocybe specimens. Integrated morphological and molecular approaches confirmed eight species from these collections, with one representing a novel taxon. This study describes and illustrates a new species and provides molecular data for seven known Calocybe species.

2. Materials and Methods

2.1. Site Description

Specimens of the newly identified species were obtained from Zhongtiao Mountain, located in southeastern Shanxi Province. This region experiences a warm-temperate monsoonal continental climate and exhibits distinct vertical zonation in vegetation. From the base to the summit, the vegetation belts include a Quercus variabilis forest zone (400–1400 m), a mixed pine–oak forest zone (1200–2000 m), a poplar–birch forest zone (1900–2200 m), and an alpine meadow zone (2000–2358 m). These vegetation zones correspond to the following soil sequence: mountainous cinnamon soil, leached cinnamon soil, mountainous brown soil, and alpine meadow soil.

2.2. Morphological Studies

Field collections from Shanxi Province were photographed fresh, dehydrated at 35–45 °C using fruit driers, and deposited in BJTC (Herbarium Biology Department, Capital Normal University) HSA (Hebarium of Shanxi Institute for Functional Foods, Shanxi Agricultural University). Macroscopic features were documented from fresh specimens, with standardized colour codes referenced via ColorHexa [http://www.colorhexa.com/ (accessed on 11 July 2025)]. For microscopic analysis, sections of dried specimens were mounted in 5% KOH, Congo Red, or Melzer’s reagent [12], observing structures under compound microscopy [12]. Notation ‘[n/m/p]’ denotes n basidiospores measured from m basidiomata across p collections. Basidiospore dimensions follow (a–)b–c(–d), where b–c covers ≥ 90% of values and a/d are extremes. Spore ratio Q = length/width (side view); Qav = mean Q ± SD [13].

2.3. DNA Extraction, PCR Amplification, and DNA Sequencing

Sample processing involved crushing basidiomata fragments (20−30 mg) in a 1.5 mL tube with a 3 mm tungsten carbide ball using a Mixer Mill MM301 (Haan, Mettmann, in North Rhine-Westphalia, Germany). Grinding was performed at 30 Hz for 45 s per cycle, repeated 2–4 times. Total genomic DNA was subsequently extracted from the resulting powder, employing the NuClean Plant Genomic DNA Kit (CWBIO, Beijing, China) according to the manufacturer’s protocol.

Two nuclear loci were targeted for amplification and sequencing: the nrDNA internal transcribed spacer region (ITS) and the nuclear large subunit ribosomal RNA gene (nrLSU). Primer pairs ITS1-F/ITS4 and LR0R/LR5 [14,15] were employed for the ITS and nrLSU loci, respectively [14,15]. PCR amplification and sequencing followed established protocols [16]. The PCR amplicons were commercially processed by Beijing Zhongkexilin Biotechnology Co., Ltd (Beijing, China). All newly generated and downloaded sequence accession numbers are detailed in

Table 1. ITS and LSU sequences employed in this study. Bold text identifies sequences that were newly generated.

Species	Collections	GenBank Accession Number
		ITS	nrLSU
Agaricus bisporus	CCBAS306	LN714517	—
Asterophora lycoperdoides	CBS17086	AF357037	AF223190
Asterophora parasitica	CBS68382	AF357038	AF223191
Calocybe aurantiaca	SYAU-FUNGI-005	KU528828	KU528833
Calocybe aurantiaca	SYAU-FUNGI-006	NR_156304	NG_058937
Calocybe badiofloccosa	HMJU00098	MN172332	MN172334
Calocybe badiofloccosa	BJTC FM2798	PX220057	—
Calocybe badiofloccosa	BJTC FM2808	PX220058	—
Calocybe betulicola	HMJAU48265	OR771918	OR771923
Calocybe betulicola	HMJAU48266	OR771919	OR771924
Calocybe betulicola	HMJAU48267	OR771920	OR771925
Calocybe buxea	EB20140228	KP885633	KP885625
Calocybe carnea	CBS55250	AF357028	AF223178
Calocybe coacta	HMJU269	OK649907	OL687156
Calocybe coacta	BJTC FM1437	PX220060	—
Calocybe coacta	BJTC FM1438	PX220061	—
Calocybe coacta	BJTC FM3594	PX220062	—
Calocybe confusa	BJTC FM3620	PX220067	PX220070
Calocybe confusa	BJTC FM4040	PX220068	PX220071
Calocybe confusa	BJTC FM4097 (Holotype)	PX220069	PX220072
Calocybe convexa	SYAU-FUNGI-007	KU528826	KU528830
Calocybe convexa	SYAU-FUNGI-008	NR_156303	NG_058936
Calocybe cyanea	K(M):56506	OM905975	—
Calocybe cystidiosa	HMJAU48268	OR771915	—
Calocybe cystidiosa	HMJAU48269(1)	OR771916	—
Calocybe cystidiosa	HMJAU48269(2)	OR771917	—
Calocybe decolorata	SYAU-FUNGI-003	KU528824	KU528834
Calocybe decolorata	SYAU-FUNGI-004	NR_156302	NG_058938
Calocybe erminea	HMJU00100	MN172331	MN172333
Calocybe favrei	HAe23497cp	AF357034	AF223183
Calocybe favrei	IE-BSC-HC 96cp4	EF421102	AF223184
Calocybe fulvipes	HMJU3027	OK649910	OK649880
Calocybe fulvipes	HMJU317	MT071590	OK649878
Calocybe fulvipes	BJTC FM3231	PX220063	—
Calocybe gambosa	HC78/64	AF357027	AF223177
Calocybe gangraenosa	Hae25197	AF357032	AF223202
Calocybe gangraenosa	BJTC FM0090	PX220064	—
Calocybe gangraenosa	BJTC FM2885	PX220065	—
Calocybe gangraenosa	BJTC FM1154	PX220066	—
Calocybe graveolens	FR2014044	KP192590	—
Calocybe graveolens	BJTC FM0585	PX220059	—
Calocybe ionides	BJTC FM0507	—	PX220073
Calocybe ionides	BJTC FM0510	—	PX220074
Calocybe ionides	BJTC FM0581	—	PX220075
Calocybe ionides	H77/133	AF357029	AF223179
Calocybe lilacea	SYAU-FUNGI-070	OM203538	OM341407
Calocybe lilacea	SYAU-FUNGI-071	OM203539	OM341409
Calocybe longisterigma	SYAU-FUNGI-066	OM203542	OM341406
Calocybe longisterigma	SYAU-FUNGI-067	OM203543	OM341408
Calocybe naucoria	AMB17094	KP885642	KP885630
Calocybe naucoria	HC80/103	AF357030	AF223180
Calocybe obscurissima	HC79/181	AF357031	AF223181
Calocybe ochracea	BSI94.cp1	AF357033	AF223185
Calocybe onychina	CAON-RH19-563	MW084664	MW084704
Calocybe onychina	CL121115-07	KP885644	KP885632
Calocybe persicolor	HC80/99	AF357026	AF223176
Calocybe pilosella	TR gmb 00697	KJ883237	—
Calocybe pseudoflammula	FR2014100	KP192649	—
Calocybe pseudoflammula	BJTC FM1450	PX220056	—
Calocybe subochraceus	SYAU-FUNGI-069	OM203541	—
Calocybe vinacea	HMJU5135	OK649908	OK649876
Calocybe vinacea	HMJU5160	OK649909	OK649877
Calocybella pudica	AMB15994	KP858000	KP858005
Calocybella semitale	EL187-09	HM572552	—
Calocybella semitale	HC85/13	AF357049	AF042581
Gerhardtia borealis	AMB15993	KP858004	KP858009
Ossicaulis lignatilis	D604	DQ825426	AF261397
Tephrocybe boudieri	BSI96/84	AF357047	DQ825430
Tephrocybe gibberosa	CBS328.50	AF357041	AF223197
Tephrocybe tylicolor	BSI92/245	AF357040	AF223195
Tricholomella constricta	HC84/75	DQ825429	AF223188

.

2.4. Phylogenetic Analysis

This study employed a concatenated ITS-nrLSU matrix to characterize the new species and resolve their phylogenetic position within Calocybe. Sequence alignment for both ITS and nrLSU was conducted using the online MAFFT 7.110 platform [17]. The resulting alignments were then manually refined in BioEdit v.7.0.9 [18].

Maximum likelihood (ML) and Bayesian inference (BI) were applied for phylogenetic analyses. The ML analyses utilized RAxML 8.0.14 [19] with the GTRGAMMAI model, applying all default parameters. For ML analysis, a rapid bootstrap procedure with 1000 replicates was executed to identify the best-scoring tree. BI analysis was performed in MrBayes 3.1.2 [20]. The analysis employed a partitioned mixed model, treating ITS and nrLSU sequences as independent partitions with distinct parameter sets. Optimal substitution models for each locus were selected using MrModeltest v2.3 [21] under the Akaike Information Criterion (AIC), resulting in the GTR + I + G model for both the ITS and nrLSU. Two independent MCMC runs (each with four chains) proceeded for 405,000 generations under default conditions. At run termination, the average standard deviation of split frequency (ASDSF) was well under 0.01. Sampling occurred every 100 generations following burn-in, and a 50% majority-rule consensus tree was built.

Significant clade support thresholds were set at MLBS ≥ 70% and BPP ≥ 0.95 [22,23]. All resulting phylogenies were examined using TreeView [24].

3. Results

3.1. Phylogenetic Analysis

In this study, 20 sequences were newly generated from our collections. The final combined ITS-nrLSU dataset consisted of 118 sequences from 71 collections and contained 1516 (614 for ITS; 902 for nrLSU) characters after the exclusion of poorly aligned sites. Maximum likelihood (ML) and Bayesian inference (BI) analyses produced congruent tree topologies; consequently, only the phylogeny inferred from the ML analysis is presented (Figure 1). The members of Calocybe formed a monophyletic lineage with strong support. Phylogenetic analysis revealed eight strongly supported clades among our sequenced collections, representing eight distinct species. Of them, seven clades corresponded well to the previously described species, i.e., Calocybe badiofloccosa J.Z. Xu & Yu Li, C. coacta J.Z. Xu & Yu Li, C. fulvipes J.Z. Xu & Yu Li, C. gambosa, C. gangraenosa (Fr.) V. Hofst., Moncalvo, Redhead & Vilgalys, C. ionides (Bull.) Donk, and C. pseudoflammula (J.E. Lange) M. Lange ex Singer. The remaining clade represented an undescribed species.

3.2. Taxonomy

Calocybe confusa L. Fan, N. Mao & T. Li, sp. nov. Figure 2 and Figure 3.

MycoBank: 860463

Etymology: confusa, referring to the fact that the new species is confused with C. gangraenosa in micromorphology.

Holotype: China. Shanxi Province, Xia County, 6 October 2023, on the ground in broadleaf forest dominated by Quercus sp., J.Z Cao, CF2333 (BJTC FM4097).

Diagnosis: This species is characterized by the following combination of features: basidiomata turning black when exposed, cream grey, grey yellowish white to pale brown pileus and narrowly ellipsoid to subcylindrical basidiospores. It is most similar to Calocybe gangraenosa but differs by habitats associated with broadleaf forests and relatively broader basidiospores.

Description: Pileus 27–65 mm diam, at first convex, later broadly convex to plano-convex with age, cream grey (#e6e5cf), grey yellowish white (#f7f7f1) to pale brown (#cebba7), darker in the centre and paler towards the margin; surface felty, not smooth, dry; margin incurved when young, explanate at maturity, occasionally cracking with age. Lamellae adnate to sinuate, crowded, up to 4 mm, cream white (#fffff8), then yellowish brown (#d7bd91) with age, turning black (#3d3d3d) when exposed, lamellulae in 1–3 tiers, concolorous with lamellae. Stipe 35–82 mm long, 5–11 mm in diam, cylindrical to clavate, equal, concolorous with the pileus, surface longitudinally striate. Context white (#ffffff), turning black (#3d3d3d) when exposed. Odour unrecorded. Taste not recorded.

Basidiospores 5–7 × 3–4 μm, Q = 1.71–2.19, Q_av = 1.92 (±0.17) [60/3/2], narrowly ellipsoid to subcylindrical, sparsely and minutely warty, inamyloid, cyanophilic. Basidia 20–30 × 5–7 μm, clavate, hyaline in water and 5% KOH, thin-walled, 4-spored. Hymenial cystidia not observed. Hymenophoral trama regular, consisting of cylindrical and subinflated hyphae, hyphae 4–10 μm wide. Pileipellis a cutis, composed of parallel cylindrical hyphae of 3–10 µm wide, yellowish-brown in water and 5% KOH. Stipitipellis a cutis, composed of parallel cylindrical hyphae of 4–8 µm wide, yellowish-brown in water and 5% KOH. Clamp connections present in all tissues.

Ecology and habitat: Scattered or gregarious on the ground in broadleaf or coniferous forest, currently only known in Shanxi Province, northern China.

Additional specimens examined: China. Shanxi Province: Qinshui County, Lishan Mountain, on the ground in broadleaf forest dominated by Quercus sp., 6 October 2023, J.Z Cao, CF2270 (BJTC FM4040); ibid., Jiexiu County, Mianshan Mountain, on the ground in coniferous forest, 20 September 2023, Y. Li, MS590 (BJTC FM3620).

4. Discussion

Our multilocus phylogenetic analysis and morphological data indicated that the new species belongs to Calocybe. Calocybe gangraenosa is the most similar species to the new species in terms of its basidiomata turning black when exposed, pileus colour, and the structure of the pileipellis [25,26]. However, C. gangraenosa differs from the new species by its relatively narrower basidiospores (7–8.5 × 3–3.2 µm; Q = 2.3–2.6) [25,26]. Calocybe coacta J.Z. Xu & Yu Li, a species recently described from Liaoning Province in north-eastern China [27], is phylogenetically closely related to the new species. Both species have a cream pileus and narrowly ellipsoid basidiospores. However, C. coacta is distinguished from C. confusa by its larger basidiomata (pileus 60–115 mm in diam, stipe 40–58 mm long and 20–30 mm wide), smooth basidospores, and hymenial cystidia. Moreover, notably, in our three samples strongly supported to be conspecific to C. coacta by the present analysis (Figure 1), the basidiomata is clearly bruised black, the context is somewhat light blue when exposed, and the lamellae are sinuate to nearly distant (Figure 2), all of which is not described in original description by Xu et al. [27]. The other two species recently described from China, C. betulicola, and C. cystidiosa, are easily distinguished from C. confuse, because C. betulicola has a violet pileus and oval basidiospores, and C. cystidiosa has pink-tinted basidiomata and hymenial cystidia [28].

Eight species of Calocybe have been confirmed in Shanxi Province, northern China, in this study, i.e., Calocybe badiofloccosa, C. coacta, C. confusa, C. fulvipes, C. gambosa, C. gangraenosa, C. ionides, and C. pseudoflammula. Geographically, Calocybe badiofloccosa, C. coacta, C. confusa, and C. pseudoflammula are found in southern regions of Shanxi Province and associated with a warm-temperate forest dominated by Pinus tabuliformis Carrière and/or Quercus spp. The remaining species, C. fulvipes, C. gangraenosa, C. gambosa and C. ionides, are found in subalpine areas of the northern regions and associated with coniferous forest dominated by Larix gmelinii var. principis-rupprechtii (Mayr) Pilg.