Multiple Analyses Reveal Evidence for Three New Species of Collybia (Clitocybaceae, Basidiomycete) from China

Abstract

Three new species of Collybia in China, Collybia clavipes, C. carnea and C. violea, are originally reported and described based on morphological characteristics and molecular data. This study provides detailed morphological descriptions of these three new species of Collybia, which can be accurately distinguished from other species within the genus Collybia. Phylogenetic relationships of Clitocybaceae were analyzed using a four-loci combined dataset (ITS-nrLSU-rpb2-tef1-α), and the results show that the three newly discovered species of Collybia form three distinct lineages, respectively. Based on the combination of morphological and molecular methods, these three newly collected species of Collybia are confirmed as new to science. A theoretical basis is provided for the species diversity of Collybia.

1. Introduction

Based on phylogenetic analyses over the past two decades, many new genera have been established to accommodate traditional Clitocybe species [1], such as Cleistocybe Ammirati, A.D. Parker & Matheny [2], Trichocybe Vizzini [3], Atractosporocybe P. Alvarado, G. Moreno & Vizzini, Leucocybe Vizzini, P. Alvarado, G. Moreno & Consiglio and Rhizocybe Vizzini, G. Moreno, P. Alvarado & Consiglio [4], Pulverulina Matheny & K.W. Hughes 2020 [5] and Spodocybe Z.M. He & Zhu L. Yang [6]. However, it is noteworthy that Clitocybe remains polyphyletic [1,4,7,8,9,10,11,12,13]. When He et al. [14], based on phylogenetic and phylogenomic analyses, reconstructed the phylogenetic framework of Clitocybaceae and recircumscribed the genera within the family, a large number of traditional Clitocybe and Lepista species were transferred to different clades of the genus Collybia, and the two genera became monophyletic groups.

Species of Collybia have a widespread distribution all over the world. However, new taxa of Collybia had rarely been reported and described in China in recent years until He et al. [14], who proposed fourteen new Collybia species in China. In this present study, several interesting specimens of Collybia were collected from southwestern and northeastern China. To identify whether these specimens of Collybia are new to science, detailed phylogenetic and morphological examinations were conducted, resulting in possibly novel additions to the genus Collybia.

2. Materials and Methods

2.1. Specimens and Morphological Studies

Specimens of Collybia were collected from southwestern and northeastern China from 2019 to 2024. Some basidiomes were dehydrated with silica gel and used for DNA extraction; the remaining basidiomes were dehydrated with an electric drying oven at 50 °C and stored in the Fungal Herbarium of Shenyang Agricultural University (SYAU-FUNGI). Detailed information is given in

Table 1. Information on newly generated sequences in this study.

Species	Voucher Numbers	GenBank Accession Numbers				Location
		ITS	nrLSU	rpb2	tef1-α
Collybia clavipes	SYAU-FUNGI-087	PQ594916	PQ594905	PQ801137	PQ801139	Xizang, China
	SYAU-FUNGI-088	PQ594917	PQ594906	PQ801138	PQ801140	Xizang, China
C. carnea	SYAU-FUNGI-089	PQ594919	PQ594908	PQ609669	PQ609675	Xizang, China
	SYAU-FUNGI-090	PQ594920	PQ594909	PQ609670	PQ609676	Xizang, China
C. violea	SYAU-FUNGI-052	MT482103	MT482097	PQ817427	MT510970	Liaoning, China
	SYAU-FUNGI-053	MT482104	MT482098	PQ817428	MT510971	Liaoning, China
	SYAU-FUNGI-054	MT482105	MT482099	PQ817429	MT510972	Liaoning, China

. Methods for morphological observation followed Qi et al. [15]. The color codes followed the Methuen Handbook of Color [16]. For observation of the spore surface, the gills of the dried specimens were coated with gold and examined with a ZEISS Ultra Plus Scanning Electron Microscope (SEM) (Oberkochen, Germany).

2.2. DNA Extraction, PCR, and Sequencing

Genomic DNA was extracted from dried specimens using the cetyltrimethylammonium bromide (CTAB) method [17]. Polymerase chain reaction (PCR) was performed to amplify sequences of the ITS, nrLSU, rpb2, and tef1-α regions. Primers ITS1 and ITS4 [18] were used for the ITS region; primers LROR and LR5 [19] were used for the nrLSU region; primers bRPB2-6F and bRPB2-7.1R [20] were used for the rpb2 region; and primers EF1-983F and EF1-1953R [21] were used for the tef1-α region. The PCR procedure was performed following Qi et al. [15].

2.3. Phylogenetic Analyses

Based on the latest phylogenetic studies on Clitocybaceae [14], high-quality sequences of Clitocybaceae species were retrieved and aligned with the newly generated sequences in this study that were checked and edited using Bioedit v7.0.9 [22]. Alignments were generated for single ITS, nrLSU, rpb2, and tef1-α datasets using MAFFT v7.313 [23]. Then, a four-locus combined dataset for Clitocybaceae, containing sequences of ITS, nrLSU, rpb2, and tef1-α, was generated by the PhyloSuite v1.2.1 software platform [24]. ModelFinder [25] was used for the selection of the best-fitting evolution model for the combined dataset. Maximum Likelihood (ML) analysis was performed with RAxML-8.2.10-WIN [26] with 1000 bootstrap replicates using the TIM2 model for the combined dataset. Bayesian Inference (BI) analysis was conducted with MrBayes v.3.2.6 [27] using the GTR+I+G model for the combined dataset, and the combined dataset was run for 8,000,000 generations. The best tree was viewed in FIGTREE v1.4.4 [28] and enhanced in Adobe Illustrator CC 2022.

3. Results

3.1. Phylogenetic Analyses

In the preliminary molecular analysis, C. clavipes sequences showed 96.4% similarity to C. hunanensis (NR_198326) with 98% query coverage for ITS, 99.4% similarity to C. tibetica (NG_243147) with 100% query coverage for nrLSU, 97.2% similarity to Clitocybe dealbata (DQ825407) with 100% query coverage for rpb2, and 80.5% similarity to C. bisterigmata (OP558049) with 97% query coverage for tef1-α. Collybia carnea sequences showed 97.8% similarity to C. pannosa (NR_198323) with 100% query coverage for ITS, 99.5% similarity to C. phyllophila (MK277723) with 99% query coverage for nrLSU, 96.2% similarity to C. xylogena (OP956582) with 92% query coverage for rpb2, and 94.6% similarity to C. xylogena (OP672233) with 60% query coverage for tef1-α. Collybia violea sequences showed 95.5% similarity to C. nuda (OP626948) with 100% query coverage for ITS, 98.7% similarity to C. sordida (OP646393) with 100% query coverage for nrLSU, 85.4% similarity to C. nuda (KJ136110) with 100% query coverage for rpb2, and 82.8% similarity to C. nuda (MG702630) with 100% query coverage for tef1-α.

In the phylogenetic analysis, this study analyzed 411 sequences (109 ITS, 108 nrLSU, 98 rpb2, 96 tef1-α) from 110 samples representing 44 species of Clitocybaceae. The combined dataset ITS-nrLSU-rpb2-tef1-α of Clitocybaceae comprised a total of 2792 positions. ML and BI resulted in almost identical tree topologies, and the BI tree was selected for display (Figure 1). The phylogenetic relationships of Clitocybaceae (Figure 1) were consistent with previous work [14].

The optimal topology indicates that Clitocybaceae represents a monophyletic lineage with high support (100% BP, 1.00 PP). As shown in the phylogenetic analyses of Clitocybaceae (Figure 1), a total of six clades can be recognized within the family as the following genera: Collybia, Pseudolyophyllum, Lepista, Dendrocollybia, Singerocybe and Clitocybe. This is in line with He et al. [14]. Sequences of Collybia species are divided into four clades, which is in line with He et al. [14]. In this present study, three new species, C. clavipes, C. carnea and C. violea, form a single clade with high support (100% BP, 1.00 PP). In detail, C. clavipes and C. carnea are located within the Collybia subgen. Collybia, and C. violea is located within the Collybia subgen. Leucocalocybe.

3.2. Taxonomy

Collybia clavipes A.G. Xu & Y. Qi, sp. nov. (Figure 2)

MycoBank: MB858330

Etymology: “clavipes” refers to the clavate stipe.

Diagnosis: Differed from other known species of the subgenus by having small and white basidiomata, a clavate stipe and ellipsoid to sublacrymoid basidiospores.

Holotype: China, Xizang, Jilong County, in a mixed forest, 3104 m alt., 2 August 2023, A.G. Xu & Y. Qi (SYAU-FUNGI-087, holotype).

Description: Basidiomata small. Pileus 1.0–3.0 cm in width, applanate to slightly depressed at center; surface glabrous, white (1A1), hygrophanous; margin not striate, slightly inrolled. Lamellae decurrent, moderately crowded, 2–3 mm wide, white (1A1), with numerous tiers of lamellulae; edge entire or slightly eroded. Stipe 2.5–4.5 cm long × 0.5–1.2 cm wide, central, subclavate, hollow, gradually becoming enlarged downwards, sometimes slightly curved, surface white (1A1), brownish (6D4–8), longitudinally striate. Context thin, white.

Basidiospores (4.0) 5.0–6.0 (6.5) × 3.0–4.0 (4.5) μm, Q = 1.32–1.87, Qav = 1.64, ellipsoid to sublacrymoid, inamyloid, surface smooth under both the light microscope and electron microscope, not adherent, almost always single. Basidia 25.6–32.0 × 6.0–9.5 μm, clavate, four-spored, sterigmata up to 3.0–6.0 μm in length. Cystidia absent. Hymenophoral trama regular, hyphae cylindrical, hyphae up to 3.5–9.6 µm wide. Pileipellis hyphae cylindrical, hyphae up to 2.0–10.5 µm wide, with many branches. Stipitipellis hyphae cylindrical, hyphae up to 3.2–9.5 µm wide. Clamp connections present.

Habitat: Generally gregarious, more rarely solitary; saprotrophic on needle and broad leaf litter, in a mixed forest; summer.

Distribution: Only known from Xizang, China.

Additional material studied: China, Xizang, Jilong County, in a mixed forest, 3014 m alt., 20 August 2024, A.G. Xu & Y. Qi (SYAU-FUNGI-088).

Notes: Within Collybia subgen. Collybia, several species also have small and white basidiomes, such as C. asiatica, C. bisterigmata and C. pannosa [14]. However, the clavate stipe and ellipsoid to sublacrymoid basidiospores of C. clavipes make it easily distinguished from these species, which generally have a cylindrical stipe and non-sublacrymoid basidiospores. Furthermore, C. asiatica differs from C. clavipes by its infundibuliform basidiome and having basidiospores always adhering in tetrads [14]; C. bisterigmata differs by having two-spored basidia and larger basidiospores (6.0–7.5 (8.0) × 4.0–6.0 μm) [14]; C. pannosa differs by having felty pileus, basidiospores sometimes adhering in tetrads and irregularly arranged pileipellis [14].

Collybia carnea A.G. Xu & Y. Qi, sp. nov. (Figure 3)

MycoBank: MB858331

Etymology: “carnea” referring to a carneous basidiome.

Diagnosis: Differed from C. dealbata by having a carneous basidiome.

Holotype: China, Xizang, Dangxiong County, Ara wetland, in a mixed forest, 3104 m alt., 2 August 2024, A.G. Xu & Y. Qi (SYAU-FUNGI-089, holotype).

Description: Basidiomata small. Pileus 2.0–3.0 cm in width, plano-convex to nearly applanate, somewhat depressed at center; surface pinkish brown (5A2–3 to 6A2), becoming light brown (6B2–3) when old, white–pruinose, sometimes slightly lobate, not hygrophanous; margin not striate, inrolled, sometimes irregular, undulating. Lamellae adnate to subdecurrent, moderately crowded, thin, 1.0–2.0 mm wide, concolorous with pileus, with numerous tiers of lamellulae; edge entire. Stipe 4.0–5.0 cm long × 0.7–1.0 cm wide, central, cylindrical, hollow, sometimes slightly curved, concolorous with pileus, longitudinally striate, base not inflated, sometimes covered with whitish mycelium. Context about 1 cm thick, fleshy.

Basidiospores 4.5–6.0 × 2.5–4.0 (4.5) μm, Q = 1.30–2.02, Qav = 1.67, ellipsoid to elongate, inamyloid, surface smooth under both the light microscope and electron microscope, often single, rarely adherent. Basidia 25.5–31.0 × 6.2–8.6 μm, clavate, four-spored, hyaline, with sterigmata up to 2.0–6.5 μm in length. Cystidia absent. Hymenophoral trama regular, hyphae cylindrical, hyphae up to 3.5–9.6 µm wide. Pileipellis hyphae cylindrical, hyphae up to 3.0–9.5 µm wide, with many branches. Stipitipellis hyphae cylindrical, hyphae up to 3.0–8.5 µm wide. Clamp connections present.

Habitat: Scattered on rich soil among grasses, at the edge of a mixed forest, summer.

Distribution: Only known from Xizang, China.

Additional material studied: China, Xizang, Dangxiong County, on rich soil among grasses at the edge of a mixed forest, 3214 m alt., 20 August 2024, A.G. Xu & Y. Qi (SYAU-FUNGI-090).

Notes: Phylogenetically, Collybia carnea represents an independent clade and is a sister taxon to C. xylogena (Figure 1). Even so, C. xylogena can be easily distinguished by its brown to grayish brown pileus, relatively larger basidiospores (5.0–8.0 × 3–4.5 μm), and growing on wood [14]. Morphologically, C. carnea is closely related to C. dealbata, which also produces a small basidiome, a white–pruinose pileus with inrolled margin and medium-sized basidiospores, which are always mostly single [29,30]). However, Collybia carnea have a carneous basidiome, while C. dealbata have a white basidiome.

Collybia violea X.D. Yu & H.B. Guo, sp. nov. (Figure 4)

MycoBank: MB858332

Etymology: “violea” refers to violet basidiome.

Diagnosis: Distinguished from C. sordida and C. nuda by its slightly darker pileus color, and slightly smaller basidiospores.

Holotype: China, Liaoning Province, Shenyang City, Dongling Park, on rich soil among grasses, 177 m alt., 17 July 2019, X.D. Yu & H.B. Guo (SYAU-FUNGI-052, holotype).

Description: Basidiomata medium. Pileus 4.0–7.0 cm in width, at first convex, then gradually applanate, sometimes slightly depressed; surface lilac to grayish lilac (15 B2–3), becoming light brown (6C2–3) to ochraceous (6D5–8) from the center outwards; margin slightly inflexed when young, sometimes uplifted, irregular, undulating when mature. Lamellae adnexed to emarginate to subdecurrent, moderately crowded, thin, 2.0–3.0 mm wide, grayish violet to violet (17 B2–4), with numerous tiers of lamellulae; edge entire or slightly eroded. Stipe 2.5–5.0 cm long × 0.4–0.8 cm wide, central, cylindrical, solid or hollow, concolorous with lamellae, longitudinally striate, base slightly inflated. Context about moderately thick, fleshy.

Basidiospores 4.5–7.0 × 3.2–4.0 μm, Q = 1.30–1.82, Qav = 1.65, ellipsoid, inamyloid, surface smooth under the light microscope, finely verruculose under the electron microscope, not adherent, almost always single. Basidia 25.5–30.5 × 7.0–9.6 μm, clavate, four-spored, with sterigmata up to 3.0–5.5 μm in length. Cystidia absent. Hymenophoral trama regular, hyphae cylindrical, hyphae up to 3.0–9.2 µm wide. Pileipellis hyphae cylindrical, hyphae up to 2.5–9.5 µm wide, with many branches. Stipitipellis hyphae cylindrical, hyphae up to 2.0–8.5 µm wide. Clamp connections present.

Habitat: Saprophytic in small groups on rich soil among grasses, on roadsides.

Distribution: Known from northeastern China.

Additional material studied: China, Liaoning Province, Shenyang City, on the campus of Shenyang Agricultural University, on rich soil among grasses, 121 m alt., 31 August 2019, X.D. Yu & H.B. Guo (SYAU-FUNGI-053); China, Liaoning Province, Shenyang City, on the campus of Shenyang Agricultural University, on rich soil among grasses, 121 m alt., 31 August 2019, X.D. Yu & H.B. Guo (SYAU-FUNGI-054).

Notes: Collybia sordida and C. nuda could be easily mistaken for C. violea because they have lilac to grayish lilac basidiomata. Collybia sordida mainly differs by its hygrophanous and more dull-colored (pale bluish violet then pinkish beige with tinge olive at the center and in concentric zones on drying) pileus [31], and slightly larger basidiospores (7 × 4 μm [31]; 6–9 × 4–5 μm [32]). C. nuda can distinguish from C. violea by having much larger basidiomata (8–15 cm [31]; 2.5–10 cm [32]; 5–15 cm [33]) and slightly larger basidiospores (8 × 5 μm [31]; 5.5–9 × 4–5.5 μm [32]; 6.5–8.5 × 3.9–4.8 μm [33]).

4. Discussion

In the combined ITS-nrLSU-rpb2-tef1-α phylogenetic tree (Figure 1), Clitocybaceae forms a monophyletic clade, which is consistent with the previous study [14]. The three new species of Collybia described in this study occupy three independent positions, respectively. Collybia clavipes forms a distinct clade within the clade containing C. dealbata, C. hunanensis, C. subtropica and C. tibetica. Collybia carnea occupies a clade with the sister taxon, C. xylogena, but morphologically, C. carnea differs from C. xylogena by having a pinkish basidiome, smaller spores and growing on soil. Collybia violea forms a sister group with C. nuda, but morphologically, C. violea can be distinguished by having a smaller basidiome and smaller spores.

In this study, two new species belongs to the Collybia subgen. Collybia, which is a species-rich subgenus, with useful diagnostic features, such as basidiospore adhesion, two-spored or four-spored basidia, muscarine existence or non-existence, and growth substrates [14]. To some extent, this study further confirmed these diagnostic features’ usefulness to separate supraspecific taxa within the subgenus. For example, C. clavipes clusters with C. subtropica, C. hunanensis, C. dealbata and C. tibetica (Figure 1), all of which have four-spored basidia, basidiospores which are almost always single and grow on leaf litter or soil [14]. However, four-spored C. carnea has basidiospores which are always single and grow on soil, while two-spored C. xylogena has basidiospores which are often in tetrads and grow on rotten wood [14], although C. carnea clusters together with C. xylogena (Figure 1). This indicates that there may be still a number of new taxa remaining to be discovered within this clade.

Collybia violea belongs to Collybia subgen. Leucocalocybe. Due to interspecific similarities and phenotypic plasticity, C. violea has always been misidentified as C. sordida or C. nuda. However, our study finds there are distinct differences between C. violea and these species in both molecular data and morphological characteristics. Furthermore, previous phylogenetic analysis [14] based on worldwide ITS sequences of this subgenus suggested that there are five subclades of C. nuda. In the future, large amounts of related specimens need to be collected and detailed morphological studies need to be conducted to resolve the puzzles concerning which subclade could represent the true C. nuda.