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Research Article
Two new Clitocella species from North China revealed by phylogenetic analyses and morphological characters
expand article infoNing Mao‡, Jing-Chong Lv‡, Yu-Yan Xu‡, Tao-Yu Zhao‡, Li Fan‡
‡ Capital Normal University, Beijing, China

Corresponding author: Li Fan ( fanli@mail.cnu.edu.cn )

Academic editor: Rui-Lin Zhao
© 2022 Ning Mao, Jing-Chong Lv, Yu-Yan Xu, Tao-Yu Zhao, Li Fan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Mao N, Lv J-C, Xu Y-Y, Zhao T-Y, Fan L (2022) Two new Clitocella species from North China revealed by phylogenetic analyses and morphological characters. MycoKeys 88: 151-170. https://doi.org/10.3897/mycokeys.88.80068
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Abstract

Two new species of Clitocella are proposed based on morphological and phylogenetic investigations. Clitocella borealichinensis sp. nov. is closely related to C. orientalis but distinguished from the latter by its slightly smaller basidiospores and hyphae of pileipellis with pale brown to brown intracellular or parietal pigment. Clitocella colorata sp. nov. is closely related to C. popinalis and C. mundula in macromorphology but is differentiated from C. popinalis by its slightly smaller basidiospores and the difference in genetic profile, and from C. mundula by its relatively colorful pileus (white to yellowish white, grayish white to grayish brown, pink white). Phylogenetic analyses based on sequence data from five different loci (ITS, nrLSU, tef1, rpb2 and atp6) support the taxonomic position of the two new species in the genus Clitocella. The illustrations and descriptions for the new taxa are provided.

Keywords

Entolomataceae, multigene, phylogeny, taxonomy

Introduction

The genus Clitocella Kluting, T.J. Baroni & Bergemann (Entolomataceae, Agaricales), with C. popinalis (Fr.) Kluting, T.J. Baroni & Bergemann as the type species, was established in 2014 (Kluting et al. 2014). The main characteristics of Clitocella are clitocyboid basidiomata, narrow and crowded, long-decurrent lamellae, central to eccentric stipe, thin-walled (<0.5 μm) basidiospores with undulate pustules or minute bumps, clamp connections absent. (Baroni 1981; Kluting et al. 2014; Jian et al. 2020). Previous studies show that Clitocella is phylogenetically closely related to the genera Clitopilus (Fr. ex Rabenh.) P. Kumm. and Clitopilopsis Maire. Clitopilus differs from Clitocella in its longitudinally ridged basidiospore ornamentation, and Clitopilopsis in its basidiospores with thickened walls (0.5–0.9 μm) and obscure irregular rounded angles of the basidiospores in polar view (Kluting et al. 2014; Baroni et al. 2020; Jian et al. 2020). There are 10 accepted species in Clitocella (Index Fungorum, http://www.Indexfungorum.org/; accessed date: 19 November 2021).

In China, the species diversity of Clitocella is scarce and only two species are described (Jian et al. 2020). Recently, several specimens of Clitocella were collected when we investigated the macrofungi in Shanxi province, North China. The morphological examination and phylogenetic analysis for these collections revealed that they represented three taxa of Clitocella, including two new species. The aim of this paper is to describe the new species and provide the DNA data to confirm the presence in China of a previously described species.

Materials and methods

Morphological studies

Collections were obtained and photographed in the field from Shanxi regions in China, and then dried in a fruit drier at 40–50 °C and deposited in BJTC herbarium (Capital Normal University, Beijing, China). The sizes of basidiomata (pileal width) used in this study are as follows: small: <30 mm; medium-sized: 30–50 mm; large: >50 mm. Standardised color values were obtained from ColorHexa (http://www.colorhexa.com/). Microscopic characters were observed in sections obtained from dry specimens mounted in 3% KOH, Congo Red, or Melzer’s reagent (Dring 1971). For scanning electron microscopy (SEM), basidiospores were scraped from the dried gleba, placed onto double-sided tape that was mounted directly on the SEM stub, coated with platinum-palladium film of 8 nm thick using an ion-sputter coater (HITACHI E-1010), and examined with a HITACHI S-4800 SEM. The term “[n/m/p]” means n basidiospores from m basidiomata of p collections. Dimensions of basidiospores are given using the following format ‘(a–)b–c(–d)’, where the range ‘b–c’ represents at least 90% of the measured values, and ‘a’ and ‘d’ are the most extreme values. Lm and Wm indicate the average basidiospore length and width (± standard deviation) for the measured basidiospore, respectively. ‘Q’ refers to the length/width ratio of basidiospores in side-view; ‘Qav’ refers to the average Q of all basidiospores ±standard deviation.

DNA extraction, PCR amplification and DNA sequencing

Dried basidiomata were crushed by shaking for 45 s at 30 Hz 2–4 times (Mixer Mill MM301, Retsch, Haan, Germany) in a 1.5 mL tube together with a 3 mm diam tungsten carbide ball. Total genomic DNA was extracted from the powdered basidiomata using NuClean Plant Genomic DNA Kit (CWBIO, China), following the manufacturer’s instructions. Primers ITS1F and ITS4 were employed for the ITS (White et al. 1990; Gardes and Bruns 1993), while LR0R and LR5 for nrLSU (Vilgalys and Hester 1990), EF1-983F and EF1-1953R for the tef1 (Rehner 2001), bRPB2-6F and bRPB2-7R2 for the rpb2 (Liu et al. 1999; Matheny 2005; Matheny et al. 2007), and ATP6-3 and ATP6-6r for the atp6 (Kretzer and Bruns 1999; Binder and Hibbett 2003). Polymerase chain reactions (PCR) for ITS region, nrLSU region, tef1 gene, rpb2 gene and atp6 gene were performed in 25 µL reaction containing 2 µL DNA template, 1 µL primer (10 µM) each, 12.5 µL of 2× Master Mix [Tiangen Biotech (Beijing) Co.], 8.5 µL ddH2O.

PCR reactions were implemented as follows: an initial denaturation at 94 °C for 5 min, then to 35 cycles of the following denaturation at 94 °C for 30 s, annealing at 52 °C for 45 s (ITS), 60 s (nrLSU), 72 °C for 1 min; and a final extension at 72 °C for 10 min. Amplification of rpb2 and tef1 sequences followed Kluting et al. (2014), which entailed a touchdown protocol: an initial incubation of 94 °C for 5 min; 12 cycles of 94 °C for 1 min, 67 °C for 1 min, decreasing 1 °C each cycle, and 72 °C for 1.5 min; 36 cycles of 94 °C for 45 s, 55 °C for 1 min, and 72 °C for 1.5 min; and a final extension period at 72 °C for 7 min. Sequences of the atp6 were amplified with a cycling protocol of 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 s, 42 °C for 2 min, and 72 °C for 1 min, and a final extension at 72 °C for 10 min. The PCR products were sent to Beijing Zhongkexilin Biotechnology Co. Ltd. for purification, sequencing, and editing. Validated sequences were deposited in the NCBI database (http://www.ncbi.nlm.nih.gov/). Other sequences of Clitocella and related species were mainly selected from those used by previous studies (Kluting et al. 2014; Vizzini et al. 2016; Baroni et al. 2020; Jian et al. 2020). The accession numbers of all sequences employed are provided in Table 1.

Table 1.
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Specimens used in molecular phylogenetic studies and their GenBank accession numbers. Newly generated sequences are in bold.

Species Voucher Locality GenBank accession No.
ITS nrLSU rpb2 tef1 atp6
Catathelasma ventricosum DAOM221514 USA KP255469 – – – –
Clitocella colorata BJTC FM1593 China OL966940 – – – –
Clitocella colorata BJTC FM1594 China OL966941 – – – –
Clitocella colorata BJTC FM1891 China OL966944 OL966955 OL989914 OL989918 OL989924
Clitocella colorata BJTC FM1892 China OL966945 OL966956 OL989915 OL989919 OL989925
Clitocella colorata BJTC FM1952 China – OL966958 OL989916 OL989920 OL989926
Clitocella fallax CBS 605.79 – AF357018 – – – –
Clitocella fallax CBS 129.63 – AF357017 AF223166 EF421018 – –
Clitocella fallax K(M): 116541 Spain – – KC816938 KC816847 KC816769
Clitocella fallax O-F88953 Norway – – KC816936 KC816845 KC816767
Clitocella fallax 25668OKM USA – – KC816937 KC816846 KC816768
Clitocella fallax ME Noordeloos 1997173 Italy – GQ289209 GQ289275 – –
Clitocella fallax ME Noordeloos 200367 Slovakia – GQ289210 GQ289276 – –
Clitocella mundula 7161 TJB USA – – KC816952 KC816862 KC816782
‘Clitocella mundula’ O-F19454 Norway – – KC816954 KC816864 KC816784
Clitocella mundula O-F71544 Norway – – KC816950 KC816860 KC816780
‘Clitocella mundula’ AFTOL-ID 521 USA – – KC816953 KC816863 KC816783
Clitocella mundula 7115 TJB USA – – KC816951 KC816861 KC816781
Clitocella mundula K(M): 164736 UK – – KC816949 KC816859 KC816779
‘Clitocella mundula’ K(M): 49620 UK – – KC816948 KC816858 KC816778
Clitocella mundula HMJAU 7274 China – MN065724 MN148161 MN166272 MN133781
Clitocella mundula HMJAU 7275 China – MN065723 MN148160 MN166271 MN133780
Clitocella mundula HMJAU 27014 China – MN065722 MN148159 MN166270 MN133779
Clitocella borealichinensis BJTC FM1618 China OL966942 OL966946 OL989912 – OL989922
Clitocella borealichinensis BJTC FM1781 China OL966943 OL966957 OL989913 OL989917 OL989923
Clitocella orientalis HKAS 75548 China MN061333 MN065727 MN148164 MN166275 MN133784
Clitocella orientalis HKAS 75664 China MN061332 MN065726 MN148163 MN166274 MN133783
Clitocella orientalis HKAS 77899 China – MN065725 MN148162 MN166273 MN133782
Clitocella orientalis HKAS 78876 China MN061334 MN065729 MN148166 MN166277 MN133786
Clitocella orientalis (Holotype) HKAS 78763 China – MN065728 MN148165 MN166276 MN133785
Clitocella orientalis BJTC FM1539 China – OL966947 OL989911 OL989921 –
Clitocella popinalis HBJU-550 India KU561066 – – – –
Clitocella popinalis CBS 481.50 UK FJ770397 – – – –
Clitocella popinalis KA12-1717 Korea KR673647 – – – –
Clitocella popinalis RA802-3b USA MK217434 – – – –
Clitocella popinalis Smith-2018 iNaturalist # 17340579 USA MK573922 – – – –
Clitocella popinalis K(M): 143166 UK – – KC816971 KC816878 KC816796
Clitocella popinalis K(M): 167017 UK – – KC816972 KC816879 KC816797
Clitocella popinalis O-F63376 Norway – – KC816974 KC816880 KC816799
Clitocella popinalis 6378 TJB Switzerland – – KC816976 KC816882 KC816801
Clitocella popinalis O-F105360 Norway – – KC816975 KC816881 KC816800
Clitocella popinalis K(M): 146162 UK – – KC816970 KC816877 KC816795
‘Clitocella popinalis’ MC2-TRENT Italy – – KC816973 – KC816798
‘Clitocella popinalis’ ME Noordeloos 9867 Austria – GQ289213 GQ289280 – –
Clitocella popinalis TB6378 USA – AF261285 GU384654 – –
Clitocella. Mundula HMJAU 7275 China MN061331 – – – –
Clitocella obscura MK09051302 Czech Republic KX271753 – – – –
Clitocella prunulus G.v. Zanen F96065 – KC885965 – – – –
Clitocella_termitophila CORT014751 Dominican Republic – – MN893319 – –
Clitopilus brunneiceps (Holotype) HKAS 104510 China – MN065684 MN148123 MN166234 MN133737
Clitopilus yunnanensis (Holotype) HKAS 104518 China – MN065698 MN148136 MN166247 MN133752
Clitopilus. Amarus A. d. Haan 98031 – KC885963 – – – –
Cltopilopsis albida (Holotype) HKAS 104519 China – MN065730 MN148167 MN166278 MN133787
Lyophyllum decastes Sundberg091007a Japan HM572548 – – – –
Mycena pura CBH371 Denmark KF913023 – – – –
Rhodocybe mellea CORT013885 Dominican Republic MN784992 – – – –
Rhodocybe mellea JBSD127402 Dominican Republic MN784993 – – – –
Rhodocybe mellea CORT014470 Belize MN784994 – – – –
Rhodocybe mellea NYBG815044 Costa Rica MN784995 – – – –

Phylogenetic analyses

The combined nrLSU-rpb2-tef1-atp6 dataset and ITS dataset were compiled to identify new species and to investigate their phylogenetic position in Clitocella. For the combined nrLSU-rpb2-tef1-atp6 dataset, Clitopilopsis albida S.P. Jian & Zhu L. Yang was chosen as outgroups for individual (nrLSU, rpb2, tef1, atp6) or combined analyses (Jian et al. 2020). For ITS dataset Mycena pura (Pers.) P. Kumm. was selected as outgroup taxon (Baroni et al. 2020). The sequences of each marker (ITS, nrLSU, rpb2, tef1, atp6) were independently aligned in MAFFT v.7.110 (Katoh and Standley 2013) under default parameters. Ambiguously aligned sites were identified by Gblocks v.0.91b (Castresana 2000; using default options except “Allowed Gap Positions” = half) with default parameters (For ITS: 1137, nrLSU: 180, rpb2: 611, tef1: 166, atp6: 25 position were deleted). The software BioEdit 7.0.9 (Hall 1999) was used to manually check the aligned sequences. To examine the conflict among topologies with maximum likelihood (ML), separate single-gene analyses were conducted. Sequences were then concatenated. The ITS alignment can be found on Suppl. material 5. For the combined analyses, a partitioned mixed model was used by defining the sequences of nrLSU, rpb2, tef1, and atp6 as four independent partitions and each gene was separately estimated by different model parameters. Maximum Likelihood (ML) and Bayesian Inference (BI) analyses were conducted on the resulting concatenated dataset.

Maximum Likelihood (ML) was performed using RAxML 8.0.14 (Stamatakis et al. 2005; Stamatakis 2006, 2014) by running 1000 bootstrap replicates under the GTRGAMMAI model (for all partitions). Bayesian Inference (BI) analysis was performed with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003) based on the best substitution models (GTR+I+G for ITS, GTR+I for nrLSU, SYM+G for rpb2, SYM+I+G for tef1, and GTR+G for atp6) determined by MrModeltest 2.3 (Nylander 2004). Two independent runs with four Markov chains were conducted for 10 M generations under the default settings. Average standard deviations of split frequency (ASDSF) values were far lower than 0.01 at the end of the runs. Trees were sampled every 100 generations after burn-in (25% of trees were discarded as the burn-in phase of the analyses, set up well after convergence), and a 70% majority-rule consensus tree was constructed.

Trees were visualized with TreeView32 (Page 2001). Bootstrap values (BS) ≥ 70% and Bayesian Posterior Probability values (BPP) ≥ 0.95 were considered significant (Hillis and Bull 1993; Alfaro et al. 2003).

Results

Phylogenetic analysis

Twenty-eight sequences were newly generated from our six collections in this study. Two datasets, nrLSU-rpb2-tef1-atp6 combined dataset and ITS dataset were compiled to investigate the phylogenetic position of these Clitocella species. For the combined dataset, the phylogenetic trees based on individual loci (including nrLSU, rpb2, tef1, atp6) showed the almost same major clades (Suppl. material 1–4: Figs S1–S4) as that of the combined dataset (Fig. 1). There was no strongly supported conflict between single gene phylogenies, except for the nrLSU phylogeny does not resolve Clitocella mundula and C. popinalis, while the atp6 phylogeny does not resolve C. orientalis and the new species C. colorata. So here the combined dataset was used to infer the phylogenetic placement of Clitocella species. The final combined nrLSU-rpb2-tef1-atp6 dataset contained 2963 total characters (905 from nrLSU, 599 from rpb2, 1010 from tef1, 449 from atp6, gaps included) and included 40 samples of 11 taxa. The topologies of ML and BI phylogenetic trees obtained in this study were practically the same, therefore only the tree inferred from the ML analysis is shown (Fig. 1). Except for the species Clitocella termitophila T.J. Baroni & Angelini, members of Clitocella in the dataset formed a monophyletic lineage with strong support (MLB = 98%, BPP = 1.00). Clitocella termitophila was sister to all other species of Clitocella but without strong support. Of our six collections, the sequences of a collection (BJTC FM1539) grouped in the clade C. orientalis S.P. Jian & Zhu L. Yang, indicating it is identity with this species. The remaining specimens fell in two strongly supported clades, one comprised of two collections was described as the new species C. borealichinensis and another comprised of three collections was described as the new species C. colorata together with a collection from USA (AFTOL-ID 521) originally labelled as C. mundula. Clitocella colorata was sister to C. orientalis with strong support, implying C. colorata is closely related to C. orientalis. Clitocella borealichinensis further clustered with C. mundula and C. popinalis (Fr.) Kluting, T.J. Baroni & Bergemann. One collection from Norway (O-F19454), which is labelled as Clitocella mundula, formed an independent clade.

Figure 1. 

Phylogeny derived from Maximum Likelihood analysis of the combined nrLSU-rpb2-tef1-atp6 dataset of Clitocella and related genera in the family Entolomataceae. Clitopilopsis albida was employed to root the tree as an outgroup. Numbers representing likelihood bootstrap support (BS≥ 70%, left) and significant Bayesian posterior probability (BPP≥ 0.95, right) are indicated above the nodes. New sequences are highlighted in bold.

The ITS dataset comprised 27 samples of 11 taxa and 662 characters. The topology of phylogenetic trees based on the ITS dataset generated from ML and BI analyses were almost identical and only the tree inferred from the ML analysis is shown (Fig. 2). The sequences of the new species C. borealichinensis formed an independent and strong support branch, like that of multilocus phylogeny (Fig. 1), supporting it is a distinct species. The sequences of the new species C. colorata together with five sequences labelled as C. popinalis from India, South Korea, UK and USA formed an independent and strong support branch, indicating they represented a distinct species.

Figure 2. 

Phylogeny derived from Maximum Likelihood analysis of the ITS sequences from Clitocella and related genera in the family Entolomataceae. Mycena pura was employed to root the tree as an outgroup. Numbers representing likelihood bootstrap support (BS≥ 70%, left) and significant Bayesian posterior probability (BPP≥ 0.95, right) are indicated above the nodes. New sequences are highlighted in bold.

Taxonomy

Clitocella borealichinensis L. Fan & N. Mao, sp. nov.

MycoBank No: 843689
Figs 3a, 4, 6a, b

Etymology

borealichinensis, referring to north China as the place of origin.

Holotype

China. Shanxi Province, Qinshui County, Lishan Mountain, 35°36.49'N, 112°11.7'E, alt. 1150m, 26 July 2021, on the ground in broad-leaved forest dominated by Quercus sp., N. Mao MNM340 (BJTC FM1781).

Figure 3. 

Basidiomata of Clitocella a Clitocella borealichinensis (BJTC FM1781, holotype) b-d Clitocella colorata (b BJTC FM1593 c BJTC FM1952 d BJTC FM1891, holotype) Scale bars: 10 mm (a–d). Photos by JingZhong Cao

Diagnosis

Clitocella borealichinensis is characterized by its clitocyboid basidiomata, globose to subglobose, occasionally broadly ellipsoid basidiospores, the absence of hymenial cystidia and clamp connection, and usually growing in broad-leaved forests. It is most similar to C. orientalis but differs from it by the slightly smaller basidiospores, non-gelatinized hyphae of pileipellis and stipitipellis with pale brown to brown intracellular or parietal pigment.

Figure 4. 

Microscopy of Clitocella borealichinensis a basidiospores b basidia c pileipellis. Scale bars: 5 μm (a); 10 μm (b, c). Drawings by Ning Mao.

Description

Basidiomata clitocyboid, small to medium-sized. Pileus 13–50 mm wide, low convex to plane convex when young, then slightly depressed at center; surface smooth, grayish white (#f2f2f2) to pale white (#ffffff), yellowish white (#ffcd9a); margin incurved, non-striate; context thin pale white, 1.0–1.2 mm thick. Lamellae decurrent, grayish white (#f2f2f2), pale yellow (#fff3e7), crowded, edges smooth, thin and fragile, lamellulae numerous and concolorous with lamellae. Stipe 20–32 × 2–8 mm, central to eccentric, occasionally lateral, cylindrical to subcylindrical, equal or sometimes slightly tapering at base, pale white (#ffffff), smooth or tomentose, usually with white rhizomorphs. Odor unrecorded. Taste not recorded. Chemical color reaction: not reacting with KOH 3% at pileus of dried specimens.

Basidiospores [60/2/2] (3.8–)4–5(–5.5) × 3.8–4.5 μm, Lm × Wm = 4.61 (± 0.42) × 4.06 (± 0.18), Q = 0.95–1.25 (Qav = 1.13 ± 0.10), hyaline, globose to subglobose, occasionally broadly ellipsoid in profile view, slightly angled in polar or face view with obscure minute pustules or bumps. Basidia 17–25 × 5–6(–7) μm, clavate, hyaline, four spored, rarely two spored; sterigmata 2–4 μm long. Lamellar trama more or less regular, composed of 3–8 μm wide hyaline hyphae, subhymenium consisting of filamentous hyphal segments. Lamellae edges fertile. Pleurocystidia and cheilocystidia absent. Pileipellis a cutis composed of more or less radially, loosely arranged, non-gelatinized, smooth, cylindrical hyphae, 2–6 μm wide and with pale brown to brown intracellular or parietal pigment; terminal hyphae subcylindric, narrowly clavate, occasionally irregular, 3–5 μm wide; subcutis made up of subparallel, compactly arranged, thin-walled, hyaline, smooth, cylindrical hyphae, 3–6 μm wide; pileal trama composed of interwoven, cylindrical hyphae, 2.5–10 μm wide. Stipitipellis a cutis composed of parallel, compactly arranged, thin-walled, non-gelatinized, hyaline hyphae, 2.5–6 μm wide. Stipititrama composed of interwoven, hyaline, cylindrical hyphae, 3–10 μm wide. Caulocystidia absent. Clamp connections absent.

Habit

Scattered or in groups on soil in broad-leaved (Quercus) forest, Shanxi province, China.

Additional specimens examined

China. Shanxi province, Xia County, alt. 970m, 7 October 2020, N. Mao MNM172 (BJTC FM1618).

Note

Clitocella borealichinensis is easily confused with C. orientalis, C. obscura (Pilát) Vizzini et al. and C. pallescens Silva-Filho & Cortez in morphology because they are all have white to grayish white pileus and decurrent lamellae. However, C. orientalis differs from C. borealichinensis by its viscid pileus and stipe when wet, gelatinized pileipellis and stipitipellis, and slightly larger basidiospores of (4–)4.5–6 × 4–5 μm (Jian et al. 2020). Clitocella obscura produce a distinctly reddish reaction when 3% KOH is placed on the pileus surface (Baroni 1981; as Rhodocybe) while C. borealichinensis has not that kind of reaction. Clitocella pallescens differs C. borealichinensis by its pale grey to yellowish white stipe (Silva-Filho et al. 2018; Jian et al. 2020).

Clitocella mundula and C. popinalis clustered with C. borealichinensis in our multilocus phylogeny (Fig. 1), indicating they are phylogenetically closely related to each other. Morphologically, C. mundula differs from C. borealichinensis by its yellowish gray or brown to dark smoke gray pileus and slightly larger basidiospores of (4–)4.5–6(–6.5) × 4–5 μm (Jian et al. 2020), C. popinalis by its brown to grayish brown pileus, bigger basidiospores of 5.5–7–5–5.5 μm, and its pileus surface produces a reddish reaction in 3% KOH (Baroni 1981; as Rhodocybe). Moreover, DNA analysis also revealed that C. borealichinensis shared less than 91.10% similarity in tef1 sequence with C. mundula and 91.20% similarity with C. popinalis, supporting their separation.

Clitocella colorata L. Fan & N. Mao, sp. nov.

MycoBank No: 843690
Figs 3b–d, 5, 6c, d

Etymology

colorata, referring to the colorful pileus.

Holotype

China. Shanxi Province, Pu County, Wulushan Mountain, 36°33.2'N, 111°11.58'E, alt. 1740 m, 28 July 2021, on the ground in coniferous forest dominated by Pinus armandii Franch., N. Mao MNM292 (BJTC FM1891).

Figure 5. 

Microscopy of Clitocella colorata a basidiospores b basidia c pileipellis. Scale bars: 10 μm (a, c); 5 μm (b). Drawings by Ning Mao.

Diagnosis

Clitocella colorata is characterized by its clitocyboid basidiomata, relatively colorful pileus (white to yellowish white, grayish white to grayish brown, pink white), globose or subglobose to broadly ellipsoid basidiospores, hyphae of pileipellis with pale yellow to yellowish brown intracellular or parietal pigment, the absence of hymenial cystidia and clamp connection. It is most similar to C. popinalis and C. mundula but differs from C. popinalis by its slightly smaller basidiospores, only appearing in the forest and genetic profile, and from C. mundula by its colorful pileus (white to yellowish white, grayish white to grayish brown, pink white).

Figure 6. 

Basidiospores of species in Clitocella. Clitocella revealed by SEM a, b Clitocella borealichinensis c, d Clitocella colorata Scale bars: 3 μm (a, b); 5 μm (c, d). Photos by Li Fan.

Description

Basidiomata clitocyboid, small to large. Pileus 20–62 mm wide, dry,convex to plano-convex, sometimes infundibuliform, with a shallow depression at the center; margin not striate, often enrolled or flat, sometimes slightly uplifted; surface white (#ffffff) to yellowish white (#ffffe7), grayish white (#f2f2f2) to grayish brown (#dba773), pink white (#fff3f5); context white (#ffffff) to grayish white (#f2f2f2), 1.0–1.5 mm thick. Lamellae decurrent, white (#ffffff) to yellowish white(#fff3e7), becoming yellowish brown (#e0b487) on drying, crowded, 1.0–2.0 mm deep, edges entire and concolorous, thin and fragile, lamellulae in 2–4 tiers of varying lengths. Stipe 22–42 × 4–10 mm, central, cylindrical, equal, pale white (#ffffff) to yellowish brown (#e0b487), smooth, usually with white rhizomorphs. Odor unrecorded. Taste not recorded. Chemical color reaction: pileal surface of dried samples negative with 3% KOH.

Basidiospores [100/5/2] (3.8–)4.5–5.5(–6.0) × (3.5–)4–4.8(–5.0) μm; Lm × Wm = 4.90 (± 0.44) × 4.29 (± 0.35), Q = 1.00–1.25 (Qav = 1.14 ± 0.09); hyaline, globose or subglobose to broadly ellipsoid in profile view, slightly angled in polar or face view with obscure minute pustules or bumps. Basidia 20–30 × (4.5–)5–6.5 μm, clavate, hyaline, with four spored, rarely two spored; sterigmata 2–3.5 μm long. Lamellar trama composed of subparallel, hyaline, cylindrical hyphae, 2.5–6 μm wide, subhymenium consisting of filamentous hyphal segments, 2–3.5 μm wide. Lamellae edges fertile. Pleurocystidia and cheilocystidia absent. Pileipellis a cutis composed of parallel, compactly arranged, non-gelatinized, smooth, cylindrical hyphae, 2–5 μm wide, with pale yellow to yellowish brown intracellular or parietal pigment; subcutis made up of interwoven, slightly loosely arranged, hyaline, smooth, cylindrical hyphae, 3–6.5 μm wide; pileal trama composed of parallel, compactly arranged, hyaline, cylindrical hyphae, 3–10 μm wide. Stipitipellis a cutis composed of parallel, compactly arranged, thin-walled, non-gelatinized, cylindrical hyphae, 2–5 μm wide, heavily or moderately encrusted with brown pigment. Stipititrama composed of parallel, compactly arranged, hyaline, cylindrical hyphae, 3–7 μm wide. Caulocystidia absent. Clamp connections absent.

Habit

Scattered or in groups on soil or rotten wood in coniferous (Pinus) or broad-leaved (Quercus) forest, Shanxi province, China.

Additional specimens examined

China. Shanxi province, Pu County, Wulushan Mountains, alt. 1750m, 28 July 2021, N. Mao MNM293 (BJTC FM1892); Wenshui County, alt. 1760m, 30 July 2021, L. Fan CF1219 (BJTC FM1952); Xia County, alt. 931m, 6 October 2020, N. Mao MNM102 (BJTC FM1593); Xia County, alt. 931m, 6 October 2020, N. Mao MNM103 (BJTC FM1594).

Notes

Morphologically, Clitocella colorata is easily confused with C. mundula and C. popinalis. However, according to Baroni (1981; as Rhodocybe), the pileus surface in C. mundula and C. popinalis can produce a reddish reaction in 3% KOH, whereas that is not exhibited in Clitocella colorata. The basidiospores of C. popinalis, 5.5–7 × 5–5.5 μm (Baroni 1981; Kluting et al. 2014; Jian et al. 2020), are broader and longer than those of C. colorata (4.5–5.5 ×4–4.8 μm). DNA analysis revealed that C. colorata shared less than 87.80% similarity in tef1 sequence with C. mundula and 86.10% similarity with C. popinalis, supporting their separation. Moreover, five ITS sequences (FJ770397, KR673647, KU561066, MK217434 and MK573922) labelled “C. popinalis” from India, Norway, South Korea, UK and USA are probably conspecific to the new species C. colorata as they clustered together with C. colorata in ITS tree (Fig. 2) and have more than 98.4% similarity in ITS region. However, these “C. popinalis” collections still need more other DNA regions and detailed morphology to support this view. One collection of “C. mundula,” namely, AFTOLID 521 from Norway, should be re-identified C. colorata as it clustered together with C. colorata in the combined nrLSU-rpb2-tef1-atp6 tree (Fig. 1) and have more than 98.1% similarity in tef1 region. These showed that the new species C. colorata maybe have a wide geographical distribution. Although C. orientalis is sister to C. colorata with strong support, these two species have obvious differences in morphology. The pileus and stipe of C. orientalis are usually viscid when wet and have gelatinized pileipellis and stipitipellis. Clitocella colorata has non-gelatinized pileipellis and stipitipellis, and its pileus is more colorful and darker (Jian et al. 2020). DNA analysis revealed that C. colorata shared less than 95.80% similarity in tef1 sequence with C. orientalis and 90.20% similarity in ITS sequence. Moreover, C. colorata has a wider distribution range than C. orientalis, which is only distributed in China.

Discussion

Three species of Clitocella are confirmed from Shanxi Province, north China in this study. Of them, C. colorata is the most commonly encountered species, which distributes across the provincial area and grows in almost all kinds of forest. Clitocella orientalis and Clitocella borealichinensis are probably limited in southern Shanxi province, and they usually occur in the Quercus spp. forests.

ITS gene is rarely used in the species classification of Clitocella in previous studies because it contains many ambiguous sites. In the contrast, the partial sequences of three protein-coding genes (the atp6, rpb2 and tef1) are usually used to infer the phylogeny of Clitocella (Kluting et al. 2014; Baroni et al. 2020; Jian et al. 2020). However, we found that ITS, rpb2, and tef1 gene tree are similar to the combined (nrLSU-rpb2-tef1-atp6) gene regions tree when we performed phylogenetic tree construction respectively using the ITS, nrLSU, rpb2, tef1 and atp6 gene of Clitocella (Fig. 2, Suppl. material 1–4: Figs S1–S4). DNA analysis also showed that the intraspecific similarity of the ITS region is ≥ 98.4% and of tef1 gene is ≥ 98.1%, the interspecific similarity of ITS region is ≤ 96.1% and of tef1 is ≤ 95.9% (Table 2, Table 3). But for the rpb2 gene, the intraspecific variation of C. mundula is more than the interspecific variation of C. colorata and C. orientalis (Table 4). Therefore, we consider that both the ITS and tef1 may be more effective for the classification of Clitocella species.

Table 2.
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Interspecific variation and intraspecific variation of ITS in Clitocella species.

Species Number (n) Intraspecific variation (%) Interspecific variation (%)
Clitocella colorata 9 < 1.6% > 3.9%
C. fallax 3 < 0.3% > 11.8%
C. mundula 1 – > 6.0%
C. borealichinensis 2 – > 9.6%
C. obscura 1 – > 6.6%
C. orientalis 3 < 0.9% > 3.9%
Table 3.
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Interspecific variation and intraspecific variation of tef1 in Clitocella species.

Species Number (n) Intraspecific variation (%) Interspecific variation (%)
Clitocella colorata 4 < 1.9% > 4.1%
C. fallax a 1 – > 9.8%
C. fallax b 2 < 0.1% > 9.8%
C. mundula 6 < 0.3% > 7.5%
‘C. mundula’c 1 – > 4.7%
C. borealichinensis 1 – > 8.4%
C. orientalis 3 < 0.1% > 4.1%
C. popinalis 7 – > 4.7%

a represents voucher 25668OKM; b represents voucher O-F88953, K(M): 116541; c represents voucher O-F19454

Our molecular phylogenetic analysis (Fig. 1) revealed that one Norway collection O-F19454, which is labelled as Clitocella mundula, formed an independent clade, and it shared less than 93.40% similarity in tef1 sequence with other Clitocella species. These show that it probably represents a new species of Clitocella. The sequences of Clitocella fallax formed two or three (in rpb2 phylogeny) independent branches in our phylogenetic analyses (Fig. 2, Suppl. material 1–4: Figs S1–S4), and the similarity between the branches is less than 90.2% in tef1 sequence and 94.9% in rpb2 sequence. These indicate that these specimens of C. fallax probably represented two or three species. The specimens of C. fallax should be therefore re-examined to resolve this taxonomic issue. Clitocella termitophila is not clustered in the genus Clitocella (Fig. 1). Moreover, in the rpb2 gene tree C. termitophila did not gather with Clitocella, Clitopilopsis or Clitopilus but formed a single branch (Suppl. material 2: Fig. S2). These indicate that Clitocella termitophila probably represents a potential taxonomic position rather than the species of Clitocella.

Table 4.
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Interspecific variation and intraspecific variation of rpb2 in Clitocella species.

Species Number (n) Intraspecific variation (%) Interspecific variation (%)
Clitocella colorata 4 < 0.7% > 1.7%
C. fallax a 1 – > 4.0%
C. fallax b 4 < 0.1% > 5.1%
C. fallax c 1 – > 4.0%
C. mundula 6 < 2.1% > 4.9%
‘C. mundula’d 1 – > 2.2%
C. borealichinensis 2 – > 5.5%
C. orientalis 6 < 0.5% > 1.7%
C. popinalis 9 < 0.4% > 2.2%
C. termitophila 1 – > 16.9%

a represents voucher 25668OKM; b represents voucher O-F88953, K(M): 116541, CBS 129.63, ME Noordeloos 1997173; c represents voucher ME Noordeloos 200367; d represents voucher O-F19454.

Key to the species of Clitocella

1 Basidiomata clitocyboid 2
– Basidiomata pleurotoid C. termitophila * (Baroni et al. 2020)
2 Pileus surface gray, dark gray, pale yellow to yellowish brown, pigments present in pileipelli 3
– Pileus surface almost white to pastel gray, pigments absent in pileipellis 8
3 Basidiospores globose to subglobose 4
– Basidiospores ellipsoid 7
4 Pileus surface of dried samples with a positive KOH reaction 5
– Pileus surface of dried samples with a negative KOH reaction 6
5 Occurring in grassland systems C. popinalis *(Baroni 1981; Kluting et al. 2014; Jian et al. 2020)
– Occurring in forested systems C. mundula *(Baroni 1981; Kluting et al. 2014; Jian et al. 2020)
6 Pileus color with pink tinges C. colorata *
– Pileus color without pink tinges C. borealichinensis *
7 Pileus color with yellow tinges, basidiospores small, 5–8 × 3.5–5.5 μm C. himantiigena (Silva-Filho et al. 2018)
– Pileus color without yellow tinges, basidiospores large, 7–9 × 5–6 μm C. ammophila (Contu 1999)
8 Basidiospores globose to subglobose or ovatae 9
– Basidiospores amygdaliform to ellipsoid 11
9 Basidia long, length > 40 μm C. nigrescens (Maire 1945)
– Basidia short, length < 28 μm 10
10 Pileus infundibuliform to plano-convex, basidiospores 4–5 × 3–4.5 μm C. pallescens (Silva-Filho et al. 2018; Jian et al. 2020)
– Pileus convex to plane, basidiospores (4–)4.5–6 × 4–5 μm C. orientalis * (Jian et al. 2020)
11 Basidiospores small, 5–6.2 × 2.5–3.6 μm C. blancii (Contu 2009)
– Basidiospores large, 6.5–8 × 4–5 μm C. fallax * (Jian et al. 2020)

Acknowledgements

We extend our appreciation to Dr. J.Z. Cao for collecting specimens and providing valuable suggestions. The study was supported by the National Natural Science Foundation of China (No. 31750001) and the Beijing Natural Science Foundation (No. 5172003).

References

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1 Indicates the presence of molecular data.

Supplementary materials

Supplementary material 1 

Figure S1

Ning Mao, Jing-Chong Lv, Yu-Yan Xu, Tao-Yu Zhao, Li Fan

Data type: JPG file

Explanation note: Phylogeny derived from Maximum Likelihood analysis of the nrLSU dataset of Clitocella and related genera in the family Entolomataceae. The bootstrap frequencies (> 70%) is shown on the supported branches. New species are highlighted in red.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (865.78 kb)
Supplementary material 2 

Figure S2

Ning Mao, Jing-Chong Lv, Yu-Yan Xu, Tao-Yu Zhao, Li Fan

Data type: JPG file

Explanation note: Phylogeny derived from Maximum Likelihood analysis of the rpb2 dataset of Clitocella and related genera in the family Entolomataceae. The bootstrap frequencies (> 70%) is shown on the supported branches. New species are highlighted in red.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (1.26 MB)
Supplementary material 3 

Figure S3

Ning Mao, Jing-Chong Lv, Yu-Yan Xu, Tao-Yu Zhao, Li Fan

Data type: JPG file

Explanation note: Phylogeny derived from Maximum Likelihood analysis of the tef1 dataset of Clitocella and related genera in the family Entolomataceae. The bootstrap frequencies (> 70%) is shown on the supported branches. New species are highlighted in red.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (1.02 MB)
Supplementary material 4 

Figure S4

Ning Mao, Jing-Chong Lv, Yu-Yan Xu, Tao-Yu Zhao, Li Fan

Data type: JPG file

Explanation note: Phylogeny derived from Maximum Likelihood analysis of the atp6 dataset of Clitocella and related genera in the family Entolomataceae. The bootstrap frequencies (> 70%) is shown on the supported branches. New species are highlighted in red.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (1.07 MB)
Supplementary material 5 

ITS alignment

Ning Mao, Jing-Chong Lv, Yu-Yan Xu, Tao-Yu Zhao, Li Fan

Data type: PHY file

Explanation note: The ITS dataset comprised 27 samples of 11 taxa and 662 characters.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (19.05 kb)
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