﻿Multiple evidence reveals two new species and new distributions of Calocybe species (Lyophyllaceae) from northeastern China

﻿Abstract The Calocybe species possess notable economic and medicinal value, demonstrating substantial potential for resource utilization. The taxonomic studies of Calocybe are lacking in quality and depth. Based on the specimens collected from northeast China, this study provides a detailed description of two newly discovered species, namely Calocybebetulicola and Calocybecystidiosa, as well as two commonly found species, Calocybedecolorata and Calocybeionides. Additionally, a previously unrecorded species, C.decolorata, has recently been discovered in Jilin Province, China. The two newly discovered species can be accurately distinguished from other species within the genus Calocybe based on their distinct morphological characteristics. The primary distinguishing features of C.betulicola include its grayish-purple pileus, grayish-brown to dark purple stipe, smaller basidiomata, absence of cellular pileipellis, and its habitat on leaf litter within birch forests. Calocybecystidiosa is distinguished by its growth on the leaf litter of coniferous forests, a flesh-pink pileus, a fibrous stipe with a white tomentose covering at the base, non-cellular pileipellis, larger basidiospores, and the presence of cheilocystidia. The reconstruction of phylogenetic trees using combined ITS, nLSU, and tef1-α sequences, employing maximum likelihood and Bayesian inference analyses, showed that C.betulicola formed a cluster with C.decurrens, while C.cystidiosa clustered with C.vinacea. However, these two clusters formed separate branches themselves, which also supported the results obtained from our morphological studies. A key to the Calocybe species reported from northeast China is provided to facilitate future studies of the genus.


Introduction
The genus Calocybe Kühner ex Donk is widely distributed in the Northern Hemisphere and has significant economic value.It belongs to the family Lyophyllaceae.However, the genus Calocybe is always neglected by researchers.The genus Calocybe was officially published in 1962 and is typified by Calocybe gambosa (Fr.)Donk (Donk 1962).At first, it was treated as a section of Lyophyllum P. Karst.(Kühner and Romagnesi 1953).Then, Singer (1962) elevated it to genus rank based on the obvious colorful pileus, separated it from Lyophyllum, and belongs to the family Lyophyllaceae.Moreover, Singer divided Calocybe into five sections, namely Sect.Calocybe Singer, Sect.Echinosporae Singer, Sect.Heterosporae Singer, Sect.Pseudoflammulae Singer, and Sect.Carneoviolaceae Sing, by the combination of three characterizes, viz. the color of pileus, spores, and types of pileipellis (Singer 1962).Later, Singer (1986) assigned Sect.Heterosporae to the genus Lyophyllum, and, thus, the genus Calocybe was divided into four sections (Singer 1986).
By applying molecular methods to research Calocybe, it was reconfirmed that Calocybe is separate from the genus Lyophyllum and belongs to the Lyophyllaceae family (Hofstetter et al. 2002;Moncalvo et al. 2002;Matheny et al. 2006;Garnica et al. 2007).However, the taxonomic systems of Calocybe were full of arguments.Hofstetter et al. (2002) and Matheny et al. (2006) revealed that Calocybe formed a monophyletic group when the combined ITS, nLSU, or mitSSU fragments were used in phylogenetic analysis.Nevertheless, Bellanger concluded from a multi-gene phylogenetic analysis that Calocybe forms a monophyletic clade with Rugosomyces Raithelh (Bellanger et al. 2015).Vizzini et al. (2015), Li et al. (2017), and Xu et al. (2021a) also conducted a familiar conclusion with Bellanger.Recent research suggests that the genus Calocybe could be divided into five main clades.Based on continuous studies, 62 species of Calocybe are listed in the Index Fungorum (www.indexfungorum.org,accessed 20 March 2023).
There have been few studies focusing on the taxonomic and molecular studies of the genus Calocybe in China until now.Tai (1979) first reported Calocybe species from China, however, under the name Lyophyllum leucophaeatum (P.Karst.)P. Karst., later, confirmed to be Calocybe gangraenosa (Fr.)V. Hofst., Moncalvo, Redhead & Vilgalys.Seven species were recorded from China (Mao 2000;Bau et al. 2003;Fan and Bau 2006).Furthermore, a preliminary taxonomic study on Calocybe was performed in recent years (Zhou 2022).And recently, more than ten new species have been described in northeast China (Li et al. 2017;Xu et al. 2021aXu et al. , 2021b;;Qi et al. 2022;Mu and Bau 2023), and proposed their perspective on the taxonomic systematic on Calocybe.As a result, a total of 19 species of Calocybe have been reported, including Calocybe aurantiaca X.D. Yu & Jia J. Li, Calocybe badiofloccosa J.Z. Xu & Yu Li, and Calocybe carnea (Bull.) Donk, etc.This study aims to describe and illustrate two new species, one new record from Jilin Province, and one common species based on both morphological and molecular data.Additionally, a key to the reported Calocybe species from northeast China is provided.

Sampling and morphological studies
The studied specimens were photographed in situ.The size of the basidiomata was measured when fresh.After examination and description of the fresh macroscopic characters, the specimens were dried in an electric drier at 40-45 °C (Hu et al. 2022a(Hu et al. , 2022b)).
Descriptions of the macroscopic characteristics were based on field notes and photographs, with the colors corresponding to the Flora of British fungi: colour identification chart (Royal Botanic Garden 1969).The dried specimens were rehydrated in 94% ethanol for microscopic examination, and then mounted in 3% potassium hydroxide (KOH), 1% Congo red (0.1 g Congo red dissolved in 10 mL distilled water), and Melzer's reagent (1.5 g potassium iodide, 0.5 g crystalline iodine, and 22 g chloral hydrate dissolved in 20 mL distilled water) (César et al. 2018); they were then examined with a Zeiss Axio lab.A1 microscope at magnifications up to 1000 ×.All measurements were taken from the sections mounted in the 1% Congo red.For each specimen, a minimum of 40 basidiospores, 20 basidia, 20 cheilocystidia, and 20 widths of pileipellis were measured from two different basidiomata.When reporting the variation in the size of the basidiospores, basidia, cheilocystidia, and width of the pileipellis, 5% of the measurements were excluded from each end of the range, and are given in parentheses.The basidiospores measurements are given as length × width (L × W).Q denotes the variation in the ratio of L to W among the studied specimens, Qm denotes the average Q value of all the basidiospores ± standard deviation.The specimens examined have been deposited in the Herbarium of Mycology of Jilin Agricultural University (HMJAU).

DNA extraction, PCR amplification and sequencing
The total DNA was extracted from dried specimens using the NuClean Plant Genomic DNA Kit (Kangwei Century Biotechnology Company Limited, Beijing, China), according to the manufacturer's instructions.Sequences of the internal transcribed spacer region (ITS), nuclear large ribosomal subunits (nLSU), and translation elongation factor (tef-1α) were used for phylogenetic analysis.The ITS sequence was amplified using the primer pair ITS4 and ITS5 (Gardes and Burns 1993), and the nLSU sequence was amplified using the primer pair LROR and LR5 (Vilgalys and Hester 1990;Cubeta et al. 1991), and tef1-α regions were using tef1-F and tef1-R (Rehner and Samuels 1994).PCR reactions (25 μL) contained dd H 2 O 9.5 μL, 2 × Taq PCR MasterMix 12.5 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, DNA sample 2 μL.Cycle parameters were as follows 2 min at 94 °C; 35 s at 95 °C, 35 s at 48 °C, 1 min at 72 °C for 30 cycles; 10 min at 72 °C; storage at 4 °C (Xu et al. 2021a(Xu et al. , 2021b)).The PCR products were visualized via UV light after electrophoresis on 1.2% agarose gels stained with ethidium bromide and purified using the Genview High-Efficiency Agarose Gels DNA Purification Kit (Gen-View Scientific Inc., Galveston, TX, USA).The purified PCR products were then sent to Sangon Biotech Limited Company (Shanghai, China) for sequencing using the Sanger method.The new sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank;Table 1).

Data analysis
Based on the results of BLAST and morphological similarities, the sequences obtained and related to these samples were collected and are listed in Table 1.The dataset of ITS, nLSU, and tef1-α resign comprised sequences from this study, with 67 representative sequences showing the highest similarity to Calocybe spp.This dataset included all Calocybe species with sequences deposited in GenBank to further explore the relationships of the newly sequenced Chinese specimens within the genus.Moreover, representative species within family Lyophyllaceae were also included to explore the relations within it.The sequences of Tricholoma terreum (Schaeff.)P. Kumm.were selected as the outgroup taxon.
Of the dataset, each gene region was aligned using Clustal X (Thonpson et al. 1997), MACSE 2.03 (Ranwez et al. 2018), or MAFFT 7.490 (Katoh and Standley 2013), and then manually adjusted in BioEdit 7.0.5.3 (Hall 1999).The datasets first were aligned, and then the ITS, nLSU, and tef1-α sequences were combined with Phylosuite 1.2.2 (Zhang et al. 2020).The best-fit evolutionary model was estimated using Modelfinder (Kalyaanamoorthy et al. 2017).Following the models, Bayesian inference (BI) algorithms were used to perform the phylogenetic analysis.Specifically, BI was calculated with MrBayes 3.2.6 with a general time-reversible DNA substitution model and a gamma distribution for rate variation across the sites (Ronquist and Huelsenbeck 2003).Four Markov chains were run for two runs from random starting trees for two million generations until the split deviation frequency value was < 0.01; the trees were sampled every 100 generations.The first 25% of the sampled trees were discarded as burn-in, while all the remaining trees were used to construct a 50% majority consensus tree and for calculating the Bayesian posterior probabilities (BPPS).RaxmlGUI 2.0.6 (Edler et al. 2021) was used for maximum likelihood (ML) analysis along with 1,000 bootstraps (BS) replicates using the GTRGAM-MA algorithm to perform a tree inference and search for the optimal topology.Then the FigTree 1.3.1 was used to visualize the resulting trees.

Phylogenetic analysis
The concatenated matrix contained 106 sequences (40 for nLSU, 58 for ITS, and eight for tef1-α) representing 61 samples were used to build a phylogenetic analysis (the concatenated matrix was deposited at treebase under the acc.no.S31166).Modelfinder selected the best-fit model for the combined dataset, and the best fit model for BI is GTR+F+I+G4.The results of the Bayesian analysis (Fig. 1) and the maximum likelihood analysis (Fig. 2) are generally in agreement.
After trimming, the combined ITS, nLSU, and tef1-α dataset represented 46 taxa and 3120 characters.The Bayesian analysis was run for two million generations and resulted in an average standard deviation of split frequencies of 0.009440.The same dataset and alignment were analyzed using the ML method.Six clades were revealed within Lyophyllaceae, representing Calocybe, Tricholomella Zerova ex Kalamees, Tephrocybe Donk, Asterophora Ditmar, Lyophyllum, and Hypsizygus Singer (Figs. 1 and 2).Moreover, from our results, the genus Calocybe was split into six independent clades, representing five sections and one newly recognized clade.Five sampled specimens formed two independent clades, representing two new species, C. betulicola and C. cystidiosa.Etymology."betulicola" refers to this species that grows on the leaf litter of Betula forests.
Basidiospores Comments.Calocybe betulicola is characterized by its grayish-purple pileus, grayish-brown to dark purple stipe, smaller basidiomata, non-cellular pileipellis, and its growth on the leaf litter in birch forests.According to these characteristics, C. betulicola is a member of Sect.Carneoviolaceae.Sect.Carneoviolaceae mainly includes four other species, viz.Calocybe decurrens J.Z. Xu & Yu Li, Calocybe fulvipes J.Z. Xu & Yu Li, Calocybe ionides (Bull.)Donk, and Calocybe coacta J.Z. Xu & Yu Li.This species is macroscopically similar to C. ionides due to the purple basidiomata.However, C. betulicola differs from C. ionides in terms of its unique habitat, subdecurrent lamellae, and wider basidiospores.Calocybe decurrens has an intimate affinity in phylogenetic analysis.However, it differed from C. betulicola by the gradual fading from pinkish purple to brownish red to grayish brown stipe, carneous pileus, and larger basidiospores ((5.8) 6.0-8.5 (9.3) × (2.1) 2.7-3.8(4.3) μm) (Xu et al. 2021b).Calocybe fulvipes differs by its tone brown to dark violet stipe, and the changes it undergoes when injured, bigger Qm, and slightly longer sterigmata (Xu et al. 2021a).Calocybe coacta can be distinguished from C. betulicola by its cream-gray pileus, the presence of hymenial cystidia, and larger basidiospores (Xu et al. 2021a).Diagnosis.This species is differentiated from other species by its fresh-pink basidiomata, uncurved margin of the pileus, whitish pink stipe covered with tomentose at the base, lager basidiospores, and the presence of cheilocystidia.
Habitat.Grows on the leaf litter in coniferous forests.
Comments.This species is characterized by its growth on the leaf litter in coniferous forests, flesh-pink pileus, fibrous stipe covered with white tomentose at the base, non-cellular pileipellis, larger basidiospores, and the presence of cheilocystidia.These characteristics suggest that C. cystidiosa belongs to Sect.Carneoviolaceae according to Singer's opinion (Singer 1986).
This species is closely related to C. carnea due to its pinkish pileus.However, this species can be distinguished from C. carnea by its unique habitat, deep color of basidiomata, light yellow lamellae, and larger basidiospores.In the Sect.Carneoviolaceae, C. vinacea J.Z. Xu & Yu Li is another species recorded from China with pinkish basidiomata.However, C. vinacea differs from this species by the curved margin of pileus, white stipe, smaller basidiospores, and the absence of cystidia (Xu et al. 2021b).
Habitat.Grows on the leaves' litter in broad-leaved forests.
Comments.This species was originally described from Liaoning Province, China by Li et al. (2017) and is mainly characterized by a brighter orange or yellow color pileus, light orange-brown stipe, and smaller basidiospores.The species was classified as a species of Sect.Carneoviolaceae based on its main morphological characteristics.
However, there are some differences between our specimen and the type specimen.The specimens observed in this study have bulbous-like terminal hyphae in the pileipellis, which were not described in the type species.
Habitat.Grows on the leaf litter in coniferous or broad-leaved forests.Comments.The main characteristics of this species are small basidiomata, a purple-blue color of the pileus, white lamellae, and a stipe that is either of the same color or lighter than the pileus.According to its main morphological characteristics, this species can be assigned to Sect.Carneoviolaceae.

Discussion
The genus Calocybe exhibits a wide distribution in China, but the full extent of its species diversity remains uncertain.This study provides a detailed description of two new species, namely C. betulicola and C. cystidiosa, as well as one previously unrecorded species, C. decolorata, found in Jilin Province.Additionally, a common species, C. ionides, was also identified in northeastern China.Moreover, the phylogenetic analysis confirmed all of the species that were previously reported.
However, our phylogenetic analysis reveals certain discrepancies when compared to the findings of Li et al. (2017) and Xu et al. (2021a).In the present study, we identified six distinct sectional clades within the genus Calocybe, supported by robust evidence.These clades have been designated as clade I to clade VI.Notably, a new sectional clade, referred to as clade VI, has been identified for the first time in this study.This clade (clade VI) is featured by the presence of a pinkish to reddish pileus and primarily consists of two newly discovered species, namely C. carnea, and C. persicolor, etc.
In addition, Clade I consists of Calocybe onychina (Fr.)Donk, Calocybe naucoria (Murrill) Singer, and Calocybe erminea J.Z. Xu & Yu Li, etc., distinguished by a pileus that ranges in color from white to yellow.The Clade II comprises primarily of Calocybe obscurissima (A.Pearson) M.M. Moser, Calocybe lilacea X.D. Yu, Ye Zhou & W.Q. Qin, Calocybe graveolens (Pers.)Singer, etc., characterized by pileus color ranging from white, yellow to violet shades.The Clade III consistent with Calocybe chrysenteron (Bull.)Singer, C. aurantiaca, and Calocybe pseudoflammula (J.E.Lange) M. Lange ex Singer, and is characterized by a yellow pileus.The main distinguishing characteristics of Clade IV, which includes C. gangraenosa and C. coacta, are the white-colored to grayish-yellow pileus.The Clade V is distinguished by the presence of a gilded pileus and includes two species, Calocybe ochracea (R. Haller Aar.) Bon and Calocybe favrei (R. Haller Aar.& R. Haller Suhr) Bon.
Based on the findings of the present study, we increased the species diversity of the genus Calocybe in China.The taxonomic system of this genus remains a subject of debate due to insufficient species sampling and the inadequate genetic variation in the DNA loci.Therefore, additional evidence is needed to contribute to a more comprehensive understanding of the genus.Furthermore, despite the recent identification of new species of Calocybe from northeast China, the true extent of its species diversity remains uncertain and calls for a comprehensive systematic analysis.

Figure 1 .
Figure 1.Bayesian analysis phylogenetic tree generated from the ITS, nLSU and tef1-α dataset.Bayesian posterior probabilities ≥ 0.95 from BI analysis are shown on the branches.Newly sequenced collections are indicated in bold, and the type specimens are denoted by (T).

Figure 2 .
Figure 2. Maximum likelihood phylogenetic tree generated from the ITS, nLSU and tef1-α dataset.Bootstrap values ≥ 75% from ML analysis are shown on the branches.Newly sequenced collections are indicated in bold, and the type specimens are denoted by (T).
cystidiosa" refers to the presence of cheilocystidia.

Table 1 .
Voucher/specimen numbers, country, and GenBank accession numbers of the specimens included in this study.Sequences produced in this study are in bold.