Morphology and Phylogeny of Lyophylloid Mushrooms in China with Description of Four New Species

The lyophylloid agarics are a group of ecologically highly diversified macrofungi, some of which are very popular edible mushrooms. However, we know little about lyophylloid species diversity in China. In this study, we described four new species from China: Lyophyllum atrofuscum, L. subalpinarum, L. subdecastes, and Ossicaulis sichuanensis. We conducted molecular phylogenetic analyses of Lyophyllaceae based on the nuclear ribosomal RNA gene (nLSU) and the internal transcribed spacer regions (ITS). Phylogenetic analyses by the maximum likelihood method and Bayesian inference showed that the four new species are unique monophyletic species. A key to the species of Lyophyllum from China and a key to Ossicaulis worldwide were given.


Introduction
While the importance of fungi as mutualists, decomposers, and pathogens is undisputed, researchers are just beginning to unravel the processes that shape their global species richness and distribution. Previous studies established the monophyly of Lyophyllaceae Jülich and positioned the family within the Tricholomatoid clade, and then Alvarado et al. revealed that Lyophyllaceae may be a putative wider concept or the existence of multiple lineages that are basal to it [1][2][3]. Several new genera established in the past decade are expected to reorganize the system, including Australocybe T.J. Baroni [1,4,5]. In addition, some species of Lyophyllum, Calocybe Kühner, Hypsizygus Singer, and Termitomyces R. Heim have edible value [6], and a few species have medicinal and significant economic importance [7][8][9][10]. In particular, Lyophyllum shimeji (Kawam.) Hongo has been recognized as a delicacy, and its price is second only to Tricholoma matsutake (S. Ito & S. Imai) Singer in Japan.
Lyophyllaceae has a worldwide distribution [11][12][13], with more than 200 species [14]. The unique characteristic that delimits Lyophyllaceae is the presence of siderophilous granulation in the basidia [15]. Lyophyllum P. Karst., is a type genus of Lyophyllaceae, and more than 40 species within this genus. The morphological classifications of Singer [16] were inconsistent with the molecular phylogenetic relationships of Lyophyllum [17,18], which explains the reason why some species of Lyophyllum used to be easily confused with Calocybe and Tephrocybe Donk [4,19]. The genus of Ossicaulis Redhead & Ginns was erected in 1985, with a north temperate distribution, and four species are known worldwide [20]. It is mainly characterized by adnate, sub-decurrent, or lamellae centrally adnexed to the eccentric stipe, tiny ellipsoid spores, and the presence of clamp connections [21].

Specimen Sampling
All the specimens used in this study were collected in 2018-2021. These samples were dried overnight using an electric oven at 40 • C and deposited in the Herbarium Mycology of Jilin Agricultural University (HMJAU).

Morphological Observation
The macro-morphological descriptions were recorded in the field, and images of the basidiocarps were taken in the field with an OLYMPUS E-P7. The color code and terminology followed Kornerup and Wanscher [31]. Tiny tissue was cut from the dried basidiomata using a sharp blade, and micro-morphological structures were observed via a light microscope (ZEISS Axioscope 5, ZEISS, Jena, Thuringia, Germany) performed in 5% KOH solution and then in Melzer's reagent solution or Acetcoarmine solution. Twenty basidiospores and basidia were measured from each specimen. Dimensions are given as (a)b-c(d), of which 'b-c' refers to the minimum of 90% of the measured values, and a or d represents the extreme values. Factor Q refers to the aspect ratio of each basidiospore in the side view; Lm/Wm defines the average length/width of all measured basidiospores ± sample standard deviation.

DNA Extraction, Amplification, and Sequencing
Total genomic DNA was extracted using the Plant Genomic DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China). The nuclear ribosomal internal transcribed spacer (ITS) and nuclear ribosomal large subunit (nLSU) sequences were amplified using primer pairs of ITS4/ITS5 and LR0R/LR5, respectively [32][33][34]. The reactions were performed with the following program: initial denaturation at 95 • C for 4 min (ITS) or 3 min (nLSU), 35 cycles at 95 • C for 40 s, 58 • C (ITS) for 40 s or 52 • C (nLSU), and 72 • C for 80 s (ITS) or 120 s (nLSU); for terminal elongation the reaction batches were incubated at 72 • C for 10 min. Then, PCR productions were sent to Sangon Biotech Co., Ltd. (Shanghai, China) to be directly sequenced using an ABI 3730xl DNA analyzer.

Phylogenetic Analyses
The newly generated sequences in this study have been deposited in GenBank (https: //www.ncbi.nlm.nih.gov/genbank/, accessed on 10 October 2022), with other similar sequences downloaded from the NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 10 October 2022) datasets, and Entoloma undatum (Gillet) M.M. Moser as the outgroup. For the datasets (see Table 1), the alignment was generated for ITS and nLSU datasets using the "L-INS-i" strategy of MAFFT v.7.017 [35]. Before performing phylogenetic analyses, start and end ambiguous sites were removed, and gaps were manually adjusted to optimize the alignment by BioEdit v7.1.3 [36] and then were combined by Phylosuite v1.2.2 [37]. The best-fit evolutionary model was estimated by using Modelfinder [38]. Phylogenetic analyses were carried out using the Bayes inference (BI) and maximum likelihood (ML). BI analysis using Markov chain Monte Carlo (MCMC) methods were carried out with MrBayes 3.2.6 [39], running in 2,000,000 generations, and sampled every 1000 generations. The initial 25% of the sampled data were discarded as burn-in, other parameters were kept at the default settings. For ML analysis, the datasets were analyzed using IQ-TREE under an ultrafast bootstrap, with 5000 replicates [40]. The posterior probability ≥ 0.95 for Bayesian inference analysis (BI-PP) and bootstrap proportions ≥ 70% for ML analysis (ML-BP) were considered significant values. Trees were edited using FigTree version 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 10 October 2022). Notes: Newly generated sequences in this study are in bold.

Molecular Phylogeny
Fourteen sequences were newly generated from specimens of Lyophyllum, and six sequences were newly generated from specimens of Ossicaulis, all of which were collected from China and deposited in GenBank (Table 1). A combined dataset of two markers, including 1616 bases, was used to execute the BI and ML analyses. Amongst the dataset, 841 were constant sites, 224 were variable and parsimony-uninformative sites, and 551 were parsimony-informative sites. Based on the Bayesian information criterion (BIC), the GTR + F + I + G4 models were selected as the substitution model for the ITS and nLSU partitions. The phylogenetic construction performed by the BI and ML analyses showed a similar topology. Therefore, we selected the ML tree as the working phylogenetic hyphothesis, with the Bayesian posterior probabilities ≥0.95 and ML bootstrap values ≥ 70% labeled along the branches (Figure 1). In the phylogram, L. subdecastes, L. decastes, L. fumosum, and L. shimeji were grouped in Clade I (sect. Difformia (Singer)). Lyophyllum ambustum (Fr.) Singer, L. anthracophilum (Lasch) M. Lange & Sivertsen, and L. atratum (Fr.) Singer were grouped in Clade II. Lyophyllum atrofuscum related to L. moncalvoanum was grouped in Clade III (sect. Lyophyllum (Singer)) with weak support. Lyophyllum subalpinarum related to L. cf. pulvis-horrei and L. semitale grouped were in Clade III (sect. Lyophyllum (Singer)) showed weak support. Pileus 2.5-9.0 cm broad, hemispherical to broadly convex when young, becoming plane to plano-concave, often with depressed, center and in-rolled margin when mature, light brown (6D4), yellowish-brown (5D8), brownish-orange (7C3), to grayish-brown (7D3), dark at the center. Sometimes margin wavy, pileus context watery soaked in wet weather conditions, brown (7E8) or reddish-brown (8D6). It was staining bluish-gray to black immediately when broken. Context white, up to 0.5 cm thick at the pileus center, is thinner toward its margin and discolored when exposed or injured. Lamellae adnate to slightly decurrent, moderately broad, tapering toward the margin white at first, becoming yellowish-white (4A2), yellowish-white (3A2), to brownish-gray (6C3) with age, usually discoloring to dark at the edge when touched or injured, with 1-3 unequal lamellulae between two entire lamellae. Stipe 3.0-10.0 cm long, 0.7-1.5 cm thick, cylindrical to clavate, usually equal, occasionally enlarged at the base, white at first, grayish-white (6B1) with brownish-gray (6C2) when mature, usually paler than the pileus, sometimes nearly black in the upper, scarcely darkening below, longitudinally fibrillose, base white-mycelioid.
Known distribution. Known to occur in the Xizang Autonomous Region at high elevations (usually above alt. 3000 m) in Southwest China.
Habit and habitat. Single to scattered on soil in forests dominated by Spruce forest, from August to October.
Phylogenetically, the new species is closely related to L. semitale and L. maleolens. However, L. semitale from Korea and North Carolina (in the United States) is bigger in size regarding the basidiospore (3.0-8.0 cm); at the same time, darker-colored pileus and pale gray to brownish-gray lamellae also help to distinguish it from the new species [53,54]. Additionally, L. maleolens from Spain is characterized by a brown to dark fuscous-brown pileus, and the stipe is wider than that of the new species [48].