Five New Species of Gymnopilus from Xizang Autonomous Region of China and Surrounding Areas

The species of Gymnopilus (Hymenogastraceae, Agricales) are commonly recognized as wood-decaying fungi. Certain members of this genus have been identified as psilocybin-producing mushrooms. Gymnopilus exhibits a diverse range and has a global distribution. In this study, a total of seventy-eight specimens were gathered from ten provinces in China. A comprehensive molecular phylogenetic analysis was conducted, employing gene sequences including ITS, nrLSU, nrSSU, rpb1, rpb2, and tef1-α. Additionally, morphological examinations were also carried out. The phylogenetic topology of Gymnopilus from this study generally agreed with previous studies and facilitated the identification of all those specimens. As a result, eleven species, including five newly discovered ones named Gy. gyirongensis, Gy. variisporus, Gy. tomentosiceps, Gy. tenuibasidialis, and Gy. aurantipileatus, were recognized. Significantly, four of the five newly identified species are native to the Xizang Autonomous Region, emphasizing their specialization in this distinctive habitat. This research contributes to our comprehension of Gymnopilus diversity and lays the groundwork for the conservation and sustainable utilization of Gymnopilus resources.

The taxonomic study of Gymnopilus started relatively late in China, and Shu-Qun Deng (1963) was the first person to record Gymnopilus species from China in his monumental work Fungi of China.Recently, Li (2012) reported a total of thirty Gymnopilus species, including six new species from the tropical region [7,21].Liu (2019) reported a species from northeast China, and she also constructed a phylogenetic tree of Gymnopilus based on ITS sequences.This phylogenetic tree, which was identical to those of previous studies, showed six stable clades [5,7,12].In terms of geographic distribution, most Gymnopilus species were found in parts of southern China, such as the Guangdong, Yunnan, Sichuan, and Hainan provinces, and some were from northeastern China (Jilin province).Generally, the species of Gymnopilus in China is poorly reported, and up to now, only thirty-one species have been recorded [7,21].
In this study, we collected seventy-eight Gymnopilus specimens from ten provinces of China and two from Thailand.Molecular phylogenetic analysis combined with morphological examination revealed they belong to eleven species, of which five are proposed as new species.

Specimen Collection and Morphological Study
We collected specimens in the field from the Xizang Autonomous Region and surrounding provinces of China and Thailand.The macroscopic features of the collected specimens were photographed and recorded, along with their odor and the fresh specimens' changes in color upon injury.The specimens were dried completely overnight at 55 • C using a food desiccator, sealed in plastic bags, and deposited in the Herbarium Mycologicum Academiae Sinicae, Beijing, China (HMAS).
The anatomical and cytological features, including lamellae, pileipellis, basidiospores, basidia, and cystidia, of the dried specimens were observed following the protocols described in [22].For microscopic characterization, the sections were mounted in 5% KOH solution.Thirty measurements of mature spores were taken for each specimen, along with twenty measurements of basidia and cystidia, which included x, the mean of length by width ± SD; Q, the quotient of basidiospore length to width; and Qm, the mean of Q-values ± SD [23].The hymenial cystidia and pileocystidia were stained with 1% aqueous Congo red solution [24].The color designation referred to methuen handbook of colour.

DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was extracted from 5 to 10 mg of dried specimen using a Broadspectrum Plant Rapid Genomic DNA Kit (Biomed, Beijing, China) and preserved at −20 • C.

Molecular Phylogenetic Study
The sequences produced from this study and some generated by a previous work and deposited in the NCBI GenBank database were used in our phylogenetic analyses [2,7,9,19,20,32,33] (Table 1).Sequences of multigene data were aligned separately using Muscle version 3.6 [34], then manually adjusted to remove ambiguous regions in BioEdit version 7.0.4[35].The six partitions were assembled in PhyloSuite v1.2.2 [36] in the order of six loci (ITS, nrLSU, nrSSU, rpb1, rpb2, tef1-α).Maximum likelihood (ML) analysis was performed using RAxmlGUI 1.3 under a GTRGAMMA model with one thousand rapid bootstrap (BS) replicates [37].Bayesian Inference (BI) analysis was performed using MrBayes v3.2.6 [38].Six Markov chains were run for 2,000,000 generations, and trees were sampled every 100th generation.Burn-ins were determined in Tracer version 1.6 with an ESS value higher than 200, and the remaining trees were used to calculate Bayesian posterior probabilities (PP).The trees were displayed in Fig Tree version 1.4.0 [39].
A total of fifty-nine ITS sequences and four nrLSU sequences of Gymnopilus were downloaded from GenBank for phylogenetic analysis.Galerina marginata (Batsch) Kühner was selected as the outgroup.The newly generated sequence samples from this study have been deposited in GenBank.
The resulting file after tree construction was used to view the phylogenetic tree using iTOL [40].Bootstrap Support (BS) ≥ 70% was considered significantly supported.Bayesian posterior probability (PP) ≥ 0.90 was regarded as significant.The bolded parts are the sequences generated in this study.

Molecular Phylogenetic Analysis Results
The seventy-eight specimens of Gymnopilus collected in this study, which represented eleven species, were included in the phylogenetic analyses with the outgroup species Galerina marginata.During our study, seventy-six ITS sequences, seventy-three nrLSU sequences, seventy-five nrSSU sequences, fifteen rpb1 sequences, sixty rpb2 sequences, and sixteen tef1-α sequences were newly generated in this study (Table 1).There was a total of five thousand and thirty-one bp (base pairs) in the final alignment after assembling those six gene sequences, of which eight hundred and ninety-one characters are from nrLSU, five hundred and forty-four characters are from tef1-α, seven hundred and six characters are from rpb2, one thousand three hundred and twenty-seven characters are from rpb1, eight hundred and ninety characters are from nrSSU, and six hundred and seventy-three characters are from ITS.The phylogenetic tree of the ML and MrBayes topology were generally the same using those six gene sequences.The Maximum Likelihood tree is shown in Figure 1, with bootstrap and posterior probability values indicated on the branches.
Microscopic description: Basidiospores (5.9-)6.2-6.8(-7.0)× (2.9-)3.4-4.3(-4.5)µm, Q = 1.6-1.8,avQ = 1.7, broadly ellipsoid, distinct suprahilar depression in side view, ornamentation moderately developed, germ pore absent.Basidia ( Notes: Gymnopilus gyirongensis sp.nov.can be easily distinguished by its orangeyellow to orange-brown pileus covered with yellowish rust squamules and umbonate center.Microscopically, this new species has medium-sized spores with moderately developed ornamentation.In the field, another proposed new species, Gy. tomentosiceps, may be confused with Gy. gyirongensis due to the fact that they both have yellowish caps; however, they have different habitats: Gy. tomentosiceps usually grows on mossy, soil under coniferous forest trees, but Gy. gyirongensis prefers fruiting on damp wood with rotting pine needles.In this study, phylogenetic analysis demonstrated that four specimens of this new species formed a cohesive cluster with full support.Additionally, they were positioned closely to Gy. penetrans, Gy. hybridus, and Gy.tomentosiceps (Figure 1).Gymnopilus penetrans is similar to Gy. gyirongensis because they share the characteristic of a smooth pileus covered with yellow and rusty brown squamules on the surface and can be found under conifers and deciduous trees [2,32].However, Gy. penetrans develop bigger basidiopores (7.2-9.9 × 4-5.5 µm) than those of Gy. gyirongensis [6].Moreover, these two species have 29 base pair differences in their ITS sequences.The four ITS sequences of this new species display four or five base pair differences.Another similar species, Gy. hybridus, can easily be distinguished from this species due to it having bigger basidiospores (6-8 × 4-4.5 µm) and basidia (20-28 × 5-8 µm) [5,7,41].
Habitat: -Scattered or clustered in meadows on moist ground of decaying pine needles, characterized by the absence of bark and cork, summer.
In this study, the macroscopic and microscopic morphology of those five new species are described in detail.Some macro-and micro-differences can be used to separate them from each other.Gymnopilus tomentosiceps is characterized by having a small and tomentose cap, while Gy.tenuibasidialis has an abrupt papilla cap.The mature spores of these five new species are all distinct, with suprahilar depressions in side view, but the shapes and ornamentation on the surface (Figures 2-6) are different among these species.The basidiospores of Gy. variisporus and Gy.tomentosiceps are ellipsoid, while those of the other species are broadly ellipsoid; Gy. aurantipileatus has developed ornamentation on basidiospores, while the ornamentation of Gy. variisporus and Gy.aurantipileatus is very variable in the process of maturity (Figures 3 and 6).In general, even though these five new species have similar macro characteristics, their characteristics in terms of spore shape and ornamentation are distinct and different from each other, as supported by our molecular phylogenetic analysis.
In the phylogenetic tree (Figure 1), twenty-nine included species of Gymnopilus segregate into three distinct clades (/penetrans, /crociphyllus, and /picreus), and these three clades are part of the six clades delineated in a previous study and are phylogenetically positioned in the same location [7,12].In the /penetrans clade, Gy. hybridus is regarded as a synonym of Gy. penetrans by Holec (2005) [6].However, in Europe, Gy. penetrans is classified as a singular species, with Gy. hybridus considered as a synonym [44,45], or as distinct and formed as two separate species (Gy.penetrans and Gy.hybridus) [46], and these species differ in color, the development of the veil, the presence of rusty spots on lamellae, and the shape of cystidia [6].In our phylogenetic tree (Figure 1), Gy. tomentosiceps, Gy. hybridus, and Gy.penetrans show a close relationship but in different phylogenetic positions.Furthermore, a total of 21 nucleotide differences were detected in the ITS region between Gy. tomentosiceps and Gy.penetrans, and 24 nucleotide differences were detected between Gy. tomentosiceps and Gy.hybridus.Thus, those three species are identified as separate species.Differences in macro characteristics exist: Gy. prnetrans typically exhibits smooth or slightly wrinkled caps and stipe with white fibrillose partial veil remnants; Gy. hybrids feature hygrophanous streaks, as well as smooth and scale-free caps; and Gy.tomentosiceps is characterized by a cap covered with tomentose scales.Gymnopilus crociphyllus (Cooke and Massee) Pegler has distinctive characteristics, such as a fasciculate fruiting body, large pileus size, and a rumpled pileus margin [43].In the /picreus clade, which includes Gy. austropicreus B.J. Rees and Gy.aurantipileatus sp.nov, this clade can be morphologcially characterized as having the same ornamentation on spores encased in a membrane which will gradually fall off with maturity.In another study [46], Gy. picreus (FT.)P. Karsten and Gy.austropicreus always emerged basal to the remainder of the species in the phylogenetic tree, which could refer to how they may be ancestral to other species in the genus [47].Gymnopilus picreus groups grow as a saprophytes on the dead wood of conifers (mostly Picea abies; less frequently Pinus sylvestris) and, rarely, deciduous trees (Betula, Fagus sylvatica) [6], but other Gymnopilus species could grow on dead wood or on mulch-rich soil [2].Thus, we suspect that the rationale for the types of wood rot they seek for nutrition is possibly ancestral in this genus.
In China, a total of thirty-one Gymnopilus species, including seven species originally described to be from China, have been published so far [7,9,21].Gymnopilus is a species-rich genus, and the previous studies from China included several provinces, such as the Guangdong, Yunnan, Sichuan, Hainan, and Jilin provinces.In this paper, we have reported species from more parts of China, namely the Xizang Autonomous Region, resulting in a total of thirty-six species of Gymnopilus that have been recorded in China.Furthermore, four of the five new species are originally from the Xizang Autonomous Region, which indicates the special nature of the species, as they are from such a unique habitat.This study complements our understanding of Gymnopilus diversity and lays a foundation for the conservation and utilization of related Gymnopilus resources.

Table 1 .
Information of the sequences generated from this study.Missing sequences are indicated by "-".