Morphological and Molecular Evidence Reveal Eight New Species of Gymnopus from Northeast China

Gymnopus is a widely distributed genus consisting of about 300 species thus far, including Gymnopus fusipes as a generic type. A total of nine species from China belong to the sect. Levipedes, including eight new species—Gymnopus longisterigmaticus, Gymnopus longus, Gymnopus macrosporus, Gymnopus striatus, Gymnopus changbaiensis, Gymnopus tomentosus, Gymnopus tiliicola, and Gymnopus globulosus—which were delimited and proposed based on morphological and molecular evidence; and one new record from Jilin Province, China—Gymnopus erythropus. Detailed descriptions and illustrations are presented, as well as comparisons to similar species. Overall, our results broaden the morphological characterization of the genus. The pileipellis of sect. Levipedes typically takes on the “Dryophila structure”, while, in our findings, pileipellis terminal hyphae inflated to spherical to prolate were observed, in addition to extremely long basidia sterigma. The phylogenies inferred from the ITS and nLSU dataset supported the Gymnopus, which was defined by Oliveira et al. as a monophyletic genus, and the novel species as separate lineages within. A key to all species described in this study is also 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 characteristics, the specimens were dried in an electric drier at 40-45 • C.
Descriptions of the macroscopic characteristics were based on field notes and photographs, with the colors corresponding to the Flora of British fungi: Color identification chart [30]. 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) [31]; 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 basidiocarps. 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, and Qm denotes the average Q value of all the basidiospores ± standard deviation. "I" refers to the number of lamellulae between every two complete lamellae, and "L" refers to the number of complete lamellae. The specimens examined are 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 by 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 (ITS) region, and nuclear large ribosomal subunits (nLSU) were used for phylogenetic analysis. The ITS sequence was amplified by using the primer pair ITS1-F (CTT GGT CAT TTA GAG GAA GTA A) and ITS4-B (CAG GAG ACT TGT ACA CGG TCC AG) [32], and the nLSU sequence was amplified by using the primer pair LROR (GTA CCC GCT GAA CTT AAG C) and LR7 (TAC TAC CAC CAA GAT CT) [33,34]. PCR reactions (25 µL) contained 8 µL 2 × EasyTaq ® PCR SuperMix (TransGen Biotech Co., Ltd., Beijing, China), 1 µL 10 µM primer L, 1 µL 10 µM primer R, 3 µL DNA solution, and 12 µL dd H 2 O. The reaction programs were as follows: for the ITS, initial denaturation at 94 • C for 4 min, followed by 30 cycles at 94 • C for 1 min, 54 • C for 1 min and 72 • C for 1 min, and a final extension of 72 • C for 10 min [35]; for the nLSU, initial denaturation at 95 • C for 3 min, followed by 30 cycles at 94 • C for 30 s, 47 • C for 45 s, and 72 • C for 90 s, and a final extension of 72 • C for 10 min [36]. The PCR products were visualized via UV light after electrophoresis on 1% agarose gels stained with ethidium bromide and purified by 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 (accessed on 17 November 2021); see 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. We used a dataset of ITS and nLSU resign comprising sequences from this study, with 49 [15].
For the dataset, each gene region was aligned by using ClustalX [37], MACSE V2.03 [38], or MAFFT 7.490 [39], and then manually adjusted in BioEdit 7.0.5.3 [40]. The datasets first were aligned, and then the ITS and nLSU sequences were combined with Phylosuit V1.2.2 [41]. The best-fit evolutionary model was estimated by using Modelfinder [42]. 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 timereversible DNA substitution model and a gamma distribution rate variation across the sites [43]. 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 remaining trees were used to construct a 50% majority consensus tree and for calculating the Bayesian posterior probabilities (BPPS). RaxmlGUI 2.0.5 [44] was used for maximum likelihood (ML) analysis, along with 1000 bootstraps (BS) replicates, using the GTRGAMMA algorithm to perform a tree inference and search for the optimal topology [45]. Then the FigTree v1.3.1 was used to visualize the resulting trees.

Phylogenetic Analyses
In the dataset, 143 sequences derived from two gene loci (ITS and nLSU) from 92 samples were used to build phylogenetic trees; 50 of these were newly generated, with 25 ITS sequences and 25 nLSU sequences. The phylogenetic construction performed via ML and BI analysis for the two combined datasets showed a similar topology. The combined ITS and nLSU dataset represented 63 taxa and 2600 characters after being trimmed. The Bayesian analysis was run for two million generations and resulted in an average standard deviation of split frequencies of 0.004989. The same dataset and alignment were analyzed by using the ML method. In the phylogenetic tree, six clades corresponding to Gymnopus, Rhodocollybia, Lnetinula, Marasmiellus, Marasmius, Mycetines, Collybiopsis, and Paragymnopus were revealed (Figures 1 and 2). Twenty-one sampled specimens formed eight new species and were clustered in a clade comprising the species of Gymnopus sect. Levipedes (Figure 2). At the same time, two sampled specimens-clustered with Gymnopus erythropus with strong support-were confirmed as new records from Jilin Province, China.
Ecology: Grows on the deciduous layer or rotten branches in coniferous and broadleaved mixed forest.
Other Note: Gymnopus longus is closed to Gymnopus erythropus, Gymnopus fagiphilus, and Gymnopus longisterigmaticus in morphological, because of the red pileus and stipe. However, Gymnopus longus differs from Gymnopus erythropus by being covered with brown farinose on the upper part, white to light reddish brown tomentose at the base, slight thin basidiospores, smaller Qm [1], and extremely long sterigmata (up to 33 µm long).
A deeper color pileus, covered with brown farinose on the stipe, smaller basidiospores, clavate with obtuse cheilocystidia, and pileipellis a translation between a cutis and a trichoderm differs Gymnopus longisterigmaticus from Gymnopus longus. Gymnopus longus differs from Gymnopus fagiphilus by a farinose stipe, deep color pileus and stipe, white lamellae, smaller basidiospores, lack of caulocystidia, and uncoralloid pileipellis [1]. Diagnosis: This species is distinguished from closed species by a convex to applanate pileus that is deep reddish brown at the center and reddish brown to yellowish brown toward the margin, with the margin beige to light yellow, striped; a deep reddish brown to reddish brown stipe with smooth, light yellow to light reddish brown tomentose at the base, coralloid pileipellis, bigger basidiospores, and extremely long basidia sterigmata. Basidiomata small-to-medium-sized, gregarious. Pileus convex to applanate, 1.2-4.6 cm diameter, smooth, hygrophanus, deep reddish brown at the center, reddish brown to yellowish brown towards margin; margin beige to light yellow, striped, entire, wavy. Context thin, fleshy, light reddish brown to light yellowish brown, odorless. Stipe center, cylindrical, 7.8-9.5 cm long and 0.2-0.5 cm wide, deep reddish brown to reddish brown, smooth, fistulose, fibrous, and light yellow to light reddish brown tomentose at the base. Lamellae adnexed to adnate or near free, light yellow, I = 1-3, L = 13-17, crowded.
Ecology: Grows on the deciduous layer or rotten branches in coniferous and broadleaved mixed forest.
Distribution Note: Gymnopus macrosporus is morphologically similar to Gymnopus longisterigmaticus and Gymnopus longus because of its reddish brown, tomentose stipe, and long sterigmata of basidia. Gymnopus macrosporus differs from Gymnopus longus due to its pileus in a darker color-pileus deep reddish brown at center, reddish brown to yellowish brown towards margin; margin beige to light yellow, striped characteristics, and smooth texture on the upper part of the stipe, coralloid and without pigment incrusting pileipellis. These two Gymnopus species have a similar basidiospore size; however, the Qm of Gymnopus macrosporus is larger than Gymnopus longus. Gymnopus longisterigmaticus differs in smooth, pale color, and unstriped pileus; pileipellis a bit wider and pigment yellowish brown incrusting in pileipellis, and it has bigger basidiospores [(6.2) 6.7-9.0 × (3.0) 3.1-4.3 (5.0) µm]. Note: Gymnopus striatus is easily confused with Gymnopus longisterigmaticus, Gymnopus longus, and Gymnopus macrosporus due to their highly similar morphology. However, Gymnopus striatus differs from those three species by its deeper color lamellae, longitudinal stripes on the stipe and stripes on the margin of pileus, bigger Qm, and short basidia sterigmata. Gymnopus striatus can be easily differentiated from Gymnopus fagiphilus by its deeper color pileus, uniform colored and longitudinally striped stipe, lack of caulocystidia, uncoralloid cheilocystidia, without pigment incrusting in pileipellis, and smaller basidiospores. Note: Gymnopus changbaiensis is significantly related to Gymnopus fagiphilus and Gymnopus striatus based on its reddish brown and tomentose stipe, and short basidia sterigmata. Gymnopus changbaiensis can be distinguished from Gymnopus fagiphilus by its lighter and depressed pileus, denser and fresh to pink lamellae, and, in terms of microscopic characteristics, smaller basidiospores, uncoralloid cheilocystidia, and lack of caulocystidia. Gymnopus changbaiensis differs from Gymnopus striatus by its pale color, striped, and depressed pileus, fresh-to-pink lamellae, non-striped stipe, a bit longer cheilocystidia, coralloid pileipellis. Basidiomata small-to-medium-sized, scattered. Pileus convex, 1.6-3.0 cm diameter, smooth, tan at the center, light brown towards margin; margin white, tomentose, entire. Context thin, fleshy, white to light yellow, odorless. Stipe center, cylindrical, 3.3-4.3 cm long and 0.2-0.5 cm wide, blackish green at apex, reddish brown below, covered with reddish brown hairs up to 1/4 (from the base upwards), fistulose, fibrous. Lamellae adnexed, yellowish green, I = 3-7, L = 19-25, crowded.
Basidiospores elliptic, (6.0) 6.9-8.0 Note: Morphologically, the rose-red to dark red pileus and stipe make Gymnopus tiliicola closed to Gymnopus erythropus. Gymnopus tiliicola differs from Gymnopus erythropus in a lighter color and striped pileus, light pink to fresh and denser lamellae. Besides, Gymnopus tiliicola grows at the base of Tilia sp., while Gymnopus erythropus grows on the deciduous layer or rotten branches. In regard to microfeatures, Gymnopus tiliicola differs from Gymnopus erythropus by a weakly coralloid pileipellis, uncoralloid cheilocystidia, bigger basidiospores, and two-or four-spored basidia. Diagnosis: This species is distinguished from closed species by a convex to applanate pileus that is deep reddish brown at the center, lighter-colored outwards, and light yellow margin, striped; white to light yellowish green lamellae, with a reddish brown and smooth stipe, pileipellis two layers and the terminal hyphae inflated to spherical to prolate, and a bit bigger basidiospores.
Basidiospores elliptic, (6. Note: In terms of morphology, Gymnopus globulosus resembles Gymnopus erythropus and Gymnopus tiliicola in its red to dark red pileus and stipe. However, Gymnopus globulosus is distinguishable from Gymnopus erythropus due to its deeper-colored pileus, light yellowish green lamellae, which is light yellow of Gymnopus erythropus. In terms of microfeature, the pileipellis of Gymnopus erythropus is between a cutis and a trichoderm, while the pileipellis of Gymnopus globulosus is layered, with the upper layer inflated to spherical to prolate hyphae and the down layer made up of branched and inflated hyphae, and bigger basidiospores. Gymnopus globulosus differs from Gymnopus tiliicola with its deeper-colored pileus, light yellowish green lamellae, grows on rotten wood, pileipellis two layers and the terminal hyphae inflated to spherical to prolate, and bigger basidiospores. New record from Jilin Province, China Gymnopus erythropus (Pers.) Antonín, Halling and Noordel. Figures 3h and 12 Basidiomata small-to-medium-sized, scattered to gregarious. Pileus convex to applanate, 1.1-3.2 cm diameter, smooth, hygrophanus, reddish brown to brown at the center, light reddish brown to yellowish brown towards margin; margin beige to light yellow, entire, wavy sometimes. Context thin, fleshy, light brown, odorless. Stipe center, cylindrical, 4.1-10.0 cm long and 0.2-0.5 cm wide, deep reddish brown to light reddish brown, paler at apex, smooth, covered with scattered light yellow to brown hairs hairy at base, fistulose, fibrous. Lamellae adnate, fresh-pink, I = 3-5, L = 14-27, crowded. Basidiospores

. New Sights on Morphological Characteristics
The genus Gymnopus is geographically widely distributed; however, in China, its species diversity is poorly known. Moreover, in China, only three species were originally described with molecular evidence. One of these is Gymnopus ramulicola T.H. Li and S.F. Deng [27] from Hainan Province, China; the second one is Gymnopus alliifoetidissimus T.H. Li and J.P. Li [25] from Guangdong Province, China; and the third is Gymnopus pallipes J.P. Li and Chun Y. Deng [25] from Guangdong and Guizhou Province, China. In our study, eight new species of Gymnopus from China are described as new species. They are well-supported by molecular phylogenetic and morphological evidence. Our newly recognized and delimited species are distributed in the broad-leaved and mixed forests, and occur in early autumn in Northeast China. The species we described here are hardly seen in the wild mushroom market; thus, their edibility is not yet known.
The description of these new species also broadens the morphological characterization of the genus Gymnopus. In the previous study, the pileipellis of the species in this genus was a cuits to trichoderm. Moreover, the pileipellis in the species of sect. Levipedes was an entangled, not radially oriented trichoderm of inflated, often lobed or coralloid elements of the "Dryophila type" [1,17]. In this study, the pileipellis of Gymnopus globulosus was divided into two layers, with the upper layer comprising hyphae inflated to spherical to prolate, differing from that of all known species in the genus, while the second layer was typical of the "Dryophila type". To our knowledge, the sterigmata of the basidia are usually not too long; however, the species Gymnopus longistrigmaticus, Gymnopus longus, and Gymnopus macrosporus had extremely long sterigmata, up to 40 µm. Thus, the structure of extremely long basidia sterigmata is traceable in our species. In addition, all the species described from this study are detailed compared in macro-and micro-features (Tables 2 and 3).

Phylogenetic Relationships of Gymnopus s.l. with Related Genera
Phylogenetic analyses of the species of Gymnopus s.l. and the related genera presented in this study confirmed that the genus Gymnopus defined by Antonín and Noordeloos, as well as Halling, is not monophyletic in a strongly supported clade. Similar results were observed with our phylogenetic analysis. Our results, thus, support the finding of Oliveira et al., promoting sect. Perforanita to the genus level, Paragymnopus, and share a close affinity with Lentinula. Moreover, sect. Vestipedes was clearly separated from Gymnopus s. str [12,15], and were closed to Marasmiellus, Collybiopsis, and Rhodocollybia. However, in their study, the species of Gymnopus sect. Vestipedes was involved with Marasmiellus; therefore, Oliveira et al. [15] proposed to transfer Gymnopus sect. Vestipedes to Marasmiellus and redefined the genus Gymnopus more strictly.
However, in our phylogenetic analyses, a different result was obtained. In our results, sect. Vestipedes did not group into one clade with Marasmiellus to form an independent clade, forming a sister clade to genus Collybiopsis. Furthermore, the taxonomic status of Collybiopsis minor R.H. Petersen still needs to be clarified; in our study, C. minor was separated far away from Collybiopsis, while being clustered with sect. Vestipedes within a single clade.
Some species of sect. Vestipedes and genus Marasmiellus have been proposed for transfer to other genera in recent years. Gymnopus cylindricus J.L. Mata and Gymnopus brunneigracilis (Corner) A.W. Wilson, Desjardin and E. Horak were suggested to be switched into Marasiellus. The type species of Marasmiellus, Marasmiellus juniperinus, and some other species within the genus, were advised to be relocated to Collybiopsis [47]. Thus, the boundaries between Gymnopus, Marasmiellus, and Collybiopsis would be more blurred, especially between Marasmiellus and Collybiopsis, as well as if these species were transferred to Collybiopsis; then it would be multiphyletic, with Rhodocollybia, Paragymnopus, and Lentinula would becoming synonyms of Gymnopus.

Nova Suggestions of Phylogenetic Relationships within Gymnopus s. str.
In our phylogenetic results, the genus Gymnopus, which was defined by Oliveira et al. [15], was mainly divided into four clades. Sect. Levipedes, sect. Gymnopus, and sect. Androsacei are somewhat more closely related, whereas they are distant from the sect. Impudicae. Before 2010, both sect. Impudicae and sect. Levipedes were subsections below the same section. However, from our results, sect. Impudicae and sect. Levipedes are more distantly related, probably due to similar environments, causing a similar appearance. In addition, the genus Mycetines and sect. Impudicae, with a strong odor, are not closely related to each other, and this is consistent with the result that they have a different pileipellis structure of pileus.
Thus, sect. Levipedes being split into two sections was supported by the phylogenetic analysis. Sect. Levipedes subsect. Levipedes was also divided into two subclades: one is Gymnopus dryophilus complex, a subclade (defined here as/dryophila) that includes all the Gymnopus dryophilus complex species reported around the world (characterized by a Gymnopus dryophilus-like appearance and arises in early spring or later in the autumn). From the result, the East Asia sequences of Gymnopus dryophilus were not clustered with the European sequences, while they were clustered with the new species-Gymnopus dryophiloides-that Antonín, Ryoo and Ka reported from Korea in 2020. Antonín et al. [51] do not accept Gymnopus lanipes (Malençon and Bertault) Vila and Llimona as a separate species and consider it to be a variant of Gymnopus dryophilus. From our phylogenetic result, it is clear that Gymnopus lanipes clusters with Gymnopus inexpectatus, which Consiglio, Vizzini, Antonín and Contu described from Europe, which, if Gymnopus lanipes is not considered an independent species, then Gymnopus dryophioides and Gymnopus inexpectatus should equally be treated as Gymnopus dryophilus. Moreover, Gymnopus erythropus complex, a subclade (defined here as/erythropus), includes Gymnopus erythropus, Gymnopus fagiphilus, and our new species (characterized by a red to reddish brown color, a smooth or scatteredto-dense tomentose stipe, and occurring in early autumn). The above results imply the need for a deeper and more extensive study on sect. Levipedes.
Based on the current study's findings, we increased the species diversity of the genus Gymnopus from China. However, probably due to the lacking of species sampling or the inadequate genetic variation in the DNA loci in our study, the deep phylogenetic relationships within the genus Gymnopus and between the related genera-Lentinula, Rhodocollybia, Mycetinis, Collybiopsis, etc.-remain unresolved. Thus, in future work, more species of this genus and similar genera will be discovered, which will provide new evidence and, thus, lead to a deeper understanding of the relationships within and among these genera.