Revealing Brownish Mycena Diversity in China: New Discoveries and Taxonomic Insights

Abstract

Within the genus Mycena, species exhibiting brownish basidiomata present considerable challenges in identification due to similar coloration. This study underscores the significance of pileipellis types and cheilocystidia characteristics as critical in delimiting brownish Mycena species. To clarify the principal taxonomic characters and their utility in distinguishing between brownish Mycena species, a morphological taxonomy and phylogenetic analysis were performed. Five new species from China were introduced and characterized through a comprehensive morphological anatomy and phylogenetic substantiation: M. campanulatihemisphaerica sp. nov., M. digitifurcata sp. nov., M. kunyuensis sp. nov., M. limitis sp. nov., and M. oryzifluens sp. nov. Discussions of these taxa are supplemented with morphological illustrations. The phylogenetic relationships were inferred using Bayesian Inference and Maximum Likelihood methods based on sequences from the internal transcribed spacer and the large subunit regions of nuclear ribosomal RNA. With the addition of these five new species, the worldwide count of brownish Mycena increases to 94, and a key to the 29 known species of brownish Mycena from China is presented.

1. Introduction

Mycena (Pers.) Roussel was established in the early 18th century, standing as one of the earliest genera among fungi [1]. Currently, there are over 500 known species, with 2418 listed in the Index Fungorum (https://www.indexfungorum.org, accessed on 27 February 2024), highlighting a remarkable species diversity [2]. Mycena species are widely distributed all over the world, and play a crucial role in the decomposition of dead branches, fallen leaves, and rotting wood, which facilitates the material circulation of forest ecosystems [3,4,5,6,7]. Moreover, some species have demonstrated a propensity for enhancing Orchidaceae seed germination, which contributes to the resilience and diversity of ecosystems [8,9,10]. The diverse morphology and color of Mycena contribute a high species diversity, while they also present significant taxonomic challenges due to subtle morphological variations and complex microstructures among species [11,12,13,14,15,16]. Taxonomists have proposed several different classification systems, reflecting a varied emphasis on different feature prioritizations [11,12,17,18,19,20]. Presently, the classification system proposed by Maas Geesteranus (1992a, 1992b) has garnered considerable consensus, which divides Mycena into 44 sections and considers the color of the pileus and stipe as a primary grouping criterion, for example, sect. Oregonenses Maas Geest., sect. Cyanocephalae Singer, etc. [11,12].

Brownish Mycena, characterized by a yellowish-brown, grayish-brown to dark brown pileus or stipe, predominantly inhabit the northern temperate zone, with fewer occurrences in tropical and subtropical areas [11,12,20,21,22,23,24,25,26,27]. They are categorized into at least nine sections, namely: sect. Amictae A.H. Sm. ex Mass Geest., sect. Basipedes (Fr.) Quél., sect. Cinerellae Singer ex Maas Geest., sect. Exornatae Maas Geest., sect. Filipedes (Fr.) Quél., sect. Fragilipedes (Fr.) Quél., sect. Hiemalis Konrad & Maubl., sect. Mycena (Pers.) Roussel, and sect. Rubromarginatae Singer ex Maas Geest. [17,21,23,26,28,29,30]. Among them, sect. Mycena and sect. Fragilipedes stand out due to substantial species diversity and typical morphological characteristics. The former is characterized by its light brown, yellowish-brown to sepia pileus, with both the cystidia and pileipellis covered with excrescences. M. galericulata (Scop.) Grayis, serving as the type species in genus Mycena and as well as in the type sect. Mycena, is a prime example of brownish Mycena’s characteristic traits. The other section, Fragilipedes, is distinguished by its grayish-brown to dark brown pileus, along with smooth cystidia and a pileipellis typically covered with excrescences [11,12,21].

In recent years, the species diversity of Mycena has received heightened attention, leading to the publication of many new species, and marking substantial advances in research [13,14,15,26,27,30,31,32]. However, brownish Mycena have remained relatively underexplored, with their phylogenetic relationships still unclear and a considerable number of species unknown. Notably, fewer than 30 brownish Mycena species have been identified in Asia, with a mere 24 documented in China, compared to the 75 cataloged in Europe and North America [17,20,21,22,23,28,29,30]. The considerable diversity, similar color, and small basidiomata present significant challenges in studying morphological structures, leading to a dominant reason for underestimating the species diversity of brownish Mycena in China and across Asia [11,12,13,16,23]. At the same time, the abundant types of cystidia and pileipellis hyphae found in brownish Mycena reflect their diverse shapes and suggest a complex evolutionary history. This indicates the species’ adaptability to a wide range of ecological pressures and its ability to thrive in diverse habitats. The diversity of ecological niches and displayed adaptability are concrete manifestations of its complex evolutionary background, reflecting the species’ versatility and flexibility in adapting to environmental changes [21,23].

During our ongoing research and fieldwork aimed at exploring the diversity of Mycena in China, five novel species were identified among 14 new specimens through comparative studies with known species. The identification of these novel species was further supported through comprehensive morphological and anatomical research, alongside molecular phylogenetic analyses. This work will expand the variety of brownish Mycena in China and provide fundamental information for the systematic study of this group.

2. Materials and Methods

2.1. Sample Collection and Morphological Description

Specimens used in this study were collected from a broad spectrum of locations across China, spanning Fujian Province, the Guangxi Zhuang Autonomous Region, Heilongjiang Province, Jilin Province, Jiangxi Province, Shandong Province, and Zhejiang Province. During fieldwork, meticulous records were maintained, noting location information, collectors, the collection date, and the ecological habits of each specimen. Simultaneously, the odor and taste of the fresh specimens were recorded. The specimens’ photos were taken using a Canon EOS 90D digital camera (Canon, Tokyo, Japan) equipped with an EF-S60 mm f/2.8 Macro USM (Canon, Tokyo, Japan) and an Olympus E-M1 Mark III camera (Olympus, Tokyo, Japan), coupled with an M. Zuiko Digital Ed 12–40 mm or 60 mm lens (Olympus, Tokyo, Japan). Macro-morphological features, encompassing basidiomata size, color, shape, and surface characteristics were detailed in records based on photographs and field notes. The nomenclature of color descriptions refers to Ridgway (1912) [33]. The extent of pileus marginal striations or sulcations was quantitatively assessed by the ratio of the striation’s or sulcation’s length to the pileus’s radius (n R) [34]. The specimens were dried in an electric oven (Stöckli, A. & J. Stöckli AG, Netstal, Switzerland) at 40 °C and were then placed in a ziplock bag with allochroic silica gel for preservation. All specimens are now preserved in the Fungarium of the Fujian Academy of Agricultural Sciences (FFAAS), China.

Microscopical features were examined with a Lab A1 microscope (Carl Zeiss AG, Jena, Germany) using dried specimens rehydrated in a 5% KOH aqueous solution and stained with a 1% Congo red aqueous solution when necessary. Melzer’s reagent was used to test the amyloidity of basidiospores and lamellar trama [35]. Basidiospores were photographed using ZEN 2.3 (Blue Edition) software (Carl Zeiss Microscopy GmbH, Jena, Germany), with at least 20 basidiospores measured per specimen. For the holotype, a more extensive examination entailed measuring 40 or more basidiospores from each basidiomata. The measured results were presented in the form [a/b/c] (d) e–f–g (h) × (i) j–k–l (m) μm [Q = (n) o–p (q), Q_m = r ± s]. The abbreviation [a/b/c] represents a number of basidiospores measured from b number of basidiomata of c number of collections; d and h represent the minimum and maximum lengths (5% extremum), while the range e–g encompasses the remaining 90% of the measured values, with f as the mean length. Width (i–m) and Q values (n–q) are analogous representations. If the difference between d and e, g and h, i and j, or l and m is less than 0.2 μm, and the difference between n and o or p and q is less than 0.01 μm, d, h, i, m, n, or q were omitted accordingly. Q represents the “length/width” ratio of a basidiospore. Q_m presents the average Q of all basidiospores ± the sample standard deviation [36]. Other microstructures examined include basidia, cheilocystidia, pleurocystidia, pileocystidia, caulocystidia, and the respective compositions of the pileipellis and stipipellis, along with lamellar trama. For each specimen, 20 of these structures were recorded, detailing their sizes, shapes, contents, and other features. The pileipellis samples were specifically cut from the mid-zone between the center and the margin of the pileus, while stipitipellis samples were procured from the middle of the stipe.

The delineation of macro- and microstructures commenced with manual sketches on A4 paper using a pencil and subsequently transposed to tracing paper using a Sakura needle pen (Sakura Color Products Corporation, Osaka, Japan). The sketches were converted to digitalization in TIF format using a Canon LiDE120 scanner (Canon, Tokyo, Japan). Photoshop was then utilized for further refinement and typesetting.

2.2. DNA Extraction, PCR Amplification, and Sequencing

DNA extraction from the dried specimens utilized the NuClean Plant Genomic DNA kit (Kangwei Century Biotechnology Co., Beijing, China). PCR amplification followed in a 25 μL reaction mix, comprising 12.5 μL 2× Utaq PCR MasterMix (ZomanBio, Beijing, China), 8.5 μL ddH₂O, 1 μL each of forward and reverse primers, and 2 μL DNA template [14]. To amplify the nuclear ITS rDNA region (ITS1–5.8S–ITS2), the primer pairs ITS1/ITS4 were employed with the following PCR thermocycling protocol: 94 °C for 4 min, then 34 cycles of 94 °C for 45 s, 52 °C for 45 s, and 72 °C for 1 min, with a final extension of 72 °C for 10 min [37,38]. The large subunit (LSU) of rDNA was amplified using LR0R/LR7 primer pairs with the PCR cycle set to 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 55°C for 30 s, and 72 °C for 1 min; followed by 72 °C for 10 min [16,39]. PCR amplification products underwent agarose gel electrophoresis for validation before the Sanger dideoxy was sequenced by the Beijing Genomics Institute (Beijing, China) [40]. The sequence quality was checked using BioEdit 7.0.9.0, with chromatograms inspected for accuracy [41,42]. Low-quality sequences were enhanced by applying plasmid vectors using VELUTE Gel Mini Purification and pBLUE-T kits (Beijing Zoman Biotechnology Co., Beijing, China) for optimal sequencing results [43]. All newly obtained sequences in this study were submitted to GenBank.

2.3. Molecular Phylogeny

The newly obtained sequences were subject to BLAST searches within the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/, accessed on 27 March 2024) to retrieve and download homologous sequences (with nucleotide identities >90%). Furthermore, sequences of species bearing a morphological resemblance to the species in this study were also sourced from GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 27 March 2024), selecting one or more representatives per species that share a morphological or phylogenetic affinity with the newly described species for preliminary phylogenetic analysis. M. tenuispinosa J. Favre and M. mucoroides Aronsen were used as outgroup taxa to the root tree. Gaps were treated as missing data after aligning the ITS and LSU datasets independently using the auto strategy (FFT-NS-1, FFT-NS-2, FFT-NS-i or L-INS-i) by MAFFT v.7.110, with gene fragments manually refined and amalgamated in MEGA 5 [44,45,46]. Both Bayesian Inference (BI) and Maximum Likelihood (ML) were utilized for phylogenetic inference. For BI analysis, the optimal nucleotide evolution was estimated using Modeltest 2.3 with the BIC criterion [47]. MrBayes 3.2.6 executed the analysis over 5,000,000 MCMC generations; four chains were conducted with the sampling every 500th generation. The first 25% of trees were discarded as burn-in after the average standard deviation of split frequencies under 0.01, with results aggregated through the “sump” and “sumt” commands [48]. Tracer v.1.7.2 served in the visualization and examination of MCMC trace files from the Bayesian phylogenetic inference [49]. Meanwhile, ML analysis was conducted by RAxMLGUI 2.0 with the GTRGAMMA model and 1000 bootstrap (BS) replicates using default parameters [50]. Finally, the phylogenetic tree was visualized in FigTree v.1.4.3 and refined in Photoshop CS4.

3. Results

3.1. Phylogenetic Analysis

The molecular analyses dataset consisted of 152 sequences in total, including 28 newly obtained sequences (14 spanning ITS and LSU) and 124 sequences downloaded from GenBank (98 ITS and 26 LSU). Detailed information is provided in

Table 1. DNA sequences of Mycena used in the phylogenetic analysis in this study.

			GenBank NO.
Species	Voucher/Strain NO.	Locality	ITS	LSU	Reference
M. abramsii	HMJAU43606	China	MH396629	MK629355	Direct sub.
M. abramsii	HMJAU43282	China	MH396626	MK629348	Direct sub.
M. aetites	CBS 221.47	Netherlands	MH856225	MH867752	[51]
M. aetites	CBS 222.47	Netherlands	MH856226	MH867753	[51]
M. aff. discobasis	DED 8211 (SFSU)	USA	MH414555	MH385331	[52]
M. aff. discobasis	BAP 658 (SFSU)	USA	MH414554	MH385330	[52]
M. aff. murina	D1.1c	Italy	KJ093496	—	[53]
M. albiceps	RA705-6	USA	MK234177	—	[54]
M. albiceps	MGW1504	USA	KY744173	MF797661	Direct sub.
M. algeriensis	HMJAU43798	China	MK733295	MK722347	Direct sub.
M. algeriensis	HMJAU59791	China	OR468696	—	Direct sub.
M. amicta	CBS:257.53	Netherlands	MH857184	MH868722	[51]
M. atkinsoniana	S.D. Russell iNaturalist # 8545962	USA	MN906081	—	Direct sub.
M. atkinsoniana	S.D. Russell MycoMap # 7612	USA	MN906082	—	Direct sub.
M. bulliformis	MF74930	USA	MT636954	—	Direct sub.
M. bulliformis	JLF9168	USA	MZ007503	—	Direct sub.
M. bulliformis	SFSU:BAP 547	USA	KX513844	KX513848	[55]
M. campanulatihemisphaerica	FFAAS1047 Holotype	China	PP706092	PP704692	This study
M. campanulatihemisphaerica	FFAAS1048	China	PP706093	PP704693	This study
M. campanulatihemisphaerica	FFAAS1049	China	PP706094	PP704694	This study
M. campanulatihemisphaerica	FFAAS1050	China	PP706095	PP704695	This study
M. chlorophos	CT15101401	China	MH400938	—	Direct sub.
M. chlorophos	CT151014	China	MH400939	—	Direct sub.
M. cinerella	O73656	Netherlands	GU234146	—	[56]
M. cinerella	H6018219	Hungary	MW540670	—	Direct sub.
M. citrinomarginata	SHXG	China	OM228755	OM228763	Direct sub.
M. citrinomarginata	S.D. Russell iNaturalist # 17474783	USA	ON416970	—	Direct sub.
M. clavicularis	HMJAU43611	China	ON791480	ON791547	Direct sub.
M. coralliformis	ACL306	Malaysia	—	KJ206962	[57]
M. cristinae	JS347	Brazil	MT921381	MT921384	[26]
M. digitifurcata	FFAAS1054	China	PP706099	PP704699	This study
M. digitifurcata	FFAAS1055 Holotype	China	PP706100	PP704700	This study
M. limitis	FFAAS1056	China	PP706101	PP704701	This study
M. limitis	FFAAS1057	China	PP706102	PP704702	This study
M. limitis	FFAAS1058 Holotype	China	PP706103	PP704703	This study
M. fulgoris	ACP1690	Malaysia	MG926694	—	[58]
M. fulgoris	XAL A. Cortes-Perez 1690	Mexico	NR_163300	—	[58]
M. galericulata	HMJAU43035	China	MW222635	—	Direct sub.
M. galericulata	HMJAU43845	China	MW222634	—	Direct sub.
M. galopus	461E	Poland	MZ078480	—	[59]
M. galopus	69	Norway	MW576941	—	[60]
M. globulispora	ACP1765	Mexico	MG926696	—	[58]
M. globulispora	ACP1704	Mexico	MG926697	—	[58]
M. haematopus	1055	Canada	KJ705181	—	Direct sub.
M. haematopus	BIOUG24046-B04	Canada	KT695316	—	[61]
M. inclinata	Champ-36	Spain	KX449443	—	[62]
M. inclinata	AH56011	Spain	ON113881	—	[63]
M. interrupta	HMJAU43791	China	MK733300	—	Direct sub.
M. interrupta	HMJAU43849	China	MK733301	—	Direct sub.
M. jingyinga	LE-BIN 4556	Russia	OP997504	—	Direct sub.
M. jingyinga	OMDL00049	USA	OR572512	—	Direct sub.
M. kunyuensis	FFAAS1045 Holotype	China	PP706090	PP704690	This study
M. kunyuensis	FFAAS1046	China	PP706091	PP704691	This study
M. laevigata	HMJAU43187	China	MK733302	—	Direct sub.
M. laevigata	HMJAU43618	China	MK733304	—	Direct sub.
M. laevigata	HMJAU43604	China	MK733303	MK722354	Direct sub.
M. maculata	CBS:235.47	Netherlands	MH856231	—	[51]
M. maculata	140 m	Norway	MW576905	—	[60]
M. maculata	CBS:239.47	Netherlands	MH856234	MH867763	[51]
M. megaspora	H6036833	Hungary	MW540680	—	Direct sub.
M. megaspora	5702	Russia	MZ754456	—	Direct sub.
M. meliigena	NSK 1014985	Russia	OQ216533	—	Direct sub.
M. meliigena	S.D. Russell ONT iNaturalist # 98315803	USA	OP455719	—	Direct sub.
M. metata	HAY-F-003966	USA	OR858777	—	Direct sub.
M. metata	HAY-F-005148	USA	OR882691	—	Direct sub.
M. mucoroides	OF76006	Sweden	NR176117	—	Direct sub.
M. mucoroides	AAronsen9-060913	Sweden	KU861561	—	[28]
M. nebula	ACP1659	Mexico	MG926685	—	[58]
M. niveipes	DA-18015	France	OM368073	—	Direct sub.
M. overholtsii	iNAT:11409406	USA	MZ146337	—	Direct sub.
M. overholtsii	S.D. Russell iNaturalist # 37566563	USA	ON561746	—	Direct sub.
M. oryzifluens	FFAAS1051 Holotype	China	PP706096	PP704696	This study
M. oryzifluens	FFAAS1052	China	PP706097	PP704697	This study
M. oryzifluens	FFAAS1053	China	PP706098	PP704698	This study
M. pasvikensis	AAronsen86-12	Sweden	KU861556	—	[28]
M. pasvikensis	AAronsen45-13	Sweden	KU861557	—	[28]
M. picta	CAFUNDIS iNaturalist 171114596	USA	OR858681	—	Direct sub.
M. picta	TUR194167	Hungary	MW540717	—	Direct sub.
M. plumbea	AFTOL-ID 1631	USA	DQ494677	DQ470813	[64]
M. pluteoides	HMJAU43771	China	MK733307	MK722357	Direct sub.
M. polycystidiata	FFAAS0418	China	ON427732	—	[14]
M. polycystidiata	FFAAS0421	China	ON427733	—	[14]
M. polygramma	CBS 243.47	Netherlands	MH856238	MH867767	[51]
M. pura	IS10/11/2000	Denmark	FN394611	—	[65]
M. renati	CBS:358.50	Netherlands	MH856658	MH868174	[51]
M. romagnesiana	388a	USA	JF908421	—	Direct sub.
M. romagnesiana	388f	USA	JF908422	—	Direct sub.
M. rubromarginata	UBC F16259	Canada	EF530939	—	Direct sub.
M. rubromarginata	CBS:265.48	Netherlands	MH856335	MH867890	[51]
M. rubromarginata	CBS:268.48	Netherlands	MH856338	MH867891	[51]
M. rufobrunnea	FFAAS0414	China	ON427728	—	[14]
M. rufobrunnea	FFAAS0415	China	ON427729	—	[14]
M. sanguinolenta	CBS:367.50	Netherlands	MH856662	—	[51]
M. sanguinolenta	KUBOT-KRMK-2020-57	India	MW446185	MW446189	Direct sub.
M. semivestipes	HMJAU43825	China	MK733308	MK722358	Direct sub.
M. semivestipes	HMJAU43830	China	MK733309	—	Direct sub.
M. seynesii	71l	USA	JF908469	—	Direct sub.
M. seynesii	71h	USA	JF908470	—	Direct sub.
M. shengshanensis	FFAAS0424	China	ON427739	—	[14]
M. shengshanensis	FFAAS0425	China	ON427740	—	[14]
M. silvae-nigrae	HMJAU43815	China	MK733310	MK722359	Direct sub.
M. sinar	ACL092	Malaysia	KF537247	—	[66]
M. sinar	ACL135	Malaysia	KF537249	—	[66]
M. subulata	FFAAS0419	China	ON427735	—	[14]
M. subulata	FFAAS0423	China	ON427737	—	[14]
M. tenuispinosa	LE 321750	Russia	MK478466	—	Direct sub.
M. tintinnabulum	H6008524	Hungary	MW540664	—	Direct sub.
M. tintinnabulum	NSK 1017255	Russia	OR242685	—	Direct sub.
M. venus	CT20160127	China	MG324368	—	[30]
M. venus	CT20160218	China	MG324369	—	[30]
M. yuezhuoi	FFAAS0347	China	MW581493	—	[15]
M. yuezhuoi	FFAAS0344	China	MW581490	—	[15]
M. zephirus	CBS:270.48	Netherlands	MH856339	MH867892	[51]

Remarks: New generated sequences are emphasized in bold; “—” show missing sequence.

. The aligned dataset culminated in a total of 1743 aligned sites, including gaps (824 ITS, 919 LSU). The average deviation of split frequencies was 0.006, the ESS (effective sample size) was 1908.3, and the average Potential Scale Reduction Factor (PSRF) parameter values = 1.000 after 5,000,000 MCMC generations. The ML phylogenetic analysis yielded a final log-likelihood value of −13,761.724187. The average bootstrap value was 78.5%, indicating a generally high confidence in the tree’s branches. The lowest bootstrap value was 50.0%, suggesting some branches may require further verification, while the highest value was 99.0%, reflecting very high confidence in certain branches. The tree topology effectively depicted the evolutionary relationships among the major taxa, with samples from each novel species clustering together as expected.

The phylogenetic evaluations using ML and BI indicated generally similar topologies for the five newly identified species, although some discrepancies between the methods were observed. Consequently, the BI tree was selected to be shown in Figure 1, incorporating only those nodes supported by bootstrap values of at least 75% and posterior probabilities exceeding 0.95. The phylogenetic structure has resolved into 13 distinct clades, each delineated by shared morphological characteristics of pileipellis hyphae or cheilocystidia among the species within them. While some clades received lower support rates, the topology of the phylogenetic tree still effectively reflects the shared characteristics of groups within these clades, such as smooth or ornamented characteristics.

The five newly identified species demonstrated high support within the phylogenetic tree, with each being robustly placed within clades 1, 2, 9, and 10. These results reinforce the taxonomic standing of these clades and confirm the systematic placement of these new species.

Clade 1 emerges as a monophyletic clade, incorporating M. oryzifluens sp. nov. supported by three specimens (FFAAS1051, FFAAS1052, and FFAAS1053). In the BI phylogenetic reconstruction, M. oryzifluens sp. nov. clusters closely with other members of Clade 1, indicating a distinct genetic lineage. Although in the ML analysis, M. oryzifluens sp. nov. and M. amicta (Fr.) Quél. span a broad clade from Clade 2 to Clade 13, their monophyletic status is firmly supported (BS/BPP = 100/1.00). Additionally, species in Clade 1 share the characteristic of non-smooth pileipellis hyphae. Clade 2 incorporates the new species M. digitifurcata sp. nov. (FFAAS1054, FFAAS1055), all members sharing the feature of non-smooth pileipellis hyphae. In the BI tree, M. digitifurcata sp. nov. is closely related to M. cristinae, although this relationship is not as strong in the ML analysis. Despite variations between the BI and ML trees, M. digitifurcata sp. nov. is consistently recognized as a monophyletic entity with high statistical support (BS/BPP = 99/1.00). Species of Clade 3 exhibit smooth pileipellis and cheilocystidia, receiving unanimous support (100%) and demonstrating high phylogenetic consistency. Clade 4, although supported at a lower rate (BS/BPP = 81/1.00), presents species with cylindrical excrescences on the cheilocystidia, providing key taxonomic insights. Clades 5 and 6 both exhibit very high support rates (BS/BPP = 100/1.00), where Clade 5 species feature smooth cheilocystidia, while Clade 6 species display excrescences on the cheilocystidia. Clades 7 and 8 also showcase specific morphological characteristics, with Clade 7 having non-smooth pileipellis and Clade 8 featuring non-smooth pileipellis, along with some species having gelatinous layers. Clade 9 includes M. limitis sp. nov. (FFAAS1056, FFAAS1057, and FFAAS1058), sharing a close phylogenetic relationship with M. niveipes (Murrill) Murrill, along with other species such as M. seynii Quél., M. bulliformis B.A. Perry & Desjardin, and M. fulgoris Cortés-Pérez & Desjardin. Although Clades 9 and 12 form independent groups in BI analysis, Clade 12 nests within Clade 9 in ML analysis. Nonetheless, the position of M. limitis sp. nov. remains well-supported (BS/BPP = 100/1.00). Clade 10 comprises the novel species M. campanulatihemisphaerica sp. nov. (FFAAS1047, FFAAS1048, and FFAAS1049) and M. kunyuensis sp. nov. (FFAAS1045, FFAAS1046), sharing characteristics of smooth cheilocystidia with other species. Clade 11, though not highly supported, includes species that exhibit smooth cheilocystidia. Clade 12 is fully supported (100%), with species displaying smooth cheilocystidia and non-smooth pileipellis, showing clear phylogenetic consistency. Clade 13, similarly supported fully (100%) in the BI tree, shares the characteristic of non-smooth pileipellis among its species.

3.2. Taxonomy

Mycena campanulatihemisphaerica R.X. Wei, X.M. Wu, J. Yu, Y.P. Ge & Q. Na, sp. nov., Figure 2, Figure 3 and Figure 4.

MycoBank no: 853739

Etymology: The epithet campanulatihemisphaerica combines the Latin feminine adjectives campanulata (bell-shaped) and hemisphaerica (half-spherical). This name accurately describes the shape of the pileus, which transitions from bell-shaped to nearly hemispherical.

Holotype: CHINA. Shandong Province, Kunyushan National Nature Reserve, Yantai City, 19 July 2019, leg. Renxiu Wei, Liming Xue, Ruichen Liu, Qin Na, and Yupeng Ge, 516 m asl, FFAAS1047 (collection no. MY022).

Diagnosis: Pileus fuscous brown, obviously sulcate, basidiospores narrowly ellipsoid to cylindrical, cheilocystidia ovate, ellipsoid, fusiform to sublageniform, some slightly tapered or forked apices, pileipellis and stipitipellis covered with cylindrical excrescences. Differs from M. venus by unbranched cheilocystidia and larger basidiospores.

Description: Pileus 3–12 mm diam., ovoid, hemispherical to campanulate, sometimes apex with an inconspicuous umbo, margin smooth; *Hair Brown (XLVI17’’’’i) at center, paler towards the margin to Pale Drab-Gray (XLVI17’’’’f), Pale Smoke Gray (XLVI21’’’’f) to White (LIII); pruinose at center, obviously sulcate towards the center up to 0.5–0.8 R, Light Drab (XLVI17’’’’b) to *Hair Brown (XLVI17’’’’i), dry. Context White (LIII), fragile, thin. Lamellae emarginate to sinuate, 0.2–0.6 mm wide, L = 12–16, l = 1–2, White (LIII), edge concolorous with face. Stipe 19–60 × 0.9–2.0 mm, central, cylindrical, hollow, fragile, *Hair brown (XLVI17’’’’i) at the base, paler toward the apex to White (LIII), finely pubescent, base covered with short, dense, White (LIII) fibrils. Odor and taste indistinctive.

Basidiospores [107/5/4] (8.5)8.8–10.0–12.1 × 4.5–5.3–6.5 μm, [Q =1.74–2.05, Q_m = 1.87 ± 0.09] [holotype (40/2/1) 8.8–10.0–12.1 × 4.5–5.3–6.5 μm, Q = 1.74–2.05, Q_m = 1.88 ± 0.09], narrowly ellipsoid to cylindrical, hyaline, smooth, thin-walled, amyloid. Basidia 21–30 × 7–10 μm, four-spored, rarely two-spored, clavate, hyaline, sterigmata 2–4 μm in length. Cheilocystidia 18–36 × 9–16 μm, ovate, ellipsoid, fusiform to sublageniform, apex 1.5–4.2 × 1.2–2.4 μm, tapered, occasionally forked, hyaline, thin-walled. Pleurocystidia absent. Pileipellis a cutis composed of two layers of hyphae, upper part of hyphae 2.0–5.2 μm diam., parallel, hyaline, thin-walled, covered with cylindrical excrescences, 2.0–7.0 × 1.0–2.4 μm; lower part of hyphae 4.3–6.8 μm diam., interwoven, hyaline, thin-walled, smooth; pileocystidia absent. Lamellae trama subcellular, composed of subglobose, ellipsoid, and narrowly ellipsoid cells, 23–43 × 17–25 μm, hyaline, dextrinoid. Stipitipellis a cutis composed of a hypha, 1.9–4.8 μm diam., hyaline, thin-walled, covered with cylindrical excrescences, 1.7–4.1 × 0.6–1.6 μm; caulocystidia absent. Clamps present in all tissues, but rarely observed in context.

Habit and habitat: Fascicled or scattered on rotten branches in mixed broadleaf–conifer forests, mainly under trees of Picea, Pinus, and Quercus.

Known distribution: Fujian Province, Jiangxi Province, Shandong Province, China.

Additional material examined: China. Fujian Province, Junzifeng National Nature Conservation Area, Mingxi County, Sanming City, 2 May 2021, leg. Renxiu Wei, Binrong Ke, Hui Zeng, Junqing Yan, Qin Na, and Yupeng Ge, 824 m asl, FFAAS1049 (collection no. MY0267); Jiangxi Province, Jiangxi Agricultural University, Nanchang City, 22 April 2021, leg. Renxiu Wei, Junqing Yan, and Qin Na, 59 m asl, FFAAS1048 (collection no. MY056); Shandong Province, Mengshan Scenic Area, Mengyin County, Linyi City, 20 July 2021, leg. Renxiu Wei, Yulan Sun, Zewei Liu, Qin Na, and Yupeng Ge, 962 m asl, FFAAS1050 (collection no. MY0389).

Notes: M. venus exhibits morphological affinities with M. campanulatihemisphaerica, particularly in the characteristics of the pileus and stipe. However, M. venus is distinguished by its branched cheilocystidia and notably smaller basidiospores, measuring (4.8) 6.8–7.7 (8.7) × (3.2) 3.8–4.8 (5.4) μm, with a Q_m of 1.69 [30]. M. silvae-nigrae Maas Geest. & Schwöbel and M. leptocephala (Pers.) Gillet share similarities with M. campanulatihemisphaerica in pileus color. Nonetheless, M. silvae-nigrae is set apart by its uniformly dark brown stipe and the presence of pleurocystidia [21,23,28]; M. leptocephala is differentiated by presenting caulocystidia and pleurocystidia [20,21,23,28,29]. Microscopically, M. abramsii (Murrill) Murrill and M. scirpicola M. Villarreal, Heykoop, Esteve-Rav. & Maas Geest. also display cheilocystidia of a similar morphology, such as clavate, fusiform, and sublageniform to cylindrical, but M. campanulatihemisphaerica is distinguishable from these species by its fuscous brown pileus and the absence of both pleurocystidia and caulocystidia [20,21,23,28,29,67,68]. Additionally, some cheilocystidia were observed with characteristics such as slightly tapered or forked apices, resembling subglobose and subclavate cells, suggesting they are in an early phase of cheilocystidia.

Mycena kunyuensis R.X. Wei, X.M. Wu, M.Z. Chen, Y.P. Ge & Q. Na, sp. nov., Figure 5, Figure 6 and Figure 7.

MycoBank no: 853782

Etymology: The epithet kunyuensis refers to the Kunyu mountain where the holotype was discovered.

Holotype: CHINA. Shandong Province, Kunyushan National Nature Reserve, Yantai City, 19 July 2019, leg. Renxiu Wei, Liming Xue, Ruichen Liu, Qin Na, and Yupeng Ge, 513 m asl, FFAAS1045 (collection no. MY021).

Diagnosis: Pileus pale olive-buff to olive-buff, basidiospores ellipsoid to narrowly ellipsoid, cheilocystidia lageniform, obpyriform, obclavate to fusiform, with a narrow neck, apex tapered, pileipellis and stipitipellis covered with cylindrical excrescences. Differs from M. abramsii by smaller basidiomata and absent pleurocystidia.

Description: Pileus 5–11 mm diam., fusiform to campanulate when young, slightly campanulate, conical, hemispherical to plano-convex with age, margin slightly serrulate, occasionally cracked when old; Citrine-Drab (XL21’’’i), Deep Quaker Drab (LI1’’’’’i) at center, paler towards margin to *Olive-Buff (XL21’’’d) when young, Citrine-Drab (XL21’’’i), Pale Mouse Gray (LI15’’’’’d) at center, gradually paler towards margin to Pale Olive-Buff (XL21’’’f), Pallid Mouse Gray (LI15’’’’’f) to White (LIII) when matured; initially pruinose, when matured glabrescent, undistinguished to clear striate towards the center up to 0.5–0.6 R, dry. Context White (LIII), fragile, thin. Lamellae sinuate, 0.3–0.6 mm wide, L = 14–18, l = 1, White (LIII), edge concolorous. Stipe 33–46 × 0.8–1.1 mm, central, clavate to cylindrical, hollow, fragile, in young stages; Deep Quaker Drab (LI1’’’’’i), *Hair Brown (XLVI17’’’’i), when matured White (LIII) in the upper part, Pale Smoke Gray (XLVI21’’’’f), Light Grayish Olive (XLVI21’’’’b) towards the base, pruinose when young, then apex to middle part finely pubescent, almost glabrous in the lower part, base slightly swollen, densely covered with long White (LIII) fibrils. Odor and taste indistinctive.

Basidiospores [65/3/2] (7.0) 7.4–9.3–11.5 (11.8) × 4.6–5.8–6.9 μm, [Q = (1.40) 1.43–1.77 (1.79), Q_m = 1.60 ± 0.09] [holotype (40/2/1) (7.0) 7.4–8.8–10.4 × 4.6–5.6–6.8 μm, Q = (1.40) 1.43–1.77, Q_m = 1.56 ± 0.08], ellipsoid to narrowly ellipsoid, hyaline, smooth (under oil), thin-walled, amyloid. Basidia 16–23 × 5–9 μm, four-spored, rarely two-spored, clavate, hyaline, sterigmata 2.1–4.2 μm in length. Cheilocystidia 28–59 × 8–20 μm, lageniform, obpyriform, obclavate to fusiform, with a narrow neck, apex tapered, smooth, sometimes neck with several excrescences, and occasionally base constricted to cylindrical, hyaline, thin-walled. Pleurocystidia absent. Pileipellis a cutis composed of parallel hyphae, 2.5–5.1 μm diam., thin-walled, densely covered with cylindrical excrescences, 1.6–9.2 × 1.0–2.6 μm, occasionally forked; pileocystidia absent. Lamellae trama cellular, composed of subglobose, ellipsoid, and narrowly ellipsoid cells, 24–48 × 21–25 μm, hyaline, dextrinoid. Stipitipellis a cutis composed of a hypha, (1.7) 2.1–4.6 μm diam., covered with cylindrical excrescences, 0.6–10.2 (15.3) × 0.9–2.9 μm, occasionally forked, hyaline, thin-walled; caulocystidia absent. Clamps present in all tissues, but rarely observed in context.

Habit and habitat: Scattered on rotten branches in mixed broadleaf–conifer forests, mainly under trees of Picea, Pinus, and Quercus.

Known distribution: Shandong Province, China.

Additional material examined: CHINA. Shandong Province, Kunyushan National Nature Reserve, Yantai City, 19 July 2019, leg. Renxiu Wei, Liming Xue, Ruichen Liu, Qin Na, and Yupeng Ge, 524 m asl, FFAAS1046 (collection no. MY023).

Notes: M. abramsii is the closest species to M. kunyuensis, sharing numerous macroscopic and microscopic characters. Nevertheless, M. abramsii is distinguished by its much larger basidiomata and the presence of pleurocystidia [20,21,23,28,29,67,68]. In addition, taxa such as M. fagetorum (Fr.) Gillet, M. metata (Fr.) P. Kumm. and M. filopes (Bull.) P. Kumm. exhibit a similar pileus color, but distinct differences are noted: M. fagetorum is distinguished by its clavate cheilocystidia, which are prominently covered by coarse excrescences [28]; M. metata is differentiated by its clavate and obovoid cheilocystidia, and presenting pleurocystidia [21,23,28,29]; and M. filopes is recognized by its excrescence-covered cheilocystidia and the presence of caulocystidia [21,23,28,29]. M. aetites (Fr.) Quél. also presents lageniform cheilocystidia, but it is uniquely identified by its pileus, which ranges from black to dark brown, and its gelatinized pileipellis [23,28]. Comparatively, M. kunyuensis and M. campanulatihemisphaerica exhibit similarities in some macroscopic and microscopic characters. However, M. campanulatihemisphaerica can be distinguished by its shorter fibrils-covered stipe; fuscous brown, markedly sulcate pileus, with its margin being slightly serrulate; larger basidiospores; and cheilocystidia that fork at the apex (Figure 8).

Mycena oryzifluens R.X. Wei, L.L. Qi, Y.P. Ge & Q. Na, sp. nov., Figure 9, Figure 10 and Figure 11.

MycoBank no: 853783

Etymology: The epithet oryzifluens is derived from the Latin word oryzi, meaning rice, and fluens, meaning flowing. This name reflects the rice-like appearance of the white, pruinose, and pubescent stipe and connects to the folklore of the type locality, where a legend speaks of a flowing rice cave, symbolizing abundance and continuity.

Holotype: CHINA. Guangxi Zhuang Autonomous Region, Qingxiushan Scenic Spot, Nanning City, 14 July 2022, leg. Renxiu Wei, LiangLiang Qi, Liying Li, Yongqiang Hu, and Yupeng Ge, 164 m asl, FFAAS1051 (collection no. MY0870).

Diagnosis: Pileus plumbeous to gray, finely white pruinose and pubescent, basidiospores subglobose to broadly ellipsoid, cheilocystidia clavate, ovoid, obpyriform, with finger-like branches in the apex, pileipellis with non-smooth terminal cells, caulocystidia thick-walled. Different from M. cretata Aronsen by clavate cheilocystidia with cylindrical branches, thick-walled caulocystidia, and absent pleurocystidia.

Description: Pileus 2.1–6.0 mm diam., campanulate when young, then hemispherical to oblate hemispherical; Dark Plumbeous (LII49’’’’’i) at center, paler towards margin to Plumbeous (LII49’’’’’b) when young, paler towards margin to French Gray (LII49’’’’’f), *Lilac Gray (LIII59’’’’’f) to White (LIII) with age; finely White (LIII) pruinose and pubescent, especially dense White (LIII) pubescence surrounds the margin, sulcate towards the center up to 0.5–0.6 R, Dark Vinaceous Gray (LII59’’’’’k), Vinaceous Gray(L69’’’’’d) when young, Light Violet Gray(LII59’’’’’b) to Deep Violet Gray (LII59’’’’’i) when matured, dry. Context White (LIII), fragile, thin. Lamellae adnate, 0.3–0.5 mm wide, L = 16–25, l = 1–2, White (LIII), edge serrulate, concolorous with face. Stipe 5–20 × 0.3–1.0 mm, center, cylindrical, hollow, fragile, Deep Purplish Gray (LIII67’’’’i) when young, then paler to Light Violet-Gray (LII59’’’’’b), Hathi Gray (LII35’’’’’b) with age, finely White (LIII) pruinose and pubescent, base densely covered with White (LIII) tomentum. Odor and taste indistinctive.

Basidiospores [96/4/3] 6.0–6.6–7.5 (7.7) × 4.6–5.3–6.3 μm, [Q = 1.10–1.45, Q_m = 1.24 ± 0.07] [holotype (40/2/1) 6.2–6.8–7.4 (7.7) × (4.8)5.0–5.6–6.3 μm, Q = 1.10–1.37, Q_m = 1.20 ± 0.05], subglobose to broadly ellipsoid, hyaline, smooth (under oil), thin-walled, amyloid. Basidia 17–27 × 7–10 μm, two- and four-spored, clavate, hyaline, sterigmata 1.7–4.7 μm in length. Cheilocystidia 15–36 × 8–18 μm, clavate, ovoid, obpyriform, with one to several finger-like branches at apex, 1.0–17.1 × 0.6–2.6 μm, sometimes branches with furcate excrescences, hyaline, thin-walled. Pleurocystidia absent. Pileipellis a cutis composed of parallel hyphae, 2.7–5.2 μm diam., hyaline, thin-walled, terminal cells often swollen, 34–103 × 5–15 μm, clavate to cylindrical, with sparse nodulose excrescences, 0.5–3.0 × 0.5–1.0 μm; pileocystidia absent. Lamellae trama cellular, composed of subglobose, ellipsoid and narrowly ellipsoid cells, 20–50 × 15–30 μm, hyaline, dextrinoid. Stipitipellis a cutis composed of a hypha, 2.8–5.7 μm diam., hyaline, thin-walled; caulocystidia 44–107 × 4–10 μm, cylindrical, with a narrow protuberance in the apex, thick-walled (0.6–1.7 μm thick), smooth, hyaline. Clamps present in all tissues, but rarely observed in context.

Habit and habitat: Scattered on litter layers and rotten branches in broad-leaved mixed forests, mainly under trees of Ficus and Parashorea.

Known distribution: Guangxi Zhuang Autonomous Region, China.

Additional material examined: China. Guangxi Zhuang Autonomous Region, Qingxiushan Scenic Spot, Nanning City, 14 July 2022, leg. Renxiu Wei, LiangLiang Qi, Liying Li, Yongqiang Hu, and Yupeng Ge, 185 m asl, FFAAS1052 (collection no. MY0871), 191 m asl, FFAAS1053 (collection no. MY0872).

Notes: Macroscopically, M. cretata shares a similar pileus color with M. oryzifluens but is distinguished by the presence of pleurocystidia and thin-walled caulocystidia, with its cheilocystidia lacking finger-like extensions [28]. Microscopically, both M. tallangattensis Grgur and M. scirpicola feature thick-walled caulocystidia. However, M. tallangattensis is identified by its thick-walled cheilocystidia and the presence of pleurocystidia [17], while M. scirpicola is noted for its elongated basidiospores and the absence of branched cheilocystidia [23,28]. Additionally, taxa M. tristis Maas Geest., M. clavularis (Batsch) Sacc., M. tenuispinosa, and M. mucor (Batsch) Quél. exhibit cheilocystidia of a similar morphology, but M. oryzifluens is distinctively characterized by its plumbeous to gray pileus, basidiospores that are subglobose to broadly ellipsoid, and thick-walled caulocystidia [28].

Mycena digitifurcata R.X. Wei, H. Zeng, Y.P. Ge & Q. Na, sp. nov., Figure 12, Figure 13 and Figure 14.

MycoBank no: 853784

Etymology: The epithet digitifurcata derives from the Latin words ‘digitus’, meaning ‘finger’, and ‘furcatus’, meaning ‘forked’. This name is chosen to describe the distinctive finger-like and forked projections at the apex of the cheilocystidia.

Holotype: CHINA. Zhejiang Province, Baiyun National Forest Park, Lishui City, 2 August 2021, leg. Renxiu Wei, Zewei Liu, Qin Na, and Yupeng Ge, 212 m asl, FFAAS1055 (collection no. MY0476).

Diagnosis: Pileus deep gray when young, light drab to drab with age, basidiospores ellipsoid to narrowly ellipsoid, cheilocystidia with finger-like branches at apex. Differs from M. cristinae by pruinose pileus, obviously decurrent lamellae, and being weakly intervenose.

Description: Pileus 4.2–12.0 mm diam., hemispherical when young, oblate hemispherical to convex with age, umbilicate at the center, margin slightly wavy, revolute, sometimes cracked with age; Castor Gray (LII35’’’’’i) to Pale Violet-Gray (LII59’’’’’d) at the center, gradually paler towards margin to *Pearl Gray (LII35’’’’’f), Smoke Gray (XLVI21’’’’d) when young, Light Drab (XLVI17’’’’b) to Hair Brown (XLVI17’’’’i) at the center, gradually paler towards margin to Pale Smoke Gray (XLVI21’’’’f), Pale Drab-Gray (XLVI17’’’’f) with age; densely covered with pruina when young, sparsely when old, striate towards the center up to 0.5–0.8 R, Castor Gray (LII35’’’’’i) when young, Hathi Gray (LII35’’’’’b), Hair Brown (XLVI17’’’’i) when matured, dry. Context White (LIII), fragile, thin. Lamellae subdecurrent to decurrent, 0.3–0.9 mm wide, L = 12–14, l = 1–3, White (LIII) to pale, edge concolorous with face, sometimes non-marginate, weak and irregularly intervened, up to 1/3–1/2 lamellae wide. Stipe 10–13 × 0.8–1.0 mm, central, clavate to cylindrical, hollow, fragile, *French Gray (LII45’’’’’f), *Pearl Gray (LII35’’’’’f) in the upper part, darker towards the base to *Cinereous (LII45’’’’’d), Hathi Gray (LII35’’’’’b), finely White (LIII) pruinose and pubescent, base slightly swollen, covered with masses of short and White (LIII) tomentum when young, rarely observed when old. Odor and taste indistinctive.

Basidiospores [108/4/2] 6.3–7.3–8.8 × 3.8–4.5–5.7 μm, [Q = 1.43–1.76, Q_m = 1.61 ± 0.08] [holotype (40/2/1) 6.4–7.5–8.8 × 3.8–4.7–5.7 μm, Q = 1.43–1.76, Q_m = 1.60 ± 0.08], ellipsoid to narrowly ellipsoid, hyaline, smooth (under oil), thin-walled, amyloid. Basidia 18–24 × 4–8 μm, two- and four-spored, clavate, hyaline, sterigmata 3.2–5.0 μm in length. Cheilocystidia 10–32 × 4–18 μm, clavate, cylindrical, apex with several to fairly numerous finger-like branches, 2.3–22.1 × 0.8–2.1 μm, branches usually with furcate excrescences at apex, 2.3–8.9 × 0.8–1.9 μm, hyaline, thin-walled. Pleurocystidia absent. Pileipellis a cutis composed of parallel hyphae, 1.9–4.6 μm diam., hyaline, thin-walled, covered with cylindrical excrescences, 1.8–17.9 (29.0) × 0.9–2.3 μm; pileocystidia absent. Lamellae trama subregular, hyphae 7–19 μm diam., hyaline, dextrinoid. Stipitipellis a cutis composed of a hypha, 2.2–4.6 μm diam., hyaline, thin-walled, covered with small cylindrical excrescences, 1.1–6.3 × 0.9–2.1 μm; caulocystidia absent. Clamps present in all tissues, but rarely observed in context.

Habit and habitat: Scattered on rotten branches in mixed broadleaf–conifer forests, mainly under trees of Liriodendron, Pseudolarix, and Pinus.

Known distribution: Zhejiang Province, China.

Additional material examined: China. Zhejiang Province, Lishui City, 1 August 2021, leg. Renxiu Wei, Binrong Ke, Zhiheng Zeng, Qin Na, and Yupeng Ge, 231 m asl, FFAAS1054 (collection no. MY0447).

Notes: M. cristinae closely resembles M. digitifurcata in pileus and stipe color, but it is distinguished by its smooth pileus, adnate lamellae, and markedly intervenose lamellae [26]. M. pasvikensis Aronsen, sharing a similar pileus color with M. digitifurcata, is differentiated by a densely fibril-covered stipe base, serrulate lamellae, and gelatinized pileipellis and stipitipellis [28]. Similar cheilocystidia shapes are observed in M. pseudopicta (J.E. Lange) Kühner and M. cinerella (P. Karst.) P. Karst., but M. pseudopicta is differentiated from M. digitifurcata by its gelatinized pileipellis and stipitipellis, and by possessing larger basidiospores [23,28]; M. cinerella is notable for its gelatinized pileipellis and the lighter color of the pileus [28,29]. Microscopic observation reveals a variation in the branch lengths of the cheilocystidia. In specimen FFAAS1054, most cheilocystidia display short (0.9–1.9 μm) branched excrescences at the apex, whereas in FFAAS1055, cheilocystidia with significantly longer (7.5–22.2 μm) branched excrescences at the apex are typically observed, indicating the various forms of cheilocystidia.

Mycena limitis R.X. Wei, L. Zou, Y.P. Ge & Q. Na, sp. nov., Figure 15, Figure 16 and Figure 17.

MycoBank no: 853786

Etymology: The epithet limitis is derived from the Latin word limes, which emphasizes the morphological similarities with related species and highlights the limited distinguishing features that set this species apart from its close relatives. Additionally, this name pays tribute to the companionship and support provided by a forest ranger, affectionately called brother on the border.

Holotype: CHINA. Heilongjiang Province, Taipinggou National Nature Reserve, Hegang City, 3 July 2023, leg. Renxiu Wei, Li Zou, Menghui Han, Nannan Geng, Tingting Sun, Xinyu Tong, Yawei Li, Zengcai Liu, and Yupeng Ge, 621 m asl, FFAAS1058 (collection no. GN1786).

Diagnosis: Pileus hair brown when young, olive brown when old, stipe with faint longitudinal stripes, cheilocystidia and pleurocystidia mainly fusiform. Differs from M. niveipes by cheilocystidia with tapered apex and stipitipellis with smooth hyphae.

Description: Pileus 10–50 mm diam., parabolic and hemispherical when young, oblate hemispherical to plano-convex with age, slightly convex at center, margin slightly serrulate, sometimes cracked at mature; *Hair Brown (XLVI17’’’’i) at center, gradually paler towards margin to Light Drab (XLVI17’’’’i), Pale Smoke Gray (XLVI21’’’’f) when young, Deep Olive (XL21’’’k) at center, gradually paler towards margin to Citrine Drab (XL21’’’i), Pale Olive Buff (XL21’’’f) when matured; covered with finely White (LIII) pubescence when young, then glabrescent, transparent sulcate towards the center up to 0.5–0.6 R, dry. Context White (LIII), fragile, thin. Lamellae adnate to slightly sinuate, 2.1–5.0 mm wide, L = 22–25, l = 1–3, White (LIII), edge concolorous with face, inconspicuous intervenose. Stipe 59–85 × 2.0–4.1 mm, central, cylindrical, White (LIII) in the upper part, Pale Smoke Gray (XLVI21’’’’f) in the middle part, *Smoke Gray (XLVI21’’’’d), *Olive-Gray (LI23’’’’’b) towards base, hollow, fragile, covered with finely White (LIII) pruina when young, almost glabrous when old, with faint longitudinal stripes, base covered with masses of short and White (LIII) fibrils. Odor and taste indistinctive.

Basidiospores [108/4/3] (8.6) 8.9–10.4–12.6 × 4.7–5.7–6.9 (7.2) μm [Q = 1.65–1.99 (2.01), Q_m = 1.82 ± 0.09] [holotype (50/2/1) 8.9–10.7–12.6 × 4.7–5.8–6.9 (7.2) μm, Q = (1.65) 1.66–1.99 (2.01), Q_m = 1.83 ± 0.09], narrowly ellipsoid, hyaline, smooth (under oil), thin-walled, amyloid. Basidia 29–37 × 8–10 μm, clavate, hyaline, two- and four-spored, sterigmata 4.1–6.0 μm in length. Cheilocystidia 43–70 × 10–21 μm, fusiform, subfusiform, lanceolate, tapering at apex, base constricted to cylindrical, hyaline, thin-walled, smooth. Pleurocystidia 54–123 × 14–26 μm, similar to cheilocystidia, hyaline, thin-walled, occasionally forked in the upper part. Pileipellis a cutis composed of parallel hyphae, 3.2–7.0 μm diam., hyaline, thin-walled, smooth; pileocystidia absent. Lamellae trama regular, hyaline, parallel, hyphae, 6–18 μm diam., dextrinoid. Stipitipellis a cutis composed of a hypha, 3.2–5.0 μm diam., hyaline, thin-walled, smooth; caulocystidia absent. Clamps present in all tissues, but rarely observed in context.

Habit and habitat: Scattered on humus layer and rotten branches in deciduous broad-leaved forests, mainly under trees of Betula, Quercus, Styphnolobium, and Tilia.

Known distribution: Heilongjiang Province, Jilin Province, China.

Additional material examined: China. Jilin Province, Changbaishan National Nature Reserve, Yanbian Korean Autonomous Prefecture, leg. Renxiu Wei, Binrong Ke, Chi Yang, Qin Na, and Yupeng Ge, 1 July 2021, 708 m asl, FFAAS1057 (collection no. MY0305); 3 July 2021, 749 m asl, FFAAS1056 (collection no. MY0341).

Notes: M. niveipes is similar to M. limitis in pileus color and pileipellis with smooth hyphae, but differs by rounded-apex cheilocystidia and the presence of swollen terminal cells in the stipitipellis [23,28]. M. abramsii and M. subcana A.H. Sm also share a comparable pileus color and cheilocystidia shape but are distinguished from M. limitis by the pileipellis and stipitipellis, which are remarkedly covered with excrescences [21,23,28,29]. Similarly, M. galericulata exhibits a pileus color similar to M. limitis, but it is differentiated by the cheilocystidia and both the hyphae of the pileipellis and stipitipellis featuring excrescences [21,23,28,29]. M. laevigata Gillet, while having comparable cheilocystidia, is distinct from M. limitis due to its pale grayish-white pileus, the absence of pleurocystidia, and a gelatinized pileipellis [28,29].

4. Discussion

Determining the identity of various brownish Mycena species based on basidiomata color imposes certain limitations, which consequently may lead to an underestimation of species diversity [11,12,13,14,21,23,28]. Specifically, M. campanulatihemisphaerica, M. limitis, and M. kunyuensis demonstrate close affiliations with M. abramsii, M. algeriensis Maire, M. galericulata, and M. maculate P. Karst., all of which are endemic to China [21]. Notably, M. campanulatihemisphaerica, M. kunyuensis, and M. abramsii exhibit a similar basidiomata coloration, with M. kunyuensis frequently misidentified as M. abramsii due to the analogous coloration of the pileus before morphological anatomy [21,23,28]. Similarly, M. limitis is indistinguishable from M. algeriensis, M. galericulata, and M. maculata when identification is based solely on the color of the basidiomata [21,28]. Moreover, species within the complex exhibit a comparable pileus coloration and are subject to variations due to changes in growth period and environment conditions, further complicating the accurate identification of these brownish Mycena species [11,12,13,14,69,70,71,72,73,74,75,76,77]. For example, the M. filopes complex, as delineated by Arnolds (2015) and Aronsen (2016), is a case in point. [28,78]. A parallel example is found in the M. pura complex. While basidiomata coloration has historically served as a criterion for subsection classification within the M. pura complex, recent insights by Liu (2023) suggest that the characteristics of the cheilocystidia and pleurocystidia, as well as the presence or absence of pleurocystidia, are critical for species differentiation [11,12,14,79]. Consequently, morphological anatomy is an effective method for the identification of species within the brownish Mycena group.

Pileipellis types and cheilocystidia characteristics are integral to the delimitation of brownish Mycena species. In the taxonomic framework proposed by Smith (1947) and Maas Geesteranus (1992a, 1992b), pileipellis types and cheilocystidia characteristics are pivotal for sectional categorization [11,12,20]. These characteristics are also crucial criteria for distinguishing various species of brownish Mycena, a delineation corroborated by molecular systematics. The phylogenetic tree constructed in this study is divided into 13 clades, and while some clades have lower support rates, the conclusions drawn from the phylogenetic tree remain consistent with those from the morphological anatomy analyses. Notably, M. campanulatihemisphaerica, M. limitis, and M. kunyuensis exhibit smooth cheilocystidia, and cluster phylogenetically into a clade and align closely with the species of sect. Fragilipedes, which also exhibit smooth cheilocystidia [17,28]. Moreover, phylogenetic analyses reveal a distinct clade comprising M. digitifurcata, which aligns closely with the species of sect. Rubromarginatae [26,57]. Sect. Rubromarginatae is characterized by marginta lamellae, ranging from deep yellow to dark greenish [11,12]. If the sectional division relies solely on this feature, M. digitifurcata would ostensibly not qualify for inclusion within the sect. Rubromarginatae due to its lamellae not being marginated [11,12]. However, its pileipellis types and cheilocystidia characteristics are congruent with those of the sect. Rubromarginatae, suggesting the need for a broader consideration of morphological characters. Accordingly, M. digitifurcata is classified within the sect. Rubromarginatae. This classification, proposed by Jadson (2021), identifies M. cristinae as a non-marginate lamellae member of the sect. Rubromarginatae [26]. Our research aligns with Maas Geesteranus’ (1992a, 1992b) interpretation of the sect. Rubromarginatae, which is further supported by the perspectives advanced by Na (2019) [11,12,21]. Specifically, it emphasizes that the delimitation of sections exhibiting colored lamellae edges should predominantly consider variations in pileipellis types and cheilocystidia characteristics. Furthermore, pileipellis and cheilocystidia are present in almost all brownish Mycena, reinforcing their utility as key diagnostic features [23,28]. For example, M. galericulata can be distinguished from its morphologic relative, M. algeriensis, by its tuberculated cheilocystidia, and M. leptocephala is distinguished from its close relative M. polygramma (Bull.) Gray by smooth pileipellis [11,12,21,23,28]. Consequently, we reaffirm the role of pileipellis types and cheilocystidia characteristics as primary taxonomic criteria within the brownish Mycena group, corroborating the classifications of Smith (1947) and Maas Geesteranus (1992a, 1992b) and substantiated by the molecular systematics of Na (2019) [11,12,20,21].

Additionally, this study suggests that M. oryzifluens may represent a prospective novel section. Phylogenetically, M. oryzifluens is closely related to three established sections, namely: sect. Exornatae, sect. Cyanocephalae, and sect. Amictae. Despite these affiliations, M. oryzifluens exhibits distinct morphological characteristics that preclude its classification within these existing sections. These distinguishing features include a dry pileus, the absence of a blue disc at the stipe base, branched cheilocystidia apex, and non-gelatinized pileipellis [11,12,21,28,57]. Consequently, M. oryzifluens does not align with any current sectional definitions and suggests the potential for defining a new section. However, due to the lack of enough specimen samples at present, it is prudent not to propose it as a new section within this publication. Future research efforts will collect and examine additional specimens to substantiate the distinctiveness of this taxonomic group.

At present, there are 24 brownish Mycena species in China. This study introduces 5 new species, increasing the total to 29. Our analysis of morphological characteristics has identified the types of pileipellis and cheilocystidia as critical distinguishing features for brownish Mycena. To facilitate future research and better species distinction, we provide an identification key for the brownish Mycena species in China.

Key to the known species of brownish Mycena from China

1. Basidiospores inamyloid	2
1. Basidiospores amyloid	3
2. Basidiospore broadly ellipsoid, Q = 1.1–1.5	Mycena hiemalis
2. Basidiospore narrowly ellipsoid, Q = 1.7–1.9	Mycena speirea
3. Pileipellis gelatinized	4
3. Pileipellis not gelatinized	10
4. Stipitipellis hyphae ornamented	5
4. Stipitipellis hyphae smooth	6
5. Pleurocystidia present	Mycena clavicularis
5. Pleurocystidia absent	Mycena polygramma
6. Caulocystidia absent	Mycena semivestipes
6. Caulocystidia present	7
7. Cheilocystidia densely covered with tuberculate excrescences	Mycena pluteoides
7. Cheilocystidia smooth	8
8. Pileus with conical spines, basal disc of the stipe present, caulocystidia with outgrowths	Mycena stylobates
8. Pileus without any spines, stipe base without disc, caulocystidia without outgrowths	9
9. Pileus convex, depressed at center, lamellae stained with yellow-brown to orange-brown spots	Mycena subpiligera
9. Pileus conical to campanulate, umbonate at center, lamellae without any spots	Mycena amicta
10. Pileipellis hyphae smooth	11
10. Pileipellis hyphae ornamented	12
11. Caulocystidia present	Mycena algeriensis
11. Caulocystidia absent	Mycena limitis
12. Lamellae flesh pink, pleurocystidia with flesh-pink contents	Mycena entolomoides
12. Lamellae white to grayish white, pleurocystidia hyaline	13
13. Caulocystidia present	14
13. Caulocystidia absent	18
14. Cheilocystidia present	15
14. Cheilocystidia absent	16
15. Cheilocystidia fusoid to lageniform, with long tapered necks, smooth	Mycena subcana
15. Cheilocystidia clavate to obpyriform, densely covered with cylindrical excrescences	Mycena mirata
16. Pileus with brown spots in age, stipitipellis hyphae ornamented	Mycena zephirus
16. Pileus without any spots in age, stipitipellis hyphae smooth	17
17. Basidiospore subglobose to broadly ellipsoid, Q = 1.1–1.4	Mycena oryzifluens
17. Basidiospore narrowly ellipsoid to cylindrical, Q = 1.6–2.1	Mycena leptocephala
18. Pleurocystidia present	19
18. Pleurocystidia absent	23
19. Cheilocystidia apically rounded or narrowed into one to several cylindrical or furcate necks	20
19. Cheilocystidia covered with cylindrical excrescences	21
20. Stipe grayish brown, basidiospore broadly ellipsoid to ellipsoid, Q = 1.4–1.7	Mycena silvae-nigrae
20. Stipe dark brown, basidiospore narrowly ellipsoid to cylindrical, Q = 1.7–2.1	Mycena abramsii
21. Pileus convex, basidiospore narrower than 4 μm	Mycena luguensis
21. Pileus conical to campanulate, basidiospore broader than 4 μm	22
22. Pileipellis with clavate to subglobose terminal cell	Mycena filopes
22. Pileipellis without clavate to subglobose terminal cell	Mycena metata
23. Cheilocystidia apex narrowed into a cylindrical or furcate neck, rarely with short apical outgrowths	24
23. Cheilocystidia densely covered with tuberculate or furcate excrescences, or apex with several branches	25
24. Basidiospore ellipsoid to narrowly ellipsoid, Q = 1.4–1.7	Mycena kunyuensis
24. Basidiospore narrowly ellipsoid to cylindrical, Q = 1.7–2.0	Mycena campanulatihemisphaerica
25. Cheilocystidia apex with several branches	26
25. Cheilocystidia densely covered with tuberculate or furcate excrescences	27
26. Pileus bell-shaped, brown sulcate present, not depressed at center, stipe brown	Mycena venus
26. Pileus oblate hemispherical to convex, brown sulcate absent, depressed at center, stipe gray	Mycena digitifurcata
27. Pileus and lamellae with red-brown spots when old, cheilocystidia sparsely covered with excrescences	Mycena maculata
27. Pileus and lamellae without any spots when old, cheilocystidia densely covered with excrescences	28
28. Basidiospore narrowly ellipsoid to cylindrical, Q = 1.9–2.2	Mycena flos-nivium
28. Basidiospore broadly ellipsoid to ellipsoid, Q = 1.1–1.7	Mycena galericulata