Phylogenetic and Morphological Perspectives on Crepidotus subg. Dochmiopus: Exploratively Unveiling Hidden Diversity in China

Abstract

Crepidotus subg. Dochmiopus contributes to more than half of Crepidotus species and exhibits highly hidden diversity. However, C. subg. Dochmiopus is challenging to study because the basidiomata of C. subg. Dochmiopus species are usually small and white, inconspicuous interspecific distinctions, and possess a familiar complex. In this study, we utilized a variety of characteristics for species identification, including habitat, presence or absence of a stipe in mature specimens, pileipellis and cheilocystidia patterns, whether the lamellae edges are fimbriated, and other characteristics. Above all, cheilocystidia and pileipellis patterns will be important in C. subg. Dochmiopus research. Based on the present specimens, we constructed a multigene phylogenetic tree (ITS + LSU) and recognized four new species: C. lamellomaculatus sp. nov., C. capitatocystidiatus sp. nov., C. succineus sp. nov., C. clavocystidiatustustus sp. nov. Detailed morphological descriptions, photographs, line drawings and comparisons with closely related taxa for the new species are provided. The current phylogenetic analysis does not support the previously classifications, indicating that the classification of Crepidotus requires re-evaluation. But the existing molecular datasets and species’ descriptions are insufficient to fully resolve the classification. Further integration of new gene segments and a comprehensive review of morphological characteristics will reveal a natural classification for Crepidotus.

1. Introduction

Crepidotus (Fr.) Staude are notable saprophytic fungi, primarily utilizing decaying wood and branches as growth substrates, characterized by small basidiomata with dark-colored basidiospores, and these mostly exhibit an underdeveloped stipe [1,2,3,4]. Since Staude elevated the Agaricus L. tribe Crepidotus Fr. to generic rank in 1857, a total of 586 Crepidotus taxa have been recorded, encompassing approximately 400 species (https://www.indexfungorum.org/Names/Names.asp, accessed on 5 October 2024). Taxonomic research about this genus has traditionally been concentrated in Europe and America [1,2,3,5,6]. Since the early 21st century, studies in Asia have gradually increased, and 34 species have been published, with 13 of these species described by Chinese scholars [7,8,9,10,11,12,13,14,15]. In 1965, based on morphological studies, Hesler and Smith classified Crepidotus into three subgenera [5], and documented 123 taxa: 19 clampless taxa in C. subg. Crepidotus Hesler & A.H. Sm, 61 elongated-spored taxa in C. subg. Dochmiopus (Pat.) Pilát, 43 globose-spored taxa in C. subg. Sphaerula Hesler & A.H. Sm. In 2008, Consiglio and Setti [6], primarily focused on European specimens, and revised the classification by dividing the genus into two subgenera and combining globose-spored species into C. subg. Dochmiopus as sect. Sphaeruli Hesler & A.H. Sm. They documented including 25 taxa in the genus Crepidotus: 6 in C. subg. Crepidotus and 19 in C. subg. Dochmiopus 12 elongated-spored and 7 globose-spored taxa [6]. Both classifications indicate that C. subg. Dochmiopus exhibited abundant species diversity, representing about 50% of taxa of the genus [5,6]. Despite different definitions of C. subg. Dochmiopus, most taxa within this subgenus are characterized by ornamented basidiospores and hyphae with clamp connections [1,5,6].

Crepidotus subg. Dochmiopus exhibits simple characteristics, limited taxonomic characteristics, insufficiently detailed morphological descriptions, and absent available molecular sequences, presenting significant challenges for precise identification and systematic classification. Initially described by Pilát in 1948, C. subg. Dochmiopus is characterized by its small and white basidiomata, non-hygrophanous, and fibrillose-covered pileus [1,5,16]. The macroscopic characteristics within this subgenus are highly similar, and only basidiospores, cystidia and pileipellis demonstrate morphological value. The restriction of utilized morphological characteristics brought obstacles to the efficiency of identification and classification. The majority of species within C. subg. Dochmiopus present cheilocystidia while lacking pleurocystidia or pileocystidia, only with a few exceptions. For instance, four species within C. subg. Dochmiopus sect. Cystiodiosi Hesler & A.H. Sm (C. pseudoflammeus Hesler & A.H. Sm., C. albatus Hesler & A.H. Sm., C. luteicolor Hesler & A.H. Sm., C. rainierensis Hesler & A.H. Sm.) possess pleurocystidia [5]. In addition, C. autochthonus J. Lang, C. herrerae Bandala & Montoya, C. malachioides Consiglio, Prydiuk & Setti, and others possess pileocystidia [6,17]. Given the high species diversity within the genus Crepidotus, the scarcity of valuable morphological characteristics further complicates identification and classification. Additionally, some species exhibit variability in cheilocystidia shape, which obscures determinations between inter- and intraspecific variations [18]. For example, C. variabilis P. Kummer, the type species of C. subg. Dochmiopus, possesses six varieties and exhibits diverse morphologies in its cheilocystidia [18]. Jančovičová studied the holotype of C. variabilis and noted that the cheilocystidia were predominantly narrowly lageniform or utriform to lobate, with a few being clavate to lageniform with forked and antler-like shapes [18]. Factors such as the maturing status of basidiomata and environmental humidity may lead to the secondary development of cheilocystidia, resulting in substantial intraspecific morphological variability [19]. Such phenomena complicate the accurate determination of the mature morphology of cheilocystidia. Furthermore, accurate recognition is further encumbered by subtle distinctions among closely related species in basidiospore ornamentations, which may vary from smooth to punctate or reticulate patterns [1,6,7]. Presently, GenBank houses fewer than 600 Crepidotus sequences, with about 65% being ITS sequences and 35% LSU sequences. These cover 96 identified species and 70 uncertain taxa (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 5 October 2024). Some sequences are less reliable, due to incorrect submissions, thereby reducing their utility. Only around 40 taxa within C. subg. Dochmiopus have associated sequences, providing limited data for taxonomic and phylogenetic study. Some species were published based on traditional morphological studies and lacked sequences, which may lead to nomenclatural synonyms and cause more difficulties for future research.

The species diversity in this genus has been consistently underestimated. In recent years, our research group has published 12 new species of Crepidotus from China [4,7,12,15,20]. However, our ongoing studies of collected specimens have revealed more unknown species. Among these, the number of C. subg. Dochmiopus is the highest, indicating significant hidden diversity. The simple morphological characteristics, the scarcity of useful taxonomic characteristics, and the limited molecular datasets pose challenges for species recognition and classification. Current phylogenetic analyses do not support the morphologically established classification, and the absence of clamp connections and smooth basidiospores is no longer an exclusive characteristic of C. subg. Crepidotus. In this study, we introduce four new species of C. subg. Dochmiopus collected from various regions in China, provide a preliminary discussion of their key taxonomic characteristics and build a multigene phylogenetic tree to discuss taxonomic problems. We consider C. subg. Dochmiopus contains a hidden species diversity, relying solely on morphological characteristics to distinguish subgenera is not suitable, and there may be new sections within C. subg. Dochmiopus. More molecular datasets and key taxonomic characteristics could potentially offer robust support to resolve C. subg. Dochmiopus’s taxonomic problems and establish a more natural classification.

2. Materials and Methods

2.1. Specimens and Morphological Examination

The studied specimens were collected from Heilongjiang Province, Jilin Province, Shandong Province, Hubei Province, and Fujian Province of China. Macro-morphological descriptions relied on field records of fresh specimens, while micro-morphological descriptions were based on observations of dry specimens. During fieldwork, high-resolution photographs of fresh basidiomata and their surrounding habitat were taken by an Olympus E-M1 Mark III camera equipped with an M. Zuiko Digital Ed 12–40 mm or 60 mm lens (Olympus, Tokyo, Japan) and a Sony ILCE-A7M3 (Sony, Tokyo, Japan) with a Sigma 70 mm f2.8 EX DG MACRO (Sigma, Kobe, Japan). Comprehensive details of the specimens were recorded, including the size, color, shape, and characteristics of pileus, lamellae, stipe, odor and taste of fresh specimens, and their location information. More characters of pileus and lamellae were recorded, including whether the pileus surface was hygrophanous, striate, smooth, villose, or scaly, and whether the lamellae edge was fimbriated. In our descriptions, the color codes and notations for the macroscopic characteristics of the basidiomata followed the work of Robert Ridgway [21]. Freshly collected specimens were dried with portable dryers at 40 °C (Stöckli, Netstal), and then sealed and stored with allochroic silica gel [22]. Microscopic examinations were carried out on dried materials rehydrated with 5% potassium hydroxide (KOH) aqueous solution and, if necessary, stained with 1% Congo red aqueous solution [23,24]. Each microstructure was observed using a Lab A1 microscope (Carl Zeiss AG, Jena, Germany), and then photographed and measured with ZEN 2.3 software [25].At least 20 matured basidiospores of each basidiomata were measured in lateral view (under oil, 1000×), and one or two basidiomata were recorded for each specimen. The notation [a/b/c] used at the beginning of each basidiospore description indicates that a number of basidiospores from b number of basidiomata of c number of specimens were measured; the dimensions (length × width) are presented as (d)e–f–g(h) × (i)j–k–l(m), in which d is the minimum length, e–g represents the range of at least 90% of values, f is the average length and h is the maximum length; the width (i–m) is expressed in the same way [23,26]. Furthermore, Q denotes the ratio of length and width, and Q_m refers to the average Q value of all basidiospores ± the sample standard deviation [24,27,28]. The type of basidiospore ornamentation referred to Consiglio & Setti’s descriptions [6]. The length and width of the hyphae of pileipellis, basidia, cheilocystidia, and pileocystidia were measured through at least 20 measurements of each species; for basidia and cystidia, the widest point value was chosen as the width, and the length of the sterigma was excluded from the measurement and measured separately. Based on the observation of the macro- and microstructures of multiple specimens, illustrations of the basidiomata and microstructures of the new species were created. First, pencil sketches were drawn on A4 paper, and then transposed to tracing paper using a needle pen. Subsequently, the line drawings on the tracing paper were digitized into TIFF format using a Canon LiDE120 scanner (Canon, Tokyo, Japan), and post-processed using Photoshop 2020. The voucher specimens used were deposited in the Fungarium of the Fujian Academy of Agricultural Sciences (FFAAS), Fuzhou, China.

2.2. DNA Extraction, PCR Amplification and DNA Sequencing

Genomic DNA was extracted from each specimen using the NuClean Plant Genomic DNA Kit (Kangwei Century Biotechnology Co., Beijing, China). The nuclear internal transcribed spacer (ITS) region (ITS1-5.8s-ITS2) and the large subunit region (LSU) of rDNA were amplified by primer pair ITS1/ITS4 and LROR/LR7 [29,30]. Amplifications were performed in a total volume of 25 μL, containing 1 μL of each primer, 12.5 μL 2 × Utaq PCR MasterMix (ZomanBio, Beijing, China), 8.5 μL ddH₂O, and 2 μL DNA template [31]. The PCR thermocycling protocol for amplification of the ITS region was 94 °C for 4 min, followed by 34 cycles of 94 °C for 45 s, 52 °C for 45 s, and 72 °C for 1 min and final extension for 10 min at 72 °C; for LSU, it was 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min and final extension for 10 min at 72 °C [23]. The PCR product was sequenced by Bgi Tech Solutions (Beijing Liuhe) Co. (Beijing, China). The BioEdit v7.0.4.1 was used to inspect chromatograms to ensure the new sequence quality [32,33]. The low-quality sequences were enhanced using plasmid vectors by using VELUTE Gel Mini Purification and pBLUE-T kits (Beijing Zoman Biotechnology Co., Beijing, China) to improve [4]. All obtained sequences in this study were submitted to GenBank. The BLAST and the GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 5 October 2024) were used to download homologous ITS and LSU sequences of Crepidotus and outgroup taxa sequences. Representative species that share a morphological or phylogenetic affinity with the new species were selected for phylogenetic analysis. Detailed information on these specimens is given in

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

Taxa	Voucher/Strain No.	Location	GenBank Sequence ID		Reference
			ITS	nLSU
C. affinis	PDD:72848	New Zealand	KY827291	–	[34]
C. alabamensis	TBGT15610	India	MK459545	MK459543	[34]
C. albolanatus	PDD:72865	New Zealand	KY827292	–	[34]
C. applanatus	SLO 2534	Slovakia	OM832521	OM832556	[35]
C. applanatus	SLO 2539	Slovakia	OM832523	OM832558	[35]
C. applanatus	SLO 2551	Slovakia	OM832526	OM832560	[35]
C. asiaticus	TJB9995	Thailand	MF077337	MF077336	[8]
C. asiaticus	TJB10018	Thailand	MF077339	MF077338	[8]
C. asiaticus	FFAAS0336	China	MW580871	–	Direct Sub.
C. asiaticus	FFAAS0338	China	MW580872	–	Direct Sub.
C. brunnescens	MCA864	Not indicated	–	AF367936	Unpublished
C. calolepis	WU 28902	Hungary	KF879617	–	[36]
C. calolepis	iNaturalist 142786935	USA	OR824560	–	Unpublished
C. calolepis	314298	USA	MH188448	–	Direct Sub.
C. capitatocystidiatus	FFAAS1310 Holotype	China	PQ061270	PQ061255	This study
C. capitatocystidiatus	FFAAS1311	China	PQ061271	PQ061256	This study
C. capitatocystidiatus	FFAAS1312	China	PQ061272	PQ061257	This study
C. caspari	FFAAS0342	China	MZ401361	MW581521	[4]
C. cinnabarinus	MCA387	USA	–	AF205686	[37]
C. circinatus	MushroomObserver. org/307011	USA	MH087459	–	Direct Sub.
C. circinatus	iNAT:66988897	USA	OQ147188	–	Direct Sub.
C. circinatus	iNaturalist 141511315	USA	OR824682	–	Direct Sub.
C. clavocystidiatus	FFAAS1316 Holotype	China	PQ061276	PQ061261	This study
C. clavocystidiatus	FFAAS1317	China	PQ061277	PQ061262	This study
C. clavocystidiatus	FFAAS1318	China	PQ061278	PQ061263	This study
C. clavocystidiatus	FFAAS1319	China	PQ061279	PQ061264	This study
C. croceotinctus	iNat31834012	USA	MN498116	–	Direct Sub.
C. croceotinctus	S.D. Russell ONT iNaturalist # 126570732	USA	OP470431	–	Direct Sub.
C. croceotinctus	S.D. Russell ONT iNaturalist 131145003	USA	OP549098	–	Direct Sub.
C. crocophyllus	SLO 2433	Slovakia	OM832529	OM832562	[35]
C. crocophyllus	SLO 2588	Slovakia	OM832530	OM832563	[35]
C. dentatus	HMJAU37097	China	MH320736	–	[12]
C. dentatus	HMJAU37161	China	MH320737	–	[12]
C. epibryus	IBNR 1997/0948	Russia	–	AF367934	[38]
C. epibryus	NL-5379	Hungary	–	MK277884	[39]
C. exiguus	TBGT17176	India	–	MK567974	[11]
C. flavobrunneus	TBGT15841	India	–	MK567981	[11]
C. fragilis	MCA 904	USA	–	AF367931	[38]
C. fraxinicola	OKM26739	USA	–	AF205699	[37]
C. fraxinicola	OKM26748	USA	–	AF205697	[37]
C. herbaceus	HMJAU37009	China	MW080327	–	[20]
C. herbaceus	HMJAU37025	China	MW080326	–	[20]
C. heterocystidiosus	HMJAU37054	China	MF461342	–	[12]
C. heterocystidiosus	HMJAU37034	China	MF461344	–	[12]
C. inhonestus	MCA638	Not indicated	–	AF205704	[40]
C. innuopurpureus	MEL 2503290	Australia	NR_182391	MZ870346	[41]
C. iqbalii	MU248	Pakistan	OQ672617	–	Direct Sub.
C. iqbalii	LAH36654	Pakistan	MT973498	MW888515	[13]
C. kangoliformis	BAP 664	São Tomé and Príncipe	KX017199	–	[42]
C. kauffmanii	MIN-F-0905412	USA	–	MK277887	[39]
C. lamellomaculatus	FFAAS1306	China	PQ061266	PQ061251	This study
C. lamellomaculatus	FFAAS1309	China	PQ061269	PQ061254	This study
C. lamellomaculatus	FFAAS1308	China	PQ061268	PQ061253	This study
C. lamellomaculatus	FFAAS1307	China	PQ061267	PQ061252	This study
C. lamellomaculatus	FFAAS1305 Holotype	China	PQ061265	PQ061250	This study
C. lanuginosus	OKM27331	USA	–	AF367940	Unpublished
C. lateralipes	PDD:72508	New Zealand	KY827293	–	[34]
C. lateralipes	PDD:72571	New Zealand	KY827294	–	[34]
C. lateralipes	PDD:98270	New Zealand	KY827295	–	[34]
C. luteolus	16834	Italy	JF907963	–	[43]
C. lutescens	HMJAU 21976	China	KU762016	–	[7]
C. lutescens	HMJAU 37002	China	KU762017	–	[7]
C. macedonicus	PV773	Hungary	MH780921	–	[44]
C. macedonicus	DB3859	Hungary	MH780922	–	[44]
C. macedonicus	MB19102501	Italy	PP131267	PP125747	Direct Sub.
C. malachioides	SLO 2578	Slovakia	OM832538	OM832568	[35]
C. malachioides	SLO 2391	Slovakia	OM832536	OM832567	[35]
C. malachius	SLO 2541	Slovakia	OM832546	OM832575	[35]
C. malachius	SLO 2091	Slovakia	OM832541	OM832571	[35]
C. malachius	SLO 2530	Slovakia	OM832543	OM832573	[35]
C. mollis	OKM26279	USA	–	AF205677	[37]
C. mollis	PBM 1036 (WTU)	USA	–	DQ986293	[45]
C. neotrichocystis	CS1150	Malta	OL672745	OL672702	[46]
C. nephrodes	OKM25896	Not indicated	–	AF205693	[37]
C. nephrodes	MCA189	Not indicated	–	AF205670	[37]
C. novae-zealandiae	PDD:95850	New Zealand	HQ533046	–	Direct Sub.
C. nyssicola	S.D. Russell MycoMap # 7426	USA	MN906237	–	Direct Sub.
C. nyssicola	S.D. Russell MycoMap # 7399	USA	MN906236	–	Direct Sub.
C. occidentalis	MUOB:367585	USA	OK376745	–	Direct Sub.
C. palodensis	TBGT16716	India	MH844890	MH310743	[10]
C. praecipuus	PDD:72624	New Zealand	KY827312	–	[34]
C. praecipuus	PDD:72481	New Zealand	KY827311	–	[34]
C. pseudomollis	HMJAU37158	China	MH320739	–	[12]
C. pseudomollis	HMJAU37163	China	MH320740	–	[12]
C. pseudomollis	HMJAU37125	China	MH320738	–	[12]
C. reticulatus	HMJAU37089	China	MF461346	–	Direct Sub.
C. roseus	TBGT15507	India	MK567976	MK567977	[11]
C. rufidulus	PDD 98272	New Zealand	NR_159823	–	[34]
C. rufofloccosus	PDD 72601	New Zealand	NR_159822	–	[34]
C. sphaerosporus	11253	Italy	JF907960	–	Direct Sub.
C. sphaerosporus	HMAS 290002	China	MK966514	–	Direct Sub.
C. stenocystis	PRM911279	Czech Republic	MF621030	MF621024	[47]
C. stenocystis	SLO 481	Slovakia	OM832552	–	[35]
C. stenocystis	SLO 2557	Slovakia	OM832553	OM832581	[35]
C. striatus	HMJAU37087	China	MH320742	–	[12]
C. striatus	HMJAU37076	China	MH320741	–	[12]
C. subfulviceps	BCN SCM B-5144	Spain	–	FJ947116	[48]
C. subfulviceps	BCN SCM B-5138	Spain	–	FJ947117	[48]
C. subverrucisporus	MCA774	USA	–	AF367948	[19]
C. succineus	FFAAS1313 Holotype	China	PQ061273	PQ061258	This study
C. succineus	FFAAS1314	China	PQ061274	PQ061259	This study
C. succineus	FFAAS1315	China	PQ061275	PQ061260	This study
C. tennesseensis	TENN 29144	USA	FJ601806	–	Unpublished
C. tennesseensis	LRH29144	USA	NR_119720	GQ892981	[49]
C. tobolensis	TCSS UB RAS9477	Russia	MK522392	–	[50]
C. tobolensis	LE 287655	Russia	MK522393	MK560762	[50]
C. tobolensis	LE313671	Russia	OL739885	–	[51]
C. tortus	TBGT17194	India	MK462161	MK462162	[11]
C. trichocraspedotus	HMJAU37250	China	MH320744	–	[12]
C. trichocraspedotus	HMJAU37138	China	MH320743	–	[12]
C. trulliformis	PDD:98274	New Zealand	KY827298	–	[34]
C. ulmicola	HMJAU37011	China	KX456184	–	[20]
C. ulmicola	HMJAU37027	China	MW080328	–	[20]
C. variabilis	SLO 2021	Slovakia	MT055889	OM832585	[18]
C. variabilis	SLO 2018	Slovakia	MT055890	OM832583	[18]
C. variabilis	SLO 2423	Slovakia	MT055887	OM832584	[18]
C. versutus	MCA381	USA	–	AF205683	[37]
C. versutus	MCA250	USA	–	AF205695	[37]
C. volubilis	TBGT15648	India	MH845231	MH310742	[11]
C. wasseri	LE 287679	Russia	MW722981	MW723022	[52]
C. wasseri	MO500187	USA	OR203555	–	Direct Sub.
C. yuanchui	FFAAS0340	China	MZ401362	–	[4]
C. yuanchui	FFAAS0341	China	MZ401363	MW581519	[4]
N. dominicanus	MCVE 26928	Dominican Republic	JN033216	JN033217	[53]
N. echinospermus	AH45884	Brazil	KY468512	KY468511	Direct Sub.
N. echinospermus	MPM 2886	Brazil	–	JN033222	[53]

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

.

2.3. Sequence Alignment and Molecular Phylogeny

This study is based on the use of combined ITS and LSU analyses to investigate the relationship of the new taxa with other species of Crepidotus. Maximum likelihood (ML) and Bayesian inferences (BI) were implemented to construct phylogenetic trees. Sequences used for phylogenetic analysis were aligned using MAFFT v7.487, strategy Auto (FFT-NS-1, FFT-NS-2, FFT-NS-i or L-INS-i), and then manually adjusted and trimmed using BioEdit v7.0.4.1. MEGA 10 was used to generate matrixes [32,33,54,55]. The raxmlGUI v2.0 inferred the best-fit substitution model for each gene partition and performed the ML analysis with 1000 bootstrap replicates [56]. For BI analysis with MrBayes v3.2.6, the best-fit substitution model for each gene partition was inferred by MrModeltest 2.3 [57,58]. MCMC generations were run 8 chains for 5,000,000 generations, sampled every 1000 generations, until the average standard deviation of split frequencies was under 0.01. In total, 25% of the trees were removed as a burn-in phase of each analysis [57]. Tracer v1.7.2 was used for the visualization and examination of MCMC trace files from Bayesian phylogenetic inference [59]. Phylogenetic trees were visualized and checked in Figtree v1.4.3 and Photoshop 2020 was used for the final editing of the trees.

3. Results

3.1. Phylogenetic Analysis

The dataset matrix was composed of 68 taxa, 126 samples, 174 sequences (104 ITS, and 70 LSU). Among these, 144 sequences were downloaded from GenBank (89 ITS, and 55 LSU), and 30 sequences (15 ITS, and 15 LSU) were amplified in this study. Neopaxillus dominicanus Angelini & Vizzini and N. echinospermus (Speg.) Singer were selected as the outgroups [53]. The aligned length, including gaps, encompassed 1782 nucleotide sites, with 861 for ITS and 921 for LSU. For BI analysis, the best-fit substitution model for both ITS and LSU was GTR + I + G. BI analysis resulted in an average standard deviation of split frequencies = 0.005577, the effective sample size (ESS) was 1333.8, and the average Potential Scale Reduction Factor (PSRF) parameter values = 1.000 after 5,000,000 MCMC generations. For ML analysis, the best-fit substitution model selected for ITS was TVM + I + G4 and that for LSU was GTR + I + G4, with a final log-likelihood value of −14,404.410766. The average bootstrap value was 87.17%, indicating generally high confidence in the tree’s branches. The BI and ML trees exhibited similar topologies, and the BI tree is shown in Figure 1. Those nodes supported by bootstrap of at least 75% and posterior probabilities exceeding 0.90 were incorporated.

According to the phylogenetic tree in Figure 1, twelve high-support clades were delineated. Clade 1 comprises eight species: C. striatus T. Bau & Y.P. Ge, C. heterocystidiosus T. Bau & Y.P. Ge, C. dentatus T. Bau & Y.P. Ge, C. alabamensis Murrill, C. pseudomollis T. Bau & Y.P. Ge, C. fraxinicola Murrill, C. mollis (Schaeff.) Staude, and C. calolepis (Fr.) P. Karst. This clade has strong support in BI analysis (BS/BPP = --/1.00); the species in this clade exhibit smooth elongated spores and their tissues do not have clamp connections. Based on Consiglio and Setti’s classification, all eight species in this clade belong to C. subg. Crepidotus sect. Crepidotus [6]. Clade 2 includes C. wasseri Kapitonov, Biketova, Zmitr. & Á. Kovács and C. lamellomaculatus, with strong support (BS/BPP = 97/1.00). The new species C. lamellomaculatus forms a well-supported clade (BS/BPP = 98/1.00).Both C. wasseri and C. lamellomaculatus possess smooth elongated spores and clamp connections [52]. Typically, species in Crepidotus with clamp connections exhibit ornamented basidiospores [4,6,12]. However, some species exhibit smooth basidiospores and clamp connections simultaneously, such as C. wasseri and C. lamellomaculatus in Clade 2, and C. novae-zealandiae Pilát, C. tortus A.M. Kumar & C.K. Pradeep, and C. trichocraspedotus T. Bau & Y.P. Ge in Clade 10 [11,12,34,52]. According to Consiglio & Setti’s classification, Clade 2 and Clade 10, characterized by clamp connections and smooth elongated spores, should be classified within C. subg. Dochmiopus sect. Autochthoni (Sennn-Irlet) Consiglio & Setii [6]. However, the current phylogenetic tree does not support this classification. Additionally, Clade 2 and Clade 10 exhibit significant differences in the morphology of their cheilocystidia. In Clade 2, the cheilocystidia of C. wasseri and C. lamellomaculatus are both lageniform and ventricose, whereas in Clade 10, the cheilocystidia are sinuously cylindrical to vine-like [52]. Clade 3 comprises C. caspari Velen., C. inhonestus P. Karst., C. sphaerosporus (Pat.) J.E. Lange, C. circinatus Hesler & A.H. Sm., C. ulmicola T. Bau & Y.P. Ge, C. occidentalis Hesler & A.H. Sm., and C. fragilis Joss. Among these, C. occidentalis and C. fragilis are classified in C. subg. Dochmiopus sect. Autochthoni, due to their smooth basidiospores and clamp connections [5,6]. The remaining taxa belong to C. subg. Dochmiopus sect. Dochmiopus. Specifically, C. sphaerosporus and C. circinatus are part of C. subg. Dochmiopus sect. Dochmiopus ser. Dochmiopus Consiglio & Setii, whereas C. caspari, C. inhonestus and C. ulmicola are classified in C. subg. Dochmiopus sect. Dochmiopus ser. Caspari Consiglio & Setii [4,6,20]. Although C. ulmicola was initially placed in C. subg. Dochmiopus sect. Dochmiopus ser. Dochmiopus when published, its smooth to nearly smooth basidiospores suggest a reclassification to C. subg. Dochmiopus sect. Dochmiopus ser. Caspari [20]. Additionally, C. sphaerosporus has been accepted as C. cesatii (Rabenh.) Sacc., and C. fragilis was combined into C. autochthonus [1,6]. Clade 4 includes three species, C. subverrucisporus Pilát, C. herbaceus T. Bau & Y.P. Ge and C. palodensis C.K. Pradeep & A.M. Kumar, all of which are regarded as C. subg. Dochmiopus sect. Dochmiopus ser. Caspari. These species are characterized by clavate cheilocystidia and a gregarious habit [1,10,20]. Clade 5 incorporates the new species C. succineus (BS/BPP = 100/1.00) along with nine other taxa. Except for C. roseus Singer and C. epibryus (Fr.) Quél., other taxa in this clade belong to C. subg. Dochmiopus sect. Dochmiopus ser. Dochmiopus. Crepidotus roseus is classified in C. subg. Dochmiopus sect. Sphaeruli due to its nearly globose basidiospores (Q_m < 1.10) [6,11]. As C. epibryus has smooth elongated spores and absent clamp connections, it is placed in C. subg. Crepidotus sect. Versuti Hesler & A.H. Sm.; C. versutus (Peck) Sacc. also belongs to this section for similar reasons [5,6]. Clade 5 contains several brightly colored species, including the yellow-to-orange C. tobolensis Kapitonov, Biketova & Zmitr., C. succineus, C. praecipuus E. Horak, C. lutescens T. Bau & Y.P. Ge, C. yuanchui Q. Na, Z.W. Liu & Y.P. Ge, and pink-to-red-colored C. innuopurpureus McMull.-Fish., T. Lebel & Senn-Irlet, and C. roseus. In Clade 6, except C. cinnabarinus Peck, the other taxa belong to C. subg. Dochmiopus sect. Dochmiopus ser. Dochmiopus, and C. kauffmanii Hesler & A.H. Sm., C. reticulatus T. Bau & Y.P. Ge and C. cinnabarinus are notably colored species. Crepidotus cinnabarinus Peck is classified in C. subg. Crepidotus sect. Cinnabarinus Hesler & A.H. Sm., due to its ornamented basidiospores and absence of clamp connections [6,27]. Additionally, we concur with the assessment by Jančovičová et al. regarding the GenBank accession numbers for C. reticulatus [60]. Upon reviewing the original publication, we confirmed that the holotype of C. reticulatus is correctly associated with the GenBank sequence MF461346. The sequence was previously misassigned, and we have submitted a correction request to GenBank to rectify this error. Among Clade 5 and Clade 6, C. epibryus, C. versutus and C. cinnabarinus, which present absent clamp connections, cannot be classified in C. subg. Crepidotus based on the current phylogenetic analysis. This suggests that the absence of clamp connections is not an exclusive characteristic of C. subg. Crepidotus, and other subgenera also include taxa without clamp connections. Therefore, the defining characteristic of C. subg. Crepidotus requires further study and refinement. All species in Clade 7 belong to C. subg. Dochmiopus sect. Dochmiopus ser. Dochmiopus. Within this clade, the new species C. capitatocystidiatus is well supported (BS/BPP = 100/1.00). Crepidotus capitatocystidiatus clusters with C. affinis E. Horak and C. volubilis C.K. Pradeep & A.M. Kumar, sharing similar guttiform basidiospores covered by irregularly punctate-verrucae [10,34]. Notably, C. asiaticus Guzm.-Dáv., C.K. Pradeep & T.J. Baroni in Clade 7, C. iqbalii A. Izhar, Usman & Khalid and C. subfulviceps (Murrill) Aime, Vila & P.-A. Moreau in Clade 9 differ from the typical Crepidotus species due to their agaricoid basidiomata, representing the stipitate group within Crepidotus. Clade 8 comprises three species within C. subg. Dochmiopus sect. Dochmiopus ser. Dochmiopus, characterized by their white basidiomata with a finely villous pileus surface. Clade 9 includes three species, C. iqbalii, C. subfulviceps and C. clavocystidiatus. The new species C. clavocystidiatus is strongly supported (BS/BPP = 98/1.00). Crepidotus clavocystidiatus belongs to C. subg. Dochmiopus sect. Dochmiopus ser. Caspari, and the other two species are classified in C. subg. Dochmiopus sect. Dochmiopus ser. Dochmiopus. The taxa of Clade 11 and Clade 12 all possess clamp connections and globose ornamented basidiospores and are classified as C. subg. Dochmiopus. sect. Sphaeruli. But in the phylogenetic tree, Clade 11 and Clade 12 do not form a monophyletic group, indicating that C. subg. Dochmiopus sect. Sphaeruli is polyphyletic. This aligns with the viewpoint proposed by Ge, which is further corroborated by our current phylogenetic analysis [61].

3.2. Taxonomy

Crepidotus lamellomaculatus M.H. Han, Q. Na, Y.P. Hu & Y.P. Ge, sp. nov., Figure 2, Figure 3 and Figure 4.

MycoBank no: 854914

Etymology: The epithet lamellomaculatae combines the Latin word ‘lamellae’ and the Latin adjective ‘maculatae’, meaning spotted. This name accurately describes the lamellae surface, which shows a few dark spots when matured.

Holotype: China. Hubei Province, Yichang City, Wufeng County, 19 June 2024, leg. Menghui Han, Jingwen Guo, Guanyu Qiu, Lijun Wang, Qin Na, and Yupeng Ge, 553 m asl, FFAAS1305 (collection number NJ5410).

Diagnosis: Pileus white, covered by short white felted tomentum, margin incurved, lamellae edge nearly smooth when matured, stipe observed in early stage; basidiospores (5.6)6.0–6.5–7.1(7.6) × (4.2)4.4–4.8–5.2(5.7) μm, broadly ellipsoid to ellipsoid, smooth, cheilocystidia narrowly lageniform with a curved elongated neck, ventricose. Differs from C. wasseri by smaller cheilocystidia and basidiospores, and absent of pileocystidia.

Description: Pileus 4–18 mm broad, in young stage conchoidal to flabelliform, White (LIII), when matured semicircular to reniform, sometimes petaloid, nearly applanate, White (LIII), when old or dry Pale Olive Buff (XL21‴f), margin incurved in all stages; dry, covered by short White (LIII) felted tomentum, margin glabrescent, non-striated, not hygrophanous, attached laterally or dorsally to the substratum, White (LIII) villosity observed near the point of attachment. Lamellae less than 1mm broad, L = 12–17, l = 3–7, subdecurrent, ventricose, when young White (LIII), edge slightly fimbriated, when matured Isabella Color (XXX19″i) to Light Brownish Olive (XXX19″k), surface with a few dark spots, and edge nearly smooth. Stipe observed in young stage, clavate to cylindrical, lateral, White (LIII), with age persisting as a knob in lateral, indistinct. Context thin (<5 mm thick), White (LIII). Odor and taste not distinctive.

Basidiospores (417/8/5)(5.6)6.0–6.5–7.1(7.6) × (4.2)4.4–4.8–5.2(5.7) μm, Q = (1.24)1.26–1.46(1.50), Q_m = 1.35 ± 0.061, [HOLOTYPE (68/2/1)(6.2)6.3–6.8–7.2(7.4) × (4.3)4.6–4.9–5.2(5.3) μm, Q = (1.30)1.32–1.47(1.49), Q_m = 1.39 ± 0.039], broadly ellipsoid to ovoid in frontal view, broadly ellipsoid to ellipsoid in lateral view, greyish orange to yellowish brown in 5% KOH aqueous solution, smooth (under oil), sometimes granular contents or large oil drop observed. Basidia 16–25 × 5–7 μm, short clavate, apex obtuse, base slightly constricted, four-spored, rarely two-spored, sterigmata 1.9–3.8 μm long, thin-walled (<0.5 μm thick), hyaline. Pleurocystidia absent. Cheilocystidia 17–41 × 3–6 μm, slightly clavate to utriform, narrowly lageniform with a curved neck, apex rounded, the middle part ventricose and slightly attenuated at the base, clumped, hyaline, thin-walled (<0.5 μm thick). Pileipellis a tomentum, composed of interwoven long cylindrical hyphae, 3–6 μm in diameter, sometimes branched, terminal cells flexuous, sometimes vertical to the pileus, thin-walled (<0.5 μm thick), hyaline, non-gelatinized. Lamellae trama subregular, composed of cylindrical hyphae, 3–6 μm in diameter, interwoven, at times inflated, non-gelatinized. Clamp connections present only in basidia and hymenium, occasionally observed in cheilocystidia.

Habit and habitat. Scattered on rotten branches and woods in the mixed broadleaf-conifer forests of temperate and subtropical zones, mainly found under Betula sp., Castanopsis sp., Cypressi sp., Cunninghamia sp., and Fraxinus sp.

Other specimens examined: China. Jilin Province, Yanbian Korean Autonomous Prefecture, Antu County, Changbaishan National Nature Reserve, 16 August 2021, leg. Menghui Han, Zewei Liu, Qin Na, and Yupeng Ge, 831m asl, FFAAS1306 (collection number GN1378), same location, 17 August 2021, leg. Menghui Han, Zewei Liu, Qin Na, and Yupeng Ge 843 m asl, FFAAS1307 (collection number GN1398), 841 m asl, FFAAS1308 (collection number GN1399); Heilongjiang Province, Hegang City, Luobei County, Taipinggou National Natural Reserve, 6 July 2023, leg. Menghui Han, Renxiu Wei, Tingting Sun, Zengcai Liu, Weiguo Chen, Xinyu Tong, Yawei Li, Nannan Geng, Li Zou, Qin Na, and Yupeng Ge, 476 m asl, FFAAS1309 (collection number GN1878).

Notes: Within the genus Crepidotus, the species with clamp connections typically exhibit ornamented basidiospores, while those lacking clamp connections generally present smooth basidiospores [1,5,6]. However, C. lamellomaculatus is an exceptional species within the genus, possessing both clamp connections and smooth basidiospores, and it is considered to belong to C. subg. Dochmiopus sect. Autochthoni. Before that, the only species in this section was C. autochthonus; it was distinguished from C. lamellomaculatus by bigger basidiospores (7.0–9.0 × 5.0–6.0 μm or 7.1–8.5 × 4.9–5.7 μm) and cylindrical to narrowly utriform cheilocystidia [1,6]. Phylogenetic analysis showed the C. wasseri is most related to C. lamellomaculatus, and there are many similarities in macroscopical, but C. wasseri can be distinguishable by its narrowly lageniform pileocystidia, bigger cheilocystidia [(33.1)37.1–59.6(74.0) × (3.9)4.6–7.3(8.4) μm] and basidiospores [(6.5)7.1–8.6(9.0) × (4.5)4.8–5.5(5.9) μm, av. = 7.8 × 5.2 μm] [50]. Crepidotus trichocraspedotus is a species discovered in China, possessing smooth basidiospores and clamp connections similar to C. lamellomaculatus, but it can be easily distinguished from bigger basidiospores [(8.8)9.1–10.5(10.7) × 6.0–6.8(7.1) μm], vine-shape with bifurcation cheilocystidia and a trichoderm pileipellis [12]. Crepidotus tortus and C. novae-zelandiae Pilát are characterized by white woolly-to-appressed squamulose pileus, smooth basidiospores, and clamp connections that resemble C. lamellomaculatus. However, both C. tortus and C. novae-zelandiae are distinguished from C. lamellomaculatus by possessing bigger basidiospores [C. novae-zealandiae (9.5)10–12 × (7)7.5–8 μm, C. tortus 8–10.5 × 5.5–6.5(6.8) μm] and flexuous cylindrical cheilocystidia [11,34].

Crepidotus capitatocystidiatus M.H. Han, H. Zeng, Q. Na & Y.P. Ge, sp. nov., Figure 5, Figure 6 and Figure 7.

MycoBank no: 854915

Etymology: The epithet capitatocystidiatus derives from the Latin words ‘capitata’, meaning capitate, and ‘cystidia’. The name is chosen to describe the cheilocystidia apex globose to capitate.

Holotype: China. Fujian Province, Nanping City, Wuyishan National Park, 13 August 2021, leg. Menghui Han, Zewei Liu, Junqing Yan, Hui Zeng, Qin Na, and Yupeng Ge, 728 m asl, FFAAS1310 (collection number NJ3779).

Diagnosis: Pileus white, margin tomentose, translucent striate, not hygrophanous; lamellae edge smooth, stipe present in young stage; basidiospores 4–6.2–7.3(7.7) × (4.1)4.3–5.0–5.7(6.2) μm, guttiform, covered by irregularly punctate-verrucae, cheilocystidia clavate with a globose apex, clamp connections present. Differs from C. palmarum Singer by pileus hygrophanous and distinguishable habitat.

Description: Pileus 6–25 mm broad, in young stage spatuliform, ungulate to conchoid, margin incurved, when matured flabelliform to rounded flabelliform, occasionally lobed, plano-convex, margin gradually straight; White (LIII), at times Chamois (XXX19″b) to Isabella color (XXX19″i) by basidiospores depositing on surface; White (LIII) pubescence when young, with age gradually sparse, when matured almost smooth, in all stages margin weakly tomentose; dry, translucent striate, not hygrophanous; pileus attached almost laterally, but some dorsally to the substratum, clear villosity near the point of attachment. Lamellae 0.5–1 mm broad, L = 11–23, l = 3–7, subdecurrent to decurrent, subventricose, edge weakly fimbriated; White (LIII) when young, becoming *Buff-Yellow (IV19d) to Baryta Yellow (IV21f), when matured Orange-Citrine (IV19k) to Buckthom Brown (XV17′i). Stipe present when young, cylindrical, when matured as a lateral knob or covered by lamellae, subtransparent. Context thin (<5 mm thick), White (LIII), hyaline. Odor and taste not distinctive.

Basidiospores (141/4/3)(5.3)5.4–6.2–7.3(7.7) × (4.1)4.3–5.0–5.7(6.2) μm, Q = (1.13)1.20–1.35(1.40), Q_m = 1.26 ± 0.051, [HOLOTYPE (80/2/1)(5.3)5.4–5.9–6.5(7.4) × (4.2)4.3–4.7–5.4(5.8) μm, Q = (1.13)1.19–1.35(1.40), Q_m = 1.25 ± 0.051], broadly fusiform to ovoid in frontal view, broadly ellipsoid to guttiform bottom slightly pointed in lateral view, greyish yellow to olive brown in 5% KOH aqueous solution, irregularly punctate-verrucose to verrucose (ornamentation verrucose type I), suprahilar depression weakly developed (under oil). Basidia 19–26 × 6–8 μm, clavate to broadly clavate, slightly constricted towards the base, four-spored, rarely two-spored, sterigmata 1.8–4.5 μm long, thin-walled (<0.5 μm thick), hyaline. Pleurocystidia absent. Cheilocystidia 29–60 × 8–18 μm, clavate to narrowly clavate with a globose to ovoid apex, middle portion more or less ventricose, constrict to base, clumped, hyaline, thin- to thick-walled (≤0.6 μm thick). Pileipellis a rectocutis, composed of cylindrical parallel hyphae, 4–8 μm diameter, sometimes interwoven, occasionally branched, thin-walled (<0.5 μm thick); some terminal cells semierect and bundled, contributed to the formation of short pubescence on pileus surface, hyaline, not-gelatinized, oleiferous hyphae observed occasionally. Lamellae trama subregular, composed of subcylindrical hyphae, 6–11 μm diameter, nearly parallel arrangement, more or less inflated, non-gelatinized. Clamp connections present in all tissues.

Habit and habitat: gregarious on deciduous twigs, rotten woods, and nutshell in mixed broadleaf-conifer forests of temperate and subtropical zones, mainly found under Betula sp., Tilia sp., Larix sp., and Pinus sp.

Other specimens examined: China. Fujian Province, Quanzhou City, Dehua County, Lingjiao Village, 30 April 2021, leg. Menghui Han, Zewei Liu, Hui Zeng, Qin Na, and Yupeng Ge, 457 m asl, FFAAS1311 (collection number GN0812); Jilin Province, Tonghua City, Baijifeng National Forest Park, 26 June 2023, leg. Menghui Han, Xiaoliang Liu, Renxiu Wei, Qin Na, and Yupeng Ge, 588 m asl, FFAAS1312 (collection number GN1547).

Notes: Crepidotus capitatocystidiatus is classified within C. subg. Dochmiopus sect. Dochmiopus ser. Dochmiopus due to its verrucose basidiospores and clamped hyphae [6]. Within this series, C. cesatii bears a resemblance to C. capitatocystidiatus, but it can be distinguished by its pileus margin striated, fimbriated lamellae edge, and irregular cheilocystidia, which generally knobby or branched [1,6]. Crepidotus palmarum also shares many similarities with C. capitatocystidiatus due to guttiform basidiospores and capitate cheilocystidia. However, C. palmarum is macroscopically distinct by its hygrophanous pileus, smooth margin, and sessile. Furthermore, C. palmarum and C. capitatocystidiatus exhibit significant differences in habitat and distribution. Crepidotus palmarum was recorded in tropical and subtropical America, gregarious on leaves and leaf petioles of Trachycarpus sp. or rotten trunk of Quercus sp., whereas C. capitatocystidiatus is a species that occurred in temperate and subtropical China, gregarious on rotten branches and woods of Betula sp., Tilia sp., Larix sp., and Pinus sp. [27,62]. Additionally, C. affinis and C. volubilis are close to the new species in phylogeny analysis, while C. affinis can be distinguished by its globose basidiospores and slender cheilocystidia [(35)45–65 × 5–10 μm] [34]. Crepidotus volubilis is different from the new species in ventricose cheilocystidia without capitate and pileus margin straight to slightly upturned [10].

Crepidotus succineus M.H. Han, L. Zou, Q. Na & Y.P. Ge, sp. nov., Figure 8, Figure 9 and Figure 10.

MycoBank no: 854916

Etymology: The epithet is from the Latin word ‘succineus’, meaning amber. The name reflects the color of the basidiomata.

Holotype: China. Heilongjiang Province, Hegang City, Luobei County, Taipinggou National Natural Reserve, 5 July 2023, leg. Menghui Han, Renxiu Wei, Tingting Sun, Zengcai Liu, Weiguo Chen, Xinyu Tong, Yawei Li, Nannan Geng, Li Zou, Qin Na, and Yupeng Ge, 454 m asl, FFAAS1313 (collection number GN1824).

Diagnosis: Pileus buff yellow to apricot yellow, covered by short villosity, margin straight, translucent striate, not hygrophanous; basidiospores (5.2)5.7–6.3–7.0(7.4) × (4.5)4.7–5.3–5.8(6.0) μm, guttiform and verrucose, cheilocystidia usually irregularly knobbed in upper portion, pileipellis a clavicutis, flexuous clavate pileocystidia observed. Differs from C. praecipuus by variable cheilocystidia and a clavicutis pileipellis with pileocystidia.

Description: Pileus 6–26 mm broad, in young stage spathuliform to rounded reniform to petaloid, margin weakly incurved, Naphthalene Yellow (XVI23′f) to *Cream Color (XVI19′f), with age flabelliform to semicircular, when matured applanate and margin progressively straight, *Buff Yellow (IV19d) to Apricot Yellow (IV19b); dry, covered by White (LIII) short tomentum when immature, transitioning to slight pubescence when matured, translucent striate, not hygrophanous; mostly dorsally attached to the substratum, attached point covered by White (LIII) tomentum. Lamellae less than 2 mm broad, L = 9–20, l = 3–9, adnate to subdecurrent, ventricose, when young *Cream Color (XVI19′f) to *Naples Yellow (XVI19′d), edge slightly fimbriated, when matured *Buff Yellow (IV19d) to Mustard Yellow (XVI19′b), surface with a few dark spots, edge smooth. Stipe obvious when young, nearly cylindrical, White (LIII), subtransparent, when matured becoming an eccentric knob in lateral. Context thin (<5 mm thick), subtranslucent. Odor and taste not distinctive.

Basidiospores (238/5/3)(5.2)5.7–6.3–7.0(7.4) × (4.5)4.7–5.3–5.8(6.0) μm, Q =(1.09)1.13–1.27(1.38), Q_m = 1.20 ± 0.045, [HOLOTYPE (146/3/1)(5.2)5.7–6.3–6.9(7.4) × (4.5)4.8–5.3–5.8(6.0) μm, Q = (1.09)1.12–1.26(1.38), Q_m = 1.19 ± 0.043], subglobose to broadly ellipsoid in frontal view, broadly ellipsoid to guttiform in lateral view, greyish yellow to olive brown in 5% KOH aqueous solution, irregularly punctate-verrucose to verrucose (ornamentation verrucose type I), suprahilar depression weakly developed (under oil). Basidia 18–26 × 5–8 μm, clavate, gradually constricted to base, four-spored, rarely two-spored, sterigmata 2.2–4.2 μm long. Pleurocystidia absent. Cheilocystidia 27–70 × 3–7 μm, predominantly contorted narrowly utriform to slender clavate, bifurcated, digitiform to irregularly knobbed branching at middle part and apex, occasionally coralline in densely branched specimens, and taper towards a constricted base, hyaline, thin-walled (<0.5 μm thick). Pileipellis a clavicutis, composed of cylindrical hyphae, 4–7 μm diameter, sometimes interwoven, occasionally branched, nearly hyaline, thin-walled (<0.5 μm thick), not-gelatinized; a few terminal cells slender and semierect, contributing to short villosity on pileus surface, majority terminal cells form pileocystidia, 24–63 × 3–9 μm, clavate, flexuous, sometimes the upper portion branched. Lamellae trama subregular, composed of cylindrical hyphae, 5–11 μm diameter, tightly arranged, slightly inflated, non-gelatinized. Clamp connections present in all tissues.

Habit and habitat: scattered on rotten branches in temperate mixed broadleaf-conifer forests, mainly found under Betula sp., Fraxinus sp., Pinus sp., and Tilia sp.

Other specimens examined: China. Jilin Province, Yanbian Korean Autonomous Prefecture, Antu County, Changbaishan National Nature Reserve, 28 June 2023, leg. Menghui Han, Renxiu Wei, Bai Wang, Qin Na, and Yupeng Ge, 1309 m asl, FFAAS1314 (collection number GN1064). Heilongjiang Province, Hegang City, Luobei County, Taipinggou National Natural Reserve, 6 July 2023, leg. Menghui Han, Renxiu Wei, Tingting Sun, Zengcai Liu, Weiguo Chen, Xinyu Tong, Yawei Li, Nannan Geng, Li Zou, Qin Na, and Yupeng Ge, 462 m asl, FFAAS1315 (collection number GN1917).

Notes: Due to clamp connections and irregularly punctate-verrucose to verrucose basidiospores, C. succineus is considered a member of C. subg. Dochmiopus sect. Dochmiopus ser. Dochmiopus [6]. In this series, C. luteolus Sacc. shares similar basidiomata, but it can be distinguished by oblong-to-cylindrical basidiospores (Q = 1.63–1.99 or Q = 1.55–2.10), cylindrical to narrowly lageniform cheilocystidia, and absent pileocystidia [6,62]. Phylogenetically, C. praecipuus is closed to the new species, resembling white tomentum at the point of attachment and guttiform basidiospores [34]. However, cheilocystidia of C. praecipuus are broadly clavate or vesiculose, and pileipellis is a cutis without pileocystidia [34]. Crepidotus tobolensis possesses similar basidiomata color and basidiospores to C. succineus, but is distinguished by hygrophanous pileus, denser lamellae, and a cutis pileipellis [50]. There are two species with similar basidiomata color to C. succineus from China, C. yuanchui and C. lutescens, but they can be easily distinguished by pileipellis type and morphology of cheilocystidia [4,7].

Crepidotus clavocystidiatus M.H. Han, Q. Na & Y.P. Ge, sp. nov., Figure 11, Figure 12 and Figure 13.

MycoBank no: 854917

Etymology: The epithet clavocystidiatus combines the Latin adjective ‘clavatus’ and the Latin word ‘cystidia’. This name describes the shape of the cheilocystidia, which is clavate to narrowly clavate.

Holotype: China. Shandong Province, Tai’an City, Culaishan National Forest Park, 23 July 2021, leg. Menghui Han, Zewei Liu, Yulan Sun, Qin Na, and Yupeng Ge, 883 m asl, FFAAS1316 (collection number GN1297).

Diagnosis: Pileus ivory yellow, covered by dense white flat villosity in all stages, white dense fibrillose hyphae observed on attachment of the substratum, non-striated; stipe present, pruinose; basidiospores (5.9)6.1–6.7–7.3(7.7) × (4.1)4.3–4.7–5.1(5.3) μm, smooth to faintly verrucose, pileipellis a rectocutis to plagiotrichoderm, clamp connections present. Differs from C. caspari var. caspari Velen. by colored stipe and smaller basidiospores.

Description: Pileus 5–14 mm broad, in young stage umbonate to conchoid or ungulate, White (LIII) to *Suphur Yellow (V25f), becoming flabelliform to semicircular, at times somewhat petaloid, when matured nearly applanate, Ivory Yellow (XXX21″f) to Naphthalene Yellow (XVI23′f), margin incurved, at times Isabella Color (XXX19″i) by basidiospores depositing on surface; dry, covered by White (LIII) flat villosity, margin weakly tomentose, non-striated, not hygrophanous, pileus attached laterally to the substratum, clearly fibrillose hyphae observed near the point of attachment. Lamellae less than 1 mm broad, L = 12–22, l = 3–9, free or subdecurrent, ventricose, edge fimbriated, when young Marguerite Yellow (XXX23″f) to Old Gold (XV19′i), becoming Pale Oliver-Buff (XL21″f) to Deep Olive-Buff (XL21″b), when matured Chamois (XXX19″b). Stipe observed in all stages, slightly clavate to cylindrical, lateral, *Olive-Buff (XL21″d) to Isabella Color (XXX19″i), pruinose, with age persisting as a bent cylinder or occasionally lateral knob. Context thin (<5 mm thick), White (LIII), subtranslucent. Odor and taste not distinctive.

Basidiospores (174/5/4)(5.9)6.1–6.7–7.3(7.7) × (4.1)4.3–4.7–5.1(5.3) μm, Q = (1.25)1.30–1.55(1.61), Q_m = 1.41 ± 0.074, [HOLOTYPE (77/2/1)(5.9)6.2–6.7–7.2(7.7) × (4.2)4.4–4.8–5.2(5.3) μm, Q = (1.25)1.31–1.52(1.59), Q_m = 1.40 ± 0.065], broadly ellipsoid to ellipsoid in frontal view, ovoid to ellipsoid in lateral view, greyish yellow to olive brown in 5% KOH aqueous solution, smooth to faintly verrucose (under oil, ornamentation rugulose-verruculose type I), sometimes granular contents or large oil drop are observed. Basidia 21–31 × 6–8 μm, clavate, four-spored, rarely two-spored, sterigmata 1.8–4.8 μm long, thin-walled (<0.5 μm thick), hyaline. Pleurocystidia absent. Cheilocystidia 28–45 × 6–12 μm, clavate to narrowly clavate, sometimes apex round or subcapitate, not forked, base contracted, flexuous, clumped, hyaline, thin-walled (<0.5 μm thick). Pileipellis a rectocutis to plagiotrichoderm, composed of densely arranged cylindrical hyphae, interwoven and branched, 5–9 μm wide, thin- to thick-walled (0.3–0.9 μm), weakly yellow-brown hyphae, some terminal cells erected, forming short-villosity on pileus surface, nearly hyaline, a few hyphae encrusted, non-gelatinized. Lamellae trama intermixed, composed of subparallel cylindrical hyphae 3–6 μm, more or less inflated, physalohyphae observed near trama base, 8–18 μm, measured up to 33 μm diameter, subregular, non-gelatinized. Clamp connections present in all tissues.

Habit and habitat: scattered on rotten twigs in temperate mixed broadleaf-conifer forests, mainly were found under Fagaceae sp., Pinaceae sp., Populus sp., and Quercus sp.

Other specimens examined: China. Shandong Province, Tai’an City, Culaishan National Forest Park, 23 July 2021, leg. Menghui Han, Zewei Liu, Yulan Sun, Qin Na, and Yupeng Ge, 909 m asl, FFAAS1317(collection number GN1294), 899 m asl, FFAAS1318 (collection number GN1296); Shandong Province, Yantai City, Luoshan National Forest Park, 19 July 2021, Menghui Han, Zewei Liu, Yulan Sun, Qin Na, and Yupeng Ge, 223 m asl, FFAAS1319 (collection number GN1157).

Notes: Crepidotus clavocystidiatus is classified into C. subg. Dochmiopus sect. Dochmiopus ser. Caspari by clamp connections and smooth to rugulose-verruculose basidiospores [6]. Within this series, C. caspari var. caspari and C. subverrucisporus share similar morphological characteristics to C. clavocystidiatus, but can be easily distinguished from C. clavocystidiatus by white to cream pileus and larger basidiospores (C. caspari var. caspari 7.1–8.9 × 4.6–5.6 μm, C. subverrucisporus 7.7–9.4 × 5.1–6.3 μm or (7.0)7.5–10.0(11.0) × (4.5)5.0–6.0(7.0) μm or 7.2–9.1 × 4.3–5.3 μm) [6,62,63]. In C. subg. Dochmiopus sect. Dochmiopus ser. Caspari, there is a species, C. furcaticystidiosus Q. Na, M.H. Han, R.X. Wei, H. Zeng & Y.P. Ge, which was discovered in Changbaishan Nature Reserve (China, Yanbian), and has faintly verrucose ellipsoid basidiospores similar to C. clavocystidiatus, but it can be readily distinguished by its furcate cheilocystidia and smooth to nearly smooth pileus surface [15]. Crepidotus subfulviceps (Murrill) Aime, Vila & P.-A. Moreau and C. iqbalii are close to the new species phylogenetically, but they are both stipitate Crepidotus, which can be readily distinguished [13,48].

4. Discussion

Crepidotus subg. Dochmiopus likely harbors a rich diversity of species in China. Despite numerous records of C. subg. Dochmiopus species in regional diversity studies in China, the documentation of new taxa remains limited [64,65,66,67,68]. For researchers new to Crepidotus, accurately identifying species is challenging due to the scarcity of molecular data and the limited number of available morphological characteristics, often leading to overlooked new taxa. Through thorough morphological analyses, the collation of key taxonomic features and the enrichment of molecular datasets can improve the accuracy of species identification, thereby increasing the likelihood of discovering new species and providing basic information to reveal the phylogenetic and taxonomic problems.

Microscopic characters are essential for identifying species within C. subg. Dochmiopus, and requiring detailed morphological examinations to reveal more referable taxonomic characters for classification. This study utilized Clémençon’s description of pileipellis types, recognized patterns such as rectocutis, plagiotrichoderm and clavicutis based on hyphae arrangement and the orientation of terminal cells, thereby enhancing the referential value of pileipellis for interspecific identification, and considered that pileipellis and cheilocystidia patterns play a role in species recognition [69]. For species that are challenging to differentiate, we attempted to employ more characteristics for identification, including, habitat, stipe present or absence in mature specimens, and whether the lamellae edge is fimbriated. For example, C. capitatocystidiatus and C. cesatii were recognized by whether the lamellae edge is fimbriated and the pileus margin is striated [6]. To identify species more conveniently and precisely, we provide an identification key for elongated-spored species of C. subg. Dochmiopus known in China, 30 elongated-spored species of C. subg. Dochmiopus, including four new species described in this study. The use of DNA sequences, however, has simplified species recognition and improved accuracy. This study contributed by sequencing 15 specimens from four new species in both ITS and LSU regions, offering valuable molecular insights for future identification efforts. Thorough morphological research combined with molecular phylogenetics is crucial for the precise identification of C. subg. Dochmiopus species and the discovery of new taxa.

The classification of Crepidotus needs to be re-evaluated, as some sections may be merged or new sections built in Crepidotus. Based on the phylogenetic tree constructed by combining ITS and LSU sequences, the phylogenetic framework of Crepidotus cannot support the classification of Hesler & Smith or Consiglio & Setti, which were proposed by morphological characteristics [5,6]. Both Hesler & Smith and Consiglio & Setti considered the distinction between C. subg. Crepidotus and C. subg. Dochmiopus to be the presence of clamp connections [5,6]. Crepidotus epibryus, C. versutus, and C. cinnabarinus were classified in subg. Crepidotus by absent clamp connections, but they are located in C. subg. Dochmiopus group in the phylogenetic tree. Additionally, the species of C. subg. Dochmiopus sect. Autochthoni possess smooth basidiospores. That hints smooth basidiospores and the absence of clamp connections may no longer be exclusive characteristics of C. subg. Crepidotus. Consiglio & Setti divided C. subg. Crepidotus into two sections and C. subg. Dochmiopus into three sections, including five series [6]. Previously, due to an incomplete comprehension of key taxonomic characteristics and the lack of phylogenetic studies, many sections were overlooked. Within this phylogenetic tree, the same sections were present in different clades. For example, Clade 2 and Clade 10 are both C. subg. Dochmiopus sect. Autochthoni. But morphologically, the cheilocystidia of Clade 2 species are lageniform, while the cheilocystidia of Clade 10 species are sinuously cylindrical to vine-like. There may be a new section among Clade 2 and Clade 10. However, relying on the current phylogenetic tree and morphological characteristics to build a new section is insufficient. More specimens, sequences and new gene segments are urgently needed to analyze and reveal the phylogenetic relationships within C. subg. Dochmiopus and Crepidotus to build a distinct classification based on morphology and phylogeny.

Key to the elongated-spored species of C. subg. Dochmiopus known in China
1 Basidiospores smooth to nearly smooth	2
1 Basidiospores ornamented	10
2 Pileocystidia present	C. autochthonus
2 Pileocystidia absent	3
3 Pileipellis gelatinous	C. betulae
3 Pileipellis non-gelatinous	4
4 Cheilocystidia vine-shaped	C. trichocraspedotus
4 Cheilocystidia not vine-shaped	5
5 Pileipellis hyphae clampless	C. albidus
5 Pileipellis hyphae clamped	6
6 Lamellae edge not fimbriate	7
6 Lamellae edge fimbriate	8
7 Pileipellis a cutis	C. caspari
7 Pileipellis a tomentose	C. lamellomaculatus
8 Basidiospores width < 4.5 μm	C. albissimus
8 Basidiospores width ≥ 4.5 μm	9
9 Pileipellis a cutis, pileus greyish white to silver-grey	C. occidentalis
9 Pileipellis a trichoderm, pileus white to light orange-yellow	C. ulmicola
10 Inhabit on living plants	C. herbaceus
10 Inhabit on rotten branches and woods	11
11 Pileus reddish	C. reticulatus
11 Pileus not reddish	12
12 Pleurocystidia present	C. luteicolor
12 Pleurocystidia absent	13
13 Pileocystidia present	14
13 Pileocystidia absent	15
14 Pileus buff yellow to apricot yellow	C. succineus
14 Pileus white	C. vulgaris
15 Basidiospores subglobose to ellipsoid, Qm ≤ 1.60	16
15 Basidiospores amygdaliform, oblong to cylindrical, Qm > 1.60	26
16 Pileus brown, at point of attachment smooth	C. payettensis
16 Pileus not brown, at point of attachment villous, fibrillose or tomentose	17
17 Pileus surface smooth	18
17 Pileus surface not smooth	20
18 Pileus translucent striate	C. capitatocystidiatus
18 Pileus not translucent striate	19
19 Lamellae edge not fimbriate, cheilocystidia apex bifurcate	C. furcaticystidiosus
19 Lamellae edge fimbriate, cheilocystidia apex branched or stag antlers	C. macedonicus
20 Pileus hygrophanous	21
20 Pileus non-hygrophanous	22
21 Cheilocystidia lageniform	C. lutescens
21 Cheilocystidia narrowly clavate to narrowly utriform	C. croceotinctus
22 Pileipellis with incrusted hyphae	23
22 Pileipellis without incrusted hyphae	24
23 Cheilocystidia clavate or ventricose, sessile	C. kauffmanii
23 Cheilocystidia clavate to narrowly clavate, pruinose stipe	C. clavocystidiatus
24 Pileus orange-yellow to brownish-yellow	C. yuanchui
24 Pileus white to cream	25
25 Basidiospores subglobose to broadly ellipsoid, Q ≤ 1.30	C. cesatii
25 Basidiospores ellipsoid to ovoid, Q > 1.30	C. subverrucisporus
26 Pileus light smoky or rusty brown	C. herbarum
26 Pileus white to light yellow or orange	27
27 Basidiospores length ≥ 8.0 μm	C. luteolus
27 Basidiospores length < 8.0 μm	28
28 Cheilocystidia with a long cylindrical protuberance at apex	C. tomentellus
28 Cheilocystidia without a long cylindrical protuberance at apex	29
29 Cheilocystidia filiform, narrowly lageniform, at times diverticular or knobby in the upper portion	C. neotrichocystis
29 Cheilocystidia clavate, utriform, strangulated, sometimes stag antlers	C. variabilis