Molecular Phylogeny and Morphology Reveal Four New Species of Conocybe (Bolbitiaceae, Agaricales) from the Qinghai-Xizang Plateau, China

Abstract

The Qinghai-Xizang Plateau, known for its high altitude, geological history of plate collision, crustal uplift, and special ecology factors, provides an ideal environment for studying fungal biodiversity in extreme environmental conditions. Some species within the Conocybe, containing secondary metabolites such as psilocybin, phallotoxins, and amatoxins, have potential medicinal value for treating psychiatric disorders and for use in drug development. This study investigates Conocybe (Bolbitiaceae, Agaricales) on the Plateau, based on specimens collected over the past decade, using morphological and molecular phylogenetic analyses. Seven species were identified, including four new species: C. alticola, C. alticoprophila, C. versicolor, and C. yadongensis. Molecular analyses, utilizing multi-gene sequence data (ITS, nrLSU, and tef-1α), support the taxonomic position of these new species within this genus as new species. Detailed descriptions, illustrations, photographs, line drawings, and comparisons with related species are provided for the new taxa. This study enriches the species diversity of Conocybe on the Qinghai-Tibet Plateau, further enhancing our understanding of fungal biodiversity in this region.

1. Introduction

The Qinghai-Xizang Plateau (abbreviation Q-X Plateau), with an average elevation exceeding 4000 m, and referred to as the “roof” of the world, is the highest and youngest plateau on earth [1]. It is known for its complex geological evolution, including plate collisions, crustal uplift, diverse climatic conditions, and unique ecological features [2,3,4,5,6]. As an “ecological island” isolated from the surrounding lower regions, the plateau has fostered a distinctive high-altitude biogeographic system [7,8]. These exceptional natural conditions have not only promoted the formation of rich and unique biodiversity in the region, but also made it an invaluable resource for global scientific research [9,10,11]. The region’s diverse plant communities and unique environmental conditions provide an ideal natural platform for studying fungal adaptive evolution, making the Q-X Plateau a critical area for exploring high-altitude ecosystems and fungal biodiversity [12,13,14]. Due to challenges posed by remote location, limited accessibility and harsh climatic conditions, research on fungal communities in this region has progressed slowly, leaving numerous valuable fungal resources yet to be explored [15,16].

Conocybe Fayod belongs to Bolbitiaceae (Agaricales) [17], is characterized by its small to medium-sized delicate basidiomata, conical pileus, adnexed to adnate and brown-rusty lamellae, powdery or pilose stipes, lecythiform cheilocystidia, and basidiospores with germ pore [18,19]. Most Conocybe species are saprophytic and are widely distributed in different vegetation types, particularly in fertile soils and herbivore dung, as well as on plant debris, decaying wood, and sawdust [18,20,21,22]. The genus Conocybe is noted for its chemical diversity and biological activities, despite some species containing neurotoxic or cytotoxic substances [23,24,25,26]. It has been believed that secondary metabolites like psilocybin, with potential therapeutic effects, and toxic cyclic peptides such as amatoxins and phallotoxins may cause severe health issues [27,28,29,30].

Conocybe is divided into more than 10 sections based on morphological characteristics, including sect. Conocybe Fayod, sect. Mixtae Singer, sect. Candidae Watling, and sect. Pilosellae Singer [18,31,32,33,34]. Currently, approximately 270 species of Conocybe have been identified worldwide, with 44 recorded in China [18,19,35,36,37]. Recent studies have focused on Conocybe specimens from various regions in China. Song and Bau (2023) described seven new species and one new record from sect. Pilosellae collected in northeastern, northern, and central–southern China [18]. In 2024, Wang et al. described C. himalayana Ke Wang, T.Z. Wei & P. Hong from sect. Pilosellae based on five specimens collected from the Q-X Plateau [16], and Lu et al. reported two new species from sect. Conocybe based on four specimens from the Yunnan-Guizhou Plateau [36]. However, research on Conocybe in the Q-X Plateau remains limited. This study investigates Conocybe specimens collected over the past decade from the plateau, combining macroscopic, microscopic, and molecular methods to explore species diversity and distribution in the region.

2. Materials and Methods

2.1. Samplings and Morphological Study

The specimens for this study were collected from Lhasa Municipality, Qamdo Municipality, Shigatse Municipality, Lingzhi Prefecture, and the Ali Region in the Xizang Autonomous Region in China between 2015 and 2024. Photographs were captured using a Canon EOS 80D camera (Tokyo, Japan), and specimens were carefully packed individually in aluminum foil to prevent mixing or damage. Specimens were dried completely using a food dryer at 50 °C, sealed in plastic bags, and deposited in the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS). Microscopic features such as basidia, basidiospores, pileipellis, and cheilocystidia were observed under an Olympus CX31 light microscope (Tokyo, Japan), with at least 30 measurements obtained for each feature. Microscopic examination followed the methodology of Largent [38]. Tissues were treated with 5% KOH and sterile water for observation. Measurements are presented as (a)b–c(d), where b–c represents at least 90% of the measured values, while a and d indicate the lowest value and the highest value, respectively. The Q value is the length/width ratio of basidiospores, with Qm representing the mean Q value ± standard deviation [39]. The color designations were determined using the Methuen Handbook of Colour [40].

2.2. DNA Extraction, PCR and Sequencing

DNA was extracted from dried specimens using the Broad-spectrum Plant Rapid Genomic DNA Kit (Biomed, Beijing, China), following the manufacturer’s instructions. The primer pairs ITS1F/ITS4 [41], LR7/LR0R [42], and EF983F/EF2218R [43] were used to amplify the nuclear internal transcribed spacer (ITS), the large subunit nuclear ribosomal DNA (nrLSU), and the translation elongation factor subunit 1 alpha (tef-1α) regions, respectively. The polymerase chain reaction (PCR) procedure was performed under the following conditions: an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 53 °C (ITS), 50 °C (nrLSU), or 55 °C (tef-1α) for 60 s, and extension at 72 °C for 90 s, with a final extension at 72 °C for 10 min [44,45,46]. The PCR products were detected by electrophoresis and sent to BGI Genomics Co., Ltd. (Beijing, China) for purification and sequencing.

2.3. Phylogenetic Analyses

Based on BLAST searches against GenBank and previous studies, we analyzed the nrITS, nrLSU and tef-1α sequences of 124 taxa, as detailed in

Table 1. Sequences used in the phylogenetic analysis in this study. Missing sequences are indicated by “–”.

Taxon Name	Voucher Number	ITS	nLSU	tef1	Country of Origin	References
Conocybe aff. ochrostriata	NL-0830	JX968236	JX968354	JX968447	Hungary	[54]
Conocybe alboradicans	NL-3226	JX968219	JX968336	JX968435	Hungary	[21]
Conocybe alboradicans	WU14678	JX968220	JX968337	–	Hungary	[54]
Conocybe alkovii	262841	JQ247196	–	–	Russia	[55]
Conocybe alticola	ZRL20240256	PQ699268	PQ699292	PQ836630	China	This study
Conocybe alticola	ZRL20240259	PQ699269	PQ699293	PQ836631	China	This study
Conocybe alticola	ZRL20240316	PQ699270	PQ699294	PQ836632	China	This study
Conocybe alticola	ZRL20240317	PQ699271	PQ699295	PQ836633	China	This study
Conocybe alticola	ZRL20240327	PQ699272	PQ699296	PQ836634	China	This study
Conocybe alticoprophila	ZRL20220041	PQ699273	PQ699297	PQ836635	China	This study
Conocybe alticoprophila	ZRL20220046	PQ699274	PQ699298	PQ836636	China	This study
Conocybe alticoprophila	ZRL20220050	PQ699275	PQ699299	PQ836637	China	This study
Conocybe ammophila	WU23983	JX968197	JX968313	JX968416	Mongolia	[19]
Conocybe anthracophila	WU14367	JX968212	JX968329	JX968430	Italy	[21]
Conocybe anthracophila var. ovispora	WU 25461	JX968237	JX968355	–	Italy	[19]
Conocybe antipus	WU19791	JX968215	JX968332	JX968432	Austria	[21]
Conocybe apala	NL-1012	JX968209	JX968326	JX968427	Hungary	[21]
Conocybe aurea	AMO16	MG663237	MT237453	–	USA	Unpublished
Conocybe aurea	WU28161	JX968184	JX968300	JX968407	Italy	[21]
Conocybe bispora	NL-2573	JX968203	JX968320	JX968423	Hungary	[21]
Conocybe bisporigera	HMJAU 45055	OP526418	–	–	China	[18]
Conocybe bisporigera	NL-1904	JX968235	JX968353	JX968446	Hungary	[19]
Conocybe brachypodii	HMJAU45017	MH141423	–	–	China	[19]
Conocybe brachypodii	NL-3169	JX968199	JX968316	JX968419	Hungary	[19]
Conocybe ceracea	HMJAU64951	OQ758110	OQ758218	OQ758305	China	[18]
Conocybe ceracea	HMJAU64952	OQ758111	OQ758219	OQ758306	China	[18]
Conocybe ceracea	HMJAU64953	OQ758112	OQ758220	–	China	[18]
Conocybe cettoiana	WU10436	JX968218	JX968335	–	Italy	[21]
Conocybe confundens	LE 313077	NR_155032	–	–	Russia	[22]
Conocybe coniferarum	LE 313009	NR_155030	–	–	Russia	[22]
Conocybe crispella	WU27367	JX968208	JX968325	JX968426	Austria	[21]
Conocybe cylindracea	WU 20796	JX968240	JX968358	JX968449	Italy	[21]
Conocybe cylindrospora	HMJAU42440	MG250375	OQ758203	–	China	[18,19]
Conocybe cylindrospora	HMJAU42441	MG250377	–	–	China	[19]
Conocybe deliquescens	HMJAU61998	OP373403	OQ758204	OQ758292	China	[18]
Conocybe deliquescens	NL-0574	JX968210	JX968327	JX968428	Hungary	[19]
Conocybe dumetorum	NL-2693	JX968201	JX968318	JX968421	Sweden	[21]
Conocybe dunensis	WU27359	JX968227	JX968345	–	Spain	[21]
Conocybe echinata	NL-1007	JX968196	JX968312	–	Hungary	[19]
Conocybe elegans	NL-0908	JX968223	JX968341	JX968437	Sweden	[21]
Conocybe enderlei	NL-0165	JX968161	JX968277	JX968389	Sweden	[21]
Conocybe enderlei	WU21272	JX968163	JX968279	–	Italy	[21]
Conocybe farinacea	NL-2173	JX968167	JX968283	–	Hungary	[21]
Conocybe fiorii	WU17793	JX968217	JX968334	JX968434	Italy	[21]
Conocybe fuscimarginata	HMJAU 45033	OQ780310	OQ758208	OQ758296	China	[18]
Conocybe fuscimarginata	NL-3668	JX968238	JX968356	JX968448	Sweden	[21]
Conocybe fuscimarginata	ZRL20235165	PQ699276	PQ699300	PQ821725	China	This study
Conocybe fuscimarginata	ZRL20235225	PQ699277	PQ699301	PQ821726	China	This study
Conocybe gigasperma	NL-3972	JX968179	JX968295	JX968403	Slovakia	[21]
Conocybe graminis	WU13466	JX968195	JX968311	–	Austria	[21]
Conocybe hausknechtii	LE253789	JQ247194	–	–	Russia	[56]
Conocybe herbarum	WU22193	JX968193	JX968309	–	Austria	[21]
Conocybe himalayana	HMAS 300530	PQ099839	PP968802	PP992938	China	[16]
Conocybe himalayana	HMAS 300531	PQ099840	PP968801	PP992937	China	[16]
Conocybe himalayana	HMAS 300532	PQ099841	PP968800	PP992939	China	[16]
Conocybe himalayana	HMAS 300534	PQ099842	PP968799	PP992936	China	[16]
Conocybe himalayana	HMAS 300535	PQ099843	PP968803	PP992940	China	[16]
Conocybe himalayana	ZRL20231801	PQ699278	PQ699302	PQ821727	China	This study
Conocybe himalayana	ZRL20240260	PQ699279	PQ699303	PQ821728	China	This study
Conocybe himalayana	ZRL20240274	PQ699280	PQ699304	PQ821729	China	This study
Conocybe himalayana	ZRL20240279	PQ699281	PQ699305	PQ821730	China	This study
Conocybe himalayana	ZRL20240281	PQ699282	PQ699306	PQ821731	China	This study
Conocybe himalayana	ZRL20240283	PQ699283	PQ699307	PQ821732	China	This study
Conocybe himalayana	ZRL20240288	PQ699284	PQ699308	PQ821733	China	This study
Conocybe himalayana	ZRL20240318	PQ699285	PQ699309	PQ821734	China	This study
Conocybe himalayana	ZRL20240328	PQ699286	PQ699310	PQ821735	China	This study
Conocybe hornana	NL-3499	JX968178	JX968294	JX968402	Slovakia	[21]
Conocybe hydrophila	HMJAU 64954	OQ758116	OQ758232	OQ758313	China	[18]
Conocybe hydrophila	HMJAU 64955	OQ758117	OQ758233	OQ758314	China	[18]
Conocybe incarnata	HMJAU 64968	OQ780316	–	–	China	[18]
Conocybe incarnata	WU 21897	JX968229	JX968347	JX968441	Finland	[21]
Conocybe incerta	LE 313017	NR_155031	–	–	Russia	[22]
Conocybe ingridiae	WU 28158	JX968244	JX968361	JX968451	Italy	[21]
Conocybe inocybeoides	NL-3589	JX968202	JX968319	JX968422	Hungary	[21]
Conocybe inopinata	WU27544	JX968165	JX968281	JX968392	Italy	[19]
Conocybe intrusa	WU25546	JX968211	JX968328	JX968429	Hungary	[21]
Conocybe juniana	NL-2105	JX968191	JX968307	JX968413	Hungary	[21]
Conocybe juniana var. sordescens	NL-2304	JX968192	JX968308	JX968414	Hungary	[21]
Conocybe karakensis	KTK-05	ON392730	–	–	Pakistan	[37]
Conocybe karinae	WU28526	JX968151	JX968268	JX968384	Hungary	[21]
Conocybe lenticulospora	HMJAU 45069	OQ780317	–	–	China	[18]
Conocybe lenticulospora	NL-0923	JX968242	JX968359	JX968450	Sweden	[21]
Conocybe leporina	NL-2380	JX968177	JX968293	JX968401	Hungary	[21]
Conocybe lobauensis	WU17826	JX968176	JX968292	JX968400	Italy	[19]
Conocybe macrocephala	WU18148	JX968182	JX968298	–	Austria	[19]
Conocybe macrospora	WU17030	JX968175	JX968291	–	Germany	[19]
Conocybe merdaria	WU25359	JX968174	JX968290	–	Austria	[19]
Conocybe mesospora	HMJAU45049	MH141419	–	–	China	[57]
Conocybe microrrhiza	NL-2180	JX968222	JX968340	JX968436	Hungary	[21]
Conocybe microspora	NL-1890	JX968160	JX968276	–	Hungary	[21]
Conocybe monikae	WU22612	JX968200	JX968317	JX968420	Hungary	[21]
Conocybe moseri	GLM-F 40421	MK412354	–	–	Germany	[18]
Conocybe moseri	HMJAU 45075	OQ780309	OQ758207	–	China	[18]
Conocybe muscicola	HMJAU64939	OQ758113	OQ758223	OQ758309	China	[18]
Conocybe muscicola	HMJAU64940	OQ758115	OQ758224	OQ758310	China	[18]
Conocybe nigrescens	WU 27557	JX968234	JX968352	JX968445	Italy	[21]
Conocybe nitrophila	WANG 140019	KR998384	–	–	China	[58]
Conocybe nitrophila	WU20916	JX968233	JX968351	JX968444	India	[21]
Conocybe ochrostriata var. favrei	WU 29786	JX968245	JX968362	JX968452	Italy	[21]
Conocybe olivaceopileata	LE 313106	NR_155028	–	–	Russia	[22]
Conocybe pallidospora	WU 7395	JX968239	JX968357	–	Austria	[21]
Conocybe pallidospora	WU17079	JX968158	–	–	Hungary	[21]
Conocybe papillata	NL-2370	JX968216	JX968333	JX968433	Hungary	[21]
Conocybe parapilosella	90551	NR_176713	–	–	Spain	[59]
Conocybe parapilosella	WU30224	MN872706	–	–	Spain	[59]
Conocybe pilosa	HMJAU 64947	OQ758122	OQ758222	OQ758307	China	[18]
Conocybe pilosa	HMJAU 64948	OQ758123	OQ758221	OQ758308	China	[18]
Conocybe pilosella	HMJAU 45062	OQ780305	OQ758205	OQ758294	China	[18]
Conocybe pilosella	HMJAU 64957	OQ780306	OQ758206	OQ758295	China	[18]
Conocybe praticola	HMJAU64965	OQ780303	–	–	China	[18]
Conocybe praticola	LE 313010	KY614060	–	–	Russia	[22]
Conocybe pseudocrispa	HMJAU 64944	OQ780308	OQ758211	OQ758298	China	[18]
Conocybe pseudocrispa	WU 18009	JX968230	JX968348	JX968442	Austria	[21]
Conocybe pseudocrispa	ZRL20240311	PQ699287	PQ699311	PQ821736	China	This study
Conocybe pseudocrispa	ZRL20240319	PQ699288	PQ699312	PQ821737	China	This study
Conocybe pubescens	NL-1986	JX968173	JX968289	JX968399	Hungary	[21]
Conocybe pubescens	WU20759	JX968170	JX968286	JX968396	Italy	[21]
Conocybe punjabensis	SKP066	MH981969	–	–	Pakistan	[54]
Conocybe punjabensis	SKP069	MH981968	–	–	Pakistan	[54]
Conocybe qujingensis	HKAS 127149	OQ755415	–	–	China	[36]
Conocybe qujingensis	HKAS 128155	OQ755416	–	–	China	[36]
Conocybe qujinguniversitatis	HKAS 128150	OQ755413	–	–	China	[36]
Conocybe qujinguniversitatis	HKAS 128151	OQ755414	–	–	China	[36]
Conocybe reniformis	HMJAU 64942	OQ758108	OQ758229	OQ758311	China	[18]
Conocybe reniformis	HMJAU 64943	OQ758109	OQ758228	OQ758312	China	[18]
Conocybe rickeniana	NL-2468	JX968198	JX968315	JX968418	Hungary	[51]
Conocybe rickenii	AH 21067	MF142238	–	–	Spain	[60]
Conocybe romagnesii	WU26605	JX968206	JX968323	JX968424	Italy	[19]
Conocybe rostellata	NL-2499	JX968162	JX968278	JX968390	Sweden	[21]
Conocybe rufostipes	HMJAU 64937	OQ758120	OQ758227	OQ758317	China	[18]
Conocybe rufostipes	HMJAU 64938	OQ758121	OQ758226	OQ758318	China	[18]
Conocybe sabulicola	WU11185	JX968186	JX968302	JX968409	Italy	[19]
Conocybe semiglobata	NL-1993	JX968181	JX968297	JX968405	Hungary	[19]
Conocybe semiglobata	WU8794	JX968188	JX968304	–	Austria	[21]
Conocybe semiglobata var. campanulata	NL-3159	JX968168	JX968284	JX968394	Austria	[21]
Conocybe semiglobata var. campanulata	WU26395	JX968169	JX968285	JX968395	Germany	[19]
Conocybe siennophylla	HMJAU 64966	OQ780312	OQ758210	OQ758297	China	[18]
Conocybe siennophylla	NL-1210	JX968246	JX968363	JX968453	Hungary	[21]
Conocybe siennophylla	WU17988	JX968243	JX968360	–	Hungary	[21]
Conocybe siliginea	NL-1211	JX968159	JX968275	–	Sweden	[21]
Conocybe siliginea	NL-2313	JX968225	JX968343	JX968438	Sweden	[21]
Conocybe singeriana	WU22129	JX968166	JX968282	JX968393	Austria	[21]
Conocybe sinobispora	HMJAU 64949	OQ758118	OQ758230	OQ758315	China	[18]
Conocybe sinobispora	HMJAU 64950	OQ758119	OQ758231	OQ758316	China	[18]
Conocybe solitaria	WU20903	JX968214	JX968331	JX968431	China	[21]
Conocybe sp.	HMJAU44988	OQ749737	–	OQ758302	China	[18]
Conocybe sp.	HMJAU64962	OQ749739	–	OQ758303	China	[18]
Conocybe sp.	HMJAU64967	OQ749741	–	–	China	[18]
Conocybe sp.	HMJAU64964	OQ749742	–	–	China	[18]
Conocybe sp.	HMJAU44994	OQ749735	–	–	China	[18]
Conocybe sp.	NL-1455	JX968194	JX968310	JX968415	Hungary	[21]
Conocybe subovalis	NL-1415	JX968190	JX968306	JX968412	Hungary	[19]
Conocybe subpubescens	NL-0162	JX968189	JX968305	JX968411	Sweden	[19]
Conocybe subpubescens	NL-2181	JX968171	JX968287	JX968397	Hungary	[19]
Conocybe subxerophytica	NL-0164	JX968187	JX968303	JX968410	Sweden	[19]
Conocybe tenera	NL-	JX968185	JX968301	JX968408	Hungary	[21]
Conocybe tenera	NL-1615	JX968180	JX968296	JX968404	Hungary	[19]
Conocybe tetrasporoides	WU 17385	JX968232	JX968350	–	New Zealand	[21]
Conocybe turkestanica	H 7034981	MH055382	–	–	Turkestan	[19]
Conocybe tuxlaensis	NL-1897	JX968164	JX968280	JX968391	Hungary	[21]
Conocybe vaginata	WU25703	JX968204	JX968321	–	Sri Lanka	[21]
Conocybe velutinomarginata	WU 28695	JX968226	JX968344	JX968439	Germany	[21]
Conocybe velutipes	HMJAU 45048	OQ780311	OQ758209	–	China	[18]
Conocybe velutipes	NL-2187	JX968228	JX968346	JX968440	Hungary	[21]
Conocybe versicolor	ZRL20152517	PQ699289	PQ699313	PQ836638	China	This study
Conocybe versicolor	ZRL20220299	PQ699290	PQ699314	PQ836639	China	This study
Conocybe volvata	WU20900	JX968205	JX968322	–	India	[19]
Conocybe volvicystidiata	1212	KY346827	–	–	France	[61]
Conocybe watlingi	WU22744	JX968172	JX968288	JX968398	Finland	[21]
Conocybe yadongensis	ZRL20220042	PQ699291	PQ699315	PQ836640	China	This study
Conocybe zeylanica	WU20185	JX968207	JX968324	JX968425	La Re’union	[21]
Conocybe zuccherellii	WU12421	JX968213	JX968330	–	Italy	[21]
Bolbitius coprophilus	HMJAU64958	OQ780315	OQ758216	–	China	[18]
Bolbitius reticulatus	WU30001	JX968249	JX968366	JX968455	Hungary	[21]
Bolbitius subvolvatus	WU28379	JX968248	JX968365	JX968454	Italy	[21]
Conobolbitina micheliana	HMJAU65015	OR995677	OR994080	PP000869	China	[62]
Conobolbitina micheliana	HMJAU65016	OR995678	OR994081	PP000870	China	[62]
Conobolbitina ochroleuca	HMJAU65017	OR995679	OR994082	PP000871	China	[62]
Conobolbitina ochroleuca	HMJAU65018	OR995680	OR994083	PP000872	China	[62]
Conocybula coprophila	SZMC-NL-2176	JX968156	JX968273	–	Hungary	[21]
Conocybula cyanopus	WU2134	JX968157	JX968274	JX968388	Austria	[21]
Galerella nigeriensis	CNF1/5859	JX968251	JX968368	JX968457	Nigeria	[21]
Descolea antarctica	NZ5182	AF325647	–	–	USA	[63]
Descolea quercina	HMJAU64959	OQ780313	OQ758213	OQ758299	China	[18]
Pholiotina changbaishanensis	HMJAU65101	OR995689	OR994092	PP000881	China	[62]
Pholiotina changbaishanensis	HMJAU65102	OR995690	OR994093	PP000882	China	[62]
Pholiotina serrata	HMJAU42442	MG250376	–	–	China	[19]
Pholiotina serrata	HMJAU62006	OP538570	OQ758217	OQ758301	China	[18]
Pholiotina eburnea	HMJAU65034	OR995693	OR994096	PP000885	China	[62]
Pholiotina eburnea	HMJAU65035	OR995694	OR994097	PP000886	China	[62]
Pholiotina excrescenticystidiata	HMJAU65021	OR995695	OR994098	PP000887	China	[21]
Pholiotina excrescenticystidiata	HMJAU65022	OR995696	OR994099	PP000888	China	[62]
Pholiotina pleurocystidiata	WU40666	NR_176740	–	–	Austria	[64]
Candolleomyces sichuanicus	HMAS 287616	PP734617	PP734628	PP729330	China	[46]
Candolleomyces sichuanicus	HMAS 287617	PP734618	PP734629	PP729331	China	[46]
Candolleomyces singeri	HMJUA37867	MG734718	MW301088	MW314077	China	[65]
Candolleomyces singeri	HMJAU37877	MW301073	MW301091	MW314080	China	[11]
Psathyrella piluliformis	HMJAU37922	MG734716	MW413364	MW411001	China	[65]
Psathyrella amygdalinospora	HMJAU37952	MW405104	MW413361	MW410999	China	[66]
Psathyrella amygdalinospora	HMJAU57044	MW405105	–	–	China	[66]

The sequences generated in this study are marked in bold.

. The sequences were aligned using Muscle version 3.6 separately [47], then manually adjusted in BioEdit version 7.0.4 to remove the ambiguous areas [48]. The aligned sequences were assembled in PhyloSuite version 1.2.3 [49]. Maximum likelihood (ML) analysis was performed on the concatenated sequences using raxmlGUI 1.3 with a GTRGAMMA model and 1000 rapid bootstrap replicates [50]. The best partitioning scheme and evolutionary models for three predefined partitions were selected using PartitionFinder2 v2.1.1 [51], based on the greedy algorithm and AICc criterion: GTR+I+G for ITS, GTR+I+G for nrLSU, and SYM+I+G for tef-1α. Bayesian Inference (BI) analysis was conducted using MrBayes v3.2.7a [52], with six Markov chains run for two million generations and trees sampled every 100th generation. The burn-ins were determined using Tracer version 1.6, with an ESS value higher than 200. The remaining trees were used to calculate Bayesian posterior probabilities (PP). The trees were displayed in Interactive Tree of Life (iTOL) version 6 [53].

3. Results

3.1. Phylogeny

In the phylogenetic analysis, 24 specimens from seven Conocybe species were included, with the following four species from the Psathyrellaceae Vilgalys, Moncalvo & Redhead selected as outgroups: Psathyrella piluliformis (Bull.) P.D. Orton, P. amygdalinospora T. Bau & J.Q. Yan, Candolleomyces sichuanicus R.L. Zhao, B. Cao & X.X. Han, and C. singeri (A.H. Sm.) D. Wächt. & A. Melzer. Furthermore, it is noteworthy that Song (2024), through morphological observations and sequence comparisons, proposes that Pholiotina pleurocystidiata Hauskn. & Krisai should be considered a synonym of Psathyrella piluliformis [62,64]. In total, 72 new sequences were generated in this study, which were from 24 specimens from Qinghai-Xizang Plateau, China, all with the nrITS, nrLSU, and tef-1α sequences. The combined dataset with 3310 characters including gaps (864 for nrITS, 1299 for nrLSU, and 1147 for tef-1α) was included in the phylogenetic analyses. The phylogenetic trees of ML and MrBayes were almost identical. The ML tree is shown in Figure 1 with bootstrap values and Bayesian posterior probabilities indicated on the branches.

3.2. Taxonomy

Conocybe alticola R.L. Zhao & X.X. Han, sp. nov., Figure 2

Fungal Names: FN 572250

Holotype: China. Xizang Autonomous Region, Ali Region, Zhada County, Rural Road 748, Qinipu, N 31°45′43″ E 79°32′49″, 4058 m asl, 5 August 2024, Xi-Xi Han, Lin-Hui Liu ZRL20240316 (holotype HMAS 287991). GenBank: PQ699270 (nrITS), PQ699294 (nrLSU), PQ836632 (tef-1α).

Etymology: alticola (Latin) refers to the habitat of the species, which is adapted to high-altitude environments.

Diagnosis: Conocybe alticola is distinguished by its hemispherical, obtusely conical to convex pileus with a non-striate surface and a pale yellow hue, the stipe slightly enlarged at base, sometimes forming a bulb. The narrowly ellipsoid to oblong basidiospores, with a germ pore. Grows on alpine meadows in summer.

Macroscopic description: Pileus 1.2–2.0 cm diam., hemispherical, obtusely conical to convex, not hygrophanous, faintly pubescent, non-striate, edge decurved, sometimes with slightly undulated edge, carnosus; yellowish white (4A2) to greyish yellow (4B3), blond (4C4) to olive brown (4D6) at the center, edge paler, pruinose surface. Context thin, fleshy, yellowish white (4A2) to blond (4C4), indistinct odor. Lamellae are distant to nearly distant, narrowly adnate to adnexed, unequal in length, orange–white (5A2) to greyish yellow (4B3) with pale edge when young, light brown (6D7) when mature. Stipes 1.3–3.8 × 0.2–0.3 cm, cylindrical, slightly enlarged at base, sometimes forming a bulb, slightly longitudinally striated, with blond (4C4) pruinose, yellowish white (4A2) to greyish yellow (4C5). Odor not distinctive. Taste indistinct.

Microscopic description: Basidiospores (9.4)9.5–10.5(11.1) × (5.5)6.0–6.7(7.1) μm, Q = 1.48–1.70, Qm = 1.59 (±0.11), narrowly ellipsoid to oblong, with germ pore, slightly thick-walled, contains oil droplets, pale yellow (4A3) to greyish yellow (4C6) in water, greyish orange (6B3) to light brown (6D7) in 5% KOH. Basidia (23.5)24.5–27.1(28.5) × (8.1)9.0–10.0(10.4) μm, clavate, sometimes with vacuolar contents, two or four spored. Cheilocystidia (14.5)16.7–21.2(25.2) × (6.9)8.0–9.8(10.7) μm, lecythiform, with 3.9–4.6 μm wide capitula. Pileipellis hymeniform, consist of broadly clavate, obovoid, or spheropedunculate elements, (25.9)29.1–42.7(54.2) × (10.2)12.3–18.8(20.4) μm. Pleurocystidia clavate or lecythiform, and rare in number. Pileocystidia not observed. Clamp connections present.

Habit and habitat: Summer solitary or scattered in the alpine meadows. So far found only in the Xizang Autonomous Region, China.

Other specimens examined: China. Xizang Autonomous Region, Ali Region, Gaer County, Zuozuo Township, N 32°23′55″ E 80°14′59″, 4409 m asl, 31 July 2024, Xi-Xi Han, Lin-Hui Liu ZRL20240256 (HMAS 287982); Xizang Autonomous Region, Ali Region, Gaer County, Zuozuo Township, N 32°23′55″ E 80°14′59″, 4409 m asl, 31 July 2024, Xi-Xi Han, Lin-Hui Liu ZRL20240259 (HMAS 287983); Xizang Autonomous Region, Ali Region, Zhada County, Rural Road 748, Qinipu, N 31°45′43″ E 79°32′49″, 4058 m asl, 5 August 2024, Xi-Xi Han, Lin-Hui Liu ZRL20240317 (HMAS 287992); Xizang Autonomous Region, Ali Region, Zhada County, Paerlong, N 31°15′39″ E 79°54′16″, 4179 m asl, 6 August 2024, Xi-Xi Han, Lin-Hui Liu ZRL20240327 (HMAS 287995).

Notes: Phylogenetically and morphologically, the new species C. alticola is closely related to C. ammophila (Figure 1) [67], but can be distinguished by its hemispherical, obtusely conical to convex, non-striate, carnosus pileus; pale yellow hue stipe; narrower, greyish orange to light brown in 5% KOH basidiospores and two or four-spored basidia, while C. ammophila, originally described from Greenland, has a pale stipe, broader spores, and four-spored basidia [67,68]. Conocybe alticola is widely distributed in the alpine and subalpine regions of the southwestern Q-X Plateau.

Conocybe alticoprophila R.L. Zhao & X.X. Han, sp. nov., Figure 3

Fungal Names: FN 572251

Holotype: China. Xizang Autonomous Region, Shigatse Municipality, Yadong County, Lower Yadong Township, N 27°13′12″ E 88°34′48″, 2872 m asl, 26 July 2022, Rui-Lin Zhao, Bin Cao ZRL20220046 (holotype HMAS 287975). GenBank: PQ699274 (nrITS), PQ699298 (nrLSU), PQ836636 (tef-1α).

Etymology: “alti-” derived from the Latin word “altus”, meaning “high-altitude”; “-coprophila” derived from the Greek words “copros” and “philos”, meaning “dung” and “fondness”, respectively.

Diagnosis: Conocybe alticoprophila is characterized by its small to medium-sized basidiomata, yellowish-brown, hygrophanous pileus, which is translucently striate to 4/5 from the edge to the center. The basidiospores are slightly amygdaliform, and basidia are two or four spored. Grows on cow dung in grasslands.

Macroscopic description: Pileus 0.2–3.0 cm diam., obtusely conical, hemispherical to conical–convex, hygrophanous, translucently striate to 4/5 from the edge to the center when moist, surface smooth, edge decurved, sometimes with slightly undulated edge; greyish orange (5B4) to yellowish brown (5E5) at the center, turning orange–white (5A2) to yellowish brown (5D8) towards edge. Context thin, and indistinct odor. Lamellae are moderately crowded, narrowly adnate to adnexed, orange–white (5A2) to yellowish brown (5D5). Stipes 0.8–5.2 × 0.1–0.3 cm, cylindrical, fragile, slightly enlarged at the base forming a weak bulb, slightly longitudinally striate, with orange–white (5A2) pruinose, orange–white (5A1) to brownish orange (5C5). Odor not distinctive. Taste indistinct.

Microscopic description: Basidiospores (15.9)16.4–17.7(18.0) × (9.7)10.0–11.0(12.0) μm, Q = 1.55–1.71, Qm = 1.63 (±0.08), narrowly ellipsoid to oblong, slightly amygdaliform, with germ pore, thick-walled, contains oil droplets, pale orange (5A3) to brownish yellow (5C7) in water, light brown (6D6) to dark brown (6F7) in 5% KOH. Basidia (26.3)27.1–31.4(33.5) × (14.2)14.7–15.8(16.6) μm, clavate to broadly clavate, sometimes with vacuolar contents, two or four spored. Cheilocystidia (19.7)21.5–27.0(30.7) × (11.2)12.2–15.5(17.5) μm, lecythiform, with 4.7–7.0 μm wide capitula. Pileipellis hymeniform, consist of clavate or spheropedunculate elements, (26.2)27.6–44.0(47.3) × (10.5)12.9–22.0(24.0) μm. Pleurocystidia lecythiform, and rare in number. Pileocystidia not observed. Clamp connections present.

Habit and habitat: Summer solitary or scattered on cow dung in grasslands. So far found only in the Xizang Autonomous Region, China.

Other specimens examined: China. Xizang Autonomous Region, Shigatse Municipality, Yadong County, Lower Yadong Township, N 27°13′12″ E 88°34′48″, 2872 m asl, 26 July 2022, Rui-Lin Zhao, Bin Cao ZRL20220041 (HMAS 287973); Xizang Autonomous Region, Shigatse Municipality, Yadong County, Lower Yadong Township, N 27°13′12″ E 88°34′48″, 2872 m asl, 26 July 2022, Rui-Lin Zhao, Bin Cao ZRL20220050 (HMAS 287976).

Notes: C. alticoprophila is a coprophilous species characterized by a small basidioma, conical and yellowish-brown pileus, and a cylindrical stipe with finely pruinose surface, it is similar to C. pubescens (Gillet) Kühner [69]. However, C. pubescens has a smaller basidiospores and pileus lack translucently striate when moist. The pileus of C. alticoprophila is obtusely conical, hemispherical to conical–convex with a cylindrical stipe that is slightly enlarged at the base forming a weak bulb. Morphologically, it resembles C. watlingii Hauskn, and is closely related phylogenetically. In contrast, the basidiospores and basidia of C. watlingii are narrower, its pileus lacks translucently striate edges when moist, and the cheilocystidia are slightly narrower with smaller capitula [70]. C. alticoprophila is phylogenetically and morphologically closely related to C. cylindrospora T. Bau & J. Liu, but the latter has smaller basidiospores that are cylindrical to ellipsoid-oblong, four-spored basidia which are also smaller, and smaller cheilocystidia [19]. The Conocybe sp. from Uruguay (PP949255 and PP949256) in the NCBI GenBank shows a high sequence similarity with the ITS of C. alticoprophila, due to partial sequence gaps and the lack of morphological information for the specimens, we cannot confirm whether they represent the same species.

Conocybe versicolor R.L. Zhao & X.X. Han, sp. nov., Figure 4

Fungal Names: FN 572252

Holotype: China. Xizang Autonomous Region, Shigatse Municipality, Yadong County, Lower Yadong Township, N 27°25′20″ E 88°55′6″, 3254 m asl, 27 July 2022, Rui-Lin Zhao, Bin Cao ZRL20220299 (holotype HMAS 287978). GenBank: PQ699290 (nrITS), PQ699314 (nrLSU), PQ836639 (tef-1α).

Etymology: versi- = variable, -color = color, referring to the basidiomata of the species having variable colors.

Diagnosis: Pileus surface of Conocybe versicolor is smooth with a slightly undulated edge. The stipe is cylindrical, hallow, slightly enlarged at the base, covered with scattered to dense pruinose, and no radicating structures were observed. Basidiospores are ellipsoid to oblong. Grow on the ground in mixed forests.

Macroscopic description: Pileus 2.9–4.6 cm diam., obtusely conical, conical–convex to campanulate with or without distinct broad umbo, hardly expanding, sometimes wrinkled, edge slightly undulated, slightly hygrophanous, not striate, surface smooth; brownish orange (5C5) to yellowish brown (5D8) at the center, edge orange–white (5A2) to greyish orange (5B4). Context thin, yellowish white (3A2) to greyish yellow (3C4), and odor indistinct. Lamellae are slightly crowded, narrowly adnate to adnexed, unequal in length, with smooth edges. Stipes 9.7–15.1 × 0.2–0.4 cm, cylindrical, hallow, slightly enlarged at the base, with scattered to dense orange–white (5A2) pruinose, and no radicating structures were observed. Odor not distinctive. Taste indistinct.

Microscopic description: Basidiospores (8.2)8.4–9.1(9.7) × (4.9)5.4–6.1(6.6) μm, Q = 1.43–1.63, Qm = 1.53 (±0.10), ellipsoid to oblong, slightly angular, with germ pore, thick-walled, contains oil droplets, yellowish white (4A2) to greyish yellow (4B4) in water, brownish orange (6C6) to brown (6E8) in 5% KOH. Basidia (18.2)19.7–22.3(23.6) × (7.4)7.8–8.6(9.3) μm, clavate, with vacuolar contents, two or four-spored. Cheilocystidia (15.1)16.9–19.6(20.9) × (6.9)7.5–9.0(10.0) μm, lecythiform, with 3.4–4.5 μm wide capitula. Pileipellis hymeniform, consist of broadly clavate, obovoid, or spheropedunculate elements, (26.9)33.3–44.9(52.4) × (14.0)17.1–23.2(26.5) μm. Pleurocystidia clavate or lecythiform, and rare in number. Pileocystidia not observed. Clamp connections present.

Habit and habitat: Summer solitary on the ground in mixed forests. So far found only in the Xizang Autonomous Region, China.

Other specimens examined: China. Xizang Autonomous Region, Lingzhi Prefecture, Lulang Town, N 29°46′18″ E 94°44′12″, 3326 m asl, 22 September 2015, Sheng-Yu Su ZRL20152517 (HMAS 287972).

Notes: Conocybe versicolor has a conical–convex to campanulate pileus, pale orange to greyish orange, resembling C. solitaria K.A. Thomas, Hauskn. & Manim. It shares similar basidiospore size and clavate basidia with C. solitaria, but the latter can be distinguished by its decurved pileus margin with faint radial striations, and larger basidia [71]. Additionally, C. versicolor has a similar stipe, basidiospores size and two or four-spored basidia to C. humicola (Thiers) Hauskn., Krisai & Voglmary, but C. humicola differs in having smaller basidiomata, shorter basidia, shorter cheilocystidia and a pileipellis consistsing of roundish-stipitate elements [67]. Phylogenetically, C. versicolor is closely related to C. antipus (Lasch) Kühner, which was originally described from North America. Both species share similar basidiospore sizes and occasionally wrinkled pileus, but C. antipus has slightly larger four-spored basidia, and long radicating stipes up to 40 mm [68,70].

Conocybe yadongensis R.L. Zhao & X.X. Han, sp. nov., Figure 5

Fungal Names: FN 572253

Holotype: China. Xizang Autonomous Region, Shigatse Municipality, Yadong County, Lower Yadong Township, N 27°13′12″ E 88°34′48″, 2872 m asl, 26 July 2022, Rui-Lin Zhao, Bin Cao ZRL20220042 (holotype HMAS 287974). GenBank: PQ699291 (nrITS), PQ699315 (nrLSU), PQ836640 (tef-1α).

Etymology: yadongensis (Latin) refers to Yadong County, Xizang, the locality of the type specimen.

Diagnosis: Conocybe yadongensis is characterized by small- to medium-sized basidiomata with a yellowish-brown pileus that is translucently striate and has a surface that is barely pruinose. The stipe is faintly longitudinally striated, and covered with strong pruinosity. The basidiospores are narrowly ellipsoid to oblong. Grows in the grassland.

Macroscopic description: Pileus 0.7–2.2 cm diam., hemispherical when young, obtusely conical, convex to plano-convex when mature, hygrophanous, translucently striate to 4/5 from the edge to the center when moist, faintly pubescent, surface hardly pruinose, edge slightly undulated; brownish orange (5C5) to yellowish brown (5E8) at the center, edge orange–white (5A2) to brownish orange (5C4). Context thin, slightly fragile, orange–white (5A2) to brownish orange (5C5), odor indistinct. Lamellae are slightly crowded, narrowly adnate to adnexed, unequal in length, surface spotted, pale orange (5A3) to light brown (5D6). Stipes 1.6–5.4 × 0.1–0.3 cm, cylindrical, hallow, slightly enlarged at the base, strongly pruinose and slightly longitudinally striate, greyish orange (5B3) to light brown (5D6).

Microscopic description: Basidiospores (8.2)8.4–9.1(9.5) × (4.8)5.0–5.5(5.7) μm, Q = 1.59–1.74, Qm = 1.66 (±0.07), narrowly ellipsoid to oblong, some lentiform, with germ pore, slightly thin walled, contains oil droplets, orange–white (5A2) to greyish orange (5B4) in water, brownish orange (5C5) to yellowish brown (5E7) in 5% KOH. Basidia (14.6)16.1–19.5(21.8) × (6.6)7.6–10.0(11.1) μm, clavate, sometimes with vacuolar contents, two or four spored. Cheilocystidia (18.3)20.4–24.4(26.5) × (9.2)9.4–11.8(13.5) μm, lecythiform, with 5.3–7.2 μm wide capitula. Pileipellis hymeniform, consist of broadly clavate, obovoid, or spheropedunculate elements, (26.9)34.3–50.9(65.9) × (13.3)17.0–27.0(34.7) μm. Pleurocystidia utriform, broadly clavate or lecythiform, and rare in number. Pileocystidia not observed. Clamp connections present.

Habit and habitat: Summer scattered in the grassland. So far found only in the Xizang Autonomous Region, China.

Notes: In the phylogenetic analyses, C. yadongensis is closely related to C. coniferarum E.F. Malysheva, C. echinata (Velen.) Singer, and C. qujingensis W.H. Lu, Karunarathna & Tibpromma (Figure 1). However, C. coniferarum has longer stipe with brownish hues, broader basidiospores and four-spored basidia [22], while C. echinata has a dark brown to dull brown pileus and larger cheilocystidia [35,72]. Additionally, C. qujingensis has a longer stipe, larger cheilocystidia, and pileipellis consists of clavate and spheropedunculate elements [36].

4. Discussion

This study revisits the taxonomy of Conocybe specimens from the Q-X Plateau based on the phylogenetic frameworks established by Tóth et al. [21], Lu et al. [36], and Song and Bau [62], alongside the morphological classification by Hausknecht [34]. We incorporated macrofungal specimens collected from the Q-X Plateau between 2015 and 2024, and employed a concatenated dataset (including ITS, nrLSU, and tef1-α sequences) to reassess the classification of Conocybe in this region. Based on the results, we describe four new species: C. alticola, C. alticoprophila, C. versicolor, and C. yadongensis, from various locations, including the Ali Region, Shigatse Municipality, and Lingzhi Prefecture. These newly identified species expand the known diversity of Conocybe on the Q-X Plateau and offer preliminary insights into the species adaptation to high-altitude, cold-climate habitats. Additionally, there are three known species, C. himalayana, C. pseudocrispa (Hauskn.) Arnolds, and C. fuscimarginata, from Lhasa Municipality, Qamdo Municipality and the Ali Region. Notably, the latter two species are reported here for the first time from the Q-X Plateau. This study broadens the understanding of Conocybe diversity in this unique high-altitude region and provides foundational data on its distribution patterns in cold environments.

The formation of basidioma is closely influenced by environmental factors, such as temperature, light, and nutrients [73]. On the Q-X Plateau, most Conocybe taxa exhibit temperate characteristics, but C. alticola, which we have newly described and has tough basidioma and relatively short stipes, is an exception. This aligns with the toughness-protection hypothesis proposed by Krah et al., which suggests that tough-fleshed basidioma reduce water loss, helping macrofungi cope with extreme microclimatic fluctuations [74]. Despite extensive surveys across various regions of Hengduan Mountains and Xizang Autonomous Region, C. alticola has only been found in the Ali region of Xizang Autonomous Region, which is known for long daylight hours, low temperatures, intense sunlight, and high altitudes (with an average elevation exceeding 4500 m) [75,76].

Phylogenetically, C. alticola is clustered with C. qujingensis, C. echinate, and C. coniferarum, forming a distinct lineage, and is sister to C. ammophila (Figure 1), which is described as being found only in cold climates with extreme temperature fluctuations and low rainfall, such as Greenland, Mongolia, and Russia. It is one of the stoutest members of the genus [68], which aligns with the characteristics of C. alticola. In contrast, C. qujingensis, C. echinata, and C. coniferarum exhibit slender stipes and relatively fragile pileus [22,35,36]. The variation in macroscopic morphology partially reveals that species within the genus may undergo adaptive evolution changes when facing extreme environmental stress. To further substantiate this conclusion, additional sampling outside the current study area is required to explore the broader distribution of these species.

Currently, a total of 15 Conocybe species have been recorded from the Q-X Plateau, including C. apala (Fr.) Arnolds, C. fragilis (Peck) Singer, C. himalayana, C. macrocephala Kühner & Watling, C. macrospora (G.F. Atk.) Hauskn., C. ochracea (Kühner) J. Favre, C. siliginea (Fr.) Kühner, C. subovalis Kühner & Watling, C. tenera (Schaeff.) Kühner, and six species identified in this study [77,78,79,80,81,82,83,84,85]. In addition, Conocybe is primarily distributed in Jilin Province and Inner Mongolia, where 26 and 9 species have been reported, respectively [36]. Limited records have also been made in regions such as Guangxi Province, Hunan Province, Hubei Province, Yunnan Province, Taiwan, Tianjin Municipality, and Xinjiang Uygur Autonomous Region [19,57,77,78,85]. However, in comparison to the total number of known species, the diversity of Conocybe in China remains relatively underrepresented.

The classification of Conocybe remains challenging, primarily due to the discordance between the clades within genus and morphological sections, as well as the presence of morphologically similar species, which complicates accurate species identification [21,62]. Traditional identification relies on morphological features such as pileus, lamellae, stipe, which are influenced by environmental conditions and growth stages, leading to variation. Additionally, some species lack sufficient and reliable molecular data, making it difficult to conduct a phylogenetic analysis for accurate classification [36]. Therefore, a combined approach using multi-gene phylogenetic analysis and morphological characteristics, along with the inclusion of multidimensional regional samples, is essential for further clarifying the distribution and classification of this genus in China.

This study describes four new species and reports the first records of two known species from the Qinghai-Xizang Plateau. By combining morphological and molecular phylogenetic analyses, it provides preliminary insights into the adaptive evolution of macrofungi in extreme high-altitude cold climates, contributing new perspectives on fungal diversity and species distribution on the Plateau. It also offers valuable data for understanding species distribution patterns and environmental adaptations in high-altitude ecosystems and provides empirical support for the toughness-protection hypothesis.