A new clitocyboid genus Spodocybe and a new subfamily Cuphophylloideae in the family Hygrophoraceae (Agaricales)

Abstract

Phylogenetically, the genera Cuphophyllus, Ampulloclitocybe and Cantharocybe are treated as basal in the family Hygrophoraceae, despite weak support. However, the exact phylogenetic positions of the three genera have remained unresolved, and taxa related to these genera are poorly known. In this study, a new clitocyboid genus Spodocybe was proposed based on multigenic phylogenetic inference datasets and morphological evidence. The analyses of ITS as well as two combined datasets ITS-nrLSU-rpb2 and ITS-nrLSU-rpb1-rpb2-tef1-α-atp6 supported that (1) Spodocybe formed a well-supported monophyletic clade; and (2) sisters Spodocybe and Ampulloclitocybe, along with Cantharocybe and Cuphophyllus also formed a monophyletic lineage, as sister to the rest of the Hygrophoraceae. Meanwhile, two new species, namely S. rugosiceps and S. bispora, from southwestern China, were documented and illustrated. These results support the new proposed genus Spodocybe, and that Spodocybe, Ampulloclitocybe, Cantharocybe and Cuphophyllus should be retained in the Hygrophoraceae as a new subfamily Cuphophylloideae.

Introduction

The widespread genus Clitocybe (Fr.) Staude currently encompasses large numbers of species with clitocyboid habit, sharing the features of saprophytic nutrition, funnel-shaped pileus, decurrent lamellae, a usually white, cream or pale colored spore-deposit and smooth and inamyloid spores (Singer 1986; Breitenbach and Kraenzlin 1991; Læssøe and Petersen 2019). As a consequence of the poor, broad and unrepresentative morphological characteristics, the genus appeared heterogeneous and was subsequently proven to be polyphyletic based on the phylogenetic analysis (Moncalvo et al. 2002; Harmaja 2003).

Based on phylogenetic analyses over the past 20 years, (i) many new genera within the Tricholomatoid clade were proposed to accommodate previous Clitocybe species deviating from the core Clitocybeae clade (Matheny et al. 2006), such as Cleistocybe Ammirati, A.D. Parker & Matheny (Ammirati et al. 2007), Trichocybe Vizzini (Vizzini et al. 2010), Atractosporocybe P. Alvarado, G. Moreno & Vizzini, Leucocybe Vizzini, P. Alvarado, G. Moreno & Consiglio and Rhizocybe Vizzini, G. Moreno, P. Alvarado & Consiglio (Alvarado et al. 2015); (ii) Several clitocyboid groups were reconfirmed as independent genera, for instance, Singerocybe Harmaja (Qin et al. 2014) and Infundibulicybe Harmaja (Binder et al. 2010); and (iii) some others were even transferred to the Hygrophoroid clade (Binder et al. 2010), such as Ampulloclitocybe Redhead, Lutzoni, Moncalvo & Vilgalys (Redhead et al. 2002) and Cantharocybe H.E. Bigelow & A.H. Sm. (Hosen et al. 2016). However, many clitocyboid taxa remain to be reclassified.

The molecular phylogenetic relationships among members of the Hygrophoraceae Lotsy were well studied by Lodge et al. (2014). In their work, the family was divided into subfamily Hygrophoroideae E. Larss., Lodge, Vizzini, Norvell & S.A. Redhead, Hygrocyboideae Padamsee & Lodge, Lichenomphalioideae Lücking & Redhead and Cuphophylloid grade. Meanwhile, the Cuphophylloid grade was retained in the Hygrophoraceae as the base comprising the genera Cuphophyllus (Donk) Bon, Ampulloclitocybe and Cantharocybe, despite weak phylogenetic support (Matheny et al. 2006; Binder et al. 2010; Lodge et al. 2014). Consequently, the taxonomic problem of the three genera on whether to be included or excluded in the Hygrophoraceae has remained unresolved.

Recently, some collections were shown to be closely related to Clitocybe trulliformis (Fr.) P. Karst. based on ITS-BLAST searches while at the same time they were surprisingly related to taxa of the genus Cuphophyllus based on nrLSU-BLAST searches. As far as we know, C. trulliformis and allied species were lacking taxonomic revision, especially regarding their molecular phylogenetic status. Furthermore, the phylogenetic delimitation of the Hygrophoraceae was ambiguous due to the uncertain positions of Cuphophyllus, Ampulloclitocybe and Cantharocybe. Hence, the aims of this study were (a) to propose and describe a new genus of the Hygrophoraceae for species related to C. trulliformis based on morphological and molecular analyses and (b) to reconstruct the phylogeny of the Hygrophoraceae for determining the exact phylogenetic placements of Cuphophyllus, Ampulloclitocybe and Cantharocybe with multi-gene data.

Materials and methods

Specimens

Twenty-three specimens of species similar to C. trulliformis and related species were collected from southwestern and northeastern China and western Germany, during 2007 to 2020. The fresh fruitbodies were dried using heat or silica gel. Voucher specimens were deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS). Detail information of these specimens is given in Table 1.

Table 1.

Specimens used in phylogenetic analysis and their GenBank accession numbers. The newly generated sequences are shown in bold.

Species	Voucher	Locality	GenBank accession number
			ITS	nrLSU	rpb2	rpb1	tef1-α	atp6
Acantholichen pannarioides	MDF352	Costa Rica	KT429795	KT429807	KT429817
Acantholichen campestris	DIC595b	Brazil	KT429798	KT429810	KT429818
Acantholichen galapagoensis	MDF058	Ecuador	KT429785	KT429800	KT429812
Ampulloclitocybe clavipes	KUN-HKAS 54426	China: Jilin	MW616462	MW600481	MW656471	MW656467	MW656461	MW656478
	AFTOL-ID 542		AY789080	AY639881	AY780937	AY788848	AY881022
	DJL06TN40	USA	FJ596912	KF381542	KF407938
Arrhenia auriscalpium	TUB 011588			DQ071732
Arrhenia acerosa	Lueck2	Germany	KP965766	KP965784
Cantharellula umbonata	CBS 398.79	France	MH861222	MH872990
Cantharocybe gruberi	AFTOL-ID 1017	USA	DQ200927	DQ234540	DQ385879	DQ435808	DQ059045
	AH24539	Spain	JN006422	JN006420
Cantharocybe brunneovelutina	DJL-BZ-1883	Belize	NR160458	NG068731
Cantharocybe virosa	TENN63483	India	KX452405	JX101471
	Iqbal-568	Bangladesh	KX452403	KF303143
Chromosera cyanophylla	AFTOL-ID 1684	USA	DQ486688	DQ457655	KF381509
Chromosera ambigua	GE18008-1	France	MK645573	MK645587	MK645593
Chromosera lilacina	GE18035	Canada	MK645577	MK645591	MK645597
Chromosera xanthochroa	GE18033	Canada	MK645576	MK645590	MK645596
Chrysomphalina chrysophylla	AFTOL-ID 1523	USA	DQ192180	DQ457656
Chrysomphalina grossula	OSC 113683		EU644704	EU652373
Clitocybe aff. costata	DJL06TN80	USA	FJ596913
Clitocybe herbarum	G0171	Hungary		MK277719
Clitocybe trulliformis	14562	Italy	JF907809
	4804	Russia	MH930178
Clitocybe cf. trulliformis	G0460	Hungary		MK277728
Clitocybe sp.	NAMA 2015-206	USA	MH910535
Clitocybe sp.	NAMA 2015-318	USA	MH910563
Clitocybe sp.	Mushroom Observer 302917	USA	MK607556
Cora pavonia	DIC215	Ecuador	KF443238	KF443261	KF443275
Cora aspera	DIC110	Bolivia	KF443230	KF443257	KF443267
Cora reticulifera	DIC119	Ecuador	KF443239	KF443262	KF443269
Cora squamiformis	DIC146	Bolivia	KF443240	KF443263	KF443273
Corella brasiliensis	MDF017	Bolivia	KF443229	KF443255	KF443276
Corella aff. Melvinii	MDF200	Brazil	KJ780569	KY861725
Cuphophyllus pratensis	Lueck7	Germany	KP965771	KP965789
	DJL-Scot-8	UK	KF291057	KF291058
Cuphophyllus aurantius	CFMR PR-6601	Puerto Rico	KF291099	KF291100	KF291102
Cuphophyllus aff. pratensis	AFTOL-ID 1682	USA	DQ486683	DQ457650		DQ435804
Cuphophyllus sp.	KUN-HKAS 105671	China: Tibet	MW762875	MW763000	MW789179	MW789163
Cyphellostereum galapagoense	CDS 41163	Ecuador	NR158415	NG068806
Cyphellostereum imperfectum	DIC115	Guatemala	KF443218	KF443243	KF443277
Dictyonema interruptum	Ertz 10475	Portugal		EU825967	KF443282
Dictyonema schenckianum	DIC113	Brazil	KF443225	KF443251	KF443285
Eonema pyriforme	G1063	Poland		MK278075
Gliophorus psittacinus	CFMR DEN-25	Denmark	KF291075	KF291076	KF291078
Gliophorus graminicolor	TJB-10048 (CORT)	Australia	KF381520	KF381545	KF407936
Gliophorus aff. laetus	CFMR PR-5408	Puerto Rico	KF291069	KF291070
Gloioxanthomyces nitidus	GDGM41710	China: Jilin	MG712283	MG712282	MG711911
Haasiella splendidissima	Herb. Roux n. 4044	France	JN944400	JN944401
	Herb. Roux n. 3666	Moldova	JN944398	JN944399
Haasiella venustissima	A. Gminder 971488	Italy	KF291092	KF291093
	E. C. 08191	Italy	JN944393	JN944394
Humidicutis marginata	JM96/33			AF042580
Humidicutis auratocephalus	AFTOL-ID 1727	USA	DQ490624	DQ457672	DQ472720	DQ447906
Humidicutis dictiocephala	QCAM6000	Ecuador	KY689661	KY780120
Humidicutis sp.	CFMR BZ-3923	Belize	KF291110	KF291111
Hygroaster nodulisporus	AFTOL-ID 2020	USA		EF561625
Hygroaster albellus	AFTOL-ID 1997	Puerto Rico	KF381521	EF551314
Hygrocybe conica	FO 46714			DQ071739
Hygrocybe cf. acutoconica	CFMR NC-256	USA	KF291117	KF291118	KF291120
Hygrocybe coccinea	AFTOL-ID 1715	USA	DQ490629	DQ457676	DQ472723	DQ447910	GU187705
Hygrocybe aff. conica	AFTOL-ID 729		AY854074	AY684167	AY803747
Hygrophorus eburneus	US97/138	Germany		AF430279
	GDGM70059	USA	MT093608
Hygrophorus chrysodon	KUN-HKAS 82501	China: Tibet	MW616463	MW600482	MW656472		MW656462	MW656479
	KUN-HKAS 112569	China: Tibet	MW762876	MW763001	MW789180	MW789164	MW773440	MW789195
Hygrophorus flavodiscus	KUN-HKAS 68013	China: Yunnan	MW616464	MW600483	MW656473	MW656468	MW656463	MW656480
	KUN-HKAS 55043	China: Yunnan	MW616465	MW600484	MW656474	MW656469	MW656464	MW656481
Hygrophorus gliocyclus	KUN-HKAS 79929	China: Tibet	MW616466	MW600485	MW656475		MW656465	MW656482
Hygrophorus hypothejus	KUN-HKAS 56550	Germany	MW616467	MW600486	MW656476	MW656470		MW656483
Hygrophorus pudorinus	AFTOL-ID 1723	USA	DQ490631	DQ457678	DQ472725	DQ447912	GU187710
Hygrophorus sp. 1	KUN-HKAS 112566	China: Yunnan	MW762877	MW763002	MW789181	MW789165	MW773441	MW789196
Hygrophorus sp. 2	KUN-HKAS 87261	China: Jilin	MW616468	MW600487	MW656477		MW656466	MW656484
Hygrophorus sp. 3	KUN-HKAS 112567	China: Tibet	MW762878	MW763003	MW789182	MW789166	MW773442	MW789197
Hygrophorus sp. 4	KUN-HKAS 112568	China: Tibet	MW762879	MW763004	MW789183	MW789167	MW773443	MW789198
Lichenomphalia hudsoniana	GAL18249	USA	JQ065873	JQ065875
Lichenomphalia meridionalis	S-270-FB1	Japan	LC428308	LC428307
Neohygrocybe ovina	GWG H. ovina Rhosisaf (ABS)	UK	KF291233	KF291234	KF291236
Neohygrocybe griseonigra	GDGM 44492	China	MG779451	MG786565
Neohygrocybe ingrata	DJL05TN62 (TENN)	USA	KF381525	KF381558	KF381516
Neohygrocybe subovina	GRSM 77065	USA	KF291140	KF291141
Spodocybe bispora	KUN-HKAS 73310	China: Yunnan	MW762880	MW763005	MW789184	MW789168	MW773444	MW789199
	KUN-HKAS 73332	China: Yunnan	MW762881	MW763006	MW789185	MW789169	MW773445	MW789200
	KUN-HKAS 112564	China: Yunnan	MW762882	MW763007	MW789186	MW789170	MW773446	MW789201
Spodocybe rugosiceps	KUN-HKAS 112561	China: Yunnan	MW762883	MW763008	MW789187	MW789171	MW773447	MW789202
	KUN-HKAS 81981	China: Yunnan	MW762884	MW763009	MW789188	MW789172		MW789203
	KUN-HKAS 69830	China: Yunnan	MW762885	MW763010	MW789189	MW789173	MW773448	MW789204
Spodocybe rugosiceps	KUN-HKAS 71071	China: Yunnan	MW762886	MW763011	MW789190	MW789174	MW773449	MW789205
	KUN-HKAS 112562	China: Yunnan	MW762887	MW763012	MW789191	MW789175	MW789159	MW789206
	KUN-HKAS 112563	China: Yunnan	MW762888	MW763013	MW789192	MW789176	MW789160	MW789207
Spodocybe sp. 1	KUN-HKAS 112560	China: Jilin	MW762889	MW763014	MW789193	MW789177	MW789161	MW789208
Spodocybe sp. 2	KUN-HKAS 112565	China: Yunnan	MW762890	MW763015	MW789194	MW789178	MW789162	MW789209
Porpolomopsis calyptriformis	CFMR ENG-3	UK	KF291242	KF291243	KF291245
Porpolomopsis aff. calyptriformis	DJL05TN80 (TENN)	USA	KF291246	KF291247	KF291249
Porpolomopsis lewelliniae	TJB-10034 (CORT)	Thailand	KF291238	KF291239	KF291241
Pseudoarmillariella ectypoides	AFTOL-ID 1557	USA	DQ192175	DQ154111	DQ474127	DQ516076	GU187733
Pseudoarmillariella bacillaris	KUN-HKAS 76377	China	KC222315	KC222316
Sinohygrocybe tomentosipes	GDGM 50075	China: Hunan	MG685873	MG696902	MG696906
	GDGM 43351	China: Sichuan	MG685872	MG696901	MG696905
Amylocorticium cebennense	CFMR HHB-2808	USA	GU187505	GU187561	GU187770	GU187439	GU187675
Aphroditeola olida	DAOM 226047	Canada	KF381518	KF381541
Macrotyphula fistulosa	IO. 14. 214	Spain	MT232352	KY224088		MT242317	MT242354
Macrotyphula juncea	IO. 14. 177	Sweden	MT232353	MT232306	MT242337		MT242355
Macrotyphula phacorrhiza	IO. 14. 167	Sweden	MT232364	MT232315	MT242348	MT242326	MT242367
	IO.14. 200	France	MT232363	MT232314	MT242347		MT242366
Phyllotopsis nidulans	IO. 14. 196	Spain	MT232308		MT242338	MT242319	MT242357
Phyllotopsis sp.	AFTOL-ID 773		DQ404382	AY684161	AY786061	DQ447933	DQ059047
Pleurocybella porrigens	UPS F-611822	Sweden	MT232355	MT232309	MT242339
Plicaturopsis crispa	AFTOL-ID 1924	USA	DQ494686	DQ470820	GU187816
Pterulicium echo	ZRL20151311		LT716065	KY418881	KY419026	KY418979	KY419076
Pterulicium gracilis	IO. 14. 142	Sweden	MT232356	MT232310			MT242358
Sarcomyxa serotina	AFTOL-ID 536	USA	DQ494695	AY691887	DQ859892	DQ447938	GU187754
Serpulomyces borealis	CFMR L-8014	USA	GU187512	GU187570	GU187782		GU187686
Tricholomopsis decora	AFTOL-ID 537		DQ404384	AY691888	DQ408112	DQ447943	DQ029195
Tricholomopsis osiliensis	ZRL20151760		LT716068	KY418884	KY419029		KY419079
Typhula capitata	IO. 15. 122	Spain	MT232357	MT232312	MT242341	MT242321	MT242360
Typhula incarnata	IO. 14. 92	Sweden	MT232362	MT232313	MT242346	MT242325
Typhula micans	IO. 14. 165	Sweden	MT232361	KY224102	MT242345	MT242324	MT242364

Morphological observation

Macroscopic characters of species were described based on the raw field record data and photographs. Colors used in description referred to Kornerup and Wanscher (1978). For the microscopic structure observation, tissue sections of dried specimens were mounted in 5% KOH solution or distilled water and structures of lamellar trama, pileipellis and stipitipellis, basidia and basidiospores were observed with a light microscopy. For the description of lamellar trama structure, seven types, including regular, subregular, divergent, pachypodial, bidirectional, tri-directional and interwoven, were used following Lodge et al. (2014). Besides, Melzer’s reagent was applied to test the amyloidity of the basidiospores. In the description of basidiospores, the abbreviation [n/m/p] represent that the measurements were made on n basidiospores from m basidiomes of p collections. The range notation (a)b–c(d) stands for the dimensions of basidiospores in which b–c contains a minimum of 90% of the measured values while a and d in the brackets stand for the extreme values. In addition, a Q value show the length/width ratio of basidiospores and a Qm value for average Q ± standard deviation. All microstructures were illustrated by hand drawing.

DNA extraction, PCR and sequencing

Total genomic DNA was extracted using the Ezup Column Fungi Genomic DNA Purificaton Kit (Sangon Biotech, Shanghai, China) according to the manual. For the PCR amplification, (1) Primers ITS5 and ITS4 (White et al. 1990) were used for the internal transcribed spacer (ITS); (2) LROR and LR5 (Vilgalys and Hester 1990) for the nuclear ribosomal large subunit (nrLSU); (3) EF1-983F and EF1-1953R (Matheny et al. 2007), designed primers SPO-TEF1-F (5’-ATTGCYGGYGGTACYGGTGA-3’) and SPO-TEF1-R (5’-TCVAGDGATTTACCTGTHCGRC-3’) or another pair of designed primers HYG-TEF1-F (5’-CTTGCCTTYACTCTYGGYGTCC-3’) and HYG-TEF1-R (5’-GCGAACTTGCASGCAATGTG-3’) for the translation elongation factor 1-α (tef1-α); (4) RPB1-Af and RPB1-Cr (Matheny et al. 2002) or designed primers SPO-RPB1-F (5’-ACGAGGTTGYGTGGTGAAAT-3’) and SPO-RPB1-R (5’-GGAGGNGGDACHGGCATNA-3’) for the DNA-directed RNA polymerase II second largest subunit 1 (rpb1); (5) RPB2-6F and RPB2-7.1R (Matheny 2005) for the DNA-directed RNA polymerase II second largest subunit 2 (rpb2); and (6) ATP6-3 and ATP-6 (Kretzer and Bruns 1999) for ATP synthase subunit 6 (atp6).

The PCR mixtures contained 1× PCR buffer, 1.5mM MgCl₂, 0.2mM dNTPs, each primer at 0.4 μM, 1.25U of Taq polymerase (Sangon Biotech, Shanghai, China), and 1 μL of DNA template in a total volume of 25 μL. Reactions were performed with the following program: initial denaturation at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 50 °C (atp6), 52 °C (nrLSU, tef1-α, rpb1 and rpb2) or 54 °C (ITS) for 30 s, and 72 °C for 30 s (ITS and atp6), 50 s (nrLSU and rpb2) or 75 s (tef1-α and rpb1), and for terminal elongation, the reaction batches were incubated at 72 °C for 10 min. All PCR products were detected by 2% agarose gel electrophoresis and then sent to the Kunming branch of Tsingke Biological Technology Co., Ltd. (Beijing, China) for sequencing.

Phylogenetic tree construction

Sequences used for phylogenetic analysis (presented in Table 1) were aligned by using MAFFT v7.471 (Katoh and Standley 2016) and then manually adjusted by using BIOEDIT v7.2.5 (Hall 1999). The intron regions of tef1-α, rpb2 and rpb1 were excluded except the conserved rpb1-intron2. Three datasets of ITS-nrLSU-rpb2, ITS-nrLSU-rpb1-rpb2-tef1-α-atp6 and ITS (Suppl. materials 1, 2 and 3) were used to construct phylogenetic trees. The two multi-gene matrixes were generated by SEQUENCEMATRIX 1.7.8 (Vaidya et al. 2011). GTR + I + G was inferred as the best-fit model for the three matrixes selected according to the AIC in MRMODELTEST v2.4 (Nylander 2004). Maximum likelihood (ML) trees with 1000 bootstrap replicates and Bayesian inferences were generated with RAXML v8.0.20 (Stamatakis 2006) and MRBAYES v3.2.7 (Ronquist and Huelsenbeck 2003), respectively.

Results

Molecular phylogenetic analysis

As shown in Table 1, a total of 393 sequences (109 ITS, 110 nrLSU, 40 tef1-α, 38 rpb1, 74 rpb2 and 22 atp6) from 118 samples were used in the phylogenetic analyses, 131 (23 ITS, 23 nrLSU, 20 tef1-α, 20 rpb1, 23 rpb2 and 22 atp6) of which were newly generated in the present study.

The combined dataset ITS-nrLSU-rpb2 comprised 221 sequences from 88 samples with a total of 3135 positions. In the three-gene tree (Fig. 1), 11 specimens from four novel Spodocybe species collected in this study, C. cf. trulliformis and C. herbarum formed a strongly supported monophyletic clade (BP = 100%, PP = 1.0), as sister to Ampulloclitocybe (BP = 63%, PP = 0.98). The phylogenetic analysis showed that the new proposed genus Spodocybe should be placed within the Hygrophoraceae, although intergeneric branched orders among Spodocybe, Ampulloclitocybe, Cantharocybe and Cuphophyllus were unstable with low support values.

Figure 1.

ML analysis of Hygrophoraceae combined ITS, nrLSU and rpb2 sequence data, with Macrotyphula juncea, Macrotyphula phacorrhiza and Phyllotopsis sp. as outgroups. Bootstrap values (BP) ≥ 50% from ML analysis and Bayesian posterior probabilities (PP) ≥ 0.90 are shown at nodes. The newly generated sequences are shown in bold.

In order to accurately determine the position of Spodocybe in the family Hygrophoraceae and better clarify the phylogenetic relationships of Spodocybe, Ampulloclitocybe, Cantharocybe and Cuphophyllus, a further six-gene matrix ITS-nrLSU-rpb1-rpb2-tef1-α-atp6 composed of 179 sequences from 54 samples with 5405 positions was used to rebuild the Hygrophoraceae tree. As revealed by the six-gene phylogenetic analysis (Fig. 2), the branch support level of the six-gene tree was obviously improved, compared with that of the previous three-gene tree. The monophyly of Spodocybe clade was strongly supported (BP = 100%, PP = 1.00), including Spodocybe rugosiceps (BP = 100%, PP = 1.00), S. bispora (BP = 100%, PP = 1.00) and two unnamed Spodocybe species. Spodocybe and Ampulloclitocybe were sister clades (BP = 78%, PP = 0.99), then further clustered with Cantharocybe (BP = 59%, PP = 0.97) and finally together with Cuphophyllus formed an independent lineage (BP = 85%, PP = 1.00). Meanwhile, this lineage (Cuphophylloideae) comprising the four genera was well-supported (BP = 83%, PP = 1.00) as sister to the rest of the Hygrophoraceae.

Figure 2.

ML analysis of Hygrophoraceae combined ITS, nrLSU, rpb1, rpb2, tef1-α and atp6 sequence data, with representatives of Amylocorticiaceae, Pterulaceae and the Hygrophoroid clade (Aphroditeola, Macrotyphula, Phyllotopsis, Pleurocybella, Sarcomyxa, Tricholomopsis and Typhula) as outgroups. Bootstrap values (BP) ≥ 50% from ML analysis and Bayesian posterior probabilities (PP) ≥ 0.90 are shown at nodes. Branches with BP ≥ 75% and PP ≥ 0.95 are bolded. The newly generated sequences are shown in bold. Lamellar trama type B for bidirectional, D for divergent, I for interwoven, P for pachypodial, R for regular, S for subregular, T for tri-directional. Lamellar trama types of specimens collected in this study were identified by ourselves and others referred to Lodge et al. (2014) and Hosen et al. (2016).

In addition, an ITS dataset (23 sequences, 1053 positions) was applied to phylogenetic analysis for displaying the relationships among Spodocybe species from this study and species of Clitocybe treated from GenBank. In the ITS tree (Fig. 3), Spodocybe species formed a highly supported monophyletic clade with C. trulliformis and related species (BP = 100%, PP = 1.00), which was also a sister clade to Ampulloclitocybe with strong support (BP = 91%, PP = 0.99).

Figure 3.

Phylogram showing the phylogenetic relationships among Spodocybe species and species of Clitocybe treated from Genbank based on ITS sequence data, with representatives of Ampulloclitocybe, Cuphophyllus and Hygrophorus as outgroups (rooted with Hygrophorus eburneus). Bootstrap values (BP) ≥ 50% from ML analysis and Bayesian posterior probabilities (PP) ≥ 0.90 are shown at nodes. The newly generated sequences are shown in bold. EA, NA and SE refer to East Asia, North America and South Europe, respectively.

Taxonomy

Cuphophylloideae

Z. M. He & Zhu L. Yang subf. nov.

C96C3465-EDF8-5E8F-B6E1-C1FDD9144C40

839377

Diagnosis.

Characterized generally by clitocyboid basidiomes, convex to funnel-shaped pileus, decurrent lamellae, absence of veils, inamyloid basidiospores and presence of clamps.

Etymology.

From the type genus Cuphophyllus.

Type genus.

Cuphophyllus (Donk) Bon.

Description.

Basidiomes small, medium-sized to large, mostly clitocyboid, rarely omphalinoid or mycenoid; veils absent. Pileus convex, applanate to funnel-shaped; surface usually dry, smooth, lubricous or rarely viscid. Lamellae decurrent to deeply decurrent. Basidiospores ellipsoid, oblong or subglobose, thin-walled and inamyloid. Pileipellis usually a cutis, sometimes ixocutis or trichoderm. Lamellar trama regular, subregular, interwoven or bidirectional. Clamp connections present.

Habitat, ecology and distribution.

Usually gregarious or caespitose on ground, rarely on wood; widespread in temperate and tropical regions.

The genera Ampulloclitocybe, Cantharocybe, Cuphophyllus and Spodocybe are included in the subfamily Cuphophylloideae, which is in correspondence with Cuphophylloid grade of Lodge et al. (2014) plus Spodocybe.

Spodocybe

Z. M. He & Zhu L. Yang gen. nov.

F15F630C-60D4-54EE-9717-AA0BF80B6081

839050

Diagnosis.

Differs from Ampulloclitocybe by its small basidiomes and subregular lamellar trama rather than medium-sized basidiomes and bidirectional lamellar trama. Differs from Cuphophyllus in the ratio of basidia to basidiospore length less than 5, and lamellar trama subregular rather than interwoven. Differs from Cantharocybe in its absence of cheilo- and caulocystidia, having small basidiomes rather than large ones and having subregular lamellar trama rather than regular one.

Etymology.

Spodo- refers to grey; -cybe refers to head; that is a Clitocybe-like genus with gray pileus.

Type species.

Spodocybe rugosiceps Z. M. He & Zhu L. Yang.

Description.

Basidiomes small, clitocyboid. Pileus convex, applanate to infundibuliform; surface dry, greyish (2B1), grey-brown (5C4) to dark grey-brown (5E4); center depressed with age. Lamellae decurrent to deeply decurrent, white (1A1) to cream (1A2), thin, moderately crowded, sometimes furcate and interveined. Stipe central, subcylindrical, concolorous with pileus. Basidiospores ellipsoid, oblong to cylindrical, colourless, hyaline, smooth, thin-walled, inamyloid; ratio of basidia to basidiospore length less than 5. Pileipellis and stipitipellis a cutis. Lamellar trama subregular. Clamp connections abundant, present in all parts of basidiome.

Habitat, ecology and distribution.

Saprophytic, usually gregarious or caespitose on the ground of coniferous or coniferous and broad-leaved mixed forest; distributed in the temperate and subtropical zones from June to November.

Spodocybe rugosiceps

Z. M. He & Zhu L. Yang sp. nov.

CCB3B84F-91A5-5DAD-B5A5-E6CC5355CF68

839052

Figs 4A, B , 5

Figure 4.

Basidiomes of described Spodocybe species. A, BSpodocybe rugosiceps (KUN-HKAS 112563, KUN-HKAS 112562, respectively) C, DSpodocybe bispora (KUN-HKAS 73332, KUN-HKAS 112562, respectively). Scale bars: 1 cm.

Figure 5.

Microscopic features of Spodocybe rugosiceps (KUN-HKAS 112563, holotype) a basidiospores b basidia c pileipellis. Scale bars: 10 μm.

Diagnosis.

Differs from S. bispora in having a rugose pileus, smaller basidiospores and 4-spored rather than 2-spored basidia. Differs from C. trulliformis in having smaller basidiospores and a rugose rather than felty-squamulose pileus.

Etymology.

rugosiceps refers to the rugose pileus.

Type.

China. Yunnan Province: Kunming City, near Yeya Lake, at 25.136658°N, 102.873027°E, alt. 2000 m, 11 Aug 2020, Z. M. He 72 (KUN-HKAS 112563, holotype).

Description.

Basidiomes small, clitocyboid. Pileus 0.5–2 cm in diam, at first nearly applanate, then concave; surface dry and rugose, gray-brown (5E2-4) to gray-black (4F2-4) in the center and gray-brown (5C2-4) or gray (5B1-2) towards margin; center often slightly umbonate; margin straight and undulating; context thin and white (1A1) to cream (1A2). Lamellae deeply decurrent, white (1A1) to cream (1A2), thin (up to 2 mm high), crowded, sometimes forked and intervenose. Stipe 2.5–6 × 0.2–0.4 cm, central, narrowly cylindrical to subcylindrical, sometimes flexuous, hollow; surface dry and nearly smooth, concolorous with pileus; context white (1A1).

Basidiospores [60/3/3] 5–6 (6.5) × (2.5)3–3.5(4) μm, Q = (1.38)1.55–1.95(2), Qm = 1.73 ± 0.14, elongate, colorless, hyaline, smooth, thin-walled, inamyloid. Basidia 20–24 × 5–6 μm, clavate, 4-spored, colorless, hyaline, thin-walled; sterigmata up to 4 μm long; ratio of basidia to basidiospore length values about 3–5. Cystidia absent. Lamellar trama subregular; hyphae colorless, hyaline, cylindrical, thin-walled, 3–10 µm wide. Pileipellis a cutis, but in places upright or trichodermial in appearance, made up with thin-walled cylindrical hyphae 3–9 µm wide. Stipitipellis a cutis, composed of thin-walled cylindrical hyphae 3–10 μm wide. Clamp connections present in all parts of basidiome.

Habitat, ecology and distribution.

Gregarious or caespitose, growing saprotrophically in forest litter, often under conifers, on the ground, known from subtropical zone of Yunnan, China; from July to October.

Additional specimens examined.

China. Yunnan Province: Dali Bai Autonomous Prefecture, Yunlong Country, Tianchi National Nature Reserve, at 25.850365°N, 99.274236°E, alt. 2509 m, 28 Sep 2019, X. H. Wang 7471 (KUN-HKAS 112561); Kunming City, Fangwang Tree Farm, at 25.063737°N, 102.870690°E, alt. 2262 m, 22 Sep 2011, Z. L. Yang 5586 (KUN-HKAS 71071); Kunming City, Kunming Institute of Botany, at 25.147081°N, 102.748855°E, alt. 1990 m, 24 Aug 2020, Z. L. Yang 6391 (KUN-HKAS 112562); Kunming City, Qiongzhu Temple, at 25.071304°N, 102.630934°E, alt. 1900 m, 28 Jul 2013, T. Guo 779 (KUN-HKAS 81981); Yulong Country, Lashi Village, at 26.883902°N, 100.234594°E, alt. 2655 m, 31 Jul 2011, L. P. Tang 1369 (KUN-HKAS 69830).

Spodocybe bispora

Z. M. He & Zhu L. Yang sp. nov.

EB4F3677-029E-51F4-A106-7B5D99A2D117

839054

Figs 4C, D , 6

Figure 6.

Microscopic features of Spodocybe bispora (KUN-HKAS 73310, holotype) a basidiospores b basidia c pileipellis. Scale bars: 10 μm.

Diagnosis.

Differs from S. rugosiceps in having a nearly smooth pileus, larger basidiospores and 2-spored rather than 4-spored basidia. Differs from C. trulliformis in having a nearly smooth rather than felty-squamulose pileus.

Etymology.

Bispora refers to 2-spored.

Type.

China. Yunnan Province: Baoshan City, Longyang District, Shuizhai Village, at 25.273967°N, 99.306216°E, alt. 2400 m, 12 Aug 2011, J. Qin 324 (KUN-HKAS 73310, holotype).

Description.

Basidiomes small, clitocyboid. Pileus 1.5–3 cm in diam, plano-convex to funnel-shaped; surface dry and nearly smooth, greyish-brown (4B2-3) to grey-brown (4E3-5); center depressed, usually with a low umbo, somewhat darker; margin generally straight and undulating, incurved when old; context thin and white (1A1). Lamellae deeply decurrent, white (1A1) to cream (1A2), thin, 1–2 mm high, relatively crowded, sometimes forked and intervenose. Stipe 1–3 × 0.2–0.4 cm, central, subcylindrical, hollow; surface dry and nearly smooth, concolorous with pileus; context white (1A1).

Basidiospores [60/3/3] (7)7.5–10.5(11.5) × 3–4 μm, Q = (2.05)2.11–3(3.33), Qm = 2.56 ± 0.3, cylindrical, colorless, hyaline, smooth, thin-walled, inamyloid. Basidia 20–30 × 4–5.5 μm, clavate, 2-spored, colorless, hyaline, thin-walled; sterigmata up to 10 μm long; ratio of basidia to basidiospore length less than 5 (about 2–4). Cystidia absent. Lamellar trama subregular, colorless, hyaline, made up of thin-walled cylindrical hyphae with 3–10 µm wide. Pileipellis a cutis, composed of thin-walled cylindrical hyphae 3–11 µm wide. Stipitipellis a cutis, composed of thin-walled cylindrical hyphae 3–10 μm wide. Clamp connections in all parts of basidiomes.

Habitat, ecology and distribution.

Saprophytic, usually gregarious on the ground of coniferous or coniferous and broad-leaved mixed forest, known from Yunnan, China; July to September.

Additional specimens examined.

China. Yunnan Province: Kunming City, Qipan Mountain, at 26.060020°N, 102.576823°E, alt. 1900 m, 25 Jul 2020, Z. M. He 35 (KUN-HKAS 112564); Nujiang City, Lanping Country, No. 311 Provincial Highway, at 26.636613°N, 99.557809°E, alt. 2660 m, 14 Aug 2011, J. Qin 346 (KUN-HKAS 73332).

Discussion

The new genus Spodocybe

In our current study, the new clitocyboid species were clustered into a monophyletic lineage (BP = 100%, PP = 1.00) in the Hygrophoraceae according to the multi-gene phylogenetic analysis (Figs 1, 2). As a result, the new generic name Spodocybe is proposed here to accommodate the new lineage, which is irrelevant to Clitocybeae of the Tricholomatoid clade (Matheny et al. 2006; Alvarado et al. 2015). The three-gene tree of the Hygrophoraceae (Fig. 1) in this study presented basically consistent topological structure with Lodge et al. (2014), and showed that Spodocybe was a sister to Ampulloclitocybe located within the family Hygrophoraceae and further confirmed by a six-gene tree (Fig. 2).

Besides the molecular analyses, morphological data also support its separation from the relative genera. Spodocybe shares clitocyboid basidiomes, decurrent lamellae, inamyloid basidiospores and the presence of clamps with the other genera Ampulloclitocybe, Cuphophyllus and Cantharocybe. However, the genus Ampulloclitocybe, typified by A. clavipes, differs from Spodocybe in having medium-sized basidiomes and bidirectional lamellar trama (Harmaja 2002; Lodge et al. 2014). Afterwards, Cuphophyllus differs from Spodocybe in having long basidia, typically 7−8 (rarely 5−6) times the length of the basidiospores, highly interwoven lamellar trama, rarely subregular (Voitk et al. 2020). Finally, Cantharocybe differs from Spodocybe in having large basidiomes, broad lamellae, cheilo- and caulocystidia, clamps but not on all hyphal septa or at the base of every basidium and more regular lamellar trama (Ovrebo 2011; Hosen et al. 2016). In view of the four genera above with different structures in lamellar trama (Fig. 2), the type of lamellar trama can become a good distinguishing microscopic character for them.

For a long time, C. trulliformis has been placed in the genus Clitocybe based on the clitocyboid feature and habit since 1879 (Karsten 1879). However, C. trulliformis shares many morphological characteristics with Spodocybe, such as the small basidioma with applanate to infundibuliform pileus, grey-brown pileus and stipe, decurrent and whitish lamellae, and smooth and inamyloid basidiospores (Bas et al. 1995). Besides, the ITS phylogenetic analysis in our study (Fig. 3) showed that C. trulliformis and related Clitocybe species were involved in the Spodocybe clade as well, indicating that C. trulliformis and related species should be placed with Spodocybe. In consequence, it is foreseeable that C. trulliformis and other related clitocyboid species will eventually be moved to Spodocybe. Accordingly, more taxonomic work is needed in future.

The placements of Spodocybe, Cuphophyllus, Ampulloclitocybe and Cantharocybe

In previous studies, Cuphophyllus, Ampulloclitocybe and Cantharocybe were treated as basal in Hygrophoraceae (Lodge et al. 2014), but their phylogenetic placements were not resolved. In a six-gene phylogenetic analysis by Binder et al. (2010) and a three-gene analysis by Wang et al. (2018), Ampulloclitocybe and Cantharocybe were located between Cuphophyllus and the rest of the Hygrophoraceae, but without support. While two four-gene analyses by Lodge et al. (2014) showed that Ampulloclitocybe and Cantharocybe were sister clades as basal to Cuphophyllus along with the rest of the Hygrophoraceae with weak support. However, in our six-gene analysis (Fig. 2), the new proposed genus Spodocybe and Ampulloclitocybe were sisters (BP = 78%, PP = 0.99) and they clustered with Cantharocybe followed by Cuphophyllus, forming a supported monophyletic sister clade to the rest of the Hygrophoraceae (BP = 83%, PP = 1.00). Hence, Spodocybe, Ampulloclitocybe, Cantharocybe and Cuphophyllus should be retained in Hygrophoraceae, and a new subfamily, Cuphophylloideae, is proposed to accommodate the lineage.

Citation

He Z-M, Yang ZL (2021) A new clitocyboid genus Spodocybe and a new subfamily Cuphophylloideae in the family Hygrophoraceae (Agaricales). MycoKeys 79: 129–148. https://doi.org/10.3897/mycokeys.79.66302

Funding Statement

Yunnan Ten-Thousand-Talents Plan - Yunling Scholar Project and Postdoctoral Directional Training Foundation of Yunnan Province

Supplementary materials

Supplementary material 1

Alignment of ITS-LSU-RPB2 dataset used in the three-gene phylogenetic analysis

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Zheng-Mi He, Zhu L. Yang

Data type

fasta file

Explanation note

ITS: 1-1380, LSU: 1381–2356, RPB2: 2357–3135.

Supplementary material 2

Alignment of ITS-LSU-RPB1-RPB2-TEF1-ATP6 dataset used in the six-gene phylogenetic analysis

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Zheng-Mi He, Zhu L. Yang

Data type

fasta file

Explanation note

ITS: 1–1217, LSU: 1218–2158, RPB1: 2159–3358, RPB2: 3359–4089, TEF1: 4090–4967, ATP6: 4968–5405

Supplementary material 3

Alignment of ITS dataset used in the single-gene phylogenetic analysis

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Zheng-Mi He, Zhu L. Yang

Data type

fasta file