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Research Article
A new clitocyboid genus Spodocybe and a new subfamily Cuphophylloideae in the family Hygrophoraceae (Agaricales)
expand article infoZheng-Mi He, Zhu L. Yang
‡ Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
Open Access

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.

Keywords

Ampulloclitocybe, Cantharocybe, Cuphophyllus, morphological characters, phylogenetic analysis, taxonomy

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.

MycoBank No: 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.

MycoBank No: 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.

MycoBank No: 839052
Figs 4A, B, 5

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.

Figure 4. 

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

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.

MycoBank No: 839054
Figs 4C, D, 6

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).

Figure 5. 

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

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).

Figure 6. 

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

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.

Acknowledgements

The authors are very grateful to their colleagues at Kunming Institute of Botany, Chinese Academy of Sciences, including Drs. Xiang-Hua Wang, Jiao Qin, Bang Feng, Qi Zhao and Master students Hua Qu and Si-Peng Jian for collecting and providing specimens; and Drs. Gang Wu, Yang-Yang Cui, Qing Cai for providing help on morphological observation and phylogenetic analysis. This study was financed by Yunnan Ten-Thousand-Talents Plan – Yunling Scholar Project and Postdoctoral Directional Training Foundation of Yunnan Province.

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Supplementary materials

Supplementary material 1 

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

Zheng-Mi He, Zhu L. Yang

Data type: fasta file

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

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.
Download file (276.51 kb)
Supplementary material 2 

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

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

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.
Download file (291.14 kb)
Supplementary material 3 

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

Zheng-Mi He, Zhu L. Yang

Data type: fasta file

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.
Download file (25.53 kb)