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Research Article
Three new species of the genus Clavulina (Hydnaceae, Cantharellales) from North China based on morphological and phylogenetic analysis
expand article infoYue Gao‡, Xin Tong§, Hao Zhou‡, Hai-Qi Wang‡, Cheng Li‡, Cheng-Lin Hou‡
‡ Capital Normal University, Beijing, China
§ Department of Life Sciences, National Natural History Museum of China, Beijing, China

Corresponding author: Cheng-Lin Hou ( chenglin_hou@sina.com )

Academic editor: Zai-Wei Ge
© 2024 Yue Gao, Xin Tong, Hao Zhou, Hai-Qi Wang, Cheng Li, Cheng-Lin Hou.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Gao Y, Tong X, Zhou H, Wang H-Q, Li C, Hou C-L (2024) Three new species of the genus Clavulina (Hydnaceae, Cantharellales) from North China based on morphological and phylogenetic analysis. MycoKeys 108: 75-94. https://doi.org/10.3897/mycokeys.108.124004
Open Access

Abstract

Clavulina possesses important ecological and economic value and has attracted extensive attention from mycologists. Macrofungal diversity is high in China, but Clavulina species have not been thoroughly studied. In this study, based on morphological evidence and phylogenetic analyses of the nucleotide sequences of three loci (nrITS, nrLSU, and rpb2), three new species of Clavulina from North China were identified. Morphologically, Clavulina chengdeensis is characterized by its white to dirty white basidiomata with somewhat pale orange tips and somewhat wrinkled hymenium. Clavulina griseoviolacea is characterized by its gray to dark grayish violet basidiomata, with a sometimes-white stipe base, monopodial or irregularly polychotomous toward branch apices. Clavulina pallida is characterized by its white to pale cream white basidiomata with somewhat orange tips. Phylogenetically, the three new species form three independent branches with high support values in the phylogenetic tree.

Key words

Clavarioid fungi, systematics, taxonomy, three new taxa

Introduction

Clavulina J. Schröt. (Hydnaceae, Cantharellales), with Clavulina cristata (Holmsk.) J. Schröt. as a type species, was established in 1888 (Schröter 1888; He et al. 2019). Clavulina is a clavarioid fungi, characterized by clavarioid to coralloid, simple or branched basidiomata with amphigenous hymenia, cylindrical to subclavate basidia with two or more cornuted sterigmata, often with postpartal septa after the release of the basidiospores, and smooth, globose to subglobose basidiospores (Corner 1950, 1970; Petersen 1988a; Thacker and Henkel 2004; Henkel et al. 2005; Olariaga et al. 2009; Henkel et al. 2011; Uehling et al. 2012a). Most Clavulina species are ectomycorrhizal fungi associated with diverse trees (Tedersoo et al. 2003; Tedersoo et al. 2010; Smith et al. 2011; Wazny 2014). Some species are edible or have medicinal value (Dai and Yang 2008; Dai et al. 2010).

The delimitation of Clavulina species is a challenge because they appear to be “simple” in morphology (Donk 1933, 1964; Corner 1950, 1970; Petersen 1988a, 1988b). Molecular phylogenetic analysis based on DNA sequences has improved the identification of species, especially those that cannot be distinguished by morphology alone (Hibbett et al. 1997; Pine and Donoghue 1999; Larsson et al. 2004; Thacker and Henkel 2004; Binder et al. 2005; Moncalvo et al. 2006). Currently, approximately 99 species of Clavulina have been described from temperate and tropical forests worldwide, with nearly half of these species found in the tropics (Wu et al. 2019; Yuan et al. 2020; de Meiras-Ottoni and Gibertoni 2023; Huang et al. 2023; Salas-Lizana et al. 2023). In China, 11 Clavulina species have been reported on the basis of morphological and molecular analyses, most of which are found in subtropical regions, viz. C. bessonii (Pat.) Corner, C. castaneipes (G.F. Atk.) Corner (= C. ornatipes (Peck) Corner), C. coralloides (= C. cristata), C. cinerea, C. rugosa (Teng 1963; Zang et al. 1996; Li et al. 2015), C. livida (He et al. 2016), C. flava, C. purpurascens P. Zhang (Wu et al. 2019), C. baiyunensis X.X. Huang & L.H. Qiu, C. minor X.X. Huang & L.H. Qiu, and C. lilaceorosea X.X. Huang & L.H. Qiu (Huang et al. 2023).

Recently, a number of Clavulina-like samples were collected during an investigation of the Yanshan Mountains in North China, a warm temperate region. Three new species were recognized on the basis of morphological and molecular data. In this paper these new species are described and illustrated. The nuclear ribosomal internal transcribed spacer (nrITS), the large subunit of the nuclear ribosomal RNA (nrLSU), and the RNA polymerase II second largest subunit (rpb2) were sequenced from dried basidiomata.

Materials and methods

Collecting and site description

The specimens were collected from 2017 to 2023 in Beijing, Hebei Province, and Tianjin, North China. These areas have a warm temperate continental monsoon climate and a diverse assortment of plants. The dominant forest types are deciduous broad-leaved forest and mixed coniferous and broad-leaved forest. The dominant trees include Quercus mongolica Fisch. ex Ledeb., Betula platyphylla Suk., Abies nephrolepis (Trautv.) Maxim., Populus tomentosa Carrière, and Pinus tabuliformis Carr. (Wang et al. 2021; Zhou et al. 2022). The annual precipitation is approximately 700 mm. The altitude ranges from 200 to 2200 m. The collected specimens were dehydrated using an electric dryer (Dorrex) at 50 °C and kept in the Herbarium of the College of Life Science, Capital Normal University, Beijing, China (BJTC).

Morphological observation

Macroscopic characteristics were documented from dried specimens and photographs, while thin sections of specimens mounted in 3% potassium hydroxide (KOH) or sterilized water were analyzed for microscopic features. The morphology and dimensions of their microscopic structures were observed and recorded using a light microscope [Olympus DP71, Tokyo, Japan]. In the description of basidiospores, the abbreviation n/m/p means that n basidiospores were measured from m basidomata of p collections. The measurements and Q values were presented in the form of (a)b–c (d), in which “b-c” contained a minimum of 90% of the measured values, and extreme values (a and d) were given in parentheses. Q represents the ratio of the length to width of basidiospores, and Qm represents the average Q value of all basidiospores measured ± the sample standard deviation (Huang et al. 2023). The nomenclatural details were submitted to MycoBank. Color designation was referred to the website colorhexa (https://www.colorhexa.com).

DNA extraction, PCR amplification and sequencing

DNA was extracted with an M5 Plant Genomic DNA Kit [Mei5 Biotechnology, Co., Ltd., China]. The obtained DNA was dissolved in 1 × TE buffer and stored at –20 °C for later use. PCRs were performed in a Bio-Rad S1000TM Thermal Cycler [Bio-Rad Laboratories, Inc., USA]. The primer set ITS1f/ITS4 (White et al. 1990) was used to amplify the nrITS region. The primer set LR5/LR0R (Vilgalys and Hester 1990) was used to amplify the nrLSU region. The primer sets 96F/938R (Uehling et al. 2012a) and RPB2-6F/fRPB2-7cR (Liu et al. 1999) were used to amplify the rpb2 region. PCRs were conducted in a 25 μL reaction volume containing 2 μL of DNA template, 1 μL of each primer (10 μM), 8.5 μL of deionized water and 12.5 μL of 2 × Master Mix [Mei5 Biotechnology, Co., Ltd., China]. The PCR amplification conditions for nrITS and nrLSU refer to Li et al. (2020) and Sui et al. (2023). The PCR amplification conditions for rpb2 refers to Salas-Lizana et al. (2023) and Huang et al. (2023). All the DNA sequences were sequenced by Sangon Biotech Co., Ltd. (Shanghai).

Molecular phylogenetic analyses

The newly obtained sequences from this study were submitted to NCBI (https://www.ncbi.nlm.nih.gov). The nrITS, nrLSU, and rpb2 sequences of the concatenated nrITS-nrLSU-rpb2 datasets were aligned with selected sequences from GenBank and previous studies. All sequences are listed in Table 1. The generated raw reads of the DNA sequences were used to obtain consensus sequences using SeqMan v.7.1.0 in the DNASTAR Lasergene Core Suite software (DNASTAR Inc., Madison, WI, USA). All sequences were aligned using MAFFT v.6 (Katoh and Toh 2010) and trimmed manually with MEGA 6 (Tamura et al. 2013). For phylogenetic analyses, newly obtained sequences and additional reference sequences of Clavulina species were included in the dataset of the combined nrITS-nrLSU-rpb2 fragment (Table 1), with Hydnum repandum L. and Hydnum rufescens Pers. as the outgroups following He et al. (2016).

Table 1.
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Specimens used in phylogenetic analysis and their GenBank accession numbers. The newly generated sequences are shown in bold.

Taxonomy Location Voucher GenBank Number
nrITS nrLSU rpb2
Clavulina alba Brazil AMO869 ON502612 – –
C. alba Brazil AMO868 ON502611 MZ484637 –
C. alba Brazil AMO867 ON502610 MZ484636 OQ305446
C. alpina Italy AMB n. 17156 (T) MH456956 MH457104 –
C. alpina Italy AMB n. 17157 MH456957 MH457105 –
C. amazonensis Guyana TH8742 – HQ680361 JN228249
C. amazonensis Colombia AMV1847 KT724111 KT724124 –
C. amazonensis Brazil AMO1143 ON502605 – –
C. amethystina Germany MTH2 MN959776 – –
C. arboreiparva Mexico FCME27282 (T) NR_185406 MT903233 MK519629
C. arboreiparva Mexico MEXU 28239 MK547192 MT903234 –
C. baiyunensis China B21062720 OP738992 OP737358 –
C. baiyunensis China B21051526 OP738990 OP737357 OP745528
C. baiyunensis China B21062712 (T) OP738991 OP737359 OP745529
C. brunneocinerea New Zealand TN42667 – JN228220 –
C. caespitosa Guyana BRG:TH8709 (T) NR_119560 DQ056370 JN228234
C. castaneipes USA OSC 116725 EU669210 EU669262 –
C. castaneipes Costa Rica TENN056432 JX287357 – –
C. castaneipes USA OSC 108705 EU669209 EU669261 –
C. cerebriformis Guyana BRG:MCA4022 (T) NR_121504 JN228222 JN228233
C. cf. amethystina Norway PRM 896664 EU862203 – –
C. cf. amethystina Norway O 62152 EU862204 – –
C. cf. amethystina USA SE-2015 KT275670 – –
C. cf. cinerea China MES427 – JN228226 JN228239
C. cf. cinerea Canada UBC:F29630 MZ868607 – –
C. cf. cinerea Canada UBC:F29600 MZ868605 – –
C. cf. cinerea Spain BIO 10304 EU862226 – –
C. cf. cinerea Spain BIO 10294 EU862225 – –
C. cf. connata Guyana TH 9586 JN247429 – JN228247
C. cf. cristata China MES426 JN228225 JN228225 JN228240
C. cf. rugosa China MHHNU 9234 MK564142 MK564132 MK564152
C. chengdeensis China BJTC TX646 (T) PP835331 PP835344 PP889517
C. chengdeensis China BJTC 0249 PP835325 PP835339 PP889513
C. chengdeensis China BJTC ZH1205 PP835335 PP835348 PP889521
C. chengdeensis China BJTC ZH1225 PP835336 PP835349 PP889522
C. cinerea USA iNat62500762 ON479751 – –
C. cinerea USA ECV4030 MG663298 MF797670 –
C. cinerea USA JKU9 JN228228 – JN228242
C. cinerea Denmark JV01-158 AJ889937 AJ889937 –
C. cinerea Finland KHL 11694 (GB) EU118616 – –
C. cinerea USA RAS494 OR471189 OR470998 OR474029
C. cinereoglebosa Guyana TH8561 (T) NR_119975 JN228232 JN228246
C. cirrhata Guyana TH8940 JQ677059 JQ677045 J0677046
C. cirrhata Guyana TH8754 JQ677050 JQ677050 –
C. cirrhata Guyana TH9266 JQ677061 – –
C. cirrhata Guyana TH9207 JQ677062 – –
C. coralloides USA iNat61787378 ON479748 – –
C. coralloides – HBAU15770 MW850398 – –
C. coralloides China 110116MFBPL0405 MW554410 – –
C. coralloides China HKAS122411 ON794279 – –
C. craterelloides Colombia AMV1401 – KT724127 –
C. craterelloides Guyana TH8234 (T) JQ911749 AY391718 –
C. cristata USA JKU8 JN228227 JN228227 JN228241
C. cristata Spain BIO 9641 EU862228 – –
C. cristata Spain BIO 10291 EU862223 – –
C. cristata Finland EL 6/00 (GB) KF218965 KF218965 –
C. cristata Sweden GB/EL95-97 AY463398 AY586648 –
C. cristata USA DUKE9312 – JN228215 JN228250
C. cristata USA RAS323 SV1 OR464379 OR460871 –
C. cristata USA RAS323 SV2 OR464380 OR460872 –
C. crystallifera Brazil AMO825 – MZ484638 –
C. crystallifera Brazil URM 95027 (T) – MZ484639 OQ305455
C. crystallifera Brazil URM 95029 – MZ484641 OQ305456
C. cystidiata Brazil URM 95030 NR_185725 MZ484642 OQ305449
C. dicymbetorum Guyana TH8730 (T) DQ056364 DQ056369 –
C. effusa Guyana TH9193 (T) – JN228230 JN228245
C. effusa Colombia AMV1837 KT724116 KT724129 –
C. effusa Guyana TH8511 JN228231 – –
C. etruriae Italy AMB n.17158 MH456958 – –
C. flava China MHHNU 9811 MK564138 MK564128 MK564148
C. flava China MHHNU9825 (T) NR_185562 MK564129 MK564149
C. floridana Mexico MEXU:28240 MK547187 MT903230 MK519625
C. floridana Mexico UNAM:FCME27277 MK547188 MT903229 MK519626
C. floridana USA Franck 4420 (T) MT894294 MT894296 –
C. griseoviolacea China BJTC ZH0998 (T) PP835334 PP835347 PP889520
C. griseoviolacea China BJTC ZH1653 PP835338 PP835352 PP889524
C. grisea Brazil URM 89966 KX811198 KX811193 –
C. grisea Brazil URM 89967 (T) KX811199 KX811194 –
C. grisea Brazil URM 89968 KX811200 KX811195 –
C. guyanensis Guyana TH9257 JQ677057 – –
C. guyanensis Guyana TH9245 (T) NR_120085 – JQ677049
C. humicola Guyana TH8737 (T) DQ056368 DQ056367 JN228244
C. incrustata Brazil AMO1300 ON502606 MZ484631 OQ305439
C. incrustata Brazil AMO800B – MZ484626 OQ305440
C. incrustata Brazil AMO802 – MZ484628 OQ305441
C. iris var. iris Cyprus ML5135C1 MN028412 MN028396 –
C. iris var. iris China QHU20388 OM970954 – –
C. iris var. occidentalis France PAM11112702 MN028408 – –
C. iris var. occidentalis France PAM12112740 MN028409 – –
C. junduensis Brazil ANMF 766 MZ092866 – –
C. kunmudlutsa Guyana MCA3117 – HQ680362 –
C. lilaceorosea China B22052918 OP738996 OP737363 OP745532
C. lilaceorosea China B21082106 OP738995 OP737362 OP745533
C. livida China MCCNNU00959 KU219603 – –
C. livida China MCCNNU00960 (T) KU219605 KU219602 –
C. mahiscolorata Mexico FCME 27660 MH542551 MN049493 –
C. mahiscolorata Mexico FCME 27665 MH542553 – –
C. mahiscolorata Mexico FCME 27662 (T) MH542554 MN049496 MN053719
C. minor China B21081646 (T) OP738993 OP737360 OP745530
C. minor China B22082717 OR149156 OR145333 OR166807
C. monodiminutiva Guyana TH8738 (T) NR_119559 DQ056372 JN228237
C. nigricans Guyana TH8284 (T) JN228224 AY391719 JN228238
C. nigricans Guyana G200 KJ786649 KJ786553 –
C. ornatipes USA iNAT:57799374 MW031157 – –
C. ornatipes USA TH9598 – JN228229 JN228243
C. ossea Brazil AMO1110 – MZ484634 OQ305444
C. ossea Brazil AMO1108 ON502607 MZ484633 OQ305443
C. pakaraimensis Guyana TH9194 (T) NR_121533 JQ677051 JQ677047
C. pakaraimensis Guyana TH9212 JQ677053 – –
C. pallida China BJTC C669 (T) PP835329 PP835342 PP889515
C. pallida China BJTC C229 PP835327 PP835341 PP889514
C. pallida China BJTC S062 PP835330 PP835343 PP889516
C. pallida China BJTC ZH0056 PP835332 PP835345 PP889518
C. pallida China BJTC ZH0810 PP835333 PP835346 PP889519
C. pallida China BJTC ZH1055 PP835337 PP835350 PP889523
C. paraincrustata Brazil URM 89969 (T) – KX811196
C. paraincrustata Brazil AMO419 KX811201 – –
C. parvispora Mexico FCME 27650 (T) MH542550 MN049492 MN053718
C. parvispora Mexico FCME 27657 MH542549 MN049491 –
C. perplexa Italy AMB 19286 OR094916 OR095022 –
C. perplexa Italy AMB 19287 OR094915 OR095021 –
C. purpurascens China MHHNU 9846 MK564136 MK564126 MK564146
C. purpurascens China MHHNU 9848 (T) MK564137 MK564127 MK564147
C. reae Mexico FCME 27623 MH542526 MN049487 MN053717
C. reae Italy AMB n. 17159 MH456959 MH457106 –
C. reae Mexico FCME 27629 MH542525 MN049488 –
C. rosiramea Guyana TH8954 (T) JQ677064 JQ677044 JQ677048
C. rugosa Tunisia H21587 KU973837 – –
C. rugosa Spain BIO 10293 EU862220 – –
C. rugosa Spain BIO 10300 EU862217 – –
C. rugosa Spain BIO 11162 EU862229 – –
C. rugosa Italy AMB 19288 OR641046 OR641045 –
C. rugosa USA RAS487 SV1 OR464375 OR460869 OR473737
C. rugosa USA RAS327 SV2 OR464364 OR460870 –
C. rugosa USA RAS327 SV1 OR464365 – OR473740
C. samuelsii USA TENN065723 JQ638712 – –
C. samuelsii New Zealand PDD:89881 GU222317 – –
C. simplex Brazil URM 95031 (T) NR_185726 MZ484643 OQ305452
C. simplex Brazil URM 95032 – MZ484644 –
C. sphaeropedunculata Mexico FCME 27661 MH542560 MK253716 –
C. sphaeropedunculata Mexico MEXU 28222 (T) MH542557 MK253717 –
C. sprucei Guyana TH9122 HQ680355 JN228223 JN228236
C. sprucei Guyana TH9120 HQ680353 – –
C. sprucei Guyana MCA3989 HQ680352 – JN228235
C. studerae Brazil URM 95036 – MZ484648 OQ305454
C. studerae Brazil URM 95033 (T) NR_185727 MZ484645 OQ305453
C. subrugosa USA TENN043395 JQ638711 – –
C. subrugosa New Zealand TN43395 JN228221 – –
C. tepurumenga Guyana MCA3116 – HQ680363 JN228248
C. terminalis Brazil AMO1109 – MZ484649 OQ305450
C. terminalis Brazil AMO1122 – MZ484651 OQ305451
C. thindii China dcy2288 MZ157027 – –
C. thindii India US_1428 MG892054 – –
C. tropica China ZP3327 ON898016 – –
C. tropica China MHHNU 9827 ON954848 – –
C. tuxtlasana Mexico MEXU 28242 MK547194 MT903236 MK519631
C. tuxtlasana Mexico MEXU 28243 MK547195 MT903237 MK519632
C. tuxtlasana Mexico UNAM:FCME27279 (T) NR_185407 MT903238 MK519633
Hydnum repandum Slovenia 031209A KU612574 KU612655 –
H. rufescens Slovenia LJU GIS 1332 AJ547868 – –
H. rufescens China HKAS82529 – KU612657 –

To estimate Maximum Likelihood (ML) phylogenetic trees, we utilized RAxML 7.4.2 Black Box software (Stamatakis 2006; Stamatakis et al. 2008; Zhou and Hou 2019; Zhou et al. 2021) with a GTRGAMMAI site substitution model (Guindon et al. 2010). Branch support was calculated with a bootstrapping (BS) method of 1000 replicates (Hillis and Bull 1993). Bayesian Inference (BI) analysis was conducted using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) with a Markov chain Monte Carlo (MCMC) algorithm (Rannala and Yang 1996). The best model was estimated using MrModeltest 2.3 (Zhou and Hou 2019; Zhou et al. 2021, 2022). The models employed for each marker of the nrITS-nrLSU-rpb2 dataset were HKY + G for nrITS, GTR + I + G for nrLSU, and HKY + I + G for rpb2. We ran two MCMC chains for 100,000,000 generations, stopping when the average standard deviation of split frequencies dropped below 0.01. Trees were saved every 1000 generations, and the initial 25% of trees were discarded as the burn-in phase for each analysis. Significant Bayesian posterior probabilities were calculated for branches in the remaining trees, as the analysis yielded relatively stable topologies, and clades with high Bayesian posterior probability (pp) values reflected the relative relationships between species (Posada and Crandall 1998).

Results

Molecular phylogeny

A total of 36 sequences, including 12 for nrITS, 12 for nrLSU and 12 for rpb2, were generated in this study. The nrITS-nrLSU-rpb2 dataset included 320 sequences (140 for nrITS, 109 for nrLSU, and 71 for rpb2), with 161 samples. The concatenated alignment contained 2188 characters, including gaps. ML and Bayesian analyses resulted in highly similar estimates of tree topologies; thus, only the tree inferred from the ML analysis is shown (Fig. 1).

Figure 1. 

Phylogenetic tree generated from a ML analysis based on combined nrITS-nrLSU-rpb2 sequences. Numbers representing Maximum Likelihood bootstrap support (MLBS ≥ 75%, right) and significant Bayesian posterior probability (BPP ≥ 0.95, left) are indicated above the nodes. Novel sequences are printed in bold. Voucher specimens and localities where the specimens were collected are provided behind the species names.

Our tree topology is similar to that of de Meiras-Ottoni and Gibertoni (2023). On the basis of our analyses, three highly supported monophyletic lineages were identified in our Clavulina samples (Fig. 1). Morphological examinations revealed that these species are morphologically different from other known species of this genus. The three clades therefore can be recognized as three new species and described in this paper, i.e., Clavulina chengdeensis sp. nov., Clavulina griseoviolacea sp. nov., and Clavulina pallida sp. nov. Moreover, Clavulina griseoviolacea (BJTC ZH0998 and BJTC ZH1653) further clustered a clade with Clavulina iris var. iris Loizides, Bellanger & P.-A. Moreau, Clavulina iris var. occidentalis Bellanger, P.-A. Moreau & Loizides, and Clavulina cinerea (MLB = 100%, BPP = 1.00). Clavulina pallida (BJTC C669, BJTC C229, BJTC S062, BJTC ZH0056, BJTC ZH0810, and BJTC ZH1055) formed one clade (MLB = 100%, BPP = 1.00) closely related to Clavulina rugosa, Clavulina mahiscolorata E. Pérez-Pazos & Villegas, and Clavulina etruriae Franchi & M. Marchetti (MLB = 100%, BPP = 1.00) (Fig. 1). The sequences of Clavulina chengdeensis (BJTC TX646, BJTC 0249, BJTC ZH1205, and BJTC 1225) formed a branch with a high support value (MLB = 100%, BPP = 1.00) and further clustered into a clade with Clavulina rugosa and Clavulina cf. cinerea (Bull.) J. Schröt.), which was well supported (MLB = 93%, BPP = 1.00).

Taxonomy

Clavulina chengdeensis Yue Gao, Hao Zhou, & C.L. Hou, sp. nov.

MycoBank No: MycoBank No: 853047
Figs 2A–C, 3A–C, 4

Diagnosis

Clavulina chengdeensis differs from known Clavulina species in its white to dirty white basidiomata with somewhat pale orange tips, somewhat wrinkled hymenium, basidiospores 6.1–9.6 × 5.6–7.9 μm, basidia 40.1–58.7 × 4.5–7.0 μm, postpartal septa present, and clamp connections present.

Figure 2. 

Morphology of Basidiomata A–C Clavulina chengdeensis (A BJTC TX646 holotype B BJTC ZH1205 C BJTC ZH1225) D–E Clavulina griseoviolacea (D BJTC ZH0998 holotype E BJTC ZH1653) F–I Clavulina pallida (F BJTC C669 holotype G BJTC S062 H BJTC ZH1055 I BJTC C229). Scale bars: 1 cm.

Etymology

The epithet “chengdeensis” refers to the specimens collected from Chengde city.

Type

China • Hebei Province, Chengde city, Xiaolabagou; 40°57'24"N, 116°27'12"E, alt. 1240 m; 13 Sep. 2023; H. Zhou, Y. Gao & X. Tong (BJTC TX646); GenBank nrITS: PP835331, nrLSU: PP835344, rpb2: PP889517.

Figure 3. 

Morphology of Basidiomata A–C Clavulina chengdeensis (A BJTC TX646 holotype B BJTC ZH1205 C BJTC 0249) D Clavulina griseoviolacea (D BJTC ZH0998 holotype) E–G Clavulina pallida (E BJTC C669 holotype F BJTC S062 G BJTC ZH0056). Scale bars: 1 cm.

Description

Basidiomata coralloid, solitary or gregarious in cespitose clusters; clusters 12–24 mm tall, 10–26 mm wide across branches and forming 3–5 ranks in multiple planes; individual basidiomata 14–40 mm tall, 4–11 mm wide, simple or sparsely branched one time, branching pattern polychotomous to dichotomous ascending, branches subterete or subclavate and somewhat flattened with age, rough, branch tips rounded when young, gradually become pointed with age; white (#ffffff) to dirty white (#fff9eb) when fresh and somewhat pale orange (#ffeab8), cream white (#fff2d2) to light grayish orange (#f6e4d0) when dry. Stipes generally distinct, 5–15 mm long, 1.5–4 mm wide, subcylindrical or flattened, concolor with branches. Hymenium amphigenous, somewhat wrinkled.

Figure 4. 

Microscopic characteristics of Clavulina chengdeensis A basidiospores B basidia C tramal hyphae. Scale bars: 10 µm.

Basidiospores [87/9/4] (5.7−) 6.1–9.6 (−11.0) × 5.6–7.9 (−8.5) μm, Q = 1.00–1.25 (−1.31), Qm = 1.04 ± 0.02, globose to subglobose, smooth, hyaline in H2O and KOH, thin-walled, inamyloid, with a 0.6–1.0 μm irregular hilar appendix, and one large oleiferous guttule. Basidia (34.7–) 40.1–58.7 (−62.3) × 4.5–7.0 (−8.3) μm, clavate to subcylindrical, tapering from apex to base; postpartal septa present in most basidia, occurring 11–30 μm below basidia tips; two sterigmata occur per basidium, 5.1–7.5 μm long, and cornute. Basidioles abundant, subclavate to subcylindrical. Tramal hyphae in stipe smooth, with slightly thickened walls, hyaline in KOH, 4.0–7.7 μm wide, sometimes inflated to 9.3 μm wide; tramal hyphae in branches hyaline, thin-walled, sometimes inflated tramal hyphae 3.7–8.5 (−10.4) μm wide; clamp connections abundant. Hyphal system monomitic. Cystidia absent.

Habit, habitat, and distribution

Solitary or gregarious caespitose in humus layers on soils in broad-leaved deciduous forests associated with Castanea Mill., Betula L., and Platanus L. Basidiomata generally occur from July to September; currently known from Hebei Province and Beijing, China.

Additional specimens examined

China • Beijing, Yanqing District, Songshan National Nature Reserve; 40°30'N, 115°48'E, alt. 652 m; 16 Sep. 2017; C.L. Hou, H. Zhou, J.Q. Li (BJTC 0249); China• Beijing, Huairou District, Labagoumen Na­ture Reserve; 40°39'24"N, 116°27'14"E, alt. 1225 m; 25 Aug. 2021; H. Zhou, X.Y. Shen, X.B. Huang (BJTC ZH1205); same location• 40°57'17"N, 116°27'3"E, alt. 1308 m; 25 Aug. 2021; H. Zhou, X.Y. Shen, X.B. Huang (BJTC ZH1225).

Note

Phylogenetically, C. chengdeensis was related to C. rugosa in our analyses (Fig. 1). However, C. rugosa has longitudinally rugulose basidiomata, relatively large basidiospores (9–14 × 8–12 µm), basidia (40–85 × 6.9–5 µm), and sterigmata 6–9 µm long (Corner 1950; Olariaga et al. 2009). Morphologically, C. chengdeensis is close to C. mahiscolorata. Clavulina mahiscolorata generally shares similar colors with basidiomata, but its basidiomata turn maize yellow to mandarin orange when dry and the branch surface is smooth (Pérez-Pazos et al. 2019).

Clavulina griseoviolacea Yue Gao, Hao Zhou, & C.L. Hou, sp. nov.

MycoBank No: MycoBank No: 853050
Figs 2D–E, 3D, 5

Diagnosis

Clavulina griseoviolacea differs from known Clavulina species in its gray to dark grayish violet basidiomata, with a white stipe, monopodial or irregularly polychotomous branches toward branch apices, basidiospores 6.5–8.0 × 6.2–7.2 μm, basidia 31.3–49.8 × 4.5–7.2 μm, postpartal septa present, and clamp connections present.

Etymology

The epithet “griseoviolacea” refers to the basidiomata being gray to dark grayish violet.

Type

China • Tianjin, Jizhou District, Jiulongshan; 40°8'51"N, 117°30'36"E, alt. 170 m; 21 Aug. 2022; H. Zhou, X.Y. Shen & X.B. Huang (BJTC ZH0998); Gen­Bank nrITS: PP835334, nrLSU: PP835347, rpb2: PP889520.

Figure 5. 

Microscopic characteristics of Clavulina griseoviolacea A basidiospores B basidia C tramal hyphae. Scale bars: 10 µm.

Description

Basidiomata coralloid, solitary or scattered; individual basidiomata 25–45 mm tall, 12–20 mm wide across branches, basidiomata sparsely branched two to three times, monopodial or irregularly polychotomous toward branch apices and dichotomous at the base, branches subclavated to fattened and somewhat flattened with age, rough, with rounded tips; gray (#a5a5a5) to dark grayish violet (#9b92a6) when fresh and tips somewhat very dark brown (#1f1605), dark brown (#4c350b) when dry. Stipes generally distinct, 10–20 mm long, 2–5 mm wide, subcylindrical or flattened, dark gray (#777777) to dark grayish purple (#7a747f) and sometimes with a white base (#ffffff). Hymenium amphigenous, generally nodulosus and farinaceous.

Basidiospores [65/2/1] 6.5–8.0 (−8.6) × (5.9−) 6.2–7.2 (−7.9) μm, Q = 1.00–1.23 (−1.27), Qm = 1.12 ± 0.06, globose to subglobose, smooth, hyaline in H2O and KOH, thin-walled, inamyloid, with a 0.6–0.9 μm irregular hilar appendix, and one large oleiferous guttule. Basidia (29.3–) 31.3–49.8 (−65.3) × 4.5–7.2 (−8.3) μm, clavate to subcylindrical, tapering from apex to base; postpartal septa present in most basidia, occurring 12–24 μm below basidia tips; two sterigmata occur per basidium, 3.8–6.7 μm long, and cornute. Basidioles abundant, subclavate to subcylindrical. Tramal hyphae in stipe smooth, with slightly thickened walls, hyaline in KOH, 2.6–5.5 μm wide, some tramal hyphae inflated; tramal hyphae in branch hyaline, thin-walled, 3.7–6.1 (−8.9) μm wide; clamp connections abundant. Hyphal system monomitic. Cystidia absent.

Habit, habitat, and distribution

Solitary or scattered humus layers on soils under Theropencedrymion, associated with Pinus L. Basidiomata generally occurring from July to August; currently known from Tianjin and Beijing, China.

Additional specimens examined

China • Beijing, Mentougou District, Baihua Mountain; 39°47'50"N, 115°33'35"E, alt. 1,223 m; 16 Aug. 2023; H. Zhou, Y. Gao & X. Tong (BJTC ZH1653).

Notes

C. griseoviolacea is phylogenetically closely related to Clavulina cinerea, Clavulina iris var. occidentalis and Clavulina iris var. iris according to phylogenetic analyses (Fig. 1.), but Clavulina cinerea has lilac-gray to gray basidiomata, larger basidiospores (7–10 × 6–8 µm) and basidia (38–65 µm long up to 7 µm wide) (Burt 1922); Clavulina iris Loizides, Bellanger & P.-A. Moreau can be distinguished by its white-pruinose basidiomata, branches sometimes partially or extensively fused, larger basidiospores (9.2–10.4 × 6.5–8.5 µm) and basidia (45–80 × 6–9 µm), and 7–9 µm wide hyphal ends (pseudocystidia) (Crous et al. 2019). C. griseoviolacea is morphologically similar to Clavulina tuxtlasana M. Villegas, Garibay-Orijel & Pérez-Pazos in the color of basidiomata, but C. tuxtlasana basidiomata are simple, rarely branching dichotomously, branches with slight longitudinal wrinkles, smaller basidiospores (6–7.5 × 5.5–7 µm), and lacking clamp connections (Salas-Lizana et al. 2023). Clavulina crystallifera Meiras-Ottoni also has similar branching, but acerose crystals are present in the medullary hyphae of its branches (de Meiras-Ottoni and Gibertoni 2023).

Clavulina pallida Yue Gao, Hao Zhou, & C.L. Hou, sp. nov.

MycoBank No: MycoBank No: 853052
Figs 2F–I, 3E–G, 6

Diagnosis

Clavulina pallida differs from known Clavulina species in its white to pale cream white basidiomata, with somewhat orange tips, basidiospores 7.0–9.7 × 6.4–8.6 μm, basidia 34.2–48.5 × 4.8–6.3 μm, postpartal septa present, and clamp connections present.

Figure 6. 

Microscopic characteristics of Clavulina pallida A basidiospores B basidia C tramal hyphae. Scale bars: 10 µm.

Etymology

The epithet “pallida” refers to the basidiomata being pale white.

Type

China • Beijing, Miyun District, Heilongtan; 40°35'30"N, 116°46'20"E, alt. 397 m; 27 Aug. 2021; C.L. Hou, G.Q. Cheng, R.T. Zhang (BJTC C669); GenBank nrITS: PP835329, nrLSU: PP835342, rpb2: PP889515.

Description

Basidiomata coralloid, solitary or scattered; individual basidiomata 17–38 mm tall, 1.8–10 mm wide across branches, simple or sparsely branched one to two times, monopodial or irregularly dichotomous toward branch apices and monopodial or dichotomous at the base, branches subterete or subclavate to fattened and somewhat flattened with age, smooth or rough, branch tips are rounded when young, gradually become pointed with age; white (#ffffff) to pale cream white (#fffaef) when fresh and tips somewhat orange (#ff9000), brownish orange (#cd8400) when dry. Stipes generally distinct, 7–14 mm long, 1.5–4 mm wide, subcylindrical or flattened, white (#ffffff) to pale cream white (#fffaef). Hymenium amphigenous, somewhat wrinkled.

Basidiospores [131/10/6] (6.6−) 7.0–9.7 (−10.1) × (6.0−) 6.4–8.6 (−10.1) μm, Q = 1.00–1.24 (−1.29), Qm = 1.10 ± 0.06, globose to subglobose, smooth, hyaline in H2O and KOH, thin-walled, inamyloid, with a 0.6–1.1 μm irregular hilar appendix, and one large oleiferous guttule. Basidia (31.8–) 34.2–48.5 (−55.3) × 4.8–6.3 (−7.6) μm, clavate to subcylindrical, tapering from apex to base; postpartal septa present in most basidia, which occurred 10–22 μm below basidia tips; two sterigmata occur per basidium, 3.7–6.2 μm long, and cornute. Basidioles abundant, subclavate to subcylindrical. Tramal hyphae in stipe smooth, thin walled, hyaline in KOH, 3.3–6.2 μm wide, not inflated; tramal hyphae in branch hyaline and thin-walled, 2.8–6.7 (−9.0) μm wide; clamp connections abundant. Hyphal system monomitic. Cystidia absent.

Habit, habitat, and distribution

Solitary or scattered in humus layers on soils in broad-leaved deciduous forests associated with Carpinus L. and Castanea Mill. Basidiomata generally occur from July to August and are currently known from Beijing and Hebei Province, China.

Additional specimens examined

China • Beijing, Pinggu District, Zhenluo Mountain Villa; 40°20'24"N, 117°8'57"E, alt. 294 m; 20 Aug. 2021; C.L. Hou, G.Q. Cheng, R.T. Zhang (BJTC C229); China • Beijing, Yanqing District, Heibei village; 40°25'48"N, 116°14'44"E, alt. 456 m; 11 Aug. 2018; X.Y. Shen, B.D. He, K.B. Huang (BJTC S062); China • Beijing, Changping District, Yanshou Tem­ple; 40°22'24"N, 116°19'22"E, alt. 276 m; 14 Aug. 2019; C.L. Hou, H. Zhou, G.Q. Cheng (BJTC ZH0056); same location • 40°22'6"N, 116°19'19"E, alt. 215 m; 17 Aug. 2021; H. Zhou, X.Y. Shen, X.B. Huang (BJTC ZH0810); China • Hebei Prov­ince, Zunhua City, Wanfo Garden; 40°11'34"N, 116°37'1"E, alt. 144 m; 22 Aug. 2021; H. Zhou, X.Y. Shen, X.B. Huang (BJTC ZH1055).

Notes

In the phylogenetic analyses (Fig. 1.), C. pallida is phylogenetically close to Clavulina mahiscolorata. C. mahiscolorata also has white basidiomata, but turns orange when dried, larger basidia (56–74 × 6–8 µm) are present (Pérez-Pazos et al. 2019). As described in the current record of the genus Clavulina, in terms of macroscopic features, Clavulina rugosa is closely related to C. pallida, with simple or sparsely branched basidiomata. However, C. rugosa differs from C. pallida in that it has longitudinally wrinkled surfaces of basidiomata and relatively large basidiospores (9–14 × 8–12 µm) (Corner 1950; Olariaga et al. 2009). Clavulina livida generally shares similar simple basidiomata with C. pallida, but the former has grayish olive to dark grayish olive basidiomata, branch tips becoming pale pinkish cinnamon to chestnut with age, larger basidiospores (11.6–12.9 × 10.7–12.5 µm) and basidia (51.5–76.7 × 7.0–12.3 µm) (He et al. 2016). Clavulina caespitosa T.W. Henkel, Meszaros & Aime differs from C. pallida in its simple basidomata in cespitose clusters, free or slightly fused basally, apex sharply acuminate, larger basidiospores (8.5–10.5 × 7–9.5 µm) and basidia (81–98 µm long, apex 6.3–7.5 µm, at base 3.5–5 µm), and postpartal septa not observed (Henkel 2005).

Discussion

In early studies on Clavulina, only brief records of the macroscopic and microscopic features of the species were made. This resulted in difficulties in distinguishing between similar genera or species and led to the occurrence of many synonymous names (Hibbett et al. 1997; Binder et al. 2005; Moncalvo et al. 2006; Larsson 2007; Uehling et al. 2012b). For example, Clavulina paraincrustata Meiras-Ottoni & Gibertoni, described only by morphology from the Brazilian Atlantic rainforest (Tibpromma et al. 2017), is treated as a synonym of C. incrustata. Wartchow found the same region (Wartchow 2012) on the basis of molecular evidence (de Meiras-Ottoni and Gibertoni 2023).

Previous studies have indicated that Clavarioid fungi, including Clavulina, are highly diverse and distributed worldwide (Corner 1950). However, to date, only 11 species of Clavulina have been described in China. China has a vast territory, complex climate, habitat types, and abundant species resources, with extremely high fungal diversity (Mcneely et al. 1990). The lack of records in this case may be due to the limited number of collections and collection areas. In this study, three new species of Clavulina were described on the basis of samples collected from North China, by means of both nrITS-nrLSU-rpb2 three-locus phylogenetic analyses (Fig. 1) and macrofungal morphological examinations, which increase understanding of the species diversity of this genus. The natural growth of Clavulina may be related to precipitation. However, the investigations and specimen collection in this study were carried out in the rainy season from August to September, with no collection in other periods. Therefore, more Clavulina species were likely to be present in the study area.

Acknowledgments

Special thanks are due to Dr. Li Fan and Dr. Qian Chen for their suggestions on the manuscript.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This study was financed by the National Natural Science Foundation of China (No. 32270012) and the Biodiversity Survey and Assessment Project of the Ministry of Ecology and Environment, China (2019HJ2096001006) and BJAST Budding Talent Program (23CE-BGS-06).

Author contributions

All authors have contributed equally.

Author ORCIDs

Yue Gao https://orcid.org/0009-0001-0831-3656

Xin Tong https://orcid.org/0000-0002-2091-6636

Hao Zhou https://orcid.org/0000-0002-4869-2187

Hai-Qi Wang https://orcid.org/0009-0004-3388-6263

Cheng Li https://orcid.org/0009-0005-0958-0456

Cheng-Lin Hou https://orcid.org/0000-0001-8162-5560

Data availability

All of the data that support the findings of this study are available in the main text.

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1Yue Gao and Xin Tong contributed equally to this work.
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