Research Article
Print
Research Article
Morphological and multi-locus phylogenetic analyses reveal three new branched species of Clavaria (Clavariaceae, Agaricales) from China
expand article infoJun Yan‡§, Li Xiong‡, Li-Xun Yang|, Zheng-Mi He§, Ping Zhang§, Ke Liao‡
‡ Hunan Provincial Demonstration Center of Forestry Seeding Breeding, Changsha, China
§ Hunan Normal University, Changsha, China
| Bureau of Forestry, Tongdao Dong Autonomous County, Huaihua, China

Corresponding author: Ping Zhang ( zhangping0000@163.net )

Corresponding author: Ke Liao ( hncslk@126.com )

Academic editor: Bao-Kai Cui
© 2025 Jun Yan, Li Xiong, Li-Xun Yang, Zheng-Mi He, Ping Zhang, Ke Liao.
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: Yan J, Xiong L, Yang L-X, He Z-M, Zhang P, Liao K (2025) Morphological and multi-locus phylogenetic analyses reveal three new branched species of Clavaria (Clavariaceae, Agaricales) from China. MycoKeys 115: 137-153. https://doi.org/10.3897/mycokeys.115.145774
Open Access

Abstract

Based on morphological and molecular evidence, 12 specimens have been identified as belonging to three previously unrecognized species of Clavaria, which are here described as C. divergens, C. orientalis, and C. tongdaoensis. Clavaria divergens is characterized by its branched, white basidiomata. Clavaria orientalis and C. tongdaoensis are very similar to C. zollingeri in the field. However, C. orientalis is distinguished by its more robust branches, while C. tongdaoensis differs by its varied or paler color of basidiomata. A concatenated sequence dataset (ITS-nrLSU-RPB2) was used for multi-locus phylogenetic analysis. The phylogenetic tree of Clavaria showed that the three branched species each formed a distinct lineage with strong support. A key to the known branched species of Clavaria in China is provided.

Key words:

Clavariaceae, morphology, phylogeny, taxonomy

Introduction

Vaillant (1727) described three clavarioid species and first used the term “Clavaria” to name them. Subsequently, Linnaeus (1753) formally established the genus Clavaria Vaill. ex L. in Species Plantarum and described five species with branched basidiomata. However, as an increasing number of species with branches were discovered, infrageneric groups of Clavaria species with branched basidiomata, such as Clavaria α Ramaria Pers., Clavaria trib. Botryoideae Fr., and Clavaria trib. Ramariae Fr., were proposed (Persoon 1801; Fries 1821). With further research, species of Clavaria with branches have been sequentially segregated as separate genera, such as Artomyces Jülich, Clavulina J. Schröt., Clavulinopsis Overeem, Ramaria Fr. ex Bonord., and Ramariopsis (Donk) Corner (Corner 1950). At present, species of Clavaria with authentically branched fruit bodies are not common, with only seven species recognized before the present study (Léveillé 1846; Corner 1967; Lazo 1972; Furtado et al. 2016; Yan et al. 2020).

Among the seven branching species, Clavaria griseolilacina P. Zhang and Clavaria sinensis P. Zhang are native to China and were described in 2020 (Yan et al. 2020). Their type specimens have been compared with the new species identified in the current study. In addition, Clavaria diverticulata A.N.M. Furtado & M.A. Neves and Clavaria martinii Corner are recorded only in the Americas, and their basidiomata are yellow (Corner 1967; Furtado et al. 2016). Therefore, the white or slightly yellowish pink Clavaria pumanquensis Lazo and the cosmopolitan Clavaria zollingeri Lév. (Corner 1950; Lazo 1972) are more directly relevant for careful differentiation from the three new species in this study.

In China, purple branching Clavaria species have often been identified as C. zollingeri in the past. However, a comparison of specimens collected during the past 20 years has revealed a clear morphological difference between species distributed in northern and southern China. In the present study, only specimens collected in Jilin Province (northern China) have larger basidiomata and longer branches and conform with C. zollingeri; specimens collected in Hubei and Hunan provinces (southern China) belong to species new to science based on morphological and molecular evidence. An additional new species, C. divergens, has a white, stably branched basidiomata, which is a very rare character combination in Clavaria.

Materials and methods

Morphological examination

Twelve specimens of the three new species were collected by the authors in Hunan or Hubei provinces between 2003 and 2022. Habitat photographs of basidiomata were taken in the field, and macromorphological data were recorded from fresh specimens. The color of the basidiomata was described with reference to color codes (Kornerup and Wanscher 1978) and color names (Ridgway 1912). Specimens were deposited in the Mycological Herbarium of Hunan Normal University (MHHNU), Changsha, China, after drying. Micromorphological features were recorded from microscopic observations. The handling of dried vouchers followed the procedures of Yan et al. (2023). The abbreviation [n/m/p] indicates that n basidiospores were measured from m basidiomata of p specimens. Dimensions of basidiospores are presented in the form (a–)b–c(–d), where a and d represent extreme values, and the range b–c comprises 90% of the measured values. All measurement data were analyzed with SPSS 14.0 (SPSS, Inc.). Q is the “length/width ratio” of a basidiospore in lateral view, and Q indicates the average Q of all basidiospores ± sample standard deviation.

DNA extraction, PCR amplification, and sequencing

Total genomic DNA was extracted from dried vouchers using the modified cetyltrimethylammonium bromide method introduced by Doyle and Doyle (1987) or the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China). The primer pairs ITS4/ITS5 (White et al. 1990) and LR0R/LR5 (Vilgalys and Hester 1990) were used to amplify the internal transcribed spacer (ITS) region and the nuclear ribosomal large subunit (nrLSU) region, respectively. The primers f RPB2-5F, f RPB2-6F, and f RPB2-7.1R (Liu et al. 1999; Matheny et al. 2007) were used to amplify the RNA polymerase II second largest subunit (RPB2) region. The PCR reaction volume and thermal-cycling conditions followed those of Yan et al. (2022) and He et al. (2023). The PCR products were examined and sequenced by Sangon Biotech. Sequences generated in this study were deposited in GenBank.

Alignment and phylogenetic analyses

The dataset used for phylogenetic analyses included the newly generated sequences and sequences downloaded from GenBank. Detailed information on the sequences is listed in Table 1.

Table 1.
Download as
CSV
XLSX

Voucher information and GenBank accession of taxa used in this study.

Identification Specimen No. GenBank No. (ITS) GenBank No. (28S) GenBank No. (RPB2) Location
Clavaria alboglobospora JAC15834 OR567635 OR567767 — New Zealand
C. amonenoides Lueck4 KP965768 KP965786 — Germany
C. amonenoides MHHNU10306 ON228386 ON231688 ON246172 China
C. amonenoides MHHNU10522 ON228387 ON231689 ON246173 China
C. appendiculata AMB 18348 MN022549 MN018833 — Italy
C. apulica AMB 150 MT853065 MT853066 — Italy
C. argillacea K(M)126733 KC759438 JQ415931 — United Kingdom
C. argillacea BRACR 16025 KC759439 JQ415930 — Slovakia
C. aspersa MHHNU32157 ON228390 ON231692 ON246176 China
C. aspersa MHHNU32397 ON228391 ON231693 ON246177 China
C. asterospora BIO-Fungi 12390 KC759440 — — Spain
C. atrofusca BRACR 13264 HQ606080 HQ606081 — Norway
C. atroumbrina K(M)143730 — JN315789 — United Kingdom
C. calabrica ZT Myc 58697 MF972889 MF972885 — Italy
C. californica AMB 18558 MT055940 — — Italy
C. californica TENN026785 HQ179660 — — USA
C. citrinorubra TENN040464 HQ179661 HQ877686 — Australia
C. crosslandii BIO-Fungi 12762 KC759441 — — Spain
C. cupreicolor TENN043696 KP257109 KP257187 — New Zealand
C. divergens MHHNU8277 PQ819508 PQ814267 — China
C. divergens MHHNU9857 PQ819509 PQ814268 PO806984 China
C. divergens MHHNU10164 PQ819510 PQ814269 PO806985 China
C. divergens MHHNU10165 PQ819511 PQ814270 PO806986 China
C. echino-olivacea TENN043686 KP257110 KP257188 — New Zealand
C. flavipes BRACR 15121 KC759450 KC759472 — Slovakia
C. flavipes TENN063740 KP257119 EF535267 — United Kingdom
C. flavostellifera BIO-Fungi 10433 KC759461 JX069828 — Slovakia
C. flavostellifera BRACR 16695 KC759462 JX069827 — Slovakia
C. fragilis MHHNU10527 ON228394 ON231696 ON246179 China
C. fragilis MHHNU32418 ON228395 ON231697 ON246180 China
C. fragilis TENN033244 KP257121 KP257195 — USA
C. fumosa MR00170 JN214482 HQ877696 — USA
C. fumosa TENN060724 KP257126 KP257199 — Russia
C. fuscata JMB08181001 KP257128 HQ877691 KP257253 USA
C. gibbsiae PDD 111979 OR567704 OR567794 — New Zealand
C. globospora TENN045945 KP257130 KP257201 — USA
C. greletii ERRO 2014102101 MF503244 — — Spain
C. greletii C(F) s/n — JN416778 — Denmark
C. griseobrunnea BIO-Fungi 12566 KY091644 — — Spain
C. griseolilacina MHHNU9722 MT028142 ON231725 ON246185 China
C. griseolilacina MHHNU10149 MT028141 ON231726 ON246186 China
C. hupingshanensis MHHNU7362 ON228396 ON231698 ON246181 China
C. incarnata AMB 18345 MK908007 MK898930 — Italy
C. incarnata BIO-Fungi 12560 KC759452 — — Spain
C. incarnata MA53113 KC759453 JQ415948 — Spain
C. lametina AMB 18933 OQ595227 OQ595225 OQ594954 Italy
C. cf. macounii PK1536 KP257131 KP257202 KP257254 Canada
C. megaspinosa JAC14897 OR567613 OR567751 — New Zealand
C. megaspinosa JAC16538 OR567650 OR567778 — New Zealand
C. messapica f. alborosea AMB 18346 MN017594 MN017499 — Italy
C. messapica AMB 12800 — KM486538 — Italy
C. messapica IHI-20Cla01 MW786738 MW786737 — Germany
C. musculospinosa PDD 82582 OR567692 OR567786 — New Zealand
C. neonigrita Ceska06112010 JN214481 JN214484 — Canada
C. orientalis MHHNU6801 PQ819512 PQ814271 — China
C. orientalis MHHNU7352 PQ819513 PQ814272 PO806987 China
C. orientalis MHHNU7586 PQ819514 PQ814273 PO806988 China
C. orientalis MHHNU7767 PQ819515 PQ814274 — China
C. orientalis MHHNU32116 PQ819516 PQ814275 PO806989 China
C. parvispora BRACR 13266 — MH727523 — Norway
C. parvispora BRACR 21309 — MH727524 — Slovakia
C. pisana AMB 18620 MW355011 MW355012 — Italy
C. pseudoincarnata AMB 17377 MN017595 MN017500 — Italy
C. pseudoincarnata AMB 17379 MN017596 MN017501 — Italy
C. pullei MONI 2018122801 MW549781 MW549780 — Spain
C. pullei SAV F3139 KP257132 KP257203 KP257255 Czech Republic
C. redoleoalii JAC14916 OR567617 OR567755 — New Zealand
C. redoleoalii JAC14917 OR567642 OR567772 — New Zealand
C. rosea TENN063100 KP257133 KP257205 KP257256 USA
C. rosea TENN065117 KP257134 KP257206 KP257257 USA
C. roseoviolacea JAC14915 OR567616 OR567754 — New Zealand
C. roseoviolacea JAC15786 OR567632 OR567764 — New Zealand
C. rubicundula JLH MyCoPortal 6603126 MK578690 — — USA
C. cf. rubicundula JMB10061005 — HQ877690 — USA
C. salentina AMB 010297 MF972892 MF972888 — Italy
C. sinensis MHHNU8198 MT028140 ON231727 ON246187 China
C. sphagnicola BRACR 13593 KC759455 KC759471 — Norway
C. sphagnicola BRNM 747282 KC759456 KC759470 — Czech Republic
C. stegasauroides JAC14852 OR567586 OR567742 — New Zealand
C. stegasauroides PBM3373 — HQ877698 KP257261 Australia
C. stellifera IHI-19Cla01 OK239673 OK239677 — Germany
C. straminea BRACR 12807 KC759449 JQ415944 — Slovakia
C. subviolacea JAC14150 OR567566 OR567726 — New Zealand
C. tenuipes ARAN-Fungi 11295 MW248489 MW248513 — Spain
C. tongdaoensis MHHNU11091 PQ819517 PQ814276 PO806990 China
C. tongdaoensis MHHNU11093 PQ819518 PQ814277 PO806991 China
C. tongdaoensis MHHNU11094 PQ819519 PQ814278 PO806992 China
C. tyrrhenica ZT Myc 58698 MF972890 MF972886 — Italy
C. ypsilonidia PDD 46673 NR174884 NG079629 — New Zealand
C. ypsilonidia TENN042411 KP257140 KP257210 KP257262 New Zealand
C. zollingeri MHHNU10528 ON228397 ON231699 ON246182 China
C. zollingeri MHHNU10548 ON228398 ON231700 ON246183 China
C. zollingeri MHHNU10550 ON228399 ON231701 ON246184 China
C. zollingeri TENN064095 KP257141 HQ877700 KP257263 USA
C. zollingeri TENN58652 AY854071 AY639882 AY480940 —
Mucronella flava IO.16.84 MT232354 MT232307 — Sweden
Mucronella sp PDD 95742 HQ533013 — — New Zealand

The ITS, nrLSU, and RPB2 sequences were respectively aligned using MAFFT v7.471 (Katoh and Standley 2016) and manually edited in BIOEDIT v7.2.5 (Hall 1999) where necessary. The combined matrix of ITS, nrLSU, and RPB2 sequences was assembled with SEQUENCEMATRIX 1.7.8 (Vaidya et al. 2011). The concatenated sequence dataset was analyzed using maximum likelihood (ML) and Bayesian inference approaches with RAXML v8.0.20 (Stamatakis 2006) and MRBAYES v3.2.7 (Ronquist and Huelsenbeck 2003), respectively. The ML analysis was conducted using the GTR+Gamma evolutionary model with 1000 bootstrap replicates. The Bayesian inference analysis ran for 1,000,000 generations using the GTR+I+G optimal evolutionary model selected with MRMODELTEST v2.4 (Nylander 2004). The phylogenetic trees were visualized using FigTree v1.4.2 (Rambaut 2012) and further refined using Adobe Photoshop CS6 and Illustrator CS5 (Adobe Systems, Inc., San Jose, CA, USA).

Results

Phylogenetic analyses

The data matrix consisted of 210 sequences (90 ITS, 88 nrLSU, and 32 RPB2) from 97 samples, among which 33 (12 ITS, 12 nrLSU, and 9 RPB2) were newly generated in the present study. The aligned concatenated ITS–nrLSU–RPB2 dataset, comprising a total of 2450 nucleotide positions, was used for the BI and ML analyses. The ML analysis yielded a tree topology with branch lengths and support values represented in Fig. 1, and the BI analysis yielded an almost identical phylogenetic construction (not shown). Bayesian posterior probabilities > 0.95 and bootstrap values > 50% are shown at the nodes in Fig. 1.

Figure 1. 

Phylogenetic relationships of Clavaria species inferred from ITS, nrLSU, and RPB2 sequences under the maximum likelihood optimality criterion. Bayesian posterior probabilities over 0.95 and bootstrap values over 50% are reported at nodes (BI/MP); the sign “–” means under the reported level. Our new species are shown in boldface text.

The ML and Bayesian analyses showed that two accessions of Mucronella Fr. (as the outgroup) and 54 species of Clavaria formed independent lineages, named Clade 1 to Clade 54 in turn. Eight main clades were resolved among the 54 species of Clavaria, which is similar to previous studies (Kautmanová et al. 2012; Birkebak et al. 2016). Clades 1 to 11 formed a well-supported (ML 100%/BI 1) clade (Clavaria fumosa clade); the Clavaria pullei clade (ML 100%/BI 1) included Clavaria atroumbrina, Clavaria lametina, and Clavaria pullei; Clavaria sensu stricto (ML 100%/BI 1) included the species Clavaria aspersa, Clavaria fragilis (the type for the genus), and Clavaria rosea; Clavaria atrofusca and Clavaria griseobrunnea formed the Clavaria atrofusca clade (ML 91%); and Clavaria greletii and Clavaria neonignta formed the Clavaria greletii clade (ML 100%/BI 1). The remaining species were resolved in three main clades; these species are united in possessing a loop-like clamp at the base of the basidium. Our three new species each formed a distinct monophyletic lineage with strong support (ML 100%/BI 1). The new species Clavaria divergens formed a distinct lineage (Clade 12) sister to the Clavaria fumosa clade, to which Clavaria crosslandii (Clade 17) and Clavaria salentina (Clade 16) were also phylogenetically close. The other two new species, Clavaria orientalis and Clavaria tongdaoensis, formed genetically distinct lineages (Clade 2 and Clade 3) that were phylogenetically closest to Clavaria zollingeri within the Clavaria fumosa clade.

Taxonomy

Clavaria divergens P. Zhang & Ju. Yan, sp. nov.

MycoBank No: 857600
Figs 2, 3

Etymology.

divergens (Latin) refers to the basidioma with dichotomous to irregularly divergent branches.

Holotype.

China • Yunnan Province: Malipo County, Donggan Town, alt. 1580 m, 23°21'41.98"N, 105°09'44.17" E, 6 August 2018, P. Zhang (MHHNU9857).

Diagnosis.

This species differs from other species within Clavaria subg. Syncoryne in its white branched basidiomata and 4-spored basidia.

Description.

Basidiomata (Fig. 2a, b) branched, brittle, scattered, or gregarious clusters; clusters 10–50 mm high, 10–30 mm broad; branches terete, 1–3 mm wide, 2–4 times, dichotomous, or irregularly divergent in the ultimate rank; branch tips subacuminate, often antler-like or claw-like. Fertile part coralloid, smooth, slightly curved, occasionally with a longitudinal depression in center, white [A1; White]. Apex white, becoming yellowish or tawny with age. Sterile part distinct, white, smooth, without tomentum and mycelial patch. Flesh concolorous with surface of basidiomata.

Figure 2. 

Basidiomata of Clavaria divergens a MHHNU9857 b MHHNU10165. Scale bars: 2 cm.

Basidiospores (Fig. 3a) [100/6/4] (4.0)4.2–5.0 × (2.4)2.7–3.8(4.0) μm [Q = (1.25)1.26–1.56(1.60), Q = 1.39 ± 0.10], mostly ellipsoid, sometimes also broadly ellipsoid, smooth, hyaline, nonamyloid, thin-walled; hilar appendix small (<1.0 μm in length). Basidia (Fig. 3b) 48–65 × 6.5–9.0 μm, clavate, 4-spored, hyaline, thin-walled or slightly thick-walled, sometimes with secondarily septated; sterigmata up to 5.2 μm long. Incrustations or crystals absent. Hyphae of the context parallel, thin-walled, hyaline, cylindrical to inflated, secondarily septated. Clamp connections absent in all parts of basidiomata.

Figure 3. 

Microscopic features of Clavaria divergens (MHHNU9857) a basidiospores b basidia.

Habitat, ecology, and distribution.

Scattered or gregarious in humus layers of soil under mixed coniferous–broadleaved forests or broadleaved forests. Basidiomata produced in summer, usually throughout the months of July to August; known from subtropical zones of Central and Southwestern China.

Additional specimens examined.

China • Hunan Province: Yongshun County, Xiaoxi National Nature Reserve, alt. 1068 m, 28°47'45.84"N, 110°12'13.89"E, 28 August 2014, P. Zhang (MHHNU8277); • Guzhang County, Gaofeng Town, alt. 573 m, 28°40'45.42"N, 110°08'28.56"E, 23 July 2020, Ju. Yan (MHHNU10164, MHHNU10165).

Clavaria orientalis P. Zhang & Ju. Yan, sp. nov.

MycoBank No: 857601
Figs 4, 5

Etymology.

orientalis (Latin), meaning eastern, refers to the occurrence of the species in East Asia.

Holotype.

China • Hunan Province: Shimen County, Hupingshan Nature Reserve, alt. 1828 m, 30°02'58.50"N, 110°31'24.90"E, 11 September 2012, P. Zhang (MHHNU7767).

Diagnosis.

Differs from Clavaria zollingeri in its stout branches, lesser degree of branching, and shorter basidia.

Description.

Basidiomata (Fig. 4a, b) branched, brittle, gregarious to caespitose clusters; clusters 50–80 mm high, 10–30 mm broad; branches terete, 1–3 mm wide, 1–4 times, dichotomous; branch tips obtuse, broadly rounded, or narrowly rounded. Fertile part coralloid, smooth, obviously curved or slightly twisted, deep amethyst [15A4-7, 15B4-6, 16A4-6; Amparo Purple, Lobelia Violet, Vinaceous Purple] to lilac [14A2-3, 15A2-3; Pale Vinaceous Purple, Pale Lobelia Violet], and changing to pale greyish purple [16A2, 17A2; Lavender Gray, Pale Payne’s Gray, Pale Verbena Violet] with age. Apex concolorous with lower part, becoming yellowish or tawny with age. Stipe distinct, sterile, smooth, often terete, semi-translucent, hygrophanous, and darker than the fertile part, sometimes flattened and pallid. Flesh concolorous or slightly paler than surface of basidiomata.

Figure 4. 

Basidiomata of Clavaria orientalis a MHHNU7767 b MHHNU32116. Scale bars: 2 cm.

Basidiospores (Fig. 5a) [100/5/5] (4.8)5.0–6.0 × 4.0–5.0(5.5) μm [Q = (1.09)1.13–1.38(1.43), Q = 1.21 ± 0.11], mostly broadly ellipsoid, sometimes ellipsoid or subglobose, smooth, hyaline, nonamyloid, thin-walled; hilar appendage present (<2.0 μm in length). Basidia (Fig. 5b) 34–48 × 5.0–8.0 μm, clavate, 4-spored, hyaline, thin-walled; sterigmata below 5.0 μm long. Incrustations or crystals absent. Hyphae of the context parallel, thin-walled, hyaline, cylindrical to inflated, secondarily septated. Clamp connections absent in all parts of basidiomata.

Figure 5. 

Microscopic features of Clavaria orientalis (MHHNU7767) a basidiospores b basidia.

Habitat, ecology, and distribution.

Gregarious to caespitose in humus layers of soil under broadleaved forests, coniferous forests, or mixed coniferous–broadleaved forests. Basidiomata produced in summer or autumn, usually throughout the months of July to September; known from subtropical zones of southern China.

Additional specimens examined.

China • Hunan Province: Sangzhi County, Badagongshan National Nature Reserve, alt. 1500 m, 29°46'58.17"N, 110°4'51.68"E, 22 July 2003, P. Zhang (MHHNU6801); • Shimen County, Hupingshan Nature Reserve, alt. 1828 m, 30°02'58.50"N, 110°31'24.90"E, 31 August 2010, P. Zhang (MHHNU7352); • 26 September 2011, P. Zhang (MHHNU7586). • Hubei Province: Hefeng County, Mulinzi National Nature Reserve, alt. 1413 m, 30°03'32.17"N, 110°12'34.35"E, 1 August 2020, Z.H. Chen (MHHNU32116).

Clavaria tongdaoensis P. Zhang & Ju. Yan, sp. nov.

MycoBank No: 857602
Figs 6, 7

Etymology.

tongdaoensis (Latin) refers to the type locality in Tongdao County, Hunan Province, China.

Holotype.

China • Hunan Province: Tongdao County, Fengshuwan Forest Park, alt. 400 m, 26°09'45.66"N, 109°46'31.52"E, 6 July 2022, P. Zhang and Li-Xun Yang (MHHNU11094).

Diagnosis.

Distinguished from Clavaria orientalis and Clavaria zollingeri by its smaller basidiomata and basidiospores.

Description.

Basidiomata (Fig. 6a, b) branched, brittle, gregarious to caespitose clusters; clusters 25–45 mm high, 30–40 mm broad; branches terete, 2–3 mm wide, 1–3 times, dichotomous; branch tips narrowly rounded or awl-shaped. Fertile part coralloid, smooth, often curved or slightly twisted, pale purple to pale purplish pink [13A2-3, 14A2-3; Pale Lavender Violet, Pale Lobelia Violet, Pale Purplish Vinaceous], turning white with age. Apex concolorous with lower part, becoming yellowish or tawny with age. Stipe distinct, sterile, smooth, terete, semi-translucent, hygrophanous, slightly darker color than the fertile part. Flesh concolorous with surface of basidiomata.

Figure 6. 

Basidiomata of Clavaria tongdaoensis a MHHNU11093 b MHHNU11094. Scale bars: 2 cm.

Basidiospores (Fig. 7a) [100/5/3] 3.5–5.0 × 3.0–4.2(4.5) μm [Q = (1.05)1.06–1.33, Q = 1.16 ± 0.08], broadly ellipsoid, sometimes subglobose, smooth, hyaline, nonamyloid, thin-walled; hilar appendage present. Basidia (Fig. 7b) 26–43 × 6.5–8.0 μm, clavate, 4-spored, hyaline, thin-walled; sterigmata below 5.0 μm long. Incrustations or crystals absent. Hyphae of the context parallel, thin-walled, hyaline, cylindrical to inflated, secondarily septated. Clamp connections absent in all parts of basidiomata.

Figure 7. 

Microscopic features of Clavaria tongdaoensis (MHHNU11094) a basidiospores b basidia.

Habitat, ecology, and distribution.

Gregarious to caespitose in humus layers of soil under broadleaved forests. Basidiomata produced in summer; known only from the type locality, China.

Additional specimens examined.

China • Hunan Province: Tongdao County, Fengshuwan Forest Park, alt. 400 m, 26°09'45.66"N, 109°46'31.52"E, 6 July 2022, P. Zhang and Li-Xun Yang (MHHNU11091, MHHNU11093).

Discussion

In this study, three new species of Clavaria within subg. Syncoryne were identified from specimens collected in China. The three species have in common the absence of a loop-like clamp connection at the base of the basidia and obvious branching of the basidiomata. Before the present study, only seven species within Clavaria were known to stably produce branched basidiomata, namely, C. diverticulata A.N.M. Furtado & M.A. Neves (Furtado et al. 2016), C. griseolilacina P. Zhang (Yan et al. 2020), C. hupingshanensis P. Zhang & Ju. Yan (Yan et al. 2022), C. martinii Corner (Corner 1967), C. pumanquensis Lazo (Lazo 1972), C. sinensis P. Zhang (Yan et al. 2020), and C. zollingeri Lév. (Léveillé 1846). Among these seven branched species, C. zollingeri is of greatest relevance in the present study.

We initially mistook C. orientalis and C. tongdaoensis to be C. zollingeri based on the purple color of their basidiomata until we collected material of C. zollingeri (MHHNU10528, Fig. 8) in Jilin Province (northern China) that matched a previous description of that species. A comparison of the specimens revealed the differences among the three species. More specifically, Corner noted that, for C. zollingeri, the basidioma is 15–75 mm high, the spores are 4.0–7.0 × 3.0–5.0 μm, the basidia are 50–60 × 7–10 μm, and the sterigmata are 4–7 μm (Corner 1950). Franchi and Marchetii (2021) noted that the basidiomata of C. zollingeri are up to 80 mm high and the basidia are 50–60 × 8–10 μm. In contrast, C. orientalis discovered in southern China has shorter basidia (34–48 × 5.0–8.0 μm) and shorter sterigmata (< 5.0 μm long) than those of C. zollingeri. An additional species collected from southern China, C. tongdaoensis, has shorter basidia (26–43 × 6.5–8.0 μm), smaller spores (3.5–5.0 × 3.0–4.2(4.5) μm), and shorter sterigmata than C. zollingeri. In addition, the branches of C. orientalis often are not as profuse as the branches of C. zollingeri, and, compared with C. zollingeri, the basidiomata of C. tongdaoensis are smaller (25–45 mm high). Clavaria divergens is quite unique within Clavaria. Based on the color of the basidiomata, C. divergens is similar to C. fragilis or C. gibbsiae, but C. fragilis and C. gibbsiae always have a simple basidiomata, occasionally once-furcate. Compared with the branched Clavaria species mentioned above, C. divergens is distinctive with its white basidiomata and subacuminate branch tips.

Figure 8. 

Basidiomata of Clavaria zollingeri (MHHNU10528). Scale bars: 2 cm.

In the phylogeny for Clavaria, 54 species are supported based on molecular data, which is much higher than previous records or predictions. For example, 28 species of Clavaria were recognized in Ainsworth and Bisby’s “Dictionary of the Fungi”, 10th edn. (Kirk et al. 2008), and Olariaga et al. (2015) estimated that the genus comprises 30–35 species. Two of the new species, C. orientalis and C. tongdaoensis, together with C. zollingeri, formed a clade with strong support (ML 100%/BI 1), a relationship consistent with the morphological similarity of the three species. In the phylogenetic analysis, sequence data for five specimens of C. zollingeri were included, two of which were collected in the United States and the other three were collected from Jilin Province, China. This finding supports the contention that C. zollingeri in North America and populations in northern China are conspecific. In contrast, eight specimens collected from southern China were genetically distinct from C. zollingeri and showed morphological differences and thus were identified as new species, named here C. orientalis and C. tongdaoensis. Moreover, C. divergens was indicated to be genetically very distinct in the phylogenetic tree. Although the phylogeny indicated that C. divergens has a close relationship with the Clavaria fumosa clade and represented a sister lineage to that clade, the node was not statistically supported. Nevertheless, the phylogenetic analysis supported its genetic distinctness and monophyly as an independent lineage and verified its identity as a previously unrecognized species. However, its phylogenetic relationships with other species of Clavaria require further research.

In summary, most species of Clavaria are unbranched, but three branching species are described in this article. Among them, C. orientalis and C. tongdaoensis are distinguished from C. zollingeri, which is considered to be distributed only in northern China. Clavaria divergens is the first species discovered in China with stable white branches. The records of these three species enrich the species diversity of the genus Clavaria and increase the number of species with branched basidiomata in the genus.

Key to branched species of Clavaria in China

1 Basidiomata white to pink 2
– Basidiomata purple 3
2 Basidiomata 10–50 mm tall, white C. divergens
– Basidiomata 35–70 mm tall, rose-white to seashell-pink C. hupingshanensis
3 Basidiomata sparsely branched C. griseolilacina
– Basidiomata profusely branched 4
4 Fruiting body usually lighter colored 5
– Fruiting body color usually darker colored 6
5 Basidiomata 30–70 mm tall, basidiospores 5.0–6.0 × 3.5–4.5 μm C. sinensis
– Basidiomata 25–45 mm tall, basidiospores 3.5–5.0 × 3.0–4.2 μm C. tongdaoensis
6 Basidiomata branching stout, 1–4 times, distribution in southern China C. orientalis
– Basidiomata branching slim, 3–5 times, distribution in northern China C. zollingeri

Acknowledgements

The authors are very grateful to Drs. Ke-Rui Huang and PhD students Peng-Tao Deng for their help with our image processing and writing. We thank Prof. Zuo-Hong Chen for providing specimens. We thank Robert McKenzie, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing a draft of this 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 financially supported by the National Natural Science Foundation of China (Grant No. 31670015, 31750001, and 32400010) and the Natural Science Foundation of Hunan Province of China (Grant No. 2024JJ6308).

Author contributions

Conceptualization: Ping Zhang and Ke Liao; methodology: Jun Yan and Li Xiong; performing the experiment: Jun Yan and Li Xiong; resources: Ping Zhang, Ke Liao, Li-Xun Yang, and Jun Yan; writing—original draft preparation: Jun Yan; writing—review and editing: Ping Zhang; supervision: Ping Zhang; project administration: Ping Zhang and Ke Liao; funding acquisition: Ping Zhang and Zheng-Mi He. All authors have read and agreed to the published version of the manuscript.

Author ORCIDs

Jun Yan https://orcid.org/0000-0002-2832-8046

Li Xiong https://orcid.org/0009-0007-3568-7737

Li-Xun Yang https://orcid.org/0009-0003-3086-4424

Zheng-Mi He https://orcid.org/0000-0001-8754-3427

Ping Zhang https://orcid.org/0000-0002-8751-704X

Data availability

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

References

  • Birkebak JM, Adamčík S, Looney BP, Matheny PB (2016) Multilocus phylogenetic reconstruction of the Clavariaceae (Agaricales) reveals polyphyly of agaricoid members. Mycologia 108(5): 860–868. https://doi.org/10.3852/15-370
  • Corner EJH (1950) A monograph of Clavaria and allied genera. Oxford University press, London, 740 pp.
  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11–15.
  • Franchi P, Marchetti M (2021) I Funghi Clavarioidi in Italia. Vol. 1. A. M. B. , Vicenza, Italy, 664 pp.
  • Fries E (1821) Systema mycologicum: sistens fungorum ordines, genera et species, huc usque cognitas. Confederatio Europaea Mycologiae Mediterraneensis AE, Berlin, 520 pp. https://doi.org/10.5962/bhl.title.5378
  • He ZM, Chen ZH, Bau T, Wang GS, Yang ZL (2023) Systematic arrangement within the family Clitocybaceae (Tricholomatineae, Agaricales): Phylogenetic and phylogenomic evidence, morphological data and muscarine-producing innovation. Fungal Diversity 123(1): 1–47. https://doi.org/10.1007/s13225-023-00527-2
  • Kautmanová I, Tomšovský M, Dueñas M, Martín MP (2012) European species of Clavaria (Agaricales, Agaricomycetes) with dark basidiomata – a morphological and molecular study. Persoonia – Molecular Phylogeny and Evolution of Fungi 29: 133–145. https://doi.org/10.3767/003158512X661543
  • Kornerup A, Wanscher JH (1978) Methuen handbook of colour, 3rd edn. Methuen, London, 252 pp.
  • Léveillé JH (1846) Descriptions des champignons de l’herbier du Muséum de Paris. Ann Sci Nat Bot, ser 3, 5: 111–167.
  • Linnaeus C (1753) Species Plantarum. Impensis Laurentii Salvii, Stockholm, 1200 pp.
  • Matheny PB, Wang Z, Binder M, Curtis JM, Hibbett DS (2007) Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi). Molecular Phylogenetics and Evolution 43(2): 430–451. https://doi.org/10.1016/j.ympev.2006.08.024
  • Nylander JAA (2004) MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.
  • Olariaga I, Salcedo I, Daniëls PP, Spooner B, Kautmanová I (2015) Taxonomy and phylogeny of yellow Clavaria species with clamped basidia—Clavaria flavostellifera sp. nov. and the typification of C. argillacea, C. flavipes and C. sphagnicola. Mycologia 107(1): 104–122. https://doi.org/10.3852/13-315
  • Persoon CH (1801) Synopsis methodica fungorum: sistens enumerationem omnium huc usque detectarum specierum, cum brevibvs descriptionibus nec non synonymis et observationibus selectis. Dieterich, 706 pp. https://doi.org/10.5962/bhl.title.166151
  • Vaidya G, Lohman DJ, Meier R (2011) Sequencematrix: Concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 27(2): 171–180. https://doi.org/10.1111/j.1096-0031.2010.00329.x
  • Vaillant S (1727) Botanicon Parisiense, ou Dénombrement par Ordre Alphabétique des Plantes, qui se Trouvent dans les Environs de Paris. J. & H. Verbeek, Leiden, Amsterdam, 205 pp. https://doi.org/10.5962/bhl.title.738
  • Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990
  • Yan J, Li GW, Liu WH, Chen ZH, Zhang P (2022) Updated taxonomy of Chinese Clavaria subg. Syncoryne (Clavariaceae, Agaricales): Description of two new species and one newly recorded species. Mycological Progress 21(8): 67. https://doi.org/10.1007/s11557-022-01815-y
  • Yan J, Wen J, Li GW, Wu SW, Zhang P (2023) Taxonomy and Phylogenetic Relationships of Clavulinopsis (Clavariaceae, Agaricales): Description of Six New Species and One Newly Recorded Species from China. Journal of Fungi 9(6): 656. https://doi.org/10.3390/jof9060656

Supplementary materials

Supplementary material 1 

ITS sequence dataset

Author: Jun Yan

Data type: fas

Explanation note: The ITS sequences were respectively aligned using MAFFT v7.471, and manually edited in BIOEDIT v7.2.5 where necessary.

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 (81.19 kb)
Supplementary material 2 

LSU sequence dataset

Author: Jun Yan

Data type: fas

Explanation note: The LSU sequences were respectively aligned using MAFFT v7.471, and manually edited in BIOEDIT v7.2.5 where necessary.

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 (93.99 kb)
Supplementary material 3 

RPB2 sequence dataset

Author: Jun Yan

Data type: fas

Explanation note: The RPB2 sequences were respectively aligned using MAFFT v7.471, and manually edited in BIOEDIT v7.2.5 where necessary.

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 (71.49 kb)
login to comment