Three new ramarioid species of Phaeoclavulina (Gomphaceae, Gomphales) from China

Peng-Tao Deng; Wen-Hao Liu; Zai-Wei Ge; Ping Zhang

doi:10.3897/mycokeys.108.128716

Abstract

Three new species of Phaeoclavulina from China are described: Phaeoclavulina bicolor, P. echinoflava, and P. jilinensis. Recognition of the new species is supported by morphological and molecular evidence. Phylogenetic analyses of concatenated ITS1–5.8S–ITS2 and nuclear large subunit sequences support the establishment of the new species and their placement within the Phaeoclavulina clade. A key to the known Phaeoclavulina species in China is provided.

Key words

Morphological characters, phylogenetic analysis, Ramarioid fungi, taxonomy

Introduction

Brinkmann (1897) first proposed the genus Phaeoclavulina Brinkmann to accommodate a coral fungus with spiny spores, and described Phaeoclavulina macrospora Brinkmann, as the type species, characterized by a branched fruiting body, ochraceous spores, and 2-spored basidia. The genus has since been expanded to include certain gomphoid fungi formerly classified in Gomphus Pers. and ramarioid fungi previously placed in Ramaria subg. Echinoramaria Corner. Thus, the morphology of the Phaeoclavulina fruiting body may be ramaroid, unipileate, or merismatoid (Giachini and Castellano 2011). Although Phaeoclavulina exhibits diversity in the morphology of the fruiting body, the microstructure is more homogeneous: monomitic hyphae, clamp connections in hyphae and basidia, and echinulate (mostly with acute spines) to verrucose, subreticulate or reticulate spores (Giachini 2004; Giachini and Castellano 2011). In the past decade, numerous new species of Phaeoclavulina have been described: 12 species were described by Franchi and Marchetti from Italy, and three species were described from China and Mexico (Franchi and Marchetti 2018, 2019, 2020; González-Vila et al. 2020; Liu et al. 2022).

Species of Phaeoclavulina have been described from all over the world, but they are mainly distributed in tropical, subtropical, and temperate regions, and grow in coniferous, broad-leaved, or mixed forest with basidiomata occurring in summer and autumn. Currently, no evidence shows that Phaeoclavulina species are mycorrhizal fungi, but their growth on decaying wood indicates that the species may be saprophytic. A number of ramarioid fungi in Phaeoclavulina are edible (Li JZ 2008; Dai YC et al. 2010), and have a delicious taste and relatively large fruiting body. Examples of edible taxa are P. abietina (Pers.) Giachini, P. longicaulis (Peck) Giachini, P. cyanocephala (Berk. & M.A. Curtis) Giachini, and P. campestris (K. Yokoy. & Sagara) Giachini.

In China, previous studies have reported the occurrence of 18 species of Phaeoclavulina: P. abietina, P. aeruginea P. Zhang, P. capucina (Pat.) Giachini, P. campestris, P. cinnamomea W.Q. Qin, P. cokeri (R.H. Petersen) Giachini, P. curta (Fr.) Giachini, P. cyanocephala, P. decolor (Berk. & M.A. Curtis) Giachini, P. eumorpha (P. Karst.) Giachini, P. flaccida (Fr.) Giachini, P. grandis (Corner) Giachini, P. longicaulis, P. macrospora, P. mutabilis (Schild & R.H. Petersen) Giachini, P. sikkimia (S.S. Rattan & Khurana) Giachini, P. viridis (Pat.) Giachini, and P. zippelii (Lév.) Overeem (Teng 1963; Li JZ 2008; Liu et al. 2022). Given that Phaeoclavulina was formerly included in Ramaria, most records of Phaeoclavulina species in China are based on previous studies of Ramaria. There is a lack of systematic research on Phaeoclavulina in China, from where only three new species have been discovered: Ramaria luteoaeruginea P. Zhang & Zhu L. Yang (assignable to Phaeoclavulina as that genus is currently circumscribed), P. cinnamomea, and P. aeruginea (Zhang et al. 2005; Liu et al. 2022). Therefore, a comprehensive survey combined with morphological and molecular research is needed to conduct a systematic analysis of Phaeoclavulina and to understand the Phaeoclavulina species diversity in China.

During research on ramarioid and coralloid fungi in China, seven specimens of Phaeoclavulina were collected. On the basis of their morphology and molecular phylogenetic analysis, these specimens were identified as three new species of Phaeoclavulina, which are formally described herein as P. bicolor, P. echinoflava, and P. jilinensis.

Materials and methods

Specimen sources

Seven specimens of Phaeoclavulina were gathered by the authors from 2004 to 2021 in Xizang Autonomous Region, Jilin Province, and Hainan Province. The fresh fruiting body characters and habitat were recorded in the field, including whether the color changed when injured. The fresh basidiomata were dried at 55–60 °C or desiccated in silica gel. The dried samples are deposited in the Mycological Herbarium of Hunan Normal University (MHHNU), Changsha, China, and the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS), Kunming, China.

Morphological observation

Macroscopic features of the newly collected specimens were described from the fresh fruiting body, record sheet, and photographs. The colors reported in the descriptions were determined following Kornerup and Wanscher (1978) and Ridgway (1912). Dried fruiting body sections were placed in 5% KOH solution, containing 1% Congo red solution and cotton blue, to observe the ornamentation of the spores. Microscopic characters were observed from a portion of a dried fruiting body with a light microscope, including spores, basidia, and hyphae. In the description of basidiospores, 60 basidiospores were measured for each species, and their size is expressed in the form (a–) b–c (–d), where ‘a’ and ‘d’ are the minimum and maximum dimensions of spores, respectively, and ‘b’ and ‘c’ are the range representing the majority of the spore dimensions. The abbreviation [n/m/p] indicates that the measurements were obtained from ‘n’ basidiospores from ‘m’ basidiomata of ‘p’ specimens. The Q value represents the length-to-width ratio of basidiospores, and the Q_m value is the average Q ± standard deviation.

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from dried specimens using the EZup Column Fungal Genomic DNA Extraction Kit (Sangon Biotech, Shanghai, China). A sample (25–30 mg) of a dried specimen was ground to powder in liquid nitrogen in accordance with the manufacturer’s instructions. The primer pairs ITS4/ITS5 and LR5/LR0R were used to amplify the nuclear rDNA ITS1–5.8S–ITS2 (ITS) and nuclear large subunit (LSU) regions, respectively (Vilgalys and Hester 1990; White et al. 1990; Gardes and Bruns 1993). The PCR amplification reactions were performed on an Eppendorf Mastercycler thermal cycler in a 25 µL volume containing 1 µL DNA, 2 µL primers, 9.5 µL ddH₂O, and 12.5 µL 2× Es Taq Master Mix. The amplification procedure consisted of pre-denaturation at 94 °C for 4 min, then 32 cycles comprising denaturation at 94 °C for 40 s, annealing at 55 °C (ITS) or 52 °C (LSU) for 40 s, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 8 min, and storage at 4 °C (Liu et al. 2022). An ABI 3730 DNA Analyzer (PerkinElmer Inc., USA) was used to sequence the PCR products. All PCR products were separated by electrophoresis in 1% agarose gel and then submitted to Sangon Biotech (Shanghai, China) for sequencing. The newly generated sequences (seven ITS and seven LSU) were deposited in GenBank (accession numbers are listed in Table 1).

Table 1.

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Details of the ITS and 28S rDNA sequences used for phylogenetic analyses. The sequences newly generated in this study are highlighted in bold.

Taxon	Voucher	GenBank No. ITS	GenBank No. LSU	Geographical origin	References
Phaeoclavulina abietina	OSC 134649	JX310378	JX287478	USA	Unpublished
P. abietina	OSC 140661	JX310379	JX287479	USA	Unpublished
P. aeruginea	MHHNU8909	ON262784	ON262781	China	Liu et al. (2022)
P. aeruginea	MHHNU6887	ON262785	ON262782	China	Liu et al. (2022)
P. alboapiculata	AMB 18590	MT055971	MT053248	Italy	Unpublished
P. alboapiculata	AMB 18585	MT055964	–	Italy	Unpublished
P. alboapiculata	AMB 18613	MT452509	–	Italy	Unpublished
*P. bicolor*	MHHNU10702	PP809798	PP800475	China	This study
*P. bicolor*	MHHNU10703	PP809799	PP800476	China	This study
P. cinnamomea	MHHNU10376	ON262786	ON262783	China	Liu et al. (2022)
P. cyanocephala	TH9064	KT339249	KT339290	Guyana	Unpublished
P. coniferarum	AMB 18562	MT055942	–	Italy	Unpublished
P. coniferarum	AMB 18531	NR_176718	NG_088119	Italy	Unpublished
P. carovinacea	AMB 18533	NR_176719	–	Italy	Unpublished
P. carovinacea	AMB 18551	MT055933	–	Italy	Unpublished
P. carovinacea	AMB 18534	MT055918	–	Italy	Unpublished
P. carovinacea	TUR-A 209584	ON561378	ON530902	Italy	Unpublished
P. clavarioides	PRM:945441	LR723647	–	Czech	Kříž et al. (2019)
P. clavarioides	PRM:945440	LR723646	LR723645	Czech	Kříž et al. (2019)
P. curta	AMB 18641	MW115423	MW092704	Italy	Unpublished
P. curta	AMB 18605	MT452501	–	Italy	Unpublished
P. curta	UBC F32034	KX236126	–	Canada	Unpublished
P. curta	HAY-F-000746	PP294846	–	USA	Unpublished
*P. echinoflava*	HKAS 45984	PP809801	PP800478	China	This study
*P. echinoflava*	HKAS 45992	PP809800	PP800477	China	This study
P. flaccida	AMB n. 18209	MF288928	MF288936	Italy	Unpublished
P. gigantea	FH109	–	AY574703	USA	Giachini et al. (2010)
P. insignis	FH104	–	AY574704	USA	Giachini et al. (2010)
*P. jilinensis*	MHHNU9149	PP809802	PP800479	China	This study
*P. jilinensis*	MHHNU9164	PP809803	PP800480	China	This study
*P. jilinensis*	MHHNU10504	PP809804	PP800481	China	This study
P. minutispora	AMB 18588	MT055969	MT053246	Italy	Unpublished
P. minutispora	AMB 18586	MT055965	MT053243	Italy	Unpublished
P. macrospora	AMB 18614	MT452510	–	Italy	Unpublished
P. pseudozippelii	BBH 43575	MG214661	MG214663	Thailand	Wannathes et al. (2018)
P. roellinii	PRM:945445	LR723649	–	Czech	Kříž et al. (2019)
Ramaria admiratia	TENN 69114	NR_137862	NG_059504	USA	Hughes et al. (2014)
R. calvodistalis	TENN 69095	KJ416132	KJ416135	USA	Hughes et al. (2014)

Alignment and phylogenetic analysis

Full sequences of the two DNA regions (ITS and LSU) obtained from the seven samples in this study, together with sequences for 31 accessions publicly accessible in GenBank, were used to construct a multigene dataset. Ramaria admiratia R.H. Petersen and Ramaria calvodistalis R.H. Petersen were selected as the outgroup owing to their phylogenetic placement external to the Phaeoclavulina clade (Xu et al. 2022). The final ITS+LSU dataset comprised 62 sequences (36 ITS and 26 LSU; Table 1) and was used for a multigene phylogenetic analysis. The sequences were aligned using MAFFT 7.471 with the default settings for gap insertion and extension penalties (Katoh and Standley 2016), then manually modified where necessary with BIOEDIT v7.2.5 (Hall 1999). The final concatenated ITS+LSU dataset was generated by SEQUENCEMATRIX v1.7.8 (Vaidya et al. 2011). Maximum likelihood (ML) analysis of the dataset was conducted with RAxML v7.2.6 (Stamatakis et al. 2005; Stamatakis 2006), using a GTR+G evolutionary model (Stamatakis et al. 2008). A bootstrap (BS) analysis was performed with 1000 replicates to assess support for the ML tree topology. Bayesian inference (BI) was performed using MRBAYES v3.2.7 (Ronquist et al. 2012). The optimal evolutionary model (GTR+I+G) was selected based on the Akaike information criterion with MRMODELTEST v2.4 (Nylander 2004). Four independent Markov chain Monte Carlo (MCMC) chains were run, with sampling every 100 generations, for a total of three million generations (standard deviation ≤ 0.01). Posterior probabilities (PP) were calculated after discarding the first 25% of generations as burn-in. FIGTREE v1.4.2 (Rambaut 2012), PHOTOSHOP CS6, and ILLUSTRATOR CS5 (Adobe Systems, Inc., San Jose, CA, USA) were used to visualize and adjust the phylogenetic trees.

Results

Phylogenetic analysis

The final ITS+LSU dataset comprised 1661 aligned positions in total. The BI phylogeny (not shown) was extremely similar in topology and branch support to the ML tree. The ML phylogeny constructed from the ITS+LSU dataset (Fig. 1) revealed that 38 samples were placed in a strongly supported (PP 1, BS 100%) Phaeoclavulina clade. The seven samples newly sequenced in this study were placed on three distinct branches, each with strong statistical support (PP 1, BS 100%). Phaeoclavulina jilinensis, P. flaccida, and P. roellinii (Schild) Giachini were grouped in a well-supported subclade (PP 0.99, BS 87%). Phaeoclavulina bicolor was placed as sister to a clade comprising the three preceding species together with P. macrospora and P. coniferarum Franchi & M. Marchetti but with no support. The sequences of P. echinoflava were placed in an isolated position sister to all other samples within the Phaeoclavulina clade. On the basis of these results, three new species of Phaeoclavulina based on the newly collected Chinese specimens are proposed.

Figure 1.

Phylogenetic relationships of Phaeoclavulina species inferred from a concatenated ITS and LSU sequence dataset under the maximum likelihood optimality criterion. Bayesian posterior probabilities (PP) > 0.90 and bootstrap values (BS) > 50% are reported at the nodes (PP/BS); “–” indicates that the support value was less than the respective threshold. The three new species from China are highlighted in bold.

Taxonomy

Phaeoclavulina bicolor P. Zhang & W.H. Liu, sp. nov.

MycoBank No: 854010

Fig. 2

Diagnosis

Differs from other Phaeoclavulina species by the yellowish white apex.

Type

China • Hainan Province, Jianfengling National Forest Park, 18°74'21"N, 108°84'81"E, 986 m asl., 30 July 2021, leg. P. Zhang (holotype MHHNU10702).

Etymology

bicolor (Latin), referring to the different color of the branches and branch tips.

Basidiomata

Coralloid, 60–90 mm tall, 30–45 mm broad, light grayish brown when young [6D3–4], dark brown in age [6E5–7]. Stipe single, 10–20 mm long, white mycelia at the base. Branches sparse, branching from the base, dichotomous to polychotomous, divided two to three times, internodes becoming gradually shorter, terminal branches short and not flat, in cross-section rounded, axils V-shaped. Tips short and blunt, yellowish white [2A2–4] or pale white [1A1–2]. Context white, fleshy. Taste and odor, and macrochemical reactions not recorded.

Figure 2.

Basidiomata and microscopic features of Phaeoclavulina bicolor (MHHNU10702) A, B basidiomata C basidia D basidiospores. Scale bars: 5 cm (A, B).

Micromorphology

Context hyphae in parallel arrangement, 3–4 µm wide, cylindric, inflated, with clamp connections but not at every septum, thin-walled, smooth, hyaline. Basidia approximately 40–50 × 5–8 µm with two sterigmata 5–6 µm long, hyaline, clavate, with clamp connection at base. Cystidia absent. Basidiospores [60/3/3] (7.6)8–10(10.5) × (3.8)4–6(6.5) µm [Q = 1.70–2.20, Q_m = 1.97 ± 0.18], long-ellipsoid or cylindrical, slightly thick-walled, pale yellow in KOH, cyanophilic, surface coarse, echinulate, spines 0.6–1.0 μm long, acute; hilar appendage acuminate.

Additional materials examined

• Hainan Province, Jianfengling National Forest Park, 18°74'32"N, 108°84'76"E, 978 m asl., 30 July 2021, MHHNU10703.

Habit and distribution

Solitary, growing on the soil of broad-leaved forest in tropical rain forest; basidiomata occur in summer. Known only from the type locality in China.

Comments

Phaeoclavulina bicolor is distinguished from other species of Phaeoclavulina by the yellowish white branch tips, and the mainly grayish brown to dark brown basidiomata. Phaeoclavulina subdecurrens (Coker) Franchi & M. Marchetti also has basidiomata with a different color at the tips, but in P. subdecurrens the branch tips are pale violet to off white. Phaeoclavulina aeruginea has unique copper-green branch tips and has relatively larger spores than P. bicolor (13–16 × 8–9 μm vs. 8–10 × 4–6 μm). In the field, P. cyanocephala has relatively larger basidiomata (8–18 × 2–7 cm) and is distributed worldwide, whereas in P. bicolor the basidiomata is 6–9 cm tall, 3–4.5 cm broad, and the species is presently known only from Hainan Province in China.

Phaeoclavulina echinoflava P. Zhang & W.H. Liu, sp. nov.

MycoBank No: 854011

Fig. 3

Diagnosis

Differs from other Phaeoclavulina species by the bright yellow basidiomata when young, brownish yellow with age.

Type

China • Xizang Autonomous Region, Jiangda County, 31°49'39"N, 98°21'88"E, 3600 m asl., 29 July 2004, Z.W. Ge 204 (HKAS 45984, holotype).

Etymology

echinoflava (Latin), referring to the bright yellow fruiting body and echinulate spores.

Basidiomata

Coralloid, 65–75 mm tall, 35–45 mm broad, bright yellow when young [1A6–7], yellow to pale brown with age [1A3–1B3]. Stipe single or falsely fasciculate, 20–30 mm long, yellowish white [1A2], smooth, densely white mycelia at the base. Many branches diverge from the stalk, dichotomous to polychotomous, divided three to four times, branches thick and sparse, terminal branches short, in cross-section rounded, bifurcation narrowly V- or U-shaped. Tips of branches concolorous, broom-form or short-digitate by maturity, short and blunt. Context whitish, fleshy. Taste and odor, and macrochemical reactions not recorded.

Figure 3.

Basidiomata and microscopic features of Phaeoclavulina echinoflava (HKAS 45984) A, B basidiomata C basidia D basidiospores. Scale bars: 5 cm (A, B).

Micromorphology

Context hyphae in parallel arrangement, 5–8 μm wide, cylindric, inflated to 12 μm wide, clamp connections present, ampulliform clamps occasional, thin-walled, smooth, hyaline. Basidia approximately 22–34 × 6–8 µm with four sterigmata 3–5 µm long, hyaline, clavate or subclavate, with clamp connection at base. Cystidia absent. Basidiospores [60/2/2] (8.0)9.0–10.5(11.0) × (3.2)3.5–4.0(4.5) μm [Q=2.39–2.86, Qm=2.61 ± 0.21] elongate obovoid, slightly thick-walled, ochraceous in KOH, cyanophilic, surface coarse, strongly cyanophilius echinulate, spines 0.8–1.5 μm long, acute; hilar appendage inconspicuous and acuminate.

Additional materials examined

• Xizang Autonomous Region, Jiangda County, 3600 m asl., 29 July 2004, Z.W. Ge 212 (HKAS 45992).

Habit and distribution

Solitary, growing on the humus layer under shrubland at high altitudes; basidiomata generally occur in summer. Known only from the type locality in China.

Comments

The bright yellow fruiting body of P. echinoflava is a distinctive character in Phaeoclavulina. In Phaeoclavulina, P. echinovirens (Corner, K.S. Thind & Dev) Giachini, P. flaccida and P. decurrens (Fr.) Giachini also possess a yellow fruiting body. However, in P. flaccida the fruiting body is beige and the basidia are relatively larger than those of P. echinoflava (38–45 × 5.5–6.5 μm vs. 22–34 × 6–8 μm). Phaeoclavulina decurrens has a ramaroid fruiting body, ochre to buff in color fading to a white stem, and has relatively shorter basidiospores than those of P. echinoflava ((4.2)4.9(5.5) × (2.5)2.9(3.2) μm vs. (8.0)9.0–10.5(11.0) × (3.2)3.5–4.0(4.5) μm). P. echinovirens differs from P. echinoflava in having larger basidiomata (7.5–13 × 4.5–7.5 cm) and orange yellow tips. The phylogenetic reconstructions placed P. echinoflava in an isolated position within the Phaeoclavulina clade, which is consistent with the unique fruiting body color of P. echinoflava.

Phaeoclavulina jilinensis P. Zhang & W.H. Liu, sp. nov.

MycoBank No: 854012

Fig. 4

Diagnosis

Differs from other Phaeoclavulina species by the citrine fruiting body and only known from Jilin Province, China.

Type

China • Jilin Province, Fusong County, 42°57'66"N, 127°97'68"E, 665 m asl., 26 August 2020, leg. P. Zhang (holotype MHHNU 10504).

Etymology

jilinensis (Latin), referring to the currently known distribution of the species in Jilin Province, China.

Basidiomata

Ramaroid, 35–90 mm tall, 15–50 mm broad, pinecone yellow [2A3–5], pale lemon-yellow [4A5–6] with age. Stipe single or falsely fasciculate, 25–35 mm long, concolorous with branches when young, ochraceous with age, smooth, many mycelia at base, not changing color on bruising. Branches dichotomous to polychotomous, divided three to five times, primary branches thick and in cross-section rounded; terminal branches short and becoming flat, bifurcation narrowly V-shaped. Apices acute, rather short, dichotomous, concolorous with branches. Context whitish, fleshy. Taste and odor, and macrochemical reactions not recorded.

Figure 4.

Basidiomata microscopic features of Phaeoclavulina jilinensis (MHHNU 10504) A, B basidiomata C basidia D basidiospores. Scale bars: 5 cm (A, B).

Micromorphology

Context hyphae compact, 3–7 μm wide, cylindric, inflated to 11 μm wide, clamp connections present, thin-walled, smooth, hyaline. Basidia approximately 20–36 × 5–7 µm with four sterigmata 3–5 µm long, hyaline, clavate or subclavate, with clamp connection at base. Cystidia absent. Basidiospores [60/3/3] (5.5)6.0–8.0(8.5) × (2.8)3.0–5.0(5.5) μm [Q = 1.67–2.40, Q_m = 1.96 ± 0.16], elongate-ellipsoid, thick-walled, pale yellow in KOH, cyanophilic, surface coarse, with short but distinct spines, spines 0.8–1.5 μm long, strongly cyanophilous, hilar appendage distinct.

Habit and distribution

Solitary on the humus layer under mixed forest; basidiomata generally occur in summer. Known only from the type locality in China.

Additional materials examined

• Jilin Province, Yanbian Korean Autonomous Prefecture, Changbai Mountain Academy of Sciences, 42°40'51"N, 128°11'48"E, 896 m asl., 6 August 2017, MHHNU 9149 • Jilin Province, Yanbian Korean Autonomous Prefecture, Changbai Mountain, 42°22'61"N, 128°19'42"E, 950 m asl., 7 August 2017, MHHNU 9164.

Comments

In the present phylogenetic analysis, P. jilinensis was closely related to accessions of P. flaccida and P. roellinii. Phaeoclavulina roellinii has an ochraceous to watery ochre-brown fruiting body and P. flaccida has a beige fruiting body. Thus, these species cannot be distinguished by color alone, but by other characters. Phaeoclavulina roellinii has long and narrowly clavate basidia (38–70 × 5–7(8) μm), whereas P. jilinensis has relatively short basidia (20–36 × 5–7 µm). In addition, P. roellinii has abundant stellate crystals adhering to the hyphae, but we have not observed this character in P. jilinensis. The color of the fruiting body, sparse branches, and relatively longer basidia (38–45 × 5.5–6.5 vs. 20–36 × 5–7 μm) distinguish P. flaccida from P. jilinensis.

Discussion

In the field, Phaeoclavulina species can be easily confused with Ramaria or Clavaria because in most Phaeoclavulina taxa the fruiting body is coralloid or ramaroid. These genera are difficult to distinguish by macroscopic characters alone. However, the following microstructural characters distinguish Phaeoclavulina from other coralloid fungi: echinulate or warty spores, strongly cyanophilius; and clamp connections are present. In the past, most species of Phaeoclavulina were classified in Ramaria subg. Echinoramaria (Corner 1970; Petersen 1981). Subsequently, Phaeoclavulina was transferred to Gomphus sensu lato (Giachini et al. 2010). However, in previous studies (Liu et al. 2022; Xu et al. 2022), a monophyletic Phaeoclavulina clade is consistently retrieved in phylogenetic analyses and Phaeoclavulina is indicated to be phylogenetically separated from the Gomphaceae. Unfortunately, the current molecular evidence does not provide strong support values. To resolve the taxonomic position of Phaeoclavulina and relationships among the genera of Gomphaceae, worldwide sampling of taxa and more comprehensive molecular data for phylogenetic analysis are needed.

The present work enriches our knowledge of the genus Phaeoclavulina in China. Three new species are described, and the ITS+LSU phylogenetic trees strongly support that each of the species is monophyletic and phylogenetically distinct from other known species of Phaeoclavulina. These three new species were collected in different regions of China: P. bicolor was collected from a tropical wet climate in Hainan province; P. echinoflava was collected in high-altitude area from Tibet; and P. jilinensis was collected from a temperate continental climate in Jilin province. Thus, the three areas differ entirely in climate and altitude, indicating that species of Phaeoclavulina are widely distributed in China and highly adaptable. Furthermore, the morphological distinctiveness of the three species is unambiguous; for example, P. echinoflava has relatively few branches and a bright yellow fruiting body. The shape and color of the Phaeoclavulina fruiting body might reflect different climates and altitudes. In addition, records of Phaeoclavulina species in China are mainly derived from fungal resource surveys and Fungi of China (Teng 1963), many species names may be misused, and there are few or no specimens to verify their authenticity. Thus, we hope that additional samples of Phaeoclavulina will be collected in China for construction of a well-supported phylogenetic tree with expanded taxonomic coverage in the future, so as to achieve a more comprehensive understanding of Phaeoclavulina and its distribution.

Key to known Phaeoclavulina species in China

1	Species pileate	2
–	Species coralloid or ramarioid	3
2	Hymenium violet	*P. grandis*
–	Pileus green	*P. viridis*
3	Spore spines connected into circular to semi-circular ridges	*P. decolor*
–	Spore spines not connected into circular to semi-circular ridges	4
4	Crystals present in trama or adhering to rhizomorphic hyphae	5
–	Crystals absent in trama or adhering to rhizomorphic hyphae	6
5	Basidiomata bruising green	*P. flaccida*
–	Basidiomata bruising red	*P. eumorpha*
6	Spores verrucose	7
–	Spores echinulate	8
7	Basidiomata bruising olive or green on handling or weathering	*P. abietina*
–	Basidiomata not bruising olive or green on handling or weathering	*P. sikkimia*
8	Hyphae with H-connection	*P. zippelii*
–	Hyphae without H-connection	9
9	Branch tips discolorous with basidiomata	10
–	Branch tips concolorous with basidiomata	11
10	Spores > 15 µm long	*P. macrospora*
–	Spores < 15 µm long	12
11	Basidiomata bright-colored	13
–	Basidiomata deep-colored	14
12	Branch tips yellowish white	*P. bicolor*
–	Branch tips green or blue	15
13	Basidiomata orange or cinnamon	16
–	Basidiomata bright-yellow or lemon-yellow	17
14	Basidiomata > 11 cm tall	*P. campestris*
–	Basidiomata < 11 cm tall	18
15	Tips copper-green	*P. aeruginea*
–	Tips blue	19
16	Spores acute spines	*P. cokeri*
–	Spores volcanic spines	*P. cinnamomea*
17	Tips blunt, broom-form or short-digitate	*P. echinoflava*
–	Tips acute, tapered	*P. jilinensis*
18	Hymenium amphigenous	*P. capucina*
–	Hymenium unilateral	20
19	Spores < 10 µm long	*P. mutabilis*
–	Spores > 10 µm long	*P. cyanocephala*
20	Spores 4.5–6.5 µm long, basidia 25–36 µm long	*P. curta*
–	Spores 7–11 µm long, basidia 40–50 µm long	*P. longicaulis*

Acknowledgements

We thank Robert McKenzie, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of 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

No funding was reported.

Author contributions

Conceptualization: PZ. Methodology: PTD, WHL. Investigation: PTD, WHL. Resources: PZ, ZWG, WHL, PTD. Writing – original draft: PTD. Writing – review and editing: PZ. Supervision: PZ. Project administration: PZ. Funding acquisition: PZ. All authors have read and agreed to the published version of the manuscript.

Author ORCIDs

Peng-Tao Deng https://orcid.org/0000-0002-8755-7965

Wen-Hao Liu https://orcid.org/0000-0002-6937-1446

Zai-Wei Ge https://orcid.org/0000-0003-3184-4604

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

Brinkmann W (1897) Vorarbeiten zu einer Pilzflora Westfalen. Jahresbericht des Westfälischen Provinzial-Vereins für Wissenschaft und Kunst 25: 195–207.

Corner EJH (1970) Supplement to “A monograph of Clavaria and allied genera”. Beihefte zur Nova Hedwigia 33: 87–92.

Dai YC, Zhou LW, Yang ZL, Wen HA, Tolgor B, Li TH (2010) A revised checklist of edible fungi in China. Mycosystema 29(1): 1–21.

Franchi P, Marchetti M (2018) Nomenclatural novelties. Index Fungorum 373: 1.

Franchi P, Marchetti M (2019) Nuove combinazioni nel Genere Phaeoclavulina. RIvis ta di Micologia 62: 19–40.

Franchi P, Marchetti M (2020) Nomenclatural novelties. Index Fungorum 457: 1–7.

Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Molecular Biology 2: 113–118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x

Giachini AJ (2004) Systematics, phylogeny, and ecology of Gomphus sensu lato. Doctoral Dissertation. Oregon State University, Corvallis.

Giachini AJ, Castellano MA (2011) A new taxonomic classification for species in Gomphus sensu lato. Mycotaxon 115(1): 183–201. https://doi.org/10.5248/115.183

Giachini AJ, Hosaka K, Nouhra E, Spatafora J, Trappe JM (2010) Phylogenetic relationships of the Gomphales based on nuc25S-rDNA, mit-12S-rDNA, and mit-atp6-DNA combined sequences. Fungal Biology 114(2–3): 224–234. https://doi.org/10.1016/j.funbio.2010.01.002

González-Vila A, Martínez-González R, Espinosa D, Estrada-Torres A (2020) Phaeoclavulina liliputiana sp. nov. (Gomphaceae, Gomphales) a new endemic species from Tlaxcala, Mexico. Phytotaxa 470: 155–164. https://doi.org/10.11646/phytotaxa.470.2.4

Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.

Hughes K, Petersen R, Justice J (2014) Two new species of Ramaria from Arkansas. MycoKeys 8: 17–29. https://doi.org/10.3897/mycokeys.8.7356

Katoh K, Standley DM (2016) A simple method to control over-alignment in the MAFFT multiple sequence alignment program. Bioinformatics 32(13): 1933–1942. https://doi.org/10.1093/bioinformatics/btw108

Kornerup A, Wanscher JH (1978) Methuen handbook of colour, 3^rd edn. Methuen, London, 1–252.

Kříž M, Jindřich O, Kolařík M (2019) Contribution to the knowledge of mycobiota of Central European dry grasslands: Phaeoclavulina clavarioides and Phaeoclav ulina roellinii (Gomphales). Czech Mycology 71(2): 137–150. https://doi.org/10.33585/cmy.71202

Li JZ (2008) Study on wild edible fungi species diversity from Hunan. Life Science Research 12(4): 314–321.

Liu WH, Yan J, Deng PT, Qin WQ, Zhang P (2022) Two new species of Phaeoclavulina (Gomphaceae, Gomphales) from Hunan Province, China. Phytotaxa 561(1): 3. https://doi.org/10.11646/phytotaxa.561.1.3

Nylander JAA (2004) MrModeltest v2.0. Program distributed by the author, Evolutionary Biology Centre, Uppsala, Sweden.

Petersen RH (1981) Ramaria subgenus Echinoramaria. Bibliotheca Mycologica 79: 1–261.

Rambaut A (2012) FigTree v1.4. University of Edinburgh, Edinburgh, UK. https://tree.bio.ed.ac.uk

Ridgway R (1912) Color standards and color nomenclature. Published by the author, Washington. https://doi.org/10.5962/bhl.title.144788

Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice a cross a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029

Stamatakis A (2006) RAxML-VI-HPC: Maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22(21): 2688–2690. https://doi.org/10.1093/bioinformatics/btl446

Stamatakis A, Ludwig T, Meier H (2005) RAxML-III: A fast program for maximum likelihood-based inference of large phylogenetic trees. Bioinformatics 21(4): 456–463. https://doi.org/10.1093/bioinformatics/bti191

Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the raxml web servers. Systematic Biology 57(5): 758–771. https://doi.org/10.1080/10635150802429642

Teng SC (1963) Fungi of China. Science Press. Beijing.

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

Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172(8): 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990

Wannathes N, Kaewketsri R, Suwannarach N, Kumla J, Lumyong S (2018) Phaeoclavulina pseudozippelii sp. nov. (Gomphales, Basidiomycota) from Northern Thailand. Phytotaxa 362: 211–219. https://doi.org/10.11646/phytotaxa.362.2.7

White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols, a guide to methods and applications. Academic Press, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1

Xu YY, Jian SP, Mao N, Yang ZL, Fan L (2022) Gomphocantharellus, a new genus of Gomphales. Mycologia 114(4): 748–756. https://doi.org/10.1080/00275514.2022.2065781

Zhang P, Yang ZL, Ge ZW (2005) Two new species of Ramaria from southwestern China. Mycotaxon 94: 235–240.

Supplementary material

Supplementary material 1

Multiple sequence alignment

Author: Peng-Tao Deng

Data type: fasta

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 (63.68 kb)

﻿Abstract

Key words

﻿Introduction

﻿Materials and methods

﻿Specimen sources

﻿Morphological observation

﻿DNA extraction, PCR amplification, and sequencing

﻿Alignment and phylogenetic analysis

﻿Results

﻿Phylogenetic analysis

﻿Taxonomy

﻿ Phaeoclavulina bicolor P. Zhang & W.H. Liu, sp. nov.

Diagnosis

Type

Etymology

Basidiomata

Micromorphology

Additional materials examined

Habit and distribution

Comments

﻿ Phaeoclavulina echinoflava P. Zhang & W.H. Liu, sp. nov.

Diagnosis

Type

Etymology

Basidiomata

Micromorphology

Additional materials examined

Habit and distribution

Comments

﻿ Phaeoclavulina jilinensis P. Zhang & W.H. Liu, sp. nov.

Diagnosis

Type

Etymology

Basidiomata

Micromorphology

Habit and distribution

Additional materials examined

Comments

﻿Discussion

﻿Key to known Phaeoclavulina species in China

﻿Acknowledgements

﻿Additional information

Conflict of interest

Ethical statement

Funding

Author contributions

Author ORCIDs

Data availability

﻿References

Supplementary material

Abstract

Introduction

Materials and methods

Specimen sources

Morphological observation

DNA extraction, PCR amplification, and sequencing

Alignment and phylogenetic analysis

Results

Phylogenetic analysis

Taxonomy

Phaeoclavulina bicolor P. Zhang & W.H. Liu, sp. nov.

Phaeoclavulina echinoflava P. Zhang & W.H. Liu, sp. nov.

Phaeoclavulina jilinensis P. Zhang & W.H. Liu, sp. nov.

Discussion

Key to known Phaeoclavulina species in China

Acknowledgements

Additional information

References