683 MycoKeys MycoKeys 108: 1-14 (2024) DOI: 10.3897/mycokeys.108.128716 Research Article Three new ramarioid species of Phaeoclavulina (Gomphaceae, Gomphales) from China Peng-Tao Deng'™®, Wen-Hao Liu™, Zai-Wei Ge*®, Ping Zhang'® 1 College of Life Science, Hunan Normal University, Changsha 410081, China 2 CAS Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, 650201, China Corresponding author: Ping Zhang (zhangping0000@163.net) OPEN Qaceess Academic editor: Bao-Kai Cui Received: 31 May 2024 Accepted: 2 August 2024 Published: 21 August 2024 Citation: Deng P-T, Liu W-H, Ge Z-W, Zhang P (2024) Three new ramarioid species of Phaeoclavulina (Gomphaceae, Gomphales) from China. MycoKeys 108: 1-14. https://doi.org/10.3897/ mycokeys.108.128716 Copyright: © Peng-Tao Deng et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). 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 morpho- logical 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 ac- commodate a coral fungus with spiny spores, and described Phaeoclavulina macrospora Brinkmann, as the type species, characterized by a branched fruit- ing 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 ra- maroid, unipileate, or merismatoid (Giachini and Castellano 2011). Although Phaeoclavulina exhibits diversity in the morphology of the fruiting body, the mi- crostructure 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; Gonzalez-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 spe- cies are mycorrhizal fungi, but their growth on decaying wood indicates that the Peng-Tao Deng et al.: New species of Phaeoclavulina 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 relative- ly large fruiting body. Examples of edible taxa are P abietina (Pers.) Giachini, P. longicaulis (Peck) Giachini, PR 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.) Giachi- ni, P. campestris, P. cinnamomea W.Q. Qin, P. cokeri (R.H. Petersen) Giachini, P. curta (Fr.) Giachini, P. cvanocephala, 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 Phaeo- clavulina 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 under- stand 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 mo- lecular 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, Chi- na, 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 solu- tion, containing 1% Congo red solution and cotton blue, to observe the orna- mentation of the spores. Microscopic characters were observed from a portion MycoKeys 108: 1-14 (2024), DOI: 10.3897/mycokeys.108.128716 5 Peng-Tao Deng et al.: New species of Phaeoclavulina 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_ 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 sam- ple (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/LROR 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 uL vol- ume containing 1 uL DNA, 2 uL primers, 9.5 pL ddH,0O, and 12.5 pL 2x 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). Alignment and phylogenetic analysis Full sequences of the two DNA regions (ITS and LSU) obtained from the sev- en 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 Phaeo- clavulina clade (Xu et al. 2022). The final ITS+LSU dataset comprised 62 se- quences (36 ITS and 26 LSU; Table 1) and was used for a multigene phylogenet- ic 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 (Bl) was performed using MRBAYES v3.2.7 MycoKeys 108: 1-14 (2024), DOI: 10.3897/mycokeys.108.128716 3 Peng-Tao Deng et al.: New species of Phaeoclavulina Table 1. Details of the ITS and 28S rDNA sequences used for phylogenetic analyses. The sequences newly generated in this study are highlighted in bold. Phaeoclavulina abietina Unpublished P. aeruginea MHHNU6887 China Liu et al. (2022) P alboapiculata AMB 18590 Italy Unpublished P. alboapiculata AMB 18585 | MT055994 | - Italy Unpublished P alboapiculata AMB 18613. | mT452509 | - | Italy Unpublished P. bicolor MHHNU10702 China This study P. bicolor MHHNU10703 China This study P. cinnamomea MHHNU10376 China Liu et al. (2022) P clavarioides | PRM:945441 | LR723647, | = - =~ | Czech Ki{z et al. (2019) P clavarioides Kifz et al. (2019) P curta AMB 18641 MWwo092704 Italy Unpublished P curta AMB 18605 | mt452501 | - | Italy Unpublished P curta UBC F32034 | Kx23612e | - | Canada Unpublished P curta HAY-F-000746 | PP204846 | - | USA Unpublished P. echinoflava HKAS 45984 China This study P. echinoflava HKAS 45992 China This study P. flaccida AMB n. 18209 Italy Unpublished P. pseudozippelii Wannathes et al. (2018) Ramaria admiratia Hughes et al. (2014) R. calvodistalis Hughes et al. (2014) (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. MycoKeys 108: 1-14 (2024), DOI: 10.3897/mycokeys.108.128716 4 Peng-Tao Deng et al.: New species of Phaeoclavulina 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 Phaeoclavulina carovinacea AMB18551 1/100, Phaeoclavulina carovinacea TURA209584 Phaeoclavulina carovinacea AMB18533 Phaeoclavulina carovinacea AMB18534 4/100 00 Phaeoclavulina minutispora AMB18588 Phaeoclavulina minutispora AMB18586 1/100| Phaeoclavulina abietina OSC 140661 17100 Phaeoclavulina abietina OSC134649 Phaeoclavulina alboapiculata AMB18613 1/100} Phaeoclavulina alboapiculata AMB18585 Phaeoclavulina alboapiculata AMB18590 Phaeoclavulina curta UBCF32034 4/100) Phaeoclavulina curta HAYFOQ00746 Phaeoclavulina curta AMB18641 Phaeoclavulina curta AMB18605 1/007 Phaeoclavulina aeruginea MHHNU6887 0.99/81 Phaeoclavulina aeruginea MHHNU8909 0.99/ a Phaeoclavulina pseudozippelii BBH43575 1/100 Phaeoclavulina cyanocephala TH9064 768 Phaeoclavulina insignis FH104 Phaeoclavulina gigantea FH109 4/100) Phaeoclavulina clavarioides PRM945440 -/52 Phaeoclavulina clavarioides PRM945441 Phaeoclavulina cinnamomea MHHNU10376 Phaeoclavulina jilinensis MHHNU9164 1/100) Phaeoclavulina jilinensis MHHNU9149 0.99/87 Phaeoclavulina jilinensis MHHNU10504 Phaeoclavulina roellinii PRM945445 4/100 0.99/93 2 Phaeoclavulina flaccida AMBn18209 Phaeoclavulina coniferarum AMB18531 -/59 -/51 | Phaeoclavulina coniferarum AMB18562 ee Phaeoclavulina macrospora AMB18614 1/100| Phaeoclavulina bicolor MHHNU10702 Phaeoclavulina bicolor MHHNU10703 1/100| Phaeoclavulina echinoflava HKAS45984 Phaeoclavulina echinoflava HKAS45992 4/100 Ramaria admiratia TENN69114 Ramaria calvodistalis TENN69095 0.05 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. MycoKeys 108: 1-14 (2024), DOI: 10.3897/mycokeys.108.128716 Peng-Tao Deng et al.: New species of Phaeoclavulina 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 posi- tion sister to all other samples within the Phaeoclavulina clade. On the basis of these results, three new species of Phaeoclavulina based on the newly collect- ed Chinese specimens are proposed. 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. PR 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, dichot- omous to polychotomous, divided two to three times, internodes becoming gradually shorter, terminal branches short and not flat, in cross-section round- ed, 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. Micromorphology. Context hyphae in parallel arrangement, 3-4 um wide, cy- lindric, inflated, with clamp connections but not at every septum, thin-walled, smooth, hyaline. Basidia approximately 40-50 x 5-8 um with two sterigmata 5-6 um long, hyaline, clavate, with clamp connection at base. Cystidia absent. Basidiospores [60/3/3] (7.6)8-10(10.5) x (3.8)4-6(6.5) um [Q = 1.70-2.20, Q_ = 1.97 + 0.18], long-ellipsoid or cylindrical, slightly thick-walled, pale yellow in KOH, cyanophilic, surface coarse, echinulate, spines 0.6-—1.0 um 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) Fran- chi & 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 aeru- ginea has unique copper-green branch tips and has relatively larger spores than P. bicolor (13-16 x 8-9 um vs. 8-10 x 4-6 um). In the field, P cyanocephala MycoKeys 108: 1-14 (2024), DOI: 10.3897/mycokeys.108.128716 6 Peng-Tao Deng et al.: New species of Phaeoclavulina 20pm 10pm Figure 2. Basidiomata and microscopic features of Phaeoclavulina bicolor (MHHNU10702) A, B basidiomata C basidia D basidiospores. Scale bars: 5 cm (A, B). has relatively larger basidiomata (8-18 x 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 ba- sidiomata 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 my- celia at the base. Many branches diverge from the stalk, dichotomous to poly- chotomous, divided three to four times, branches thick and sparse, terminal MycoKeys 108: 1-14 (2024), DOI: 10.3897/mycokeys.108.128716 7 Peng-Tao Deng et al.: New species of Phaeoclavulina 20pm 10pm Figure 3. Basidiomata and microscopic features of Phaeoclavulina echinoflava (HKAS 45984) A, B basidiomata C basidia D basidiospores. Scale bars: 5 cm (A, B). 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 reac- tions not recorded. Micromorphology. Context hyphae in parallel arrangement, 5-8 um wide, cylindric, inflated to 12 um wide, clamp connections present, ampulliform clamps occasional, thin-walled, smooth, hyaline. Basidia approximately 22-34 x 6-8 um with four sterigmata 3-5 um long, hyaline, clavate or subclavate, with clamp connection at base. Cystidia absent. Basidiospores [60/2/2] (8.0)9.0- 10.5(11.0) x (3.2)3.5-4.0(4.5) um [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 um long, acute; hilar append- age 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 ba- MycoKeys 108: 1-14 (2024), DOI: 10.3897/mycokeys.108.128716 8 Peng-Tao Deng et al.: New species of Phaeoclavulina sidia are relatively larger than those of P. echinoflava (38-45 x 5.5-6.5 um vs. 22-34 x 6-8 um). 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) x (2.5)2.9(3.2) um vs. (8.0)9.0- 10.5(11.0) x (3.2)3.5-4.0(4.5) pm). P. echinovirens differs from P. echinoflava in having larger basidiomata (7.5-13 x 4.5-7.5 cm) and orange yellow tips. The phylogenetic reconstructions placed P. echinoflava in an isolated position with- in 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 fascicu- late, 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 branch- es 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 mac- rochemical reactions not recorded. Micromorphology. Context hyphae compact, 3-7 um wide, cylindric, inflated to 11 um wide, clamp connections present, thin-walled, smooth, hyaline. Basid- ia approximately 20-36 x 5-7 um with four sterigmata 3-5 um long, hyaline, clavate or subclavate, with clamp connection at base. Cystidia absent. Basid- iospores [60/3/3] (5.5)6.0-8.0(8.5) x (2.8)3.0-5.0(5.5) um [Q = 1.67-2.40, Q_ = 1.96 + 0.16], elongate-ellipsoid, thick-walled, pale yellow in KOH, cyano- philic, surface coarse, with short but distinct spines, spines 0.8-1.5 um long, strongly cyanophilous, hilar appendage distinct. Habit and distribution. Solitary on the humus layer under mixed forest; basid- iomata generally occur in summer. Known only from the type locality in China. Additional materials examined. + Jilin Province, Yanbian Korean Auton- omous 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 re- lated 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 MycoKeys 108: 1-14 (2024), DOI: 10.3897/mycokeys.108.128716 9 Peng-Tao Deng et al.: New species of Phaeoclavulina 20pm Figure 4. Basidiomata microscopic features of Phaeoclavulina jilinensis (MHHNU 10504) A, B basidiomata C basidia D basidiospores. Scale bars: 5 cm (A, B). other characters. Phaeoclavulina roellinii has long and narrowly clavate basidia (38-70 x 5-7(8) um), whereas P jilinensis has relatively short basidia (20-36 x 5-7 um). 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 x 5.5-6.5 vs. 20-36 x 5-7 um) distinguish P. flaccida from P jilinensis. Discussion In the field, Phaeoclavulina species can be easily confused with Ramaria or Cla- varia because in most Phaeoclavulina taxa the fruiting body is coralloid or rama- roid. 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 mono- phyletic Phaeoclavulina clade is consistently retrieved in phylogenetic analy- ses 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 MycoKeys 108: 1-14 (2024), DOI: 10.3897/mycokeys.108.128716 10 Peng-Tao Deng et al.: New species of Phaeoclavulina 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 Chi- na. Three new species are described, and the ITS+LSU phylogenetic trees strong- ly support that each of the species is monophyletic and phylogenetically distinct from other known species of Phaeoclavulina. These three new species were col- lected 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 adapt- able. Furthermore, the morphological distinctiveness of the three species is un- ambiguous; 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 Phaeoclavuli- na 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 phy- logenetic 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 SPECICS DiUGATS 5.552% feed nnn Seve cetecace ell cess DE eRe steed een n deal cet rete deanyes 2 = & Specles:cOrallOldsOr RAMAN Ol cece errs paw cceecanecnees <n eocelesnavgncten spree esexamecten 3 2 FRYING VIDIGE Ag toni e ee ee et Ae P. grandis eS Bee ra LUE 9 [21517 Mar ae Pee olivate Pan is) cele: perme Ot 4 ye PoP Sa we P. viridis 3 Spore spines connected into circular to semi-circular ridges ......R. 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) r(BasicdiomatarbGaisinG:Qreen er <.cos-ccsetececacncescsanacmelentas.ccunncastepeads P. flaccida rz. a DASHIO Mabe OI GISMMG ROCA rn. ..cys.seeadicnnlWe Satan ten canshe Os manptinateedces P. eumorpha GMM SOLES VSMC OS Cotas ket Re ol Ue Re ads 0s Meas Se aS ae 7 rn eS POFESCS CHIANG. curt Ne ou eR saeeer egret Meee cv sea ee ear erate ek 8 7 Basidiomata bruising olive or green on handling or weathering.....P. abietina - Basidiomata not bruising olive or green on handling or weathering ........... besa h une eiMdst umethat als Wad Pesaran ca neNe dab yererres took caMtodatn on co obeanaehe al vena P. sikkimia 8 “Hyphae avith HacOnneCton c..6. Actes ig ad Hevea ess tec nngeertioce Lia P. zippelii —- shiyphae-withOut:- HCOMMmMeCuOn: <2) etch .2.2s corct seed temeat yetese eegeces de oars Ae 9 9 Branch tips discolorous with basidiomata ..............ccccccssscccesseeeeeeseeeeeeaees 10 - Branch tips concolorous with basidiomata ...............ccccceseceeesseeeeesteeeeees 11 LOS -SpHoresealS WiilOng! 2.4.2 128m sissy eS ee Peek ec renee P. macrospora Sey ON ONSS te. TOM NOM tas xtaen a ae tele reser ee hed terse tea aed eeewns tants Renee mails 12 ‘Teh 'Basidiomiata: brightcolored | 7... WB. ak See teraccs cute setae 13 r= Bastdiomiata-deep-COlored?. fh: nseed satutrrasdben eet eetirhectunntesabieacieneges 14 12) “ Branchcips ellOWISMWIMTE Ys. ......52.1 deca idesatetedeeanecsnoad eladeandesshentsasacted P. bicolor = | (Branch wpsigreen-or DIGS. is. 2 chr ccc tcl OP phar ancobet ts Ms sucaeodacdl oubtntvacess 15 MycoKeys 108: 1-14 (2024), DOI: 10.3897/mycokeys.108.128716 i Peng-Tao Deng et al.: New species of Phaeoclavulina TRB asidioiiata-Orange Ole CINMAMION se. necescennercecuscamedent epcecreecnence perme eater cine 16 - Basidiomata bright-yellow or lemon-yellOW...............ccesccceeesseeeeerteeeeeteeeees 17 AL a BAS ICL Fiennes MIT eM th aaiachas ha beccendaan wes olas nandeaesada aman aa kale P. campestris am edn Fos | FO1 Ae bees kO8 S | | Rn cereals PE een rete Leta age Lm 18 EAI SEO DDE OLER MM coe scxceri les a arorusne states ted crasestrt fess uleresawelds P. aeruginea ll RATS DMC A et ely des teem ety nee 3a, Rese meee oder pan cd es ede ee An, 19 LBS! SORES AC UISS DIMCS 5a cheesy ativan dtycemed bvtalVncssanuerestna vecadverareat veosh nets P. cokeri — = SPOOLS: VOICEMC SPINES S: a..01.0 .<.eaerraerieatdecd teers Wernaetees P. cinnamomea 17 Tips blunt, broom-form or short-digitate ...................ccccceeees P. echinoflava = TIPSTAGUS TAD STOO se Feces vecask avuy encterranhaeet en ance ccaxathices acany saben age P jilinensis TO a VMEMIUIMTAMIP MIG SN OUSiccecssucedecerrarenees spay eeessupaateen seavtedenuoenteaas P. capucina wa Pee aNd (WI GCAO LAT re Wie] ia Ds comes be Srareadien a cach arepben ch 1 prt Sippemka A Ak Lech freee ntact 20 OS POLS 2 Ot OMG banc teoduas ais nate macs tatec Mcrdacle tn anieedtarenkl es P. mutabilis Fs SSPOLSS > TO MIONG) 0s .0sen saactrrrenncdieecadeag thesauri secteur sens P. cyanocephala 20 Spores 4.5-6.5 um long, basidia 25-36 UM IOng............ce cece eee P. curta - Spores 7-11 um long, basidia 40-50 um long................ ee 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. Su- pervision: 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 ® hittps://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. 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