The genus Clavariadelphus (Clavariadelphaceae, Gomphales) in China

Hong-Yan Huang; Jie Zhao; Ping Zhang; Zai-Wei Ge; Xian Li; Li-Ping Tang

doi:10.3897/mycokeys.70.54149

Abstract

Clavariadelphus species (Clavariadelphaceae, Gomphales) in China were examined using morphology, molecular phylogenetic analyses of ITS data and chemical reactions. Eleven taxa were identified in China, including four species known previously to occur in China (C. griseoclavus, C. ligula, C. sachalinensis and C. yunnanensis), two new record species from China (C. elongatus and C. himalayensis), four novel species (C. alpinus, C. amplus, C. gansuensis and C. khinganensis) and one species that could not be described due to the paucity of material. Finally, we also provided a taxonomic key for the identification of Clavariadelphus species in China.

Keywords

Clavarioid fungi, taxonomy, molecular systematics, new taxa, species diversity

Introduction

Clavariadelphus Donk (Clavariadelphaceae, Gomphales, Basidiomycota), typified by C. pistillaris (L.) Donk, is a group of fungi characterised by erect, simple, club-shaped basidiomes with rhizomorphs at the stipe base, hymenium with (2–) 4-spored basidia, clavate leptocystidia, ellipsoid to amygdaliform, thin-walled, inamyloid basidiospores and clamp connections at the septa of the hyphae (Methven 1990). The genus is widely distributed in temperate regions of the Northern Hemisphere and 24 species were described before this study.

Clavariadelphus has been studied in Europe and North America and important taxonomic works are available (Corner 1950, 1970; Welden 1966; Smith and Corner 1967; Petersen 1967, 1972; Smith 1971; Petersen et al. 1974; Methven 1989; Methven and Guzmán 1989). The genus has not received as much attention in Asia, except for a couple of novel species described from Pakistan (Hanif et al. 2014; Sher et al. 2018). In China, two novel taxa have been described (Methven 1989; Lu and Li 2020). To date, only seven Clavariadelphus species have been reported in China, namely C. griseoclavus L. Fan & L. Xia, C. ligula (Schaeff.) Donk, C. pallido-incarnatus Methven, C. pistillaris, C. sachalinensis (S. Imai) Corner, C. truncatus Donk and C. yunnanensis Methven (Methven 1989, 1990; Mao et al. 1993; Yuan and Sun 1995; Zang 1996; Bau et al. 2003; Mao 2009; Tang and Yang 2014; Tang 2015; Lu and Li 2020). The studies, in which these species were identified, are comparatively brief and solely based on morphological criteria except C. griseoclavus.

Although Clavariadelphus can be readily distinguished from other members of the Gomphales, the delimitation of infrageneric taxa is difficult in many cases due to subtle variations in morphological characteristics and growth habits (Methven 1990). Recently, molecular techniques have been widely applied and have provided useful information for species delimitation in systematic fungal studies (Hibbett 2007; Yang 2011). Chemical reactions are also helpful in delimiting species of many macrofungal groups besides Clavariadelphus, including Agaricus, Boletopsis, Chroogomphus, Cortinarius, Hygrophorus, Leucoagaricus and Leratiomyces (Corner 1950; Hanif et al. 2014; Siegel and Schwarz 2016). Scanning electron microscopy (SEM) has been applied to the identification of other macrofungal groups (Zeng et al. 2013; Tang et al. 2014; Huang et al. 2018). However, SEM of structures of Clavariadelphus has not yet been reported. We mainly examined Chinese Clavariadelphus collections through analysis of morphological characteristics using light microscopy and SEM, as well as molecular phylogenetic data, ecological data and chemical reactions, to better understand species diversity of Clavariadelphus in China.

Materials and methods

Morphological studies

Aside from one collection from the Czech Republic, most specimens of Clavariadelphus in this study were collected from coniferous forests or mixed coniferous and broad-leaved forests in North (N) China, Northwest (NW) China and Southwest (SW) China during the rainy seasons (July–September). Collections and field records are deposited in the Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences (HKAS), Mycological Herbarium, Institute of Mycology, Chinese Academy of Sciences (HMAS), Mycological Herbarium of Hunan Normal University (MHHNU) and Mycological Herbarium of Pharmacy College, Kunming Medical University (MHKMU) (Appendix 1). Specimens and their habitats were photographed in situ. Relevant metadata, such as altitude, latitude, longitude and nearby tree associates were recorded in the field. Detailed notes on macro-morphological descriptions were taken from fresh material and colour codes were from Kornerup and Wanscher (1981).

Light microscopy

Micro-morphological characteristics were observed under a light microscope (Leica DM 2500). Preparations were made from dried specimens. Tissue fragments of dried materials were sectioned, mounted in 10% KOH and observed. The abbreviation [n/m/p] means n basidiospores measured from m basidiomes of p collections. Dimensions for basidiospores are given as (a) b–c (d). The range of b–c contains a minimum of 90% of the measured values. Extreme values, i.e. a and d are given in parentheses. Q is used to denote the length/width ratio of basidiospores in the side view, whereas Q_m refers to the average Q value of all basidiospores ± standard deviation.

Scanning electron microscopy

The material was sampled and directly used from herbarium collections. The hymenium and basal mycelium from dried specimens were mounted on to aluminium stubs coated with gold palladium. Basidiospores and hyphae of the basal mycelium were observed and micrographs were taken with a ZEISS Sigma 300 scanning electron microscope at 7.0 kV accelerating voltage.

Chemical reactions

Seven chemical reagents were used: 10% (w/v) KOH, 10% (w/v) FeCl₃, 10% (w/v) FeSO₄, 10% NH₄OH, 10% (w/v) phenol, Melzer’s reagent and 95% (v/v) ethanol. Small slices of tissue were taken from the hymenium of the basidiomes. The reagents were systematically added to the depression in plates so that each piece of tissue was submerged in several drops of a single reagent. Positive colour reactions were recorded immediately following the application of reagents.

DNA extraction, PCR and DNA sequencing

Total genomic DNA was isolated from dried materials using a modified CTAB method (Doyle 1987) with a prolongation of the extraction period as necessary. For PCR reactions, the nuclear ribosomal DNA internal transcribed spacer (ITS) region was amplified using primers ITS5 and ITS4 (White et al. 1990). The PCR amplification mix consisted of a total volume of 25 μl containing 2.5 μl of 10 × amplification buffer (with MgCl₂), 0.5 μl dNTP (200 μM), 0.2 μl Taq DNA polymerase (5 U/μl), 1 μl of each primer (10 μM), 1 μl DNA template and 18.8 μl sterile water. PCR reactions were performed with an initial denaturation at 94 °C for 4 min; 38 cycles of denaturation at 94 °C for 40 s, annealing at 54 °C for 40 s, extension at 72 °C for 60 s; and a final extension at 72 °C for 8 min. PCR products were checked on 1% agarose gel. Successful reactions were sequenced using an ABI 3730 DNA Analyzer (Sangon, Shanghai, China) with both PCR primers. The DNA sequences were used as queries in NCBI BLAST searches to rule out contamination. The forward and reverse sequences were assembled with SeqMan (DNASTAR Lasergene 9) and their quality controlled according to the guidelines of Nilsson et al. (2012). Novel and already available sequences were aligned by using MAFFT version 7 (Katoh and Standley 2013). The alignment was manually adjusted in BioEdit version 7.0.9 (Hall 1999) and trimmed in trimAl version 1.2 (Capella-Gutiérrez et al. 2009).

Phylogenetic analyses

Two phylogenetic tree inference methods, Randomised Accelerated Maximum Likelihood (RAxML) and Bayesian Analysis (BA), were used to analyse the ITS sequence data. The programme RAxML version 7.0.3 (Stamatakis et al. 2008) was used to infer a maximum likelihood tree with bootstrap support values and the GTRGAMMA was selected as a default model. The programme MrBayes version 3.2.6 (Ronquist et al. 2012) was run using a Markov Chain Monte Carlo (MCMC) tree sampling procedure. The ITS1, 5.8S and ITS2 loci were extracted from the aligned ITS dataset, allowing the selection of substitution models for each partition. Aligned sequences were partitioned into ITS1 (1–270), 5.8S (271–429) and ITS2 (430–703). Nucleotide substitution models based on the Akaike Information Criteria (AIC) data were obtained in PartitionFinder 2 (Lanfear et al. 2016). The selected models were GTR+G for ITS1, K80 for 5.8S and HKY+G for ITS2. After four simultaneous Markov chains running with 7,000,000 generations and sampling every 100 generations, the average deviation of split frequencies was 0.004022 at the end of the run. Burn-in values were determined in Tracer v1.7 (Rambaut et al. 2018). Effective sample sizes were well over 200 for all sampled parameters for each run and the initial 20% of the samples was discarded. Bayesian Posterior Probabilities (PP) were calculated for a majority consensus tree of the retained Bayesian trees.

Results

Taxonomic identification based on morphological data

Fifty specimens of Clavariadelphus were examined in this study. Six species were previously reported from China, except the late described one, C. griseoclavus. However, the re-examination of available vouchers confirmed the occurrence of only three of these species, specifically C. ligula, C. sachalinensis and C. yunnanensis. Our morphological observations revealed that nine taxa, including three species previously identified in China (C. ligula, C. sachalinensis and C. yunnanensis), two species that have not been previously reported from China (C. elongatus and C. himalayensis) and four novel species (C. alpinus, C. amplus, C. gansuensis and C. khinganensis), were identified on the basis of morphological characters. So far, there are ten described taxa in China, including C. griseoclavus which is recently published.

Taxonomic identification based on molecular data

The ITS dataset comprised 27 ingroup taxa including the type species C. pistillaris and three outgroup taxa, with 64 sequences in total. The length of the alignment was 703 aligned bases (TreeBASE accession 24163). Three species of Lentaria Corner and Kavinia Pilát were chosen as outgroups in the dataset, based on a previous study (Giachini et al. 2010).

In the phylogeny, based on ITS sequences, few differences in the topology of major clades were detected between the ML and Bayesian analyses. Twenty-seven phylogenetic species were recovered, amongst which, eleven species were from China, including one with a GenBank accession JQ991679 from Zhejiang Province, China, which might represent a separate species in the tree (Fig. 1). Clavariadelphus sachalinensis formed a distinct lineage with high support and was sister to the rest of the genus. Seven Chinese lineages, namely C. amplus, C. elongatus, C. griseoclavus, C. himalayensis, C. ligula, C. khinganensis and C. yunnanensis, were strongly supported as monophyletic groups. The other two species from China, namely C. alpinus and C. gansuensis, were each represented by only one specimen in the phylogenetic tree. The sister of each Chinese taxon is discussed below.

Figure 1.

Phylogenetic tree of Clavariadelphus based on ITS sequence data. RAxML BP values (≥ 50%) are shown above branches, Bayesian posterior probabilities (≥ 0.90) are shown above branches; new taxa are marked in red.

Taxonomic identification based on chemical reactions

Steglich et al. (1984) proposed that a positive ferric salts reaction of the basidiomes was indicative of the presence of pistillarin in the basidiomes of Clavariadelphus. To a large extent, Methven’s study (1990) supported this hypothesis, excluding one exception (C. cokeri V.L. Wells & Kempton). Methven (1990) mentioned the negative ferric salts reaction of some species might be the result of pistillarin being present in too low concentrations or the result of samples affected by pesticides during storage. In our study, most species have positive reactions with four reagents (FeCl₃, KOH, NH₄OH and phenol), but all species from China showed a negative reaction to FeSO₄, Melzer’s reagent and ethanol. The results of the chemical testing in this study are summarised in Table 1. As those specimens are preserved, pesticides are used regularly. Thus, we agree with Methven’s argument (1990).

Table 1.

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XLSX

Chemical reactions of representative species of Clavariadelphus from China.

Taxa	KOH	FeCl₃	NH₄OH	Phenol	Ethanol	Melzer’s reagent	FeSO₄
C. alpinus	3B8	–	6A8	–	–	–	–
C. amplus	12A4	1A8	2A8	2A5	–	–	–
C. elongatus	2A5	1A8	6A8	–	–	–	–
C. gansuensis	9B7	1A8	2A8	2A8	–	–	–
C. himalayensis	5B7	30A8	6A8	–	–	–	–
C. khinganensis	2A5	–	–	–	–	–	–
C. ligula	3B8	–	6A8	–	–	–	–
C. sachalinensis	2A5	30A8	6A8	–	–	–	–
C. yunnanensis	5B7	30A8	2A8	2A5	–	–	–

Taxonomy

1. Clavariadelphus alpinus J. Zhao & L.P. Tang, sp. nov.

MycoBank No: MycoBank No: 830258

Figs 2a, 3a, 4a, 5a, 6a, b

Diagnosis

This species is distinguished from other taxa in Clavariadelphus by the light yellow, clavate basidiomes with enlarged apex, broadly ellipsoid basidiospores, hyphae of the basal mycelium with nipple-shaped protuberances and basidiomes turning lemon-chiffon in KOH.

Figure 2.

Clavariadelphus species in China. a C. alpinus (HKAS 57396, holotype) b, c C. amplus (HKAS 54876, holotype) d, e C. elongatus (d from HKAS 50742 e from HKAS 76589) f C. gansuensis (HKAS 76487, holotype) g C. himalayensis (HKAS 58811) h, i C. khinganensis (h from MHHNU 7789, holotype i from MHKMU H.Y. Huang 368) j C. sachalinensis (MHHNU 7816) k, l C. yunnanensis (k from HKAS 49398 l from HKAS 58789).

Figure 3.

Basidiospores of Clavariadelphus under light microscope. a C. alpinus (HKAS 57396, holotype) b C. amplus (HKAS 54876, holotype) c C. elongatus (HKAS 76589) d C. gansuensis (HKAS 76487, holotype) e C. himalayensis (HKAS 58811) f C. khinganensis (MHHNU 7789, holotype) g C. ligula (HKAS 35954) h C. sachalinensis (MHHNU 7816) i C. yunnanensis (HKAS 57659).

Etymology

Latin “alpinus” refers to this species occurring in high-altitude areas.

Description

Basidiomes up to 12 cm high, 0.9 cm diam. at the base, enlarged upwards to 2 cm diam., simple, initially cylindrical to subcylindrical, then narrowly clavate to clavate, laterally compressed in age; hymenium initially smooth, then longitudinally rugose, light yellow (4A4–5) to yellow or yellowish-orange, apricot-yellow, light orange-yellow (4A6–7) or (5A5–6); apex subacute to obtuse, smooth to rugose, concolorous with the hymenium; surface not staining when cut or bruised; base terete, smooth, white to cream; mycelial hyphae white; flesh initially solid, then soft and spongy upwards as the apex enlarges, white not staining on exposure. Odour and taste not recorded. Spore deposit not recorded.

Hymenium extending over the apex of basidiomata, composed of basidia and leptocystidia. Basidia 65–85 × 8–10 μm, clavate, hyaline, thin-walled, (2–, 3–) 4-spored, sterigmata 8–12 μm in length. Basidiospores [20/1/1] (7.4–) 7.8–9.6 (–10.1) × 5.5 (–5.1)–7.4 μm, Q = 1.25–1.55 (–1.58), Q_m = 1.38 ± 0.10, broadly ellipsoid, ovate or amygdaliform, with a small apiculus, inamyloid, thin-walled, hyaline in KOH, smooth. Leptocystidia 45–55 × 2.8–4.2 μm, scattered amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity and at times, with apical or subapical branches. Mycelial hyphae 2–4 μm diam., interwoven or aggregated into rhizomorphic strands, branched, clamped; hyphal walls echinulate with light microscopy, covered with massive nipple-shaped protuberances without crystals with SEM.

Chemical reactions

(dried basidiomes). KOH = positive, lemon-chiffon; NH₄OH = positive, orange; ethanol, FeCl₃, FeSO₄, Melzer’s reagent and phenol = negative.

Known distribution and ecology

SW China, Yunnan Province. Solitary on the ground in forests dominated by conifers (e.g. Abies georgei) at elevations of approximately 3700 m.

Materials examined

China. Yunnan Province: Shangri-la Prefecture, Bita Lake, 24 August 2009, approximately 3700 m elev., B. Feng 667 (HKAS 57396, Holotype).

Comments

Clavariadelphus alpinus is well characterised by its yellow basidiomes, broadly ellipsoid basidiospores, hyphae of the basal mycelium with nipple-shaped protuberances, the apex of the basidiomes having a positive reaction to NH₄OH and KOH and distribution at high elevations in SW China in association with conifers.

Morphologically, this taxon is similar to C. khinganensis. However, C. khinganensis has light brown-tan basidiomes, more elongated basidiospores (Q = 1.6–2.2), negative reaction to NH₄OH and distribution at lower elevations in NE China.

In the ITS phylogeny, this species is a sister species of C. truncatus with strong support (Fig. 1). However, C. truncatus differs from C. alpinus by having dark coloured basidiomes from yellow to cinnamon-brown or brown, broader apices (up to 3.5 cm) and larger basidiospores (10.3–12.6 × 5.5–7.1 μm from neotype; Methven 1990).

2. Clavariadelphus amplus J. Zhao, L.P. Tang & Z.W. Ge, sp. nov.

MycoBank No: MycoBank No: 830271

Figs 2b, c, 3b, 4b, 5b, 7a, b.

Diagnosis

This species is unique in its pink-orange basidiomes with enlarged, truncate and sterile apices, ellipsoid basidiospores, hyphae of the basal mycelium with nipple-shaped protuberances and prism-like crystals and basidiomes turning cherry-red in KOH. It differs from C. truncatus by the latter’s darker coloured basidiomes, narrower apices and larger basidiospores.

Figure 4.

Basidiospores of Clavariadelphus under SEM. a C. alpinus (HKAS 57396, holotype) b C. amplus (HKAS 54876, holotype) c C. elongatus (HKAS 76589) d C. gansuensis (HKAS 76487, holotype); e, f C. himalayensis (HKAS 58811) g C. khinganensis (MHHNU 7789, holotype) h C. yunnanensis (HKAS 57659).

Figure 5.

Hyphae of basal mycelium from Clavariadelphus under SEM. a C. alpinus (HKAS 57396, holotype) b C. amplus (HKAS 54876, holotype) c C. elongatus (HKAS 76589) d C. gansuensis (HKAS 76487, holotype) e C. himalayensis (HKAS 58811) f, g C. sachalinensis (f from HKAS 33844; g from MHHNU 7816) h C. yunnanensis (HKAS 57659).

Etymology

Latin “amplus” refers to the enlargement of the apex of the basidiomes.

Description

Basidiomes up to 15 cm high, 0.5–1 cm diam. at the base, enlarged upwards to 3–7.5 cm diam. near apex; hymenium initially smooth, longitudinally rugulose in age, pruinose, pinkish-orange (7A5–7), paler downwards, greyish-orange (5B4–5); apex initially obtuse or broadly rounded, finally truncate, depressed, surface rugose to rugulose, more or less darker than the hymenium, apricot-yellow (5B6–7) to pink-orange, reddish-orange (7A7–8) or red-orange (7B7–8) at maturity; surface slowly staining light brown or light leather-brown (7D6–7) to brown (7E6–7) when cut or bruised, staining more conspicuously downwards; base simple, terete, nearly smooth, cylindrical to subcylindrical, pruinose; mycelial hyphae interwoven, white; flesh solid initially, then soft and spongy upwards as the apex enlarges, white, slowly staining light leather-brown (7D6–7) to brown (7E6–7) on exposure. Odour pleasant. Taste not distinctive. Spore deposit not recorded.

Hymenium limited to the sides of basidiomes, composed of basidia and leptocystidia; the apex of basidiomata is composed of sterile elements 18–28 × 5–8 μm, clavate, thin-walled, smooth, clamped. Basidia 85–95 × 8–12 μm, clavate, hyaline, thin-walled, (2–) 4-spored, sterigmata 9–11 μm in length. Basidiospores [40/2/2] 8.2–11.0 × 5.1–6.4 μm, Q = (1.36–) 1.38–2.00 (–2.18), Q_m = 1.75 ± 0.17, ellipsoid to broadly ellipsoid, ovate or amygdaliform, with a small apiculus, inamyloid, thin-walled, hyaline in KOH, smooth. Leptocystidia 45–70 × 2.8–3.8 μm, scattered amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity and at times, with apical or subapical branches. Mycelial hyphae 2–4 μm diam., parallel, interwoven or aggregated into rhizomorphic strands, branched, clamped; walls thin or irregularly slightly thickened, the hyphal walls echinulate with light microscopy, covered with nipple-shaped protuberances, as well as encrusted with prism-like crystals (up to 6 μm long) that are insoluble in KOH.

Chemical reactions

(dried basidiomes): FeCl₃ = positive, green-yellow; KOH = positive, cherry-red or pink; NH₄OH = positive, golden-rod or vivid yellow; phenol = positive, light yellow; ethanol, FeSO₄, and Melzer’s reagent = negative.

Known distribution and ecology

NW China and SW China, and India. Gregarious habit on the ground in conifer or mixed conifer forests (e.g. Abies spp. and Picea spp.) at elevations ranging from 3000–3950 m.

Materials examined

China. Gansu Province: Zhouqu Prefecture, under Abies spp., 6 August 2005, X.T. Zhu 728 (HKAS 76577). Qinghai Province: Qilian mountains, 38°6.00'N, 100°7.03'E, alt. 3000 m, 21 August 2004, H.A Wen 4305 (HMAS 132008); same location and date, Q.B. Wang 438 (HMAS 97090). Sichuan Province: Seda Prefecture, Picea-Juniperus forests, 31°43.20'N, 100°43.17'E, alt. 3775–3925 m, 6 August 2005, Z.W. Ge 783 (HKAS 49278); Litang Prefecture, 5 August 2007, Z.W. Ge 1712 (HKAS 53797). Tibet: Linzhi City, 29°20.07'N, 094°18.00'E, alt. 3850 m, 19 July 2004, Y.H. Wang 125 (HMAS 97248); Jilong Prefecture, on the ground in coniferous woods, 12 September 1990, J.Y. Zhuang 3814 (HMAS 59867); Chengdu City, under forests dominated by Picea spp., 31°30.43'N, 097°20.07'E, alt. 3480–3550 m, 17 August 2004, Z.W. Ge 381 (HKAS 46160); Riwoqe Prefecture, under Picea spp., 31°14.27'N, 096°31.92'E, alt. 3890 m, 12 August 2004, Z.W. Ge 340 (HKAS 46120). Yunnan Province: Shangri-La Prefecture, Haba Snow Mountains, alt. 2800 m, 15 August 2008, L.P. Tang 645 (HKAS 54876, Holotype); Shangri-La Prefecture, 27°28.13'N, 099°25.03'E, alt. 3600 m, 15 August 2008, T.Z. Wei 172 (HMAS 250466).

Comments

Clavariadelphus amplus is distinctive by its pink-orange to red-orange, bright basidiomes, obviously enlarged, truncate, depressed, sterile apices (up to 7.5 m diam.) at maturity, large basidiospores (8.2–11.0 × 5.1–6.4 μm), gregarious habit at high elevations, base mycelial hyphae with nipple-shaped protuberances and prism-like crystals and a cherry-red staining reaction to KOH. It is sold as an edible mushroom in markets in SW China. This taxon has a wide distribution in NW and SW China, including Gansu, Qinghai, Sichuan, Tibet and Yunnan Provinces. The data from GenBank (accession MT012805) also indicated its distribution of India.

This species was previously referred to as either C. pallido-incarnatus (Yuan and Sun 1995) or C. truncatus (Mao et al., 1993; Zang 1996; Mao 2009; Tang and Yang 2014; Tang 2015). Clavariadelphus pallido-incarnatus, a species described from the Pacific Northwest in North America, has pale pinkish-cinnamon basidiomes with fertile, non-truncated apices, no reactivity to KOH and habitat preference for coastal forests of Sequoia sempervirens and Picea sitchensis (Methven 1990). Clavariadelphus truncatus from Europe is readily confused with C. amplus as they have similar size and truncate sterile apex. However, C. truncatus has dark coloured basidiomes from yellow to cinnamon-brown or brown, narrower apices (up to 3.5 cm) and larger basidiospores (10.3–12.6 × 5.5–7.1 μm from neotype; Methven 1990). Clavariadelphus unicolor (Berk. & Ravenel) Corner, is also from North America and has enlarged sterile apices, but it is distinct in its reddish-brown to violet-brown basidiomes, narrow basidiospores with Q_m 2.1, a golden-yellow reaction to KOH and association with deciduous trees (Methven 1990).

So far, there are two species with sterile apices found in China, C. amplus and C. gansuensis. However, C. gansuensis has a narrower apex (up 1.6 cm), slightly broader basidiospores with a lower Q value (8.3–10.1 × 5.3–6.3 μm, Q = 1.47 –1.78, Q_m = 1.60) and a solitary growth habit. Except for the mentioned species, C. amplus is also similar to C. pakistanicus. Clavariadelphus pakistanicus, another species also from Asia, is distinct in smaller basidiomes (up to 12 cm high), with narrower fertile apices (up to 1.4 cm), smaller basidiospores (7.5–9.2 × 4.0–5.6 μm), solitary growth habit at lower elevations and violet-brown staining reactions to KOH (Hanif et al. 2014).

In the ITS tree, C. amplus exhibits a sister relationship with C. pakistanicus with strong support (Fig. 1).

Figure 6.

Microscopic features of Clavariadelphus alpinus (HKAS 57396, holotype). a Basidia b Leptocystidia.

Figure 7.

Microscopic features of Clavariadelphus amplus (HKAS 54876, holotype). a Leptocystidia and immature basidia b Basidia.

3. Clavariadelphus elongatus J. Khan, Sher & Khalid, Phytotaxa 365: 184, 2018

Figs 2d, 2e, 3c, 4c, 5c, 8a, 8b

Note

The following description is taken from Sher et al. (2018), field notes of the Chinese material including macro-morphology, growth habit, distribution, host plants and our examination of the specimens.

Figure 8.

Microscopic features of Clavariadelphus elongatus (HKAS 76589). a Leptocystidia and immature basidia b Basidia.

Description

Basidiomes up to 28 cm high, 0.5–1.0 cm diam. basally, enlarged upwards to 1.5 cm diam., subcylindrical to fusiform, simple or occasionally branched, laterally compressed in age; hymenium longitudinally rugose, plum colour (13C2–4) or light purple to greyish-purple (14C2–3) or dull-lilac (15D2–3); apex tapered, subacute to obtuse, initially smooth, rugulose in age, caramel-brown to sandy-brown or sienna (6C5–6); base terete, smooth, white; mycelial hyphae scant, white; flesh initially solid, then soft and spongy in age. Odour and taste not recorded.

Hymenium extending over the apex of the basidiomata, composed of basidia and leptocystidia. Basidia 75–95 × 6–10 μm, clavate, hyaline, thin-walled, 4-spored, sterigmata 7–10 μm in length. Basidiospores [40/2/2] (8.3–) 9.0–11.0 (–12.0) × (5.5–) 5.7–7.4 μm, Q = (1.43–) 1.44–2.04 (–2.31), Q_m = 1.71 ± 0.16, narrowly ellipsoid to ellipsoid, ovate or amygdaliform, with a small apiculus, inamyloid, thin-walled, hyaline in KOH, smooth. Leptocystidia 70–75 × 3.5–4.5 μm, scattered amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity, at times with apical or subapical branches. Mycelial hyphae 2–3 or 6–8 μm diam., interwoven or aggregated into rhizomorphic strands, branched, clamped; the hyphal walls echinulate with light microscopy, encrusted with massive triangular or irregular, flaky crystals up 1 μm high, which are insoluble in KOH.

Chemical reactions

(dried basidiomes): KOH = positive, light yellow; FeCl₃ = positive, green-yellow; NH₄OH = positive, orange; ethanol, FeSO₄, phenol and Melzer’s reagent = negative.

Known distribution and ecology

NW and SW China (in this study), Pakistan (Sher et al. 2018). Solitary to scattered on the ground in coniferous woods (Abies spp. and Picea spp.) or mixed with broad-leaved trees (Quercus spp., Rhododendron spp. and Salix spp.) at elevations ranging from 3000–4350 m.

Materials examined

China. Gansu Province: Zhouqu Prefecture, Shatan National Forest Park, Abies spp. woods, 16 August 2012, X.T Zhu 740 (HKAS 76589). Sichuan Province: Litang Prefecture, Gaowa, Kobresia-Bistorta meadows with extensive areas of dwarf Rhododendron and Salix scrub with Picea spp., 30°10.10'N, 100°35.12'E, alt. 4300–4350 m, 8 August 2006, Z.W. Ge 1221 (HKAS 50801); Yajiang Prefecture, meadows with shrub thickets and Picea spp. forests, 30°2.67'N, 101°18.48'E, alt. 3850–3870 m, 4 August 2006, Z.W. Ge 1162 (HKAS 50742). Yunnan Province: Yulong Prefecture, Lizui Village, mixed coniferous and broad-leaved forests of Picea spp. and Quercus spp., alt. 3000 m, 23 August 2007, Y. Zhang 36 (HKAS 52425); Shangri-La Prefecture, 27°29.00'N, 99°25.00'E, alt. 3600 m, 13 August 2008, T.Z. Wei 150 (HMAS 260746).

Comments

Clavariadelphus elongatus was originally described from Pakistan (Sher et al. 2018). In this study, it was found in NW and SW China. This species is unique in its greyish-purple basidiomes with acute to subacute, non-enlarged apex, hyphae of the basal mycelium encrusted with massive, flaky crystals and basidiomes having a light yellow reaction to KOH. Clavariadelphus himalayensis, another Asian taxon, might be confused with C. elongatus since both have a tinge of grey-purple when young. However, C. himalayensis is distinct in having smaller basidiomes, pastel-red colouration at maturation, shorter basidiospores (8.2–9.4 × 5.0–5.5 μm), hyphae of the basal mycelium covered nipple-shaped protuberances without crystals and basidiomes having a brown-yellow reaction to KOH.

Phylogenetically, C. elongatus is related to C. pistillaris and the sequence of “C. occidentalis” from GenBank with weak support (Fig. 1).

4. Clavariadelphus gansuensis J. Zhao & L.P. Tang, sp. nov.

MycoBank No: MycoBank No: 830272

Figs 2f, 3d, 4d, 5d, 9a, b

Diagnosis

This species is characterised by its orange, clavate basidiomes with slightly enlarged, truncate, sterile apex, broadly ellipsoid to ellipsoid basidiospores, hyphae of the basal mycelium with nipple-shaped protuberances and prism-like crystals and basidiomes that turn pink or light cherry-red in KOH. It differs from C. truncatus by the latter’s robust, darker basidiomes with enlarged apices, and larger basidiospores.

Figure 9.

Microscopic features of Clavariadelphus gansuensis (HKAS 76487, holotype). a Leptocystidia and immature basidia b Basidia.

Etymology

Latin “gansuensis” refers to the holotype location in Gansu Province.

Description

Basidiomes up to 9 cm high, enlarged upwards to 1.6 cm diam., simple, clavate; hymenium longitudinally rugose, pruinose, light yellow to greyish-orange at maturity; apex initially obtuse or broadly rounded, flattening laterally, then truncate, slightly rugose, light orange or melon-orange (5A5–7) to orange (6A6–7) in age; base terete, smooth, pruinose, dirty white or pallid where covered, otherwise pruinose, pale orange or light orange (5A3–4); mycelial hyphae white; flesh initially solid, then soft and spongy upwards as the apex enlarges, white to pallid. Odour and taste not recorded.

Hymenium limited to the side of basidiomata, composed of basidia and leptocystidia; the apex of basidiomata composed of sterile elements 15–25 × 5–7 μm, clavate, thin-walled, smooth, clamped. Basidia 75–90 × 8–10 μm, clavate, hyaline, thin-walled to thick-walled, 4-spored, sterigmata 7–10 μm in length. Basidiospores [20/1/1] 8.3–10.1 (–10.3) × 5.3–6.3 (–6.4) μm, Q = (1.34–) 1.47 –1.78 (–1.83), Q_m = 1.60 ± 0.09, ellipsoid to broadly ellipsoid, ovate or amygdaliform, with a small apiculus, inamyloid, thin-walled, hyaline in KOH. Leptocystidia 50–65 × 3–5 μm, scattered amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity, at times with apical or sub-apical branches. Mycelial hyphae 2–3 μm diam., interwoven or aggregated into rhizomorphic strands, branched, clamped; the hyphal walls echinulate with light microscopy, covered with massive nipple-shaped protuberances, as well as encrusted with prism-like crystals up 5 μm long that are insoluble in KOH.

Chemical reactions

(dried basidiomes): KOH = positive, pink, light coral or light cherry-red; FeCl₃ = positive, green-yellow; NH₄OH = positive, golden-rod or vivid yellow; phenol = positive, yellow; ethanol, FeSO₄ and Melzer’s reagent = negative.

Known distribution and ecology

NW China, Gansu Province. Solitary on the ground in coniferous woods (Abies spp.) or mixed with broad-leaved trees (Betula spp. and Rosaceae) at elevations of approximately 3000 m.

Materials examined

China. Gansu Province: Lintan Prefecture, Yeliguan National Forest Park, coniferous woods (Abies spp.) or mixed with Betula spp. and Rosaceae plants, alt. 3000 m, 10 August 2012, X.T. Zhu 638 (HKAS 76487, Holotype); Wudu Prefecture, September 1992, M.L. Tian M6465 (HMAS 63052).

Comments

Clavariadelphus gansuensis, currently known only from NW China, is distinct by its slender, clavate, orange basidiomes with truncate apex, ellipsoid basidiospores (8.3–10.1 × 5.3–6.3 μm), pink staining reaction to KOH, hyphae of the basal mycelium with nipple-shaped protuberances and prism-like crystals and solitary growth habit in coniferous or mixed forests.

This species is most likely to be confused with several taxa, including C. amplus, C. pallido-incarnatus, C. pakistanicus, C. truncatus and C. unicolor. The comparison between C. gansuensis and C. amplus can be found in our treatment of C. amplus.

According to our phylogenetic analyses, C. gansuensis is allied with the sequence of “C. truncatus” from GenBank with strong support (Fig. 1).

5. Clavariadelphus himalayensis Methven, Mem. New York Bot. Garden 49: 152, 1989

Figs 2g, 3e, 4e, f, 5e, 10a, b

Note

The following description is mainly from Methven (1989), combined with our field notes, including macro-morphology, growth habit, distribution, host plants and examination.

Figure 10.

Microscopic features of Clavariadelphus himalayensis (HKAS 58811). a Leptocystidia and immature basidia b Basidia.

Description

Basidiomes up to 15 cm high, 1–1.5 cm diam. basally, slightly enlarged towards to 2 cm diam., simple, narrow clavate, ligulate to spathulate, laterally compressed in mature specimens; hymenium initially smooth, longitudinally rugose in age, greyish-red to pastel-red; apex obtuse, smooth, concolorous with the hymenium; surface not staining where cut or bruised; base terete, smooth, pruinose, pallid-white; mycelial hyphae interwoven, white to pallid; flesh soft and spongy, hollow apically in age, white to cream colour, not staining on exposure. Odour and taste not recorded.

Hymenium extending over the apex of basidiomata, composed of basidia and leptocystidia. Basidia 75–95 × 8–11 μm, clavate, hyaline, thin-walled, (2–) 4-spored, sterigmata 8–10 μm in length. Basidiospores [20/1/1] (7.8–) 8.2– 9.4 (–9.6) × (4.6–) 5.0–5.5 (–6.0) μm, Q = 1.50–1.82 (–1.90), Q_m = 1.56 ± 0.08, ellipsoid to broadly ellipsoid or ovate, with a small apiculus, inamyloid, thin-walled, hyaline in KOH, smooth. Leptocystidia 50–70 × 2.5–3.5 μm, scattered amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity, at times with apical or subapical branches. Mycelial hyphae 1–2 or 3–5 μm diam., interwoven or aggregated into rhizomorphic strands, branched, clamped; walls thin or irregularly slightly thickened, the hyphal walls echinulate under light microscopy, covered nipple-shaped protuberances with SEM.

Chemical reactions

(dried basidiomes): KOH = positive, golden-yellow; FeCl₃ = positive, green-yellow; NH₄OH = positive, orange; ethanol, FeSO₄, Melzer’s reagent and phenol = negative.

Known distribution and ecology

SW China (in this study) and India (Methven 1989). Solitary to gregarious habit on the ground in mixed woods at elevations above 3200 m.

Materials examined

China. Yunnan Province: Shangri-La Prefecture, mixed coniferous (Pinus spp.) and broad-leaved forests (Caragana spp., dwarf Quercus monimotricha and Sanguisorba spp.), 27°28.55'N, 99°53.05'E, alt. 3280 m, 27 June 2006, Z.W. Ge 1113 (HKAS 50684). Lijiang Prefecture, mixed conifers, alt. 3300 m, 27 August 2009, Q. Cai 146 (HKAS 58811).

Comments

Clavariadelphus himalayensis was originally described from India (Methven 1989). It is the first report from China. Chinese collections match the original descriptions except for slightly smaller basidiospores (8.2–9.4 × 5.0–5.5 μm). The difference in basidiospore size might be from measurement error or the collections being from different geographical regions. Clavariadelphus himalayensis is distinct by its pastel-red to greyish-red, ligulate to spathulate basidiomes flesh that does not stain where bruised or cut, broadly ellipsoid basidiospores (9–11 × 5–6 μm from the holotype; Methven 1989), hyphae of the basal mycelium with nipple-shaped protuberances and a negative reaction with phenol. Other taxa from Asia, which might be confused with C. himalayensis include C. mirus (Pat.) Corner and C. yunnanensis. Although similar in size to those of C. himalayensis, the basidiomes of C. mirus are light brown to brown and produce broadly ovate, larger basidiospores (10–13 × 6–8 μm; Methven 1990). Clavariadelphus yunnanensis, known from northern India and SW China, is distinct by its larger basidiomes that are light brown, larger basidiospores (10–13.5 × 6.5–8 μm), hyphae of the basal mycelium covered by massive nipple-shaped protuberances and a light yellow staining reaction with phenol. Additionally, the flesh of C. himalayensis does not stain where bruised or cut, whereas the flesh of C. mirus and C. yunnanensis slowly stains brunnescent to russet on exposure.

The phylogenetic analyses show that C. himalayensis is allied with the sequence of “C. pistillaris” and Clavariadelphus (JQ991679 from Zhejiang Province, China) from GenBank with weak support (Fig. 1). More data are needed for understanding the phylogenetic relationship of the three species.

6. Clavariadelphus khinganensis J. Zhao, L.P. Tang & P. Zhang, sp. nov.

MycoBank No: MycoBank No: 830273

Figs 2h–i, 3f, 4g, 11a, b

Diagnosis

This species is distinct from other taxa in Clavariadelphus by the yellowish-brown, clavate basidiomes with slightly enlarged apex, narrowly ellipsoid basidiospores and basidiomes that turn very light yellow in KOH.

Figure 11.

Microscopic features of Clavariadelphus khinganensis (MHHNU 7789, holotype). a Leptocystidia and immature basidia b Basidia.

Etymology

Latin “khinganensis” refers to the holotype location, Greater Khingan Mountains or Da Xing’an Ling, in NE China.

Description

Basidiomes up to 12.5 cm high, around 0.8 cm diam. basally, 2.5 cm diam. apically, simple, initially subcylindrical to subfusiform, enlarged upwards in age, then clavate to broadly clavate, finally irregularly laterally compressed; hymenium initially smooth, longitudinally rugose to rugulose in age, pale yellow-brown (4A3) or pale orange (5A 4–6) to greyish-orange (5B4–5, 6B4–5); apex obtuse or broadly rounded, rugose, concolorous with the hymenium at maturity; base terete, smooth, white to pallid when covered, otherwise pale yellow (4A4–5) to light orange (5A4–6); mycelial hyphae interwoven, white; flesh initially solid, becoming soft and spongy upwards as the apex enlarges in age, dirty white. Odour and taste not recorded. Spore deposit not recorded.

Hymenium extending over the apex of basidiomata, composed of basidia and leptocystidia. Basidia 85–105 × 8–11 μm, clavate, hyaline, thin-walled, 4-spored, sterigmata 9–10 μm in length. Basidiospores [20/1/1] 9.2–12.0 × 4.6–6 μm, Q = 1.6–2.2, Q_m = 1.97 ± 0.17, narrowly ellipsoid or amygdaliform, with a small apiculus, inamyloid, thin-walled, hyaline in KOH, smooth. Leptocystidia 60–70 × 3–4 μm, scattered amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity, at times with apical or subapical branches. Mycelial hyphae lacking material.

Chemical reactions

(dried basidiomes): KOH = positive, very light yellow; ethanol, FeCl₃, FeSO₄, phenol, Melzer’s reagent and NH₄OH = negative.

Known distribution and ecology

N China. Solitary on the ground in broad-leaved forests at around 800 m altitude.

Materials examined

CHINA. Jilin Province: Antu Prefecture, Er-dao-bai-he Town, Changbai Mountains, mainly broad-leaved forests (Betula platyphylla, Corylus mandshurica, and Quercus monimotricha), mixed with the coniferous tree (Pinus koraiensis), 42°24.05'N, 128°6.00'E, alt. 753 m, 18 August 2019, H.Y. Huang 368 (MHKMU H.Y. Huang 368). Inner Mongolia: De-er-bu-er Town, Greater Khingan Mountains, alt. 800 m, 6 August 2013, P. Zhang 1289 (MHHNU 7789 Holotype); Ku-ti-he Town, Zha-lan-tun City, 24 July 1985, W. Huang s. n. (HMAS 49920).

Comments

Clavariadelphus khinganensis, known from broad-leaved forests in N China, is distinct by its solitary habit at low elevations (around 800 m), small size, pale brown-orange basidiomes, ellipsoid basidiospores and very pale yellow reaction in KOH.

Morphologically, C. khinganensis is quite similar to two Asian taxa, C. mirus and C. yunnanensis. However, C. mirus was originally described from northern Vietnam and has larger basidiomes, broader basidiospores and a tropical distribution (Butan, India, Nepal; Methven 1990). Clavariadelphus yunnanensis is unique in its habit, growing with conifers at high elevations (above 3000 m), has darker colouration and larger basidiomes (up to 20 cm high), broader basidiospores and basidiomes with yellow reactivity in KOH.

Interestingly, C. khinganensis is clustered with a collection labeled as “C. truncatus” from Canada, the GenBank accession DQ097871 (Durall et al. 2006) and there are no genetic differences on ITS (Fig. 1). It indicates C. khinganensis may be distributed in Canada. More data from North America are needed to confirm the distribution pattern of this species. The sister relationship of C. khinganensis cannot be resolved according to the present data.

7. Clavariadelphus ligula (Schaeff.) Donk, Rev. Niederl. Homob. Aphyll. 2: 73, 1933

Figs 3g, 12a, b

Note

The following taxonomic description is drawn from Methven (1990) and our observations.

Figure 12.

Microscopic features of Clavariadelphus ligula (HMAS 35954). a Leptocystidia and immature basidia b Basidia.

Description

Basidiomes up to 10 cm high, 0.2–0.8 cm diam. basally, slightly enlarged upwards, simple, narrowly clavate to clavate; hymenium longitudinally rugose in age, light yellow, brownish-orange to light brown at maturity; apex subacute to obtuse or broadly rounded, surface slightly rugulose, concolorous with the hymenium; surface slowly staining brownish-orange to brownish-grey where cut or bruised; base terete, initially pale yellow to light yellow, then brownish-orange to light brown to brown; mycelial hyphae white to pallid; flesh initially solid, becoming soft and spongy upwards as the apex enlarges in age, white to pallid. Odour not distinctive. Taste not distinctive or slightly sweet. Spore deposit yellowish-white to light buff in mass.

Hymenium extending over the apex of basidiomata, composed of basidia and leptocystidia. Basidia 45–85 × 8–11 μm, clavate, hyaline, thin-walled, 4-spored, sterigmata 9–10 μm in length. Basidiospores 11.0–14.0 × 4.0–5.5 μm, Q = 2.4–3.1, Q_m = 2.7, narrowly ellipsoid, with a small apiculus, inamyloid, thin-walled, hyaline in KOH, smooth. Leptocystidia 40–80 × 2.5–5 μm, scattered amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity, at times with apical or subapical branches. Mycelial hyphae 2–4 μm diam., interwoven or aggregated into rhizomorphic strands, branched, clamped. Insufficient material to perform SEM.

Chemical reactions

(dried basidiomes): KOH = positive, lemon-chiffon; NH₄OH = positive, orange; ethanol, FeCl₃, FeSO₄, Melzer’s reagent and phenol = negative.

Known distribution and ecology

Widespread in the Northern Hemisphere, including in North America, Bulgaria, NE China, England, Estonia, Finland, Germany, India, Italy, Sweden and Switzerland (Methven 1990). Scattered to gregarious habit on the ground in mixed woods (Abies, Picea, Pinus, Thuja and Tsuga).

Materials examined

China. Heilongjiang Province: Linkou Prefecture, 19 August 1972, X.L. Mao, s. n. (HKAS 35954); same location, Q.X. Wu, s. n. (HMAS 51688). Czech: 2 September 1960, M. Geesteranus 13290 (HMAS 41146).

Comments

Clavariadelphus ligula was originally described from Germany, but was also reported in China (Mao 2009). Our study confirms that this species is mainly found in N China, whereas our data do not support the previous report of the distribution in SW China (Mao et al. 1993; Mao 2009). The basidiospores of Chinese collections (11.0–14.0 × 4.0–5.5 μm, Q = 2.4–3.1, Q_m = 2.7) are smaller and broader than the neotype of C. ligula from Germany (12.0–16.5 × 3.5–4.5 μm, Q = 2.9–4.6, Q_m = 3.7; Methven 1990).

Morphologically, C. ligula and C. sachalinensis are similar in the field. However, C. sachalinensis has more elongated, narrower basidiospores (21–24 × 4–6 μm, Q = 3.5–5.0, Q_m = 4.2). Additionally, C. ligula lacks any reaction with FeCl₃, whereas C. sachalinensis turns green-yellow in FeCl₃. Clavariadelphus yunnanensis is likely to be confused with C. ligula when young. However, C. yunnanensis differs in that it has larger basidiomes (up to 20 cm high), smaller and broader basidiospores (9.0–11.0 × 4.6–6.4 μm, Q = 1.32–1.72, Q_m = 1.56) and a positive reaction with phenol.

The phylogenetic analyses show that C. ligula is allied with the sequence of C. americanus from GenBank with strong support (Fig. 1).

8. Clavariadelphus sachalinensis (S. Imai) Corner, Ann. Bot. Mem. I: 282, 1950

Figs 2j, 3h, 5f, g, 13a, b

Note

The following taxonomic description is drawn from Methven (1989), combined with our field notes, including macro-morphology, growth habit, distribution, host plants and observations.

Figure 13.

Microscopic features of Clavariadelphus sachalinensis (MHHNU 7816). a Basidia b Leptocystidia and immature basidia.

Description

Basidiomes up to 8 cm high, 0.3–0.6 cm diam. basally, slightly enlarged upwards 0.8–1.2 cm diam., simple, initially cylindrical to subcylindrical, then narrowly clavate to clavate; hymenium longitudinally rugose in age, tawny or light walnut-brown to light brown at maturity; apex subacute, obtuse to broadly rounded, surface smooth to slightly rugulose, concolorous with the hymenium; surface slowly staining, brown or dark brown where cut or bruised, staining more conspicuously; base terete, pubescent to tomentose, initially pale yellow to light yellow, then brownish-orange to light brown; mycelial hyphae greyish to pallid; flesh initially solid, becoming soft and spongy upwards, white to pallid, staining on exposure. Odour and taste not distinctive. Spore deposit yellowish-white to light buff.

Hymenium extending over the apex of basidiomata, composed of basidia and leptocystidia. Basidia 65–105 × 8–12.5 μm, clavate, hyaline, thin-walled, (2–) 4-spored, sterigmata 8–10 μm in length. Basidiospores 21–24 × 4–6 μm, Q = 3.5–5.0, Q_m = 4.2, narrowly ellipsoid, boletoid or sway-backed in profile, with a small apiculus, inamyloid, thin-walled, hyaline in KOH, smooth. Leptocystidia 50–70 × 2.5–5 μm, scattered amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity, at times with apical or subapical branches. Mycelial hyphae 2–8 μm diam., interwoven or aggregated into rhizomorphic strands, branched, clamped; hyphal walls smooth with light microscopy and SEM.

Chemical reactions

(dried basidiomes): KOH = positive, light yellow; FeCl₃ = positive, green-yellow; NH₄OH = positive, orange; ethanol, phenol, FeSO₄ and Melzer’s reagent = negative.

Known distribution and ecology

N China (in this study) and SW China (Methven 1990). Gregarious habit on fallen needles and other debris under conifers, especially pine at elevations ranging from 2000–3600 m.

Materials examined

China. Inner Mongolia: Mo er dao ga National Forests, Great Khingan Mountains, 8 August 2013, P. Zhang 1316 (MHHNU 7816). Sichuan Province: Hongyuan Prefecture, Kangle Town, alt. 3600 m, 14 August 1998, M.S. Yuan 3361 (HKAS 33844).

Comments

Clavariadelphus sachalinensis was proposed by Imai, based on Japanese collections as a species of Clavaria and then transferred to genus Clavariadelphus (Imai 1930, Corner 1950). In China, C. sachalinensis was previously reported with distribution in SW China (Methven 1990; Zang 1996) and is also found in northern regions of China. Clavariadelphus sachalinensis is similar to C. ligula and C. yunnanensis. Their differences are described in our discussion of C. ligula.

9. Clavariadelphus yunnanensis Methven, Mem. New York Bot. Garden 49: 156 1989

Figs 2j–l, 3i, 4h, 5h, 14a, b

Note

The following taxonomic description is mainly drawn from Methven (1989). Field notes including macro-morphology, growth habit, distribution and host plants, SEM characteristics and chemical tests are from this study.

Figure 14.

Microscopic features of Clavariadelphus yunnanensis (HKAS 57731). a Leptocystidia and immature basidia b Basidia.

Description

Basidiomes up to 20 cm high, 0.5 cm diam. basally, enlarged upwards 2 cm diam., simple, initially cylindrical to subcylindrical, then narrowly clavate, subolanceolate; hymenium initially smooth, longitudinally rugose to rugulose in age, light brown to cinnamon at maturity; apex obtuse, smooth to rugose, concolorous with the hymenium; surface slowly staining, russet to umber; base terete, smooth, pale cinnamon or pale ochraceous-buff; mycelial hyphae white; flesh initially solid, becoming soft and spongy upwards as the apex enlarges, white to pinkish-buff. Odour not distinctive. Taste slightly bitter. Spore deposit white.

Hymenium extending over the apex of the basidiomata, composed of basidia and leptocystidia. Basidia 70–80 × 8–9 μm, clavate, hyaline, thin-walled, (2–) 4-spored, sterigmata 7–10 μm in length. Basidiospores [40/2/2] (8.8–) 9.0–11.0 × 4.6–6.4 (–7.4) μm, Q = (1.29–) 1.32–1.72 (–1.76), Q_m = 1.56 ± 0.11, ellipsoid to broadly ellipsoid, ovate or amygdaliform, smooth. Leptocystidia 40–60 × 2.5–3.5 μm, scattered amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity, at times with apical or subapical branches. Mycelial hyphae 2–4 μm diam., parallel, interwoven or aggregated into rhizomorphic strands, branched, clamped; walls thin or irregularly slightly thickened, the hyphal walls echinulate with light microscopy, covered with massive nipple-shaped protuberances and lacking crystals with SEM.

Chemical reactions

(dried basidiomes): KOH = positive, golden-yellow; FeCl₃ = positive, green-yellow; NH₄OH = positive, golden-rod or vivid yellow; phenol = positive, light yellow; ethanol, FeSO₄ and Melzer’s reagent = negative.

Known distribution and ecology

SW China and northern India (Methven 1989). Either solitary, scattered or gregarious habit on the ground in mixed deciduous-coniferous forests in association with several genera (e.g. Abies, Berberis, Picea, Pinus, Quercus, Rosa and Salix) at elevations ranging from 2200–3600 m.

Materials examined

China. Sichuan Province: Hongyuan Prefecture, Shuajing Temple, Picea, alt. 3400 m, 3 August 1996, M.S. Yuan 2375 (HKAS 30752); Kangding Prefecture, Liuba, alt. 3500 m, 9 September 1996, M.S. Yuan 2686 (HKAS 31136); Kangding Prefecture, Zheduo Mountains, shrubs dominated by Berberis, Quercus, Rosa, Salix, alt. 3585 m, 14 August 2008, Z.W. Ge 903 (HKAS 49398). Yunnan Province: Shangri-La Prefecture, 19 August 2008, 28°18.00'N, 98°33.00'E, alt. 3100 m, T.Z. Wei 270 (HMAS 250510); Deqing Prefecture, Xiaruo, 18 September 2010, HBB2010-D15 (HKAS 62644); Jianchuan Prefecture, Shibao Mountains, 14 August 2003, Z.W. Ge 4 (HKAS 43816); same location, 30 August 2009, G. Wu 199 (HKAS 57731); Kunming City, Yeya Lake, alt. 2200 m, 22 September 2012, Z.L. Yang 5629 (HKAS 77288); Shangri-La Prefecture, Haba Mountains, 13 August 2008, L.P. Tang 618 (HKAS 54849); Shangri-La Prefecture, 16 August 2008, T.Z. Wei 271 (HMAS 250466); Shangri-La Prefecture, Bita Lake, 24 August 2009, Q. Cai 122 (HKAS 58789); same location and date, G. Wu 127 (HKAS 57659); Weixi Prefecture, Qizong, 19 September 2010, HBB2010-W21 (HKAS 61417); Yulong Prefecture, Yulong Snow Mountains, Sandaowan, under Abies spp., alt. 3200 m, 1 August 1995, M. Zang 12514 (HKAS 30038); Yulong Prefecture, Lizui Village, 20 August 2008, Q. Zhao 8262 (HKAS 55244); Yulong Prefecture, Jiuhe, 20 August 2010, G. Wu 327 (HKAS 63558); Yulong Prefecture, Yulong Snow Mountains, Ganhaizi, under Picea spp., alt. 3100 m, 3 September 1986, M. Zang 10739 (HKAS 17788); Yulong Prefecture, Yulong Snow Mountains, Yu Lake, Abies forests, alt. 3000 m, 1 August 1985, M. Zang 10220 (HKAS 15063); Yulong Prefecture, Yulong Snow Mountains, 6 September 1986, R.H. Petersen s. n. (HKAS 20067); same location and date, R.H. Petersen s. n. (HKAS 20068); Yulong Prefecture, Wenhai, 17 September 2012, G. Wu 1054 (HKAS 77226).

Comments

Clavariadelphus yunnanensis is quite common in SW China where it was previously reported as C. ligula or C. pistillaris (Mao et al. 1993; Yuan and Sun 1995; Zang 1996; Mao 2009). It is well characterised by its cinnamon buff, large basidiomes, broadly ellipsoid basidiospores, hyphae of the basal mycelium covered with nipple-shaped protuberances and occurrence at high elevation forests. This taxon is also similar to C. ligula and C. sachalinensis, but differs microscopically in the size and shape of the basidiospores (see the comments under C. ligula). Immature fruit bodies of C. yunnanensis are similar to C. griseoclavus. However, the latter can be distinguished from smaller basidiomata (less than 13 cm high), narrower apex (less than 1.5 cm diam.) and narrower basidiospores (Q_m 1.89) (Lu and Li 2020). Although C. yunnanensis might be confused with the Asian taxon C. mirus, the latter is distinct by its slender cylindrical, light brown basidiomes and broader basidiospores (Methven 1990). The presence of C. mirus in China needs to be ascertained. In the phylogenetic analyses, C. yunnanensis has a joint relationship with C. elongatus, C. pistillaris and the sequence of “C. occidentalis” from Tunisia, but the sister relationship cannot be resolved (Fig. 1).

Taxonomic key to species of Clavariadelphus in China

1	Basidiospores narrowly ellipsoid, Q_m > 2	2
–	Basidiospores broadly ellipsoid to ellipsoid, Q_m < 2	3
2	Basidiospores 11.0–14.0 × 4.0–5.5 μm, Q_m 2.7	*C. ligula*
–	Basidiospores 21–24 × 4–6 μm, Q_m 4.2	*C. sachalinensis*
3	Basidiomes orange; apex sterile, truncate	4
–	Basidiomes without orange tinge; apex fertile, not truncate	5
4	Basidiomata apex 3–7.5 cm diam	*C. amplus*
–	Basidiomata apex < 2 cm diam	*C. gansuensis*
5	Basidiomes usually 20–30 cm high	6
–	Basidiomes usually < 20 cm high	7
6	Basidiomes grey-purple; basidiospores narrowly ellipsoid, 9.0–11.0 × 5.7–7.4 μm, Q_m 1.71	*C. elongatus*
–	Basidiomes cinnamon; basidiospores broadly ellipsoid, 9.0–11.0 × 4.6–6.4 μm, Q_m 1.56	*C. yunnanensis*
7	Basidiomes greyish-red to pastel-red	*C. himalayensis*
–	Basidiomes grey or yellow, without red colouration	8
8	Basidiomes grey; basidiospores ellipsoid 10–11 × 5–6.5 μm, Q_m 1.89	*C. griseoclavus*
–	Basidiomes yellow colouration	9
9	Basidiomes yellow; basidiospores broadly ellipsoid 7.8–9.6 × 5.5–7.4 μm, Q_m 1.38	*C. alpinus*
–	Basidiomes pale yellow-brown; basidiospores narrowly ellipsoid 9.2–12.0 × 4.6–6 μm, Q_m 1.97	*C. khinganensis*

Discussion

The taxonomic importance of comprehensive data in Clavariadelphus

Many studies have verified that molecular methods are effective in resolving relationships in complicated groups of fungi (Zeng et al. 2013; Tang et al. 2017; Huang et al. 2018; Yang et al. 2018). In a pre-study analysis, we evaluated four DNA gene markers: ITS, large subunit of nuclear ribosomal RNA (nrLSU), translation elongation factor 1α gene (tef1-α) and DNA-directed RNA polymerase II second subunit (rpb2). Compared to the others, ITS offered the highest probability of successful identification in Clavariadelphus. By contrast, other markers displayed a lower success rate of PCR amplification or inferior species resolution in some close or sibling taxa. ITS sequences acquired from this study are listed in Appendix 1.

Macro-morphological, micro-morphological and SEM characteristics are very important in the taxonomy of Clavariadelphus. Although Clavariadelphus can be readily distinguished from other clavarioid genera, the delimitation of infrageneric taxa is difficult in many cases, especially without critical observation and examination (Methven 1990). Basidiomata colour is a diagnostic characteristic, although it must be used in conjunction with other morphological features. In China, basidiomata colour ranges from yellow to orange, grey-purple, pastel-red or brown. Despite the inherent variability in shape, size and other characteristics of the basidiomes, these features are important diagnostic characteristics in Clavariadelphus, as well as the size and shape of basidiospores. The basidiospores of Chinese taxa of this genus are summarised in Fig. 3 and 4. Additionally, some SEM characteristics, especially hyphae of the basal mycelium, are of taxonomic value. The variations of basal hyphae of Chinese taxa range from smooth, having nipple-shaped protuberances to crystals or both at the same time (Fig. 5).

Chemical reactions also are helpful in distinguishing Clavariadelphus species. Doty (1948) distinguished this genus using FeSO₄ reactions. Corner reported that chemical reactions, especially KOH, are useful for delimiting Clavariadelphus taxa (Corner 1950). In this study, chemical colour reactions were conducted using seven chemical reagents (Table 1). Amongst these, four reagents were found to be discriminatory with some species, specifically KOH, FeCl₃, NH₄OH and phenol. Three additional reagents, namely ethanol, Melzer’s reagent and FeSO₄, were found to lack discriminatory value.

Metadata supply taxonomic information, such as habit, distribution and host plants. The growth habit of Chinese taxa includes solitary, scattered and gregarious. Growth habit is of taxonomic value only when used in conjunction with other features (Methven 1990). The Chinese specimens were collected in mixed or coniferous forests in association with Abies, Berberis, Quercus, Pinus, Picea, Rhododendron, Rosa, Salix, Thuja and Tsuga. The distribution of a species usually correlates with that of its host plant. Although the Chinese taxa exhibit no apparent preference of host plants, the so-called cosmopolitan species within Clavariadelphus seem to be rare in this study.

Clavariadelphus species diversity in China

Many new fungal taxa have been discovered in the last ten years in China (Zhang et al. 2005; Zeng et al. 2013; Tang et al. 2014; Huang et al. 2018; Yang et al. 2018). However, there are still a large number of undescribed fungal taxa in this country. This study indicates that there are at least ten known taxa of Clavariadelphus in China, including four previously described (C. griseoclavus, C. ligula, C. sachalinensis and C. yunnanensis), two not previously reported in China (C. elongatus and C. himalayensis) and four novel species (C. alpinus sp. nov., C. amplus sp. nov., C. gansuensis sp. nov. and C. khinganensis sp. nov.). Several taxa, previously reported from China, need to be confirmed, including C. mirus, C. pistillaris and C. truncatus. In China, there are still some species that need to be discovered, such as GenBank accession JQ991679. To date, with the four novel taxa described in this study, there are twenty-eight species of Clavariadelphus worldwide. Although the taxonomy of Clavariadelphus has received much attention in the past, this group needs to be further examined with molecular methods. More reliable sequence data, especially those species from North America and Europe, are needed to understand phylogenetic relationships better.

Acknowledgements

We thank the curators and collectors of the herbarium of HMAS and HKAS. Financial support for fieldwork and experiment expenses were from the following projects: the National Natural Science Foundation of China (Nos. 31960007, 31560004, 31670015, 31670024), The Program Innovative Research Team in Science and Technology in Yunnan Province (No. 2019HC008), Yunnan Applied Basic Research Projects-joint special project (No. 2014FB016), the Science Research Foundation of Department of Education, Yunnan Province (No. 2015Y147), the Program Innovative Research Team in Science and Technology in Yunnan Province (No. 2019HC008) and the Open Research Foundation of Yunnan Key Laboratory of Pharmacology for Natural Products (No. 2015G003). We are greatly indebted to Dr. Andrew S. Methven and other anonymous reviewers for their valuable suggestions and comments. Many thanks are also due to the linguistic assistance provided by TopEdit (www.topeditsci.com) during the preparation of this manuscript.

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Appendix 1

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Sequences used or produced in our phylogenetic analyses of Clavariadelphus in China.

Taxon	Voucher	Locality	GenBank Accession No. (ITS)
*Clavariadelphus alpinus*	HKAS 57396	China, Yunnan	MK705888*
C. americanus	MycoMap # 1288	USA, Indiana	MK575228
C. amplus	HKAS 76577	China, Gansu	MK705851*
*C. amplus*	HKAS 54876	China, Yunnan	MK705857*
C. amplus	HMAS 132008	China, Qinghai	MK705852*
C. amplus	HMAS 97090	China, Qinghai	MK705853*
C. amplus	HKAS 49229	China, Sichuan	MK705854*
C. amplus	HKAS 49278	China, Sichuan	MK705855*
C. amplus	HKAS 53797	China, Sichuan	MK705856*
C. amplus	HMAS 250466	China, Yunnan	MK705858*
C. amplus	HMAS 97248	China, Tibet	MK705859*
C. amplus	HMAS 59867	China, Tibet	MK705860*
C. amplus	HKAS 46160	China, Tibet	MK705861*
C. amplus	HKAS 46120	China, Tibet	MK705862*
C. elongatus	HKAS 76589	China, Gansu	MK705842*
C. elongatus	HKAS 50742	China, Sichuan	MK705843*
C. elongatus	HKAS 50801	China, Sichuan	MK705844*
C. elongatus	HMAS 260746	China, Yunnan	MK705845*
C. elongatus	HKAS 52425	China, Yunnan	MK705846*
C. elongatus	LAH 31397	Pakistan, Khyber Pakhtunkhwa	MG768847*
C. elongatus	SWAT 000559	Pakistan, Khyber Pakhtunkhwa	MG768848*
*C. gansuensis*	HKAS 76487	China, Gansu	MK705847*
C. griseoclavus	BJTC FM964	China, Shanxi	MT302370
C. griseoclavus	BJTC FM965	China, Shanxi	MT302371
C. himalayensis	HKAS 50684	China, Yunnan	MK705863*
C. himalayensis	HKAS 58811	China, Yunnan	MK705864*
*C. khinganensis*	MHHNU 7789	China, Inner Mongolia	MK705865*
C. khinganensis	MHKMU H.Y. Huang 368	China, Jilin	MT447468*
C. ligula	HMAS 51688	China, Heilongjiang	MK705848*
C. ligula	HMAS 35954	China, Heilongjiang	MK705849*
C. ligula	HMAS 41146	Czech, –	MK705850*
C. mucronatus	OSC 1064138	USA, Oregon	EU526000
C. occidentalis	OSC 104664	USA, the Pacific Northwest	EU669308
C. occidentalis	OSC 112861	USA, the Pacific Northwest	EU669202
C. occidentalis	OSC 114250	USA, the Pacific Northwest	EU834202
C. occidentalis	OSC 114281	USA, the Pacific Northwest	EU846242
C. occidentalis	H21536	Tunisia, Aïn Draham	KU973835
C. pistillaris	NAMA 2017-123	USA, Wisconsin	MH979250
C. pistillaris	AMB 18611	Italy, Aquila	MT452507
C. pakistanicus	MH 129901	Pakistan, Khyber Pakhtunkhwa	HQ379937
C. pakistanicus	SR1742	India, –	MT012805
C. sachalinensis	MHHNU 7816	China, Inner Mongolia	MK705866*
C. sachalinensis	p061i	USA, the Pacific Northwest	EU624408
C. sachalinensis	p059i	USA, the Pacific Northwest	EU624410
C. sachalinensis	p058i	USA, the Pacific Northwest	EU624411
C. sachalinensis	OSC 96213	USA, the Pacific Northwest	EU834196
Clavariadelphus sp.	src121	USA, California	DQ974709
Clavariadelphus sp.	OSC 105674	USA, the Pacific Northwest	EU669206
Clavariadelphus sp.	HC-PNNT-268	Mexico, Mexico State	KT874982
Clavariadelphus sp.	ECM54	China, Zhejiang	JQ991679
Clavariadelphus sp.	MushroomObserver.org/254047	Mexico, Queteraro	MH304404
Clavariadelphus sp.	Montri-108	Switzerland, Montricher	MK028378
C. subfastigiatus	OSC 119587	USA, the Pacific Northwest	EU669207
C. subfastigiatus	MICH 73554	USA, Clackamas County	JX275756
C. truncatus	MA-Fungi 48062	Spain, –	AJ292288
C. truncatus	OUC99108	Canada, British Columbia	DQ097871
C. truncatus	SIM278	Canada, British Columbia	HQ650728
C. truncatus	AMB 18612	Italy, Belluno	MT452508
C. unicolor	Mushroom Observer #112193	USA, Indiana	MN906166
C. yunnanensis	HKAS 49398	China, Sichuan	MK705867*
C. yunnanensis	HKAS 31136	China, Sichuan	MK705868*
C. yunnanensis	HKAS 54849	China, Yunnan	MK705869*
C. yunnanensis	HKAS 63558	China, Yunnan	MK705870*
C. yunnanensis	HKAS 57731	China, Yunnan	MK705871*
C. yunnanensis	HKAS 58789	China, Yunnan	MK705872*
C. yunnanensis	HKAS 55244	China, Yunnan	MK705873*
C. yunnanensis	HMAS 250510	China, Yunnan	MK705874*
C. yunnanensis	HMAS 250471	China, Yunnan	MK705875*
C. yunnanensis	HKAS 62644	China, Yunnan	MK705876*
C. yunnanensis	HKAS 61417	China, Yunnan	MK705877*
C. yunnanensis	HKAS 43816	China, Yunnan	MK705878*
C. yunnanensis	HKAS 30752	China, Yunnan	MK705879*
C. yunnanensis	HKAS 30083	China, Yunnan	MK705880*
C. yunnanensis	HKAS 20068	China, Yunnan	MK705881*
C. yunnanensis	HKAS 20067	China, Yunnan	MK705882*
C. yunnanensis	HKAS 17788	China, Yunnan	MK705883*
C. yunnanensis	HKAS 15063	China, Yunnan	MK705884*
C. yunnanensis	HKAS 57659	China, Yunnan	MK705885*
C. yunnanensis	HKAS 77226	China, Yunnan	MK705886*
C. yunnanensis	HKAS 77288	China, Yunnan	MK705887*
Lentaria byssiseda	TENN 61159	USA, Tennessee	FJ596788
Kavinia himantia	CFMR:DLL2011-079	USA, Wisconsin	KJ140598
K. alboviridis	CFMR:DLL2011-131	USA, Wisconsin	KJ140634