The genus Clavariadelphus (Clavariadelphaceae, Gomphales) in China

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.


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, 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) FeCl 3 , 10% (w/v) FeSO 4 , 10% NH 4 OH, 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 MgCl 2 ), 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 . 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.

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.
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 (FeCl 3 , KOH, NH 4 OH and phenol), but all species from China showed a negative reaction to FeSO 4 , 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).
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.
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 NH 4 OH 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 NH 4 OH and distribution at lower elevations in NE China.
This species was previously referred to as either C. pallido-incarnatus (Yuan and Sun 1995) or C. truncatus Zang 1996;Mao 2009;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 cinnamonbrown 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 goldenyellow reaction to KOH and association with deciduous trees (Methven 1990).
In the ITS tree, C. amplus exhibits a sister relationship with C. pakistanicus with strong support (Fig.1).
Phylogenetically, C. elongatus is related to C. pistillaris and the sequence of "C. occidentalis" from GenBank with weak support (Fig. 1). 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.
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).
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. 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.
Etymology. Latin "khinganensis" refers to the holotype location, Greater Khingan Mountains or Da Xing'an Ling, in NE China.
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. Note. The following taxonomic description is drawn from Methven (1990) and our observations.
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.
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). 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 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).
The phylogenetic analyses show that C. ligula is allied with the sequence of C. americanus from GenBank with strong support (Fig. 1).

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. 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 brownishorange 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.
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.
Taxonomic key to species of Clavariadelphus in China

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 FeSO 4 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, FeCl 3 , NH 4 OH and phenol. Three additional reagents, namely ethanol, Melzer's reagent and FeSO 4 , 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;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.