Updated taxonomy of Chinese Clavaria subg. Syncoryne (Clavariaceae, Agaricales): description of two new species and one newly recorded species

Species of Clavaria (Clavariaceae, Agaricales) collected from China were studied using morphological and molecular methods. Two species, C. aspersa and C. hupingshanensis, are here described as new to science; C. aspersa possesses simple, white basidiomata in gregarious or scattered fascicles, whereas C. hupingshanensis possesses simple, rose-white to seashell-pink basidiomata in gregarious to caespitose clusters. In addition, C. amoenoides is described as a newly recorded species for China; this species is characterized by simple, very pale orange-yellow to picric-yellow basidiomata. A phylogenetic analysis was conducted based on a combined dataset of internal transcribed spacer, nuclear ribosomal RNA large subunit, and the RNA polymerase II second largest subunit sequences. The phylogenetic reconstruction resolved accessions of the three species into three independent lineages within the Clavaria. The morphology of the three species is described in detail and is illustrated with line drawings and photographs. Holotypes of the new species are deposited in the Mycological Herbarium of Hunan Normal University. The sequences newly generated in this study have been deposited in GenBank. An updated key to the known Clavaria species in China is provided.


Introduction
The genus Clavaria Vaill. ex L. is a member of the family Clavariaceae Chevallier (1826). The name "Clavaria" was first proposed by Vaillant (1727) and was subsequently used by Linnaeus (1753) for all species of fungi with an erect, clubshaped, or branched fruit body. As now circumscribed, the genus is characterized by its simple clavarioid or branched basidiomata and the absence of clamps in contextual hyphae. The species are classified into two subgenera: Syncoryne Fr. lacks clamps at the basidium base, and Holocoryne (Fr.) Quél. has a broad, loop-like clamp at the base of the basidium (Coker 1923;Corner 1950Corner , 1970Petersen 1978Petersen , 1988. The color of the basidiomata is an important character for classification in Clavaria, which comprises species that produce basidiomata with a high diversity of colors, including white, pink, and purple (Burt 1922;Corner 1950). In recent years, many studies on the color of basidiomata in Clavaria have been undertaken; for example, Roberts (2007) studied black and brown Clavaria species in the British Isles, Kautmanová et al. (2012) focused on European species of Clavaria with dark basidiomata, and Olariaga et al. (2015) studied yellow Clavaria species with a loop-like clamp at the base of the basidium.
Before the present study, eight species of Clavaria were recorded in China and approximately 35 species were recognized worldwide (Tai 1979;Olariaga et al. 2015;Chen & Zhang 2019;Yan et al. 2020). In China, only Clavaria acuta Sowerby and Clavaria gibbsiae Ramsb. have a clamp at the base of the basidium; the other species native to China are classified in subg. Syncoryne. Since 2010, we have collected a number of Clavaria specimens with clampless basidia in several provinces of China. Morphological and molecular studies showed that these specimens belong to two species that are new to science, and to one species not previously recorded in China. Descriptions and illustrations of the three species are presented herein.

Sample sources
Specimens of the two new species, C. aspersa and C. hupingshanensis, were collected by the authors in Hunan Province or Anhui Province, Central China, between 2010 and 2020. Material of the newly recorded species in China, C. amoenoides, was collected in Jilin Province between 2019 and 2020. The dried vouchers are housed in the Mycological Herbarium of Hunan Normal University (MHHNU), Changsha, China. We extracted DNA from the dried specimens, and amplified and sequenced three DNA regions: the internal transcribed spacer (ITS), the nuclear ribosomal RNA large subunit (nrLSU), and the RNA polymerase II second largest subunit gene (RPB2). A dataset comprising the concatenated 27 ITS, 26 nrLSU, and 22 RPB2 sequences, combined with selected sequences downloaded from GenBank, were used for phylogenetic analyses. Voucher information, GenBank accession numbers, and other relevant information are listed in Table 1.

Morphological descriptions
The macromorphological data were based on field notes and habitat photographs. Micromorphological characters were recorded from microscopic observation. The color of basidiomata was described using color codes (Kornerup & Wanscher 1978) and color terms (Ridgway 1912). Dried material was sectioned, rehydrated with 5% KOH solution, and stained with 1% Congo red solution. The stained basidiospores, basidia, and hyphae were observed in 5% KOH solution with a light microscope. The notation [n/m/p] indicates that n spores were measured from m basidiomata of p specimens. The basidiospore dimensions are described using the notation (a-)b-c(-d), where the range b-c includes a minimum of 90% of the measured values, and extreme values (i.e., a or d) are provided in parentheses. Q is the length/width ratio of a basidiospore in lateral view. Q is the average Q of all basidiospores ± sample standard deviation.

DNA extraction, PCR amplification, and sequencing
Total genomic DNA was extracted from dried specimens using the modified cetyltrimethylammonium bromide method of Doyle and Doyle (1987). The following primer pairs were used to amplify the selected DNA regions: ITS4 and ITS5 (Vilgalys & Hester 1990;White et al. 1990;Gardes & Bruns 1993) for ITS region; LR0R and LR5 (Vilgalys & Hester 1990) for nrLSU region; and fRPB2-5F, fRPB2-6F, and fRPB2-7.1R (Liu et al. 1999;Matheny et al., 2007) for RPB2 gene. Each PCR amplification was conducted using an Eppendorf Mastercyler thermal cycler (Eppendorf Inc., Germany) in a 25-μL reaction volume. The thermal-cycling protocol was as follows: initial denaturation at 94°C for 4 min; denaturation for 34 cycles of 94°C for 40 s, annealing at an appropriate temperature (55°C for 40 s for ITS and nrLSU; 50°C for 1 min for RPB2), and extension at 72°C for 1 min; and a final extension at 72°C for 8min (Liu et al. 2017;Wu et al. 2019). The PCR products were separated by 1% agarose gel electrophoresis (Sangong Inc., China). The purified PCR products were sequenced using an ABI 3730 DNA Analyzer (PerkinElmer Inc., USA). The same primers used for PCR amplification were used for sequencing reactions. Newly generated sequences were deposited in GenBank (accession numbers are listed in Table 1).

Taxonomy
Habitat: Gregarious to caespitose in humus layers of soil in broad-leaved forest or on soil in pine-oak forest. Basidiomata produced in summer or autumn, usually throughout August to September.
Habitat: Scattered to gregarious or weakly fascicled in humus layers of soil in broad-leaved forest or on the ground covered with moss. Basidiomata produced in summer or autumn, usually throughout July to September. Comments:-Clavaria aspersa is primarily characterized by the simple, gregarious or weakly fascicled, white basidiomata and ellipsoid hyaline basidiospores. Before this study, more than 10 white species have been reported in Clavaria, which represents the largest group of species in the genus. Clavaria gibbsiae and Clavaria tenuipes Berk. & Broome are consistent with C. aspersa in lacking secondarily septate hyphae, but they have a loop-like basal clamp at the base of the basidium and belong to subg. Holocoryne; Clavaria acuta is also classified in subg. Holocoryne and is secondarily septate; Clavaria alliacea Corner and Clavaria fuscata Oudem. are similar to C. aspersa macromorphologically but they differ in producing 2-spored basidia; and Clavaria filiola Corner and Clavaria fossicola Corner are white tone species, but both are extremely small species and easy to distinguish (Corner 1950(Corner , 1970.
Habitat: Fasciculate to caespitose in humus layers on soils in coniferous forest. Basidiomata generally produced from August to September.
Distribution: Known only from the type locality in Hunan Province, China.
Comments: Clavaria hupingshanensis is mainly characterized by the simple, gregarious, or caespitose clusters of rosewhite to seashell-pink basidiomata. Within the genus Clavaria, pink tone species are not uncommon. Corner (1950) (Agnello et al. 2014, Franchi and Marchetti, 2021, Agnello & Papetti 2020. In 2020, we described one new species, Clavaria sinensis P. Zhang, with pink basidiomata from central China (Yan et al. 2020). However, C. hupingshanensis is clearly distinct from these species. In contrast to C. apulica, C. barlae, and C. zollingeri, C. hupingshanensis does not produce branched basidiomata and the basidiomata color is paler. Compared with C. appendiculata, C. incarnata, C. messapica, and C. pseudoincarnata, C. hupingshanensis, as a member of subg. Syncoryne, lacks basidia with a loop-like clamp. Clavaria helicoides is the most unique among the pink species; its spores are pink, which can be distinguished from C. hupingshanensis with white spores. Clavaria rosea and C. sinensis have been reported in China (Tai 1979;Yan et al. 2020). In morphology, C. rosea is darker than C. hupingshanensis, and C. sinensis can produce branched basidiomata. With regard to phylogenetic relationships, C. rosea is closely related to C. fragilis within /Clavaria sensu stricto (Birkebak et al. 2016), and C. hupingshanensis and C. sinensis cannot form a sister lineage within the /fumosa clade.

Phylogenetic analyses
The alignment of concatenated sequences, which were 2516bp long, was used for BI and ML analyses. The matrix comprised 77 sequences (28 ITS, 27 nrLSU, and 22 RPB2) representing 11 species. Clavaria taxa and two species of Mucronella Fr. as the outgroups. The ML analysis yielded the phylogeny shown in Figure 7. The BI phylogeny (not shown) was extremely similar in topology to the ML tree. Bayesian posterior probabilities greater than 0.90 and bootstrap values exceeding 50% are shown at the relevant nodes. The ML and BI analyses resolved two clades among the species of Clavaria: /Clavaria sensu stricto and /fumosa clade. Clavaria aspersa, C. fragilis, and C. rosea were grouped in the well-supported /Clavaria sensu stricto (BI 1/ML 97%). The /fumosa clade (BI 1/ML 100%) comprised six clampless species: C. amoenoides, Clavaria fumosa Pers, Clavaria  griseolilacina P. Zhang, C. hupingshanensis, C. sinensis, and C. zollingeri. The results were consistent with the previous findings (Kautmanová et al. 2012;Birkebak et al. 2016). The two new species and one newly recorded species form a distinct monophyletic lineage in the tree.

Discussion
In this study, two new Clavaria species and one newly recorded Clavaria species in China (C. amoenoides) were identified in China. Yellow Clavaria species are rare in China. Morphologically, C. amoenoides is similar to other yellow Clavaria or Clavulinopsis species, but can be distinguished from most of these species because it lacks a clamp at the base of the basidium. It is distinguishable from C. straminea by the basidiospores shape and stem characteristics. Phylogenetically, our specimens formed a well-supported (BI 1/ML 100%) lineage with a previously sequenced C. amoenoides accession. Therefore, we confirmed that the distribution range of C. amoenoides includes China. Clavaria aspersa and C. hupingshanensis are described here as new species. The former is similar to C. gibbsiae and C. fragilis, and C. hupingshanensis is distinguishable from other species in the /fumosa clade. In the field, C. aspersa is scattered to gregarious or weakly fascicled, whereas C. gibbsiae and C. fragilis are densely caespitose. With regard to micromorphology, C. aspersa produces clampless basidia and the hyphae are not secondarily septated, whereas C. gibbsiae has a clamp at the base of the basidium and C. fragilis has secondarily septated hyphae. In the present phylogenetic reconstructions, C. aspersa was closely related to C. fragilis and C. rosea, which formed a sister lineage with stronger support (BI 1/ML 97%). Clavaria hupingshanensis may be mistaken for other species in the /fumosa clade, but the macromorphological data and molecular analyses confirmed that the specimen belonged to a species new to science. In the phylogenies, C. hupingshanensis formed a distinct earlydiverging monophyletic lineage in the /fumosa clade.
In China, before this study, eight Clavaria species were formally reported, namely C. acuta, C. fragilis, C. fumosa, C. gibbsiae, C. griseolilacina, C. rosea, C. sinensis, and C. zollingeri (Tai 1979;Chen and Zhang 2019;Yan et al. 2020). Thus, so far, the species diversity of Clavaria is considerably lower than that elsewhere in the world, of which more species need uncovering. In addition, the limited availability of sequences for Clavaria taxa restricts assessment of Fig. 7 Phylogenetic relationships of Clavaria species inferred from a concatenated sequence (ITS, nrLSU, and RPB2) dataset under the maximum likelihood optimality criterion. Bayesian posterior probabilities over 0.90 and bootstrap values over 50% are reported at nodes (BI/MP); the sign "-" means under the reported level. Two new species and one newly recorded species are shown in boldface text phylogenetic relationships within the genus. In this study, two new species and one newly recorded species of Clavaria from China are documented, which enriches the species diversity of this genus in China; forty-five newly generated sequences (14 ITS, 17 nrLSU, and 16 RPB2) for Clavaria taxa have been deposited in GenBank, which provides reliable data for future phylogenetic studies of Clavaria. Notably, the present study is the first to use a DNA region other than ITS and nrLSU to explore phylogenetic relationships in Clavaria.