A Contribution to the Knowledge of Hydnum (Hydnaceae, Cantharellales) in China, Introducing a New Taxon and Amending Descriptions of Five Known Species

: Hydnum (Hydnaceae, Cantharellales), one of the edible ectomycorrhizal mushrooms, is of considerable ecological and economic importance. Although previous studies have focused on the genus in China, the diversity still remains incompletely understood. In the present study, in addition to the known species from China being reviewed, six phylogenetic species from the country were described/redescribed, which included a new species: H. erectum , and five known taxa: H. cremeoalbum , H. minus , H. orientalbidum , H. tenuistipitum , and H. treui ; H. treui is new to China. Detailed descriptions, color photographs of fresh basidiomata, and line drawings of microstructures of them are presented. A key to the accepted species of Hydnum in China is also provided.

Previous studies indicated that Hydnum species were mainly found in temperate regions of the world; few taxa were reported from subtropical/tropical forests [1,4,9,12,[23][24][25][26][27][28][29][30].It is well-known that subtropical/tropical China is a biodiversity hotspot, and with more field investigations in the region, more species of the genus will be uncovered.Recently, several collections of Hydnum were made from subtropical/tropical China, and these specimens were studied using morphological and molecular phylogenetic analyses, aiming to (i) describe new taxa and uncover more species of Hydnum in China; (ii) amend descriptions of some Hydnum species; (iii) provide an overview the Hydnum species in China.

Morphological Studies
The studied specimens were collected from Hainan, Hunan, and Zhejiang Provinces in China and deposited in the Fungal Herbarium of Hainan Medical University (FHMU), Haikou City, Hainan Province, China.Fresh specimens were used to make field records and digital photographs.Color documentation of fresh materials followed Kornerup and Wanscher [31].Observations and measurements of microscopic features were made in 5% KOH solution and stained with 1% Congo Red [4].Sections of the pileipellis were taken from the pileus between the center and margin.The number of measured basidiospores is presented as n/m/p, where "n" represents the total number of basidiospores measured from "m" basidiomata of "p" collections.Dimensions of basidiospores were presented in the form (a-) b-e-c (-d), where the range b-c contains at least 90% of the measured values (5th to 95th percentile), "a" and "d" are the extreme values, and "e" refers to the average length/width of basidiospores.Q refers to the length/width ratio of basidiospores; Qm refers to the average Q of basidiospores and is presented with standard deviation [32,33].The analyses of basidiospore size were constructed using SPSS Statistics Version 17.0 [34].The terms referring to the size of basidioma were based on Bas [35].

Molecular Procedures
Total genomic DNA was obtained with the Plant Genomic DNA Kit (KANGWEI Company, Taizhou, China) from materials dried with silica gel, according to the manufacturer's instructions.The primer pairs used for amplification were: LR0R/LR5 [36,37] for the nuclear ribosomal large subunit RNA (28S) and the nuclear rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS), with ITS5/ITS4 [38].PCR conditions followed the program of Zhang et al. [32].PCR products were checked on 1% (w/v) agarose gels, and positive reactions with a bright single band were purified and directly sequenced using an ABI 3730xl DNA Analyzer (Guangzhou Branch of BGI, Guangzhou, China) with the same primers used for PCR amplifications [32,33].DNA sequences were compiled by BioEdit [39] and then deposited in GenBank.

Dataset Assembly
Twenty-four DNA sequences (twelve of 28S and twelve of ITS) from thirteen specimens were newly generated.For the concatenated dataset, the sequences of 28S and ITS from the new collections were aligned with sequences of taxa of Hydnum from previous studies and GenBank/UNITE (Table 1).Sequences of H. elatum Massee, H. sp.(FRI62832), H. sp. 17 BF-2016 (PDD93275, PDD98029, PERTH07830742, PERTH08072957, and GD1588), H. sp.18 BF-2016 (PDD94968, GD1590, and GD1589), and H. sp.19 BF-2016 (PERTH07608543, PERTH08018413, PERTH08091676, and PERTH08093865) were excluded from analyses due to the distinct genetic divergences of their ITS sequences, which was also noted by Feng et al., Niskanen et al.,and Sugawara et al. [1,9,14].Sistotrema muscicola (Pers.)S. Lundell was chosen as outgroup following Cao et al. [15].To test for phylogenetic conflict between the two genes in the combined dataset, the phylogenetic trees based on 28S and ITS datasets were analyzed and conducted using the ML method to detect the topologies of genes used.The results of analyses showed that the different gene fragments were not in conflict.Then, the two datasets (28S and ITS) were aligned with MUSCLE v3.6 [40] and concatenated using Phyutility v2.2 for further analyses [41].

Phylogenetic Analyses
The combined nuclear dataset (28S + ITS) was analyzed with maximum likelihood (ML) and Bayesian Inference (BI).Maximum likelihood tree generation and bootstrap analyses were performed with the program RAxML 7.2.6,running 1000 replicates combined with an ML search.Bayesian analysis with MrBayes 3.1, implementing the Markov Chain Monte Carlo (MCMC) technique and parameters predetermined with MrModeltest 2.3, was performed [32,33].The best-fit likelihood model for 28S and ITS was GTR + I + G and GTR + I + G, respectively.Bayesian analysis of the combined nuclear dataset (28S + ITS) was run for 45 million generations, and the average deviation of split frequencies was 0.005618.Trees sampled from the first 25% of generations were discarded as burn-in, and Bayesian posterior probabilities (PP) were then calculated for a majority consensus tree of the retained Bayesian trees [32,33].

Molecular Data
The combined dataset (28S + ITS) consisted of 255 taxa with 1454 nucleotide sites, and the alignment was submitted to TreeBASE (S31163).The phylogram with branch lengths generated from RAxML and support values (BS and PP) are shown in Figure 1.The topologies of phylogenetic trees generated from ML and BI analyses were identical, although statistical support for some branches showed slight differences.

Molecular Data
The combined dataset (28S + ITS) consisted of 255 taxa with 1454 nucleotide sites, and the alignment was submitted to TreeBASE (S31163).The phylogram with branch lengths generated from RAxML and support values (BS and PP) are shown in Figure 1.The topologies of phylogenetic trees generated from ML and BI analyses were identical, although statistical support for some branches showed slight differences.Habitat-Gregarious on the ground in forests dominated by fagaceous trees.Known distribution-Japan, southern China (Hainan and Guangdong Provinces), and central China (Hubei Province); probably found in Hunan Province and Chongqing Municipality (Figure 1). of subcylindric hyphae measuring 2-8 µm wide, interwoven to subparallel, and yellowish in KOH; terminal hyphae 16-38 × 2-5.5 µm, thin-walled (less than 5 µm), slightly inflated at the apex or septum.Clamp connections present in all tissues.
Basidiospores Notes-Hydnum cremeoalbum was originally described from Japan [1], then recently reported from Guangdong and Hubei Provinces, China [22].In the present study, it was also found to be distributed in Hainan and Yunnan Provinces of China.The species is characterized by a cream white to cream, infundibuliform, and large pileus, a robust and branched stipe, a decurrent hymenophore, long spines (up to 4 mm), subglobose to ovoid basidiospores, and association with fagaceous trees.In addition, intraspecific variations of H. cremeoalbum were observed; for example, the Japanese specimens have smaller basidiomata (pilei 3-7 cm diameter), non-branched stipitis, and larger basidiospores measuring 5-7 × 3.5-5.5 µm [1].2c,d and  4).Habitat-Solitary and concrescent on the ground in forests dominated by fagaceous trees.
Habitat-Gregarious on the ground in forests dominated by fagaceous trees.Known distribution-Japan, southern China (Hainan Province), southwestern China (Yunnan Province), and central China (Hunan Province).
Notes-Hydnum minus was originally described from Japan [5] and subsequently reported from Hunan Province, central China [15].In the present study, it was also found to be distributed in Hainan and Yunnan Provinces of China.The species is characterized by a small, slightly umbilicate, cream-white pileus with a lobed to wavy margin, a translucent or watery, white context turning reddish-brown to red slowly when injured, long spines (up to 4 mm), a pale cream stipe turning pale brown gradually when injured, and broadly ellipsoid to ellipsoid basidiospores.In addition, intraspecific variations of H. minus were observed; for example, the Japanese specimens have smaller basidiomata (pilei 1-2.5 cm diameter) and shorter spines (0.5-1.7 mm) [5].
broadly ellipsoid to ellipsoid basidiospores.In addition, intraspecific variations of H. minus were observed; for example, the Japanese specimens have smaller basidiomata (pilei 1-2.5 cm diameter) and shorter spines (0.5-1.7 mm) [5].Notes-Hydnum orientalbidum was originally described from Japan [14] and subsequently reported from Sichuan and Zhejiang Provinces and Chongqing Municipality, China [22].In the present study, it was also found to be distributed in Hainan, southern China.The species is characterized by a small, creamy yellow to pale orange pileus with a white margin, short spines, a white and short stipe, ovoid to broadly ellipsoid basidiospores, and association with fagaceous trees.In addition, intraspecific variations of H. orientalbidum were observed,; for example, the Japanese specimens have larger basidiomata (pilei up to 5.5 cm diameter) and longer spines (up to 6 mm) [14].Habitat-Solitary or gregarious on the ground in forests dominated by Pinus massoniana Lamb.
Odor not distinctive.

Other known species from China
one species is new to science, viz.H. erectum (lineage 32), and one is new to China, viz.

H. treui (lineage 22).
Recent phylogenetic analyses of worldwide Hydnum samples have provided some new insights into the cloudy phylogeny and geography of this genus [9].Our phylogenetic tree was constructed using two-locus DNA sequences (28S + ITS) with reliable sequences of already published species and those from new Chinese collections, which have contributed to our new knowledge of the genus.Hydnum minus was previously assigned to the subgen.Alba s. l. by Niskanen et al. [1], whereas our phylogenetic study indicated that H. minus is phylogenetically related to H. brevispinum, the type species of subgen.Brevispina [15], with 94% RAxML likelihood bootstrap (Figure 1).Moreover, H. minus is morphologically similar to H. brevispinum [15].Therefore, H. minus was transferred to the subgen.Brevispina in the present study (Table 2).Hydnum tenuistipitum and H. microcarpum were previously clustered into the subgen.Brevispina [15,22].However, our phylogenetic tree demonstrated that they are located in an isolated unidentified branch (Figure 1).Hence, H. tenuistipitum and H. microcarpum were removed from the subgen.Brevispina (Table 2).The subgenus classification of H. tenuistipitum and H. microcarpum needs to be further elucidated.It is worth noting that two Chinese collections (IFP 019480 and IFP 019481) were previously identified as H. albomagnum by Cao et al. [15].However, our phylogenetic study showed that they clustered together with the holotype of H. cremeoalbum, forming a well-supported lineage (Figure 1: lineage 19).The two specimens are expected to be further identified by morphological studies.Our phylogenetic analysis also demonstrated that H. subberkeleyanum is a later synonym of H. ventricosum (Figure 1: lineage 8).Moreover, there are no essential morphological differences between the two taxa, judging from their protologues [14,15].It is worth noting that the holotype of H. pallidomarginatum and the holotype of H. flabellatum grouped together without statistical support (Figure 1: lineage 31); their taxonomic relationship should be further assessed.In addition, although many section or subsection classifications of Hydum have been defined in this study, there is no sufficient support for many clades (Figure 1).Hence, more gene fragments (TEF1 and RBP2) are needed to further construct molecular phylogenetic trees in the future.
Our phylogenetic study provided new perspectives into the geography of Hydnum.For example, H. umbilicatum is shared by East Asia, Europe, and North America [1,4]; H. boreorepandum, H. melitosarx, and H. mulsicolor occur in both East Asia and Europe [1,12,22]; H. cremeoalbum, H. minus, H. orientalbidum, H. pinicola, and H. ventricosum (synonymy: H. subberkeleyanum) are found in both China and Japan (Figure 1) [14,15,22]; H. treui is distributed in both tropical China and Papua New Guinea (Figure 1).It is worth noting that H. jussii and H. vesterholtii were reported to be distributed in China, according to previous studies [9,22].However, they were just identified by molecular data; the specimens of the two species from China should be further studied morphologically in the future.Our phylogenetic tree also indicated that H. berkeleyanum and H. umbilicatum are probably distributed in China (Figure 1).Similarly, the Chinese collections of the two taxa should be further defined using morphological studies.
Therefore, H. berkeleyanum, H. jussii, H. umbilicatum, and H. vesterholtii were excluded in Table 2 and the key, and they should be further studied morphologically in the future.

Conclusions
In this study, a total of six Hydnum species from China are described, including a new species: H. erectum and five known taxa: H. cremeoalbum, H. minus, H. orientalbidum, H. tenuistipitum, and H. treui.Among them, H. treui is new to China.Hydnum subberkeleyanum is a later synonym of H. ventricosum; H. minus is confirmed to be a member of subgen.Brevispina.

Table 1 .
Taxa, vouchers, locations, and GenBank/UNITE accession numbers of DNA sequences used in this study.

Table 2 .
Subgenera, sections, subsections, and accepted species of Hydnum in China.