Multi-Gene Phylogeny and Taxonomy of Hydnellum (Bankeraceae, Basidiomycota) from China

The genus Hydnellum is an important group of stipitate hydnaceous fungi which can form ectomycorrhiza with many species of woody plants. In recent decades, the frequency and number of basidiocarps observed in China have been declining significantly. So far, however, we know little about the species diversity of Hydnellum in China. In this study, we conducted molecular phylogenetic analyses based on sections of multiple loci, including the large subunit of nuclear ribosomal RNA gene (nLSU), the internal transcribed spacer regions (ITS), the small subunit of nuclear ribosomal RNA gene (SSU) and the second-largest subunit of RNA polymerase II gene (RPB2), as well as morphological studies, of collected samples of Hydnellum from China. We also inferred Maximum Likelihood and Bayesian phylogenies for the order Thelephorales from the dataset of the combined nLSU and ITS. This study has revealed the phylogenetic position of Hydnellum in the order Thelephorales, and phylogenetically confirmed ten major clades in Thelephorales; Twenty-nine taxa are proposed, described or reported, including 10 new subgenera (Hydnellum subgenus Hydnellum, subg. Caesispinosum, subg. Croceum, subg. Inflatum, subg. Rhizomorphum, subg. Scabrosum, subg. Spongiosum, subg. Subindufibulatum, subg. Violaceum and subg. Zonatum), 11 new species (Hydnellum atrorubrum, H. atrospinosum, H. bomiense, H. brunneorubrum, H. fibulatum, H. granulosum, H. inflatum, H. rubidofuscum, H. squamulosum, H. sulcatum and H. yunnanense), 3 newly recorded species (H. caeruleum, H. peckii and H. spongiosipes) and 5 notable specimens (Hydnellum sp 1, H. sp 2, H. sp 3, H. sp 4 and H. sp 5). A classification system based on the morphological characteristics (especially the hyphal structure types) and molecular analyses is proposed to accommodate most species in Hydnellum. The distinguishing characters of the subgenera and the new species with their closely related taxa are discussed. A key to the species of Hydnellum from China is provided.


Introduction
The genus Hydnellum, together with Bankera, Phellodon and Sarcodon, are a homogenous group of soil-inhabiting Basidiomycota (with the common characteristic of a hymenophore with a spinulose hymenium) that belongs to the Bankeraceae, Thelephorales [1,2].
All species of Bankeraceae are considered ectomycorrhizal and are associated with woody plants, mainly members of Pinaceae and Fagaceae [3][4][5][6][7], and colonize natural or relatively undisturbed forests [8]. These fungi can absorb organic substances from host plants and also transport nutrients and water from the soil to the plants, which improves the stability of forest ecosystems [9,10]. In addition, some species of Hydnellum have important medicinal functions, including cholesterol-lowering, antioxidant, anti-inflammatory, antitumor, immune enhancement, etc. [11]. For instance, Lee et al. [12] suggested that H. concrescens extracts prevents the expression of NDV-HN glycoprotein on the cell surface by loid and dextrinoid reactions were tested in Melzer's reagent (IKI): 1.5 g KI (potassium iodide), 0.5 g I (crystalline iodine), 22 g chloral hydrate, 20 mL distilled water; IKI− = neither amyloid nor dextrinoid reaction. Sections were mounted in 5% KOH and studied at magnifications up to 1000× using a Nikon Eclipse E600 microscope (Tokyo, Japan) with phase contrast illumination. Dimensions were measured by the ruler in the eyepiece, with accuracy within 0.1 µm. In presenting basidiospore size ranges, 5% of the measurements at each end of the range are given in parentheses. The following abbreviations are used in the text: Lm = mean spore length, Wm = mean spore width, Q = range of length/width ratios for specimens studied, and n = total number of basidiospores measured from a given number of specimens. The surface morphology for the basidiospores was observed with a Phenom Prox scanning electron microscope (ESEM, Phenom Prox, FEI, The Netherlands) at an accelerating voltage of 20 kV. A thin layer of gold was coated on the samples to avoid charging. Special color terms are from Rayner [39] and Munsell [40].

Molecular Procedures and Phylogenetic Analyses
Fungal taxa and strains used in this study are listed in Table 1. Phire Plant Direct PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA) procedures were used to extract total genomic DNA from the basidiocarps. Polymerase chain reactions (PCR) was performed on a Bio-Rad T100 TM Thermal cycler (Bio-RAD Inc., Hercules, CA, USA). Amplification reactions were performed in a 30 µL reaction mixture using the following final concentrations or total amounts: 0.9 µL template DNA, 15 µL of 2× Phire Plant PCR buffer, 1.5 µL of each primer, 0.6 µL Phire HS II DNA Polymerase, and 10.5 µL ddH 2 O (double distilled water). Primer sequences for the used genes are provided in Table 2. The PCR lthermal cycling program condition was set as follows: initial denaturation at 98 • C for 5 min, followed by 39 cycles at 98 • C for 30 s, × • C (the annealing temperatures for LROR/LR7, ITS1-F/ITS4, NS1/NS4, and bRPB2-6F/bRPB2-7.1R were 47.2 • C, 57.2 • C, 48 • C and 57.2 • C, respectively) for 30 s, 72 • C for 30 s, and a final extension at 72 • C for 1 min. PCR amplification was confirmed on 1% agarose electrophoresis gels stained with ethidium bromide [41]. DNA sequencing was performed at the Beijing Genomics Institute (BGI). All newly generated sequences were submitted to GenBank. Additional LSU rDNA, ITS rDNA, SSU rDNA and RPB2 rDNA sequences in the dataset used to establish phylogenetic relationships were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/php (accessed on 10 August 2021)) and UNITE (https://unite.ut.ee/index.php (accessed on 10 August 2021)) ( Table 1). Nuclear ribosomal RNA genes were used to determine the phylogenetic position of the new species. After PCR amplification, the products were sequenced in both directions and the sequences were assembled using DNAMAN 8.0. DNA sequences were aligned using MAFFT 7.110 [42]. To ensure the repeatability of the results, alignments were not manually adjusted. The best-fit evolutionary models selected by jmodeltest-2.1.10 for genes were GTR+I+G (nLSU), K80+G (ITS1), K80 (5.8S), JC+G (ITS2), TrN+I+G (SSU), K80+G (RPB2) in the first dataset (Hydnellum and Sarcodon dataset) and GTR+I+G (nLSU), K80+G (ITS1), K80+G (5.8S), K80+G (ITS2) in the second dataset (Thelephorales dataset). These models were applied in Bayesian analyses. All gaps were treated as missing data. Maximum Likelihood (ML) analysis was performed in RAxML v8.2.4 with GTR+I+G model [43]. The best tree was obtained by executing 100 rapid bootstrap inferences and thereafter a thorough search for the most likely tree using one distinct model/data partition with joint branch length optimization [44]. Bayesian analyses with MrBayes 3.2.4 [45] implementing the Markov Chain Monte Carlo (MCMC) technique and parameters predetermined with MrMODELTEST2.3 [46,47] were performed. Four simultaneous Markov chains were run starting from random trees, keeping one tree every 100th generation until the average standard deviation of split frequencies was below 0.01. The value of burn-in was set to discard 25% of trees when calculating the posterior probabilities. Bayesian posterior probabilities were obtained from the 50% majority rule consensus of the trees kept. Then the FigTree v1.3.1 were used to visualize the resulting trees.

Phylogenetic Analyses
In the first dataset, 272 sequences derived from four gene loci (nLSU, ITS, SSU and RPB2) were used to build phylogenetic trees; 108 of them were newly generated, including 25 of nLSU, 37 sequences of ITS, 25 of SSU and 21 of RPB2. The phylogenetic construction performed with maximum likelihood and Bayesian Inference (BI) analyses for two combined datasets showed similar topology. The combined LSU-ITS-SSU-RPB2 dataset represented 70 taxa and 3629 characters after being trimmed. Polyozellus mariae and P. multiplex were used as the outgroups according to phylogenetic analysis of Thelephorales. Bayesian analysis ran for 8 million generations and resulted in an average standard deviation of split frequencies of 0.005062. The same dataset and alignment were analysed using the ML method. The Maximum Likelihood tree is shown in Figure 1. In the phylogenetic tree, ten clades which correspond to subgenus Hydnellum, subg. Caesispinosum, subg. Croceum, subg. Inflatum, subg. Rhizomorphum, subg. Scabrosum, subg. Spongiosum, subg. Subindufibulatum, subg. Violaceum and subg. Zonatum were revealed. Twenty-eight sampled specimens formed 11 new species (Hydnellum atrorubrum, H. atrospinosum, H. bomiense, H. brunneorubrum, H. fibulatum, H. granulosum, H. inflatum, H. rubidofuscum, H. squamulosum, H. sulcatum and H. yunnanense) and clustered in a clade that comprised most species of Hydnellum. Four sampled specimens (Wei1474a, Yuan13708 and Yuan13720, Yuan14517) that were confirmed as new records from China clustered with Hydnellum caeruleum, H. peckii and H. spongiosipes with strong support. In addition, five notable specimens, Hydnellum sp 1, Hydnellum sp 2, Hydnellum sp 3, Hydnellum sp 4 and Hydnellum sp 5, formed five separate clades, and need further verification. In the second dataset, the combined ITS and nLSU gene also included sequences from 129 specimens representing 58 taxa of Thelephorales, as well as Steccherinum ochraceum and S. murashkinskyi, which were chosen as outgroups according to previous study [31]. The average standard deviation of split frequencies in the Bayesian analyses reached 0.007357 after running for 8 million generations. The calculated values based on the dataset analysed using the ML method. The Maximum Likelihood tree is shown in Figure 2. It revealed that the Hydnellum clade occupies an independent phylogenetic position. The Hydnellum clade is sister to the Sarcodon clade. According to the phylogenetic tree, ten major clades, Amaurodon clade, Boletopsis clade, Hydnellum clade, Lenzitopsis clade, Odontia clade, Phellodon/Bankera clade, Pseudotomentella/Polyozellus clade, Sarcodon clade, Thelephora/Tomentella clade and Tomentellopsis clade, were identified within the Thelephorales ( Figure 2). Therefore, in order to use the maximum amount of genetic information when defining new species, we conducted the first dataset. Meanwhile, ITS trees of Hydnellum and Sarcodon were constructed and produced a topology similar to that generated by the first dataset (see Supplementary, Figure S1). The purpose of executing the second dataset was to demonstrate the phylogenetic position of Hydnellum species in the Thelephorales.

Taxonomy
Hydnellum subg. Hydnellum MycoBank MB841191 Etymology. Hydnellum (Latin), refers to the subgenus in which the type species of the genus is located.
Included species: Hydnellum atrospinosum, H. suaveolens Type species: Hydnellum suaveolens (Scop.) P. Karst. Notes: This subgenus consists of the genus type Hydnellum suaveolens and our new species H. atrospinosum; they share the characteristics of dark blue context, decurrent and dark spines, clamped generative hyphae in the context and the spines trama and irregularly oblong, tuberculate basidiospores of similar size. Furthermore, both species occur in coniferous forests [27,34]. Basidiocarps annual, solitary to gregarious or concrescent, leathery when fresh, becoming hard and light in weight upon drying; taste mild, odor fragrant when dry. Pileus irregularly ellipsoid to circular, later flabelliform or semicircular and applanate with age, up to 75 mm diam and 6-11 mm thick at center. Pileal surface light orange (5A4) to yellow-

Taxonomy
Hydnellum subg. Hydnellum MycoBank MB841191 Etymology. Hydnellum (Latin), refers to the subgenus in which the type species of the genus is located.
Included species: Hydnellum atrospinosum, H. suaveolens Type species: Hydnellum suaveolens (Scop.) P. Karst. Notes: This subgenus consists of the genus type Hydnellum suaveolens and our new species H. atrospinosum; they share the characteristics of dark blue context, decurrent and dark spines, clamped generative hyphae in the context and the spines trama and irregularly oblong, tuberculate basidiospores of similar size. Furthermore, both species occur in coniferous forests [27,34].
Notes: Hydnellum atrospinosum and H. suaveolens have a close phylogenetic relationship with full support (100% in ML and 1.00 BPP). Morphologically, they both have single to gregarious basidiocarps with glabrous to rugose pileal surface, woody and dark blue context, an eccentric and terete stipe with a bulbous base, conical, decurrent and dark spines, clamped generative hyphae and irregularly oblong, tuberculate basidiospores of similar size. However, H. suaveolens differs from H. atrospinosum by longer spines (up to 6 mm vs. 2.5 mm in H. atrospinosum), context tissues turning light blue to green in KOH and presence of inflated hyphae in the context [27,34]. A special characteristic of H. atrospinosum is that the clamped generative hyphae are present in all parts of the basidiocarp; this trait can also be observed in H. cruentum, H. cyanopodium, H. geogenium and H. scleropodium. H. cruentum differs from H. atrospinosum by plushy to tomentose pileal surface, grayish blue and slightly longer spines (up to 3.5 mm vs. 2.5 mm in H. atrospinosum) and subglobose basidiospores [26,27,51]. H. cyanopodium and H. scleropodium obviously differs in blue spines [27,33,51]. H. geogenium differs in reflexed-multiplex and yellow basidiocarps, pale yellow to brown spines and subglobose basidiospores [1, 27,34].
Notes: The phylogenetic analyses showed that the studied sample matched with Hydnellum caeruleum (EBendiksen584-11) with full support (100% in ML and 1.00 BPP) (Figure 1). ITS sequence BLAST also revealed it is 100% identical to H. caeruleum. Besides, our collection shares identical characters with H. caeruleum described by Maas Geesteranus [26] in morphology. This is the first report of this species from China.
Notes: Hydnellum caeruleum and H. ferrugipes have an adjacent phylogenetic relationship with H. fibulatum according to the phylogenetic tree (Figure 1). H. caeruleum and H. fibulatum have similar morphological characteristics, such as a flat and velutinous pileus when immature, white pileal margin when fresh, central, terete and tomentose stipe, olivaceous context tissue in KOH, presence of occasional clamp-connections in the context and simple-septate hyphae in the spines. However, H. caeruleum can be distinguished by having a larger pileus (up to 80 mm vs. 45 mm in H. fibulatum), rough or colliculose pileal surface when mature, duplex and zonate context [2,26]. H. ferrugipes resembles H. fibulatum in having a white pileal margin when fresh, tomentose and orange to brown stipe, orange-white to brown spines when dry, regularly arranged and occasionally clamped hyphae in the context, unclamped hyphae in the spines and basidia sterigmata with similar size. However, H. ferrugipes differs from H. fibulatum in the infundibuliform pileus with pitted to subnodulose or subcolliculose pileal surface, blue-gray or grayish orange context, considerably longer spines (up to 6 mm vs. 1.5 mm in H. fibulatum) and wider basidiospores (5-6 µm vs. 4.1-4.9 µm in H. fibulatum) [27,34].
Based on microscopical hyphal structure species of Hydnellum and Sarcodon can be divided into five groups, as shown in Table 3 One species, H. regium, has not been classified into these five groups because the information is not available from the original description [33].
In the phylogenetic tree, ten subgenera with moderate to high support in Hydnellum have been distinguished (Figure 1) Group V corresponds to all hyphae clamped in the pileus and the spines, incorporating two subgenera of Hydnellum and six Sarcodon species; the subgenus Hydnellum has dark spines and includes H. atrospinosum and H. suaveolens; the subgenus Caesispinosum has blue spines and includes H. cyanopodium and H. scleropodium; H. geogenium is morphologically related to other species in Group V, but is distantly related in the phylogenetic tree; Sarcodon clade S. aspratus, S. imbricatus, S. leucopus, S. quercinofibulatus, S. scabripes and S. squamosus have the characteristics of long spores compared with the Hydnellum species. Therefore, the classification system using hyphal structure type and phylogenetic subgenera can fix the positions for most species in Hydnellum and Sarcodon, except for some species in Groups III, IV and those without phylogenetic support.
The specimens involved in this study were collected from the northeast, northwest and southwest regions of China, where industrial pollution is relatively low and vegetation is relatively abundant. The forests are primarily dominated by Pinaceae and Fagaceae trees such as Pinus spp., Picea spp., Quercus spp., Lithocarpus spp. and a small portion of other tree families. Thus, we speculated that these species may form an ectomycorrhizal association with Pinaceae and Fagaceae host trees. The species diversity and basidiocarps richness of stipitate hydnoid fungi represented by Hydnellum and Sarcodon species have shown a declining trend across Europe and some regions of North America during the 1970s to 2000s [6,[55][56][57]; the phenomenon is most probably caused by forest management, air pollutants, forest soil acidification, nitrogen deposition and forest succession, among other causes [19,[58][59][60][61]. Many stipitate hydnoid fungi have been included in national Red Lists in Europe [27,[62][63][64]. With the rapid industrialization in China over the past four decades, a significant decline of basidiocarps has also been observed during the course of our field investigation. The identification and description of stipitate hydnoid fungi in this paper will contribute to the understanding of species diversity and provide baseline data for the evaluation and protection of these fungi in China. Table 3. Hyphal-septa type observations in the context of the pileus and the spine trama in the species of Hydnellum and Sarcodon.  Others H. regium hyphae with few simple-septa and with a few clamp-connections Harrison 1964 [33] Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof7100818/s1, Figure S1: Maximum likelihood tree illustrating the phylogeny of Hydnellum and Sarcodon based on ITS sequence dataset. Data Availability Statement: Publicly available datasets were analyzed in this study. All resulting alignments were deposited in TreeBASE (http://www.treebase.org (accessed on 17 August 2021); accession number S28676). All newly generated sequences were deposited in GenBank (https: //www.ncbi.nlm.nih.gov/genbank/ (accessed on 23 September 2021); Table 1). All new taxa were deposited in Mycobank.