Phylogenetic Analyses and Morphological Studies Reveal Four New Species of Phellodon (Bankeraceae, Thelephorales) from China

Phellodon is a genus of ectomycorrhizal fungi with important ecological roles and exploitable biological activities. In this study, four new species of Phellodon, P. caesius, P. henanensis, P. concentricus and P. subgriseofuscus, are described from China based on morphological characters and molecular evidence. The phylogenetic analyses of Phellodon were carried out based on the ITS + nLSU gene regions and the combined sequence dataset of ITS + nLSU + nSSU + RPB1 + RPB2 gene regions. Phellodon caesius is characterized by its dark bluish-grey, dark grey to black grey pileus, ash grey to dark bluish-grey spines, and the presence of both simple septa and clamp connections on generative hyphae of stipe. Phellodon concentricus is characterized by its zonate pileal surface, dark grey context in pileus, and spongy basidiomata. Phellodon henanensis is characterized by its ash grey, light vinaceous grey to light brown pileal surface, thin context in pileus, and the presence of both simple septa and clamp connections on generative hyphae of spines. Phellodon subgriseofuscus is characterized by its fuscous to black pileal surface, white to light brown spines, and vinaceous grey context. Illustrated descriptions and the ecological habits of the novel species are provided.

Species in Phellodon are a group of ectomycorrhizal fungi with important ecological roles [3]. The symbiotic relationship between mycorrhizal and host plants plays an essential role in nutrient cycling, energy flow, community species composition, biodiversity, and ecosystem change in forest ecosystems [4]. As significant ectomycorrhizal fungi, stipitate hydnoid fungi connected with plant roots can reflect the conservation state of forest ecosystems [5]. They can promote the absorption of nutrients by plants, which in turn promotes the circulation of materials in the ecosystem [3]. In addition, some species in Phellodon have exploitable biological activity. Stadler and Anke [6] conducted a study on Phellodon melaleucus (Sw. ex Fr.) P. Karst. and isolated a new antibiotic Phellodonic Acid from it. Reekie et al. [7] isolated a biologically active and highly functionalized hirsute derivative from the Tasmanian fungus Phellodon melaleucus and proposed the chemoenzymatic total synthesis of phellodonic acid. Fang et al. [8] isolated cyathane diterpenoids and nitrogenous terphenyl derivative from the fruiting bodies of basidiomycete Phellodon niger. Therefore, taxonomic and phylogenetic studies on Phellodon can lay the foundation for exploring their ecological functions and biological activities.
Fries originally placed the species of Phellodon in his tribe Mesopus, section Lignosa, which was made to include all tough mesopodous species of the Hydnaceae [9]. At that time, species of Phellodon were considered members of Hydnaceae. In 1961, Donk established Bankeraceae and made Bankera Coker and Beers and Phellodon members of the family [10]. Baird et al. [11] recombined Bankera fuligineoalba (J.C. Schmidt) Pouzar, the typified species of Bankera, to Phellodon. Since then, Bankera has been incorporated into Phellodon. From 1956 to 2005, morphological characteristics of Phellodon were systematically and deeply studied in North America and Europe [2,[12][13][14][15][16][17][18][19][20][21][22]. Subsequently, with the development of molecular systematics, DNA sequence analysis was gradually introduced into the taxonomic and phylogenetic studies of the Bankeraceae [11,[23][24][25]. However, these studies only focus on the internal transcribed Spacer (ITS) sequences, and there are still many unanswered questions. Baird et al. [11] reevaluated the species of stipitate hydnums from the southern United States and identified 41 distinct taxa of Hydnellum, Phellodon, and Sarcodon. They conducted a phylogenetic study based on ITS sequence and proved that Phellodon is independent of Hydnellum and Sarcodon. Li [26] conducted a systematic study of the Bankeraceae in Korea using ITS, the large subunit of nuclear ribosomal RNA gene (nLSU), and the second largest subunit of RNA polymerase II (RPB2) sequences, and 17 species were determined including the genus Phellodon. It was the first analysis of the family based on multigene sequences, but the number of species included in this phylogenetic analysis is relatively limited because many species do not have available sequences. In recent years, taxonomic and phylogenetic studies of Phellodon have been carried out in China, and multiple gene fragments of Phellodon have been provided. Song et al. [27] described four new species of Phellodon from southern China and provided the available sequences of nLSU, the small subunit of nuclear ribosomal RNA gene (nSSU), the small subunit of mitochondrial rRNA gene (mtSSU), the largest subunit of RNA polymerase II (RPB1), and RPB2 genes of Phellodon. Phylogenetic trees were constructed based on the combined ITS + nLSU + nSSU + RPB1 + RPB2 sequences, which confirmed the affinities of three new species and reveal the relationships of Phellodon species [28]. About 33 species have been described and transferred to the genus according to Index Fungorum (http://www.indexfungorum.org/ (accessed on 26 April 2022)). So far, eight species of Phellodon have been described in China [27][28][29], which means that the genus may have a relatively large distribution in China.

Morphological Studies
The specimens used in this study were collected during the annual growing season of macrofungi. At the same time, the specimen information, host trees, ecological habits, location, altitude, collector, and date were recorded, and photos of the fruiting bodies and growth environment were taken. The location information and ecological habits of the specimens mentioned above are stated in the results section. All samples examined in this study were deposited at the herbaria of the Institute of Microbiology, Beijing Forestry University, China (BJFC). Micro-morphological data were obtained from dried specimens and observed under a light microscope (Nikon Eclipse E 80i microscope, Nikon, Tokyo, Japan) following methods in Liu et al. [38].
Samples for microscopic examination were mounted in Cotton Blue, Melzer and 5% potassium hydroxide (KOH), separately. Basidiospores were measured from sections cut from the spines. The following abbreviations are used: IKI, Melzer's reagent; IKI-, neither amyloid nor dextrinoid; KOH, 5% potassium hydroxide; CB, Cotton Blue; CB-, acyanophilous; L = mean spore length, W = mean spore width, Q = L/W ratio, n (a/b) = number of spores (a) measured from given number of specimens (b). A field Emission Scanning Electron Microscope (FESEM) Hitachi SU-8010 (Hitachi, Ltd., Tokyo, Japan) was used to film the spore's morphology, and the materials were studied at up to 1800 times magnification, according to the method by Sun et al. [45].

DNA Extraction, PCR Amplification, and Sequencing
A CTAB plant genome rapid extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd.) was employed for DNA extraction from dried specimens. The extracted DNA were used to perform the polymerase chain reaction (PCR) according to the manufacturer's instructions with some modifications [34,40]. The primer pairs ITS5/ITS4, LR0R/LR7, NS1/NS4, AF/Cr, and 5F/7Cr were used to amplify ITS, nLSU, nSSU, RPB1, and RPB2 sequences [27,28]. The concentration of all primers is 1 g per mL. The final Polymerase Chain Reaction (PCR) volume was 30 µL; each tube contained 1 µL each primer, 1 µL extracted DNA, 12 µL ddH2O, and 15 µL 2 × EasyTaq PCR Supermix (TransGen Biotech Co., Ltd., Beijing, China). PCRs were performed on S1000™ Thermal Cycler (Bio-Rad Laboratories, CA, USA). The PCR procedure for ITS was: initial denaturation at 95 • C for 3 min, followed by 34 cycles of denaturation at 94 • C for 40 s, annealing at 56 • C for 45 s and extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min. The PCR process for nLSU and nSSU was as follows: initial denaturation at 94 • C for 1 min, followed by 35 cycles at 94 • C for 30 s, 50 • C for 1 min, 72 • C for 90 s, and a final extension of 72 • C for 10 min. The PCR process for RPB1 and RPB2 was as follows: initial denaturation at 94 • C for 2 min, 9 cycles at 94 • C for 45 s, 60 • C for 45 s, followed by 36 cycles at 94 • C for 45 s, 53 • C for 1 min, 72 • C for 90 s and a final extension of 72 • C for 10 min. The PCR products were purified and sequenced at the Beijing Genomics Institute, China, with the same primers. All sequences analyzed in this study were deposited at GenBank and listed in Table 1.

Phylogenetic Analyses
The phylogenetic relationships of Phellodon were analyzed by the datasets of combined ITS + nLSU sequences and ITS + nLSU + nSSU + RPB1 + RPB2 sequences. The ITS + nLSU sequences were used to infer the phylogeny of Phellodon. The 5-gene datasets more specifically showed the differences between Phellodon species. The sequences generated in this study and retrieved from GenBank were combined with ITS, nLSU, nSSU, RPB1, and RPB2 sequences of Phellodon and outgroups. Amaurodon aquicoeruleus Agerer (UK 452) and A. viridis (Alb. and Schwein.) J. Schröt (TAA 149664) were used as the outgroups, according to Song et al. [28]. The datasets were aligned in MAFFT 7 [46] and manually adjusted in BioEdit [47]. Alignments were spliced in Mesquite v. 3.2. [48]. The congruences of the 5-gene (ITS, nLSU, nSSU, RPB1, and RPB2,) were evaluated with the incongruence length difference (ILD) test [49] implemented in PAUP* version 4.0b10 [50], under heuristic search and 1000 homogeneity replicates. The best-fit evolutionary model was selected with AIC (Akaike Information Criterion) using jModelTest for each partition [51,52]. Phylogenetic analyses were carried out according to previous studies [31,40].
Maximum parsimony (MP) analysis was performed in PAUP*version 4.0b10 [50] with the heuristic search. All characters were equally weighted and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees was set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap analysis with 1000 replicates [53]. Descriptive tree statistics, tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each Maximum Parsimonious Tree (MPT) generated. Only the Maximum Parsimony best tree from all searches was kept. Maximum Likelihood (ML) analysis was performed in RAxmL v.7.2.8 with a GTR + G + I model [54]. All model parameters were estimated by the program, but only the best maximum likelihood tree from all searches was kept. MrModeltest 2.3 [55,56] was used to determine the best-fit evolution model for each dataset for Bayesian inference (BI). BI was performed using MrBayes 3.2.6 on Abe through the Cipres Science Gateway (www.phylo.org, accessed on 23 April 2022) with 2 independent runs, each one beginning from random trees with 4 simultaneous independent Chains, performing 2 million replicates, sampling one tree every 100 generations [57]. The first 25% of the sampled trees were discarded as burn-in and a majority rule consensus tree of all remaining trees was calculated.
Branches that received bootstrap supports for MP, ML greater than or equal to 50% and Bayesian inference (BI) greater than or equal to 0.95 were considered as significantly supported. Phylogenetic trees were visualized using FigTree v1.4.2.

Phylogenetic Analyses
The combined ITS + nLSU dataset included sequences from 81 fungal samples representing 35 taxa. The dataset had an aligned length of 2244 characters, including gaps (865 characters for ITS, 1379 characters for nLSU), of which 1564 characters were constant, 69 were variable and parsimony-uninformative, and 611 were parsimony-informative. Maximum parsimony analysis yielded 1205 equally parsimonious trees (TL = 1894, CI = 0.548, RI = 0.843, RC = 0.462, HI = 0.452). The best models for each region of the combined ITS + nLSU sequence dataset estimated and applied in the Bayesian analysis were both GTR + I + G models. Bayesian and ML analysis resulted in a topology similar to that from MP analysis. The Bayesian analysis resulted in a concordant topology with an average standard deviation of split frequencies = 0.005071. Only the MP tree is provided in Figure 1, and the MP (≥50%), ML (≥50%), and BI (≥0.95) are shown at the nodes.
The combined 5-gene ITS + nLSU + nSSU + RPB1 + RPB2 dataset included sequences from 81 fungal samples representing 35 taxa. The dataset had an aligned length of 5597 characters, including gaps (865 characters for ITS, 1379 characters for nLSU, 1070 characters for nSSU, 1204 characters for RPB1, 1079 characters for RPB2), of which 4513 characters were constant, 199 were variable and parsimony-uninformative, and 885 were parsimony-informative. Maximum parsimony analysis yielded 2389 equally parsimonious trees (TL = 2389, CI = 0.615, RI = 0.857, RC = 0.527, HI = 0.385). The best-fit evolutionary models applied in Bayesian analyses were selected by jModelTest for each region of the five genes, the model for ITS, nLSU, nSSU, RPB1, and RPB2 was GTR + I+ G with an equal frequency of nucleotides. Bayesian and ML analysis resulted in a topology similar to that from MP analysis. The Bayesian analysis resulted in a concordant topology with an average standard deviation of split frequencies = 0.004330. Only the MP tree is provided in Figure 2, and the MP (≥50%), ML (≥50%), and BI (≥0.95) are shown at the nodes.
Both the ITS + nLSU dataset and the ITS + nLSU + nSSU + RPB1 + RPB2-based phylogenetic tree (Figures 1 and 2) confirmed the affinities of the four new species within Phellodon. The four new species P. caesius, P. concentricus, P. henanensis, and P. subgriseofuscus formed distinct well-supported lineages distant from other species of Phellodon.

Taxonomy
Phellodon caesius B.K. Cui & C.G. Song, sp. nov., Figures 3a, 4a and 5. MycoBank: 846978 Diagnosis-Differs from other Phellodon species by its bluish-grey, dark grey to black grey pileus, ash grey to dark bluish-grey spines, and the presence of both simple septa and clamp connections on generative hyphae of the surface layer of stipe.
Fruitbody-Basidiomata annual, centrally or eccentrically stipitate, single to concrescent, with a light fenugreek odor when dry. Pileus slightly convex in the middle, plicate, up to 3.6 cm in diam, and 0.7 cm thick at the center. Pileal surface bluish-grey, dark bluish-grey to black grey when fresh and becoming pale mouse grey to mouse grey upon drying, azonate, fibrillose to spongy; margin white to ash grey when fresh, and becoming pale mouse grey upon drying, up to 2 mm wide. Context tough, dark violet to dark grey upon drying, up to 3 mm thick. Spines soft, white, ash grey to dark bluish-grey when fresh, becoming fragile, pale mouse grey to ash grey upon drying, up to 2 mm long. Stipe cylindrical, glabrous, dark grey to black in outer layer, black in the inner layer, up to 2.2 cm long, 1.2 cm in diam.
Additional specimen (paratype) examined-CHINA. Fruitbody-Basidiomata annual, centrally or eccentrically stipitate, single to concrescent, with a strong fenugreek odor when dry. Pileus depressed, circular to irregular, up to 4.5 cm in diam, 0.3 cm thick at the center. Pileal surface deep olive to mouse grey upon drying, zonate, fibrillose to spongy at the center; margin fuscous to black upon drying, up to 5 mm wide. Context tough, dark grey upon drying, up to 1 mm thick. Spines soft when fresh, becoming fragile, ash grey upon drying, up to 2.5 mm long. Stipe cylindrical, spongy, deep olive, fuscous to black, up to 2.5 cm long, 1 cm in diam.          Hyphal structure-Hyphal system monomitic; generative hyphae with simple septa; all the hyphae IKI-, CB-; tissues turned light yellow-green to olive green in KOH. Generative hyphae in context dark yellowish-green, thick-walled, rarely branched, regularly arranged, 3-6.5 µm in diam. Generative hyphae in spines yellowish-brown to dark brown, slightly thick-walled, branched, regularly arranged, 2-4.5 µm in diam. Generative hyphae in stipe dark olive-green to black, thick-walled, rarely branched, regularly arranged, 2-6 µm in diam.
Spores-Basidiospores subglobose to globose, hyaline, thin-walled, echinulate, IKI-, CB-, 5-6.2 × 4.5-5. Fruitbody-Basidiomata annual, eccentrically stipitate, usually solitary, with a fenugreek odor when dry. Pileus depressed or shallow infundibuliform, up to 2.2 cm in diam, 0.3 cm thick at the center. Pileal surface ash grey, light vinaceous grey to light brown when fresh and becoming dark brown to black upon drying, azonate, fibrillose; margin cream to light brown when fresh, and becoming apricot-orange upon drying, up to 3 mm wide. Context tough, greyish-brown, up to 1 mm thick. Spines soft, ash grey to light brown when fresh, becoming fragile, vinaceous grey to greyish-brown upon drying, up to 1 mm long. Stipe cylindrical, glabrous, pale greyish-brown to pale mouse grey, up to 1.3 cm long, 0.2 cm in diam.
Fruitbody-Basidiomata annual, eccentrically stipitate, single to concrescent, with a fenugreek odor when dry. Pileus circular to irregular, up to 4.8 cm in diam, 1.2 cm thick at the center. Pileal surface fuscous to black when fresh and becoming dark brown to fuscous upon drying, zonate, glabrous, with radially aligned stripes; margin white to dark brown when fresh, and becoming white to cream upon drying, up to 3 mm wide. Context tough, vinaceous grey upon drying, up to 3 mm thick. Spines soft, white to light brown when fresh, becoming fragile, cream to buff-yellow upon drying, up to 2.5 mm long. Stipe cylindrical, glabrous, greyish-brown, dark brown to fuscous, up to 3.3 cm long, 1.5 cm in diam.

Discussion
Based on the phylogenetic analyses, 29 species of Phellodon grouped together (Figures 1 and 2), including four new species from China: P. caesius, P. concentricus, P. henanensis, and P. subgriseofuscus. Our phylogenetic results are consistent with previous observations [27,28], and further information on the phylogeny and taxonomy of Phellodon is supplied. During the investigations of Phellodon, information on distribution areas and ecological habits was also obtained ( Table 2).
Phellodon subgriseofuscus is closely related to P. griseofuscus B.K. Cui and C.G. Song in our phylogenetic analyses (Figures 1 and 2). Morphologically, P. griseofuscus is similar to P. subgriseofuscus in having dark brown or black pileal surface and white to light brown spines. However, P. griseofuscus can be distinguised by its shorter spines (up to 1 µm), and clamp connections in generative hyphae of pileus and stipe [28].
The diversity and evolutionary relationships of Phellodon species can be objectively revealed by combining traditional morphological observation with molecular systematics methods. In the past, only a few numbers of publications had used phylogenetic analyses of the Phellodon genus, and the majority of those studies had only used the ITS sequences of a few species [11,24,25,29]. Song et al. [27,28] conducted phylogenetic analysis of Phellodon based on 5-gene sequences (ITS + nLSU + nSSU + RPB1 + RPB2), which undoubtedly filled in the blank of multiple gene fragments of Phellodon. In this study, both ITS + LSU and ITS + LSU + SSU + RPB1 +RPB2 datasets share a similar topology with Song et al. [27,28] but with discrepant bootstrap values.
Phellodon species frequently grow beneath pine needles or oak leaves, which serve to prevent water loss, in damp woodlands covered in dense mosses. Specimens collected in China were gathered from forests of pinaceae, fagaceae, or mixed trees (Table 2). It revealed that Phellodon species are host-biased, providing an additional foundation for species discovery and identification. The specimens were collected from northeast, southwest, northwest, and central China at elevations ranging from 870 to 3320 m, which indicated that the genus is a widespread species.
With the addition of the species discussed above, there are now 12 taxa in Phellodon known from China. The identification and descriptions of stipitate hydnoid fungi in this paper can enrich the species diversity of Phellodon and promote the taxonomy and phylogeny of the genus. The combination of morphological and phylogenetic methods will contribute to the exploration of species diversity. Additionally, it suggested that other Phellodon species might be discovered by combining the evidence of morphological characters, molecular data, and ecological habits. A fully resolved phylogeny for species in Phellodon requires evolutionary information from more samples.