Phylogenetic and Taxonomic Analyses of Three New Wood-Inhabiting Fungi of Xylodon (Basidiomycota) in a Forest Ecological System

Wood-inhabiting fungi are a cosmopolitan group and show a rich diversity, growing in the vegetation of boreal, temperate, subtropical, and tropical regions. Xylodon grandineus, X. punctus, and X. wenshanensis spp. nov. were found in the Yunnan–Guizhou Plateau, China, suggested here to be new fungal species in light of their morphology and phylogeny. Xylodon grandineus is characterized by a grandinioid hymenophore and ellipsoid basidiospores; X. punctus has a membranous hymenophore, a smooth hymenial surface with a speckled distribution, and absent cystidia; X. wenshanensis has a grandinioid hymenophore with a cream to slightly buff hymenial surface and cystidia of two types. Sequences of the ITS and nLSU rRNA markers of the studied samples were generated, and phylogenetic analyses were performed using the maximum likelihood, maximum parsimony, and Bayesian inference methods. After a series of phylogenetic studies, the ITS+nLSU analysis of the order Hymenochaetales indicated that, at the generic level, six genera (i.e., Fasciodontia, Hastodontia, Hyphodontia, Lyomyces, Kneiffiella, and Xylodon) should be accepted to accommodate the members of Hyphodontia sensu lato. According to a further analysis of the ITS dataset, X. grandineus was retrieved as a sister to X. nesporii; X. punctus formed a monophyletic lineage and then grouped with X. filicinus, X. hastifer, X. hyphodontinus, and X. tropicus; and X. wenshanensis was a sister to X. xinpingensis.

To accomplish the genome evolution and reconstruction of the phylogenetic relationships of fungi, an increasing number of taxa have been used for the fungal tree of life by using genome-scale data in the molecular systematics by mycologists [16], and both the species diversity and the classification of fungi are still in great, flux mainly in the more basal branches of the tree topology. The true diversity will come to light from genomic analyses and more region surveys worldwide based on some unique fungal groups.
These pioneering studies of the genus Xylodon were just the prelude to the molecular systematics period [16]. Hyphodontia s.l. was shown to be a polyphyletic genus, in which Xylodon and Kneiffiella P. Karst are the most species rich [10,12,14]. Due to a lack of rDNA sequences for many taxa, the molecular data were not enough to separate many genera clearly; therefore, a broad concept of Hyphodontia s.l. was employed by mycologists [10,12,14,32,34]. Yurchenko et al. described two clades: the Xylodon-Lyomyces-Rogersella clade and the Xylodon-Schizopora-Palifer clade, and they suggested to mix the species of Xylodon, Schizopora Velen., Palifer Stalpers and P.K. Buchanan, Lyomyces P. Karst., and Rogersella Liberta and A.J. Navas within both clades. The research comprised the representative sequences and taxa of Hyphodontia s.l., such as Xylodon, Schizopora, Palifer, Lyomyces, and Rogersella, in which the result demonstrated that it was hard to distinguish the two genera Xylodon and Schizopora on the basis of the morphological and phylogenetic information; therefore, the authors proposed that Xylodon and Schizopora should be united into the genus Xylodon [12]. For the phylogenetic relationship of the Xylodon species, it was confirmed that the two genera Lagarobasidium Jülich and Xylodon should be synonymous based on molecular data from the ITS and nLSU regions, in which the three species X. pumilius (Gresl. and Rajchenb.) K.H. Larss., X. magnificus (Gresl. and Rajchenb.) K.H. Larss., and X. rickii (Gresl. and Rajchenb.) K.H. Larss. were combined into Xylodon [36]. All of the members of the genera Odontipsis Hjortstam and Ryvarden and Palifer were placed in the genus Xylodon based on the molecular analyses of 28S and ITS data, in which they proposed four new species of Xylodon as X. exilis Yurchenko, Riebesehl and Langer, X. filicinus Yurchenko and Riebesehl, X. follis Riebesehl, Yurchenko and Langer, and X. pseudolanatus Nakasone, Yurchenko and Riebesehl [14]. Based on the multiple loci in Hyphodontia s.l., Fasciodontia Yurchenko and Riebesehl, Hastodontia (Parmasto) Hjortstam and Ryvarden, Hyphodontia J. Erikss., Lyomyces, Kneiffiella, and Xylodon in the order Hymenochaetales, they were divided into four clades [41]. The phylogeny of the Xylodon species based on the ITS and nLSU sequences proposed three new taxa from China, in which X. gossypinus C.L. Zhao and K.Y. Luo and X. brevisetus (P. Karst.) Hjortstam and Ryvarden grouped together [40]. Based on the morphological descriptions and molecular analyses, three new species: Xylodon angustisporus Viner and Ryvarden, X. dissiliens Viner and Ryvarden, and X. laxiusculus Viner and Ryvarden, were described and placed in Xylodon, which were found in Africa [42]. A phylogenetic and taxonomic study on Xylodon (Hymenochaetales) described three new species of this genus from southern China, inferred from 61 fungal specimens representing 55 species, which enriched the fungal diversity of this areas [43].
During investigations on wood-inhabiting fungi in the Yunnan-Guizhou Plateau of China, three additional Xylodon species were collected. To clarify the placement and relationships of the three species, we study carried out a phylogenetic and taxonomic study on Xylodon, based on the ITS and nLSU sequences.

Sample Collection and Herbarium Specimen Preparation
The fresh fruiting bodies of the fungi growing on fallen angiosperm branches and fallen Pinus armandii branches were collected from Honghe, Wenshan, and Yuxi of Yunnan Province, China. The samples were photographed in situ, and fresh macroscopic details were recorded. Photographs were recorded by a Jianeng 80D camera. All of the photos were focus stacked and merged using Helicon Focus software. Macroscopic details were recorded and transported to a field station where the fruit body was dried on an electronic food dryer at 45 • C. Once dried, the specimens were sealed in an envelope and zip-lock plastic bags and labeled [43]. The dried specimens were deposited in the herbarium of the Southwest Forestry University (SWFC), Kunming, China.

Morphology
The macromorphological descriptions were based on field notes and photos captured in the field and lab. The color terminology follows that of Petersen [44]. The micromorphological data were obtained from the dried specimens after observation under a light microscope with a magnification of 10 × 100 oil [27]. The following abbreviations are used: KOH = 5% potassium hydroxide water solution, CB− = acyanophilous, IKI− = both inamyloid and indextrinoid, L = mean spore length (arithmetic average for all spores), W = mean spore width (arithmetic average for all spores), Q = variation in the L/W ratios between the specimens studied, and n = a/b (number of spores (a) measured from given number (b) of specimens).

Molecular Phylogeny
The CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to obtain genomic DNA from the dried specimens according to the manufacturer's instructions. The nuclear ribosomal ITS region was amplified with ITS5 and ITS4 primers [45]. The nuclear nLSU region was amplified with the LR0R and LR7 primer pair (http://lutzonilab.org/nuclear-ribosomal-dna/, accessed on 22 January 2022). The PCR procedure for ITS was as follows: initial denaturation at 95 • C for 3 min, followed by 35 cycles at 94 • C for 40 s, 58 • C for 45 s and 72 • C for 1 min, and a final extension of 72 • C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94 • C for 1 min, followed by 35 cycles at 94 • C for 30 s, 48 • C for 1 min and 72 • C for 1.5 min, and a final extension of 72 • C for 10 min. The PCR products were purified and sequenced at Kunming Tsingke Biological Technology Limited Company (Kunming, China). All of the newly generated sequences were deposited in NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 22 January 2022) ( Table 1).
The sequences were aligned in MAFFT version 7 [56] using the G-INS-i strategy. The alignment was adjusted manually using AliView version 1.27 [57]. The dataset was aligned first, and then the sequences of ITS and nLSU were combined with Mesquite version 3.51. The alignment datasets were deposited in TreeBASE (submission ID 29411). ITS+nLSU sequences and ITS-only datasets were used to infer the position of the three new species in the genus Xylodon and related species. Sequences of Hymenochaete cinnamomea (Pers.) Bres. and H. rubiginosa (Dicks.) Lév. retrieved from GenBank were used as an outgroup in the ITS+nLSU analysis ( Figure 1); sequences of Lyomyces orientalis Riebesehl, Yurch. and Langer, and L. sambuca (Pers.) P. Karst. retrieved from GenBank were used as an outgroup in the ITS analysis ( Figure 2) [41].
Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) analyses were applied to the combined three datasets following a previous study [58], and the tree construction procedure was performed in PAUP* version 4.0b10 [59]. All of the characters were equally weighted, and gaps were treated as missing data. Using the heuristic search option with TBR branch swapping and 1000 random sequence additions, trees were inferred. Max trees were set to 5000, branches of zero length were collapsed, and all parsimonious trees were saved. Clade robustness was assessed using bootstrap (BT) analysis with 1000 replicates [60]. Descriptive tree statistics, tree length (TL), the consistency index (CI), the retention index (RI), the rescaled consistency index (RC), and the homoplasy index (HI) were calculated for each maximum parsimonious tree generated. The multiple sequence alignment was also analyzed using maximum likelihood (ML) in RAxML-HPC2 [61]. Branch support (BS) for ML analysis was determined by 1000 bootstrap eplicates.  [62] was used to determine the best-fit evolution model for each dataset for Bayesian inference (BI), which was performed using MrBayes 3.2.7a with a GTR+I+G model of DNA substitution and a gamma distribution rate variation across sites [63]. A total of four Markov chains were run for two runs from random starting trees for 1.2 million generations for ITS+nLSU ( Figure 1) and 9 million generations for ITS ( Figure 2) with trees and parameters sampled every 1000 generations. The first one-fourth of all of the generations were discarded as burn-ins. The majority-rule consensus tree of all of the remaining trees was calculated. Branches were considered significantly supported if they received a maximum likelihood bootstrap value (BS) of >70%, a maximum parsimony bootstrap value (BT) of >70%, or Bayesian posterior probabilities (BPP) of >0.95. sion 3.51. The alignment datasets were deposited in TreeBASE (submission ID 29411). ITS+nLSU sequences and ITS-only datasets were used to infer the position of the three new species in the genus Xylodon and related species. Sequences of Hymenochaete cinnamomea (Pers.) Bres. and H. rubiginosa (Dicks.) Lév. retrieved from GenBank were used as an outgroup in the ITS+nLSU analysis ( Figure 1); sequences of Lyomyces orientalis Riebesehl, Yurch. and Langer, and L. sambuca (Pers.) P. Karst. retrieved from GenBank were used as an outgroup in the ITS analysis ( Figure 2) [41].  Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) analyses were applied to the combined three datasets following a previous study [58], and the tree construction procedure was performed in PAUP* version 4.0b10 [59]. All of the characters were equally weighted, and gaps were treated as missing data. Using the heu-  Etymology-grandineus (Lat.): Referring to the hymenial surface grandinioid of the specimens.
Phylogenetically, the molecular relationships of Xylodon and related genera located in Hyphodontia s.l. (Hymenochaetales), on the basis of the combined datasets of ITS, nLSU, and mt-SSU regions, indicated that seven families-Chaetoporellaceae Jülich, Coltriciaceae Jülich, Hymenochaetaceae Donk, Neoantrodiellaceae Y.C. Dai, B.K. Cui, Jia J. Chen and H.S. Yuan, Nigrofomitaceae Jülich, Oxyporaceae Zmitr. and Malysheva, and Schizoporaceae-were monophyletic lineages, which nested in the order Hymenochaetales, in which some genera grouped into Hyphodontia s.l. as independent genera, including Xylodon [41]. In the present study (Figure 1), four families in the order Hymenochaetales were analyzed by the ITS+nLSU data, which showed that the genus Xylodon nested into the family Schizoporaceae.
The macromorphology of the basidiomata and hymenophore construction do not reflect monophyletic groups based on a higher-level phylogenetic classification of polypores [82]. The current phylogeny (Figure 2) shows that the morphological characteristics do not follow the phylogenetic grouping of different taxa in Xylodon based on the ITS datasets.
Wood-inhabiting fungi are a characteristic group of Basidiomycota, which has a number of corticioid, poroid, and hydnoid genera based on the results of morphological, phylogenetic, and cytological studies in China [8,9]. To date, thirty-six species of Xylodon have been recorded in China [12,14,33,36,40,41,43,46,52,83], but the species diversity of Xylodon is still not well known in China, especially in the country's subtropical and tropical areas. This paper enriches our knowledge of fungal diversity in this area, and it is likely that more new taxa will be found with further fieldwork and molecular analyses.