A Phylogenetic and Taxonomic Study on Xylodon (Hymenochaetales): Focusing on Three New Xylodon Species from Southern China

Three wood-inhabiting fungal species, Xylodon laceratus, X. montanus, and X. tropicus spp. nov., were collected from southern China, here proposed as new taxa based on a combination of morphological features and molecular evidence. Xylodon laceratus is characterized by the resupinate basidiomata with grandinioid hymenophore having cracked hymenial surface, and ellipsoid basidiospores; X. montanus is characterized by the annual basidiomata having the hard, brittle hymenophore with cream hymenial surface, and ellipsoid to broadly ellipsoid basidiospores (3.9–5.3 × 3.2–4.3 µm); and X. tropicus is characterized by its grandinioid hymenophore with buff to a pale brown hymenial surface and subglobose basidiospores measuring 2–4.8 × 1.6–4 µm. Sequences of ITS and nLSU rRNA markers of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony, and Bayesian inference methods. The ITS+nLSU analysis of the order Hymenochaetales indicated that the three new species clustered into the family Schizoporaceae, located in genus Xylodon; based on further analysis of ITS dataset, X. laceratus was a sister to X. heterocystidiatus; X. montanus closely grouped with X. subclavatus and X. xinpingensis with high support; while X. tropicus was retrieved as a sister to X. hastifer.


Introduction
Wood-inhabiting fungi are noteworthy components of woody plant ecosystems and take an active part in the decomposition and turnover of nutrients from wood, in which the corticioid fungi are one of the major groups of wood-inhabiting Basidiomycota [1]. Hymenochaetales is one of the most important orders in Basidiomycota because many species in this order are medicinal fungi, and some of them are forest pathogens [2]. Within Hymenochaetales, commonly accepted families Chaetoporellaceae, Coltriciaceae, Hymenochaetaceae, Hyphodontiaceae, Neoantrodiellaceae, Nigrofomitaceae, Oxyporaceae, and Schizoporaceae are supported as individual monophyletic lineages [3]. Schizoporaceae was introduced by Walter Jülich with Schizopora Velen. as the type genus and included the other three genera Fibriciellum J. Erikss. & Ryvarden, Fibricium J. Erikss. and Fibrodontia Parmasto [4].

Morphology
Macromorphological descriptions are based on field notes and photos captured in the field and lab. Color terminology follows Petersen [35]. Micromorphological data were obtained from the dried specimens after the observation under a light microscope with a magnification 10 × 100 oil [17]. The following abbreviations were 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, P.R. China) was used to obtain genomic DNA from the dried specimens using the manufacturer's instructions. The nuclear ribosomal ITS region was amplified with primers ITS5 and ITS4 [36]. The nuclear nLSU region was amplified with primer pair LR0R and LR7 (http://lutzonilab.org/nuclear-ribosomal-dna/, accessed on 5 November 2021). 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 (Yunnan Province, P.R. China). All newly generated sequences were deposited in NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 20 October 2021) ( Table 1). The sequences were aligned in MAFFT 7 (https://mafft.cbrc.jp/alignment/server/, accessed on 5 November 2021) using the "G-INS-i" strategy for the ITS and ITS+nLSU dataset. The alignment was adjusted manually using BioEdit [48]. The dataset was aligned first, and then ITS and nLSU sequences were combined with Mesquite version 3.51. Alignment datasets were deposited in TreeBASE (submission ID 29060). ITS + nLSU sequences and ITS-only datasets were used to infer the position of the three new species among Xylodon and related taxa. 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 mascarensis Riebesehl, Yurchenko & Langer, and L. sambuca (Pers.) P. Karst. retrieved from GenBank were used as an outgroup in the ITS-only analysis ( Figure 2) [3].
Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) analyses were applied to the combined three datasets following a previous study [49], and the tree construction procedure was performed in PAUP* version 4.0b10 [50]. All 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 [51]. 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 generated. The multiple sequence alignment was also analyzed using maximum likelihood (ML) in RAxML-HPC2 through the Cipres Science Gateway [52]. Branch support (BS) for ML analysis was determined by 1000 bootstrap replicates. MrModeltest 2.3 [53] was used to determine the best-fit evolution model for each data set 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 [54]. A total of 4 Markov chains were run for 2 runs from random starting trees for 6 million generations for ITS + nLSU ( Figure 1) and 7.5 million generations for ITS ( Figure 2) with trees and parameters sampled every 1000 generations. The first one-fourth of all generations were discarded as burn-in. The majority-rule consensus tree of all remaining trees was calculated. Branches were considered as significantly supported if they received maximum likelihood bootstrap value (BS) >70%, maximum parsimony bootstrap value (BT) >70%, or Bayesian posterior probabilities (BPP) >0.95.   Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) analyses were applied to the combined three datasets following a previous study [49], and the tree construction procedure was performed in PAUP* version 4.0b10 [50]. All 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 [51]. 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 generated. The multiple sequence alignment was also analyzed using maximum likelihood (ML) in RAxML-HPC2           Etymology-montanus (Lat.): referring for species collected in montane habitat in Yunnan-Guizhou Plateau.
Phylogenetically, the molecular relationships of species belonging to Hyphodontia s.l. within Hymenochaetales inferred from the combined dataset of ITS, nLSU, and mt-SSU regions, showed that the phylogeny employed by the dataset strongly supported Hymenochaetales as an independent order, in which seven families, Chaetoporellaceae,
Phylogenetically, the molecular relationships of species belonging to Hyphodontia s.l. within Hymenochaetales inferred from the combined dataset of ITS, nLSU, and mt-SSU regions, showed that the phylogeny employed by the dataset strongly supported Hymenochaetales as an independent order, in which seven families, Chaetoporellaceae, Coltriciaceae, Hymenochaetaceae, Neoantrodiellaceae, Nigrofomitaceae, Oxyporaceae, Schizoporaceae were as seven monophyletic lineages, that several genera nested into Hyphodontia s.l. were supported as independent genera [3]. In the present study (Figure 1), four related families in the order Hymenochaetales were carried out by the ITS+nLSU analysis, which showed that the six genera, Fasciodontia, Hastodontia, Hyphodontia, Kneiffiella, Lyomyces, and Xylodon nested into related families, and our three new taxa clustered into genus Xylodon, belonging to the family Schizoporaceae.
Miettinen et al. [71] analyzed a higher-level phylogenetic classification of polypores and showed that the macromorphology of fruiting bodies and hymenophore construction did not reflect monophyletic groups. The current phylogeny shows that the morphological characters do not follow the phylogenetic group on different taxa in this genus. However, several characters lead the key role, e.g., the hyphal system monomitic, basidiospores ellipsoid, and thin-walled ( Figure 2).