Phylogenetic and Taxonomic Analyses of Five New Wood-Inhabiting Fungi of Botryobasidium, Coltricia and Coltriciella (Basidiomycota) from China

In this present study, five new wood-inhabiting fungal taxa, Botryobasidium gossypirubiginosum, Botryobasidium incanum, Botryobasidium yunnanense, Coltricia zixishanensis, and Coltriciella yunnanensis are proposed. Botryobasidium gossypirubiginosum is distinguished by its slightly rubiginous hymenial surface, monomitic hyphal system, which branches at right angles, and subglobose, smooth basidiospores (14–17.5 × 13–15.5 µm); B. incanum is characterized by its white to incanus basidiomata having a hypochnoid hymenial surface, and ellipsoid, smooth basidiospores (6.5–8.5 × 3.5–5 µm); B. yunnanense is characterized by its buff to slightly yellowish hymenial surface, monomitic hyphal system, and broadly ellipsoid to globose, smooth, thick-walled basidiospores (11.5–14.5 × 9.5–10.5 µm); Coltricia zixishanensis differs in its rust brown pileal surface, and ellipsoid, thick-walled basidiospores (5–6.5 × 4–4.5 µm). Coltriciella yunnanensis is distinguished by its tiny pilei, short stipe, and navicular, verrucose basidiospores (10.5–12.5 × 6–7 µm). Sequences of ITS and nLSU genes were used for phylogenetic analyses using the maximum likelihood, maximum parsimony, and Bayesian inference methods. The phylogenetic results inferred from ITS sequences revealed that B. gossypirubiginosum was closely related to B. robustius; the species B. incanum was grouped with B. vagum; B. yunnanense was related to B. indicum. The species C. zixishanensis was grouped with C. confluens and C. perennis. ITS sequences revealed that C. zixishanensis was grouped into the genus Coltriciella, in which it was grouped with Co. globosa and Co. pseudodependens.


Introduction
Wood-inhabiting fungi are a vital component of forest ecosystems, playing several significant ecological roles [1,2].They play a pivotal role in carbon storage and the regulation of nutrient cycling [3].In fact, a variety of fungi, plants, and animals have different degrees of association with wood-inhabiting fungi, providing appropriate microenvironments for growth, reproduction, shelter and, sources of nutrients [4].The genus Botryobasidium Donk (1931: 116) belonged to the family Botryobasidiaceae (Cantharellales, Basidiomycota), typified by B. subcoronatum (Höhn.& Litsch.)Donk (1931: 117) [5].Based on the Index Fungorum (www.indexfungorum.org;accessed on 27 December 2023), the genus Botryobasidium has 106 specific and registered names with 78 species having been accepted worldwide [6].Based on nLSU data analysis, this research demonstrated that the genus Botryobasidium formed a well-supported monophyletic group, as previously demonstrated by its micromorphological and ultrastructural characteristics [7,8].
In this contribution, our main goal is to describe five new species collected from Yunnan Province, China, providing a detailed description of their morphology and molecular characterizations.We present the morphological characteristics and molecular analyses with ITS and nLSU DNA markers that support the taxonomy and phylogenetics of Botryobasidium, Coltricia and Coltriciella species.

Sample Collection and Herbarium Specimen Preparation
Fresh fruiting bodies of fungi growing on the branches and above-ground from angiosperms were collected in Qujing, Puer, Chuxiong, and Dali of Yunnan Province, China.The samples were photographed in situ, and fresh macroscopic details were recorded.Photographs were taken using a Jianeng 80D camera (Tokyo, Japan).All of the photos were focus-stacked and merged using Helicon Focus Pro7.7.5 software.Specimens were dried in an electric food dehydrator at 40 • C, then sealed and stored in an envelope bag, and deposited in the herbarium of the Southwest Forestry University (SWFC), Kunming, Yunnan Province, China.

Morphology
Our macroscopic morphological descriptions are based on field notes and photographs taken outdoors and in the laboratory, and follow Petersen's color terminology [20].The micromorphologic data of dried specimens were observed under a light microscope.The following abbreviations were used: KOH = 5% potassium hydroxide water solution; CB+ = cyanophilous; CB = cotton clue; CB− = acyanophilous; IKI = Melzer's reagent; 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 a given number (b) of specimens).

DNA Extraction and Sequencing
The EZNA HP Fungal DNA Kit (Omega Biotechnologies Co., Ltd., Kunming, China) was used to extract DNA, with some modifications, from the dried specimens.The ITS and nLSU regions were amplified with the ITS5/ITS4 [21] and LR0R/LR7 [22] primer pairs, respectively.The PCR procedure for ITS and nLSU followed that in a previous study [22].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, at 58 • C for 45 s, and at 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, at 48 • C for 1 min, and at 72 • C for 1.5 min, and a final extension of 72 • C for 10 min.The PCR products were purified and directly sequenced at Kunming Tsingke Biological Technology Limited Company, Yunnan Province, China.All of the newly generated sequences were deposited in GenBank (Table 1).

Phylogenetic Analyses
The DNA sequences were aligned in MAFFT version 7 using the G-INS-i strategy [34].The alignment was adjusted manually using AliView version 1.27 [35].The sequence of Lyomyces pruni (Lasch) Riebesehl & Langer fetched from GenBank was used as an outgroup in ITS (Figure 1) analysis following a previous study's analysis [31].The sequence of Russula begonia G.J. Li, T.Z.Liu & T.Z.Wei retrieved from GenBank was used as an outgroup in ITS + nLSU (Figure 2) analysis following a previous study's analysis [33].The sequence of Fomitiporia chinensis (Pilát) Y.C. Dai, X.H. Ji & Vlasák retrieved from GenBank was used as an outgroup in ITS (Figures 3 and 4) analysis following a previous study's analysis [31].

Phylogenetic Analyses
The DNA sequences were aligned in MAFFT version 7 using the G-INS-i strategy [34].The alignment was adjusted manually using AliView version 1.27 [35].The sequence of Lyomyces pruni (Lasch) Riebesehl & Langer fetched from GenBank was used as an outgroup in ITS (Figure 1) analysis following a previous study's analysis [31].The sequence of Russula begonia G.J. Li, T.Z.Liu & T.Z.Wei retrieved from GenBank was used as an outgroup in ITS + nLSU (Figure 2) analysis following a previous study's analysis [33].The sequence of Fomitiporia chinensis (Pilát) Y.C. Dai, X.H. Ji & Vlasák retrieved from GenBank was used as an outgroup in ITS (Figures 3 and 4) analysis following a previous study's analysis [31].Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) analyses were applied to the combined three datasets.Approaches to the phylogenetic analysis process followed those of Zhao and Wu [36].MP analysis was performed in PAUP* version 4.0b10 [37].All of the 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 were set to 5000, branches of zero length were collapsed, and all most-parsimonious trees were saved.Clade robustness was assessed using bootstrap (BT) analysis with 1000 replicates [38].Descriptive tree statistics, such as 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 most-parsimonious tree generated.ML was inferred using RAxML-HPC2 through Cipres Science Gateway (www.phylo.org(accessed on 10 January 2024)) [39].Branch support (BS) for ML analysis was determined using 1000 bootstrap replicates and evaluated under the gamma model.
MrModeltest 2.3 [40] was used to determine the best-fit evolution model for each data set for Bayesian inference (BI), which was performed using MrBayes 3.2.7awith a GTR + I + G model of DNA substitution and a gamma distribution rate variation across sites [41].Four Markov chains were run for 2 runs from random starting trees, for 1 million generations (Figure 1), 2 million generations (Figure 2), 1 million generations (Figure 3), and 2 million generations (Figure 4), and trees were sampled every 100 generations.The first one-fourth of all generations was discarded as a burn-in.The majority-rule consensus tree of all remaining trees was calculated.Branches were considered significantly supported if they received maximum likelihood bootstrap values (BS) > 70%, maximum parsimony bootstrap values (BT) >70%, or Bayesian posterior probabilities (BPP) > 0.95.

Molecular Phylogeny
The dataset based on ITS (Figure 1) comprises sequences from 24 fungal samples representing 12 species.The dataset had an aligned length of 673 characters, of which 234 characters were constant, 89 characters were variable and parsimony-uninformative, and 350 characters were parsimony-informative.Maximum parsimony analysis yielded one equally parsimonious tree (TL = 935, CI = 0.7134, HI = 0.2866, RI = 0.8617, RC = 0.6147).Bayesian analysis and ML analysis resulted in a similar topology to that resulting from MP analysis with an average standard deviation of split frequencies = 0.004023 (BI), and the effective sample size (ESS) across the two runs was double the average ESS (avg ESS) = 1232.5.
The dataset based on ITS + nLSU (Figure 2) comprises sequences from 104 fungal specimens representing 56 species.The dataset had an aligned length of 2471 characters, of which 1097 characters were constant, 221 characters were variable and parsimony-uninformative, and 1153 characters were parsimony-informative.Maximum parsimony analysis yielded 35 equally parsimonious trees (TL = 7468, CI = 0.3502, HI = 0.6498, RI = 0.6488, RC = 0.2618).Bayesian analysis and ML analysis resulted in a similar topology to that resulting from MP analysis with an average standard deviation of split frequencies = 0.005527 (BI), and the effective sample size (ESS) across the two runs was double the average ESS (avg ESS) = 197.
The dataset based on ITS (Figure 3) comprises sequences from 67 fungal specimens representing 33 species.The dataset had an aligned length of 782 characters, of which 109 characters were constant, 153 characters were variable and parsimony-uninformative, and 520 were parsimony-informative.Maximum parsimony analysis yielded 216 equally parsimonious trees (TL = 3166, CI = 0.4166, HI = 0.5834, RI = 0.6414, RC = 0.2672).Bayesian analysis and ML analysis resulted in a similar topology to that resulting from MP analysis with an average standard deviation of split frequencies = 0.007467 (BI), and the effective sample size (ESS) across the two runs was double the average ESS (avg ESS) = 372.5.
The dataset based on ITS (Figure 4) comprises sequences from 18 fungal specimens representing 12 species.The dataset had an aligned length of 779 characters, of which 273 characters Were constant, 215 characters are variable and parsimony-uninformative, and 291 characters were parsimony-informative.Maximum parsimony analysis yielded two equally parsimonious trees (TL = 1044, CI = 0.7241, HI = 0.2759, RI = 0.6488, RC = 0.4698).Bayesian analysis and ML analysis resulted in a similar topology as MP analysis with an average standard deviation of split frequencies = 0.004052 (BI), and the effective sample size (ESS) across the two runs was double the average ESS (avg ESS) = 2563.5.
The phylogram based on the ITS rDNA gene regions (Figure 1) demonstrated that three new species were grouped into the genus Botryobasidium, in which B.  Etymology-gossypirubiginosum (Lat.): from the Latin gossypium, referring to its cottony and rubiginous basidiomata surface.
Basidiomata-Annual, centrally stipitate, pendent, solitary or adnate, without odor or taste when fresh, becoming soft corky when dry. Pilei tiny, circular, up to 5 mm in diameter and 1 mm thick at center, fibrillose, hirsute, pilei surface fawn to grayish brown, margin thin and obtuse, curved down when dry. Pore surface light brown, angular, 1-3 per mm, dissepiments thin, entire.Context rust brown, soft, spongy, up to 0.4 mm thick.Tubes dark brown, up to 0.6 mm thick.Stipe short, reddish brown, corky, up to 4 mm long, 0.5 mm in diameter.Etymology-yunnanensis (Lat.):referring to the locality (Yunnan Province) of the type specimen.

Discussion
In the several previous studies, molecular data confirmed phylogenetic relationships, in which the genus Botryobasidium nested in the cantharelloid clade, and was grouped with related genera: Cantharellus, Craterellus, Hydnum, and Clavulina [5,7].Based on the molecular systematics study of Coltricia and Coltriciella, the result supported that both genera belonged to the family Hymenochaetaceae, and that both of them shared similar morphological features and a close molecular relationship [41,44].
In the present study, from the phylogram created based on inferences from the ITS data (Figure 1

Discussion
In the several previous studies, molecular data confirmed phylogenetic relationships, in which the genus Botryobasidium nested in the cantharelloid clade, and was grouped with related genera: Cantharellus, Craterellus, Hydnum, and Clavulina [5,7].Based on the molecular systematics study of Coltricia and Coltriciella, the result supported that both genera belonged to the family Hymenochaetaceae, and that both of them shared similar morphological features and a close molecular relationship [41,44].
As wood-inhabiting fungi efficiently degrade lignocellulose in wood, they play a vital ecological role in the material circulation and energy flow of forest ecosystems, as well as leading to major economic value [46,48].Therefore, they are important strategic biological resources [49,50].Wood-inhabiting fungi are an extensively studied group of Basidiomycota, but their diversity is still unknown in China, where many of the recently described taxa of this ecogroup were found [51][52][53][54][55][56][57][58].Based on morphological and molecular phylogenetic analysis, we described five new species from Yunnan Province, China.This study enriches our understanding of the diversity of wood-inhabiting fungi worldwide.

Figure 1 .
Figure 1.Maximum parsimony strict consensus tree illustrating the phylogeny of three new species and related species in Botryobasidium based on ITS sequences.Branches are labeled with maximum likelihood bootstrap values > 70%, parsimony bootstrap values > 50%, and Bayesian posterior probabilities > 0.95.The new species are in bold.

Figure 1 .
Figure 1.Maximum parsimony strict consensus tree illustrating the phylogeny of three new species and related species in Botryobasidium based on ITS sequences.Branches are labeled with maximum likelihood bootstrap values > 70%, parsimony bootstrap values > 50%, and Bayesian posterior probabilities > 0.95.The new species are in bold.

Figure 2 .
Figure 2. Maximum parsimony strict consensus tree illustrating the phylogeny of two new species of Coltricia and Coltriciella based on ITS + nLSU sequences.Branches are labeled with maximum likelihood bootstrap values > 70%, parsimony bootstrap values > 50%, and Bayesian posterior probabilities > 0.95.The new species are in bold.

Figure 2 . 1 Figure 3 .
Figure 2. Maximum parsimony strict consensus tree illustrating the phylogeny of two new species of Coltricia and Coltriciella based on ITS + nLSU sequences.Branches are labeled with maximum likelihood bootstrap values > 70%, parsimony bootstrap values > 50%, and Bayesian posterior probabilities > 0.95.The new species are in bold.

Figure 4 .
Figure 4. Maximum parsimony strict consensus tree illustrating the phylogeny of the Coltriciella yunnanensis and related species in Coltriciella based on ITS sequences.Branches are labeled with maximum likelihood bootstrap values > 70%, parsimony bootstrap values > 50%, and Bayesian posterior probabilities > 0.95.The new species are in bold.
), three new species were grouped into the genus Botryobasidium, in which B. gossypirubiginosum clustered with B. robustius; B. incanum was closely related to B. vagum; B. yunnanense was grouped with B. indicum.From the molecular tree created based on inferences from the ITS + nLSU data (Figure 2), both genera, Coltricia and Coltriciella, clustered into Hymenochaetaceae.According to the ITS data (Figure 3), C. zixishanensis clustered into the genus Coltricia, in which it was grouped with two taxa, C. confluens and C. perennis.In the phylogram created based on inferences from the ITS data (Figure 4), Coltriciella yunnanensis clustered into the genus Coltriciella, in which it was grouped with two taxa, Co. globosa and Co. pseudodependens.However, morphologically, B. robustius differs from B. gossypirubiginosum in its smooth hymenophore and subnavicular to amygdaliform smaller basidiospores (7-9 × 3-4 µm) [5]; the species B. vagum is distinguished
), three new species were grouped into the genus Botryobasidium, in which B. gossypirubiginosum clustered with B. robustius; B. incanum was closely related to B. vagum; B. yunnanense was grouped with B. indicum.From the molecular tree created based on inferences from the ITS + nLSU data (Figure 2), both genera, Coltricia and Coltriciella, clustered into Hymenochaetaceae.According to the ITS data (Figure 3), C. zixishanensis clustered into the genus Coltricia, in which it was grouped with two taxa, C. confluens and C. perennis.In the phylogram created based on inferences from the ITS data (Figure 4), Coltriciella yunnanensis clustered into the genus Coltriciella, in which it was grouped with two taxa, Co. globosa and Co. pseudodependens.However, morphologically, B. robustius differs from B. gossypirubiginosum in its smooth hymenophore and subnavicular to amygdaliform smaller basidiospores (7-9 × 3-4 µm) [5]; the species B. vagum is distinguished from B. incanum through its yellowish to greyish hymenial surface, basidia with six sterigmata, and navicular basidiospores [5]; B. indicum differs from B. yunnanense in yellow velvety hymenial surface and pyriform basidiospores [45].Coltricia confluens is distinct from C.

Table 1 .
List of species, specimens, and GenBank accession numbers of sequences used in this study.New species are in bold.