Phylogenetic and Taxonomic Analyses Reveal Three New Wood-Inhabiting Fungi (Polyporales, Basidiomycota) in China

Three new wood-inhabiting fungal species, Cerioporus yunnanensis, Perenniporiopsis sinensis, and Sarcoporia yunnanensis, are proposed based on a combination of the morphological features and molecular evidence. Cerioporus yunnanensis is characterized by the pileate basidiomata having a fawn brown to black pileal surface, a dimitic hyphal system with clamped generative hyphae, and the presence of the fusoid cystidioles and cylindrical basidiospores (9–12.5 × 3.5–5 µm). Perenniporiopsis sinensis is distinct from the osseous pileus with verrucose, an orange-yellow to dark reddish-brown pileal surface with a cream margin, a trimitic hyphal system with clamped generative hyphae, and the presence of the fusiform cystidioles and ellipsoid basidiospores (9–11 × 5.5–6.5 µm). Sarcoporia yunnanensis is typical of the pileate basidiomata with a salmon to reddish-brown pileal surface, a monomitic hyphal system with clamped generative hyphae, and the presence of the ellipsoid basidiospores (4–5.5 × 2.5–4 µm). Sequences of ITS + nLSU + mt-SSU + TEF1 + RPB1 + RPB2 genes were used for the phylogenetic analyses using maximum likelihood, maximum parsimony, and Bayesian inference methods. The multiple genes with six loci analysis showed that the three new species nested within the order Polyporales, in which C. yunnanensis and P. sinensis nested into the family Polyporaceae, and S. yunnanensis grouped into the family Sarcoporiaceae.


Introduction
Fungi are eukaryotic microorganisms that play fundamental ecological roles as decomposers and mutualists of dead and living plants and animals, in which they drive carbon cycling in forest soils, mediate the mineral nutrition of plants, and alleviate the carbon limitations of other soil organisms [1,2].Wood-inhabiting fungi form an ecologically important branch of the tree of life, based on their distinct and diverse characteristics [3].Taxonomy and phylogeny of the Polyporales are updated continuously by mycologists with the frequent inclusion of data from DNA sequences [4,5].In recent years, the mycologist revised the species and taxonomic status of the genus Cerioporus using ITS + nLSU + TEF1 datasets, while the research established the new genus Perenniporiopsis using ITS + nLSU + mt-SSU + TEF1 datasets and two new species have also been discovered and grouped in the genus Sarcoporia [6][7][8][9][10].

Morphology
Macromorphological descriptions are based on field notes and photos captured in the field and laboratory and follow the color terminology of Petersen [11].Micromorphological data were obtained from the dried specimens following observation under a light microscope [12].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 = means spore length (arithmetic average for all spores), W = means 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).

Phylogenetic Analyses
The DNA sequences were aligned in MAFFT version 7 using the G-INS-i strategy [44].The alignment was adjusted manually using AliView version 1.27 [45].Sequence of Heterobasidion annosum (Fr.)Bref.retrieved from GenBank was used as an outgroup in ITS + nLSU + mt-SSU + TEF1 + RPB1 + RPB2 (Figure 1) analysis following a previous study [5].Sequence of Trametes hirsuta (Wulfen) Lloyd retrieved from GenBank was used as an outgroup in ITS (Figure 2) analysis following a previous study [27].Sequence of Pyrofomes demidoffii (Lév.)Kotl.and Pouzar retrieved from GenBank was used as an outgroup in ITS + nLSU (Figure 3) analysis following a previous study [9].Maximum parsimony (MP), Maximum Likelihood (ML), and Bayesian Inference (BI) analyses were applied to the combined three datasets.Approaches to the phylogenetic analyses process was followed by Zhao and Wu [46].MP analysis was performed in PAUP* version 4.0b10 [47].All of the characters were equally weighted, and gaps were treated as missing data.Clade robustness was assessed using bootstrap (BT) analysis with 1000 replicates [48].ML was inferred using RAxML-HPC2 through the Cipres Science Gateway (www.phylo.org(accessed on 28 November 2023)) [49].
MrModeltest 2.3 [50] was used to determine the best-fit evolution model for each dataset 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 [51].Four Markov chains were run for two runs from random starting trees, for eight million generations (Figure 1), one million generations (Figure 2), and 0.85 million generations (Figure 3), and trees were sampled every 100 generations.
The dataset based on ITS (Figure 2) comprises sequences from 23 fungal specimens representing 13 species from GenBank.The dataset had an aligned length of 631 characters, of which 354 characters are constant, 42 are variable and parsimony-uninformative, and 235 are parsimony-informative.Maximum parsimony analysis yielded two equally parsimonious trees (TL = 676, CI = 0.6080, HI = 0.3920, RI = 0.6989, RC = 0.4249).
The phylogenetic tree (Figure 1) inferred from ITS + nLSU + mt-SSU + TEF1 + RPB1 + RPB2 sequences revealed that Cerioporus yunnanensis and Perenniporiopsis sinensis nested into the family Polyporaceae, and Sarcoporia yunnanensis clustered into the family Sarcoporiaceae.The phylogram based on the ITS gene regions (Figure 2 3), it revealed that P. sinensis grouped into genus Perenniporiopsis, in which it was retrieved as a sister to P. minutissima.Based on ITS + nLSU + mt-SSU + TEF1 + RPB1 + RPB2 gene regions (Figure 1), it revealed that S. yunnanensis divided into genus Sarcoporia, in which it grouped with S. longitubulata Vlasák and Spirin, and then clustered with S. polyspora P. Karst.
Basidiomata-Annual, pileate, odorless when fresh, hard corky when dry. Pileus applanate to triquetrous, up to 2 cm long, 1 cm wide, and 5 mm thick at base.Pileal surface fawn brown to black, distinctly sulcate, and margin obtuse.Pore surface white when fresh, becoming white to cream when dry, sterile margin distinct, up to 1 mm wide, pores round, 2-3 per mm.Context cinnamon brown to fawn brown, corky, up to 2 mm thick.Tubes cream to brown, hard corky, distinctly stratified, up to 3 mm long.
Basidiomata-Annual, pileate, odorless when fresh, hard corky when dry. Pileus applanate to triquetrous, up to 2 cm long, 1 cm wide, and 5 mm thick at base.Pileal surface fawn brown to black, distinctly sulcate, and margin obtuse.Pore surface white when fresh, becoming white to cream when dry, sterile margin distinct, up to 1 mm wide, pores round, 2-3 per mm.Context cinnamon brown to fawn brown, corky, up to 2 mm thick.Tubes cream to brown, hard corky, distinctly stratified, up to 3 mm long.Etymology-sinensis (Lat.):Referring to the locality (China) of the type specimen.Basidiomata-Annual, pileus solitary or imbricate, osseous, without odor or taste when fresh, projecting up to 3 cm, 3 cm wide, and 1 cm thick at base.Pileal surface orangeyellow to dark reddish-brown, verrucose; margin cream, obtuse.Pore surface white when fresh, becoming pale yellowish-to yellowish-brown when dry, pores round, 4-6 per mm; Context cream, rigidly osseous, up to 7 mm thick; tubes pale cream to honey yellow, rigidly osseous, up to 3 mm long.
Basidiomata-Annual, pileate, corky when fresh, brittle and hard when dry, odorless, and up to 4 cm long, 3 cm wide, and 1.5 cm thick.Pileal surface salmon to reddish-brown; margin cream, obtuse.Pore surface orange-yellow, pores angular, 2-4 per mm; context orange-brown, cottony, up to 1 cm thick; tubes pinkish-buff, up to 5 mm, extremely brittle and shattering easily when dry.

Discussion
In the present study, three new species, Cerioporus yunnanensis, Perenniporiopsis sinensis and Sarcoporia yunnanensis are described based on phylogenetic analyses and morphological characteristics.
An outline of all genera of Basidiomycota, including three phylogenetic analyses with combined nLSU, SSU, 5.8S, RPB1, RPB2, and TEF1 datasets for the subphyla Agaricomycotina, Pucciniomycotina and Ustilaginomycotina, revealed that the genera Cerioporus and Perenniporiopsis nested into the family Polyporaceae Fr. ex Corda (Polyporales, Agaricomycetes), and the genus Sarcoporia clustered into the family Sarcoporiaceae (Polyporales, Agaricomycetes) [4].In the present study, based on the ITS + nLSU + mt-SSU + TEF1 + RPB1 + RPB2 data (Figure 1), Cerioporus yunnanensis and Perenniporiopsis sinensis nested into the family Polyporaceae, while Sarcoporia yunnanensis clustered into the family Sarcoporiaceae, and the present results are similar to the previous topology research.Our results of the phylogram inferred from the ITS data, showed that C. yunnanensis grouped into Cerioporus (Figure 2), in which it grouped with two taxa, C. scutellatus and C. subtropicus, and then closely grouped with C. tibeticus.Based on the ITS + nLSU topology (Figure 3), it was revealed that P. sinensis was retrieved as a sister species to P. minutissima.Based on the ITS + nLSU + mt-SSU + TEF1 + RPB1 + RPB2 topology (Figure 1), it was revealed that S. yunnanensis grouped with S. longitubulata and S. polyspora.However, morphologically, C. scutellatus is distinct from C. yunnanensis by smaller pores (3-5 per mm), cyanophilous

Discussion
In the present study, three new species, Cerioporus yunnanensis, Perenniporiopsis sinensis and Sarcoporia yunnanensis are described based on phylogenetic analyses and morphological characteristics.
In ecological and biogeographical studies, wood-inhabiting fungi are an extensively studied group of Basidiomycota, in which Polyporales species are an important group, mainly found on hardwood, although a few species grow on coniferous wood [54][55][56][57][58][59][60].Further studies should focus on the relationships between the host and Cerioporus, Perenniporiopsis, and Sarcoporia species.We believe more species of Polyporales will be found in the oriental realm, since wood-inhabiting fungi are a cosmopolitan group and they are rich in the oriental realm [61][62][63][64], and it is very possible that the same phenomenon occurs for Cerioporus, Perenniporiopsis, and Sarcoporia.

Figure 2 .
Figure 2. Maximum parsimony strict consensus tree illustrating the phylogeny of the new species of Cerioporus 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 3 .
Figure 3. Maximum parsimony strict consensus tree illustrating the phylogeny of Perenniporiopsis and related species in Perenniporia s.l.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.

Table 1 .
Names, voucher numbers and corresponding GenBank accession numbers of the taxa used in this study.The new species are in bold, NA refers to data not available.