Four New Fungal Species in Forest Ecological System from Southwestern China

Four new wood-inhabiting fungi were found in Southwestern China within the genera Phanerochaete, Phlebiopsis, Asterostroma, and Vararia of the families Phanerochaetaceae and Peniophoraceae, belonging to the orders Polyporales and Russulales individually. Combined with their morphological characteristics and molecular biological evidence, the present study describes them as new fungal taxa. Asterostroma yunnanense is characterized by the resupinate, membranaceous to pellicular basidiomata with a cream to salmon-buff hymenial surface, hyphal system dimitic bearing simple-septa, thin- to thick-walled, yellowish brown asterosetae with acute tips, and thin-walled, echinulate, amyloid, globose basidiospores. Phanerochaete tongbiguanensis is characterized by the resupinate basidiomata with a white to cream hymenial surface, a monomitic hyphal system with simple-septa generative hyphae, the presence of subclavate cystidia covered with a lot of crystals, and oblong ellipsoid basidiospores (6–9 × 3–4.5 µm). Phlebiopsis fissurata is characterized by the membranaceous, tuberculate basidiomata with a buff to slightly brown hymenial surface, a monomitic hyphal system with simple-septa, conical cystidia, and broadly ellipsoid. Vararia yingjiangensis is characterized by a corky basidiomata with a pinkish buff to cinnamon-buff hymenial surface, cracking, yellowish dichohyphae with slightly curved tips, subulate gloeocystidia, and thick-walled, ellipsoid basidiospores (6.5–11.5 × 5–7 µm). The phylogenetic analyses of ITS + nLSU revealed that the two new species were nested into the genera Phanerochaete and Phlebiopsis within the family Phanerochaetaceae (Polyporales), in which Phanerochaete tongbiguanensis was sister to P. daliensis; Phlebiopsis fissurata was grouped with P. lamprocystidiata. Two new species were clustered into the genera Asterostroma and Vararia within the family Peniophoraceae (Russulales), in which Asterostroma yunnanense was sister to A. cervicolor; Vararia yingjiangensis formed a single branch.

During the investigations on wood-inhabiting fungi in Yunnan province, China, four new species were found, which could not be assigned to any described species.We present the morphological and molecular phylogenetic evidence that support the recognition of these four new species in Phanerochaetaceae and Peniophoraceae based on the internal transcribed spacer (ITS) regions and the large subunit nuclear ribosomal RNA gene (nLSU) sequences.

Sample Collection and Herbarium Specimen Preparation
Fresh fruiting bodies of basidiomycetous macrofungi were collected from Lincang, Dehong, Yunnan province, P.R. China.Specimens were dried in an electric food dehydrator at 40 • C and then sealed and stored in an envelope bag and deposited in the herbarium of Southwest Forestry University (SWFC), Kunming, Yunnan province, P.R. China.Macromorphological descriptions are based on field notes and photos captured in the field and lab.

Molecular Phylogeny
Macromorphological descriptions and color terminology are based on field notes and photos captured in the field or lab, and they follow those of a previous study [54].The micromorphological data were obtained from the dried specimens based on observing them under a light microscope following a previous study [55].The following abbreviations are used: KOH = 5% potassium hydroxide water solution, CB = Cotton Blue, 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 given number (b) of specimens).

DNA Extraction and Sequencing
According to the manufacturer's instructions, we used the CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Kunming, China) to obtain genomic DNA from dried specimens [62].A total of 3 µL of DNA was evenly mixed with 3 µL 5 × bromophenol blue indicator and a 3 µL DNA sample to be tested, and the samples were placed on a 1.5% agarose gel plate (containing 0.5 µg/mL EB).The DNA molecular weight was labeled DL 2000 with a molecular weight of 560-23,130 bp, and the pressure was stabilized at 90 V. Electrophoresis occurred for 30 min.The nuclear ribosomal ITS region was amplified with primers ITS5 (GGA AGT AAA AGT CGT AAC AAG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) [62].The nuclear nLSU region was amplified with primer pair LR0R (ACC CGC TGA ACT TAA GC) and LR7 (TAC TAC CAC CAA GAT CT) [62].The basic amplification reaction system of ITS and nLSU is shown in Table 1.And the newly generated sequences were deposited in NCBI GenBank (Table 2).
Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) analyses were applied to the combined three datasets [66].BS (Branch Support) for ML (maximum likelihood) analysis was determined by 1000 bootstrap replicates, and bootstrap values were >70% [66].MP (maximum parsimony) analysis was performed in PAUP* version 4.0b10, and parsimony bootstrap values were >50% [67].BI (Bayesian inference) and clade robustness were assessed using bootstrap (BT) analysis with 1000 replicates, and Bayesian posterior probabilities were >0.95 [68,69].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 parsimonious trees were saved.Clade robustness was assessed using bootstrap (BT) analysis with 1000 replicates [68].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 [69].The best-fit evolution model for each dataset for BI (Bayesian inference) was determined by using MrModeltest 2.3 [84].BI was calculated with MrBayes3.1.2with a general time reversible (GTR + I + G) model of DNA substitution and a gamma distribution rate variation rate variation across sites [85].A total of four Markov chains were run for two runs from random starting trees for 2 million and 0.5 million generations for ITS + nLSU (Figures 1 and 2), respectively, and based on ITS for 5 million generations (Figure 3), 0.5 million generations (Figure 4), for 0.5 million generations (Figure 5), and 0.2 million generations (Figure 6), with trees and parameters sampled every 1000 generations.The best-fit evolution model for each dataset for BI (Bayesian inference) was determined by using MrModeltest 2.3 [84].BI was calculated with MrBayes3.1.2with a general time reversible (GTR + I + G) model of DNA substitution and a gamma distribution rate variation rate variation across sites [85].A total of four Markov chains were run for two runs from random starting trees for 2 million and 0.5 million generations for ITS + nLSU (Figures 1 and 2), respectively, and based on ITS for 5 million generations (Figure 3), 0.5 million generations (Figure 4), for 0.5 million generations (Figure 5), and 0.2 million generations (Figure 6), with trees and parameters sampled every 1000 generations.

Molecular Phylogeny
The ITS + nLSU dataset (Figure 1) included sequences from 32 fungal specimens representing 29 species.The dataset had an aligned length of 2550 characters, of which 1682 characters are constant, 424 are variable and parsimony-uninformative, and 444 are parsimony-informative.Maximum parsimony analysis yielded one equally parsimonious tree (TL = 2330, CI = 0.5408, HI = 0.4592, RI = 0.4861, RC = 0.2629).The best model for the ITS + nLSU dataset estimated and applied in the Bayesian analysis was GTR + I + G (lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1).Bayesian analysis and ML analysis resulted in a similar topology to MP analysis with an average standard deviation of split frequencies = 0.008198 (BI), and the effective sample size (ESS) across the two runs is the double of the average ESS (avg ESS) = 1887.The phylogeny (Figure 1) based on the combined nLSU sequences includes six genera within the family Peniophoraceae: Bjerkandera, Phaeophlebiopsis, Phanerochaete, Phlebiopsis, Rhizochaete Gresl.and Nakasone & Rajchenb.and Terana Adans.Our current two new species were clustered into genera Phanerochaete and Phlebiopsis.
The ITS dataset of the genus Asterostroma (Figure 3) included sequences from 18 fungal specimens representing 10 species.The dataset had an aligned length of 1560 characters, of which 983 characters are constant, 246 are variable and parsimony-uninformative, and 331 are parsimony-informative.Maximum parsimony analysis yielded one equally parsimonious tree (TL = 814, CI = 0.8710, HI = 0.1290, RI = 0.8930, RC = 0.7778).The best model for the ITS dataset estimated and applied in the Bayesian analysis was GTR + I + G (lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1).Bayesian analysis and ML analysis resulted in a similar topology to MP analysis with an average standard deviation of split frequencies = 0.009408 (BI).The phylogenetic tree indicated that A. yunnanense was grouped with the close taxa A. cervicolor (Berk.& M.A. Curtis) Massee.
The ITS dataset of the genus Phanerochaete (Figure 4) included sequences from 96 fungal specimens representing 60 species.The dataset had an aligned length of 880 characters, of which 319 characters are constant, 77 are variable and parsimony-uninformative, and 484 are parsimony-informative.Maximum parsimony analysis yielded one equally parsimonious tree (TL = 2187, CI = 0.4015, HI = 0.5985, RI = 0.6231, RC = 0.2501).The best model for the ITS dataset estimated and applied in the Bayesian analysis was GTR + I + G (lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1).Bayesian analysis and ML analysis resulted in a similar topology to MP analysis with an average standard deviation of split frequencies = 0.001737 (BI).The phylogenetic tree indicated that P. tongbiguanensis was grouped with the close taxa P. daliensis J. Yu & C.L. Zhao.
The ITS dataset of the genus Phlebiopsis (Figure 5) included sequences from 33 fungal specimens representing 20 species.The dataset had an aligned length of 665 characters, of which 392 characters are constant, 82 are variable and parsimony-uninformative, and 191 are parsimony-informative.Maximum parsimony analysis yielded six equally parsimonious trees (TL = 685, CI = 0.5650, HI = 0.4350, RI = 0.6543, RC = 0.3697).The best model for the ITS dataset estimated and applied in the Bayesian analysis was GTR + I + G (lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1).Bayesian analysis and ML analysis resulted in a similar topology to MP analysis with an average standard deviation of split frequencies = 0.003384 (BI).The phylogenetic tree indicated that P. fissurata was grouped with the close taxa P. lamprocystidiata (Sheng H. Wu) Sheng H. Wu & Hallenb.
The ITS dataset of the genus Vararia (Figure 6) included sequences from 52 fungal specimens representing 40 species.The dataset had an aligned length of 796 characters, of which 148 characters were constant, 116 were variable and parsimony-uninformative, and 532 were parsimony-informative.Maximum parsimony analysis yielded one equally parsimonious tree (TL = 4063, CI = 0.3104, HI = 0.6896, RI = 0.4313, and RC = 0.1339).The best model for the ITS dataset estimated and applied in the Bayesian analysis was GTR + I + G.The Bayesian and ML analyses resulted in a similar topology to that of the MP analysis with split frequencies = 0.000442 (BI).The phylogram inferred from ITS sequences (Figure 6) revealed that V. yingjiangensis was grouped with six close taxa, namely V. ambigua Boidin, Lanq.& Gilles, V. ellipsospora G. Cunn., V. fragilis L. Zou & C.L. Zhao, V. gallica (Bourdot & Galzin) Boidin, V. ochroleuca (Bourdot & Galzin) Donk and V. tropica A.L. Welden.
Fruiting body-Basidiomata annual, resupinate, membranaceous to pellicular, soft, without odor and taste when fresh, up to 110 mm long, 60 mm wide, and 280 µm thick.Hymenial surface smooth, cream when fresh, cream to salmon-buff, sometimes cracked when dried.Sterile margin thinning out, becoming indistinct and concolorous with hymenophore surface, up to 1 mm.
Fruiting body-Basidiomata annual, resupinate, thin, adnate, leather, without odor and taste when fresh, up to 70 mm long, 10 mm wide, 70-130 µm thick.Hymenial surfaces are smooth, white to cream when fresh, to cream to slightly buff upon drying.Sterile margins are distinct, whitish, and up to 1 mm.
Fruiting body-Basidiomata annual, adnate, corky, without odor and taste when fresh, up to 80 mm long, 40 mm wide, 80-120 µm thick.Hymenial surface smooth, cream to pinkish buff when fresh, pinkish buff to cinnamon-buff when dry, cracking with age.Sterile margin thin, indistinct, slightly cream to pinkish buff, up to 2 mm.

Discussion
The family-level classification for the order Polyporales (Basidiomycota) revealed that the two taxa of Phanerochaete daliensis and Phlebiopsis lamprocystidiata nested into the family Phanerochaetaceae within the residual polyporoid clade based on the molecular systematics study amplifying the ITS, nLSU, RPB1, and RPB2 genes [21,27].Seven genera, Asterostroma, Dichostereum, Gloiothele, Peniophora, Scytinostroma, Vararia, and Vesiculomyces E. Hagstr., were grouped together and clustered within the family Peniophoraceae [18].In the present study, four new species were nested into the families Phanerochaetaceae

Discussion
The family-level classification for the order Polyporales (Basidiomycota) revealed that the two taxa of Phanerochaete daliensis and Phlebiopsis lamprocystidiata nested into the Institutional Review Board Statement: Not applicable for studies involving humans or animals.
Informed Consent Statement: Not applicable for studies involving humans.

Figure 1 .
Figure 1.Maximum parsimony is a strict consensus tree illustrating the phylogeny of two new species and related genera in the family Phanerochaetaceae based on ITS + nLSU sequences.The new species are marked with asterisks.

Figure 1 .
Figure 1.Maximum parsimony is a strict consensus tree illustrating the phylogeny of two new species and related genera in the family Phanerochaetaceae based on ITS + nLSU sequences.The new species are marked with asterisks.

Figure 2 .
Figure 2. Maximum parsimony is a strict consensus tree illustrating the phylogeny of two new species and related genera in the family Peniophoraceae based on ITS + nLSU sequences.The new species are marked with asterisks.

Figure 2 .
Figure 2. Maximum parsimony is a strict consensus tree illustrating the phylogeny of two new species and related genera in the family Peniophoraceae based on ITS + nLSU sequences.The new species are marked with asterisks.

Figure 2 .
Figure 2. Maximum parsimony is a strict consensus tree illustrating the phylogeny of two new species and related genera in the family Peniophoraceae based on ITS + nLSU sequences.The new species are marked with asterisks.

Figure 3 .
Figure 3. Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Asterostroma based on ITS sequences.Figure 3. Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Asterostroma based on ITS sequences.

Figure 3 .
Figure 3. Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Asterostroma based on ITS sequences.Figure 3. Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Asterostroma based on ITS sequences.

Figure 4 .
Figure 4. Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Phanerochaete based on ITS sequences.Figure 4. Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Phanerochaete based on ITS sequences.

Figure 4 .
Figure 4. Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Phanerochaete based on ITS sequences.Figure 4. Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Phanerochaete based on ITS sequences.

Figure 5 .
Figure 5. Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new s cies and related species in the genus Phlebiopsis based on ITS sequences.

Figure 5 .
Figure 5. Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Phlebiopsis based on ITS sequences.

Figure 6 .
Figure 6.Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Vararia based on ITS sequences.

Figure 6 .
Figure 6.Maximum parsimony is a strict consensus tree illustrating the phylogeny of the new species and related species in the genus Vararia based on ITS sequences. .
editing, C.Z. and Y.D.; visualization, C.Z.; supervision, C.Z.; project administration, C.Z.; funding acquisition, C.Z. and J.Z.All authors have read and agreed to the published version of the manuscript.Funding: The research was supported by the National Natural Science Foundation of China (Project No. 32170004, U2102220), Forestry Innovation Programs of Southwest Forestry University (Grant No: LXXK-2023Z07), and the High-level Talents Program of Yunnan province (YNQR-QNRC-2018-111).

Table 1 .
PCR reaction system and reaction conditions.

Table 2 .
List of species, specimens, and GenBank accession numbers of sequences used in this study.The new species are in bold.
* Is shown in holotype.