Four New Wood-Inhabiting Fungal Species of Peniophoraceae (Russulales, Basidiomycota) from the Yunnan-Guizhou Plateau, China

Four new fungi of the family Peniophoraceae, viz., Peniophora roseoalba, P. yunnanensis, Vararia daweishanensis, and V. fragilis are herein proposed, based on a combination of morphological features and molecular evidence. Peniophora roseoalba is characterized by resupinate, membranaceous basidiomata with a rose pink to pale pinkish grey hymenophore, a monomitic hyphal system with clamped generative hyphae, the presence of cystidia, and ellipsoid basidiospores. However, P. yunnanensis differs in being tuberculate, with a pale cream to cream hymenial surface, small lamprocystidia (18–29 × 4.5–7 µm), and subcylindrical basidiospores. Vararia daweishanensis is characterized by resupinate, membranous basidiomata with a pale yellowish hymenial surface, a dimitic hyphal system with clamped generative hyphae, strongly dextrinoid dichohyphae, and allantoid basidiospores; V. fragilis is characterized by resupinate, brittle basidiomata, with a buff to ochraceous hymenial surface and small ellipsoid basidiospores measuring 3.5–5.5 × 2.5–3.5 µm. Sequences of the ITS and nLSU rRNA markers of the studied samples were generated, and phylogenetic analyses were performed with the maximum likelihood, maximum parsimony, and Bayesian inference methods. The nLSU analysis revealed that the four new species can be clustered into the family Peniophoraceae (Russulales), in the genera Peniophora and Vararia. Further studies based on the ITS dataset showed that four fungi of the family Peniophoraceae were new to science.


Introduction
The family Peniophoraceae (Russulales) is a large and rather heterogeneous family with seven genera accepted; two genera, Peniophora Cooke and Vararia P. Karst., have the highest number of taxa in this family, in which they play fundamental ecological roles to drive carbon cycling in forest soils, acting as decomposers [1,2].

Figure 2.
A maximum parsimony strict consensus tree, illustrating the phylogeny of two new species and the related species in the genus Peniophora, based on the ITS sequences. The branches are labeled with maximum likelihood bootstrap values higher than 70%, parsimony bootstrap proportions that are higher than 50%, and Bayesian posterior probabilities of more than 0.95, respectively.

Molecular Phylogeny
The nLSU dataset ( Figure 1) included sequences from 55 fungal specimens, representing 55 species. The dataset had an aligned length of 1415 characters, of which 923 characters are constant, 152 are variable and parsimony-uninformative, and 340 are parsimony-informative. The maximum parsimony analysis yielded one equally parsimonious tree (TL = 860, CI = 0.3233, HI = 0.6767, RI = 0.6123, RC = 0.1979). The best model for the ITS+nLSU dataset, which was estimated and applied in the Bayesian analysis, was GTR+I+G (lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). The Bayesian analysis and ML analysis resulted in a similar topology to the MP analysis, with an average standard deviation of split frequencies = 0.009575 (BI); the effective sample size (ESS) across the two runs is the double of the average ESS (avg ESS) = 200.5. The phylogeny (Figure 1), based on the combined nLSU sequences, includes six families within the order of Russulales, which indicated that nine genera, comprising  Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) analyses were applied to the three combined datasets, following the technique used in a previous study [42], and the tree construction procedure was performed in PAUP*, version 4.0b10 [46]. All 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. The max trees were set to 5000, branches of zero length were collapsed, and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates [47]. The descriptive tree statistics were tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI); these were calculated for each maximum parsimonious tree generated. The multiple sequence alignment was also analyzed using the maximum likelihood (ML) in RAxML-HPC2, through the Cipres Science Gateway (www.phylo.org; accessed on 13 October 2022) [48]. Branch support (BS) for the ML analysis was determined by 1000 bootstrap replicates. MrModeltest 2.3 [57] was used to determine the best-fit evolution model for each dataset, using Bayesian inference (BI), which was performed using MrBayes 3.2.7a, with a GTR+I+G model of the DNA substitution and a gamma distribution rate variation across the sites [58]. Four Markov chains were run for 2 runs, beginning from random starting trees for 0.9 million generations for nLSU (Figure 1), for 1.5 million generations for ITS ( Figure 2) with trees, and 1 million generations for ITS ( Figure 3) with trees, with the parameters sampled every 1000 generations. The first one-quarter of all generations were discarded as the burn-in. The majority rule consensus tree of all the remaining trees was calculated. Branches were considered significantly supported if they received a maximum likelihood bootstrap value (BS) > 70%, a maximum parsimony bootstrap value (BT) > 70%, or Bayesian posterior probabilities (BPP) > 0.95.

Molecular Phylogeny
The nLSU dataset ( The ITS-alone dataset of the genus Peniophora ( Figure 2) included the sequences from 83 fungal specimens, representing 52 species. The dataset had an aligned length of 607 characters, of which 353 characters were constant, while 64 were variable and parsimonyuninformative, and 190 were parsimony-informative. The maximum parsimony analysis yielded 12 equally parsimonious trees (TL = 1681, CI = 0.3111, HI = 0.6889, RI = 0.4496, RC = 0.1399). The best model for the ITS dataset that was 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 = 0.009599 (BI). The phylogenetic tree indicated that P. roseoalba can be grouped with two close taxa, P. versicolor and P. ovalispora, whereas P. yunnanensis can be grouped with a clade comprising P. lycii and P. violaceolivida.
The ITS-only dataset of the genus Vararia ( Figure 3) included sequences from 63 fungal specimens, representing 39 species. The dataset had an aligned length of 1128 characters, of which 511 characters were constant, 133 were variable and parsimony uninformative, and 484 were parsimony informative. Maximum parsimony analysis yielded 6 equally parsimonious trees (TL = 4589, CI = 0.2805, HI = 0.7195, RI = 0.4174, and RC = 0.1171). 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 = 0.0096082 (BI). The phylogram inferred from the ITS sequences ( Figure 3) revealed that Vararia daweishanensis could be grouped with four close taxa: V. gomezii, V. rhombospora, V. sigmatospora, and V. trinidadensis, whereas the other species of V. fragilis could be grouped with a clade comprising V. ambigua and V. ellipsospora, with a low level of support. Etymology-Roseoalba (Lat.): referring to the rose to pale pinkish grey color of the hymenial surface of the specimens.

Taxonomy
Fruiting body-Basidiomata are annual, resupinate, membranaceous, without odor and taste when fresh, up to 90 mm long, 20 mm wide, 70-100 µm thick. The hymenial surface is smooth, occasionally cracked, and rose to pale pinkish grey. The sterile margin is indistinct and is rose to pinkish grey.
Fruiting body-Basidiomata are annual, resupinate, and coriaceous, without odor and taste when fresh, up to 100 mm long, 25 mm wide, and 70-100 µm thick. The hymenial surface is tuberculate and is pale cream to cream. The sterile margin is indistinct and slightly cream-colored.
Fruiting body-Basidiomata are annual, resupinate, and coriaceous, without odor and taste when fresh, up to 100 mm long, 25 mm wide, and 70-100 µm thick. The hymenial surface is tuberculate and is pale cream to cream. The sterile margin is indistinct and slightly cream-colored.
Hyphal system-Dimitic, generative hyphae with clamp connections, colorless, thinto thick-walled, occasionally branched, interwoven, 2-4 µm in diameter, IKI−, CB+, tissues are unchanged in KOH; dichohyphae in subhymenium abundant, yellowish, capillary, distinctly thick-walled; dichotomously to irregularly branched, with the main branches up to 4 µm in diameter and with acute tips, moderately dextrinoid when in Melzer's reagent; more frequently branched with more narrow and shorter branches in the hymenium, with slightly curved tips and a stronger dextrinoid reaction.
Basidiospores-The basidiospores are allantoid, colorless, thin-walled, and smooth, with oil droplets inside, IKI−, CB−, ( are unchanged in KOH; dichohyphae in subhymenium abundant, yellowish, capillary, distinctly thick-walled; dichotomously to irregularly branched, with the main branches up to 4 µm in diameter and with acute tips, moderately dextrinoid when in Melzer's reagent; more frequently branched with more narrow and shorter branches in the hymenium, with slightly curved tips and a stronger dextrinoid reaction.
Fruiting body-Basidiomata are annual, resupinate, adnate, thin, membranous, and fragile, without odor and taste when fresh, up to 85 mm long, 40 mm wide, and 30-100 µm thick. The hymenial surface is smooth, buff when fresh, buff to ochraceous on drying and cracking. The sterile margin is indistinct, attached, and is cream to buff.
Hyphal system-Dimitic, generative hyphae, bearing simple septa, colorless, thin-to thick-walled, occasionally branched, interwoven, 1.5-3.5 µm in diameter, IKI−, CB+, and with tissues unchanged in KOH; the dichohyphae in the subhymenium are abundant, predominantly yellowish, capillary, distinctly thick-walled, and dichotomously to irregularly branched, with the main branches up to 2 µm in diameter and with acute tips; moderately dextrinoid in Melzer's reagent; more frequently branched, with more narrow and shorter branches in the subiculum, with slightly curved tips and a stronger dextrinoid reaction.
Basidiospores-The basidiospores are broad from ellipsoid to ellipsoid, colorless, thin-walled, smooth, IKI−, CB−, 3.5-5. predominantly yellowish, capillary, distinctly thick-walled, and dichotomously to irregularly branched, with the main branches up to 2 µm in diameter and with acute tips; moderately dextrinoid in Melzer's reagent; more frequently branched, with more narrow and shorter branches in the subiculum, with slightly curved tips and a stronger dextrinoid reaction.

Discussion
Four genera, Gloiothele, Peniophora, Scytinostroman, and Vararia have been grouped together and clustered within the family Peniophoraceae, as inferred from a dataset with 178 terminal taxa [37]. In the present study, based on the nLSU data (Figure 1), four new species were classified in the family Peniophoraceae and were then classified within the genera of Peniophora and Vararia.
In the current study, based on the further ITS phylogenetic tree (Figure 3), two new taxa have been grouped within the genus Vararia. These are V. daweishanensis and V. fragilis, in which V. daweishanensis was grouped with four close taxa, namely, V. gomezii, V. rhombospora, V. sigmatospora and V. trinidadensis, while V. fragilis was grouped with a

Discussion
Four genera, Gloiothele, Peniophora, Scytinostroman, and Vararia have been grouped together and clustered within the family Peniophoraceae, as inferred from a dataset with 178 terminal taxa [37]. In the present study, based on the nLSU data (Figure 1), four new species were classified in the family Peniophoraceae and were then classified within the genera of Peniophora and Vararia.