Morphological and molecular identification of Phlebia wuliangshanensis sp. nov. in China

A new white-rot fungus, Phlebia wuliangshanensis, is proposed based on a combination of morphological features and molecular evidence. The species is characterized by an annual growth habit, resupinate basidiocarps with a smooth to tuberculate hymenial surface, a monomitic hyphal system with thinto thick-walled generative hyphae bearing simple septa, presence of cystidia, and narrow ellipsoid to ellipsoid basidiospores (5–6 × 3–3.7 μm). Our phylogenetic analyses of ITS and LSU nrRNA sequences performed with maximum likelihood, maximum parsimony, and Bayesian inference methods support P. wuliangshanensis within a phlebioid clade in Meruliaceae (Polyporales). ITS+nLSU sequence analyses of additional Phlebia taxa strongly support P. wuliangshanensis within a monophyletic lineage grouped with P. chrysocreas and P. uda. Key words—Basidiomycota, Ceriporiopsis, taxonomy, wood-inhabiting fungi, Yunnan Province Introduction Phlebia Fr. (Meruliaceae, Polyporales) is typified by P. radiata Fr. (Fries 1821). Basidiocarps are resupinate or rarely pileate with a subceraceous to subgelatinous consistency when fresh and membranaceous to coriaceous when dry. The hymenophore may be smooth, tuberculate, phlebioid,

During our investigations of wood-inhabiting fungi in southern China, we found an additional taxon that could not be assigned to any described species. In examining the taxonomy and phylogeny of this new species, we employed a two-gene molecular phylogenetic approach using internal transcribed spacer (ITS) and long subunit (nLSU) plus an expanded sampling of Phlebia isolates.

Materials & methods
The specimens studied are deposited at the herbarium of Southwest Forestry University (SWFC). Macro-morphological descriptions are based on field notes. Colour terms follow Petersen (1996). Micro-morphological data were obtained  from the dried specimens and observed under light microscopy following Dai (2012). Abbreviations are: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB-= acyanophilous, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios among the specimens studied, n (a/b) = number of spores (a) measured from given number (b) of specimens. We extracted genomic DNA from dried specimens using HiPure Fungal DNA Mini Kit II (Magen Biotech Co.) according to the manufacturer's instructions with some modifications. A small piece (c. 30 mg) of dried fungal material was ground to powder with liquid nitrogen. The powder was transferred to a 1.5 ml centrifuge tube, suspended in 0.4 ml of lysis buffer, and incubated in a 65 °C water bath for 60 min, after which 0.4 ml phenol-chloroform (24:1) was added to each tube and the suspension was shaken vigorously. After centrifugation at 13,000 rpm for 5 min, 0.3 ml supernatant was transferred to a new tube and mixed with 0.45 ml binding buffer. The mixture was then transferred to an adsorbing column (AC) for centrifugation at 13,000 rpm for 0.5 min. Then, 0.5 ml inhibitor removal fluid was added in AC for centrifugation at 12,000 rpm for 0.5 min. After washing twice with 0.5 ml washing buffer, the AC was transferred to a clean centrifuge tube, and 100 ml elution buffer was added to the middle of adsorbed film to elute the genomic DNA. The ITS region was amplified with primer pairs ITS5 and ITS4 (White & al. 1990). Nuclear LSU region was amplified with primer pairs LR0R and LR7 (Vilgalys 2018). The ITS was amplified by 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 nLSU was amplified by initial denaturation at 94 °C for 1 min, followed by 35 cycles at 94 °C for Table 1, concluded 30 s, 48 °C 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 directly sequenced at Kunming Tsingke Biological Technology Limited Company. All newly generated sequences were deposited in GenBank (Table 1).
Phylogenetic analyses of the ITS+nLSU sequences were performed using maximum parsimony, maximum likelihood, and Bayesian inference methods. Maximum parsimony (MP) analyses followed Zhao & Wu (2017), and tree construction was performed in PAUP* version 4.0b10 (Swofford 2002). All 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. Maxtrees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using bootstrap (BP) analysis with 1,000 replicates (Felsenstein 1985). 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. Sequences were analyzed using Maximum Likelihood (ML) with RAxML-HPC2 through the Cipres Science Gateway (Miller & al. 2009). Branch support (BS) for ML analysis was determined by 1000 bootstrap replicates.
MrModeltest 2.3 (Posada & Crandall 1998;Nylander 2004) was used to determine the best-fit evolution model for each data set for Bayesian inference (BI). Bayesian inference was calculated with MrBayes_3.1.2 using a general time reversible (GTR+G) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist & Huelsenbeck 2003). Four Markov chains were run for 2 runs from random starting trees for 8 million generations (ITS+nLSU) in Fig. 1, for 5 million generations (ITS+LSU) in Fig. 2 and trees were sampled every 100 generations. The first 25% of the generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for maximum likelihood (BS), maximum parsimony (BP) and Bayesian posterior probabilities (BPP) greater than or equal to 75 % (BP) and 0.95 (BPP) were considered significantly supported, respectively.
The second ITS+nLSU dataset (Fig. 2) comprising sequences from 51 fungal specimens representing 22 Phlebia species plus the outgroup had an aligned length of 2102 characters, of which 1588 characters were constant, 142 variable  1,1,1,1). Here also Bayesian analysis and ML analysis generated a similar topology as MP analysis (average standard deviation of split frequencies = 0.002221 (BI). The phylogeny (Fig. 2)  Basidiomata annual, resupinate, easily separable from the substratum, ceraceous to gelatinous, without odor or taste when fresh, becoming membranaceous upon drying, ≤12 cm long, 200-700 µm thick. Hymenial surface smooth to tuberculate, white to cream to pale brown when fresh, cream to pale brown upon drying. Sterile margin distinct, white.
Type of rot: white.

Discussion
We describe a new species, Phlebia wuliangshanensis, based on phylogenetic analyses and morphological characters.