Morphological and Phylogenetic Evidence for Recognition of Two New Species of Phanerochaete from East Asia

Two new corticioid fungal species, Phanerochaete pruinosa and P. rhizomorpha spp. nov. are proposed based on a combination of morphological features and molecular evidence. Phanerochaete pruinosa is characterized by the resupinate basidiomata with the pruinose hymenial surface, a monomitic hyphal system with simple-septate generative hyphae and subcylindrical basidiospores measuring as 3.5–6.7 × 1.5–2.7 µm. Phanerochaete rhizomorpha is characterized by having a smooth hymenophore covered by orange hymenial surface, the presence of rhizomorphs, subulate cystidia, and narrower ellipsoid to ellipsoid basidiospores. Sequences of ITS+nLSU nrRNA gene regions of the studied specimens were generated and phylogenetic analyses were performed with maximum likelihood, maximum parsimony, and Bayesian inference methods. These phylogenetic analyses showed that two new species clustered into genus Phanerochaete, in which P. pruinosa was sister to P. yunnanensis with high supports (100% BS, 100% BT, 1.00 BPP); morphologically differing by a pale orange to greyish orange and densely cracked hymenial surface. Another species P. rhizomorpha was closely grouped with P. citrinosanguinea with lower supports; morphologically having yellow to reddish yellow hymenial surface, and smaller cystidia measuring as 31–48 × 2.3–4.8 µm.

Molecular studies involving Phanerochaete based on the ribosomal DNA (rDNA) sequences, revealed the phylogenetic distribution of resupinate forms across the major clades of mushroom-forming fungi, in which P. chrysosporium Burds. nested into phlebioid clade in Polyporales [20]. Revisiting the taxonomy of Phanerochaete (Polyporales, Basidiomycota) using a four gene dataset and extensive ITS sampling indicated that Phanerochaete sensu lato was polyphyletic and distributed across nine lineages in the phlebioid clade, in which six lineages were associated to described genera [21]. Miettinen et al. [22]. explored the DNA-phylogeny-based and morphology-based to reconcile the polypores and genus concepts in the family Phanerochaetaceae, which the macromorphology of fruiting bodies and hymenophore construction did not reflect monophyletic groups, and Ceriporia inflata B.S. Jia and B.K. Cui was combined into Phanerochaete. Amplifying nrLSU, nrITS, and rpb1 genes across the Polyporales revealed that eleven genera clustered into family Phanerochaetaceae, and two families Hapalopilaceae and Bjerkanderaceae were placed as synonyms of Phanerochaetaceae [23]. Recently, the research supported by morphological studies and the phylogenetic analyses, showed that many new taxa of Phanerochaete s.s. were found and displayed the taxonomic status for the new taxa within genus Phanerochaete [14,19].
In 2018-2019, we collected the material supposedly belonging to the two undescribed species of corticioid fungi from Yunnan Province, China. We present the morphological and molecular phylogenetic evidence that support the recognition of two new species within the Phanerochaete s.s., based on the internal transcribed spacer (ITS) and regions nLSU sequences.

Morphology
The fruiting bodies were observed growing on the ground of broad-leaved treemixed forest. The fruiting bodies were dried in an electric food dehydrator at 40 • C, then sealed and stored in an envelope bag. They were then transported to Kunming where microscopic morphology and phylogeny to be studied at the mycology laboratory of Southwest Forestry University, Kunming, Yunnan Province, China. The for-study specimens were deposited at the herbarium of Southwest Forestry University (SWFC), Kunming, Yunnan Province, China. Macromorphological descriptions were based on field notes and photos captured in the field and lab. Color terminology follow Petersen [24]. Micromorphological data were obtained from the dried specimens, and observed under a light microscope following Dai [25]. The following abbreviations were 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, n = a/b (number of spores (a) measured from given number of specimens (b)).

Molecular Phylogeny
CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to obtain genomic DNA from dried specimens, according to the manufacturer's instructions followed previous study [26]. ITS region was amplified with primer pair ITS5 and ITS4 [27]. nLSU region was amplified with primer pair LR0R and LR7 (http://lutzonilab.org/nuclear-ribosomal-dna/; accessed on 28 September 2021). 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, 58 • C for 45 s, and 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, 48 • C for 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, Kunming, Yunnan Province, China. All newly generated sequences were deposited in NCBI GenBank (Table 1). Sequences were aligned in MAFFT 7 (https://mafft.cbrc.jp/alignment/server/; accessed on 28 September 2021) using G-INS-i strategy for ITS+nLSU combined dataset, and manually adjusted in BioEdit [38]. Aligned dataset was deposited in TreeBase (submission ID 28442). Phlebiopsis gigantea Fr. and Rhizochaete radicata (Henn.) Gresl., Nakasone and Rajchenb were selected as an outgroup for phylogenetic analyses of combined dataset following a previous study [19]. The taxon sampling strategy for the selection of sequences for phylogenetic trees was to choose (1) in a larger scale, focusing on the related genera in the families Phanerochaetaceae and Irpicaceae in Figure 1; (2) the related taxa based on BLAST search in GenBank within Phanerochaete s.l.; and (3) all species of Phanerochaete s.s.
Maximum parsimony analysis was applied to the combined (ITS+nLSU) dataset. Its approaches followed Zhao and Wu [26], and the tree construction procedure was performed in PAUP* version 4.0b10 [39]. 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. 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 [40]. 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. Datamatrix was also analyzed using Maximum Likelihood (ML) approach with RAxML-HPC2 through the CIPRES Science Gateway (www.phylo.org; accessed on 28 September 2021) [41]. Branch support (BS) for ML analysis was determined by 1000 bootstrap replicates.
MrModeltest 2.3 [42] was used to determine the best-fit evolution model for the dataset for Bayesian inference (BI). BI was calculated with MrBayes 3.1.7a [43]. Four Markov chains were run for 2 runs from random starting trees for 10 million generations for ITS+nLSU (Figure 2). The first one-fourth of all generations was discarded as burn-in. The majority rule consensus tree of all remaining trees was calculated. Branches were considered as significantly supported if they received maximum likelihood bootstrap value (BS) >70%, maximum parsimony bootstrap value (BT) >70%, or Bayesian posterior probabilities (BPP) >0.95. 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 [40]. 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. Datamatrix was also analyzed using Maximum Likelihood (ML) approach with RAxML-HPC2 through the CIPRES Science Gateway (www.phylo.org; accessed on 28 September 2021) [41]. Branch support (BS) for ML analysis was determined by 1000 bootstrap replicates. MrModeltest 2.3 [42] was used to determine the best-fit evolution model for the dataset for Bayesian inference (BI). BI was calculated with MrBayes 3.1.7a [43]. Four Markov chains were run for 2 runs from random starting trees for 10 million generations for ITS+nLSU ( Figure 2). The first one-fourth of all generations was discarded as burn-in. The majority rule consensus tree of all remaining trees was calculated. Branches were considered as significantly supported if they received maximum likelihood bootstrap value (BS)

Molecular Phylogeny
The ITS+nLSU dataset (Figure 1) included sequences from 86 fungal specimens representing 50 species. The dataset had an aligned length of 2368 characters, of which 1170

Molecular Phylogeny
The ITS+nLSU dataset (Figure 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 = 0.038487 (BI), and the effective sample size (ESS) across the two runs is the double of the average ESS (avg ESS) = 303.
Habitat and ecology-Climate of the sample collection site is a transition between tropical and subtropical climate, and the forest type is the tropical monsoon evergreen broad-leaved forest, and the samples were collected on an angiosperm trunk.

Discussion
In the present study, two new species, Phanerochaete pruinosa C.L. Zhao and D.Q. Wang and P. rhizomorpha C.L. Zhao and D.Q. Wang spp. nov., are described based on phylogenetic analyses and morphological characters. The nucleotide differences of phylogenetically similar species to Phanerochaete pruinosa and P. rhizomorpha.
In the ecology and biogeography, the taxa of Phanerochaete are a typical example of wood-rotting fungi, which are mainly distributed in Asia, Europe, and America, and the substrata are mostly hardwood [1,25], and this genus is an extensively studied group of Basidiomycota; nonetheless, the wood-rotting fungi diversity is still not well known in the subtropics and tropics [44][45][46][47][48]. The two new species, Phanerochaete pruinosa and P. rhizomorpha spp. nov., were found in subtropics, which enriches the diversity of woodrotting fungi.