Taxonomy and Phylogeny of Meruliaceae with Descriptions of Two New Species from China

Two new wood-inhabiting fungi Hermanssonia fimbriata sp. nov. and Phlebia austroasiana sp. nov. in the Meruliaceae family are described and illustrated from southwestern China based on molecular and morphological evidence. The characteristics of H. fimbriata include annual, resupinate basidiomata, the absence of cystidia and cystidioles, oblong ellipsoid basidiospores of 5–6 × 2.4–3 μm, and growth on rotten gymnosperm wood in the east Himalayas. Its basidiomata change drastically upon drying, from being a light-coloured, juicy, papillose-to-wrinkled hymenophore, to a dark-coloured, corky-to-gelatinous, and more or less smooth hymenophore. The characteristics of Ph. austroasiana include annual, resupinate basidiomata, a hydnoid hymenophore, 2–3 spines per mm, the presence of tubular cystidia of 20–25 × 3–3.5 µm, oblong ellipsoid basidiospores of 4.4–5.2 × 2.1–3 μm, and growth on angiosperm wood in tropical forests in the southern Yunnan Province. The phylogenetic analyses based on the combined 2-locus dataset (ITS1-5.8S-ITS2 (ITS) + nuclear large subunit RNA (nLSU)) confirm the placement of two new species, respectively, in Hermanssonia and Phlebia s. lato. Phylogenetically, the closely-related species to these two new species are discussed.


Introduction
The phlebioid clade within Polyporales includes three lineages at a family level, namely Phanerochaetaceae, Irpicaceae, and Meruliaceae [1,2]. The taxonomy of many of the genera belonging to these families is not currently settled, and a case in point example is the genus Phlebia. In a recent study, Chen et al. [3] concluded that Phlebia s.l. is still polyphyletic, with members addressed in all families of the phlebioid clade. Based on their multigene phylogenetic analysis, the core Phlebia clade belongs to the Meruliaceae with three additional clades: the Hydnophlebia clade, the Mycoacia clade, and the Sarcodontia clade. The core Phlebia clade included the genera Aurantiopileus Ginns et al., Aurantiporus Murrill, Pappia Zmitr., and Phlebia s.s., as well as some species of Ceriporiopsis Domański s.l. and Mycoacia s.l. [3].
Four resupinate phlebioid specimens were collected from southwestern China (Tibet and Yunnan Province) during studies on wood-inhabiting fungi, and their morphology corresponded to concepts of Hermanssonia and Phlebia. Phylogenetic analyses based on the ITS1-5.8S-ITS2 (ITS) and nuclear large subunit RNA (nLSU) rDNA sequences were conducted to confirm their affinity. Both morphological and molecular evidence demonstrated that these four specimens represent two undescribed species of Meruliaceae. Thus, they are described in this paper.

Morphological Studies
Macro-morphological descriptions were based on voucher specimens and field notes. Microscopic structures were prepared from slide preparations of dried tissues stained with Cotton Blue and Melzer's reagent as described by Wu et al. [8]. The following abbreviations are used in the description: CB = Cotton Blue; CB-= acyanophilous in Cotton Blue; IKI = Melzer's reagent; IKI-= neither amyloid nor dextrinoid in Melzer's reagent; KOH = 5% potassium hydroxide; L = mean spore length (arithmetic average of basidiospores); W = mean spore width (arithmetic average of basidiospores); and 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). When the variation in spore size is shown, 5% of the measurements were excluded from each end of the range, and these values are shown in parentheses. Special colour terms follow Petersen [9] and herbarium abbreviations follow Thiers [10]. The voucher specimens for the present study are deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC), Beijing, China.

DNA Extraction, PCR, and Sequencing
Total genomic DNA was extracted from dried specimens using a CTAB Rapid Plant Genome Extraction Kit (Aidlab Biotechnologies Company, Ltd., Beijing, China) according to the manufacturer's instructions with some modifications [11]. The ITS regions were amplified with primers ITS4 and ITS5 [12]. The nLSU regions were amplified with primers LR0R and LR7 [13].
The polymerase chain reaction (PCR) procedure for the ITS was as follows: initial denaturation at 95 • C for 3 min, followed by 35 cycles at 94 • C for 40 s, 54 • C for 45 s, 72 • C for 1 min, and a final extension of 72 • C for 10 min. The PCR procedure for the 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 [14]. The purification and sequencing of the PCR products was conducted by the Beijing Genomics Institute, Beijing, China, with the same primers used in the PCR reactions. Species were identified by sequence comparison with accessions in the NCBI databases using the BLAST program.

Phylogenetic Analyses
Phylogenetic trees were constructed using ITS + nLSU rDNA sequences, and phylogenetic analyses were performed with the Maximum Likelihood (ML), Maximum Parsimony (MP), and Bayesian Inference (BI) methods. Sequences of the species and strains were primarily adopted from ITS-based and 28S-based tree topology, as described by Huang et al. [5] and Chen et al. [3]. New sequences generated in this study, along with reference sequences retrieved from GenBank (Table 1), were aligned by MAFFT 7 (Katoh et al. [15]; http://mafft.cbrc.jp/alignment/server/, accessed on 18 April 2022) using the "G-INS-i" strategy and manually adjusted in BioEdit v. 7.2.5 [16]. Unreliably aligned sections were removed before the analyses, and efforts were made to manually inspect and improve the alignment. The data matrix was edited in Mesquite v3.70 (https://www.mesquiteproject.org/ (accessed on 18 April 2022). [17]. The sequence alignment was deposited at TreeBase. Sequences of Hyphoderma mutatum (Peck) Donk and H. setigerum (Fr.) Donk obtained from GenBank (https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 18 April 2022) were used as outgroups to root the trees in the ITS + nLSU analysis. Maximum Parsimony analysis was applied to the ITS + nLSU dataset sequences. The approaches to phylogenetic analysis utilized those conducted by Chen and Cui [47], and the tree was constructed using PAUP* version 4.0 beta 10 [49]. All the characters were equally weighted, and gaps were treated as missing data. Trees were inferred using the heuristic search option with tree bisection and reconnection (TBR) branch swapping, and 1000 random sequence addition maxtrees were set to 5000. Branches of zero length were collapsed, and all the parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates [50]. Descriptive tree statistics, including the Consistency Index (CI), Homoplasy Index (HI), Rescaled Consistency index (RC), Retention Index (RI), and tree length (TL), were calculated for each Maximum Parsimonious Tree (MPT) generated.
The research using ML was conducted using RAxML-HPC v. 8.2.3 [51] and RAxML-HPC through the CIPRES Science Gateway ( [52]; http://www.phylo.org, accessed on 18 April 2022). Statistical support values (BS) were obtained using nonparametric bootstrapping with 1000 replicates. The BI analysis was performed with MrBayes 3.2.7a [53]. Four Markov chains were run for two runs from random starting trees for 3 million generations until the split deviation frequency value < 0.01, and the trees were sampled at every 1000 generation. The first 25% of the sampled trees were discarded as burn-in, and the remaining ones were used to reconstruct a majority rule consensus tree and calculate the Bayesian Posterior Probabilities (BPP) of the clades.
A total of 24 models of evolution were scored using PAUP* version 4.0 beta 10 [49]. Optimal substitution models for the combined dataset were then determined using the Akaike Information Criterion (AIC) implemented in MrModeltest 2.3 [54,55]. The model GTR + I + G was selected for use in the Maximum Likelihood (ML) and Bayesian Inference (BI) analyses.
Branches that received bootstrap support for Maximum Likelihood (BS), Maximum Parsimony (BP), and Bayesian Posterior Probabilities (BPP) > 75% (BS), 50% (BP), and 0.9 (BPP) were considered to be significantly supported. In addition, the ML analysis resulted in the best tree, and only the ML tree is shown along with the support values from the MP and BI analyses. FigTree v1.4.4 [56] was used to visualize the resulting tree.

Phylogenetic Analyses
The combined ITS + nLSU dataset included sequences from 110 specimens representing 61 taxa (Table 1). The dataset had an aligned length of 2349 characters, of which 1503 were constant, 195 were variable but parsimony-uninformative, and 651 were parsimony-informative. MP analysis yielded nine equally parsimonious trees (TL = 3586, CI = 0.377, RI = 0.752, RC = 0.283, HI = 0.623). The best model for the ITS + nLSU dataset estimated and applied in the Bayesian analysis was GTR + I + G. Bayesian analysis and MP analysis resulted in a similar topology to the ML analysis, with an average standard deviation of split frequencies of 0.006112 (BI).

Discussion
Chen et al. [3] divided the taxa of Meruliaceae into four clades: the core Phlebia clade, the Hydnophlebia clade, the Mycoacia clade, and the Sarcodontia clade. Two new species, Hermanssonia fimbriata and Phlebia austroasiana, are described in this study, based on morphological characters and phylogenetic analyses. Phylogenetically, they are nested in the core Phlebia clade, based on the ITS + nLSU sequence data ( Figure 1).