Taxonomy and Phylogeny of the Wood-Inhabiting Fungal Genus Hyphoderma with Descriptions of Three New Species from East Asia

Three new wood-inhabiting fungi, Hyphoderma crystallinum, H. membranaceum, and H. microporoides spp. nov., are proposed based on a combination of morphological features and molecular evidence. Hyphoderma crystallinum is characterized by the resupinate basidiomata with smooth hymenial surface scattering scattered nubby crystals, a monomitic hyphal system with clamped generative hyphae, and numerous encrusted cystidia present. Hyphoderma membranaceum is characterized by the resupinate basidiomata with tuberculate hymenial surface, presence of the moniliform cystidia, and ellipsoid to cylindrical basidiospores. Hyphoderma microporoides is characterized by the resupinate, cottony basidiomata distributing the scattered pinholes visible using hand lens on the hymenial surface, presence of halocystidia, and cylindrical to allantoid basidiospores. Sequences of ITS+nLSU rRNA gene regions of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony, and Bayesian inference methods. These phylogenetic analyses showed that three new species clustered into Hyphoderma, in which H. crystallinum was sister to H. variolosum, H. membranaceum was retrieved as a sister species of H. sinense, and H. microporoides was closely grouped with H. nemorale. In addition to new species, map to show global distribution of Hyphoderma species treated in the phylogenetic tree and an identification key to Chinese Hyphoderma are provided.


Introduction
Fungi are an ecologically important branch of the tree of life based on its distinct and diverse characteristics, in which these organisms play a vital role in ecosystems as diverse as soil, forest, rocks, and ocean, but their roles are primarily enacted behind the scenes, literally as hidden layers within their substrate [1]. On the basis of the nature of their intertwined partners in numerous symbiotic interactions, they have mostly marched via stepwise codiversification with the plants [2]. Fungi have evolved numerous strategies to degrade hard-to-digest substrates for outcompeting with other microbes, while combating competitors using an arsenal of bioactive metabolites, such as the familiar antibiotics, ethanol, and organic acids [3]. Taxonomy plays a central role in understanding the diversity of life, discovering into systems of names that capture the relationships between species, and translating the products of biological exploration [4]. Despite the early embrace of the molecular systematics by mycologists, both the discovery and classification of fungi are still in great flux, particularly among the more basal branches of the tree, in which the true diversity is only now coming to light from genomic analyses and environmental DNA surveys [1]. The researches revealed that perhaps less than 5% of the estimated two to four million species have been formally described, therefore, the hidden and microscopic nature of many fungi also means that their diversity is undersampled [5,6].
The studied specimens are deposited at the herbarium of Southwest Forestry University (SWFC), Kunming, Yunnan Province, China. Macromorphological descriptions are based on field notes and photos captured in the field and laboratory. Color terminology follows Petersen [20]. Micromorphological data were obtained from the dried specimens, which were observed under a light microscope following Dai [21]. 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, n = a/b (number of spores (a) measured from given number (b) of specimens).

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 [22]. ITS region was amplified with primer pair ITS5 and ITS4 [23]. Nuclear nLSU region was amplified with primer pair LR0R and LR7 (http: //lutzonilab.org/nuclear-ribosomal-dna/; accessed on 16 April 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 10 April 2021) using the "G-INS-i" strategy for ITS+nLSU and manually adjusted in BioEdit [32]. The dataset was aligned first and then, ITS and nLSU sequences were combined with Mesquite. Alignment datasets were deposited in TreeBASE (submission ID 27983). Climacocystis borealis (Fr.) Kotl. and Pouzar and Diplomitoporus crustulinus (Bres.) Domański were selected as an outgroup for phylogenetic analysis of ITS+nLSU phylogenetic tree ( Figure 1) following a previous study [19].
Maximum parsimony analysis was applied to the combined (ITS+nLSU) dataset. Its approaches followed previous study [22], and the tree construction procedure was performed in PAUP* version 4.0b10 [33]. 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 [34]. 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 8 April 2021) [35]. Branch support (BS) for ML analysis was determined by 1000 bootstrap replicates. MrModeltest 2.3 [36] was used to determine the best-fit evolution model for each data set for Bayesian inference (BI). BI was calculated with MrBayes 3.1.2 [37]. Four Markov chains were run for 2 runs from random starting trees for 6 million generations for ITS+nLSU ( Figure 1). 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 ( Clade names follow previous study [19]. The new species are in bold. Clade names follow previous study [19].
The phylogram inferred from ITS+nLSU sequences (Figure 1) demonstrated that three new species are clustered into genus Hyphoderma, in which H. crystallinum was sister to H. variolosum Boidin, Lanq. and Gilles, H. membranaceum was retrieved as a sister species of H. sinense C.L. Zhao and Q.X. Guan, and H. microporoides was closely grouped with H. nemorale K.H. Larss. (100% BS, 100% BP, and 1.00 BPP).    Fruiting body-Basidiomata annual, resupinate, adnate, without odor and taste when fresh, membranaceous when fresh, becoming hard membranaceous upon drying, and up to 15 cm long, 3 cm wide, and 30-100 µm thick. Hymenial surface white to pale gray when fresh, pale gray to slightly cream upon drying, with scattered nubby crystals. Margin sterile indistinct and white.
Fruiting body-Basidiomata annual, resupinate, adnate, without odor and taste when fresh, cottony when fresh, fragile upon drying, and up to 22 cm long, 2.5 cm wide, and 50-100 µm thick. Hymenial surface smooth with scattered pinholes visible under hand lens, cream to pale buff when fresh, and slightly buff upon drying. Margin sterile, indistinct, and white to cream. Etymology-microporoides (Lat.): referring to the scattered pinholes on the hymenophore that are visible under hand lens.
Fruiting body-Basidiomata annual, resupinate, adnate, without odor and taste when fresh, cottony when fresh, fragile upon drying, and up to 22 cm long, 2.5 cm wide, and 50-100 µm thick. Hymenial surface smooth with scattered pinholes visible under hand lens, cream to pale buff when fresh, and slightly buff upon drying. Margin sterile, indistinct, and white to cream.

Discussion
In the present study, three new species, Hyphoderma crystallinum, H. membranaceum, and H. microporoides are described based on phylogenetic analyses and morphological characteristics. Phylogenetically, the family-level classification of the Polyporales (Basidiomycota) amplified nLSU, nITS, and rpb1 genes across the Polyporales, was employed, in which four species Hyphoderma macaronesicum, H. medioburiense (Burt) Donk, H. mutatum (Peck) Donk, and H. setigerum, nested into family Hyphodermataceae within the residual polyporoid clade [19]. In the present study, three new taxa clustered into Hyphoderma, in which Hyphoderma crystallinum was sister to H. variolosum, H. microporoides grouped
(H.S. Jacks.) Donk [8,18,25,27,29,40,41,46]. Further studies should focus on the relationships between the host and Hyphoderma species, as well as trying to better understand the evolutionary directions between plant and Hyphoderma species. The researches on the phylogeny of Hyphoderma, as well as many fungal studies on the molecular systematics [47][48][49], will be useful to push the further research on fundamental research and applied research of fungi. More species of Hyphoderma should be found in subtropical and tropical Asia as it was shown that wood-inhabiting fungi are rich in tropical China [50,51].