Morphological and phylogenetic evidence for recognition of two new species of Hyphoderma (Basidiomycota) from southern China, with a key to all Chinese Hyphoderma

Abstract Wood-inhabiting fungi play crucial roles as decomposers in forest ecosystems and, in this study, two new wood-inhabiting corticioid fungi, Hyphodermapuerense and H.tenuissimumspp. nov., are proposed, based on a combination of morphological features and molecular evidence. Hyphodermapuerense is characterised by effused basidiomata with smooth to floccose hymenial surface, a monomitic hyphal system with clamped generative hyphae and ellipsoid basidiospores. Hyphodermatenuissimum is characterised by resupinate basidiomata with tuberculate to minutely-grandinioid hymenial surface, septate cystidia and cylindrical to allantoid basidiospores. Sequences of ITS and nLSU rRNA markers of the studied samples were generated and phylogenetic analyses were performed with Maximum Likelihood, maximum parsimony and Bayesian Inference methods. These analyses showed that the two new species clustered into Hyphoderma, in which H.puerense grouped with H.moniliforme and H.tenuissimum formed a singleton lineage. In addition, an identification key to Chinese Hyphoderma is provided.


Introduction
Fungi are eukaryotic microorganisms that play fundamental ecological roles as decomposers and mutualists of plants and animals. They drive carbon cycling in forest soils, mediate mineral nutrition of plants and alleviate carbon limitations of other soil organisms (Tedersoo et al. 2014). Fungi form an ecologically important branch of the tree of life, based on their distinct and diverse characters (James et al. 2020).
Hyphoderma has been studied using molecular data, particularly the internal transcribed spacer (ITS) region and the large subunit nuclear ribosomal RNA gene (nLSU). Larsson (2007) showed that H. obtusum J. Erikss. and H. setigerum  (Yurchenko and Wu 2014b). A revised family-level classification of the Polyporales revealed that four Hyphoderma species grouped into the residual polyporoid clade, belonging to Hyphodermataceae in that they grouped with three related genera in Meripilaceae: Meripilus P. Karst., Physisporinus P. Karst. and Rigidoporus Murrill (Justo et al. 2017).
In this study, two undescribed species of corticioid fungi from forest ecosystems were collected in the Yunnan Province, China. We present morphological and molecular phylogenetic evidence that support the recognition of two new species in Hypho derma, based on the nuclear ribosomal internal transcribed spacer region (ITS1, 5.8S and ITS2) and the nuclear ribosomal nLSU (28S) gene.

Morphology
The studied specimens are deposited at the Herbarium of Southwest Forestry University (SWFC), Kunming, Yunnan Province, P.R. China. Macromorphological descriptions are based on field notes and photos captured in the field and lab. Colour terminology fol-lows Petersen (Petersen 1996). Micromorphological data were obtained from the dried specimens when observed under a light microscope following Dai (2012). 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 and n = a/b (number of spores (a) measured from given number (b) of specimens).

Molecular phylogeny
The CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain genomic DNA from the dried specimens following the manufacturer's instructions (as done in Zhao and Wu 2017). The nuclear ribosomal ITS region was amplified with the primers ITS5 and ITS4 (White et al. 1990). The nuclear ribosomal LSU gene was amplified with the primers LR0R and LR7 (Vilgalys and Hester 1990;Rehner and Samuels 1994). 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 sequenced at Kunming Tsingke Biological Technology Limited Company, Kunming, Yunnan Province, P.R. China. All newly-generated sequences were deposited in NCBI GenBank (Table 1).
The sequences were aligned in MAFFT version 7 (Katoh et al. 2019) using the G-INS-i strategy. The alignment was adjusted manually using AliView version 1.27 (Larsson 2014). Each dataset was aligned separately at first and then the ITS1, 5.8S, ITS2 and nLSU regions were combined with Mesquite version 3.51. The combined dataset was deposited in TreeBASE (submission ID 28564). Climacocystis borealis (Fr.) Kotl. and Pouzar and Diplomitoporus crustulinus (Bres.) Domański were selected as outgroup ( Fig. 1) as inspired by a previous study (Justo et al. 2017).
Maximum parsimony analysis in PAUP* version 4.0a169 (http://phylosolutions.com/paup-test/) was applied to the combined ITS1+5.8S+ITS2+nLSU dataset. 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 1,000 random sequence additions. Max-trees were set to 5,000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using bootstrap (BT) analysis with 1,000 pseudoreplicates (Felsenstein 1985). Descriptive tree statistics -tree length (TL), composite consistency index (CI), composite retention index (RI), composite rescaled consistency index (RC) and composite homoplasy index (HI) -were calculated for each maximum parsimonious tree generated. The combined dataset was also analysed using Maximum Likelihood (ML) in RAxML-HPC2 through the CIPRES Science Gateway (Miller et al. 2012). Branch support (BS) for the ML analysis was determined by 1,000 bootstrap pseudoreplicates. MrModeltest 2.3 (Nylander 2004) was used to determine the best-fit evolution model for each dataset (ITS1+5.8S+ITS2+nLSU) for Bayesian Inference (BI). BI was calculated with MrBayes version 3.2.7a (Ronquist et al. 2012). Four Markov chains were run for two runs from random starting trees for 3 million generations (Fig. 1). The first 25% of all generations was discarded as burn-in. A majority rule consensus tree was computed from the remaining trees. Branches were considered as significantly supported if they received a maximum likelihood bootstrap support value (BS) of > 70%, a maximum parsimony bootstrap support value (BT) of > 70% or a Bayesian posterior probability (BPP) of > 0.95.

Molecular phylogeny
The ITS1+5.8S+ITS2+nLSU dataset comprised sequences from 86 fungal specimens representing 46 taxa. The dataset had an aligned length of 2,034 characters, of which 1,360 characters were constant, 131 were variable and parsimony-uninformative and 543 (35%) were parsimony-informative. Maximum parsimony analysis yielded 108 equally parsimonious trees (TL = 3,317, CI = 0.3361, HI = 0.6946, RI = 0.7051 and RC = 0.2370). The best model of nucleotide evolution for the ITS1+5.8S+ITS2+nLSU dataset estimated and applied in the Bayesian analysis was found to be GTR+I+G. Bayesian analysis and ML analysis resulted in a similar topology as in the MP analysis. The Bayesian analysis had an average standard deviation of split frequencies = 0.008952 (BI) and the effective sample size (ESS) across the two runs is double the average ESS (avg. ESS) = 1,771. The Bayesian tree is shown here (Fig. 1). New species is shown in bold; * type material. The phylogram inferred from ITS1+5.8S+ITS2+nLSU sequences (Fig. 1) highlights the two undescribed species in Hyphoderma; H. puerense as a sister to H. mo niliforme and H. tenuissimum that forms an independent monophyletic lineage (100% parsimony bootstrap support, 100% likelihood bootstrap support and 1.00 BPP).  Description. Basidioma annual, resupinate, adnate, byssoid, without odour and taste when fresh, up to 15 cm long, 3 cm wide, 100-260 µm thick. Hymenial surface smooth to floccose, cream when fresh, cream to slightly buff on drying. Margin sterile, thinning out, narrow, cream.
Habitat and ecology. Climate of the sample collection site is subtropical monsoon climate area, the forest type is evergreen angiosperm forest and samples were collected on fallen angiosperm branches.   Description. Basidioma annual, resupinate, adnate, membranaceous when fresh, hard membranaceous upon drying, up to 20 cm long, 3 cm wide, 30-100 µm thick. Hymenial surface tuberculate to minutely-grandinioid, slightly buff when fresh, buff upon drying, cracking. Margin sterile, slightly buff, 1 mm wide.
Habitat and ecology. Climate of the sample collection site is subtropical monsoon climate area, the forest type is evergreen angiosperm forest and samples were collected on fallen angiosperm branches.

Discussion
In the present study, two new species, Hyphoderma puerense and H. tenuissimum are described, based on phylogenetic analyses and morphological characters.
Phylogenetically, the two new taxa were found to belong to Hyphoderma, in which H. puerense forms a sister species to H. moniliforme and H. tenuissimum forms an independent monophyletic lineage (100% BS, 100% BP and 1.00 BPP).
In the current phylogenetic tree, two partially annotated GenBank sequences (KJ668522 and KJ714002) of Hyphoderma sp. (South Korea) cluster closely with four sequences of the new species Hyphoderma tenuissimum, although whether they really belong to this species remains to be assessed. It is certainly conceivable that they do, which would mean that Hyphoderma tenuissimum has been collected and sequenced at least six times in Asia. Regarding the new taxon H. puerense (Fig. 1), four partially annotated GenBank sequences (KR868735, KR868736, KR868737 and DQ340327) form a reasonably wellsupported clade together with our two specimens of H. puerense. We interpret this to mean that all six taxa represent H. puerense. All of the samples in this clade are from Asia, which supports the point of the importance of vicariance in the evolution in this genus.