Elaphroporia ailaoshanensis gen. et sp. nov. in Polyporales (Basidiomycota)

Abstract A new poroid wood-inhabiting fungal genus, Elaphroporia, typified by E. ailaoshanensis sp. nov., is proposed based on a combination of morphological features and molecular evidence. The genus is characterised by an annual growth habit, resupinate basidiocarps, becoming rigid and light-weight up on drying, a monomitic hyphal system with thick-walled generative hyphae bearing both clamp connections and simple septa, slightly amyloid, CB+ and ellipsoid, hyaline, thin-walled, smooth and IKI–, CB– basidiospores. Sequences of ITS and LSU nrRNA gene regions of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony and bayesian inference methods. The phylogenetic analysis based on molecular data of ITS+nLSU sequences showed that Elaphroporia belonged to the residual polyporoid clade and was closely related to Junghuhnia crustacea. Further investigation was obtained for more representative taxa in the Meruliaceae based on ITS+nLSU sequences, in which the result demonstrated that the genus Elaphroporia formed a monophyletic lineage with a strong support (100 % BS, 100 % BP, 1.00 BPP) and then grouped with Flaviporus and Steccherinum.


Introduction
The Polyporales is a large group of Agaricomycetes and includes more than 1800 taxa at species level belonging to 216 genera and 13 families (Kirk et al. 2008). Species in Polyporales are the key players amongst the wood-rotting fungi because of their importance in the carbon cycle (Floudas et al. 2012) and the pathogenic and potential application in biomedical engineering and biodegradation (Dai et al. 2009, Levin et al. 2016. Molecular systematics has played a powerful role in inferring phylogenies within fungal groups since the early 1990s (White et al. 1990, Hibbett et al. 2007, Larsson 2007, Binder et al. 2013, Choi and Kim 2017. Recently, molecular studies involving Meruliaceae P. Karst. have been carried out (Binder et al. 2005, Miettinen and Rajchenberg 2012, Miettinen et al. 2016. Larsson (2007) introduced a new division for part of the Polyporales, effectively renaming the phlebioid and residual polyporoid clades as the Meruliaceae, Phanerochaetaceae Jülich, and Byssomerulius Parmasto families. A phylogenetic study of Meruliaceae employing multi-genes suggested that 1) this family included species with both poroid and hydnoid hymenophore configurations, and 2) the genera of Flabellophora G. Cunn., Flaviporus Murrill, Junghuhnia Corda, Steccherinum Gray and Xanthoporus Audet belong to this family ). Moreover, further study employing a six-gene (5.8S, nrLSU, nrSSU, rpb1, rpb2, tef1) dataset has constructed a phylogenetic and phylogenomic overview of the Polyporales, which showed that the species of Meruliaceae fall into the residual polyporoid clade (Binder et al. 2013).
Wood-rotting fungi is a cosmopolitan group and it has a rich diversity on the basis of growing on boreal, temperate, subtropical, and tropical vegetations (Gilbertson and Ryvarden 1987, Núñez and Ryvarden 2001, Dai 2012, Ryvarden and Melo 2014. During investigations on wood-inhabiting fungi in southern China, an additional taxon was found which could not be assigned to any described genus. It produces annual, resupinate basidiocarps, a monomitic hyphal system with generative hyphae bearing both simple septa and clamp connections, slightly amyloid, CB+ and ellipsoid, hyaline, thin-walled, smooth basidiospores. These characters make it distinguishable from all known poroid and hydnoid wood-inhabiting fungal genera (Gilbertson and Ryvarden 1987, Núñez and Ryvarden 2001, Bernicchia and Gorjón 2010, Ryvarden and Melo 2014. In this study, the authors expand samplings from previous studies to examine taxonomy and phylogeny of this new genus within the Polyporales, based on the internal transcribed spacer (ITS) regions and the large subunit nuclear ribosomal RNA gene (nLSU) sequences.

Materials and methods
Morphological studies. The specimens studied are deposited at the herbarium of Southwest Forestry University (SWFC). Macro-morphological descriptions are based on field notes. Special colour terms follow Petersen (1996). Micro-morphological data were obtained from the dried specimens and observed under a light microscope following Dai (2010). The following abbreviations were used: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB-= acyanophilous, IKI = Melzer's reagent, IKI-= both inamyloid and indextrinoid, IKI+ = amyloid, 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 between the specimens studied, n (a/b) = number of spores (a) measured from given number (b) of specimens.
DNA extraction and sequencing. CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain genomic DNA from dried specimens, according to the manufacturer's instructions with the modification that a small piece of dried fungal specimen (about 30 mg) 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 that, 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 a 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 the adsorbed film to elute the genome DNA. The ITS region was amplified with primer pairs ITS5 and ITS4 (White et al. 1990). The nuclear LSU region was amplified with primer pairs LR0R and LR7 (https://sites.duke.edu/vilgalyslab/ rdna_primers_for_fungi/). 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. All newly generated sequences were deposited at GenBank (Table 1).
Maximum parsimony analysis was applied to the ITS+nLSU dataset sequences. Approaches to phylogenetic analysis followed Li and Cui (2013) and the tree construc- tion procedure 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. Max-trees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1,000 replicates (Felsenstein 1985). 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 (MPT) generated. Sequences were also analysed using Maximum Likelihood (ML) with RAxML-HPC2 through the Cipres Science Gateway (www.phylo.org; Miller et al. 2009). Branch support for ML analysis was determined by 1000 bootstrap replicates. MrModeltest 2.3 (Posada andCrandall 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 with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist and Huelsenbeck 2003). Four Markov chains were run for 2 runs from random starting trees for 5 million generations (Fig. 1), for 3 million generations (Fig. 2) and trees were sampled every 100 generations. The first one-fourth 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) respectively, were considered as significantly supported.
The phylogenetic tree (Fig. 1), inferred from ITS+nLSU sequences, demonstrated seven major clades for 60 sampled species of the Polyporales. The new genus Elaphroporia fell into the Meruliaceae within the residual polyporoid clade. It was closely related to Junghuhnia crustacea (Jungh.) Ryvarden with a good support (95% BS, 89% BP, 0.97 BPP).
A further phylogeny (Fig. 2) inferred from the combined ITS+nLSU sequences was obtained for 48 fungal specimens representing 31 taxa within the residual polyporoid clade and demonstrated that the new genus formed a monophyletic entity with a high 100 % BS, 100 % BP and 1.00 BPP and sisters to Junghuhnia crustacea and then grouped with Flaviporus and Steccherinum.
Basidiocarps. Annual, resupinate, easy to separate from substrate, soft corky when fresh, without odour or taste when fresh, becoming rigid and light-weight up on drying, up to 5 cm long, 3.5 cm wide, 4 mm thick at centre. Pore surface cream to pale yellow when fresh, turning to yellow upon drying; pores angular, 7-9 per mm; dissepiments thin, entire. Sterile margin narrow, cream, up to 1 mm wide. Subiculum thin, cream, corky, up to 0.2 mm thick. Tubes concolorous with pore surface, hard corky, up to 3.8 mm long.

Discussion
In the present study, a new genus, Elaphroporia, is described based on phylogenetic analyses and morphological characters. The genus has unique morphological characters in Meruliaceae.
Previously, seven clades were found in the Polyporales: antrodia clade, core polyporoid clade, fragiliporia clade, gelatoporia clade, phlebioid clade, residual polyporoid clade and tyromyces clade (Binder et al. 2013, Zhao et al. 2015. According to these results based on the combined ITS+nLSU sequence data (Fig. 1), the new genus is nested into the residual polyporoid clade with strong support (100 % BS, 100 % BP, 1.00 BPP).  analysed a higher-level phylogenetic classification of the residual polyporoid clade morphological plasticity in a group of the polypores, and showed that the natural genera could mostly be characterised morphologically and poroid and hydnoid species belong to separate genera. The current phylogeny shows that the genus Elaphroporia falls into the residual polyporoid clade and belongs to the family Meruliaceae (Figs 1, 2). Furthermore, the new genus is closely related to Junghuhnia and then grouped with Flaviporus and Steccherinum based on ITS+LSU-nrRNA gene regions with a strong support (100 % BS, 100 % BP, 1.00 BPP; Fig. 1). However, morphologically Junghuhnia differs from Elaphroporia by a dimitic hyphal system and presence of cystidia (Núñez andRyvarden 2001, Ryvarden andMelo 2014). Flaviporus is separated from Elaphroporia by the dark brown to bay pileus, a dimitic hyphal system and presence of the metuloid cystidia (Murrill 1905). Steccherinum differs in its odontioid to hydnoid hymenophore and cyanophilous basidiospores (Bernicchia and Gorjón 2010).
Morphologically, Elaphroporia resembles Ceriporia Donk and Phlebiporia Jia J. Chen, B.K. Cui & Y.C. Dai. Ceriporia is similar to Elaphroporia in an annual growth habit with poroid hymenophore, a monomitic hyphal structure and hyaline, thinwalled and smooth basidiospores. In addition, both genera cause a white rot. However, Ceriporia differs from Elaphroporia by the generative hyphae IKI-, CB- (Jia et al. 2014). Additionally, in molecular studies, Ceriporia fell into the phlebia clade , Miettinen and Rajchenberg 2012, Binder et al. 2013 which is also the same as in the authors' study (Fig. 1). Phlebiporia is similar to Mellipora by having the poroid hymenophore and the generative hyphae bearing both simple septa and clamp connections, but it is separated from Elaphroporia by having dextrinoid generative hyphae, tissues becoming brownish in KOH and presence of thin-walled quasi-binding hyphae in the subiculum .
Polypores are an extensively studied group of Basidiomycota (Gilbertson and Ryvarden 1987, Núñez and Ryvarden 2001, Dai 2012, Ryvarden and Melo 2014, but the Chinese polypore diversity is still not well known, especially in subtropics and tropics, from where many recently described taxa of polypores were discovered (Song et al. 2014, Nie et al. 2017, Yuan et al. 2017). The new genus in the present study, Elaphroporia, is also from the subtropics. It is possible that new polypore taxa will be found after further investigations and molecular analyses.