Fomitiporia rhamnoides sp. nov. (Hymenochaetales, Basidiomycota), a new polypore growing on Hippophae from China

Abstract Based on morphology and phylogenetic analysis, Fomitiporia rhamnoides sp. nov. is described. It is characterised by perennial, pileate basidiomata, distinctly shining poroid surface, a zonate context, 11–13 pores per mm, parallel tramal hyphae and regularly arranged contextual hyphae, the presence of cystidioles, globose, hyaline, thick-walled, smooth, dextrinoid, strongly cyanophilous basidiospores measuring 5.8–7 × 5.4–6.5 µm and growing on Hippophae rhamnoides in northern China. Fomitiporia rhamnoides differs from other Fomitiporia species growing on Hippophae by its smaller pores (11–13 per mm vs. <10 per mm).


Introduction
Fomitiporia Murrill (Murrill 1907), typified by F. langloisii Murrill, is an important genus in Hymenochaetaceae because some species are pathogens of trees (Dai et al. 2007, Rajchenberg and Robledo 2013, Ota et al. 2014 whereas some other species are claimed to be medicinal (Dai et al. 2009). Fomitiporia is easy to distinguish from other members of Hymenochaetaceae in having subglobose to globose, hyaline, thickwalled, strongly dextrinoid and cyanophilous basidiospores (Fiasson and Niemelä 1984, Amalfi and Decock 2013, Chen and Cui 2017.
During investigations on wood-inhabiting fungi in northern China, in Hebei and Shanxi provinces, some specimens of a Fomitiporia species were collected on living Hippophae rhamnoides. They are characterised by distinctly small pores which make them different from other Fomitiporia species growing on Hippophae.
To understand their taxonomic placement, phylogenetic analysis was carried out based on the nuc rDNA regions of the 5.8S rDNA (ITS) and nuc 28S rDNA D1-D2 domains. Molecular analyses showed that the sampled specimens are clustered into a lineage representing an unknown species of Fomitiporia.

Materials and methods
The studied specimens are deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). The microscopic procedure follows Zhou et al. (2016a). The following abbreviations are used: IKI = Melzer's reagent, IKI− = both inamyloid and indextrinoid, KOH = 5% potassium hydroxide, CB = Cotton Blue, CB+ = cyanophilous, CB− = acyanophilous, 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 and n = number of spores measured from a given number of specimens. Special colour codes followed Petersen (1996).
CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co. Ltd, Beijing) was used to obtain PCR products from dried specimens according to the manufacturer's instructions with some modifications. Two DNA gene fragments, ITS and 28S were amplified using respectively the primer pairs ITS5/ITS4 (White et al. 1990) and LR0R/LR7 (http://www.biology.duke.edu/fungi/mycolab/primers.htm). The PCR procedures for ITS and 28S followed Zhou et al. (2016b). DNA sequencing was performed at the Beijing Genomics Institute and newly generated sequences were deposited in the GenBank database.
Sequences generated for this study and additional sequences downloaded from Gen-Bank were aligned using BioEdit (Hall 1999) and ClustalX (Thompson et al. 1997).
In the study, nuclear ribosomal RNA genes were used to determine the phylogenetic position of the new species. Phellinus uncisetus Robledo, Urcelay & Rajchenb. was designated as an outgroup following Decock et al. (2007).
Maximum parsimony analysis was applied to the combined dataset of ITS+28S sequences using 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. Maxtrees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using bootstrap analysis with 1000 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 generated. The Maximum likelihood (ML) tree was constructed using raxmlGUI 1.2 (Stamatakis 2006, Silvestro andMichalak 2012) with GTR+I+ G model and auto FC option (Pattengale 2010) in bootstrap (BS) replicates.
MrModeltest 2.3 (Posada andCrandall 1998, Nylander 2004) was used to determine the best-fit evolution model for the combined dataset of ITS+28S sequences for running Bayesian inference (BI). BI was calculated with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). Four Markov chains were run for two runs from random starting trees for 2 million generations for the combined dataset of ITS+28S sequences and trees were sampled every 100 generations. The first quarter of the generations were discarded as burn-in. The majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for Maximum parsimony (BP), Maximum likelihood (BS) and Bayesian posterior probabilities (BPP) greater than or equal to 50% (BP/BS) and 0.95 (BPP), respectively, were considered as significantly supported.

Phylogeny results
The combined ITS+28S dataset includes 78 specimens and resulted in an alignment of 1737 characters, of which 1124 characters are constant, 98 are variable and parsimony-uninformative and 515 are parsimony-informative. Maximum parsimony analysis yielded 28 equally parsimonious trees (TL = 1515, CI = 0.549, HI = 0.451, RI = 0.813, RC = 0.446). The best model for the combined dataset, estimated and applied in the Bayesian analysis, is GTR+I+G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (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 = 0.007191 (BI). Therefore, only the MP tree was presented and BP, BS and BPP values simultaneously above 50%, 50% and 0.95, respectively, were indicated at the nodes (Fig. 1). The phylogeny shows that the three newly sequenced specimens gathered with F. guoshangensis S. Guo & L. Zhou in a single, isolated, variably supported (68%/71%/1.00) clade (Fig. 1).  Etymology. Rhamnoides (Lat.) refers to the species growing on Hippophae rhamnoides. Basidiomata perennial, pileate, solitary or a few imbricated, hard corky and without odour or taste when fresh, woody hard and medium in weight when dry; pilei dimidiate to ungulate, triquetrous in section, projecting up to 5 cm, 7 cm wide and 2.5 cm thick at base; pileal surface yellowish-brown, greyish-brown to dark brown, concentrically sulcate, at first velutinate, becoming glabrous and slightly cracked with age; margin obtuse. Poroid surface clay-buff to yellowish-brown when fresh, becoming  orange brown to snuff brown when dry, shining; sterile margin yellowish-brown, up to 3 mm wide; pores circular, 11-13 per mm, dissepiments entire. Context yellowishbrown, zonate, woody hard, up to 1.5 cm thick; tubes greyish-brown, paler than context, hard corky to brittle, up to 1 cm long, annual layers indistinct.
Additional specimens ( Type of rot. Causing a white rot.

Discussion
Fomitiporia rhamnoides is characterised by its very small pores (11-13 per mm) and growing on Hippophae rhamnoides. It has the same sequences of Fomitiporia guoshangensis, an illegitimate name (art. 7, 8, 32A, code of nomenclature) also described based in Chinese collections (Guo et al. 2016 (Chen et al. 2016, Chen and Cui 2017, Ryvarden and Melo 2017. Amongst them, F. hippophaëicola has a distribution in Europe whereas F. norbulingka and F. subhippophaëicola have, so far, been found in Tibet, China (Chen et al. 2016). Fomitiporia hippophaëicola was previously recorded in China (Dai 2010), but the voucher specimens were re-identified as F. subhippophaëicola. The main characters of F. hippophaëicola, F. norbulingka and F. subhippophaëicola were given by Chen et al. (2016).