Species of Hymenochaete ( Hymenochaetales , Basidiomycota ) on bamboos from East Asia , with descriptions of two new species

Six species of Hymenochaete are found on bamboos in East Asia. Among them, H. bambusicola and H. orientalis are described and illustrated as new to science. Hymenochaete bambusicola is found exclusively on dead bamboos in northern Thailand and southwestern China, and characterized by the presence of dendrohyphidia and skeletal hyphae. It is phylogenetically and morphologically closely related to H. innexa, H. koeljalgii and H. tropica. Hymenochaete orientalis is found on bamboos in Taiwan and on angiosperm branches in southern China. It is distinguished by the absence of a hyphal layer and by having relatively large, oblong-ellipsoid to cylindrical basidiospores. Hymenochaete orientalis is morphologically similar to H. longispora and H. cinnamomea, and forms a distinct lineage close to H. cinnamomea in the ITS+nrLSU based phylogenetic analyses. An identification key to the six species on bamboos is given.


Introduction
Species of Hymenochaete Lév. are readily recognized by having brown basidiomes turning black in contact with potassium hydroxide, characteristic setae, generative hyphae without clamp connections, and smooth, thin-walled basidiospores.Hymenochaete is a large, morphologically heterogenous genus that includes more than one hundred and twenty species (Léger 1998, Parmasto 2004, Parmasto and Gilbertson 2005, He and Li 2011a, b, He and Dai 2012, Parmasto 2012).Wagner and Fisher (2002) separated the genus Pseudochaete T. Wagner & M. Fisch.from Hymenochaete according to phylogenetic analyses of nuclear large subunit ribosomal DNA sequences.Subsequently, this separation was supported by several molecular studies, and more species were described in or transferred to Pseudochaete (Larsson et al. 2006, He and Dai 2012, He and Li 2013, Parmasto et al. 2014).Yang et al. (2016) proposed Hymenochaetopsis S.H.He & Jiao Yang to replace Pseudochaete, since the latter has been used for algae since 1903.On the other hand, species in Cyclomyces Kunze ex Fr. and Hydnochaete Bres., with poroid/cyclolamellate and hydnoid hymenophores, were nested within the clades Hymenochaetopsis and Hymenochaete in the phylogenetic studies (Wagner and Fisher 2002, He and Dai 2012, Baltazar et al. 2014, Parmasto et al. 2014).Now, the former two genera are treated as synonyms of Hymenochaete (Fischer andWagner 2001, Baltazar et al. 2014).
Although Hymenochaete s.l. is widely distributed in subtropical and tropical areas on angiosperm substrates, only one species, H. muroiana I. Hino & Katum., has been reported on bamboos in eastern Asia to date (Léger 1998, Parmasto 2005, Parmasto and Gilbertson 2005).In 2015 and 2016, several field trips were carried out in southern China, northern Thailand and central Taiwan, and many corticioid fungal specimens including those of Hymenochaete on bamboos were collected.Morphological and molecular studies of the specimens revealed six species of Hymenochaete on bamboos, two of which, H. bambusicola and H. orientalis, are described here as new.

Materials and methods
Morphological studies.Voucher specimens are deposited in the herbaria of Beijing Forestry University, Beijing, China (BJFC), the Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand (MFLU), and the National Museum of Natural Science, Taichung, Taiwan (TNM).Samples for microscopic examination were mounted in cotton blue and 2% potassium hydroxide (KOH).The following abbreviations are used: L = mean spore length, W = mean spore width, Q = L/W ratio, n (a/b) = number of spores (a) measured from given number of specimens (b).Color codes and names follow Kornerup and Wanscher (1978).
DNA extraction and sequencing.A CTAB plant genome rapid extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd) was employed for DNA extraction and PCR amplification from dried specimens.The ITS region was amplified with the primer pair ITS5 and ITS4 (White et al. 1990) using the following procedure: initial denaturation at 95 °C for 4 min, followed by 34 cycles at 94 °C for 40 s, 58 °C for 45 s and 72 °C for 1 min, and final extension at 72 °C for 10 min.The nrLSU gene region was amplified with the primer pair LR0R and LR7 (Vilgalys andHester 1990, Lapeyre et al. 1993) using the following procedure: initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 50 °C for 1 min and 72 °C for 1.5 min, and final extension at 72 °C for 10 min.DNA sequencing was performed at Beijing Genomics Institute, and the sequences were deposited in GenBank (Table 1).
Maximum likelihood (ML) and maximum parsimony (MP) analyses were conducted for the dataset.MP analysis were performed using PAUP* 4.0b10 (Swofford 2002).Gaps in the alignments were treated as missing data.Trees were generated using 100 replicates of random stepwise addition of sequence and tree-bisection reconnection (TBR) branch-swapping algorithm, with all characters given equal weight.Branch supports for all parsimony analyses were estimated by performing 1000 bootstrap replicates (Felsenstein 1985) with a heuristic search of 10 random-addition replicates for each bootstrap replicate.Max-trees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved.The tree length (TL), consistency indices (CI), retention indices (RI), rescaled consistency indices (RC) and homoplasy index (HI) were calculated for each generated tree.RAxML v.7.2.6 (Stamatakis 2006) was used for ML analysis.Default setting were used for all parameters in the ML analysis, and statistical support values were obtained using nonparametric bootstrapping with 1000 replicates (Hillis and Bull 1993).

Phylogeny results
The ITS+nrLSU sequences dataset contained 66 ITS and 69 nrLSU sequences from 69 samples representing 59 ingroup taxa and two outgroup taxa (Table 1).Eight ITS and 21 nrLSU sequences were newly generated.The dataset had an aligned length of 2226 characters, of which 611 were parsimony informative.MP analysis yielded 13 equally parsimonious trees (TL = 3172, CI = 0.397, RI = 0.734, RC = 0.291, HI = 0.603).ML analysis resulted in a topology similar to that of MP analysis.Only the MP tree is provided with both parsimony and likelihood bootstrap ≥ 70% labeled along the branches (Fig. 1).Alignments and trees are deposited at TreeBASE (submission ID: 20657).In the tree, samples of H. bambusicola and H. orientalis formed two distinct lineages.For H. innexa G. Cunn., H. orientalis and H. rhabarbarina (Berk.)Cooke, samples collected from bamboos and wood clustered together with high bootstrap values (Fig. 1).Etymology."Bambusicola" (Lat.)refers to growing on bamboo.
Etymology."Orientalis" (Lat.)refers to the known distribution in East Asia.

Discussion
As shown in previous studies (Larsson et al. 2006, He and Dai 2012, He and Li 2013, Parmasto et al. 2014), species of Hymenochaetopsis formed a highly supported clade in our phylogenetic tree (Fig. 1).Four species, Hymenochaete bambusicola, H. innexa, H. koeljalgii and H. tropica clustered in a fully supported clade.Morphologically, these species have some similar features, such as strictly resupinate basidiomes, presence of abundant hyphidia, and oblong-ellipsoid to short cylindrical basidiospores.In the phylogenetic tree of Parmasto et al. (2014) Until now, six species, H. bambusicola, H. innexa (Fig. 6A), H. muroiana (Fig. 6B), H. orientalis, H. rhabarbarina (Fig. 6C) and H. tropica (Fig. 6D) have been found on bamboos in East Asia.Among these species, H. muroiana, originally reported from Japan is a common species in East Asia.Parmasto (2012) described H. muroiana subsp.africana Parmasto on bamboo from Kenya, which is very similar to H. muroiana subsp.muroiana in morphology.However, it is not clear whether they are conspecific in phylogeny.In addition to H. muroiana, H. bambusicola is also found exclusively on dead bamboos based on present materials.Hymenochaete innexa, H. rhabarbarina and H. tropica are here reported on bamboos for the first time.

Figure 1 .
Figure1.Strict consensus tree obtained from maximum parsimony analysis of combined ITS and nrLSU sequence data of taxa of Hymenochaete and Hymenochaetopsis.Branches are labeled with parsimony bootstrap (before slash) ≥ 70% and likelihood bootstrap (after slash) ≥ 70%.

Table 1 .
Taxa with locality and GenBank accession numbers for ITS and nrLSU sequences used in the phylogenetic analysis.
, H. koeljalgii clustered with H. floridea Berk.& Broome, but in present analyses the latter species grouped with H. sphaericola Lloyd and H. cruenta (Pers.)Donk.The topology of these and other closely related species is still not completely resolved.Hymenochaete orientalis nested within a highly supported clade with H. cinnamomea, H. minuscula, H. acerosa S.H.He & Hai J. Li, and H. epichlora (Berk.& M.A. Curtis) Cooke.This clade includes species of the H. cinnamomea group (Parmasto 2001, He and Li 2011a).In Parmasto et al. (2014), H. cinnamomea formed a fully supported clade with the generic type H. rubiginosa (Dicks.)Lév. and H. ochromarginata P.H.B. Talbot; however, in our tree, H. cinnamomea group and H. rubiginosa group are in a large clade that is not supported.This may be because our phylogenetic analysis includes more taxa related to H. cinnamomea and H. rubiginosa.