Steccherinum tenuissimum and S. xanthum spp. nov. (Polyporales, Basidiomycota): New species from China

Two new wood-inhabiting fungal species, Steccherinum tenuissimum and S. xanthum spp. nov. are described based on a combination of morphological features and molecular evidence. Steccherinum tenuissimum is characterized by an annual growth habit, resupinate basidiomata with an odontioid hymenial surface, a dimitic hyphal system with clamped generative hyphae, strongly encrusted cystidia and basidiospores measuring 3–5 × 2–3.5 μm. Steccherinum xanthum is characterized by odontioid basidiomata and a monomitic hyphal system with generative hyphae bearing clamp connections and covering by crystals, colourless, thin-walled, smooth, IKI–, CB–and has basidiospores measuring 2.7–5.5 × 1.8–4.0 μm. Sequences of the ITS and nLSU 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 analyses based on molecular data of ITS + nLSU sequences showed that two new Steccherinum species felled into the residual polyporoid clade. Further investigation was obtained for more representative taxa in Steccherinum based on ITS + nLSU sequences, which demonstrated that S. tenuissimum and S. xanthum were sister to S. robustius with high support (100% BP, 100% BS and 1.00 BPP).

Molecular studies related to Steccherinum have been carried out [4][5][6][7][8]. Larsson [4] analysed the classification of corticioid fungi, and suggested that S. ochraceum was nested in the Meruliaceae and grouped with Junghuhnia nitida (Pers.) Ryvarden and hydnoid genera Antrodiella Ryvarden & I. Johans., Junghuhnia Corda, and Steccherinum (Polyporales, Basidiomycota) was studied utilizing sequences of the gene regions ITS, nLSU, mtSSU, atp6, rpb2, and tef1, which revealed that the genus Steccherinum was shown to contain both hydnoid and poroid species, and the taxa from Junghuhnia and Steccherinum grouped together mixed within Steccherinum clade [5]. A molecular study based on multi-gene datasets demonstrated that Steccherinum belonged to the residual polyporoid clade and the generic type (S. ochraceum) was grouped with J. nitida [6]. A revision of the family-level classification of the Polyporales, including eighteen families, showed that, Steccherinum was grouped with Cerrena Grey and Panus Fr. [7]. On the basis of a re-evaluation of Junghuhnia s.  [8].
During investigations on wood-inhabiting fungi in southern China, two taxa which could not be assigned to any described species of Steccherinum, were found. To confirm the placement of the undescribed species in this genus, morphological examination and phylogenetic analyses based on the internal transcribed spacer (ITS) regions and the large subunit nuclear ribosomal RNA gene (nLSU) sequences were carried out.

Morphological studies
The specimens studied are deposited at the herbarium of Southwest Forestry University (SWFC), Kunming, Yunnan Province, P.R. China. The macromorphological descriptions are based on field notes. The colour terms follow Petersen [9]. Micromorphological data were obtained from the dried specimens, and observed under a light microscope (Nikon Eclipse E 100, Tokyo, Japan) following a previous study [10]. The following abbreviations were used for the microscopic characteristic descriptions: KOH = 5% potassium hydroxide, CB = cotton blue, CB-= acyanophilous, IKI = Melzer's reagent, IKI-= both non-amyloid and non-dextrinoid, 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.

Molecular procedures and phylogenetic analyses
The 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 some modifications: a small piece of dried fungal specimen (approximately 30 mg) was ground to a 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. Then, 0.4 mL of phenol-chloroform (24:1) was added to the tube, and the suspension was shaken vigorously. After centrifugation at 13,000 rpm for 5 min, 0.3 mL of supernatant was transferred to a new tube and mixed with 0.45 mL of 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 of inhibitor removal fluid was added to the AC, and the solution was centrifuged at 12,000 rpm for 0.5 min. After washing twice with 0.5 mL of washing buffer, the AC was transferred to a clean centrifuge tube, and 0.1 mL of elution buffer was added to the middle of the adsorbed film to elute the genomic DNA. The ITS region was amplified with primer pairs ITS5 and ITS4 [11]. The PCR procedure for the ITS region 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 at 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 in GenBank (Table 1). Sequencher 4.6 (GeneCodes, Ann Arbor, MI, USA) was used to edit the DNA sequences. The sequences were aligned in MAFFT 7 (http://mafft.cbrc.jp/alignment/server/) using the "G-INS-I" strategy and manually adjusted in BioEdit [12]. The sequence alignment was deposited in TreeBase (submission ID 27218). Sequences of Heterobasidion annosum (Fr.) Bref. and Stereum hirsutum (Willd.) Pers. obtained from GenBank was used as an outgroup to root trees following previous study [6] in the ITS + nLSU analysis (Fig 1), and Byssomerulius corium (Pers.) Parmasto and Irpex lacteus (Fr.) Fr. were used as an outgroup in the ITS + nLSU (Fig 2) analyses following previous study [8].
Maximum parsimony analyses were applied to the ITS + nLSU dataset sequences. The approaches used for the phylogenetic analysis followed previous study [13], and the tree construction procedure was performed in PAUP � version 4.0b10 [14]. 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 was 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 1000 replicates [29]. Descriptive tree statistics, including tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each maximum parsimony tree generated. The sequences were also analysed using maximum likelihood (ML) with RAxML-HPC2 through the Cipres Science Gateway (www.phylo.org) [30]. Branch support (BS) for the ML analysis was determined by 1000 bootstrap replicates.
MrModeltest 2.3 [31] was used to determine the best-fit evolution model for each dataset through Bayesian inference (BI). Bayesian inference was calculated with MrBayes 3.1.2, with a general time reversible (GTR+I+G) model of DNA substitution and gamma distribution rate variation across sites [32]. Four Markov chains were run for 2 runs from random starting trees for 800 thousand generations (Fig 1), for 1100 thousand generations (Fig 2) and trees were sampled every 100 generations. The first one-fourth of the generations were discarded as burn-in. A majority rule consensus tree of all the remaining trees was calculated. The branches were considered significantly supported if they received maximum likelihood bootstrap values (BS) >75%, maximum parsimony bootstrap values (BT) >75%, or Bayesian posterior probabilities (BPP) >0.95.

Nomenclature acts
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In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www. mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: PubMed Central and LOCKSS.

Molecular phylogeny
The ITS + nLSU dataset (Fig 1) included sequences from 49 fungal specimens representing 45 species. The dataset had an aligned length of 1820 characters, of which 995 characters are constant, 202 are variable and parsimony-uninformative, and 623 are parsimony-informative. The maximum parsimony analysis yielded 2 equally parsimonious trees (TL = 4151, CI = 0.3404, HI = 0.6596, RI = 0.5438 and RC = 0.1851). The best model for the ITS + nLSU dataset estimated and applied in the Bayesian analysis was GTR+I+G (lset nst = 6, rates = invgamma, prset statefreqpr = dirichlet (1,1,1,1)). The Bayesian analysis and ML analysis resulted in a similar topology as the MP analysis, with an average standard deviation of split frequencies of 0.006728 (BI). The phylogenetic tree (Fig 1) inferred from the ITS + nLSU sequences, demonstrated seven major clades for 45 sampled species in Polyporales. Two new Steccherinum species nested into the residual polyporoid clade. Steccherinum xanthum grouped with S. tenuissimum and was closely related to S. robustius (J. Erikss. & S. Lundell) J. Erikss.
The ITS + nLSU dataset (Fig 2) included sequences from 40 fungal specimens representing 21 species. The dataset had an aligned length of 2117 characters, of which 1587 characters are constant, 151 are variable and parsimony-uninformative, and 379 are parsimony-informative. The maximum parsimony analysis yielded 576 equally parsimonious trees (TL = 1322, CI = 0.551, HI = 0.449, RI = 0.798 and RC = 0.440). The best model for the ITS + nLSU dataset estimated and applied in the Bayesian analysis was GTR+I+G (lset nst = 6, rates = invgamma, prset statefreqpr = dirichlet (1,1,1,1)). The Bayesian analysis and ML analysis resulted in a similar topology as the MP analysis, with an average standard deviation of split frequencies of 0.009054 (BI).
The phylogenetic tree (Fig 2) inferred from the ITS + nLSU sequences had 19 species of Steccherinum and revealed that Steccherinum tenuissimum and S. xanthum were sister to S. Etymology: tenuissimum (Lat.): referring to the relatively thin basidiomata. Basidiomata: Annual, adnate, without odour or taste when fresh, becoming membranaceous up on drying, very thin, up to 20 cm long, 3 cm wide, 50-100 μm thick. Hymenial surface odontioid, with round aculei, 3-4 per mm, 0.2-0.5 mm long, white to cream when fresh, turning to cream to olivaceous buff upon drying.

Discussion
In the present study, two new species, Steccherinum tenuissimum and S. xanthum spp. nov., are described based on phylogenetic analyses and morphological characters. Phylogenetically, seven clades were found in Polyporales: the residual polyporoid clade, the phlebioid clade, the antrodia clade, the tyromyces clade, the fragiliporia clade, the core polyporoid clade and the gelatoporia clade [6,27]. According to our result based on the combined ITS + nLSU sequence data (Fig 1), two new species are nested into the residual polyporoid clade with strong support (100% BS, 100% BP, 1.00 BPP).
Steccherinum tenuissimum and S. xanthum were closely related to S. robustius based on rDNA sequences (Fig 2). However, morphologically S. robustius differs from the two new species by having a reddish orange to pale orange or brown hymenial surface and pale yellowish cystidia [2]. S. tenuissimum differs from S. xanthum by the cream to olivaceous hymenial surface and a dimitic hyphal system.
Geographically Steccherinum subglobosum H.S. Yuan & Y.C. Dai and S. subulatum H.S. Yuan & Y.C. Dai were described as new to science in P.R. China, but morphologically, S. subglobosum differs in its effuse-reflexed to pileate basidiomata with velutinate to tomentose hymenial surface and subglobose basidiospores (3.9-4.6 × 3.3-3.9 μm), S. subulatum differs from the two new taxa in the resupinate to effuse-reflexed basidiomata with longer hymenophore spines [33]. Wood-rotting fungi are an extensively studied group of Basidiomycota [2,5,6,10,[34][35][36][37], but Chinese wood-rotting fungal diversity is still not well known, especially in the subtropics and tropics. Many recently described taxa of wood-rotting fungi are from subtropical and tropical areas in China [38][39][40][41][42]. The two new species in the present study are also from the subtropics. It is possible that new taxa will be found after further investigations and molecular analyses.