Scytinostromabambusinum sp. nov. (Russulales, Basidiomycota) in China evidenced by morphological characteristics and phylogenetic analyses

Abstract Background Wood-rotting fungi as an important group within the Basidiomycota are known for their ecological role in the forest ecosystem in terms of decaying living and dead trees and recycling nutrients in forest ecosystems. Many new species were revealed in the last five years. In the present study, during an ongoing study on Scytinostroma, a new species of Scytinostroma was found from China. It is described and illustrated on the basis of the morphological and phylogenetic evidence. New information Scytinostromabambusinum sp. nov. is described as a new species, based on morphological and molecular evidence. It is characterised by annual, resupinate and broadly ellipsoid basidiomata with white to cream hymenophore, a dimitic hyphal structure with generative hyphae bearing simple septa, the presence of cystidioles and amyloid basidiospores measuring 5.5–7 × 4–5.3 µm. Phylogeny, based on molecular data of ITS and nLSU sequences, shows that the new species forms an independent lineage and is different in morphology from the existing species of Scytinostroma.

During a survey for wood-decaying fungi from China, two samples were collected from Jiangxi Province and their morphological characters fit Scytinostroma well.To confirm their taxonomic affinity and the evolutionary relationships amongst representative species of Scytinostroma, phylogenetic analysis was carried out, based on ITS and nLSU sequences.Both morphological and molecular data support these two samples to represent a new species.In this paper, we give an illustrated description for the new species S. bambusinum.

Morphological studies
The studied specimens were deposited in the Mycological Herbarium of Jiujiang University (MHJU), China.Microscopic examination follows Dai (2010) and colour terms follow Petersen (1996).Spores were measured from sections cut from the tubes.Five percent of measurements were excluded from each end of the range and are given in parentheses.Abbreviations include IKI = Melzer's reagent, IKI-= negative in Melzer's reagent, 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 = the L/W ratio and n = number of spores measured from the given number of specimens.

Molecular study and phylogenetic analysis
A CTAB-based rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain genomic DNA from dried specimens.The DNA was amplified with the following primers: ITS4 and ITS5 for ITS (White et al. 1990) and LR0R and LR7 for nLSU.The PCR procedure for ITS amplification was as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles at 94°C for 40 s, 54°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 of 94°C for 30 s, 50°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 the Changsha Genomics Institute, China, with the same primers.The newly-generated sequences were deposited in GenBank (Table 1).1999).Prior to phylogenetic analyses, ambiguous regions at the start and the end were deleted.
Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI) analyses were performed for the ITS and nLSU dataset.MP analysis was performed using PAUP* 4.0b10 (Swofford 2002) with gaps in the alignments treated as missing data.Trees were inferred using 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 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 generated.Sequences were also analysed using ML with RAxML-HPC2 on Abe through the CIPRES Science Gateway (www.phylo.org).BI was calculated with MrBayes3.1.2with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist and Huelsenbeck 2003).MrModelTest2.3(Nylander 2004) was used to determine the best-fit evolution model for the dataset.
The BI was conducted with MrBayes 3.2.6 in two independent runs, each of which had four chains for 10 million generations and started from random trees (Ronquist and Huelsenbeck 2003).Trees were sampled every 1,000 generations.The first 25% of the sampled trees were discarded as burn-in and the remaining ones were used to reconstruct a majority rule consensus and calculate Bayesian Posterior Probabilities (BPP) of the clades.
The three phylogenetic analyses algorithms generated nearly identical topologies for the dataset; thus, only the topology from the MP analysis is presented along with statistical values from the ML, MP and BI algorithms (Bootstrap support < 50% and Bayesian posterior probabilities < 0.9 are not shown) at the nodes.Tree was visualised in TreeView 1.6.6 (Page 1996).

Description
Basidiomata (Fig. 1) -Annual, resupinate, coriaceous, not separable from substrate, up to 30 cm long, 4 cm wide and less than 0.3 mm thick at centre.Hymenial surface smooth to tuberculate, white to cream when fresh, cream upon drying; margin concolorous with hymenial surface, thinning out and adnate.

Etymology
Bambusinum (Lat.):refers to the species growing on dead bamboo.

Analysis
Two newly-generated ITS and nLSU sequences of the new species are deposited at GenBank.The accession numbers of the sequences in this study are labelled in the phylogenetic tree (Fig. 3).The ITS and nLSU dataset has 66 taxa and resulted in an alignment of 1653 characters, of which 748 characters are constant, 67 are variable and parsimony-uninformative and 838 are parsimony-informative.Maximum parsimony analysis yielded 15 equally parsimonious trees (TL = 3274, CI = 0.520, HI = 0.480, RI = 0.859, RC = 0.447).Best model for the ITS and nLSU estimated and applied in the Bayesian analysis is: GTR+I+G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1, 1, 1, 1).The Bayesian and ML analyses produced similar topologies compared to the MP analysis, with an average standard deviation of split frequencies = 0.009737 (BI).The phylogenetic tree (Fig. 3) shows that the two newly-sequenced specimens form a distinct with full statistical supports (100% BS, 100% BP, 1.00 BPP) and this lineage occupies a separate position from known species of Scytinostroma (Fig. 3).Phylogeny of Scytinostroma inferred from the ITS and nLSU dataset.Topology is from the MP tree and statistical values (MP/ML/BI) are indicated for each node that simultaneously received BS from MP and ML not below 50% and BPPs from BI not below 0.9.

Discussion
The new species, Scytinostroma bambusinum, is described, based on morphological differences and molecular phylogenetic analyses in this study.The morphological characters and phylogeny, based on ITS and nLSU sequences (Fig. 3), show the position of the new species in the genus Scytinostroma.