﻿Two new brown rot polypores from tropical China

﻿Abstract Brown-rot fungi are types of fungi that selectively degrade cellulose and hemicellulose from wood and are perhaps the most important agents involved in the degradation of wood products and dead wood in forest ecosystem. Two new brown-rot species, collected from southern China, are nested within the clades of Fomitopsis sensu stricto and Oligoporus sensu stricto, respectively. Their positions are strongly supported in the Maximum Likelihood phylogenetic tree of the concatenated the internal transcribed spacer (ITS) regions, the large subunit of nuclear ribosomal RNA gene (nLSU), the small subunit of nuclear ribosomal RNA gene (nuSSU), the small subunit of mitochondrial rRNA gene (mtSSU), the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2) and the translation elongation factor 1-α gene (TEF1) sequences. Fomitopsisbambusae, only found on bamboo, is characterised by its resupinate to effused-reflexed or pileate basidiocarps, small pores (6–9 per mm), the absence of cystidia, short cylindrical to oblong-ellipsoid basidiospores measuring 4.2–6.1 × 2–2.3 μm. Oligoporuspodocarpi is characterised by white to pale cream pore surface, round or sometimes angular pores (5–6 per mm), broadly ellipsoid to reniform basidiospores measuring 3.8–4.2 × 2–2.3 μm and growing on Podocarpus. Illustrated descriptions of these two novel species, Fomitopsisbambusae and Oligoporuspodocarpi, are provided.


Introduction
Wood-inhabiting basidiomycota can be grouped into two categories, white-rot and brown-rot fungi, according to their ability for decaying or decomposing wood. Brownrot fungi selectively degrade cellulose and hemicellulose from wood and decayed mate-rial becomes reddish-brown or tan, crisp, causing massive cracks in the middle of a longitudinal crisscross. However, white-rot fungi can degrade all the components of wood and decayed material, become white or pale-yellow or light reddish-brown and expose the fibrous structure. The number of brown rot fungi is remarkably smaller compared to white rot fungi (Zhang 2003;Wu et al. 2020). Gilbertson (1981) has estimated that approximately 6% of the wood-rotting basidiomycetes in North America give a brown rot. On the other hand, Dai (2012) demonstrated that 14% of Chinese polypores in northern China can cause a brown rot . Brown-rot fungi are perhaps the most important agents involved in the degradation of wood products and in the degradation of dead wood in forest ecosystems. It is worth emphasising that the diversity of brown rot fungi is higher in high-latitude areas than in low-latitude areas and the number of brown rot fungi decreases from north to south in China (Zhou and Dai 2012;Dai et al. 2015), so that brown-rot fungi are infrequent in tropical areas.
During our investigations of brown-rot fungi in China, eight specimens were collected from Hainan Province in tropical China. Morphological examination shows these collections to represent two brown-rot polypores, corresponding to Fomitopsis s.s. and Oligoporus s.s. After phylogenetic analyses of the internal transcribed spacer (ITS) regions, the large subunit of nuclear ribosomal RNA gene (nLSU), the small subunit of nuclear ribosomal RNA gene (nuSSU), the small subunit of mitochondrial rRNA gene (mtSSU), the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2) and the translation elongation factor 1-α gene (TEF1) sequences, two new species were confirmed as belonging to Fomitopsis s.s. and Oligoporus s.s.. In this paper, we describe and illustrate these two new species.

Morphological studies
The examined specimens were deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC) in Beijing, China. Macro-morphological descriptions were based on the field notes and measurements of herbarium specimens. Colour terms followed Petersen (1996). Micro-morphological data were obtained from the dried specimens and observed under a light microscope following Chen et al. (2017) and Shen et al. (2019). Sections were studied at a magnification up to 1000× using a Nikon Eclipse 80i microscope with phase contrast illumination (Nikon, Tokyo, Japan). Drawings were made with the aid of a drawing tube. Microscopic features, measurements and drawings were made from slide preparations stained with Cotton Blue and Melzer's Reagent. Spores were measured from sections cut from the tubes. To present the variation of spore size, 5% of measurements were excluded from each end of the range and are given in parentheses. The following abbreviations are used: IKI = Melzer's Reagent, IKI-= neither amyloid nor dextrinoid, KOH = 5% potassium hydroxide, CB = Cotton Blue, 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, n (a/b) = number of basidiospores (a) measured from given number (b) of specimens.

DNA extraction and sequencing
A cetyltrimethylammonium bromide rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing, China) was used to extract the total genomic DNA from dried specimens according to the manufacturer's instructions with some modifications (Song and Cui 2017;Xing et al. 2018). The ITS regions were amplified with the primer pairs ITS5 (GGA AGT AAA AGT CGT AAC AAG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) (White et al. 1990). The nLSU regions were amplified with the primer pairs LR0R (ACC CGC TGA ACT TAA GC) and LR7 (TAC TAC CAC CAA GAT CT) (http://www.biology.duke.edu/fungi/mycolab/primers. htm). The nuSSU regions were amplified with the primer pairs NS1(CCG GAG AGG GAG CCT GAG AAA C) and NS4 (CCC GTG TTG AGT CAA ATT A) (White et al. 1990). The mtSSU regions were amplified with the primer pairs MS1 (CAG CAG TCA AGA ATA TTA GTC AAT G) and MS2 (GCG GAT TAT CGA ATT AAA TAA C) (White et al. 1990). RPB1 was amplified with the primer pairs RPB1-Af (GAR TGY CCD GGD CAY TTY GG) and RPB1-Cr (CCN GCD ATN TCR TTR TCC ATR TA) (Matheny et al. 2002). RPB2 was amplified with the primer pairs fRPB2-5F (GAY GAY MGW GAT CAY TTY GG) and fRPB2-7CR (CCC ATR GCT TGY TTR CCC AT) (Matheny 2005). TEF1 was amplified with the primer pairs EF1-983F (GCY CCY GGH CAY CGT GAY TTY AT) and EF1-1567R (ACH GTR CCR ATA CCA CCR ATC TT) (Rehner and Buckley 2005). The PCR procedure followed that of Liu et al. (2019). The PCR products were purified with a Gel Extraction and PCR Purification Combo Kit (Spin-column) in Beijing Genomics Institute, Beijing, P.R. China. The purified products were then sequenced on an ABI-3730-XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA) using the same primers as in the original PCR amplifications. The sequence quality was checked following Nilsson et al. (2012). All newly-generated sequences were submitted to GenBank and were listed in Tables 1 and 2.
Maximum Parsimony (MP) analysis was applied to those two phylogenies and trees construction procedure were performed in PAUP* version 4.0b10 (Swofford 2002). Settings for phylogenetic analyses in this study followed the approach of Zhu et al. (2019) and Song and Cui (2017). 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 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 (MPT) generated.
Maximum Likelihood (ML) analysis was conducted with RAxML-HPC252 on Abe through the CIPRES Science Gateway (www.phylo.org) and involved 100 ML searches. All model parameters were estimated by the programme. Only the best Maximum Likelihood tree from all searches was kept. The Maximum Likelihood bootstrap values (ML-BS) were performed using a rapid bootstrapping with 1000 replicates. The phylogenetic tree was visualised using Treeview (Page 1996).
MrModeltest 2.3 (Posada and Crandall 1998;Nylander 2004) was used to determine the best-fit evolution model for two combined matrices to reconstruct phylogenetic analyses as a 6-gene dataset (ITS+nLSU+nuSSU+mtSSU+RPB2+TEF1) and a 7-gene dataset Fomitopsis ostreiformis Fomitopsis ostreiformis (ITS+nLSU+nuSSU+mtSSU+RPB1+RPB2+TEF1) for Bayesian Inference (BI). Bayesian Inference was calculated with MrBayes 3.2.6 (Ronquist et al. 2012), with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites. Four Markov chains were run for two runs from random starting trees for one million generations and trees were sampled every 100 generations. The burn-in was set to discard 25% of the trees. A majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Posterior Probabilities (BPP) greater than or equal to 75% (MP and ML) and 0.95 (BPP) were considered as significantly supported.
In our phylogenies (Figs 1 and 2), five samples on bamboo formed an independent lineage in the Fomitopsis s.s. clade with strong support (100% ML, 100% MP, 1.00 BPPs) and are distant from other taxa in the genus. Both morphology and rDNA sequence data confirmed that the five samples represent a new species in Fomitopsis. Meanwhile, three samples on Podocarpus were nested in the Oligoporus s.s. clade and formed an independent lineage with a robust support (100% ML, 100% MP, 1.00 BPPs). Both morphology and rDNA sequence data confirmed that the three samples represent a new species in Oligoporus. Diagnosis. Fomitopsis bambusae is characterised by resupinate to effused-reflexed or pileate, soft corky basidiocarps with bluish-grey pores, small pores measuring 6-9 per mm, cylindrical to oblong ellipsoid basidiospores measuring 4.2-6.1 × 2-2.3 μm and growing on dead bamboo.
Etymology. Bambusae (Lat.): refers to the species growing on bamboo. Fruiting body. Basidiocarps annual, resupinate to effused-reflexed or pileate, separable from the substrate, without odour or taste and soft corky when fresh, corky and light in weight when dry. Pilei semicircular, projecting up to 1 cm, 1.5 cm wide and 5 mm thick at base; resupinate part up to 14 cm long, 6 cm wide and 2 mm thick at centre. Pileal surface bluish-grey when fresh, pale mouse-grey to greyish-sepia when dry, glabrous to slightly velutinate, rough, azonate; margin acute, incurved when dry. Pore surface bluish-grey to pale mouse-grey when fresh, becoming mouse-grey to dark grey when dry; sterile margin up to 1 mm wide; pores round to angular, 6-9 per mm; dissepiments thin, entire. Context white to cream, corky, up to 3.5 mm thick. Tubes paler than pore surface, corky, up to 1.5 mm long.