﻿Phylogenetic analyses and morphological characters reveal two new species of Ganoderma from Yunnan province, China

﻿Abstract Ganodermadianzhongensesp. nov. and G.esculentumsp. nov. are proposed as two new species based on both phenotypic and genotypic evidences. Ganodermadianzhongense is characterized by the stipitate basidiomata, laccate and oxblood red pileus, gray white pore surface, duplex context and broadly ellipsoid basidiospores (9.0–12.5 × 6.5–9.0 μm) with coarse interwall pillars. Ganodermaesculentum is characterized by its basidiomata with slender stipe, white pore surface, homogeneous pileus context, and slightly truncate, narrow basidiospores (8.0–12.5 × 5.0–8.0 µm). Phylogenetic analyses were carried out based on the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF1-α) and the second subunit of RNA polymerase II (RPB2) sequence data. The illustrations and descriptions for the new taxa are provided.

Ganoderma has long been regarded as one of the most important medicinal fungi in the world (Paterson 2006); they have been used as medicine for over two millennia in China (Dai et al. 2009). Several Ganoderma species are known to be prolific sources of highly active bioactive compounds, especially polysaccharides, protein, sterols, and triterpenoids (Ahmadi and Riazipour 2007;Chan et al. 2007). These compounds are known to possess extensive therapeutic properties, such as antioxidant, antitumor, and antiviral agents, and improve sleep function (De Silva et al. 2013).
Species diversity of Ganoderma is abundant in China and more than 30 species have been described (Zhao et al. 2000;Wang et al. 2009;Cao et al. 2012;Li et al. 2015;Xing et al. 2016;Hapuarachchi et al. 2018;Liu et al. 2019;He et al. 2019;Wu et al. 2020). Yunnan province is considered as one of the hot-spots for studying biodiversity of polypores, and some new Ganoderma species have been described (Zhao 1989;Wang et al. 2010;Cao and Yuan 2013).
During our investigation into the diversity of Ganoderma in Yunnan province, several specimens of Ganoderma were collected from central and southern Yunnan. Phylogenetic analysis showed that the seven collections formed two distinct lineages and can be recognized as new species, hence two new species, namely G. dianzhongense and G. esculentum are introduced based on morphology and phylogeny.

Sample collection
Seven Ganoderma specimens were collected during the rainy season from July 2016 to August 2019 in Yunnan Province of China. The samples were then photographed and transported back to the laboratory where their fresh macroscopic details were described. The specimens were deposited in the herbarium of Kunming Institute of Botany Academia Sinica (KUN-HKAS).

Morphological studies
Macro-morphological characters were described based on fresh material field notes, and the photographs provided here. Color codes are from Kornerup and Wanscher (1978). Micro-morphological data were obtained from the dried specimens and observed by using a microscope following Li et al. (2015). Sections were studied at magnification of up to 1000× using a NiKon E400 microscope and phase contrast illumination. Microscopic features and measurements were made from slide preparations stained with 5% potassium hydroxide (KOH) and 2% Melzer's reagent. Basidiospore features, hyphal system, color, sizes and shapes were recorded and photographed. Measurements were made using the Image Frame work v.0.9.7 to represent variation in the size of basidiospores, 5% of measurements were excluded from each end of the range and extreme values 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+ = Cyanophilous (Xing et al. 2018). The abbreviation for basidiospores measurements (n/m/p) denote "n" basidiospores measured from "m" basidiomata of "p" specimens. Basidiospore dimensions (and "Q" values) are given as (a) b-av-c (d), where "a" represents the minimum, "d' the biggest, "av" the average "b" and "c" covers a minimum of 90% of the values. "Q", the length/width ratio of a spore in side view, and "Q m " for the average of all basidiospores ± standard deviation .

DNA extraction, PCR amplification, and sequencing
Total genomic DNA was extracted from dried pieces of pileus with tubes with modified CTAB protocol Doyle (1987). The genes ITS, TEF1-α and RPB2 were amplified by polymerase chain reaction (PCR) technique. The primers ITS1F / ITS4, TEF1-983 / TEF1-1567, and RPB2-6f / fRPB2-7cR were used to amplify the ITS, TEF1-α, RPB2 region, respectively (White et al. 1990;Liu et al. 1999;Matheny et al. 2007). PCR reactions (25 μL) contained mixture: 2.5 μL PCR reaction buffer, 2.5 μL 0.2% BSA, 2 μL dNTP (2.5 mm), 0.5 μL each of primer, 0.2 μL 5 U/μL Taq DNA polymerase, 1-1.5 μL DNA solution and 16 μL sterilized distilled H 2 O. The PCR cycling for ITS was as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 sec, 53 °C for 30 sec and 72 °C for 50 sec and a final extension of 72 °C for 10 min. The PCR cycling for TEF1-α was as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 sec, 55 °C for 30 sec and 72 °C for 50 sec and a final extension of 72 °C for 10 min. The PCR cycling for RPB2 was as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 sec, 50 °C for 30 sec and 72 °C for 50 sec and a final extension of 72 °C for 10 min. The PCR products were visualized via UV light after electrophoresis on 1% agarose gels stained with ethidium bromide. Successful PCR products were sent to Sangon Biotech Limited Company (Shanghai, China), using forward PCR primers. When sequences have heterozygous INDELS or ambiguous sites, samples were sequenced bidirectionally to make contigs of the amplified regions or verify the ambiguous sites . Raw DNA sequences were assembled and edited in Sequencher 4.1.4 and the assembled DNA sequences were deposited in GenBank (Table 1).

Sequencing and sequence alignment
Sequence data of three partial loci Internal transcribed spacer region (ITS), RNA polymerase II subunit 2 (RPB2), and translation elongation factor 1-alpha (TEF1-α) were used in the phylogenetic analyses. Besides the sequences generated from this study, other reference sequences were selected from GenBank for phylogenetic analyses (Table 1). Sequences were aligned using the online version of MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013) and adjusted using BioEdit v.7.0.9 by hand (Hall 1999) to allow maximum alignment and minimize gaps. Ambiguous regions were excluded from the analyses and gaps were treated as missing data. The phylogeny website tool "ALTER" (Glez-Peña et al. 2010) was used to convert the alignment fasta file to Phylip format for RAxML analysis and AliView and PAUP 4.0b 10 were used to convert the alignment fasta file to a Nexus file for Bayesian analysis (Swofford 2003). Phylogenetic analyses were obtained from Maximum Likelihood (ML) and Bayesian analysis (BI).

Molecular phylogenetic analyses
The maximum likelihood (ML) and Bayesian inference (BI) methods were used to analyze the combined dataset of ITS, TEF1-α and RPB2 sequences. Maximum likelihood analysis was conducted with RAxML-HPC2 on the CIPRES Science Gateway (Miller et al. 2010), involved 100 ML searches; all model parameters were estimated by the program. The ML bootstrap values (ML-BS) were obtained with 1000 rapid bootstrapping replicates. Maximum likelihood bootstrap values (ML) equal to or greater than 70% are given above each node (Figure 1).
Bayesian analysis was performed with MrBayes v3.2 (Ronquist et al. 2012), with the best-fit model of sequence evolution estimated with MrModeltest 2.3 (Nylander et al. 2008) to evaluate posterior probabilities (PP) (Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002) by Markov Chain Monte Carlo (MCMC) sampling. Six simultaneous Markov chains were run for 10,000,000 generations, trees were sampled every 500 th generation, and 2,000 trees were obtained. The first 5000 trees, represent- ing the burn-in phase of the analyses, were discarded, while the remaining 1500 trees were used for calculating posterior probabilities in the majority rule consensus tree (the critical value for the topological convergence diagnostic is 0.01). The phylogenetic tree was visualized with FigTree version 1.4.0 (Rambaut 2012) and made in Adobe Illustrator CS5 (Adobe Systems Inc., USA). Sequences derived in this study were deposited in GenBank (http://www.ncbi.nlm.nih.gov). The final sequence alignments and the phylogenetic trees are available at TreeBase (http://www. treebase.org, accession number: 28875).
Habit. On decaying hardwood trees or bamboo roots, accompanied in humus rich soil with over heavily rotted litter on the ground.

Discussion
Ganodermataceae is a large family of polypores, and has received great attention from mycologists for over many decades. However, species identification and circumscriptions have been unclear and taxonomic segregation of the genera has been controversial because of different viewpoints among mycologists (Moncalvo et al. 1995;Moncalvo and Ryvarden 1997;Costa-Rezende et al. 2020). Ganodermataceae was treated as a synonym of Polyporaceae and classify the genus Ganoderma into Polyporaceae by Justo et al. (2017). Later, Cui et al. (2019) excluded Ganoderma from Polyporaceae, due to Ganoderma having unique double-walled basidiospores. In addition, recent studies have clarified someuncer-tainties of generic delimitation and classification of polypores with ganodermatoid basidiospores, and proved that Ganodermataceae is a monophyletic group . More collections of this family are needed in order to estimate the attributes of this taxon better.