Two new species of Ganoderma (Ganodermataceae, Basidiomycota) from Southwest China

Ganoderma is a large and diverse genus containing fungi that cause white rot to infect a number of plant families. This study describes G. phyllanthicola and G. suae as new species from Southwest China, based on morphological and molecular evidence. Ganoderma phyllanthicola is characterized by dark brown to purplish black pileus surface with dense concentric furrows, pale yellow margin, irregular pileipellis cells, small pores (5–7 per mm) and ellipsoid to sub-globose basidiospores (8.5–10.0 × 6.0–7.5 µm). Ganoderma suae is characterized by reddish brown to oxblood red pileus surface and lead gray to greyish-white pore surface, heterogeneous context, wavy margin and almond-shaped to narrow ellipsoid basidiospores (8.0–10.5 × 5.0–7.0 μm). The phylogeny of Ganoderma is reconstructed with multi-gene sequences: the internal transcribed spacer region (ITS), the large subunit (nrLSU), translation elongation factor 1-α gene (TEF-1α) and the sec - ond subunit of RNA polymerase II (RPB2). The results show that G. suae and G. phyllan-thicola formed two distinct line-ages within Ganoderma . Descriptions, illustrations and phylogenetic analyses results of the two new species are presented.


Introduction
Ganodermataceae is one of the main families of polypores with fourteen accepted genera: Amauroderma Murrill Sun (Costa-Rezende et al. 2020;Sun et al. 2022), of which most species are classified in the genus Ganoderma.
The word Ganoderma is derived from the Greek words "Gano", meaning "shiny", and "derma", meaning "skin" (Loyd et al. 2018).The genus Ganoderma (Polyporales, Basidiomycota) was described by Fries (1821) based on Polyporus lucidus (Curtis) Fr. and typified by Ganoderma lucidum (Curtis) P. Karst.from Europe (Fries 1821;Karsten 1881).Ganoderma is a globally distributed genus of wood-decaying fungi that encompass important species for forestry, medicine, food, and cultural traditions, in which morphological delimitation has been challenging due to its large plasticity and wide distribution across various regions (Luangharn et al. 2021).In the past few decades, DNA or amino acid sequence analyses have provided effective tools for taxonomists to combine data.These modern techniques have helped to clarify the distribution of different species complexes in the genus Ganoderma, and have revealed some instances of misidentification (Kinge et al. 2015;Fryssouli et al. 2020).
The genus is characterized by laccate or non-laccate basidiocarps, sessile to stipitate basidiomata, white to pale yellow margin, and red-brown colored truncate double-walled basidiospores, an apical germinal pore, thin and colourless external wall (exosporium), with a brown to dark brown interwall pillars (endosporium), and the ability to cause white rot in woody plants (Karsten 1881;Moncalvo and Ryvarden 1997).Furthermore, these species hold different characteristics, such as the shape and the color of the fruit body, host specificity, and geographical origin, which are used to identify individual members of the species.The species concept in the genus Ganoderma is thus not universally accepted nor well established due to the highly variable morphological features of the species (Wang et al. 2014;Náplavová et al. 2020).
Currently, based on credible morphological and phylogenetic evidence, 191 species of Ganoderma have been described worldwide (He et al. 2022;Sun et al. 2022;Vinjusha et al. 2022;Cabarroi-Hernández et al. 2023).Ganoderma is economically important, due to the fact that members of the genus are regarded as valuable medicinal mushrooms (Hapuarachchi et al. 2018a).Several Ganoderma species are known to be prolific sources of a high number of natural bioactive compounds such as polysaccharides, triterpenoids, sterols, and secondary metabolites (Richter et al. 2015).Approximately 45 species of Ganoderma are recorded in Chinese Fungi (Sun et al. 2022), of which Ganoderma lucidum "lingzhi" and G. sinense which used to be listed in Chinese Pharmacopeia to prevent and treat many diseases and are listed in Chinese Pharmacopeia, and which are included in the homologous list of medicine and food (Li et al. 2018).Furthermore, Ganoderma was included in the American Herbal Pharmacopoeia and Therapeutic Compendium (Hapuarachchi et al. 2018b).They are commonly named as "Lingzhi" or "Rui-zhi" in China, "Youngzhi" in Korea, "Reishi" in Japan and "Ganoderma" in the USA (Liu et al. 2015).These natural bioactive compounds are used to treat and remedy many pathological diseases, including traditional medicine for treating neurasthenia, debility of prolonged illness, insomnia, arthritis, asthma, anorexia, dizziness, chronic hepatitis, hypercholesterolemia, mushroom poisoning, coronary heart disease, hypertension, prevention of acute mountain sickness, deficiency fatigue', carcinoma, and bronchial cough in the elderly (Wang et al. 2020).In addition, Ganoderma products come in the form of various commercial products of Ganoderma such as powders, dietary supplements, coffee, tea, spore products, drinks, syrup, toothpaste, soap, lotion, and capsules, and have been commercialized as effective food and drug supplements for health benefits (Lai et al. 2004).
Southwest China contains some of the highest concentrations of fungal biodiversity in the world, and Yunnan Province, in particular, has a varied topography, environmental conditions, and a variety of habitats for a diverse range of fungi (He et al. 2022).Despite the advancement in taxonomic studies of Ganoderma species diversity, many novel species are still being discovered (He et al. 2021;He et al. 2022).During our investigations of macrofungi in Southwest China, a couple of specimens of Ganoderma were collected.In the current study, the phylogenetic analyses of Ganoderma were carried out based on the combined sequence dataset of ITS + nLSU + TEF1-α + RPB2 gene regions.Subsequent morphological and molecular studies uncovered two undescribed species.These species are illustrated and described below.

Specimen collection
During the rainy season from June 2019 to September 2023, four Ganoderma specimens were collected in southwest China.They were photographed in the field, then macro-mophology was described on fresh basidiomata, on the same day of collection.Specimens were there after thoroughly dried at 45 °C (Hu et al. 2022), in a thermostatic drier, stored in sealed plastic bags, and deposited in the herbarium of Kunming Institute of Botany, Chinese Academy of Sciences Academia Sinica (KUN-HKAS).

Morphological studies
Colour codes were determined following Kornerup and Wanscher (1978).For microscopic characteristics, anatomical and cytological characteristics including basidia, basidiospores, hyphal system, and pileipellis were observed and photographed using a Nikon ECLIPSE Ni-U microscope (Nikon, Japan) at magnifications up to × 1000.Tarosoft(R) Image Frame Work (IFW) was used for the measurement of photomicrographs, and Adobe Photoshop CS5 software was used to process images for making photo plates (He et al. 2021).
The following abbreviations are used: IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, KOH = 10% potassium hydroxide, CB = Cotton Blue, CB+ = cyanophilous.The notation [n/m/p] specifies that measurements were made on "n" basidiospores from "m" basidiomata and "p" collections.Basidiospore dimensions are given as (a) b-av-c (d).Where a and d refer to the lower and upper extremes of all measurements, respectively, b-c the range of 95% of the measured values, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q is the length/width ratio of basidiospores, Qm denotes the average of n measured basidiospores and SD is their standard deviation.Results are presented as Q = Qm ± SD.

DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from dry specimens using the Ezup Column Fungi Genomic DNA Purification Kit following manufacturer instructions.Primers pairs for PCR were respectively ITS1F/ITS5 (White et al. 1990), LR5/LR0R (Liu et al. 1999), TEF1-983 / TEF1-1567R (Matheny et al. 2007), and RPB2-6f / fRPB2-7cR (Liu et al. 1999), respectively.Primer sequences are available in the WASABI database at the AFTOL website (aftol.org).The PCR mixture was prepared in a 30 μL final volume, with 15 μL 2× Taq Plus Master Mix II (Sangon Biotechnology Co., Kunming, China), 12 μL ddH2O, 0.5 μL 10 μM of forward and reverse primers, 2 μL DNA.The PCR thermal cycle program for ITS and nrLSU amplification was conducted using the following profiles: 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 53 °C for 50 s, 72 °C for 1 min, and 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 s, 55 °C for 30 sec and 72 °C for 1 min, and 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 s, 50 °C for 50 s and 72 °C for 1 min, and 72 °C for 10 min.PCR products were checked on 1% agarose gels stained with ethidium bromide under UV light.The PCR products were purified and sequenced by the Sangon Biotech Limited Company (Shanghai, China).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
Sequences newly generated in this study and sequences obtained from Gen-Bank (Table 1) were analyzed.The related sequences were determined by using a BLAST search to reveal the closest matches with taxa in Ganoderma and recent relevant publications (Sun et al. 2022).Sequences were aligned using MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/)(Katoh and Standley 2013) and then checked visually and manually optimized using BioEdit v.7.0.9 (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 4.0 b 10 were used to convert the alignment fasta file to a Nexus file for Bayesian analysis (Swofford 2003).

Phylogenetic analyses
A maximum likelihood (ML) analysis was performed at the CIPRES web portal (Miller et al. 2010) using RAxML v.8.2.12 as part of the "RAxML-HPC2 on TG" tool (Miller et al. 2010).A general time-reversible model (GTR) was applied with a discrete gamma distribution and four rate classes.Fifty thorough ML tree searches were conducted out in RAxML v.8.2.11 under the same model.One thousand non-parametric bootstrap iterations were run with the GTR model and a discrete gamma distribution.The resulting replicates were plotted onto the best scoring tree obtained previously.Since no supported conflict (BS ≥ 60%) was detected among the topologies, the four single-gene alignments were concatenated using SequenceMatrix (Vaidya et al. 2011).
The Bayesian analyses were performed using PAUP v.4.0b10 and MrBayes v.3.2 (Ronquist et al. 2012), and the best-fit model of sequences evolution was estimated via MrModeltest 2.3 (Guindon and Gascuel 2003;Nylander 2004;Darriba et al. 2012).Markov Chain Monte Carlo (MCMC) sampling approach was used to calculate posterior probabilities (PP) (Rannala and Yang 1996).Bayesian analyses of six simultaneous Markov chains were run for one million generations and trees were sampled every 100 th generation with a total of 10,000 trees.The first 2000 trees were discarded and the remaining trees were used for calculating posterior probabilities in the majority rule consensus tree.
Table 1.Names, voucher numbers, origins, and their corresponding GenBank accession numbers of the taxa used in the phylogenetic analyses.The new species sequences generated sequences is show in bold, after the species name and the type specimens show "T" after the number.

Species
Voucher/strain Origin GenBank accession numbers

Species
Voucher/strain Origin GenBank accession numbers Diagnosis.Differs from other species in the genus by its sessile and coriaceous basidiomata, dark brown to purplish black pileus surface with dense concentric furrows, pale yellow margin, irregular pileipellis cells, broadly ellipsoid to subglobose basidiospores and truncated apex, exospore walls smooth, endospore walls with dense spinules.
Ganoderma aridicola described from South Africa is similar to G. phyllanthicola in the sessile basidiomata with dark brown pileus surface, homogeneous context, small pores and ellipsoid basidiospores.However, G. aridicola differs by the distinctly stratified tubes and lacks branched or protuberant apical cells (Xing et al. 2016).Besides, the phylogenetic analyses separated G. aridicola and G. phyllanthicola (Fig. 1).Ganoderma multiplicatum also has pale yellow margin and irregular pileipellis cells., but it differs from G. phyllanthicola by the photo brown to reddish brown pileus surface, short stipe (1.8-3 cm) and ellipsoid basidiospores (6.0-10.0× 4.5-7.0μm, Gottlieb and Wright 1999).

Ganoderma suae J. He & S.H. Li, sp. nov.
MycoBank No: 853506 Fig. 3 Diagnosis.Differs from other species in the genus by its large and substipitate basidiomata, reddish brown to oxblood red pileus surface with concentric furrows and radial rugose, whitish and wavy margin, almond-shaped basidiospores, heterogeneous context and non-stratified tubes.
Culture characteristics.Initially, white to yellowish white, pale yellow when growing, become orange white, pale orange, light orange and some reddish yellow to dark brown around the plugged circle of active mycelium after incubation for 3 weeks.

Discussion
Ganoderma has long been regarded as one of the most important genera of medicinal fungi worldwide with more than 45 species described in China.(Zhao et al. 1983;Cao et al. 2012;Li et al. 2015;Hapuarachchi et al. 2019;He et al. 2021;He et al. 2022;Sun et al. 2022;Yang et al. 2022).During the last five years, the diversity of Ganoderma in Southwest China was mainly reported from Yunnan Province and Guizhou Province (Hapuarachchi et al. 2019;Luangharn et al. 2021;He et al. 2022;Sun et al. 2022).These studies show that there is an unrecognized diversity of Ganoderma species in southwest China.More potential new species of Ganoderma may be discovered in the future.
In this study, two new species viz G. phyllanthicola and G. suae from Southwest China are introduced based on morphology and multigene phylogeny.Ganoderma phyllanthicola and G. suae satisfied the generic concept of the genus Ganoderma (Karsten 1881).They comprise subglobose to ellipsoid or ovoid basidiospores, truncated, double-walled with thick walls, exospore wall semi-reticulate, endospore wall smooth or with conspicuous spinules, homogeneous or heterogeneous context and laccate with variable ornamentation pileus surface.When compared with each other, G. phyllanthicola and G. suae occupied distinct and distant positions in the multilocus phylogenetic tree, and the morphology of their basidiomata also exhibits distinct macro-and microscopic characters that can further differentiate the two species.Thus, based on convergent results from morphology and molecular data analyses, G. phyllanthicola and G. suae are considered to be new species to science.
Ganoderma phyllanthicola was closely related to G. castaneum, G. philippii and G. tropicum in the phylogeny inferred from the concatenated sequence data set.Morphologically, they are easily distinguishable by some macroand microscopic characters of their basidiomata.Contrary to G. phyllanthicola, G. castaneum has a broadly attached, flabelliform, chestnut brown pileus surface with wide concentric ridges, heterogeneous context, regular palisade pileipellis cells, and broadly ellipsoid basidiospores not obviously truncated with smooth endospore walls (Sun et al. 2022; Table 2).Ganoderma philippii and G. tropicum, contrary to the new species, is characterized by flabelliform to circular, non-coriaceous basidiomata and very much smaller basidiospores (Steyaert 1972;Moncalvo and Ryvarden 1997; Table 2).Moreover, Ganoderma enigmaticum can be easily distinguished from G. phyllanthicola by the stipitate basidiomata and regular pileipellis cells (Coetzee et al. 2015).Ganoderma orbiforme has biannual or perennial basidiospores and longer pileipellis cells than those of G. phyllanthicola (Ryvarden 2000; Table 2).
Ganoderma resinaceum is known to be a Northern Hemisphere species, mainly occurring in Europe (Patouillard 1889;Moncalvo et al. 1995;Ryvarden and Melo 2014).The European specimens are easily recognized in the field by thick, soft and pale context.The first signs of genetic diversity within  G. resinaceum were observed by Moncalvo (2000), and Loyd et al. (2018) showed that G. resinaceum sensu American auctores encompassed at least two distinct species, viz.G. polychromum and G. sessile.Cabarroi-Hernández et al. (2019) studies confirmed that G. resinaceum sensu auctores from China, East Africa, Europa, and both North and South America represented a species complex.Study of phylogenetic inferences based on multilocus sequences by Hernand éz et al. ( 2019) also showed that G. resinaceum represents a species complex.
Our results based on polygenic phylogenetic analysis also confirm that Ganoderma resinaceum represents a species complex, encompassing several distinct species, namely G. platense, G. polychromum, G. sessile, and G. suae.Ganoderma suae emerges as a newly recognized species within the G. resinaceum sensu complex group (Fig. 1).Ganoderma suae is characterized by its annual basidiomata, reddish brown to oxblood red pileus surface, heterogeneous context without resinous incrustations (without black melanoid lines), wavy margin and almond-shaped basidiospores not obviously truncated, endospore walls with dense spinules (8.0-10.5 × 5.0-7.0 μm), can be easily distinguished from G. resinaceum (Ryvarden 2000).Náplavová et al. 2020 confirmed the presence of two distinct genotypes (genotype A and genotype B) in European G. resinaceum by comparing partial sequences of the TEF1-α region and the 25 s LSU rRNA gene.Their study also showed that basidiospore sizes range between 9.6-14.4× 6.0-8.4 µ m in genotype A and 6-12.0 × 7.2-9.6µ m in genotype B. Besides, specimens of both genotypes share the same pileus surface (glossy with resinous layer) and almost identical coloration.Only the context color was lighter brown beige to sand yellow in genotype A and darker brown beige to ochre brown in genotype B. Ganoderma resinaceum from Europe has a special laccate and glossy with resinous layer pileus surface, homogeneous context, and larger basidiospores cells than those of G. suae.(Ryvarden 2004;Náplavová et al. 2020).Thus, Ganoderma suae from China and G. resinaceum from Europe should be recognized as two different species.Table 2 presents a morphological comparison between the new species and its closest phylogenetic neighbors.Although we are of the opinion that G. suae well represent a species on its own, more material, ideally from various localities, and DNA sequences, is necessary to reveal the species diversity and kinship of G. resinaceum complex groups.
Recent studies have shown that the specimen G. resinaceum collected from China is inconsistent with the original description; therefore, it is clear that G. resinaceum is not distributed in China (Sun et al. 2022).It's noteworthy that we have also collected a sample (HL199) from Yunnan Province, which differs from both G. resinaceum and G. suae in terms of its distinct macro-morphology and multi-gene sequences.Regrettably, the specimens were sterile and micromorphological data were missing.In the future, collecting additional specimens will be crucial for revealing the true distribution and diversity of G. resinaceum complex groups in China.

Figure 1 .
Figure1.Maximum likelihood (ML) tree based on combined ITS + nrLSU + TEF1-α + RPB2 sequence data.Bootstrap support values with a maximum likelihood (ML) equal to or greater than 60% and Bayesian posterior probabilities (PP) equal to or greater than 0.90 are given above the nodes, shown as "ML/PP".New species are indicated in bold blue.

Table 2 .
Morphological comparison of Ganoderma phyllanthicola sp.nov., and G. suae sp.nov., with their closest relatives in the combined phylogeny.