Additions to the knowledge of Ganoderma in Thailand: Ganoderma casuarinicola, a new record; and Ganoderma thailandicum sp. nov.

Abstract Ganoderma is a cosmopolitan genus of mushrooms, which can cause root and butt rot diseases on many tree species. Members of this genus are particularly diverse in tropical regions. Some Ganoderma spp. are medicinally active and therefore are used to treat human diseases or as a dietary supplement. In this study, three Ganoderma strains were collected in tropical southern Thailand. Phylogenetic analyses of combined ITS, LSU, TEF1α and RPB2 sequence data indicated that the three strains grouped in a distinct lineage within laccate Ganoderma. One strain was collected from Surat Thani Province clustered in the G. casuarinicola clade with high statistical support (MLBS = 100% / MPBS = 98% / PP = 0.96), while the other two strains of Ganoderma, collected from Nakhon Si Thammarat Province, formed a distinct well-supported clade (MLBS = 100% / MPBS = 100% / PP = 1.00) and are described here as a new species. Ganoderma casuarinicola is reported here as a new record to Thailand. Morphological differences of the two taxa and their closely related taxa are discussed. Colour photographs of macro and micro morphological characteristics and a phylogenetic tree to show the placement of the new record and new species are provided.


Introduction
Ganoderma, a genus of the Ganodermataceae, was established by Karsten (1881) with G. lucidum (Curtis) P. Karst. as the type species. Justo et al. (2017) treated Ganodermataceae as a synonym of Polyporaceae, while Cui et al. (2019) state that Ganoderma was not included in Polyporaceae because their double-walled basidiospores are quite different from Polyporaceae. Relevant characteristics for Ganoderma species delimitation are unique to laccate and non-laccate basidiocarps: truncated double walled basidiospores, an apical germinal pore, a thin and colourless external wall (exosporium) and a dark brown internal wall (endosporium) (Moncalvo and Ryvarden 1997;Zhao 1989;Núñez and Ryvarden 2000;Ryvarden 2004). Ganoderma is a cosmopolitan genus and some of the species are pathogenic, causing white rot diseases on rotting stumps, roots and living trunks (Moncalvo and Ryvarden 1997;Pilotti et al. 2004). Ganoderma are distributed in both tropical and temperate regions, but are particularly diverse in the tropical regions (Cao and Yuan 2013). Index Fungorum records 451 taxa (http://www.indexfungorum.org/; accessed date: 1 June 2019) and MycoBank records 387 taxa (http://www.mycobank.org/; accessed date: 1 June 2019). Ganoderma can be a confusing genus to study due to the highly variable morphological features of the species in this group, including intra-species variations (Ryvarden 2000;Papp et al. 2017;Hapuarachchi et al. 2018a, c;Hapuarachchi et al. 2019a, b).
The genus Ganoderma is economically important, as the members of the genus are regarded as valuable medicinal mushrooms (Dai et al. 2009;Hapuarachchi et al. 2018b). Ganoderma spp. have been used in traditional medicines for hundreds of years in Asian countries. Several Ganoderma species are known to be prolific sources of highly active bioactive compounds such as polysaccharides, proteins, steroids and triterpenoids, such as ganoderic acids (Shim et al. 2004;Qiao et al. 2005;Wang and Liu 2008;Teng et al. 2011;De Silva et al. 2012a, b;De Silva et al. 2013;Li et al. 2018). Those bioactive compounds have a therapeutic potential to treat and remedy many pathological diseases (Sanodiya et al. 2009;Richter et al. 2015;Hapuarachchi et al. 2018b).
In Thailand, several Ganoderma species have been reported based on both morphological characteristics and molecular data, including G. australe (Luangharn et al. 2017), G. sichuanense (Thawthong et al. 2017) and G. tropicum (Luangharn et al. 2019). The aims of the present study are to report G. casuarinicola as a new record to Thailand and describe G. thailandicum as a new species from Thailand, based on both morphological characteristics and phylogenetic data.

Mushroom collections and morphological study
Three specimens of Ganoderma were photographed at the collecting sites: one from a tropical climate at Surat Thani Province and the other two from Prachuap Khiri Khan Province in Thailand during the rainy season. The detailed morphological characteristics of the specimens were recorded, based on fresh materials (Luangharn et al. 2017). Specimens were subsequently dried at 40 °C for 24 hours, covered with wax papers, kept in sealed plastic bags with anhydrous silica gel (Luangharn et al. 2017) and deposited in the Mae Fah Luang University herbarium (MFLU herb.), while being duplicated in the Herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (HKAS).
Morphological characteristics were determined following the methodology described by Lodge et al. (2004). Colour changes on bruising were recorded in the field. Colours were recorded following Ridgeway (Ridgeway 1912). Micro-morphological characteristics were observed using a compound Carl Zeiss™ SteREO Discovery.V8 Microscope, while basidiospores were photographed using a Scanning Electron Microscope (SEM). Microscopic features and measurements were made from glass slide preparations, staining the tissues with 3-5% potassium hydroxide (KOH), 2% Melzer's reagent and 3% Congo red reagent (Kreisel and Schauer 1987). Measurements were made using the Tarosoft Image Framework programme v. 0.9.0.7. Basidiospore features, hyphal system, colour, sizes and shapes were recorded and photographed. The description of basidiospore measurements was done by using at least 50 basidiospores from each basidiomata (Miettinen and Larsson 2006). The basidiospore quotient was followed [Q = L/W], where Q, the quotient of basidiospore length to width (L/W) of a basidiospore in side view and Qm, the mean of Q-values ± SD, was calculated considering the mean value of the lengths and widths of basidiospores (Tulloss 2005). The basidiospore size was measured with and without the myxosporium and given as (a-)b-c(-d) (Tulloss 2005).

DNA extraction, PCR amplification and sequencing
Dried internal tissues of the fruiting bodies were used to extract DNA by using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux), following the manufacturer's instructions. Total reaction mixtures (25 μl) contained 9.5 μl ddH 2 O, 12.5 μl of PCR master mix, 1 μl of DNA template and 1 μl of each primer (10 μM). The primers used in PCR amplification were: ITS4/ITS5 for internal transcribed spacer gene region (ITS); LROR/LR5 for partial large subunit rDNA gene region (LSU) (Vilgalys and Hester 1990;White et al. 1990); 983F/2218R for partial translation elongation factor 1-alpha gene region (TEF1α) (Sung et al. 2007); and fRPB2-5f/fRPB2-7cR for partial RNA polymerase II second largest subunit gene (RPB2) (Liu et al. 1999). PCR amplification conditions were 3 min at 94 °C, followed by 35 cycles of 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min, followed by a final extension at 72 °C for 10 min for ITS and LSU. The amplification condition for TEF1α consisted of initial denaturation at 5.30 min at 95 °C, followed by 35 cycles of 94 °C for 1 min, 57 °C for 30 s and 72 °C for 1.30 min, followed by a final extension at 72 °C for 10 min and 3 min at 94 °C followed by 35 cycles of 95 °C for 1 min, 52 °C for 2 min and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min for RPB2. PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China.

Phylogenetic analyses
Sequence data, retrieved from GenBank based on previous studies, are listed in Table 1. The sequences were subjected to standard BLAST searches in GenBank to determine the primary identity of the fungal isolates. Amauroderma rugosum Cui 9011 (Li and Yuan 2015) and Tomophagus colossus (Zhou et al. 2015) were selected as the outgroup taxa. All the newly generated sequences were aligned with the combined datasets of ITS, LSU and TEF1α with MAFFT v. 7.309 (Katoh and Standley 2013) and manually adjusted using Bioedit v. 7.2.5 (Hall 1999). Gaps were treated as missing data. Maximum parsimony (MP) analysis was performed with PAUP v. 4.0b10 (Swofford 2002). Maximum likelihood analyses (ML) were estimated by using the software on the CIPRES Gateway platform (Miller et al. 2010) and performed using RAxML-HPC2 on XSEDE (v. 8.2.8) (Stamatakis 2014), then carried out using the raxmlGUI version v. 1.3.1 (Silvestro and Michalak 2011).
MrModeltest v. 2.3 was used to determine the best-fitting substitution model for each single gene partition and the concatenated dataset for Bayesian analyses (Nylander 2004). Bayesian inference posterior probabilities (PP) with a GTR+I+G model was used for each partition. MrBayes v. 3.2.2 (Huelsenbeck and Ronquist 2001) was used to evaluate PP by Markov Chain Monte Carlo sampling (BMCMC) (Rannala and Yang 1996;Zhaxybayeva and Gogarten 2002). The number of generations was set at 4,000,000, with trees being sampled every 100 generations and a total of 40,000 trees obtained, resulting in an average standard deviation of split frequencies below 0.01. Based on the tracer analysis (Rambaut et al. 2014), the first 20% of trees (8,000 trees) were discarded as the burn-in phase of the analyses represented. The remaining 32,000 trees were used for calculating PP in the majority rule consensus tree (Larget and Simon 1999). ML and MP bootstrap values, equal to or greater than 70% and Bayesian Posterior Probabilities (BP) equal to or greater than 0.95 are presented above each node (

Discussion
In this study, we describe a new species of Ganoderma growing on Pinus sp. in tropical southern Thailand, in a well-researched genus. This is not surprising as Hyde et al. (2018) found that up to 96% of species discovered in northern Thailand were new to science. Ganoderma casuarinicola was collected on a Pinus kesiya stump in a pine forest at Surat Thani Province in Thailand, while two collections of Ganoderma thailandicum were collected on Pinus merkusii stumps from Kanom District, Nakhon Si Thammarat Province in Thailand. All three collections grouped as sister taxa to the laccate Ganoderma clade, their morphological characteristics and molecular analyses providing insights to resolve species delimitation. In this study, we introduce G. casuarinicola (HKAS 104639) as a new record to Thailand which grouped with the holotype from Guangdong, China ( Fig. 1) with high statistical support (MLBS = 100% / MPBS = 98% / PP = 0.96) and G. thailandicum is described as a new species, the two collections of G. thailandicum (HKAS 104640 and HKAS 104641) grouping together as a distinct clade with 100% ML, 100% MP and 1.00 PP support.
Our findings are consistent with Xing et al. (2018), who demonstrated that G. casuarinicola forms a sister clade with G. aridicola J.H. Xing & B.K. Cui, from South Africa and G. enigmaticum M.P.A. Coetzee, Marinc., M.J. Wingf., from Africa (Coetzee et al. 2015). The morphological differences of these three Ganoderma species were detailed in Xing et al. (2018). Moreover, our study allows us to compare the holotypes of G. casuarinicola from Guangdong and our collection from Thailand. The Guangdong's G. casuarinicola shows its distinctive sectorial to shell-shaped, 10 cm long and 7 cm wide pileus (Xing et al. 2018), while the Thai G. casuarinicola shows its annual, applanate to dimidiate shape, 3-16 cm long and 1.5-3 cm wide pileus, larger than the Guangdong collection. Our G. casuarinicola collections show longer tubes of 6-14 mm, while the tubes of the Guangdong collection are 9 mm long; however, our collections show a thinner margin (0.8-1.2 cm thick) than the Guangdong collection (2 cm thick) (Xing et al. 2018). Macro-morphological characteristics of our G. casuarinicola share similarities with the holotype collection, such as strongly laccate, shallow sulcate, reddish-brown pileus surface, lateral stipe shape, white pore surface and brown context.
Micro-morphological characteristics of the context layers of both Guangdong and Thai G. casuarinicola share similar characteristics, such as the dense light brown to brown context layers; thin to thick-walled generative hyphae; thin-walled binding hyphae; and thick-walled skeletal hyphae. Although both type specimens and our collection of G. casuarinicola collection have mostly distinctive yellowish-brown basidiospores, Thai G. casuarinicola collections have a smaller size range of (8.7)10. 8-13.5(14.4) × (6.6)7.6-8.9(9.8) μm than the type of G. casuarinicola (8.3-)9.0-10.2(-11.5) × (4.5-)5.0-6.0(-7.0) μm (including myxosporium). However, the type of G. casuarinicola does not have the melanoid band (Xing et al. 2018), while our collection has a dark brown, melanoid band. Although both type specimens and our G. casuarinicola collections are grouped in the same clade, macro-morphologically, their pilei are very different, most probably due to geographical and climatic changes. Boddy et al. (2014) also mentioned that climate change and geography affect fungi in many ways, especially regarding phenological changes of fungal fruiting and the spatial and temporal distribution of hosts.
According to our phylogenetic analyses (Fig. 1), collections of G. thailandicum were grouped as a sister to G. aridicola, G. casuarinicola, and G. enigmaticum as a wellsupported clade of 100% ML, 100% MP and 1.00 PP statistical supports. Ganoderma aridicola, G. casuarinicola, G. enigmaticum and G. thailandicum share morphological similarities of laccate to strong laccate upper pileus surface and ellipsoid to broadly ellipsoid basidiospores at maturity. Ganoderma aridicola , G. casuarinicola (Xing et al. 2018) and G. enigmaticum (Coetzee et al. 2015) are considered as members of the G. lucidum complex and our G. thailandicum is also clustered within the G. lucidum complex, according to the results of the phylogenetic analyses. Our phylogenetic tree showed G. thailandicum clustered together with G. casuarinicola. Although G. thailandicum and G. casuarinicola form a distinctive laccate pileus surface, their macro-and micro-morphological characteristics are quite different. Ganoderma thailandicum can be easily distinguished from G. casuarinicola, by its deep magenta colour near the stipe, brownish-red colour at the centre of the pileus surface and vivid yellow colour at the actively-developed margin, while the fruiting bodies of G. casuarinicola are homogenously brownish-red to reddish-brown at maturity. Ganoderma thailandicum also has a smaller sized pileus (3-9 cm long, 3-6 cm width, 0.4-1.8 cm thick), while G. casuarinicola has a larger pileus (up to 10 cm long, 4-9 cm width, up to 2 cm thick). Ganoderma thailandicum has a smaller pore size (4-8 per mm) than G. casuarinicola (4-6 per mm) and G. thailandicum has narrower basidiospores (6.93 × 9.11 μm; including myxosporium) than G. casuarinicola (8.25 × 11.68 μm; including myxosporium). The basidiopore shapes of G. thailandicum are distinctive, with ellipsoid to broadly ellipsoid or some globose, while basidiospores of G. casuarinicola are mostly ellipsoid to broadly ellipsoid at maturity. Both G. thailandicum and G. casuarinicola are quite similar by having angular to round pore shapes. The differences of G. aridicola and G. enigmaticum have been described in Xing et al. (2016). Ganoderma mbrekobenum can be differentiated from G. casuarinicola and G. thailandicum by its woody to corky texture when dried, with dimitic hyphal system, ovoid and bitunicate basidiospores (Crous et al. 2016).
Casuarina has been reported as a host genus for G. casuarinicola (Xing et al. 2018), which is found in coastal areas, while our G. casuarinicola collection was found on dead Pinus kesiya wood, thus this is the first Pinus host recorded for G. casuarinicola. Based on comprehensive morphological characteristics and molecular analyses, we report G. casuarinicola as a new record to Thailand, with G. thailandicum as a new species from Thailand.