Ganoderma sichuanense (Ganodermataceae, Polyporales) new to Thailand

Ganoderma sichuanense (Ganodermataceae) is a medicinal mushroom originally described from China and previously confused with G. lucidum. It has been widely used as traditional medicine in Asia since it has potential nutritional and therapeutic values. We collected 8 specimens of Ganoderma species from Thailand and show that they represent the first record of G. sichuanenese for Thailand. In this paper, we describe our specimens of Ganoderma sichuanense based on fresh basidiomes, and provide line drawings and photographs. The data from macroand microscopic features are consistent with the characteristics of the species. Analysis of ITS sequence data indicates that the Thai collections cluster in same species clade as the epitype of G. sichuanense.


Ganoderma sichuanense (Ganodermataceae, Polyporales) new to Thailand Introduction
The genus Ganoderma was established by Karsten (1881) based on Ganoderma lucidum (Curtis) P. Karst.The genus Ganoderma includes the subgenera Ganoderma (which in turn includes sections Ganoderma and Phaenema), Elfvingia, and Trachyderma (Zhao and Zhang 2000).Many members of this genus are found in subtropical and tropical regions and appear to thrive in hot and humid conditions (Pilotti et al. 2004).Ganoderma species grow as facultative parasites of trees but can also live as saprobes on rotting stumps and roots (Turner 1981, Pilotti 2005).Basidiomes are commonly in the form of a bracket (Pilotti et al. 2004).Bioactive compounds from Ganoderma show a huge structural and chemical diversity (Deepalakshmi and Mirunalini 2011).These bioactive constituents are reported to be responsible for anti-cancer, anti-inflammatory, anti-tumor, anti-oxidant, immunomodulatory, immunodeficiency, anti-diabetic, anti-viral, anti-bacterial, anti-fungal, anti-hypertensive, anti-atherosclerotic, anti-aging, anti-androgenic, hepatoprotective, radical scavenging properties, neuroprotection, sleep promotion, cholesterol synthesis inhibition, preventing hypoglycemia, inhibition of lipid peroxidation/oxidative DNA damage, maintenance of gut health, prevention of obesity, and stimulation of probiotics (Paterson 2006, De Silva et al. 2012a, b, De Silva et al. 2013, Bishop et al. 2015, Hapuarachchi et al. 2016a, 2016b).The traditional taxonomy of Ganoderma is based on morphological traits, and the genus was divided into two distinct groups, the laccate (G.lucidum complex) and the non-laccate (G.applanatum complex) groups, which correspond to the subgenera Ganoderma and Elfvingia, respectively (Zheng et al. 2007).There are 437 epithets listed in Index Fungorum (2017) for Ganoderma, of which 414 are accepted by Species Fungorum in May, 2017) (http://www.speciesfungorum.org/Names/Names.asp)."Lingzhi" is the Chinese name mainly referring to G. lucidum (Curtis) P. Karst, which has been widely used in China for medicinal purposes for over two millennia (Sliva 2006).However, this species was originally described from Europe (Ryvarden and Gilbertson 1993).Patouillard (1907) reported G. lucidum from China for the first time and Teng (1934) described collections of G. lucidum from different regions in China.Liu (1974) compiled a monograph of traditional Chinese medicinal fungi, and he reported G. lucidum in his book.Since then, G. lucidum was accepted as the scientific binomial of "Lingzhi" in many reports on Chinese edible and medicinal mushrooms (Ying et al. 1987, Mao 1998, Dai et al. 2009).Moncalvo et al. (1995) mentioned that G. lucidum sensu stricto was distributed in northern and southern Europe, and probably extended to China.However, their further studies confirmed that the species named G.lucidum from both Europe and mainland China was not conspecific based on analyses of ITS and 25S ribosomal DNA sequences.Later, other authors (Pegler and Yao 1996, Smith and Sivasithamparam 2000, Hong and Jung 2004) have confirmed the same idea.Hawksworth (2005) suggested to conserve the name G. lucidum for an Asian type and introduce a new name for the European species.Later, it was found that G. lucidum from tropical Asia is not conspecific with G. lucidum sensu stricto, and not even conspecific with the real "Lingzhi" distributed in East Asia, and was named G.multipileum Ding Hou, (Wang et al. 2009).Cao et al. (2012) named the medicinal species G. lucidum from China as G. lingzhi.Among the Chinese Ganoderma species, G. flexipes Pat, G. multipileum D. Hou, G. sichuanense J.D. Zhao and X.Q.Zhang, G. tropicum (Jungh.)Bres.and G. tsugae Murrill are the most similar species to G. lingzhi.However, the validity of the separation of G. lingzhi and G. sichuanense has been debated recently.Wang et al. (2012) proposed that 'G.lucidum' for Chinese species is incorrect and this should be corrected to Ganoderma sichuanense.Furthermore, Cao et al. (2012) proposed the name G. lingzhi for "Lingzhi" species which has an eastern Asian distribution based on strong morphology and molecular evidence.Yao et al. (2013) proposed G. lingzhi and G. sichuanense as synonyms based on morphological data from an epitype of G. sichuanense.However, Zhou et al. (2015) again challenged this opinion, with G. lingzhi and G. sichuanense being an independent and taxonomically valid species by stressing that species types depends on their ecological environments.Richter et al. (2015) stated that the new taxon G. lingzhi is taxonomically superfluous because the rules of fungal nomenclature require that the oldest valid name of any given taxon should be given preference.In 2016, Mark Stadler annotated this record in Mycobank (http://www.mycobank.org/).Now G. lingzhi is regarded as a later synonym of G. sichuanense in Species Fungorum (http://www.indexfungorum.org/names/names.asp)and Mycobank.Despite all this taxonomic work, the Chinese "Lingzhi" has continuously been referred to as G. lucidum in monographs of Ganodermataceae in China (Hapuarachchi et al. 2015).
The aim of this study is to report and illustrate the new findings of this medicinal species in Thailand and further, to improve the understanding of species delimitation in the genus Ganoderma.

Sample collection
Eight Ganoderma specimens growing up from soil were collected in a single site in Mae On District, Chiang Mai Province, northern Thailand (18°52.02'N,99°18.18'E)during the rainy season between June 2015 and September 2015.

Macroscopic and microscopic characterization
Macro-morphological characters were described based on fresh material, and on the photographs provided here.Colour codes (e.g.3A3) are from Kornerup and Wanscher (1978).Specimens were dried and placed separately in plastic bags.Material was deposited at Mae Fah Luang University herbarium (MFLU), Chiang Rai, Thailand.Living cultures were not obtained in this study.For micro-morphological examina-tion, basidiomes were examined under a stereo dissecting microscope (Motic SMZ 168 series) and sections were cut with a razor blade, mounted in 5% KOH, and then observed, measured, and illustrated under a compound microscope (Nikon ECLIPSE 80i) equipped with a camera (Canon 600D).Measurements were made using Tarosoft (R) Image Frame Work v. 0.9.7.At least 20 basidiospores were measured from each mature specimen except for very scanty materials.The basidiospore size was measured both with and without the myxosporium based on those with collapsed apex, but only spore sizes with myxosporium were used for comparisons.The cuticle sections were taken from the mature pileus portion and mounted in Melzer's reagent for observations.In the description of the basidiospores: n indicates the number of spores which were measured; L m is the mean spore length over a population of spores; W m the mean spore width over a population of spores; Q the length/width ratio (L/W) of a spore in side view; and Q m the average Q of all spores measured.The Facesoffungi number is provided as explained in Jayasiri et al. (2015).

DNA Extraction, PCR and sequencing
Dried samples of basidiome were used to extract genomic DNA.Genomic DNA was extracted using an EZgene Fungal gDNA Kit (Biomiga, CA, USA) according to the manufacturer instructions.DNA concentrations were estimated visually in agarose gel by comparing band intensity with a DNA ladder 1Kb (Invitrogen Biotech).The nuclear ribosomal internal transcribed spacer (ITS) was amplified using primers ITS5 and ITS4 (White et al. 1990).Reaction mixtures (20 µl) contained 1 µl template DNA (ca.10 ng), 10 µl distilled water, and 1 µl (10 µM) of each primer (ITS5/ITS4) and 7 µl 2× BenchTop Taq Master Mix (Biomigas).Amplification conditions were 35 cycles of 95 °C for 30 s, 59 °C for 30 s and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min.Amplified PCR products were verified by 1% agarose gel electrophoresis stained with ethidium bromide in 1x TBE.The PCR products were sequenced by Invitrogen Biotechnology (Beijing).

Sequence alignment and phylogenetic analysis
Other sequences used in the analyses (Table 1) were obtained from GenBank based on ITS BLAST searches in GenBank (Benson et al. 2017) and recently published data.Sequences that had possibly been contaminated by fungi or other unnamed species (such as those with aff. in the species name) were discarded, ambiguous regions were excluded and gaps were treated as missing data in the analysis (Nilsson et al. 2012).110 strains representing 40 species of Ganodermataceae from Asia, America and Europe were retrieved and those retrieved sequences and the newly generated sequences were aligned with MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html;Katoh and Standley 2013).The resulting alignment was improved manually when necessary using BioEdit v. 7.0.5.2 (Hall 1999).The Maximum Likelihood (ML) analyses were performed using RAxML-HPC2 (Stamatakis 2014) on the CIPRES Science Gateway V. 3.3 (Miller and Blair 2009), with default settings except that the number of bootstrap replicates was set to 1,000.A partitioned model analysis was performed with ITS1+ITS2 and 5.8S.For Bayesian analysis (BY), the GTR+I+G model of nucleotide evolution was selected with the help of MrModeltest 2.2 (Nylander 2004) as the best-fit model and posterior probabilities (PP) (Rannala and Yang 1996) were determined by Markov Chain Monte Carlo sampling (BMCMC) using MrBayes v3.1.2(Ronquist et al. 2012).BY analyses were conducted with six simultaneous Markov chains and trees were summarized every 100th generation.The analyses were stopped after 5,000,000 generations when the average standard deviation of split frequencies was below 0.01.The convergence of the runs was checked using TRACER v1.6 (Rambaut et al. 2013).
The first 25% of the resulting trees were discarded as burn-in, and PP were calculated from the remaining sampled trees.In both ML and BY analyses, Tomophagus colossus was selected as the outgroup.ML bootstrap values and BY posterior probabilities greater than or equal to 70% and 0.95, respectively, were considered as significant support.The phylogenetic tree was visualized with FigTree version 1.4.0 (Rambaut 2012) available at http://tree.bio.ed.ac.uk/software/figtree/.

Phylogeny
The tree topologies obtained from ML and BY were identical.Therefore, only the ML tree is shown (Fig. 1).Six major clades were identified in Ganoderma (Fig. 1).Our eight collections of Ganoderma sichuanenese from Thailand clustered with all G. sichuanenese sequences, including the epitype, in a well-supported clade (BS=98%; BPP=1.0)..37 (n = 20).L m = 9.09 µm, W m = 6.27µm,Cutis 4-12 mm thick, pale brown streaks the cutis, a closely-packed palisade, yellowish brown, clavate terminal elements, about 15-30 µm long.Stipe flattened or sub cylindrical to cylindrical, lateral to horizontally lateral or eccentric, (6-9) × (1.5 along stipe) cm, dark brown (8F5).Material examined.THAILAND, Chiang Mai Province, Mae On District, (18°52.02'N,99°18.18'E),eight specimens (MFU 16-2667, MFU 16-2668, MFU 16-2669, MFU 16-2670, MFU 16-2671, MFU 16-2672, MFU 16-2673, MFU 16-2709).However, one of the strains of G. sichuanense (Cui7691) of which we retrieved a sequence from GenBank clustered in the G. lucidum species complex.This strain was most likely wrongly identified.The specimens have been collected at some geographical distance (min.100 m between two collection points), which makes it unlikely that all come from the same mycelium.Nevertheless it is interesting to note that all new isolates cluster together and could not be segregated based on our phylogenetic analyses (Fig. 1).Given the phylogenetic results obtained herein where our new collections are found in a clade with G. sichuanense -including the type specimen -we believe that it would taxonomically more appropriate to establish them as new records of G. sichuanense.Furthermore, the deep nodes are not supported well in the tree, but this does not affect the final conclusions of the study.However, to obtain a better view of the evolution of the genus, a phylogeny with more genes, and in particular single-copy nuclear genes such as tef1 or rpb2 would be recommended.Ganoderma sichuanense was originally described from the Sichuan Province in 1983 and was diagnosed as having a distinctly radially rugose pileus, with a verrucose or tuberculose upper surface; pore surface yellowish when young, becoming brown or black when bruised; and small spores (Fig. 2) distinguished from other Ganoderma species (Zhao and Zhang 2000).The size range of basidiospores was described as (7.4-9.5 × 5-7) µm cum myxosp., in the original description (Zhao et al. 1983).Later, this range was updated to (7.8-10.4× 5.2-6.4)µm cum myxosp.(Zhao et al. 1989, Zhao andZhang 2000) and (9-11.5 × 6.5-8) µm cum myxosp.(Wang et al. 2012).In this study basidiospores were (8.2)8.3-9.8(10.2) × (5.6)5.7-6.8(7.3)µm cum myxosp., which lies within the range given by the original authors and is not distinct from those of basidiospores found in other specimens.Cao et al. (2012) stated that G. sichuanense differs from G. lingzhi in its sessile basidiocarps and smaller basidiospores (7.4-9.2 × 5-6.6) µm (Fig. 3).Furthermore, they revealed that the original description was a mixture of G. sichuanense and G. weberianum especially with the small spores and smooth  or slightly echinulate eusporium.Ganoderma curtisii, originally described from North America (Moncalvo and Ryvarden 1997) is a sister taxon to G. sichuanense in the phylogenetic estimate.Ganoderma flexipes, G. multipileum and G. tropicum are also closely related with G. sichuanense and are reported from China.

Conclusion
Macroscopic, microscopic, and molecular data all confirm that the collections from Thailand belong to G. sichuanense.This is the first discovery of the species in Thailand.The study of more collections of this species is needed to better estimate the variability of this taxon.

Figure 1 .
Figure 1.Phylogram generated by maximum likelihood analysis of 5.8S-ITS rDNA sequences.Bootstrap support values for maximum likelihood (in black) greater than 70% and Posterior Probabilities (PP) from Bayesian Inference (in blue) ≥0.95 are given above branches.The tree was rooted with Tomophagus colossus.The strain numbers are mentioned after the species names.Specimens of the newly recorded species are indicated in red and type specimens are indicated in black bold.(Treebase ID 20740)

Table 1 .
Sequences used in the phylogenetic analysis.
Basidiome annual to perennial, with distinctly contracted base to stipitate, corky, becoming hard corky to woody hard when dry.
Habitat.Rotten wood, in dry dipterocarp forest and in upper mixed deciduous forest and growing up from soil.Distribution.Tropical and temperate regions of China; Thailand (this study).Ganoderma strains used in this study were clustered in six major clades (G.applanatum, G. sichuanense, other laccate and non laccate Ganoderma, G. lucidum species complex, G. orbiforme and Ganoderma species).Sequences obtained from the eight Thai collections clustered in the well-supported G. sichuanense group.The Thai specimens are closely related to the originally described Chinese G. lingzhi taxa (Cui 9166, Cui 6982 and Dai 12426) and the epitype of G. sichuanense (CGMCC5.2175),forming a monophyletic group with G. sichuanense from China with 98% bootstrap support.