Two new species of Fulvifomes (Hymenochaetales, Basidiomycota) from America

Two new species of Fulvifomes are described from samples collected in America based on morphological characteristic and molecular evidence: F. centroamericanus Y.C. Dai, X.H. Ji & Vlasák, sp. nov. and F. krugiodendri Y.C. Dai, X.H. Ji & Vlasák, sp. nov. The former is characte rized by perennial and sessile basidiocarps, a concentrically sulcate and cracked pileal surface, a homogeneous context, small pores (7–9 per mm), a dimitic hyphal system, subglobose, yellowish and thick-walled basidiospores 4.3–5 × 4–4.5 μm. Macroscopically it resembles F. merrillii, which differs in having larger basidiospores (5–6 × 4–5 μm). F. centroamericanus is similar to F. robiniae in sharing applanate basidiocarps and subglobose, yellowish and thick-walled basidiospores 3.9–4.5 × 3.7–4.2 μm, whereas F. robiniae has larger basidiospores (5–6 × 4.5–5 μm). In nuclear large subunit rDNA (nLSU) and internal transcribed spacer (ITS) based phylogenies, the two new species formed two distinct lineages in the Fulvifomes clade.


Molecular study
A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain PCR products from dried specimens. Primer pair LR0R and LR7 (Vilgalys and Hester 1990) was used to amplify nLSU sequences, while ITS region was amplified using primers ITS5 and ITS4 (White et al. 1990). The PCR procedure for ITS was as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles at 94°C for 40 s, 54°C for 45 s and 72°C for 1 min, and a final extension of 72°C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94°C for 1 min, followed by 35 cycles at 94°C for 30 s, 50°C for 1 min and 72°C for 1.5 min, and a final extension of 72°C for 10 min. The PCR products were purified and sequenced in Beijing Genomics Institute, China, with the same primers.

Phylogenetic analysis
In this study, thirteen new sequences were generated (Table 1). Other sequences for phylogenetic analysis were downloaded from GenBank which were used in the previous study (Zhou 2015), the nLSU dataset with Stereum hirsutum (Willd.) Pers. and Bondarzewia montana (Quél.) Singer as the outgroup (Wagner and Fischer 2002) was used to confirm the generic position of the newly sequenced specimens. The ITS dataset was used to further clarify the interspecific relationships of Fulvifomes with Phellinus laevigatus (P. Karst.) Bourdot & Galzin, and P. populicola Niemelä as the outgroup (Wagner and Fischer 2002).
Sequences were aligned with BioEdit  and ClustalX (Thompson et al. 1997). Prior to phylogenetic analysis, ambiguous sequences at the start and the end were deleted and gaps were manually adjusted to optimize the alignment. Sequence alignment was deposited at TreeBase (submission ID 19989; www.treebase.org). Phy-logenetic analysis was carried out according to previous studies (Zhou 2015). Maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) were employed to perform phylogenetic analysis of the two aligned datasets. The three phylogenetic analysis algorithms generated nearly congruent topologies for each dataset, and, thus, only the topology from the MP analysis is presented along with statistical values from the ML, MP and BI algorithms (simultaneous BS not less than 50 % and BPP not less than 0.8) at the nodes.
In the phylogeny inferred from the ITS sequences (Fig. 2), four newly sequenced specimens (JV0904/1, JV0312/24.10-J, JV1008/21 as Fulvifomes krugiodendri and JV0611/III as Fulvifomes centroamericanus) clustered together with all sampled species of Fulvifomes and Inonotus porrectus in the Fulvifomes clade. The two new species formed lineages that had full statistical supports and were separated from other sampled species. Etymology. Centroamericanus (Lat.): referring to the distribution of species. Description. Basidiocarps perennial, sessile, broadly attached, solitary, without odor or taste, woody hard, light in weight when dry. Pilei dimidiate, applanate, projecting up to 15 cm, 20 cm wide and 8 cm thick at the base. Pileal surface dark grey, crusted, uncracked; margin cinnamon-buff, obtuse. Pore surface pale yellow, shining; sterile margin distinct, yellowish brown, up to 3 mm wide; pores circular, 8-10 per mm; dissepiments thick, entire. Context yellowish brown, woody hard, up to 5 cm thick. Tubes yellowish brown, woody hard, up to 3 cm thick, tube layers distinctly stratified with intermittent context layers, individual tube layer up to 2 mm thick. Figure 1. Phylogeny of Fulvifomes inferred from nLSU dataset. Topology is from MP tree and statistical values (MP/ML/BI) are indicated for each node that simultaneously received BS from ML and MP not below 50%, and BPP from BI not below 0.8.

Figure 2.
Phylogeny of Fulvifomes inferred from ITS dataset. Topology is from MP tree and statistical values (ML/MP/BI) are indicated for each node that simultaneously received BS from ML and MP not below 50%, and BPP from BI not below 0.8. Hyphal structure. Hyphal system dimitic; generative hyphae simple septate; skeletal hyphae dominant; tissue darkening but otherwise unchanged in KOH.

Discussion
Fulvifomes krugiodendri and F. centroamericanus fit well in Fulvifomes (emended by Zhou 2014) with perennial and pileate basidiocarps with a homogeneous, dimitic hyphal system, lack of hyphoid and hymenial setae, and subglobose, yellowish to brown and thick-walled basidiospores. Besides, they formed distinct lineages within the Fulvifomes clade in the phylogenies inferred from nLSU and ITS datasets (Figs 1 and 2).
Inonotus porrectus Murrill and I. luteoumbrinus (Romell) Ryvarden are nested within the Fulvifomes clade (Figs 1 and 2). Data from Sakayaroj et al. (2012) indicated that I. luteoumbrinus and I. porrectus fell into the Fulvifomes clade. However, both species have annual and soft fruiting bodies, a monomitic hyphal structure, and dark brown to black basidiospores, these characters do not correspond to Fulvifomes, and for the time being we still keep them in Inonotus P. Karst.
Lack of setae and relatively uniform basidiospores (shape, color and size) in most Fulvifomes species reduce the number of scorable traits substantially so that the mor-phological determination is problematic. Fulvifomes fastuosus (type from Singapore) and F. merrillii (type from Philippines) were described from tropical Asia and their similar kins from Central and South America were simply classified under these old names because of lack of discriminating characters. Nevertheless, our broadly based sequencing of about 50 recent collections from Central America (not shown in the Figs 1 and 2) revealed F. fastuosus or F. merrillii not existed in Central America but several more or less distinct clades of related but different species which will be published in the coming papers.

Kalbionora palaeotropica, a new genus and species from coastal forests in southeast Asia and Australia (Malmideaceae, Ascomycota) introduction
Coastal forests in the tropics, especially mangroves, are species-rich habitats and constitute an important part of tropical biodiversity (Donato et al. 2011;Friess 2016). These forests are comprised of unique plant, fungal, and animal species in the interface between marine, estuarine, and terrestrial ecosystems of the tropical and subtropical regions (Hyde et al. 1998;Rangsiruji et al. 2016;Sethy et al. 2012;Stevens 1979). Despite their importance for tropical biodiversity, mangroves are at great risk, with alarming rates of deforestation, especially in Southeast Asia Polidoro et al. 2010;Richards and Friess 2016).
Recent studies on the diversity of lichen-forming fungi in Thailand have dramatically increased our knowledge of these organisms in Southeast Asia, with numerous new records and new species discovered in a number of different habitats, including coastal forests (Aptroot et al. 2007;Kalb et al. 2012Kalb et al. , 2016aKalb et al. , 2016bKantvilas et al. 2010;Luangsuphabool et al. 2016aLuangsuphabool et al. , 2016bNaksuwankul et al. 2016;Neuwirth et al. 2014Neuwirth et al. , 2016Papong and Lumbsch 2011;Papong et al. 2014;Pitakpong et al. 2015;Rangsiruji et al. 2016;Sutjaritturakan and Kalb 2015;Buaruang et al. 2017).
During a recent survey of crustose lichens in mangrove habitats of eastern Thailand, the first author collected a species that appeared undescribed and while superficially resembling the common, pantropical Lecanora caesiorubella, showed similarities to the genera Eugeniella and Malmidea, currently placed in Malmideaceae and Pilocarpaceae, respectively (Jaklitsch et al. 2016;Lücking et al. 2016). This species was also collected by Klaus Kalb in Northeastern Australia, who kindly sent us the material. In addition, revision of material of a record of Dirina paradoxa from Vietnam (Joshi et al. 2014) turned out to represent this species as well. A new species and genus is described below based on molecular and phenotypical data.

Morphological and chemical analysis
Specimens were studied from the herbaria F, KoLRI, RAMK, and the private herbarium of Klaus Kalb (Neumarkt). Morphological characters were studied using a Leica Wild M 8 dissecting microscope. Observations and measurements of ascospores were made in water at 630× magnification with a Zeiss Axioscope microscope.

Molecular methods
Total genomic DNA was extracted from thallus fragments following the manufacturers' instructions using the ZR Fungal/Bacterial DNA Miniprep Kit (Zymo Research Corp., Irvine, CA). PCR reactions were performed and primers were used as described previously (James et al. 2006;Schmitt et al. 2010). PCR products were sequenced using an ABI PRISM™ 3730 DNA Analyzer (Applied Biosystems). New sequences were assembled and edited using Geneious v8.1.7 (http://www.geneious.com).
RPB2 and nuLSU sequences were aligned to each locus independently in the Miadlikowska et al. (2014) alignment (TreeBase no. 156552) using the '--add' option in the program MAFFT v7 . For the analysis focusing on Malmideaceae, nuLSU and mtSSU sequences were aligned using the 'E-INS-I' alignment algorithm in Mafft v7, with the remaining parameters set to default values. A group I intron in the nuLSU and present in a limited number of nuLSU sequences was not alignable and removed from the data matrix. Ambiguous positions of the mtSSU alignment were removed using Gblocks 0.91b (Castresana 2000). Phylogenetic analyses were performed using RAxML-HPC BlackBox 8.2.6 (Stamatakis 2006) and MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003) on the Cipres Science Gateway (http://www.phylo.org; Miller et al. 2010). The model for each locus used in the phylogenetic analysis was estimated using jModelTest v2.1.9 (Darriba et al. 2012;Guindon and Gascuel 2003). In the ML analysis, the GTR+G+I model was used as the substitution model with 1000 pseudoreplicates. The data was partitioned according to the different genes. Two parallel Markov chain Monte Carlo (MCMC) runs were performed each using 8,000,000 generations and sampling every 1,000 steps. A 50% majority rule consensus tree was generated from the combined sampled trees of both runs after discarding the first 25% as burn-in. The tree files were visualized with FigTree 1.4.2 (http:// tree.bio.ed.ac.uk/software/figtree/).  Diagnosis. Characterized by having asci of the Catillaria-type, yellowish brown, granulose epihymenium, exciple consisting of prosoplectenchymatous cells, dark brown hypothecium, hyaline, 1-3 transversely septate ascospores, and the presence of atranorin, zeorin, and the stictic and arthothelin chemosyndromes.

Results and discussion
Etymology. The specific epithet refers to the occurrence of the species in the Paleotropics, whereas the genus is named after our colleague Klaus Kalb who has made tremendous contributions to our knowledge of tropical lichens and who has been enormously helpful to colleagues in Thailand.
Distribution and ecology. The new species was found in coastal forests in eastern Thailand, Vietnam, and northeastern Australia (Queensland), growing on bark. It is known only from a few localities but is expected to be more common and potentially overlooked in mangrove forests of Southeast Asia and Australia.
Notes. Morphologically similar is the genus Malmidea -some species have similar ascoma morphology and the ascus in this genus also lacks amyloid structures in the thallus. However, this genus can be easily separated by having non-septate, halonate, thick-walled ascospores, and lacking depsidones. Further, molecular evidence suggests that the genera are only distantly related. Another morphologically similar genus is Eugeniella and both Eugeniella and the new genus also share similar ascospore septation. However, these taxa readily distinguished by the ascus-type (Byssolomatype in Eugeniella), the exciple (composed of moniliform hyphae in Eugeniella), and the epihymenium (usually indistinct in Eugeniella) (Breuss and Lücking 2015;Cáceres et al. 2013a). The new genus might be confused in the field with the superficially similar, common, pantropical Lecanora caesiorubella or has been confused with Dirina paradoxa, but is readily distinguished by numerous anatomical characters and a different chemistry.

Phylogenetic analysis
Sequences of RPB2 and nuLSU rDNA were generated (Genbank nos. KY926780-KY926790) from the type specimen of the new species and added to an alignment used by Miadlikowska et al. with over 1300 representatives in Lecanoromycetes (downloaded from https://treebase.org -study no. 156552; Miadlikowska et al. 2014). In a second analysis focusing on Malmideaceae, we aligned nuLSU and mtSSU sequences from three specimens of the new species with all Malmideaceae sequences used in Ertz et al. (2013). Based on the phylogenetic relationship of the new species to other taxa within Lecanoromycetes inferred in this study and published results from Ertz et al. (2013), we selected two species in the genus Frutidella and Miriquidica garovaglii as outgroups to assess relationships within Malmideaceae.
In our phylogenetic analysis assessing the relationship of Kalbionora palaeotropica within the Lecanoromycetes (Suppl. material 1), the type specimen did not cluster with Pilocarpaceae but in Malmideaceae as circumscribed by Ertz et al. (2013). Hence we performed a second analysis focusing on Malmideaceae. In the resulting tree (Fig. 2), the three specimens representing the new species clustered together in a strongly supported monophyletic group, supporting our re-identification of the Vietnamese material [recorded as Dirina paradoxa (Joshi et al. 2014)] as belonging to our new species. The new species, which is below described as Kalbionora palaeotropica, formed a strongly supported sister-group relationship to a clade including Malmidea, Savoronala, Lecidea plebeja, and L. cyrtidia.
In Malmideaceae, Lecidea plebeja and L. cyrtidia are temperate species occurring in North America and/or Europe and are poorly known. The morphology and distribution of the saxicolous L. cyrtidia has been discussed in the literature (Coppins and Muhr 1997;Hertel 1969), and it was suggested that it is closely related to the lignicolous L. plebeja, based on shared traits, such as an indistinct thallus, ascus-type, paraphyses with brown apical caps, ascospores of similar dimensions, and similar hypothecium and excipulum. Currently, these two species are poorly understood and additional sampling is necessary to evaluate the relationship of these two taxa. The genus Savoronala was recently described to accommodate a single species from coastal Erica heathland in Madagascar (Ertz et al. 2013), from which ascomata are unknown. This genus is morphologically characterized by having small, placodioid thalli, sporodochia at the apices of stipes, and brown conidia dispersed with an algal cell. It contains zeorin and usnic acid. The genus Malmidea was recently described (Kalb et al. 2011) to accommodate the bulk of corticolous and foliicolous, crustose tropical lichens previously included in the large, polyphyletic genus Lecidea, but differing in numerous characters, including the ascus-type (Hafellner 1984). Species in the genus were previously placed in the distantly related, now monotypic genus Malcolmiella and includes about 50 species with a thallus usually composed of goniocysts, usually paraplectenchymatous excipulum, prosoplectenchymatous hypothecium, and an ascus of the Catillaria-type, i.e. a tholus with no tubular structures to observe (Breuss and Lücking 2015;Cáceres et al. 2012Cáceres et al. , 2013bKalb et al. 2011Kalb et al. , 2012. Species in Malmidea often contain atranorin, sometimes in addition anthrachinones or biphenyls. Kalbionora palaeotropica differs morphologically by having a thallus not composed of goniocysts, transversely septate ascospores, and a different chemistry. Molecular data (Fig. 2) support that it is distinct from Malmidea and hence a new genus is described here to accommodate this new species.

Ganoderma sichuanense (Ganodermataceae, Polyporales) new to Thailand
Anan Thawthong 1,2,3 , Kalani K. Hapuarachchi 1,2,3 , Ting-Chi Wen 1 , Olivier Raspé 5,6 , Naritsada Thongklang 2 , Ji-Chuan Kang 1 , Kevin D. Hyde 2,4 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. 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; . The resulting alignment was improved manually when necessary using BioEdit v. 7.0.5.2 . 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).  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).

Discussion
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. 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.