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Species clarification of the prize medicinal Ganoderma mushroom “Lingzhi”

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“Lingzhi” is a mushroom that has been renowned in China for more than 2,000 years because of its claimed medicinal properties plus its symbolic fortune. “Lingzhi” has high economic value mostly as a dietary supplement in the modern market especially in East Asia, and its medicinal functions have become a hot study topic. For over a century, the highly prized medicinal fungus, known as “Lingzhi” in East Asia, has been assigned to Ganoderma lucidum, a species originally described from Europe. Molecular studies in recent years have revealed that the commercially cultivated ‘G. lucidum’ (“Lingzhi”) in East Asia is a different species from the true G. lucidum. The present study aims to clarify the species identity of “Lingzhi” based on morphological studies and analysis of rDNA nuc-ITS sequences, and additional gene fragments of mt-SSU, RPB1, RPB2, and TEF1-α of “Lingzhi” were provided. All Ganoderma species that mostly resemble “Lingzhi” in phylogeny and /or morphology were included for analysis. We propose a new species G. lingzhi for “Lingzhi”, which has an East Asia distribution. The most striking characteristics which differentiate G. lingzhi from G. lucidum are the presence of melanoid bands in the context, a yellow pore surface and thick dissepiments (80–120 μm) at maturity. G. curtisii is most closely related to G. lingzhi in phylogeny and is from North America. Ganoderma flexipes, G. multipileum, G. sichuanense, G. tropicum and ‘G. tsugae’, are also closely related with G. lingzhi and are reported from China. These species are compared and discussed. ‘Ganoderma tsuage’ reported from China is determined as conspecific with G. lucidum, hence the distribution of G. lucidum extends from Europe to northeastern China.
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Species clarification of the prize medicinal Ganoderma
mushroom Lingzhi
Yun Cao &Sheng-Hua Wu &Yu-Cheng Dai
Received: 11 May 2012 / Accepted: 22 May 2012 /Published online: 10 June 2012
#Mushroom Research Foundation 2012
Abstract Lingzhiis a mushroom that has been renowned
in China for more than 2,000 years because of its claimed
medicinal properties plus its symbolic fortune. Lingzhi
has high economic value mostly as a dietary supplement in
the modern market especially in East Asia, and its medicinal
functions have become a hot study topic. For over a century,
the highly prized medicinal fungus, known as Lingzhiin
East Asia, has been assigned to Ganoderma lucidum,a
species originally described from Europe. Molecular studies
in recent years have revealed that the commercially culti-
vated G. lucidum(Lingzhi) in East Asia is a different
species from the true G. lucidum. The present study aims to
clarify the species identity of Lingzhibased on morpho-
logical studies and analysis of rDNA nuc-ITS sequences,
and additional gene fragments of mt-SSU, RPB1, RPB2,
and TEF1-αof Lingzhiwere provided. All Ganoderma
species that mostly resemble Lingzhiin phylogeny and /or
morphology were included for analysis. We propose a new
species G. lingzhi for Lingzhi, which has an East Asia
distribution. The most striking characteristics which differ-
entiate G. lingzhi from G. lucidum are the presence of
melanoid bands in the context, a yellow pore surface and
thick dissepiments (80120 μm) at maturity. G. curtisii is
most closely related to G. lingzhi in phylogeny and is from
North America. Ganoderma flexipes,G. multipileum, G.
sichuanense,G. tropicum and G. tsugae,are also closely
related with G. lingzhi and are reported from China. These
species are compared and discussed. Ganoderma tsuage
reported from China is determined as conspecific with G.
lucidum, hence the distribution of G. lucidum extends from
Europe to northeastern China.
Keywords Ganodermataceae .Ganoderma lingzhi .
G. lucidum .Medicinal fungus .Phylogeny .Taxonomy
Introduction
The Chinese word Lingzhitranslates as the polypore
genus Ganoderma P. Ka r s t . in a broad sense, and in a
narrow sense it represents the highly prized medicinal
Ganoderma species distributed in East Asia. In this paper,
we treat Lingzhiin its narrow sense: the widely medici-
nally applied species. Lingzhi, also known as Chi-zhior
Rui-zhiin China, is one of the worlds most important
medicinal fungi. In the Orient, it is viewed as herb of
spiritual potencyor mushroom of immortality, and sym-
bolizes sanctity, success, goodness and longevity (Gao and
Zhou 2003; Wasser 2005; Lin 2009; De Silva et al. 2012).
The earliest report about Lingzhis medicinal value appeared
in the oldest Chinese medicinal monograph Shennongs
Y. Cao :Y.-C. Dai
State Key Laboratory of Forest and Soil Ecology,
Institute of Applied Ecology, Chinese Academy of Sciences,
Shenyang 110164, China
Y. Cao
Graduate University of the Chinese Academy of Sciences,
Beijing 100049, China
S.-H. Wu
Department of Botany, National Museum of Natural Science,
Taichung 404, Taiwan
S.-H. Wu (*)
Department of Plant Pathology, National Chunghsing University,
Taichung 402, Taiwan
e-mail: shwu@mail.nmns.edu.tw
Y.-C. Dai (*)
Institute of Microbiology, Beijing Forestry University,
Beijing 100083, China
e-mail: yuchengd@yahoo.com
Fungal Diversity (2012) 56:4962
DOI 10.1007/s13225-012-0178-5
Compendium of Material Medica about 100 B.C. The
knowledge of Lingzhiwas also noted and renewed in
the subsequent medicinal literatures including the famous
Chinese Compendium of Materia Medica complied by Shi-
Zhen Li in 1590 during the Ming Dynasty (Yu and Shen
2003).
Modern medicinal studies indicate that Lingzhiis ef-
fective in preventing or relieving various human diseases
(Lin 2007; Dai et al 2009). Its medicinal properties include
anti-aging, lowering blood pressure, improving immunity,
and preventing and treating various cancers, chronic bron-
chitis, diabetes mellitus, gastric ulcers, hepatitis, hyperlipid-
emia, hypertension, neurasthenia and thrombosis (Lin 2007;
Dai et al. 2009; Aly et al. 2011; De Silva et al. 2012). The
fungus is also used in various cosmetics (Hyde et al. 2010).
The medicinal effects of Lingzhicome from its metabo-
lites including polysaccharides, triterpenes, lucidenic acids,
LZ-8 protein, adenosine, ergosterol, glucosamine, cerebro-
sides et al. (Jong and Birmingham 1992; Gao and Zhou
2003; Lin 2009). Currently, Lingzhiis among the most
sort after medicinal mushrooms in the world market. Vari-
ous Lingzhiproducts made from the cultivated fruiting
bodies have been commercialized as dietary supplements
worldwide and especially in Asian countries; its estimated
annual global turnover is approximately US$2.16 billion
(Lai et al. 2004; Wachtel-Galor et al. 2004).
Ganoderma lucidum (Curtis) P. Karst., the generic type,
was originally reported from Peckham, London, UK
(Moncalvo and Ryvarden 1997). Unfortunately, the holo-
type of G. lucidum was lost and attempts to seek a neotype
from the type locality also failed (Steyaert 1972; Moncalvo
and Ryvarden 1997). At present, this taxon has been
reported to have a worldwide distribution based on gross
similarity of morphological features, e.g., Europe (Steyaert
1972; Ryvarden and Gilbertson 1993), Asia (Hongo and
Izawa 1994; Núñez and Ryvarden 2000; Zhao and Zhang
2000), America (Bazzalo and Wright 1982; Gilbertson and
Ryvarden 1986), Oceania (Quanten 1997), and Africa
(Ryvarden and Johansen 1980).
Patouillard (1907) first reported G.lucidum from China,
based on collections from Guizhou Province. Later, the
Chinese mycologist SC Teng (Teng 1934) reported more
collections of G. lucidum from different regions of China. In
recent decades, G.lucidum has been reported from China by
other Chinese mycologists, e.g. Tai (1979), Zhao and Zhang
(2000), Wu and Dai (2005) and Dai (2012).
A cluster of Lingzhispecimens were collected from
Laoshan, in Shandong Province, China in 1969 and deter-
mined as G. lucidumby the mycologists of Institute of
Microbiology, Chinese Academy of Sciences (Beijing).
With these specimens as mother strains, the first successful
cultivation of fruiting bodies of Lingzhiwas performed in
the same year (Yu and Shen 2003). Since then, the
cultivation of Lingzhihas been popularized in China
and its adjacent countries, and G. lucidum has always been
adopted as the scientific binomial for the commercially
cultivated Lingzhi.
Liu (1974) compiled a monograph of Chinese medicinal
fungi, and he assigned G.lucidum to Lingzhiin his book.
Since then, G. lucidum was accepted as the scientific bino-
mial of Lingzhiin many reports on the Chinese edible and
medicinal mushrooms (Ying et al. 1987; Mao 1998; Dai et
al. 2009). Presently, G.lucidum has been widely used for
naming the commercialized Lingzhiproducts in the world
market of mushroom industry (Lai et al. 2004). Meanwhile,
the oriental Lingzhimaterials used for biochemical, me-
dicinal and pharmaceutical studies were also labelled G.
lucidum (Kino et al. 1989;Minetal.2000; Bao et al.
2002; Hu et al. 2002; Sliva et al. 2002; Zhang et al. 2002).
Studies of Moncalvo et al. (1994,1995) indicated that the
collections of G. lucidumreported from different regions
of the world (Europe, America, South and East Asia) do not
represent a single species, based on analysis of sequence
data from the internal transcribed spacers of nuclear rDNA
(nuc-ITS) and the divergent domain D2 of the large ribo-
somal subunit gene of nuclear rDNA (D2 of nuc-LSU).
Smith and Sivasithamparam (2000) further detected that
the materials of G. lucidumfrom Australia cluster with
G. lucidumfrom tropical Asia and they differ from Euro-
pean G. lucidum through nuc-ITS analysis. Hong and Jung
(2004) reached a similar conclusion with Moncalvo et al.
(1994,1995) in their study of Ganoderma phylogeny based
on the nearly complete mitochondrial small-subunit ribo-
somal DNA sequences (mt-SSU).
Smith and Sivasithamparam (2003) proposed a new spe-
cies, G. steyaertanum B.J. Smith & K. Sivasithamparam, to
replace the mistakenly named G. lucidum in Australia and
Indonesia. Wang et al. (2009b) showed that the G. lucidum
distributed in tropical Asia is G. multipileum Ding Hou,
which is neither conspecific with the true G. lucidum dis-
tributed in Europe, nor conspecific with the real Lingzhi
distributed in East Asia.
Their phylogenetic analyses (Moncalvo et al. 1994,1995;
Smith and Sivasithamparam 2000; Hong and Jung 2004;
Wang et al. 2009b) also clearly indicated that the G. luci-
dumfrom East Asia represents a different species from the
true G. lucidum. From the morphological perspective,
Pegler and Yao (1996) noticed that Lingzhibears a more
slender basidiocarp as compared to G. lucidum from
Europe. In recent years, Szedlay (2002), Wasser et al.
(2006) and Wasser (2011) also considered that G. lucidum
was mistakenly applied to this famous medicinal fungus
Lingzhi. Obviously, the clear recognition of the scientific
binomial to represent the Lingzhispecies remains un-
known before our study. To determine the correct identity
of the Lingzhispecies, we performed a detailed
50 Fungal Diversity (2012) 56:4962
morphological and phylogenetic study of nine Ganoderma
species that mostly resemble Lingzhi.
Materials and methods
To search the most similar species with Lingzhi, we have
referred to numerous literatures published during 1902
2011 with special emphasis on those with recent descrip-
tions from the type or authentic specimens (Steyaert 1972,
1980; Ryvarden and Johansen 1980; Ryvarden 1981,1983,
1985,2004; Gilbertson and Ryvarden 1986; Ryvarden and
Gilbertson 1993; Hattori and Ryvarden 1994; Moncalvo and
Ryvarden 1997; Gottlieb and Wright 1999; Núñez and
Ryvarden 2000; Smith and Sivasithamparam 2003; Wang
and Wu 2008; Welti and Courtecuisse 2010). In particular,
we also studied the type specimens of Ganoderma species
described from China that share similar morphological fea-
tures with Lingzhi. We also consulted the studies of
Moncalvo et al. (1994) and Wang et al. (2009b) to select
the species for the phylogenetic analysis. Molecular data can
be used to infer relationships amongst groups of morpho-
logically similar basidiomycetes (Ge et al. 2010; Zhao et al.
2010; Yang 2011; He and Dai 2012; Zhao et al. 2012).
Samples for the analysis included wild collections, com-
mercially cultivated fruiting bodies or strains, and sequences
derived from GenBank (Table 1). The commercially culti-
vated materials G.lucidumwere obtained from the major
cultivation bases in ten provinces of China. Most wild speci-
mens were collected by the authors from 15 provinces of
China over the last decade. All specimens used in this study
are mainly deposited at the herbaria of Institute of Applied
Ecology, Chinese Academy of Sciences (IFP), Shenyang
and Beijing Forestry University (BJFC), Beijing. Some
were from the Mycological Herbarium, Institute of Micro-
biology, Chinese Academy of Sciences (HMAS), Beijing,
Herbarium of the National Museum of Natural Science
(TNM), Taichung, and Botanical Museum, University of
Helsinki (H), Helsinki.
The microscopic procedure used in this study follows Dai
(2010) with some minor amendments. Sections were exam-
ined at magnification up to×1000 under a Nikon Eclipse E
80i microscope and phase contrast illumination. Drawings
were made with the aid of a drawing tube. As the turgid
vesicular appendix at distal end of basidiospores in Gano-
derma are collapsed at maturity, the size of spores was
measured with the turgid vesicular appendix excluded and
included respectively (Niemelä and Miettinen 2008). In
presenting the variation in the size of spores, 5 % of the
measurements were excluded from each end of the range,
and are given in parentheses. The following abbreviations
used in the text include: IKI 0Melzers reagent, IKI0neg-
ative in Melzers reagent, KOH05 % potassium hydroxide,
CB0Cotton Blue, CB+0cyanophilous, L 0mean spore
length (arithmetic average of all spores), W 0mean spore
width (arithmetic average of all spores), Q 0variation in the
L/W ratios between the specimens studied, n 0number of
spores measured from given number of specimens. Special
colour terms are from Anonymous (1969)andPetersen
(1996).
The nuc-ITS sequences were amplified for Lingzhiand
its related species for phylogenetic analysis. The mt-SSU
sequences were also obtained for the Chinese G. tsuageto
study its relationship with the true G. lucidum. In addition,
the gene fragments of mt-SSU, RPB1, RPB2, and TEF1-α
were amplified for Lingzhi. Total genomic DNA was
extracted from dried specimens or living cultures using the
Phire® Plant Direct PCR Kit (Finnzymes Oy, Finland)
according to the manufacturers instructions with slight
modifications. The primer pairs, ITS5/ITS4 (White et al.
1990) or ITS1F/ITS4B (Gardes and Bruns 1993) were used
to amplify the nuc-ITS region. For some specimens depos-
ited in herbarium for more than 10 years, three new Gano-
derma-specific primers, G-ITS-F1 (ACC CTG TCG CTG
AGA ACT TGA), G-ITS-R1 (AGC ACT GGT AGT CCG
TGT CA) and G-ITS-R2 (TTG AGA GCG CAT CAC AAA
GC), were designed to select for Ganoderma specimens
rather than possible internal inhabitats. Combined with the
published primers for nuc-ITS amplification, the primer
pairs G-ITS-F1/G-ITS-R2 and G-ITS-F1/ITS4B were se-
lected for successful amplification. The primers for ampli-
fying the mt-SSU gene fragment were BMS05/BMS173
(Hong and Jung 2004). The RPB1 gene fragment was am-
plified using the primer pairs RPB1-2.2f/RPB1-Cr
(Matheny et al. 2002; Binder et al. 2010). The RPB2 gene
fragment was amplified with the primer pair fRPB2-5F/
bRPB2-7R2 (Liu et al. 1999; Matheny et al. 2007). The
TEF1-αgene fragment was amplified using the primer pair
EF1-983F/EF1-2218R (Rehner and Buckley 2005). The
obtained genomic DNA was diluted 110 times if necessary.
PCR reactions were performed in 50 μL reaction mixtures
containing 25 μL of 2×Phire® Plant PCR buffer, 1μL
Phire® Hot Start II DNA Polymerase, 5 μL of each PCR
primer (10 μM), 25μl dilution protocol and total volume
was adjusted to 50 μL with sterile deionized water. The
following PCR procedure for Phire® Plant Direct PCR Kit
was: initial denaturation for 5 min at 98 °C, followed by 39
cycles at 98 °C for 5 s, annealing temperature for 5 s and
72 °C for 20 s, and a final extension of 72 °C for 2 min. PCR
amplification was performed at the following annealing
temperatures: 59 °C (ITS5/ITS4), 60 °C (ITS1F/ITS4B),
69.5 °C (G-ITS-F1/ITS4B), 66.5 °C (G-ITS-F1/ G-ITS-
R2), 55 °C(BMS05/BMS173), 50 °C (RPB1-2.2f/RPB1-
Cr), 55 °C (fRPB2-5F/bRPB2-7R2) and 59 °C (EF1-
983 F/EF1-2218R). The successful PCR products were sent
to Beijing Genomics Institute, China for purification and
Fungal Diversity (2012) 56:4962 51
Table 1 Species used in nuc-ITS rDNA analysis, along with their specimen/strain numbers, locality and GenBank accession numbers
Accepted names Names from
specimens/GenBank
Specimen/
strain
numbers
Locality GenBank No. Reference
Amauroderma rudevar.
intermedium
JMM ASP.1 Chinese Taiwan X78753&X78774 Moncalvo et al. 1994
Ganoderma curtisii G. curtisii CBS 100131 North Carolina, USA JQ781848 This study
G. curtisii G. curtisii CBS 100132 North Carolina, USA JQ781849 This study
Ganoderma flexipes G. flexipes Wei 5491 Hainan, China JQ781850 This study
G. flexipes G. flexipes Wei 5494 Hainan, China JN383979 Cao and Yuan 2012
Ganoderma lucidum G. lucidum Dai 2272 Sweden JQ781851 This study
G. lucidum G. lucidum RYV 33217 Norway Z37096&Z37073 Moncalvo et al. 1994
G. lucidum G. lucidum CBS 270.81 France Z37049&Z37099 Moncalvo et al. 1994
G. lucidum G. lucidum CBS 176.30 UK AF094511&AF044490 Park et al. (unpublished)
G. lucidum G. lucidum Dai 11593 Finland JQ781852 This study
G. lucidum Ganoderma tsugae Dai 3937 Jilin, China JQ781853 This study
G. lucidum G. tsugae Yuan 5649 Jilin, China JQ781854 This study
G. lucidum G. tsugae Zhang 0981 northern China X78748&X78769 Moncalvo et al. 1994
Ganoderma lingzhi G. tsugae RSH J2* Japan X78746&X78767 Moncalvo et al. 1994
G. lingzhi G. tsugae RSH 1109 Japan X78747&X78768 Moncalvo et al. 1994
G. lingzhi G. tsugae RSH BLC* Chinese Taiwan Z37097&Z37078 Moncalvo et al. 1994
G. lingzhi G. lucidum HMAS 60537 China Z37050&Z37074 Moncalvo et al. 1994
G. lingzhi G. lucidum ACCC 5.65 China X87354&X87364 Hseu et al. 1996
G. lingzhi G. lucidum WD-565 Japan EU021455 Wang et al. 2009b
G. lingzhi G. lucidum WD-2038 Japan EU021456 Wang et al. 2009b
G. lingzhi G. lucidum Dai 12573* Liaoning, China JQ781855 This study
G. lingzhi G. lucidum Cui 4018 Jiangsu, China JQ781856 This study
G. lingzhi G. lucidum Cui 10165 Zhejiang, China JQ781857 This study
G. lingzhi G. lucidum Wu 1006-38
(holotype)
Hubei, China JQ781858 This study
G. lingzhi G. lucidum Cui 9164 Shandong, China JQ781859 This study
G. lingzhi G. lucidum Dai 10631 Anhui, China JQ781860 This study
G. lingzhi G. lucidum Dai 12438* Henan, China JQ781861 This study
G. lingzhi G. lucidum Cui 6982 Tianjin, China JQ781862 This study
G. lingzhi G. lucidum Li 245 Henan, China JQ781863 This study
G. lingzhi G. lucidum Dai 12479* Anhui, China JQ781864 This study
G. lingzhi G. lucidum IFP 01021* Hubei, China JQ781865 This study
G. lingzhi G. lucidum Dai 12443* Guangdong, China JQ781866 This study
G. lingzhi G. lucidum Dai 12374 Yunan, China JQ781867 This study
G. lingzhi G. lucidum Dai 3583 Hunan, China JQ781868 This study
G. lingzhi G. lucidum Dai 12441* Zhejiang, China JQ781869 This study
G. lingzhi G. lucidum Dai 12426* Sichuan, China JQ781870 This study
G. lingzhi G. lucidum Dai 12425* Shandong, China JQ781871 This study
G. lingzhi G. lucidum Dai 12447* Fujian, China JQ781872 This study
G. lingzhi G. lucidum Dai 12449* Jiangsu, China JQ781873 This study
Ganoderma multipileum G. lucidum JMM P93-1 Philippines X78745&X78766 Moncalvo et al. 1994
G. multipileum G. lucidum BCRC 36123 India EU021459 Wang et al. 2009b
G. multipileum G. lucidum BCRC 37043 Chinese Taiwan EU021460 Wang et al. 2009b
G. multipileum G. lucidum Dai 9521 Hainan, China JQ781874 This study
Ganoderma oregonense G. oregonense CBS 265.88 Oregon, USA JQ781875 This study
G. oregonense G. oregonense CBS 266.88 Washington, USA JQ781876 This study
G. resinaceum G. resinaceum CBS 194.76 Netherlands X78737&X78758 Moncalvo et al. 1994
52 Fungal Diversity (2012) 56:4962
sequencing with the same primers used in PCR procedure
and/or the additional newly designed primers in this study.
G-Intron F1 (GGA ACC TAA GGA AGA CTA TTA C), G-
Intron R1 (TCA GGG ATG TTA GTT TCT ACA) and G-
Intron R2 (GGG TAT CTA ACC GTG GAA TCA GA) were
newly designed for sequencing the intron of mt-SSU region.
Two additional primers G-RPB2-F1 (CAT CGA GTT CTT
GGA GGA GTG G) and G-RPB2-R1 (CGG AAT GAT
GCT GGC ACA GAC A) were for RPB2. G-TEF1-F1
(GGT GAG TTC GAG GCT GGT ATC T) and G-TEF1-
R1 (CGG GTA CTC GTT GTA AGA CTC) were for
TEF1-α.
The newly-generated sequences were assembled and
modified manually according to the chromatograms in Con-
tigExpress (Vector NTI Suite 6.0, InforMax Inc.) and then
submitted to GenBank. The phylogenetic analysis included
nuc-ITS sequences from our lab work and GenBank
(Table 1). Amauroderma rude var. intermedium J.S. Furtado
and Tomophagus colossus (Fr.) Murrill were selected as
outgroup (Moncalvo et al. 1994; Wang et al. 2009b). All
sequences were aligned with Clustal X 2.0 (Larkin et al.
2007) using default settings and further optimized manually
using BioEdit 7.0.5.3 (Hall 1999) to allow maximum align-
ment and minimize gaps. The alignment was deposited at
TreeBASE (Accession No: 12548).
Phylogenetic analysis was performed using maximum
parsimony (MP), maximum likelihood (ML) and Bayesian
algorithm (BA). The parameters were set as follows: 1) MP:
the analysis was performed in PAUP* 4.0b10 (Swofford
2002). Heuristic search with TBR branch swapping was
implemented with 1000 replicates of random-addition se-
quence. All characters were equally weighted and gaps were
treated as missing data. MAXTREES was set to auto-
increase. Bootstrap analysis was carried out with 1,000
replicates using the heuristic search (Felsenstein 1985). Tree
length (TL), consistency index (CI), retention index (RI),
rescaled consistency index (RC), and homoplasy index (HI)
were calculated for all parsimony trees. 2) ML: The analysis
was conducted in PhyML3.0 (Guindon and Gascuel 2003).
The best-fit model was selected by jModelTest (Posada
2008) according to Corrected Akaike Information Criterion
(AICc). Bootstrap analysis was performed with 100 repli-
cates. 3) BA: The analysis was run in MrBayes3.1.2
(Ronquist and Huelsenbeck 2003). The best-fit model of
nucleotide evolution was selected by Hierarchical Likeli-
hood Ratio Tests (hLRT) in MrModeltest 2.3 (Nylander
2004). Markov Chain Monte Carlo (MCMC) algorithm
(Larget and Simon 1999) was conducted to calculate Bayes-
ian posterior probabilities. Four Markov chains were run for
3,000,000 generations with the trees sampled every 100
th
generation. The average standard deviation of split frequen-
cies was restricted to be below 0.01. The option of burnin
was set to discard 10 % trees (Hall 2004).
Results
Phylogeny
The nuc-ITS dataset contains 53 sequences representing 12
taxa. Amplification of the nuc-ITS region yielded approxi-
mately 650 bp fragments. After deleting the ambiguous sites
at both ends and 5.8S region in the alignment, 409 charac-
ters remained for phylogenetic analysis.
For MP analysis, 286 characters are constant, 33 charac-
ters are variable, but parsimony-uninformative, and 90
characters are parsimony-informative. MP analysis yielded
2992 most parsimonious trees (CI00.769608, RI00.928463,
RC 00.714552, HI 00.230392), and one of them is shown in
Fig. 3. For ML analysis: the best-fit model was HKY +G
selected by AICc in jModelTest. For BA analysis, the best-
fit model was HKY+G selected in MrModeltest 2.3. Four
simultaneous Monte Carlo chains were run for 3,000,000
generations, and the average standard deviation of split
Table 1 (continued)
Accepted names Names from
specimens/GenBank
Specimen/
strain
numbers
Locality GenBank No. Reference
G. resinaceum G. resinaceum CBS 152.27 UK Z37062&Z37085 Moncalvo et al. 1994
Ganoderma
sichuanense
G. sichuanense HMAS 42798
(holotype)
Sichuan, China, JQ781877 This study
G. sichuanenseG. sichuanense Cui 7691 Guangdong, China JQ781878 This study
Ganoderma tropicum G. tropicum Dai 9724 Hainan, China JQ781879 This study
G. tropicum G. tropicum Yuan 3490 Yunnan, China JQ781880 This study
G. tropicum G. tropicum BCRC 37122 Chinese Taiwan EU021457 Wang et al. 2009b
Tomophagus colossus CBS 216.36 Unknown Z37071&Z37091 Moncalvo et al. 1994
* indicates the specimens or strains were artificially cultivated.
Fungal Diversity (2012) 56:4962 53
frequencies was 0.006618. As the trees generated by ML
and BA analyses shared nearly identical topology with that
of MP analysis, MP bootstrap, ML bootstrap and Bayesian
Posterior Probabilities were respectively labelled near the
nodes in the same tree (Fig. 1).
In the phylogenetic tree, the samples named G. lucidum
separated into three distinct clades. They were respectively
listed as Clade I, Clade II and Clade III in Fig. 1.
Clade I includes 22 wild and cultivated samples named
G. lucidum, as well as three samples named G. tsugae, all
from East Asia. These taxa form a monophyletic lineage
with strong support (MP/ML/BA 095/97/1.00), and are dis-
tantly related to G. lucidum from Europe in Clade III.
Samples of G. curtisii (Berk.) Murrill from North America
form a well-supported clade (MP/ML/BA084/92/0.95) and
serves as the sister clade to Clade I.
Clade II comprises four samples named G. lucidum
from tropical Asia, and represents a separate lineage of G.
lucidumfrom Asia and has high bootstrap support value
and posterior probability value (MP/ML/BA 097/94/1.00).
All four samples in this clade represent G. multipileum
(Wang et al. 2009b).
Clade III contains five samples of G. lucidum from
Europe as well as three samples labelled G. tsugaefrom
northern China. This clade has quite low support (ML/
Bayes071/0.73), but forms a robust group with high support
(MP/ML/BA0100/99/1.00) with its sister clade containg G.
oregonense Murrill from America.
In addition to G. multipileum and G. tsugaementioned
above, another three similar species, G. flexipes, G. sichua-
nense and G. tropicum, form the well-supported clades (G.
flexipes, MP/ML/BA 0100/100/1.00; G. sichuanense MP/
ML/BA0100/100/1.00; G. tropicum MP/ML/BA0100/98/
1.00; Fig. 1). They are not closely related to G. lucidum
from East Asia in phylogeny.
Taxonomy
Ganoderma lingzhi Sheng H. Wu, Y. Cao & Y.C. Dai, sp.
nov. (Figs. 2,3)
MycoBank no.: MB 564240
CHINA. Hubei Province, Wuhan, Jiufeng National For-
est Park, alt. 90 m, on the ground in forest of Quercus,29
June 2010, Wu 100638 (holotype in TNM, isotype in
BJFC, IFP).
Etymology lingzhi (Lat.): referring to the Chinese name
Lingzhi, a highly prized Ganoderma fungus in Chinese
folklore.
Fruitbody Basidiocarps annual, stipitate, corky and without
odor, but bitter when fresh, becoming hard corky to woody
hard when dry. Pilei variable, from semicircle, shell-like,
reniform to circular, occasionally lobed, corky, projecting up
to 16 cm, 17 cm broad and 2.7 cm thick. Pileal surface
weakly laccate when juvenile, strongly laccate with age,
cinnamon-buff to clay-buff when juvenile, orange-brown
to reddish brown with age, often with more or less concen-
tric zones especially near the margin and fine furrows in the
middle; margin acute to obtuse, sometimes with slight
waves, color variable, orange-brown, yellowish brown,
cinnamon-buff to buff when juvenile, becoming orange-
brown to reddish brown with age. Stipe flattened or sub-
cylindrical, lateral, dorso-lateral, horizontally lateral or ec-
centric, orange-yellow to yellowish brown when juvenile,
becoming reddish brown to vinaceous brown with age, up to
22 cm long and 3.5 cm thick. Pore surface white when
juvenile, turning sulphur yellow at maturity, turning brown
to dark brown when bruised, usually straw-colored when
dry; pores circular or angular, mostly entire, (4)56(7) per
mm, (40)80140(160) μm in diam; dissepiments (60)
80120(140) μm. Context not completely homogeneous in
color, the upper part buff, the lower part clay-buff, corky,
without concentric growth zones, up to 0.5 cm thick at base,
often with 12 black melanoid bands in the mature basidio-
carps. Tubes clay-buff, woody hard, not stratified, up to
2.2 cm long.
Hyphal structure Hyphal system trimitic; generative hyphae
bearing clamp connections, hyaline, thin-walled, infrequent;
skeletal hyphae dominant, thick-walled to subsolid, fre-
quently dichotomous branched; binding hyphae thick-
walled with a narrow lumen to subsolid; all the hyphae
IKI, CB+; tissues darkening in KOH.
Context Generative hyphae not observed; skeletal hyphae
dominant, pale brown to brown, thick-walled to subsolid,
frequently branched, interwoven, 34.5 μm in diam; bind-
ing hyphae abundant, brownish, thick-walled with a narrow
lumen to subsolid, flexuous, interwoven, 12.1 μm in diam.
Tubes Generative hyphae hyaline, thin-walled, moderately
branched and clamped, 1.32.8 μm in diam; skeletal hyphae
dominant, pale brown to distinctly brown, thick-walled with a
medium or narrow lumen to subsolid, frequently branched,
strongly interwoven, 24μm in diam; binding hyphae brown-
ish, thick-walled to almost solid, frequently branched, flexuous,
interwoven, 11.8 μm in diam; basidia barrel-shaped with 4
sterigmata and a basal clamp connection, 1418× 610 μm;
basidioles in shape similar to basidia, but slightly smaller.
Cutis Composed of a vertical and closely-packed palisade
of cells; cells clavate, brown, thick-walled to subsolid, oc-
casionally with blunt outgrowths or protuberances in the
apical or lateral parts, moderately to strongly amyloid at
maturity, 3050× 510 μm.
54 Fungal Diversity (2012) 56:4962
Spores Basidiospores ellipsoid, truncate at maturity, yellow-
ish brown, IKI, CB+, double-walled, exospore smooth,
endospore with moderate to coarse echinulae, sometimes
even to short ridges up to 1.2 μm long, (8)910.7(12)×
(5.2)5.87(7.5) μm, L09.74 μm, W06.38 μm, Q01.45
1.59 (n0300/10; the exospore included and the turgid ve-
sicular appendix excluded); (9.2)9.511.2(13)× (5.2)
5.67(7.8) μm, L010.27 μm, W 06.28 μm, Q 01.54
1.74 (n090/6; the exospore included and the turgid vesicu-
lar appendix included); (6.4)78.2(9) × (4.5)4.95.7
(6.2) μm, L07.58 μm, W05.19 μm, Q 01.381.56
(n0300/10; the exospore excluded).
Additional specimens (paratypes in IFP or mentioned by
herbarium code) studied. China. Anhui Province, Hefei,
on dead tree of Quercus, 25 September 2008, Dai 10631.
Hubei Province, Wuhan, Maanshan National Forest Park, on
the ground in forest of Quercus, 30 June 2010, Wu 1006-75
(TNM), Wu 1006-76 (TNM). Hunan Province, Changsha,
Yuelushan, on root of Castanopsis, 14. July.2011 Dai
12458; on rotten angiosperm stump, 5 July 2002, Dai
3583; Yizhang County, Mangshan Nature Reserve, on
stump of Castanea, 26 June 2007, Dai 8172. Jiangsu Prov-
ince, Nanjing, Zijinshan, on angiosperm stump, 22 August
2006, Cui 4018. Jiangxi Province, Jiujiang County, South
Lake Park, on angiosperm stump 10 October 2008, Cui
6109; Fenyi County, Dagangshan Nature Reserve, on an-
giosperm wood, 18 September 2008, Dai 10457; on living
tree of Quercus, 22 September 2009, Cui 7848 & 7849.
Shandong Province, Taian, Taishan Nature Reserve, on fall-
en trunk of Quercus, 2 August 2010, Cui 9166; on rotten
wood of Quercus, 2 August 2010, Cui 9164; Yantai,
Kunyushan Nature Reserve, on stump of Quercus, 23 Au-
gust 2010, Dai 11701. Sichuan Province, Dujiangyan Coun-
ty, Qingchenshan, on angiosperm stump, 13 September
2010, Cui 9223; Mianyang, Longshan, on stump of Cyclo-
balanopsis, 10 October 2009, Dai 11958. Tianjin, Ji County,
Panshan, on root of Quercus, 1 August 2009, Cui 6982.
Yunnan Province, Tengchong County, Gaoligongshan Na-
ture Reserve, on angiosperm stump, 24 October 2009, Cui
8032; Puer County, Laiyanghe Nature Reserve, on root of
Castanea,9June2011,Dai 12374. Zhejiang Province,
Fig. 1 One of the most parsimonious trees illustrating the phylogeny
of Ganoderma lingzhi and related species within the genus based on
nuc-ITS sequences. Clade stabilities were calculated from MP (50 %),
ML (50 %) and BA (0.70). Branches that got strong support from all
three analyses (MP, ML and BA) are in bold
Fungal Diversity (2012) 56:4962 55
Yongjia County, Longwan Lake Forest Park, on angiosperm
stump, 21 August 2011, Cui 10165.
Molecular data In addition to nuc-ITS sequences (Table 1)
for phylogenetic analysis, another four gene fragments of
mt-SSU, RPB1, RPB2, and TEF1-αare supplied in Gen-
Bank for further comparison with the other related species
(mt-SSU:Cui9166, Shandong, JX029987; Dai12479, culti-
vated, Anhui, JX029988; Wu 1006-38, Hubei, JX029989;
Dai12574 cultivated, Liaoning, JX029990. RPB1: Cui9166,
Shandong, JX029982; Dai12479, cultivated, Anhui,
JX029983; Wu 1006-38, Hubei, JX029984; Dai12574, cul-
tivated, Liaoning, JX029985. RPB2: Cui9166, Shandong,
JX029978; Dai12479, cultivated, Anhui, JX029979; Wu
1006-38, Hubei, JX029980; Dai12574, cultivated, Liaon-
ing, JX029981. TEF1-α: Cui9166, Shandong, JX029974;
Dai12479, cultivated, Anhui, JX029975; Wu 1006-38,
Hubei, JX029976; Dai12574, cultivated, Liaoning,
JX029977). Although the above mentioned gene sequences
are available for G. lingzhi, it is impossible to carry out
multigene analysis for phylogeny, because these genes are
not available for other species of the genus.
56 Fungal Diversity (2012) 56:4962
Fig. 2 Basidiocarps of Ganoderma lingzhi.af. Cultivated fruting bodies of Ganoderma lingzhi go. Wild fruting bodies of Ganoderma lingzhi
(gh. holotype; io. paratypes)
Discussion
Analysis of nuc-ITS sequences shows that the strains of
wild and cultivated G. lingzhi from East Asia (Fig. 1)
are nested in a strongly-supported Clade I (MP/ML/
BA098/98/1.00). Clade I is clearly separated from
Clade III in phylogeny, where the European G. lucidum
is nested (Fig. 1). Therefore, the newly introduced spe-
cies, G.lingzhi, which is widely distributed and culti-
vated in East Asia, is not conspecific with G. lucidum
from Europe.
Ganoderma lingzhi was long assigned to G. lucidum as
both species have a reddish brown pileal surface, similar-
sized basidiospores and mostly regular clavate cuticle cells.
However, several morphological features separate G. lingzhi
from the European G. lucidum. In mature basidiocarps, G.
lingzhi bears 12 black melanoid bands in the context while
G. lucidum lacks this structure. G. lingzhi normally has a
sulphur-yellow to straw-colored pore surface and thick dis-
sepiments (80120 μm) at maturity. In contrast, G. lucidum
has a white pore surface and thin dissepiments (4080 μm).
The cuticle cells of G.lucidum are also usually longer (47
70 μm) than that of G. lingzhi.
In addition to the identification of our materials of G.
lingzhi and G. lucidum, we also conducted morphological
studies for the species similar to G. lucidum.Zhaoand
Zhang (2000)andWuandDai(2005)alsohavemen-
tioned that G. lucidum(G. lingzhi) from China has
yellow pore surface, while the European G. lucidum al-
ways has a white pore surface (Ryvarden and Gilbertson
1993; Ryvarden 1994). Kim et al. (2001) also found that
the specimens labelled G. lucidum from Korea have pale
brownish thread-like tissues in the centre of context (i.e.,
melanoid bands). These descriptions of G. lucidumfrom
East Asia are consistent with our observation of G.
lingzhi.
After studying type specimens of Ganoderma species
from China that share similar morphological features with
G.lingzhi, we found that they can be divided into 3 catego-
ries: 1) Ganoderma species with concentric growth zones in
Fig. 3 Microscopic structures
of Ganoderma lingzhi a.
basidiospores; b. basidia and
basidioles; c. apical cells from
the cuticle; d. hyphae from
trama. e. hyphae from context
(scale bars: 10 μm)
Fungal Diversity (2012) 56:4962 57
the context: G.chenghaiense J.D. Zhao and G.simaoense
J.D. Zhao. G.chenghaiense has been resolved as G. multi-
pileum by Wang et al. (2009a). Based on our study, G.
simaoense is a tropically-distributed species due to its con-
centric growth zones in the context, which can separate this
species from G. lingzhi clearly. 2) Ganoderma species with
small pilei and slender stipes: G.atrum J.D. Zhao et al., G.
calidophilum J.D. Zhao et al., G.hainanense J.D. Zhao et
al. and G.parviungulatum J.D. Zhao & X.Q. Zhang. They
were all reported from Hainan Province of China, and they
are conspecific with G. flexipes Pat. (Data to be published in
another paper). 3) G. sichuanense is an accepted species and
its morphological differences from G. lingzhi are discussed
below.
Among the Chinese Ganoderma species, G.flexipes
Pat., G. multipileum Ding Hou, G. sichuanense J.D.
Zhao & X.Q. Zhang, G. tropicum (Jungh.) Bres. and
G. tsugaeMurrill are the mostly similar species to G.
lingzhi because they share a reddish brown pileal sur-
face, similar basidiospores and cuticle cells (Fig. 4).
However, the morphological characteristics can distin-
guish G. lingzhi from these five species through careful
observation. G. flexipes is separated from G. lingzhi by
its mostly small pileus, slender and long stipe, and
yellow brown to dark brown context when mature. G.
multipileum is distinguished from G. lingzhi by its dis-
tinct concentric growth zones in context at maturity, and
finely echinulate basidiospores. Ganoderma lingzhi is
often associated with Fagaceae (e.g., Castanea,Cyclo-
balanopsis and Quercus) and is distributed in temperate
to subtropical East Asia, while G. multipileum inhabits
Fabaceae trunks (e.g., Acacia,Delonix) and is distribut-
ed in tropical and subtropical Asia (Wang et al. 2009b).
Ganoderma sichuanense differs from G. lingzhi in its
sessile basidiocarps and smaller basidiospores (7.49.2×
56.6 μm). Ganoderma tropicum differs from G. lingzhi
by its mostly sessile basidiocarps, dark brown context,
concentric growth zones in the context and mostly ir-
regular cuticle cells. Similar to G. multipileum,G. tro-
picum also inhabits Fabaceae trees. The Chinese G.
tsugaeis separated from G. lingzhi by the absence of
melanoid bands in the context, white pore surface and
thin dissepiments (2060 μm) when mature. The feature
differences among G. lingzhi and some other similar
species are listed in Table 2.
Ganoderma curtisii, a species originally described from
North America (Moncalvo and Ryvarden 1997), was found
to be the most closely phylogenetically related species to G.
lingzhi (MP/ML/BA099/100/1.00, Fig. 3). Two strains of
G.curtisii (CBS 100131, 100132) from the type locality
were cultivated to obtain their fruiting bodies for comparison.
Fruiting bodies of G.curtisii resemble G.lingzhi when
juvenile as they share a yellow to buff pileal surface
and white pore surface. At maturity, G.curtisii still
bears yellowish brown to olivaceous buff pileal surface
withathinandeasilyscaledoffsoftcrust,whileG.
lingzhi often bears a reddish brown pileal surface with a
hard and non-breakable crust. In addition, G.curtisii
usually has a white pore surface and thin dissepiments
(4080 μm) when mature. In contrast, G. lingzhi has a
yellow pore surface when mature and its dissepiments
are thick (80120 μm). Microscopically, G.curtisii has
more inflated, loosely-arranged cuticle cells with wider
apical parts ((6)812(14) μm, W09.98 μm), while G.
lingzhi often has more slender, closely-packed cuticle
cells with narrower apical parts ((5)69(10) μm,
W07.34).
Wang and Yao (2009) reported that G. sichuanense can
represent G. lucidumin China. Our analysis indicates that
G. sichuanense is distantly related to G. lingzhi, but it is
closely phylogenetically related to G. resinaceum (Fig. 1).
Coupled with the obvious morphological differences from
G. lingzhi (Fig. 4, Table 2), we confirm that G. sichuanense
is a distinct species.
The nuc-ITS sequences obtained from four samples of
G. lucidum(ACCC 5.65 and HMAS 60537 from China;
WD565 and WD2038 from Japan) in previous studies
(Moncalvo et al. 1994,1995; Wang et al. 2009b) are also
Fig. 4 Basidiocarps of Ganoderma lingzhi and its most similar species
in China. a.Ganoderma flexipes;b.Ganoderma lingzhi (holotype); c.
Ganoderma lucidum (Ganoderma tsugae). d.Ganoderma multipi-
leum;e.Ganoderma sichuanense (holotype); f.Ganoderma tropicum
58 Fungal Diversity (2012) 56:4962
included in this study. They are also nested in Clade I
(Fig. 1), which is G. lingzhi.
The mt-SSU sequences of G. lucidum(IFO 31863
(KCTC 6729) from Japan; IMSNU 30042, KCTC 6366,
KCTC 6530b, KCTC 6531 and KCTC 6532 from Korea)
in the study of Hong and Jung (2004) share very high
similarity (>99 %) with that of G. lingzhi in our study. These
Japanese and Korean G. lucidumare determined as G.
lingzhi in this study. Accordingly, G. lingzhi has a distribu-
tion in China, Japan and Korea.
Moncalvo et al. (1995) concluded that an Asian spe-
cies was mistaken for G. tsugae from his phylogenetic
study of the cultivated Ganoderma species labelled G.
tsugae (RSH J2, RSH 1109, RSH BLC) from East Asia.
These sequences were also included in our analysis and
these strains are nested in the well-supported Clade I
(Fig. 3). Consequently, they are determined in this study
as G. lingzhi.
We investigated 35 specimens or strains of cultivated
Lingzhifrom 10 representative production sites in China.
In spite of the highly variable basidiocarps of the different
Lingzhivarieties, the analysis of nuc-ITS sequences
shows that they share high similarity (>99 %) and clustered
in a single clade with a robust support. This indicates that
the strains of Lingzhiwidely cultivated in China represent a
single species (i.e., G.lingzhi).
The strain labelled G. tsugae (Zhang 0981) from studies of
Moncalvo et al. (1994,1995) and two specimens named G.
tsugae(Dai 3937, Yuan 5649) collected on Larix from north-
eastern China were also analyzed. The results showed that
these samples are nested in Clade III and cluster with G.
lucidum from Europe. Similar results were mentioned by
Moncalvo et al. (1994;1995). The Chinese G. tsugae(Yuan
5649, nuc-ITS:JQ781854, mt-SSU:JX029986) also shares
Table 2 Morphological characters of Ganoderma lingzhi and its most similar species in China
Species Stipe Melanoid bands in
mature fruiting
body
Context color Concentric
growth zones
in context
Dissepiments
(μm)
Cuticle cells Basidiospores (μm) Pore color
(at maturity)
Ganoderma flexipes present present not completely homogeneous;
yellow brown to dark brown
absent thick; 80120
(140)
mostly regular; clavate (8.5)910.3(11)× ( 5)
5.37
white to pale sulphur
yellow
Ganoderma lingzhi present present not completely homogeneous;
light buff, buff, clay buff
to snuff buff
absent thick; (60)80
120(140)
mostly regular; clavate (8)910.7(12)× (5.2)
5.87(7.5)
pale yellow, sulphur
yellow to straw-
colored
Ganoderma lucidum
(0G. tsugaefrom China)
present absent not completely homogeneous;
white, cream to pale clay
brown
absent thin; 2060(80) mostly regular; clavate (8.8)910.7(11)× (5.3)
5.76.6(7)
white
Ganoderma multipileum present, rarely
absent
present not completely homogeneous;
clay buff to fulvous
present thick; 60120
(160)
mostly regular; clavate (8)8.810.4(11.3)×(5)
5.66.9(7.2)
cream to straw-
colored
Ganoderma sichuanense absent present not completely homogeneous;
buff to pale clay buff
absent thick; (80)100
140(200)
mostly regular; clavate (7)7.49.2(9.3)× (4.6)
56.6(6.8)
buff yellow
Ganoderma tropicum absent, sometimes
present
present homogeneous; fulvous present thick; (60)80
140(160)
mostly irregular; clavate,
often with blunt outgrowths
or protuberances
(8.3)8.810.7(11.2)×
(5)5.46.3(6.8)
cream to pale straw-
colored
Table 3 The studied Ganoderma species from China in this paper
Species Names
Accepted
species
G. flexipes Pat.
G. lingzhi Sheng H. Wu, Y. Cao & Y.C. Dai
G. lucidum (Curtis) P. Karst.
G. multipileum Ding Hou
G. sichuanense J.D. Zhao & X.Q. Zhang
G. tropicum (Jungh.) Bres.
Unaccepted
species
G. atrum J.D. Zhao, L.W. Hsu & X.Q. Zhang
G. calidophilum J.D. Zhao, L.W. Hsu & X.Q. Zhang
G. chenghaiense J.D. Zhao
G. hainanense J.D. Zhao, L.W. Hsu & X.Q. Zhang
G. parviungulatum J.D. Zhao & X.Q. Zhang
G. simaoense J.D. Zhao
G. tsugae Murrill
Fungal Diversity (2012) 56:4962 59
high similarity (>99 %) in nuc-ITS and mt-SSU sequences with
G. lucidum from UK (CBS 176.30, nuc-ITS:
AF094511&AF044490, mt-SSU:AF248324&AF248325).In
addition, the Chinese G. tsugaehas stipitate basidiocarps, a
reddish brown pileus, a white pore surface and thin dissepi-
ments (2060(80) μm) when mature, but lacks melanoid
bands in the context. All of these macro-morphological char-
acters fit that of G. lucidum from Europe with such micro-
morphological features as regular cuticle cells and similar basi-
diospores. Thus, the G. tsugaesamples from conifers in
northeastern China are identified as G. lucidum in this study.
Studies of Moncalvo et al. (1995) indicated that G. lucidum
sensu stricto was distributed in northern and southern Europe,
and probably extended to China. Based on more molecular and
morphological evidences from more samples, our study con-
firmed that the distribution of G. lucidum includes northeastern
China. Geographic distribution of G. lucidum resembles the
Europe (-Siberia)-Northeast Asia (-Southwestern China) distri-
bution pattern of Heterobasidion Bref. and Chroogomphus
(Singer) O.K. Mill (Dai et al. 2003;Lietal.2009).
Ganoderma species have been widely cultivated in Asia
and consumed worldwide, among which most commercial
materials are named as G. lucidum. In addition to the Asian
origin, some commercial strains are from America, e.g.
Fungi Perfecti G. lucdium, which was also determined as
another different species from the true G. lucidum (Smith
and Sivasithamparam 2000). As misidentification of Gano-
derma strains may hinder strategies for drung discovery
(Wasser et al. 2006) and create complications for publica-
tions, patents and products (Wasser 2011), the correct iden-
tification of commercial and research-oriented Ganoderma
strains especially those labelled G. lucidum is obviously
important. The species identity of the G. lucidumcollec-
tions outside Eurasia should be further studied. The species
of Ganoderma studied and accepted in this study from
China can be found in Table 3.
Specimens of related Ganoderma species studied G. cur-
tissi.USA. North Carolina, Durham, Duke gardens, on the
base of living tree of Quercus, November 1996, JM 96/80
(CBS 100131); Durham, yard Alabama street, on the base of
living hardwood tree, 6 December 1996, JM 96/81(CBS
100132). G. flexipes.China. Fujian Province, Wuyishan
County, Tianyoufeng, on the ground of mixed forest, 26
August 2006, Cui 4122. Hainan Province, Baoting County,
Diaoluoshan Nature Reserve, on the ground of angiosperm
forest, 30 June 2010, Wei 5491 &5494; Ledong County,
Jianfengling Nature Reserve, on the root of dead angio-
sperm, 25 June 2010, Wei 5200; on the ground of forest,
15 November 2007, Yang 407.G. lucidum.Finland. Espoo,
Westend, Varsasaari, on the stump of Picea, 1 August 2005,
Stella (H). Helsinki, on the dead tree of Quercus 4 Septem-
ber 2004, Dai 5857. Vantaa, 18 October 2009, Dai 11593.
Varsinais-Suomi, on ground, Salo 11212 (H). Romania.
Transsilvania Distr., on Quercus, 16 September 1955, Sila-
ghi (H). Sweden. Halland, Västergotland, on rotten wood of
Corylus, 22 August 1996, Dai 2272.Skane,Brunby,on
Fagus, 29 September 1985, Nordin (H). UK. England,
Windsor Great Park, X 2003, HMAS 86598 (HMAS). G.
lucidum (G. tsugae).China. Jilin Province, Antu County,
Changbaishan Nature Reserve, on fallen gymnosperm trunk,
6 September 1993, Dai 1115; on the stump of Pinus,21
September 2002, Dai 3935; on the fallen trunk of Larix,21
September 2002, Dai 3937, 12 December 2007, Dai 9028,
10 August 2011, Yuan 5649.G. multipileum.China.
Hainan Province, Haikou, on angiosperm stump, 21 May
2008, Yuan 4146; on the stump of Acacia, 23 May 2008,
Dai 9523 &9524; on the stump of Hevea, 4 June 2008, Dai
10042.G. sichuanense.China. Guangdong Province,
Guangzhou, South China Botanical Garden, on angiosperm
stump, 19 September 2009, Cui 7691. Sichuan Province,
Panzhihua, on angiosperm stump, 1976, HMAS 42798 (ho-
lotype, HMAS). G. tropicum.China. Guangxi Autonomous
Region, Chongzuo County, Nonggang Nature Reserve, on
dead angiosperm root, 7 July 2007, Zhou 269.Hainan
Province, Baoting County, Tropical Botanical Garden, on
angiosperm stump, 27 May 2008, Dai 9724. Yunnan Prov-
ince, Xishuangbanna, Menglun Green Stone Forest Park, on
angiosperm stump, 4 August 2005, Dai 6721; Menglun
Tropical Botanical Garden, on living tree of Cycas,10
September 2005, Yuan 2300; on the living angiosperm, 12
September 2007, Yuan 3490.
Acknowledgements We thank Drs. T.Z. Wei and X.C. Wang (HMAS,
China), Prof. L. Ryvarden and Dr. K.-H. Larsson (O, Norway) for loans
of specimens. We are grateful to Prof. S. P. Wasser (UOH, Israel) for
offering some important literatures and to CBS for offering some Gano-
derma cutltures. We are indebted to Dr. D.M. Wang (GIM, China) and Dr.
J.M. Moncalvo (ROM, Canada) for offering suggestions during this
study. We appreciate Dr. H.S. Yuan (IFP, China) for improving the
drawings, and Dr. S.H. He (BJFC, China) for offering some Ganoderma
images. Y.C. Dai and S.H. Wu are deeply indebted to Dr. T. Niemelä (H,
Finland) for his friendly support of their studies at Botanical Museum of
the University of Helsinki. We are grateful to Prof. K.D. Hyde (Chiang
Rai, Thailand) for improving the manuscript. The research was financed
by the National Natural Science Foundation of China (Project Nos.
30910103907, 31070022).
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... Ganoderma is one of the most taxonomically scrutinized genera among the Ganodermataceae and even in Polyporales (Richter et al. 2015;Costa-Rezende et al. 2020). Most Ganoderma species are wood decomposers, found in all temperate and tropical regions (Pilotti et al. 2004;Cao et al. 2012;Zhou et al. 2015). ...
... 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). ...
... The dataset composed of ITS, TEF1-α and RPB2 genes, comprising a total of 2092 characters including gaps, ITS (1-656 bp), TEF1-α (657-1192 bp) and RPB2 (1193-2092 bp), including 57 taxa with Tomophagus colossus (Fr.) C.F. Baker as the outgroup taxon (Wang et al. 2009;Cao et al. 2012). Best model for the combined 3-gene dataset estimated and applied in the Bayesian analysis was GTR+I+G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). ...
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Ganoderma dianzhongense sp. nov. and G. esculentum sp. nov. are proposed as two new species based on both phenotypic and genotypic evidences. Ganoderma dianzhongense 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. Ganoderma esculentum 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.
... Karsten introduced the genus Ganoderma in 1881 to encompass only one species, Ganoderma lucidum (Curtis) P. Karst. Steyaert (1961) later designated an illustration as its lectotype; but efforts to find a specimen as an epitype have been unsuccessful so far (Steyaert 1972;Moncalvo and Ryvarden 1997;Cao et al. 2012). Despite reports of this species across Europe, Asia, America, Africa, and Oceania, it is found worldwide. ...
... The first clade, consisting of tropical collections, was identified as G. multipileum, while the second clade remained unknown. Wang et al. (2012) recognized this unknown clade as G. sichuanense, but concurrent study by Cao et al. (2012) found discrepancies, suggesting unknown clade as a new species called G. lingzhi. Recently, Du et al. (2023) proposed that the correct scientific name for this species is G. sichuanense, with G. lingzhi identified as a later synonym. ...
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Ganoderma lucidum, known as the “mushroom of immortality,” is a white rot fungus renowned for its medicinal properties, attributed to its bioactive compounds. Although species with similar morphological traits to G. lucidum are found across the globe, precise identification is made possible through DNA barcoding and molecular phylogenetic analysis. Global cultivation and wild harvesting of G. lucidum are both done in response to the growing market needs. Artificial cultivation is typically performed on sawdust, but other woody substrates and the wood log method are also employed. This cultivation leverages the fungus’s ecological role in converting industrial and agricultural solid wastes into biomass, thereby producing functional food and potential pharmaceutical sources. The review consolidates research on various aspects of, including cultivation methods (sawdust, agricultural waste, wood logs, and submerged fermentation), and the current global market conditions.
... Используемые в медицинских, химических, геномных исследованиях изоляты G. lingzhi (син. G. sichuanense), а также коммерчески культивируемые штаммы до сих пор объединяют под общим названием G. lucidum, хотя около 20 лет назад было показано, что образцы G. lucidum из Европы и Восточной Азии в большинстве своем не являются конспецифическими [3], а вид G. lingzhi отличается от близкородственного вида G. lucidum [4]. ...
... Экстракты плодовых тел, мицелия, культуральная жидкость, споры G. lucidum и G. lingzhi обладают широким спектром фармакологических свойств и используются в качестве источника биологически активных веществ (БАВ) [5], оказывающих иммуномодулирующее, противоопухолевое [6,7], противовирусное [8], антимикробное [9], гиполипидемическое, гипогликемическое [10], гепатопротекторное [11] и противовоспалительное действия [12]. Стоимость препаратов, получаемых из грибов рода Ganoderma, в настоящее время ежегодно оценивается почти в два млрд $ [4]. Плодовые тела Ganoderma spp. ...
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... The genus Ganoderma has a worldwide distribution but is predominantly found in tropical and temperate regions including Africa, America, Europe and Asia (Pilotti et al., 2005, Cao et al., 2012and Wang et al., 2012 [18,2,28] . Many Ganoderma species have been used as a medicinal herb for thousands of years in many Asian countries. ...
... The genus Ganoderma has a worldwide distribution but is predominantly found in tropical and temperate regions including Africa, America, Europe and Asia (Pilotti et al., 2005, Cao et al., 2012and Wang et al., 2012 [18,2,28] . Many Ganoderma species have been used as a medicinal herb for thousands of years in many Asian countries. ...
... It is well-known that several highly conserved genes, such as nrSSU and nrSSU, tef 1-α, β-tubulin, rpb2, mtSSU and mtLSU, were extensively utilized in the phylogenetic analysis of Ganoderma [15][16][17][18][19][20][21]. However, these sequences represented partial CDS (incomplete gene sequences) that were commonly acquired through amplification sequencing, employing the Sanger method. ...
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Ganoderma is the most important genus in the family Ganodermataceae; many species have attracted much attention and widely cultivated because of their medicinal values, but so far, not a sequenced mitogenome derived from dikaryon strains has been explicitly recorded. Herein, four novel mitogenomes of commonly cultivated Ganoderma (G. leucocontextum H4, G. lucidum G6, G. sinense MZ96 and G. tsugae SS) were de novo assembled and given detail functional annotations. Collinearity analysis revealed that the four mitogenomes shared 82.93–92.02% similarity with their corresponding reference mitogenomes at the nucleotide level. A total of 15 core protein-coding genes (PCGs), along with rrnL and rrnS (mtLSU and mtSSU) were chosen as potential candidates for constructing their individual phylogenetic trees. These trees were compared with those derived from the concatenated sequences of 15 core PCGs. And finally, we found that the atp9 and nad4L were the most reliable markers for the phylogenetic analysis of Ganoderma and chosen as standard sequences to generate new DNA barcodes. This finding was further verified by comparing it against almost all available Ganoderma mitogenomes in the NCBI, with Trametes versicolor (Polyporaceae) and Rigidoporus microporus (Meripilaceae) as two outgroups. A total of 52 mitogenomes from three families were highly conserved, with identical gene lengths for atp9 (222 bp) and nad4L (267 bp). These genes were capable of distinguish distinctly different various species, which are grouped into separate clades within the phylogenetic trees. The closest related clades (I and II), including at least 30 samples of the three classical taxonomic species (G. lingzhi, G. sichuanense and G. lucidum), differed in only one SNP. The single base mutation rate increased with the evolutionary divergence of the phylogenetic clades, from two to three SNPs in earlier clades (e.g., clade IV containing G. leucocontextum) to five to six SNPs in later clades (e.g., clade X containing G. sinense). Despite these variations between species, the atp9 and nad4L genes of Ganoderma mitogenomes consistently encoded the same ATP synthase F0 subunit c (73 aa) and NADH dehydrogenase subunit 4L (88 aa). These two genes have been identified as reliable markers of new DNA barcodes, offering valuable insights and contributing significantly to understanding the evolutionary relationships and phylogeny of the Ganoderma genus and even the Ganodermataceae family.
... DNA was extracted from the mycelium of the remaining strains using the Fungi Genomic DNA Extraction Kit (Beijing Solarbio Science, Beijing, China) as per the manufacturer's instructions. Two universal primers-ITS4 (5 ′ -TCCTCCGCTTATTGATATGC-3 ′ ) and ITS5 (5 ′ -GGAAGTAAAAGTCGTAACAAGG-3 ′ )-were used to amplify the internal transcribed spacer (ITS) region [16,17]. The extracted samples were sent to Sangon Biotech (Shanghai) Co., Ltd. ...
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Sarcomyxa edulis is a characteristic low-temperature, edible mushroom in Northeast China. It has a delicious taste and rich nutritional and medicinal value. The artificial cultivation of S. edulis has been increasing in recent years. However, the number of S. edulis varieties is scarce, and strain degradation is a serious issue, affecting the yield and quality of S. edulis. Therefore, we collected 21 wild strains of S. edulis (Y1–21) in this study, aiming to develop strains of S. edulis. Five strains without antagonistic reaction were eliminated via the antagonism test, and the remaining sixteen strains were identified as S. edulis using internal transcribed spacer (ITS) marker identification. The mycelial growth rate, mildew resistance, fruiting body yield, agronomic traits, and nutrient content of the 16 strains were determined. The results demonstrate that Y12, Y13, Y14, and Y15 exhibited a rapid mycelial growth rate (6.43–6.8 mm·day−1). Their colony density was moderate; their edges were neat; and their colonies were leathery and had obvious pigmentation. Moreover, they exhibited strong mildew resistance and a low Trichoderma contamination rate (<40%). Their fruiting body yield (281.15–342.03 g) and biological efficiency (56.23–68.40%) were high. Their fruiting body shape was good. Their polysaccharide and crude protein contents were higher, while their crude fiber, ash, and crude fat contents were lower. Overall, these four S. edulis strains exhibited high yield, excellent traits, and good quality for commercial production and food production with high nutrient contents. This study provides a foundation for the further cross-breeding and matrix improvement of S. edulis.
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