The Species Identity of the Widely Cultivated Ganoderma, ‘G. lucidum’ (Ling-zhi), in China

Ling-zhi, a widely cultivated fungus in China, has a long history in traditional Chinese medicine. Although the name ‘Ganoderma lucidum’, a species originally described from England, has been applied to the fungus, their identities are not the same. This study aims to clarify the identity of this medicinally and economically important fungus. Specimens of Ling-zhi from China (field collections and cultivated basidiomata of the Chinese ‘G. lucidum’), G. lucidum from UK and other related Ganoderma species, were examined both morphologically and molecularly. High variability of basidioma morphology was found in the cultivated specimens of the Chinese ‘G. lucidum’, while some microscopic characters were more or less consistent, i.e. short clavate cutis elements, Bovista-type ligative hyphae and strongly echinulate basidiospores. These characters were also found in the holotype of G. sichuanense, a species originally described from Sichuan, China, and in recent collections made in the type locality of the species, which matched the diagnostic characters in the prologue. For comparison, specimens of closely related species, G. lucidum, G. multipileum, G. resinaceum, G. tropicum and G. weberianum, were also examined. DNA sequences were obtained from field collections, cultivated basidiomata and living strains of the Chinese ‘G. lucidum’, specimens from the type locality of G. sichuanense, and specimens of the closely related species studied. Three-gene combined analyses (ITS+IGS+rpb2) were performed and the results indicated that the Chinese ‘G. lucidum’ shared almost identical sequences with G. sichuanense. Based on both morphological and molecular data, the identity of the Chinese ‘G. lucidum’ (Ling-zhi) is considered conspecific with G. sichuanense. Detailed morphological descriptions and illustrations are provided in addition to discussion of nomenclature implications.


Introduction
Ling-zhi is a famous fungus for its medicinal values well documented in the Chinese literature which can be dated back nearly two thousand years to the Shen Nong Materia Medica (102-200AD [1]). It symbolises happiness, good fortune, good health and even immortality in Chinese traditional culture [2]. There are thousands of publications relating to this fungus and it is now commercially cultivated on a large scale. Isolates used in medicinal studies and the commercially cultivated strains are generally named 'Ganoderma lucidum'.
Ling-zhi ('G. lucidum') has been reported by numerous studies to possess properties relating to its anti-tumour, antiviral, antibacterial, anti-inflammatory, anti-oxidant, anti-platelet aggregation, hepatoprotective, hypotensive, immuno-modulating, immunosuppressive effects [3][4][5][6] and, especially, the activity against HIV/AIDS, improving the quality of life of the affected in Africa in recent years [7]. Many kinds of health care products and medicines derived from this fungus are produced, traded and consumed in large quantities each year. The world trade market value of 'G. lucidum' products reached US$2.5 billion in 2003 [8]. The cultivation of Lin-zhi ('G. lucidum') has become a growth industry in China and other East-Asian countries, and is spreading to other areas of the world.
Ganoderma lucidum (Curtis) P. Karst. was described by Curtis [9] based on material from England and the epithet was sanctioned by Fries [10]. The fungus is widely distributed in Europe, especially in the UK. Moncalvo et al. [11,12] revealed that the species named G. lucidum from both Europe and mainland China was not conspecific based on analyses of nuclear ribosome DNA regions. Based on morphological examination of collections from both China and Britain, Pelger and Yao [13] also found that there was no Chinese collection referable to true G. lucidum. Moreover, 'G. lucidum' collected from mainland China and tropical Asia was also separated into two lineages in molecular studies [11,12,14,15]. The misidentification of the fungus in most pharmacological studies was also recognized by Wasser [16].
Recently, one of the two lineages revealed by molecular studies has been re-identified as G. multipileum D. Hou [17], whilst the correct identity of the most widely cultivated species of Ganoderma in mainland China, Japan, Korea and now spreading to other parts of the world, has not yet been determined and is still named 'G. lucidum' in many scientific articles and commercial reports. In view of the importance of the species, Hawksworth [18] proposed to conserve the name Ganoderma lucidum with an Asian type and introduce a new name for the European species.
The first record of Ganoderma from China, in modern scientific research, was made by Teng in 1934 [19] with four species and one variety. One of the species recorded was named 'G. lucidum'. A further 26 species were added to the Chinese records of this group in two genera, Ganoderma and Amauroderma, nearly 30 years later in 1963 [20] and some 38 species were listed in the two genera by Tai in 1979 [21]. The name G. lucidum, introduced to China by Teng [19], was widely accepted by his contemporary and later Chinese mycologists [21][22][23][24][25]. The members of Ganodermataceae reported from China was increased to 86 species in four genera -Ganoderma, Amauroderma, Haddowia and Humphreya -in the 1980s [23]. A total of 98 species in the four genera were finally assembled by Zhao and Zhang [24] in 'Flora Fungorum Sinicorum 18: Ganodermataceae', including 58 new species based on collections from China. Of the 58 new species, 10 belong to Amauroderma and 48 to Ganoderma. Apart from 12 nonlaccate and 16 laccate species with dark brown context of the 48 new species of Ganoderma, 20 laccate species with light-coloured context were classified in the same section with the Chinese 'G. lucidum'. Recently, Wu and Dai [25] were able to distinguish morphologically 103 Chinese species in Ganodermataceae, adding four more new species from China, including two in Ganoderma (one laccate species with dark brown context and the other nonlaccate). Additionally, one new laccate variety with lightcoloured context was added by Wasser et al. [26] based on collections from northeastern China.
In total, there are 20 new laccate species and one new laccate variety with light-coloured context described from China. Among them, 13 can be distinguished from the widely cultivated Chinese 'G. lucidum' (Ling-zhi) based on basidiospore characteristics, i.e. subglobose spore shape in G. bicharacteristicum X.Q. Zhang and G. kunmingense J.D. Zhao different from ovoid shape in the latter; large spore size (up to 13.0-15.0 mm long) in G. albomarginatum S.C. He, G. shandongense J.D. Zhao & L.W. Hsu, G. stratoideum S.C. He and G. xingyiense S.C. He and small size (less than 9.0 mm long) in G. daiqingshanense J.D. Zhao and G. jianfenglingense X.L. Wu compared with medium size (9.0-11.0 mm long) in 'G. lucidum' (Ling-zhi); slightly echinulate ornamentation in G. microsporum R.S. Hseu, G. multipileum, G. ramosissimum J.D. Zhao, G. tenue J.D. Zhao et al. and G. theaecolum J.D. Zhao, but strongly echinulate in 'G. lucidum' (Ling-zhi) [24,26] The aim of this study is to clarify the identity of the Chinese 'G. lucidum' (Ling-zhi) based on both morphological and molecular data. Detailed morphological descriptions and illustrations are presented below with discussion of nomenclature implications.

Ethics Statement
No specific permits were required for the described field sampling because the locations are not privately-owned or protected in any way and the field studies did not involve endangered or protected species.

Fungal Materials
Some 113 collections, including 48 samples of commercially cultivated 'G. lucidum' (Ling-zhi) from China, 55 field collections identified as the same species, and 10 specimens of G. sichuanense (types, authentic material and recent collections from the type locality) were examined morphologically. In addition, five related laccate species, i.e. G. multipileum, G. tropicum (Jungh.) Bres., G. weberianum (Sacc.) Steyaert, G. resinaceum Boud. as well as the true G. lucidum, were selected for comparison according to published molecular phylogeny of Ganoderma [17,27] and the similar results derived from analyses of cumulated molecular data (unpublished) by this group. Ganoderma multipileum and G. tropicum were the sister groups of 'G. lucidum' (Ling-zhi) in the same clade and distributed in China; and G. resinaceum and G. weberianum were the representatives of another clade which also comprised laccate species with light-coloured context. A total of 45 collections of these five species were examined and one representative collection of each species was used for molecular analyses and illustration. Extraction of genomic DNA from 118 collections and from three living strains named 'G. lucidum' and one strain of Tomophagus colossus (Fr.) Murrill (to serve as an outgroup) deposited in the China General Microbiological Culture Collection Center (CGMCC) was performed. Amplification of DNA fragments from 28 collections and the four living strains was successfully obtained. These collections and living strains were included in phylogenetic analyses. Sources of the specimens and strains used in this study are listed in Table 1. Voucher specimens were preserved in the Fungarium, Institute of Microbiology, Chinese Academy of Sciences (HMAS). The strains were stored at 4uC on potato dextrose agar (PDA) medium and sub-cultivated at 25uC in liquid PDA medium for 14 days to collect the mycelia for DNA extraction.

Morphological Observations
Morphological observations mainly followed the methods described previously by Wang et al. [28]. A 5% KOH solution was used as the mounting medium. Microscopic characters were observed using a light microscope (Zeiss Axiophot). Images were captured with a Zeiss Axiocam MRc digital camera using Differential Interference Contrast (DIC) microscopy and the AxioVision Rel.4.6.3 acquisition software (Zeiss). At least 30 basidiospores of each mature specimen were measured and the basidiospore size was given both with and without the myxosporium in the species description. The Q-value (length: breadth ratios) for each spore was calculated and the mean value was used in the description.

DNA Isolation, PCR Amplification and Sequencing
Genomic DNA from specimens and fresh fungal cultures was isolated by using the modified cetyltrimethylammonium bromide (CTAB) method as described by Jiang and Yao [29]. The ITS region, including the intervening 5.8S gene, was amplified from the total DNA using the primers ITS5 and ITS4 [30]. The primers CNL12 and 5SA-Anderson were used to amplify the IGS region [31]. For the amplification of rpb2, the primers bRPB2-6F and bRPB2-7.1R [32] were used. PCR amplification was carried out according to the procedures described by Wang and Yao [33]. Purified PCR products were sequenced by the cyclic reaction termination method on an ABI Prism 3730 genetic analyzer (Applied Biosystems). Each fragment was sequenced in both directions for confirmation, and the sequences of the two strands were assembled with the software ContigExpress (Vector NTI Suite 6.0, InforMax Inc.).

Phylogenetic Analyses
Five ITS sequences from the five related laccate species with light-coloured context examined morphologically above, and one from Tomophagus colossus (as outgroup) were retrieved from GenBank and aligned with those sequences of the same region obtained in this study for confirmation of species identity. Sequences of all the three gene partitions obtained were compiled into a combined matrix. The sequences were aligned using Clustal X 1.81 [34] and then further manually adjusted using BioEdit 5.0.6 [35]. Maximum parsimony (MP) analyses were performed using a heuristic search in PAUP 4.0b10 for Macintosh [36], with the random addition of sequences with 1,000 replicates, tree bisection-reconnection as the branch-swapping algorithm, one tree held at each step during stepwise addition, and the MULTREES option off. Gaps were treated as missing data. Bootstrap values were calculated from 1,000 replicates, with 10 heuristic searches per replicate. Bayesian Metropolis coupled Markov chain Monte Carlo (B-MCMCMC) analyses were implemented in MrBayes 3.1.2 [37] and Modeltest 3.7 [38] was used to select the best-fit models and the parameters of DNA substitution. Bayesian analyses involved 1,000,000 generations, two independent runs with four Markov chains and sampling trees every one-hundredth generation. The average split frequencies were checked to determine optimal convergence of the chains below 0.01. A 50% majorityrule consensus tree was constructed after the exclusion of the first 25% of trees from the first stage of the run (burn-in).

Morphological Observations
High variability was observed in macroscopic characters of the Chinese 'G. lucidum' (Ling-zhi), especially in those cultivated samples (Fig. 1). Seven characters of basidiomata were examined for their variability. The results showed that the shape of pileus varied from reniform to subcircular and also from convex to concave (Figs. 1-A-1 to 1-A-4); the length of stipe was from very short (less than the pileus radius, inconspicuous in Fig. 1-B-2) to long (more than the pileus radius, Fig. 1-B-1); attachment of the stipe to the pileus varied from lateral to nearly central (Figs. 1-C-1 and 1-C-2); the surface of pileus was either with radial furrows or with concentrically sulcate zones (Figs. 1-D-1 and 1-D-2); the thickness of pileus appeared from one layer (thinner than 1 cm) to several layers (often thicker that 1 cm; Figs. 1-E-1 and 1-E-2); the colour of pore surface varied from whitish to yellowish (Figs. 1-F-1, 1-F-2, 1-C-1 and 1-C-2); the length of tube layer was from short (less than one third of the pileus thickness) to long (more than one third of the pileus thickness; Figs. 1-G-1 and 1-G-2). The morphological variability was found not only in different specimens but also often seen in a single collection (Fig. 2). Attachment of stipe to the pileus varied from nearly central to lateral (Figs. 2-A and 2-B) and pileus thickness varied from very thin to considerably thick (Figs. 2-C and 2-D).
The shape of the pileus of the five related laccate species examined in this study varied from flabelliform and concave (G.

Phylogenetic Analyses
A total of 32 ITS sequences from 28 specimens (five field collections determined as 'G. lucidum' by S.C. Teng or J.D. Zhao, 13 cultivated Chinese 'G. lucidum', five field collections of G. sichuanense from its type locality and five related laccate Ganoderma species) and four living strains (including the out group) were obtained. Attempts for DNA extraction from the holotype, paratype and authentic specimens of G. sichuanense failed. Several methods of DNA extraction, such as WizardH Genomic DNA Purification Kit (Promega, U.S.A.), Chelex 100 Resin (Solarbio, China) and CTAB method, were employed for the holotype, but the specimen was found to be contaminated by an ascomycete, a species of Eurotium, through ITS sequencing. A basidiomycetespecific primer pair (ITS1F and ITS4B) [39] was also used to obtain ITS sequence from the holotype in PCR amplifications but no positive reaction resulted from this work. IGS sequences were successfully obtained from 20 samples, including four field collections of G. sichuanense, seven cultivated Chinese 'G. lucidum', five related species and four living strains. Some 13 rpb2 sequences were obtained from three field collections of G. sichuanense, one cultivated Chinese 'G. lucidum', five related species and four living strains. All the sequences generated in this study were submitted to GenBank with accession numbers of JF915393-915436, JN197275-197290 and JQ081068-081070 (Table 1).
The combined 3-gene dataset with 38 taxa consisted of 2179 base pairs (ITS 561 bp, IGS 1037 bp and rpb2 581 bp), of which 324 were parsimony informative (ITS 85 bp, IGS 161 bp and rpb2 78 bp). A total of 13 taxa (one cultivated Chinese 'G. lucidum', three G. sichuanense field collections, five related species and four living strains) were complete for all the three genes partitions (Table 1). Maximum-parsimony (MP) analyses of the dataset yielded 947 equally parsimonious trees (length = 897, CI = 0.8016, RI = 0.7764). Tamura-Nei (TrN) model of DNA substitution with gamma-distributed rate variation across invariant sites was determined as best-fit for the combined 3-gene Bayesian analyses. Since the topology of the Bayesian consensus tree was nearly identical to that of MP analyses, one of the 947 equally parsimonious trees was presented in Fig. 5 with bootstrap values of MP analyses and posterior probabilities of Bayesian analyses.
Six major terminal clades were recognized from the combined 3-gene analyses, i.e. the Chinese 'G. lucidum' (including the field collections of G. sichuanense), G. multipileum, G. tropicum, G. weberianum, G. resinaceum and the true G. lucidum clades (Fig. 5). The five additional ITS sequences of the related laccate species and the one of Tomophagus colossus retrieved from GenBank clustered well with those sequences of corresponding species obtained in this study, forming the terminal clades. All the clades received support of over 85% bootstrap proportions (BP) in MP analyses and of 1.00 posterior probabilities (PP) in Bayesian analyses. Ganoderma multipileum and G. tropicum further formed a group with support of BP = 93% and PP = 1.00, sister to the lineage of the Chinese 'G. lucidum', and G. resinaceum and G. weberianum formed another group (BP = 100%, PP = 1.00).
In the Chinese 'G. lucidum' clade, a total of 26 samples included five field collections of 'G. lucidum' named by S.C. Teng or J.D. Zhao, 13 Chinese cultivated 'G. lucidum', three living strains of 'G. lucidum' and five G. sichuanense field collections. The clade received very strong support by both MP and Bayesian analyses (BP = 100%, PP = 1.00) and was phylogenetically separated from the true G. lucidum from England in the 3-gene analyses (Fig. 5).
Morphological similarities and differences of the six species of Ganoderma are presented in the table in Fig. 5. Letters A to G in the table represent seven macroscopic characters of basidiomata as depicted in Fig. 1, showing the high variability of the Chinese 'G. lucidum' in macroscopic morphology. It was noted that samples of the Chinese 'G. lucidum' varied in each of the seven characters. Letters H and I in the table represent macroscopic similarities of the six species in the appearance of basidiomata (laccate or nonlaccate) and the colour of the context (light-coloured or dark brown). Letters J to N indicate microscopic differences of the six species in the length and shape of cutis elements, presence of Bovista-type ligative hyphae and the length and ornament of the basidiospores.

Taxonomy
Based on both morphological and molecular data obtained in this study, the cultivated Chinese 'G. lucidum' (Ling-zhi) is clarified here as conspecific with G. sichuanense. Since the original description of G. sichuanense was based on very limited specimens and some published data were in contradiction with that obtained during this study, a new full description of G. sichuanense is provided here: Ganoderma sichuanense J.D. Zhao & X.Q. Zhang, Acta Mycologica Sinica 2:159. 1983. Figs. 1, 2, 3