Abstract 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.

Citation: Wang X-C, Xi R-J, Li Y, Wang D-M, Yao Y-J (2012) The Species Identity of the Widely Cultivated Ganoderma, ‘G. lucidum’ (Ling-zhi), in China. PLoS ONE 7(7): e40857. https://doi.org/10.1371/journal.pone.0040857 Editor: Olivier Lespinet, Université Paris-Sud, France Received: May 7, 2012; Accepted: June 14, 2012; Published: July 20, 2012 Copyright: © 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by the National Natural Science Foundation of China (30970015, 30270006), the Chinese Academy of Sciences (KSCX2-YW-G-074-04, KSCX2-SW-101C), the scheme of Introduction of Overseas Outstanding Talents operated by the Chinese Academy of Sciences, the National Science Funds for Distinguished Young Scholars from the National Natural Science Foundation of China (30025002), and the Science and Technology Planting Project of Guangdong Province (2010BO90300040). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

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, anti-bacterial, anti-inﬂammatory, anti-oxidant, anti-platelet aggregation, hepatoprotective, hypotensive, immuno-modulating, immunosuppressive effects [3]–[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]–[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 light-coloured 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 µm 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 µm long) in G. daiqingshanense J.D. Zhao and G. jianfenglingense X.L. Wu compared with medium size (9.0–11.0 µm 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]. There are six species, i.e. G. atrum J.D. Zhao et al., G. calidophilum J.D. Zhao et al., G. cantarelloideum M.H. Liu, G. hainanense J.D. Zhao et al., G. mongolicum Pilát and G. tsugae var. jannieae Wasser et al., lacking Bovista-type ligative hyphae, a well preserved character in all the collections of ‘G. lucidum’ (Ling-zhi), while the cutis structure in G. rotundatum J.D. Zhao et al. is composed of irregularly parallel or interwoven hyphae, different from hymeniodermiformic cutis in ‘G. lucidum’ (Ling-zhi) [24], [26]. The remaining species, G. sichuanense J.D. Zhao & X.Q. Zhang is morphologically similar to the Chinese ‘G. lucidum’ in all the above characters. 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.

Materials and Methods 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 4°C on potato dextrose agar (PDA) medium and sub-cultivated at 25°C 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% majority-rule consensus tree was constructed after the exclusion of the first 25% of trees from the first stage of the run (burn-in).

Discussion Based on both morphological observations and molecular analyses the Chinese ‘G. lucidum’ (Ling-zhi) is confirmed to be distinct from the true G. lucidum and proved to be conspecific with G. sichuanense. Although the name ‘G. lucidum’ has been widely applied to the Chinese species of Ling-zhi in recent decades, it is a misapplied name. Ganoderma sichuanense, originally described from Sichuan Province in 1983, is the correct name for the widely cultivated Ganoderma species (Ling-zhi) even though it had not been well recognized since it was published. The name Ganoderma lucidum was first introduced to the Chinese mycota by Teng [19] for collections from several provinces, i.e. Anhui, Fujian, Guangxi, Guizhou, Hainan, Jiangsu, Sichuan, Yunnan and Zhejiang. The name was then adapted by contemporary Chinese mycologists [22] and used for the Ganoderma species widely cultivated in China after the 1970s [21], [23]–[25], [41]. Cultivation of the species became popular in the 1980s and 1990s and the name G. lucidum appeared in many publications and commercial catalogs which have surged in numbers since then. In fact, G. lucidum is not only incorrectly recorded in China, but is similarly reported incorrectly from around the world. The name G. lucidum has been applied to collections from East Africa (Ghana, Kenya and Tanzania [42]), Oceania (Australia [14]), North America (Canada and U.S.A. [15], [43]), South America (Argentina, Brazil and Uruguay [44]), South and Southeast Asia (India, Indonesia, Philippines, Thailand and Vietnam [14], [45], [46]), East Asia (China, Japan and Korea [15], [46]) as well as Europe (almost all the European countries [47]). However, the collections named as G. lucidum from different parts of the world have appeared in several separated lineages in phylogenetic analyses of the genus [11], [14], [15], [43], [48]. Apparently, those collections are also likely misidentified, especially if they are not from Europe, and are in need of further taxonomic investigations. One such examples is that of G. lucidum reported as a pathogen, causing the basal stem rot disease of oil palms (Elaeis guineensis) in southeastern Asia since the 1930s [49], but later studies, both morphological [50] and molecular [51], indicated that the fungus is, in fact, another species of Ganoderma, G. boninense Pat., which is further treated as a synonym of G. orbiforme (Fr.) Ryvarden [52]. Ganoderma sichuanense was described by Zhao et al. [40] based on collections from Panzhihua City (as ‘Dukou City’ before, and spelled as ‘Dokou shi’ in the protologue), Sichuan Province. In addition to the holotype (HMAS 42798), a paratype (HMAS 43728) was also cited in the protologue. The species was diagnosed as having ‘a distinctly and radially rugose pileus, with the upper surface verrucose and tuberculose; pore surface yellowish when young, becoming brown or black when bruised; and small spores distinguished from other Ganoderma species’ [24], [40]. It became obvious in this study that the original description was a mixture of G. sichuanense and G. weberianum, as represented by the paratype (HMAS 43728), especially in the small spores and smooth or slightly echinulate eusporium. Since the original publication, G. sichuanense has been rarely recorded in China, either with uncertainty [25], or with small basidiospores (7.5–10×5.5–7.25 µm cum myxosp. [53]), or without any supporting specimen [54]. The name G. sichuanense is almost a forgotten name, possibly as a result of the inaccurate description. Morphological observations of the holotype of G. sichuanense and an authentic specimens of topotype (HMAS 47694) revealed that the species was the same as the widely cultivated Chinese ‘G. lucidum’ (Ling-zhi), and this identity was further confirmed by the molecular evidence from recent collections (HMAS 251145–251148) from the type locality sharing primary morphological characters with the holotype. Although the doubt on the identity of the Chinese ‘G. lucidum’ (Ling-zhi) was expressed by some researchers, e.g. Moncalvol et al. [11], [12], Pegler and Yao [13], and Wasser [16], the misapplication of this name had not been corrected. The proposal to conserve the name G. lucidum for the Chinese species [18] could be an option, but this would involve synonymising G. sichuanense with the former and introducing a new name for the well recognised European species. The misapplication of G. lucidum to the Chinese species has a relatively short history, although it has become dominant in the last few decades since the successful cultivation and intensive medicinal exploitation of the species. As stated above, G. lucidum has been misapplied to many different species of Ganoderma around the world, and conservation of this name for Ling-zhi will not provide a universal solution. The name ‘G. lucidum’ as used for the Chinese species is erroneous and should be corrected; G. sichuanense is the correct name to use.

Acknowledgments The authors are grateful to Profs Xiao-Hui Zheng (Xichang College), Lin Guo, Jian-Yun Zhuang and Xiao-Qing Zhang (Institute of Microbiology, CAS), Mrs. Yi Zeng, Ji-Yong Yang and Zi-Hao Li (Xichang College) for their assistance in field collection; to Ms. Hong-Mei Lü and Dr. Tie-Zheng Wei (Institute of Microbiology, CAS) for providing the fungarium specimens.

Author Contributions Conceived and designed the experiments: YJY. Performed the experiments: XCW. Analyzed the data: XCW YJY. Contributed reagents/materials/analysis tools: XCW RJX YL DMW YJY. Wrote the paper: XCW YJY.