Identification of commercial Ganoderma (Lingzhi) species by ITS2 sequences

Background DNA barcoding can be used to authenticate Ganoderma species for safe use. This study aims to identify commercial products containing Ganoderma using DNA barcoding. Methods We used 63 internal transcribed spacer (ITS) 2 sequences of Ganoderma species from 33 newly-sequenced wild samples, crude drugs, mycelia, spores, and authentic extracts and spore oils collected from various locations and markets, as well as 30 sequences from GenBank. Sequences were assembled and aligned using CodonCode Aligner V3.71. Intra- and inter-specific distances were estimated by MEGA 6.0, and phylogeny reconstruction was based on the K2P model. SNP(s) and RNA secondary structure of ITS2 were analyzed and compared among closely related Ganoderma species. Results G. lucidum cultivated in China was different from those cultivated in Europe. “Chizhi” (G. lucidum) and “Zizhi” (G. sinense) were clustered into two clades that were separated from the other Ganoderma species. The fruiting bodies and commercial products of G. lucidum and G. sinense were successfully distinguished from those of other Ganoderma species by comparing the ITS2 sequences and RNA secondary structures. Conclusion The DNA barcoding method is applicable to the authentication of commercial products containing Ganoderma species.


Background
Ganoderma (Lingzhi) is widely used in health products for its anti-tumor, anti-aging, anti-bacterial, immune system-enhancing, and anti-hypertension activities [1][2][3][4][5]. Lingzhi and its derivative products have a world trade value of approximately four billion US dollars [6]. Lingzhi products are popular in the market because of their high demand and potential profits.
There are approximately 76 Ganoderma species in China [7], but only approximately 20 of the species are used for medical purposes [8]. Moreover, only Ganoderma lucidum (Leyss. ex Fr.) Karst., 1881 (Chizhi) and G. sinense Zhao, Xu et Zhang, 1979 (Zizhi) are officially described in the Chinese Pharmacopoeia [9], and they are the most common types of Lingzhi on the market. They are difficult to distinguish because of the intra-species diversity of morphological features [8].
G. lucidum was collected from the UK. Karsten (1881) established the genus Ganoderma based on G. lucidum [10], which was reported in China in 1934 and was first successfully artificially cultivated in 1969 [11]. Cao et al. [11] proposed a new species name, G. lingzhi in 2012 for the Lingzhi that is distributed in East Asia. However, Wang et al. [12] determined that the widely cultivated G. lucidum in China was, in fact, G. sichuanense based on morphological and molecular evidence. Although they provided descriptions for the Lingzhi species in China, they did not obtain sequences from type specimens of G. sichuanense. The genome sequence of G. lucidum in China was first published by our research group [13]. The taxonomy of Lingzhi in China is still under dispute.
The internal transcribed spacer (ITS) region was proposed as a global DNA barcode sequence for identification of fungi at the fourth International Barcode of Life Conference [14]. Chen et al. [15] proposed the nuclear ribosomal DNA second internal transcribed spacer (ITS2) locus as a novel universal DNA barcode to identify herbs based on 6600 samples that represented 4800 species. Han et al. [16] compared the ITS and ITS2 regions and found that ITS2 was more suitable for species identification because of its short length and high efficiency for PCR amplification of this region. Moreover, the sequences and secondary structures of ITS2 could be considered as molecular morphological characteristics for species identification [17]. Considering DNA degradation in Lingzhi products, especially Lingzhi extracts and spore oil, the shorter sequence of ITS2 would likely provide a higher amplification and identification efficiency.
This study aims to authenticate commercial products containing Ganoderma using the DNA barcoding method.

Sample collection and data acquisition
Sixty-three specimens belonging to 11 Ganoderma species were analyzed. Specimens included 33 samples of commercially cultivated fruiting bodies, strains, slices, spore powders, extracts and spore oils collected in this study, and 30 sequences obtained from GenBank (Table 1). Twenty-six samples of G. lucidum, five strains of G. sinense, and two samples of G. resinaceum were collected in this study. Voucher samples were deposited in the herbarium of the Institute of Medicinal Development at the Chinese Academy of Medicinal Science, Beijing, China. Other published Ganoderma ITS2 sequences were downloaded from GenBank and were also analyzed for their ability to identify species in this study. We screened 348 ITS sequences named G. lucidum (or G. lingzhi). Sequences that met the following criteria were selected: (1) the sequences had already been published; (2) the sequences had complete ITS2 regions; and (3) sequences with original samples that were not from East Asia and Europe or if the original location of the sample was unknown, would be abandoned. The original samples of European G. lucidum for ITS2 sequences that we selected were identified based on morphological features by Yun Cao during previous Ganoderma research [11] and were stored in the Mycological Herbarium, Institute of Microbiology, Chinese Academy of Sciences (HMAS).

DNA extraction, PCR amplification, cloning and sequencing
Specimens were divided into three groups. One group included fruiting bodies, slices, spore powders and extracts. Samples of approximately 30 mg were needed and were ground into powder using a Retsch MM400 (Retsch Co., Germany). Strains (50 mg) were homogenized in liquid nitrogen. Spore oil (300 µL) was first centrifuged at 10,625×g for 10 min using a Sigma 1-14K (Sigma Co., Germany), and the pellet was used for DNA extraction. Total genomic DNA was subsequently extracted using the Plant Genomic DNA kit (Tiangen Biotech Co., China) following the recommended protocol. One pair of primers, 156 (5′-AACCATCGAGTCTTT GAACGC-3′) and 157 (5′-CCTTGTAAGTTTCTTTTC CTCC-3′), were designed for PCR amplification of the ITS2 region of Ganoderma. PCR was performed in 25-µL reaction mixtures, containing 12.5 µL of 2 × PCR buffer (Aidlab Biotechnologies Co., China), 1 µL of each PCR primer (2.5 µM), and 2 µL of DNA extract, and the total volume was adjusted to 25 µL with sterile deionized water. PCR amplification was conducted according to the following procedure: 94 °C for 5 min, 40 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min, and a final extension at 72 °C for 10 min. PCR products were analyzed by electrophoresis in a 1 % agarose gel. The PCR products were purified using the PCR Purification Kit (Tiangen Biotech Co., China) and sequenced bidirectionally using a ABI 3730XL sequencer (Applied Biosystems Co., USA) based on the Sanger sequencing method at the Genome Center, Chinese Academy of Agricultural Sciences.

Phylogenetic analysis
The sequences were edited and assembled manually using CodonCode Aligner V3.71 (CodonCode Co., USA). The new sequences obtained in this study were deposited in GenBank. ITS sequences from Gen-Bank were annotated using the Hidden Markov model (HMM) [18] to obtain the ITS2 sequences. All ITS2 sequences were included in the phylogenetic analysis by MEGA 6.0 [19]. All of the sequences were aligned using the MUSCLE method [20]. A neighbor-joining (NJ) [21] tree was constructed with the following parameters: the bootstrap method was conducted with 1000 replicates, the substitution model was Kimura-2-parameter (K2P) [22], and gaps were treated as missing data (complete deletion). Maximum parsimony (MP) [23] trees were constructed with the following parameters: the bootstrap method was conducted with 1000 replicates, the MP search method was subtree pruning and regrafting [24], the number of initial trees was ten (random addition), and gaps were treated as missing data (complete deletion). Sequence divergence was also calculated using the K2P model, and gaps were treated as missing data. Tomophagus colossus was selected as the outgroup. The secondary structure of ITS2 was predicted at the ITS2 database website (http://www.its2.bioapps.biozentrum. uni-wuerzburg.de/) [25].

ITS2 sequence analysis and intra-and inter-species variations
The PCR product sizes for the ITS2 region ranged from 469 to 566 bp. The length of ITS2 was 218 bp after deletion of the 5.8S and 28S rDNAs and alignment using the MUSCLE method. The average G-C and A-T contents of the ITS2 region were 49.4 and 50.6 %, respectively. The aligned ITS2 rDNA sequences are shown in Fig. 1. The 26 newly collected samples of G. lucidum from China had seven intraspecific variable sites (Fig. 1), and 10 of these 26 samples had identical sequences. The ITS2 regions of G. sinense resulted in ambiguous sequences with direct sequencing of the PCR products; thus, a cloning method was used. Nucleotide analysis of the ITS2 region could provide more information about inter-and intra-species divergences. The average intraspecific genetic distances calculated by the Kimura-2-parameter model [22] were 0.007 for G. lucidum from East Asia. No variable sites were detected among the ITS2 regions of nine G. sinense samples collected from Shandong, Hubei and Hainan. The interspecific diversities ranged from 0.035 to 0.047 between G. lucidum from Europe and G. lucidum from East Asia, from 0.097 to 0.111 between G. lucidum from East Asia and G. sinense, and from 0.035 to 0.123 between G. lucidum from East Asia and the other species examined. In this study, the intra-species distances of the Ganoderma species were lower than the inter-species distances except for in G. sichuanense and G. weberianum (Fig. 2).
SNP-based molecular barcodes have been used for identification studies in closely related species [26]. There were seven stable SNPs existing between G. lucidum from East Asia and G. lucidum from Europe, including sites of deletion/insertion, and 25 stable SNPs between G. lucidum from East Asia and G. sinense including four sites of deletion/insertion (Fig. 1). At positions 23, 76, 108 and 196 bp, all G. lucidum samples from Europe contained C, G, A, and G, respectively. Meanwhile, all G. lucidum samples from East Asia contained T, A, G, and T, respectively.

Phylogenetic analysis
Species of the genus Ganoderma, including G. lucidum (from Europe), G. sinense, G. applanatum, G. fornicatuma, G. multipileum, G. resinaceum, G. sichuanense, G. weberianum, G. tenue, and G. tropicum, which are closely related to G. lucidum (from East Asia), were used to study the relationships between Lingzhi species. Sixty-three ITS2 sequences were analyzed. A total of 218 characters were included for phylogenetic analysis, of which 61 were variable and 53 were parsimony informative characters. The consistency index was 0.6914, the retention index was 0.9324, and the composite index was 0.6614 for all sites and parsimony-informative sites (in parentheses).
The topologies of the NJ and MP trees (Fig. 3) were similar. The high level of nucleotide substitution in the ITS2 rDNA resulted in six clades. Although most sequences of either G. lucidum or G. sinense had identical ITS2 sequences, G. lucidum from Europe did not fit into these clades. In the phylogenetic trees, Group 1 consists of collections from G. lucidum from East Asia; 28 sequences of G. lucidum from China and Korea and five sequences of G. lucidum from Europe were clustered into two distinct clades, which were separate from  . G. sichuanense, G. weberianum, and G. tenue were grouped into one well-supported clade (94.3 %), but the relationships among these species require further study. Two samples of G. multipileum from China clustered together and formed a clade with G. lucidum from Asia. G. multipileum, a species for which there was a holotype specimen from Taiwan, was suggested as the correct name for the tropical Ganoderma

Efficiency of species identification
BLAST1 was used to further evaluate the efficiency of ITS2. The barcode sequences obtained in this study were used to build corresponding reference sequence libraries as described previously [28]. The results showed that ITS2 successfully identified 100 % of the commercial Lingzhi products collected in this study.
The ITS2 sequence-structure provided the most accurate phylogenetic analysis [29], and ITS2 sequencestructure information was correlated with the biological species concept [30]. Thus, the RNA secondary structures of ITS2 were analyzed to differentiate the species of G. lucidum. The three closely-related species have similar secondary structures of ITS2 sequences. Stem-loops I, II, and III were conserved, whereas stem-loop IV of the three species varied. The three species could be identified directly based on the RNA secondary structure of ITS2 (Fig. 4).

Discussion
G. lucidum is one of the most economically important species of fungi; thus the stability of its taxonomy is highly important. Different researchers have different views on the scientific binomial of "Chizhi" [11,12,[31][32][33]. In China, Lingzhi has long been misnamed G. lucidum. We identified commercial Lingzhi products based on the ITS2 rDNA marker, and our phylogenetic analysis clearly indicated that Chizhi, or G. lucidum, from East Asia, is not the same species as G. lucidum from Europe. The nucleotide divergence among G. lucidum from Europe and G. lucidum from East Asia, as well as the high bootstrapping support, indicated that they were different species. Analysis of RNA secondary structure further supported these results.
Identifying Ganoderma products (such as spore oil and extracts) according to morphological characteristics alone is difficult. The triterpenes of G. lucidum and G. sinense show significant differences in terms of types and content, and a distinction should be made between the medical uses of the two species [34][35][36][37][38]. The use of systematic methods for species identification and classification would be useful. The evolutionary context of the related species should be studied first to identify the biological species. An ideal barcode sequence should possess high inter-species divergence but low intra-species divergence to readily identify different species. ITS2 has a wider taxonomic coverage than was previously assumed because of the high sequence variability and conserved core secondary structure [16]. Moreover, ITS2 had  accurately identify commercial Lingzhi products. Our results showed that most Ganoderma products, including Chizhi (G. lucidum), Zizhi (G. sinense) and other Ganoderma species, could be successfully identified using ITS2 sequences. Our results also support the suggestion that G. sinense and G. japonicum should be considered synonymous because of their high sequence similarity. In this study, regardless of the complicated taxonomy of Ganoderma, the sequence-based phylogeny supported the hypothesis that G. lucidum species originating in Europe and East Asia are not the same species.

Conclusion
The DNA barcoding method is applicable to the authentication of commercial products containing Ganoderma species.