Re-Examination of the Holotype of Ganoderma sichuanense (Ganodermataceae, Polyporales) and a Clarification of the Identity of Chinese Cultivated Lingzhi

The widely cultivated Chinese Lingzhi is a famous fungus with significant medicinal and economic value, which has commonly been misidentified as Ganoderma lucidum for a long period of time. The scientific binomial of the fungus is always a hotly debated question that revolves around G. lingzhi and G. sichuanense. To interpret the species concept of the taxon, six specific primers for G. sichuanense and one universal primer were designed. Through directed and nested PCRs, we obtained nine ITS sequences from the holotype (HMAS 42798) of G. sichuanense. By genome sequencing, the ITS sequence of the first cultivated Lingzhi (HMAS 25103) was assembled. Based on a phylogenetic study of the genus Ganoderma, the correct name for widely cultivated Ganoderma species in China was confirmed as G. sichuanense, and G. lingzhi should be a later synonym.


Introduction
Ganoderma P. Karst. is a cosmopolitan genus established by Karsten [1], based on the generic type G. lucidum (Curtis) P. Karst from England [2]. The use of Ganoderma mushroom in China can be traced back to 6800 years ago [3], and species of Ganoderma have had a considerable impact on Chinese history [4]. According to Tai [5], Patouillard first identified G. lucidum in China in 1907 by the specimens collected from Guizhou Province.
However, based on the morphological characters, such as the thickness of context and diameter of the stipe, Pegler and Yao suggested that the widely cultivated "G. lucidum" (as "Lingzhi" or "Ruizhi" in Chinese) in China was not conspecific with the species described in Europe [6]. The result was also supported by phylogenetic evidence [7][8][9][10][11]. In 1959, the first successful cultivation of the fruit bodies of Lingzhi was developed by the Institute of Microbiology, Chinese Academy of Sciences. Approximately 10 years later, under the vigorous promotion of the Ganoderma research group at the Institute of Microbiology [12], the cultivation of Lingzhi became an important industry in China and other adjacent countries. At present, this fungus is famous as a traditional Chinese medicine possessing great economic value [13][14][15][16][17].
The scientific binomial for this economically and medicinally important fungus, Lingzhi, has long been controversial, which was considered to be G. lucidum for a long period in China. Using molecular phylogeny, Wang et al. highlighted that the Asian G. lucidum specimens were separated from the European G. lucidum by two individual clades, and the tropical collections from Asian areas represented G. multipileum D. Hou 1950, while the classification status of the other collections obtained from mainland China, Japan, and Korea was uncertain [11]. Wang et al. recognized the uncertain clade as G. sichuanense J.D. Zhao & X.Q. Zhang [18,19], which is the Ganoderma species widely cultivated in China. However, Cao et al. proposed it as a new species G. lingzhi Sheng H. Wu, Y. Cao & Y.C. Dai based on a single available internal transcribed spacer (ITS) sequence from the holotype (HMAS 42798) of G. sichuanense [20]. Yao et al. designated an epitype (HMAS 252081) to interpret the species concept of Lingzhi and secured the position of the holotype of G. sichuanense (HMAS 42798), both morphologically and molecularly [21]. However, based on the holotype sequence from Cao et al. [20], the epitype was not accepted by some researches [22][23][24]. Yao et al. re-clarified the typification of G. sichuanense and demonstrated that the epitype of G. sichuanense was appropriately designated to support the holotype of the name [25].
To clarify the confusion of this important fungus, we designed six specific primers for G. sichuanense and one universal primer to obtain the ITS sequence from the G. sichuanense holotype (HMAS 42798) (by directed PCR and nested PCR), which is the key point of the hot topic. For the fruit bodies of the first cultivated Lingzhi (HMAS 25103), its DNA had been largely degraded, so genome sequencing was chosen. Based on all these representative sequences, we performed a phylogenetic study of the genus Ganoderma, including the type materials of G. sichuanense (holotype, epitype, and topotype) and G. lingzhi (holotype). As a result, we can confirm that G. lucidum is a name mistakenly applied to the widely cultivated Ganoderma species in China, that the scientific binomial for Lingzhi is G. sichuanense, and that the designation of the epitype is necessary to support the holotype because of its poor DNA status. G. lingzhi is the later synonym of G. sichuanense.

Specimens
The fungal collections are deposited in the Fungarium of the Institute of Microbiology (HMAS) of the Chinese Academy of Sciences; including the holotype of G. sichuanense (HMAS 42798), the topotype (HMAS 244431) collected from Panzhihua City (previously "Dukou Shi") in Sichuan Province, and the first cultivated Lingzhi (HMAS 25103), performed by Zhuang Deng, the daughter and assistant of Professor Shu-Chün Teng, in 1959.

DNA Samples
A total of 24 genomic DNA samples were extracted from the holotype (HMAS 42798) by Xin-Cun Wang and Li Yi in 2010 using various methods, including the CTAB method described in Jiang and Yao [26], the Wizard ® Genomic DNA Purification Kit (Promega, U.S.A.), and Chelex 100 Resin (Solarbio, China). The DNA samples were kept in ultra-low temperature freezer (below −80 • C) until use. The additional sampling of the topotype (HMAS 244431) was performed by Zhuo Du separately to avoid any possible contamination, using a DNA Extracting Kit (Cat#: NEP023-1) distributed by Beijing Dingguochangsheng Biotechnology Co. Ltd. (Beijing, China), following the instructions of the manufacturer.

Specific Primer Design, Amplification, and Sequencing
The specific primers for G. sichuanense were designed based on the sequence alignment of ITS sequences obtained from G. lucidum, G. multipileum, G. resinaceum, G. sichuanense, G. tropicum, and G. weberianum. Primer 3 v. 0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/, accessed on 15 June 2018) software was employed in combination with manual adjustments. The primers were selected and tested using Primer 5 v. 5.00 and then utilized to perform amplifications ( Table 1). The suggested annealing temperature of Primer 5 v. 5.00 was tested in PCR and compared to the conventional temperature of 55 • C, and the latter was adopted throughout the experiment.
The primers used in the amplifications included ITS5, ITS1, and ITS4 for both ends, ITS2 and ITS3 were utilized for the internal positions of the whole length of ITS1-5.8S-ITS2 [27]. ITSGs1-1, ITSGs1-2, ITSGs2-1, ITSGs2-2, ITSGs2-4, and ITSGs4-2, are specific to G. sichuanense and ITSGs4-4 is a universal primer, all of which were designed in the present study. The sequences and locations of the newly designed primers are presented in Table 1 and Figure 1. study. The sequences and locations of the newly designed primers are presented in Table  1 and Figure 1.  Both directed and nested PCRs with various combinations of primer pairs were used to obtain better results. PCR thermal cycling was performed in 25 μL reaction mixtures containing 1 μL of DNA template, 12.5 μL of 2 × PCR Master Mix, 1 μL of each PCR primer (10 μM), and 9.5 μL of double-distilled H2O. For the nested PCR, the second round of reactions consisted of a template at a 1:10 dilution of the first round PCR product. The PCR protocol comprised denaturation at 95 °C for 4 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a final cycle at 72 °C for 10 min.
1. Directed PCR: the following 4 primer combinations were used: ITS1/ITSGs1-2, ITSGs1-1/ITS2, ITS3/ ITSGs2-2, and ITSGs2-1/ ITS4. 2. Nested PCR: ITS5/ITS4 was followed by 6 different primer pairs as internal primers ( Table 2).  Both directed and nested PCRs with various combinations of primer pairs were used to obtain better results. PCR thermal cycling was performed in 25 µL reaction mixtures containing 1 µL of DNA template, 12.5 µL of 2 × PCR Master Mix, 1 µL of each PCR primer (10 µM), and 9.5 µL of double-distilled H 2 O. For the nested PCR, the second round of reactions consisted of a template at a 1:10 dilution of the first round PCR product. The PCR protocol comprised denaturation at 95 • C for 4 min, followed by 30 cycles of 95 • C for 30 s, 55 • C for 30 s, 72 • C for 1 min, and a final cycle at 72 • C for 10 min.

2.
Nested PCR: ITS5/ITS4 was followed by 6 different primer pairs as internal primers ( Table 2). Based on the aim of obtaining a full-length ITS sequence, all the PCR products of directed PCRs and two groups of nested PCRs (internal primer sets: ITS1/ITSGs4-2 and ITSGs1-1/ITSGs4-4) were selected to create a sequence. DNA sequencing was performed using an ABI PRISM ® 3730XL DNA Analyzer with a BigDye ® Terminator Kit v3.1 at the Tsingke Biological Technology Company (Beijing, China)

Genome Sequencing
The first artificially cultivated Lingzhi (HMAS 25103) was performed in 1959. Due to the long time preservation, the surface was contaminated with other fungi. The DNA condition of the specimen was very poor and heavily disintegrated. All the methods mentioned above used to amplify the DNA fragments from this specimen were unsuccessful; therefore, genome sequencing was performed to obtain the ITS region sequence.
Approximately 50 mg of ground tissue from HMAS 25103 was sent to the Shanghai Biozeron Biotechnology Company (Shanghai, China). DNA extraction was performed by the company using E.Z.N.A. ® Stool DNA Kits (OMEGA Bio-tek, Norcross, GA, USA), and the quality was tested on 1% agarose by Covaris M220. Paird-end (PE) libraries were developed using the TruSeq™ DNA Sample Prep Kit. Then a cBot Truseq PE Cluster Kit v3-cBot-HS was utilized to accomplish bridge PCR. Additionally, Illumina sequencing was carried out by a Truseq SBS Kit (300 cycles).

Phylogenetic Analyses
A total of 185 DNA sequences generated by each forward and reverse primer were used to obtain consensus sequences using Seqman v.7.1.0 in the DNASTAR laser gene core suite software (DNASTAR, Madison, WI, USA). The single gene ITS sequences were initially aligned with Clustal W and implemented in MEGA 6 and improved by MAFFT v.7 [28,29]. Tomophagus colossus (Fr.) Murrill was selected as the outgroup taxon for all analyses [18,21]. The aligned matrices used for the phylogenetic analyses were maintained in TreeBASE (www.treebase.org; accession number: 30118).
RAxML-HPC BlackBox v.8.2.10 was performed to construct a maximum likelihood (ML) tree, employing a GTRGAMMA substitution model with 1000 bootstrap replicates [30]. The branch support of ML analyses was evaluated using bootstrapping (BS) method for 1000 replicates. Bayesian inference (BI) was performed using a Markov Chain Monte Carlo (MCMC) algorithm to construct the topology of the tree [31]. Two MCMC chains were run from random trees for 10,000,000 generations and stopped when the average standard deviation of the split frequencies fell below 0.01. From each 1000 generations, the trees were saved. The first 25 % of trees were discarded as the burn-in phase of each analysis, and the posterior probabilities (BPP) were calculated to assess the remaining trees [32]. Phylograms were presented using Figtree v. 1.3.1 and processed by Adobe Illustrator CS v.5. Reference sequences were selected based on the type materials available in GenBank and published papers. The sequence data of the present study were deposited in GenBank, and the GenBank accession numbers of all accessions included in the phylogenetic analyses are listed in Table 3.

Results of PCR, Sequencing Using Different Primers
Both directed and nested PCRs were adopted for 24 DNA samples. The results of various combinations of primer pairs are presented in Table 4. Throughout the directed PCR procedure, using published primer pair ITS5/ITS4, only sample 1 was successfully sequenced, but it proved to be Aspergillus sp. For ITS1/ITS4, the sequencing result for sample 1 was the same as when using primer pair ITS5/ITS4, and sample 4 was Gymnopus sp. When using the ITS1/ITS2, samples 1 and 22 were all Aspergillus sp. The ITS3/ITS4 results were the same as using the primer pair ITS1/ITS4. When using specific primer pairs designed in this study for G. sichuanense (ITS1/ITSGs1-2, ITSGs1-1/ITS2, ITS3/ITSGs2-2, and ITSGs2-1/ITS4) from 24 DNA samples, we obtained 2, 6, 3, and 1 sequences respectively, 13 short fragments in total, which can assemble into one complete G. sichuanense sequences (HMAS 42798-d, Gene Bank number OP805623).
In the nested PCR experiment, ITS5/ITS4 was selected as the external primer pair, when using ITS1/ITS4 as the internal primer pair, the PCR amplification resulted in the presence of polymorphic bands. Only eight samples produced sequencing results (Tsingke Biological Technology). The results of the taxonomic groups belong to Aspergillus, Astraeus, Cercospora, Cladosporium, Cryptococcus, and Pleosporales. When using the ITS1/ITSGs4-4 primer pair as the internal primers, the agarose electrophoresis results were the same.

Results of Genome Sequencing and Assembly
Genome sequencing produced 7,230,782 raw reads (1,084,617,300 bp) for HMAS 25103, resulting in 4,121,318 clean reads (612,283,701 bp) following filtration. Filtration refers to removing adapter sequences, low-quality reads, and reads higher than a certain proportion (10%) of N (ambiguous sites). Cleaned reads were assembled using MegaHit [33]. The assemblies contained 15,034 contigs (N50 = 6656 bp), the lengths of which were 31,314,762. The ITS sequence alignments included 61 species of Ganoderma were used as queries to search the possible target sequences obtained from the genome assemblies. A sole 556 bp nuclear ribosomal DNA (nrDNA) fragment was obtained, which contained a partial 5.8S ribosomal RNA gene, complete ITS2, and partial large subunit ribosomal RNA gene sequence. Additionally, the sequence was submitted to GenBank (Gene Bank number OP805628).

Results of Phylogenetic Analyses
The ITS dataset from 185 strains was analyzed to infer the interspecific relationships within Ganoderma. The sequences were clustered in 62 groups representing 61 known species of Ganoderma with 49 type isolates and 1 outgroup taxon Tomophagus colossus. The topologies resulting from the ML and BI analyses of the concatenated dataset were congruent. A total of 14 sequences obtained from the present study were formed in 1 individual clade (clade A) representing the species G. sichuanese (Figure 3).

Results of Genome Sequencing and Assembly
Genome sequencing produced 7,230,782 raw reads (1,084,617,300 bp) for HMAS 25103, resulting in 4,121,318 clean reads (612,283,701 bp) following filtration. Filtration refers to removing adapter sequences, low-quality reads, and reads higher than a certain proportion (10%) of N (ambiguous sites). Cleaned reads were assembled using MegaHit [33]. The assemblies contained 15,034 contigs (N50 = 6656 bp), the lengths of which were 31,314,762. The ITS sequence alignments included 61 species of Ganoderma were used as queries to search the possible target sequences obtained from the genome assemblies. A sole 556 bp nuclear ribosomal DNA (nrDNA) fragment was obtained, which contained a partial 5.8S ribosomal RNA gene, complete ITS2, and partial large subunit ribosomal RNA gene sequence. Additionally, the sequence was submitted to GenBank (Gene Bank number OP805628).

Results of Phylogenetic Analyses
The ITS dataset from 185 strains was analyzed to infer the interspecific relationships within Ganoderma. The sequences were clustered in 62 groups representing 61 known species of Ganoderma with 49 type isolates and 1 outgroup taxon Tomophagus colossus. The topologies resulting from the ML and BI analyses of the concatenated dataset were congruent. A total of 14 sequences obtained from the present study were formed in 1 individual clade (clade A) representing the species G. sichuanese (Figure 3).    Notes: A total of 43 sequences of G. sichuanense were used in this study and diverged into two different evolutionary branches. Except for two sequences (JQ781877 and JQ781878) grouped with G. weberianum, other sequences, including nine sequences obtained from the holotype of G. sichuanense (HMMAS 42798) in this study, were all gathered in clade A. Additionally, all sequences of the name "G. lingzhi" were also mixed in clade A. It is worth noting that clade A included many sequences from the type or pivotal materials of the two species mentioned above, as follows: (1) the sequence of G. lingzhi (Wu 1006-38) holotype and all other G. lingzhi sequences used in the paper of Cao et al. [20]; (2) nine sequences of the G. sichuanense (HMAS 42798) holotype obtained using two different independent PCR methods; (3) the sequence obtained from G. sichuanense (HMAS 252081) epitype [21] and four sequences obtained from different fruit bodies of topotype (HMAS 244431); (4) the sequence of first cultivated Lingzhi fruit bodies (HMAS 25103) in China. Our research suggests that clade A should be the authentic G. sichuanense. The new species G. lingzhi was proposed on the premise that ITS sequence JQ781877 was unquestionably obtained from the holotype of G. sichuanense [20], but the sequence JQ781877 was obtained only once [20,34]. The reasons why we insisted that the sequences of the holotype presented in this research are reliable are (1) the DNA samples of the G. sichuanense holotype were extracted using different methods, the CTAB method, the Wizard ® Genomic DNA Purification Kit (Promega, U.S.A.), and Chelex 100 Resin (Solarbio, China); (2) by directed and nested PCRs, the sequences obtained from different DNA samples from the G. sichuanense (HMAS 42798) holotype were clustered together in the phylogenetic analysis; (3) the first cultivated Lingzhi (HMAS 25103), which represents the widely cultivated Chinese Ganoderma species, was also clustered in the evolutionary branch of G. sichuanense.
Complying with the International Code of Nomenclature for algae, fungi, and plants (Art.11.3), the earliest legitimate name of a taxon should be given priority [35]. Ganoderma sichuanense [19] has more preference than G. lingzhi [20]. The current results treat G. lingzhi as a later synonym of G. sichuanense.

Discussion
Although gene conversion [36] and unequal cross-over [37] are two of the most commonly proposed concerted evolution events [38], numerous studies have revealed that ITS is a multicopy gene and does not subscribe to evolution perfectly. Intra-strain and intra-species variations exist in several fungal taxa, such as Fusarium [39], Laetiporus [40], Ophiocordyceps [41], Scutellospora [42], Xanthophyllomyces [43], and also Ganoderma [44]. The results of our phylogenetic analysis demonstrated the ITS sequence heterogeneity within the holotype of G. sichuanense (HMAS 42798). This is the first report of ITS heterogeneity for G. sichuanense; heterogeneity occurs in three parts (ITS1, ITS2, and 5.8S) of the ITS region. This might explain why small divisions exist in the whole, large clade of G. sichuanense (Clade A). For nested PCR, the results are identical when using different primer pairs as second-round primer sets (ITS1/ITSGs4-2 and ITSGs1-1/ITSGs4-4). Therefore, we speculate that this phenomenon might be the nature of the species G. sichuanense.
In the specimen box of the holotype, two fruit bodies existed (Figure 4). We can confirm that the sequence OP805618 (sample 5, HMAS 42798-5) was obtained from the big fruit body, and the sequence OP805619 (sample 6, HMAS 42798-6) was obtained from the small one. For the other DNA samples from the holotype, depending on the information on the lid of the DNA sample container and the lab notebook of the operator, we were unable to verify which fruit body was taken for (the DNA samples were extracted in 2010), but samples 5 and 6 ensured that all the fruit bodies of the holotype obtained ITS sequence successfully.
We provided ITS sequences to perform a phylogenetic study based on the root of the long-standing discussion and the obtainable experimental results. The aim of the research was to resolve the problem of the scientific binomial for the widely cultivated Lingzhi in China, the discussion of which has centered around the controversial ITS sequence JQ781877 since 2012 [20]. We designed seven primers, using different PCR methods to successfully obtain repeatable and reliable ITS sequences from the holotype. Additionally, in our experiment, the attempts to amplify the other gene sequences from the holotype failed due to the largely disintegrated DNA. In some research papers concerning Ganoderma, even though other gene loci were applied, for the specimen HMAS 42798, only ITS sequence JQ781877 was available [20,22,45]. Therefore, based on the fact that the crux of the dispute was the key ITS sequence, and no other gene sequences of the holotype were available, the ITS phylogenic tree presented in this study can break the present deadlock. Though the ITS gene can perform species division in the genus Ganoderma [20,21] and is the most abundant gene region in Ganodermataceae [45], depending on a single gene does not address all the problems in the classification of Ganoderma. For the complex groups in Ganoderma or the higher rank classification of Ganodermataceae, multigene phylogenetic analyses are essential [22,[45][46][47][48][49][50][51][52].
For the old specimens and DNA samples used in this research, any preserved DNA was present only in small amounts and in various states of degradation. Therefore, we explored various approaches for DNA amplification from the important, old samples, including designing new primers for directed and nested PCRs and genome sequencing. All the methods mentioned in this paper may also be applied to type material of other important species. China, the discussion of which has centered around the controversial ITS sequence JQ781877 since 2012 [20]. We designed seven primers, using different PCR methods to successfully obtain repeatable and reliable ITS sequences from the holotype. Additionally, in our experiment, the attempts to amplify the other gene sequences from the holotype failed due to the largely disintegrated DNA. In some research papers concerning Ganoderma, even though other gene loci were applied, for the specimen HMAS 42798, only ITS sequence JQ781877 was available [20,22,45]. Therefore, based on the fact that the crux of the dispute was the key ITS sequence, and no other gene sequences of the holotype were available, the ITS phylogenic tree presented in this study can break the present deadlock. Though the ITS gene can perform species division in the genus Ganoderma [20,21] and is the most abundant gene region in Ganodermataceae [45], depending on a single gene does not address all the problems in the classification of Ganoderma. For the complex groups in Ganoderma or the higher rank classification of Ganodermataceae, multigene phylogenetic analyses are essential [22,[45][46][47][48][49][50][51][52].
For the old specimens and DNA samples used in this research, any preserved DNA was present only in small amounts and in various states of degradation. Therefore, we explored various approaches for DNA amplification from the important, old samples, including designing new primers for directed and nested PCRs and genome sequencing. All the methods mentioned in this paper may also be applied to type material of other important species.  Institutional Review Board Statement: Not applicable for studies involving humans or animals.

Informed Consent Statement:
Not applicable for studies involving humans.

Data Availability Statement:
The sequences in the present study were submitted to the NCBI website (https://www.ncbi.nlm.nih.gov/), and the accession numbers are listed in Table 3.

Conflicts of Interest:
The authors declare no conflict of interest.  Institutional Review Board Statement: Not applicable for studies involving humans or animals.

Informed Consent Statement:
Not applicable for studies involving humans.