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.

Deep Sequencing, Nested PCR, and Denaturing Gradient Gel Electrophoresis Reveal a Wider Distribution of Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes), in Native Soil Types.

Ophiocordyceps sinensis appears as stroma emerging from underground sclerotium enclosed by the skeleton of Thitarodes moth larvae. However, the actual distribution of the fungus in soil still remains unclarified. In this study, 40 soil samples were used for detection of O. sinensis to confirm its distribution in native habitats using denaturing gradient gel electrophoresis, nested internal transcribed spacer (ITS) PCR, and 454 pyrosequencing methods. The soil samples included six types: Os, where both stromata and host moth larvae were found; NL, representing no signs of stromata, but where moth larvae were found; NOs, where neither stroma nor moth larvae were found; BS, with bare soil without the presence of stroma of O. sinensis or moth larvae; AF, from soil surrounding the stroma; and MP, soil particles firmly wrapping the sclerotium of O. sinensis. Of 40 samples tested, 36 showed positive detection of O. sinensis by at least one of the three detection methods, with positive detection in all six sample types at all five sites. The results showed that traces of O. sinensis can be detected in locations with no macroscopically visible evidence of the fungus or its host and at least 100 m away from such locations.

RIP mutated ITS genes in populations of Ophiocordyceps sinensis and their implications for molecular systematics.

Different hypotheses have been proposed to interpret the observed unusual ITS (internal transcribed spacer) sequences in Ophiocordyceps sinensis. The coexistence of diverged ITS paralogs in a single genome was previously shown by amplifying the ITS region from mono-ascospore isolates using specific primers designed for different ITS paralog groups. Among those paralogs, are AT-biased ITS sequences which were hypothesized to result from repeat-induced point mutation (RIP). This is a process that detects and mutates repetitive DNA and frequently leads to epigenetic silencing, and these mutations have been interpreted as pseudogenes. Here we investigate the occurrence and frequency of ITS pseudogenes in populations of O. sinensis using large-scale sampling, and discusses the implications of ITS pseudogenes for fungal phylogenetic and evolutionary studies. Our results demonstrate a wide distribution of ITS pseudogenes amongst different geographic populations, and indicate how ITS pseudogenes can contribute to the reconstruction of the evolutionary history of the species.

On the Typification of Ganoderma sichuanense (Agaricomycetes)-the Widely Cultivated Lingzhi Medicinal Mushroom.

The well-known and widely cultivated lingzhi has had a significant impact on Chinese culture and is now an important fungal crop providing medicinal benefits to human health and economic value to social development within China and around the world. The European mushroom name, Ganoderma lucidum, has been misapplied to this species for over 100 years until recently reidentified as G. sichuanense. Soon after this, a new species name, G. lingzhi, was also proposed for the fungus because of an unusual internal transcribed spacer (ITS) sequence purportedly of the holotype of G. sichuanense. This extraordinary ITS sequence, which apparently belongs to another species, created an inconsistency between morphological characteristics and molecular data of the holotype making it "demonstrably ambiguous"; this led to an epitypification to support the holotype for the precise application of the name, according to the International Code of Nomenclature for algae, fungi, and plants. However, arguments concerning the names G. sichuanense and G. lingzhi are still heating up, including attempts to reject the epitype of G. sichuanense. To clarify the confusion, the typification of G. sichuanense is reviewed here to demonstrate that the epitype of G. sichuanense was appropriately designated for the purpose to support the holotype of the name, the fact that both G. sichuanense and G. lingzhi are conspecific, and that the name G. lingzhi was based on the unwarranted ITS sequence claimed to be of the holotype of G. sichuanense. Suggestions are made for this case to make a way forward, especially re-examination of relevant fungarium collections to reach a consensus to stabilize the use of the name.

Classification, Biological Characteristics and Cultivations of Ganoderma.

Species of Ganoderma (Ling-zhi) have been widely researched and cultivated due to their highly prized medicinal value, which is famous as a traditional Chinese medicine. The aims of this chapter are to (1) review the historical taxonomy of the family Ganodermataceae, (2) provide an account of the genera and species of Ganoderma together with the distributions and habitats, (3) evaluate morphological features and phylogenetic methods to define the genera and species and (4) present two commonly used cultivated methods (wood-log cultivation and substitute cultivation) for Ganoderma.

Evaluation of the ribosomal DNA internal transcribed spacer (ITS), specifically ITS1 and ITS2, for the analysis of fungal diversity by deep sequencing.

The nuclear ribosomal DNA internal transcribed spacer (ITS) has been widely used to assess the fungal composition in different environments by deep sequencing. To evaluate the ITS in the analysis of fungal diversity, comparisons of the clustering and taxonomy generated by sequencing with different portions of the whole fragment were conducted in this study. For a total of 83,120 full-length ITS sequences obtained from the UNITE database, it was found that, on average, ITS1 varied more than ITS2 within the kingdom Fungi; this variation included length and GC content variations and polymorphisms, with some polymorphisms specific to particular fungal groups. The taxonomic accuracy for ITS was higher than that for ITS1 or ITS2. The commonly used operational taxonomic unit (OTU) for evaluating fungal diversity and richness assigned several species to a single OTU even with clustering at 99.00% sequence similarity. The clustering and taxonomic capacities did not differ between ITS1 and ITS2. However, the OTU commonality between ITS1 and ITS2 was very low. To test this observation further, 219,741 pyrosequencing reads, including 39,840 full-length ITS sequences, were obtained from 10 soil samples and were clustered into OTUs. The pyrosequencing results agreed with the results of the in silico analysis. ITS1 might overestimate the fungal diversity and richness. Analyses using ITS, ITS1 and ITS2 yielded several different taxa, and the taxonomic preferences for ITS and ITS2 were similar. The results demonstrated that ITS2 alone might be a more suitable marker for revealing the operational taxonomic richness and taxonomy specifics of fungal communities when the full-length ITS is not available.

rRNA Pseudogenes in Filamentous Ascomycetes as Revealed by Genome Data.

The nuclear ribosomal DNA (rDNA) is considered as a paradigm of concerted evolution. Components of the rDNA tandem repeats (45S) are widely used in phylogenetic studies of different organisms and the internal transcribed spacer (ITS) region was recently selected as a fungal DNA bar code. However, rRNA pseudogenes, as one kind of escape from concerted evolution, were reported in a wide range of organisms, especially in plants and animals. Moreover, large numbers of 5S rRNA pseudogenes were identified in several filamentous ascomycetes. To study whether rDNA evolves in a strict concerted manner and test whether rRNA pseudogenes exist in more species of ascomycetes, intragenomic rDNA polymorphisms were analyzed using whole genome sequences. Divergent rDNA paralogs were found to coexist within a single genome in seven filamentous ascomycetes examined. A great number of paralogs were identified as pseudogenes according to the mutation and secondary structure analyses. Phylogenetic analyses of the three rRNA coding regions of the 45S rDNA repeats, i.e., 18S, 5.8S, and 28S, revealed an interspecies clustering pattern of those different rDNA paralogs. The identified rRNA pseudogenic sequences were validated using specific primers designed. Mutation analyses revealed that the repeat-induced point (RIP) mutation was probably responsible for the formation of those rRNA pseudogenes.

New Germplasms of the Culinary-Medicinal Button Mushroom, Agaricus bisporus (Agaricomycetes): Two Wild Strains from the Tibetan Plateau (China).

Agaricus bisporus is one of the most important commercially cultivated culinary-medicinal mushrooms worldwide. In China, most of the cultivated strains of the fungus were introduced from other countries and cultivated in the eastern provinces. In this study, 2 wild strains of A. bisporus, 2091 and 2094, isolated from fresh specimens collected from the Tibetan Plateau, were domesticated and cultivated alongside a commercial hybrid strain, As2796, in Lhasa, China, for comparison in order to provide new germplasms for cultivation. Basic characteristics, mushroom yield, dry weight, polysaccharide contents, and antioxidant activities of the tested strains were analyzed. Compared with strain As2796, the 2 wild strains displayed good values for mycelial growth, time to fruiting, mushroom yield, dry weight, and polysaccharide contents, and their basidiomata had distinct morphological characteristics (e.g., brown or pale brown caps with some white scales). In addition, the antioxidant activities (reducing power and DPPH radical scavenging effect) of strain 2094 were significantly higher than those of the other 2 strains. Domestication of the 2 wild strains would add more genetic variation into the germplasm of A. bisporus for cultivation, especially in China, and might help to address the problem inherent to the nearly monoculture crop lacking genetic diversity in China.

Total Phenolic Content and Antioxidant Activity of Mycelial Extracts from the Medicinal Fungus Paecilomyces hepiali (Ascomycetes).

Paecilomyces hepialid, a fungus originating in the Tibetan Plateau, has been used as a substitute for Ophiocordyceps sinensis because the primary chemical compounds and pharmacological effects of P. hepialid are similar to those of O. sinensis. P. hepialid has been developed into a dietary supplement and pharmaceutical products. The antioxidant activity of extracts using 2 solvents (water and ethanol) from mycelia obtained from 2 cultivation modes (solid-state and submerged cultivation) were evaluated in this study. Four strains of P. hepialid obtained from Qinghai, Sichuan, and Yunnan Provinces were included; the total phenolic content and in vitro antioxidant activity of mycelial extracts were compared. The total phenolic content and antioxidant activity of strains were found to be affected by the cultivation mode and extraction solvent. The ethanol extracts of solid-state cultivation of strain 2138, obtained from Sichuan Province, exhibited the highest antioxidant activity. The results showed that different strains might require different cultivation modes and extraction solvents for better antioxidant activity. However, solid-state cultivation and ethanol extraction are generally recommended based on the analyses conducted. Strain 2138 may be a good candidate for the purpose of producing functional foods. The results suggest that strain selection is important when P. hepialid is used to manufacture pharmaceutical products.

Beauveria medogensis sp. nov., a new fungus of the entomopathogenic genus from China.

Beauveria is among the most ubiquitous genera of entomopathogenic fungi throughout the world. A previously unknown species of the genus was recently discovered from a soil sample collected from Tibetan Plateau, China and is here described as new to science, B. medogensis sp. nov. The new species is distinguished from its closest relatives based on both morphological characterization and molecular phylogenetic analyses. Beauveria medogensis is characterized by globose to subglobose conidia, morphologically similar to some other species of in the genus, but was conclusively separated from those species in the phylogenetic analyses including sequences of four nuclear genes (RPB1, RPB2, TEF1 and Bloc). The new species was clustered in the analyses in a single terminal lineage which was grouped with B. australis sequences together as a sister clade to the B. brongniartii terminal clade. Although molecularly closely related, the new species is distinct morphologically from its closest sisters, B. australis and B. brongniartii, in producing globose to subglobose conidia rather than subglobose, broadly ellipsoid to ellipsoid conidia or ellipsoidal to cylindrical conidia. As isolated from a soil sample, the entomopathogenicity of the new species has been confirmed using Helicoverpa armigera and Tenebrio molitor larvae.

Comparison of different sequencing and assembly strategies for a repeat-rich fungal genome, Ophiocordyceps sinensis.

Ophiocordyceps sinensis is one of the most expensive medicinal fungi world-wide, and has been used as a traditional Chinese medicine for centuries. In a recent report, the genome of this fungus was found to be expanded by extensive repetitive elements after assembly of Roche 454 (223Mb) and Illumina HiSeq (10.6Gb) sequencing data, producing a genome of 87.7Mb with an N50 scaffold length of 12kb and 6972 predicted genes. To test whether the assembly could be improved by deeper sequencing and to assess the amount of data needed for optimal assembly, genomic sequencing was run several times on genomic DNA extractions of a single ascospore isolate (strain 1229) on an Illumina HiSeq platform (25Gb total data). Assemblies were produced using different data types (raw vs. trimmed) and data amounts, and using three freely available assembly programs (ABySS, SOAP and Velvet). In nearly all cases, trimming the data for low quality base calls did not provide assemblies with higher N50 values compared to the non-trimmed data, and increasing the amount of input data (i.e. sequence reads) did not always lead to higher N50 values. Depending on the assembly program and data type, the maximal N50 was reached with between 50% to 90% of the total read data, equivalent to 100× to 200× coverage. The draft genome assembly was improved over the previously published version resulting in a 114Mb assembly, scaffold N50 of 70kb and 9610 predicted genes. Among the predicted genes, 9213 were validated by RNA-Seq analysis in this study, of which 8896 were found to be singletons. Evidence from genome and transcriptome analyses indicated that species assemblies could be improved with defined input material (e.g. haploid mono-ascospore isolate) without the requirement of multiple sequencing technologies, multiple library sizes or data trimming for low quality base calls, and with genome coverages between 100× and 200×.

Complete mitochondrial genome of the medicinal fungus Ophiocordyceps sinensis.

As part of a genome sequencing project for Ophiocordyceps sinensis, strain 1229, a complete mitochondrial (mt) genome was assembled as a single circular dsDNA of 157,510 bp, one of the largest reported for fungi. Conserved genes including the large and small rRNA subunits, 27 tRNA and 15 protein-coding genes, were identified. In addition, 58 non-conserved open reading frames (ncORFs) in the intergenic and intronic regions were also identified. Transcription analyses using RNA-Seq validated the expression of most conserved genes and ncORFs. Fifty-two introns (groups I and II) were found within conserved genes, accounting for 68.5% of the genome. Thirty-two homing endonucleases (HEs) with motif patterns LAGLIDADG (21) and GIY-YIG (11) were identified in group I introns. The ncORFs found in group II introns mostly encoded reverse transcriptases (RTs). As in other hypocrealean fungi, gene contents and order were found to be conserved in the mt genome of O. sinensis, but the genome size was enlarged by longer intergenic regions and numerous introns. Intergenic and intronic regions were composed of abundant repetitive sequences usually associated with mobile elements. It is likely that intronic ncORFs, which encode RTs and HEs, may have contributed to the enlarged mt genome of O. sinensis.

Stucturally Diverse Sesquiterpenes Produced by a Chinese Tibet Fungus Stereum hirsutum and Their Cytotoxic and Immunosuppressant Activities.

Two new heterodimeric sesquiterpenes, sterhirsutins C (1) and D (2), along with eight new sesquiterpenoid derivatives, sterhirsutins E--L (3-10), were isolated from the culture of Stereum hirsutum. The absolute configuration of 1 was assigned by a single-crystal X-ray diffraction experiment. Compounds 1 and 2 possessed an unprecedented chemical skeleton with a 5/5/5/6/9/4 fused ring system. Compound 10 is the first sesquiterpene coupled with a xanthine moiety. Compounds 1-10 showed cytotoxicity against K562 and HCT116 cell lines. Compound 9 induced autophagy in HeLa cells. Compound 5 inhibited the activation of IFNß promoter in Sendai virus infected cells.

Gloeophyllins A-J, Cytotoxic Ergosteroids with Various Skeletons from a Chinese Tibet Fungus Gloeophyllum abietinum.

Ten new ergosteroids, gloeophyllins A-J (1-10), have been isolated from the solid cultures of Gloeophyllum abietinum. The absolute configurations of 1, 2, and 9 were determined by X-ray crystallographic analysis. Compound 1 has a rare C-nor-D-homosteroid skeleton. Compound 9 possesses an unusual ergostane skeleton having a 10-oxabicyclo [4.3.1] decane moiety replacing 6/5 fused C/D rings. Compound 10 represents the first ergosteroid featuring the cleavage of a C8-C14 bond. The cytotoxicity of 1-10 was tested against the human cancer cell lines K562 and HCT116. The biosynthetic pathway for 1-10 is postulated.

Bacterial diversity in native habitats of the medicinal fungus Ophiocordyceps sinensis on Tibetan Plateau as determined using Illumina sequencing data.

Ophiocordyceps sinensis is one of the most well-known traditional Chinese medicinal fungi. In this study, bacterial diversity in the soils of native habitats of O. sinensis was investigated using Illumina sequencing data. A total of 525,000 sequences of V6-16S rRNA were analyzed. The number of OTUs from each sample ranged from 13,858 to 15,978 at 97% sequence similarity cut-off. The results demonstrated that the deep sequencing approach provides improved access to rare genotypes. Richness indices and Shannon's diversity index did not differ significantly between samples collected from locations where O. sinensis was present (Os1-3) and not present (NOs1-3). Classified bacterial sequences were grouped into 23 phyla including Proteobacteria, Actinobacteria, Acidobacteria, Verrucomicrobia, etc. The Venn diagram revealed that 7183 OTUs belonging to 14 phyla were shared by Os, NOs and MP (mycelial pellicle wrapping the sclerotium of O. sinensis) samples, possibly representing a core microbiome existing in native habitats of O. sinensis, and that 863 belonging to 12 phyla were shared by Os and MP samples, possibly related to the occurrence of O. sinensis. Overall, the results revealed a high bacterial diversity in the soil samples and the relationships between the bacterial diversity and O. sinensis merit further investigation.

Sterhirsutins A and B, two new heterodimeric sesquiterpenes with a new skeleton from the culture of Stereum hirsutum collected in Tibet Plateau.

Two new heterodimeric sesquiterpenes, sterhirsutins A (1) and B (2), and two new sesquiterpenes, hirsutic acids D-E (3 and 4), were identified from the culture of Stereum hirsutum. The absolute configurations in 1 and 2 were confirmed by single-crystal X-ray diffraction experiments and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are likely biosynthesized from a hirsutane-type sesquiterpene and α-humulene by a hetero-Diels-Alder cycloaddition. Compounds 1-4 showed cytotoxicity against K562 and HCT116 cell lines.

Phylogenetic-based nomenclatural proposals for Ophiocordycipitaceae (Hypocreales) with new combinations in Tolypocladium.

Ophiocordycipitaceae is a diverse family comprising ecologically, economically, medicinally, and culturally important fungi. The family was recognized due to the polyphyly of the genus Cordyceps and the broad diversity of the mostly arthropod-pathogenic lineages of Hypocreales. The other two cordyceps-like families, Cordycipitaceae and Clavicipitaceae, will be revised taxonomically elsewhere. Historically, many species were placed in Cordyceps, but other genera have been described in this family as well, including several based on anamorphic features. Currently there are 24 generic names in use across both asexual and sexual life stages for species of Ophiocordycipitaceae. To reflect changes in Art. 59 in the International Code of Nomenclature for algae, fungi, and plants (ICN), we propose to protect and to suppress names within Ophiocordycipitaceae, and to present taxonomic revisions in the genus Tolypocladium, based on rigorous and extensively sampled molecular phylogenetic analyses. When approaching this task, we considered the principles of priority, monophyly, minimizing taxonomic revisions, and the practical utility of these fungi within the wider biological research community.

Clarification of the concept of Ganoderma orbiforme with high morphological plasticity.

Ganoderma has been considered a very difficult genus among the polypores to classify and is currently in a state of taxonomic chaos. In a study of Ganoderma collections including numerous type specimens, we found that six species namely G. cupreum, G. densizonatum, G. limushanense, G. mastoporum, G. orbiforme, G. subtornatum, and records of G. fornicatum from Mainland China and Taiwan are very similar to one another in basidiocarp texture, pilear cuticle structure, context color, pore color and basidiospore characteristics. Further, we sequenced the nrDNA ITS region (ITS1 and ITS2) and partial mtDNA SSU region of the studied materials, and performed phylogenetic analyses based on these sequence data. The nrDNA ITS sequence analysis results show that the eight nrDNA ITS sequences derived from this study have single-nucleotide polymorphisms in ITS1 and/or ITS2 at inter- and intra-individual levels. In the nrDNA ITS phylogenetic trees, all the sequences from this study are grouped together with those of G. cupreum and G. mastoporum retrieved from GenBank to form a distinct clade. The mtDNA SSU sequence analysis results reveal that the five mtDNA SSU sequences derived from this study are clustered together with those of G. cupreum retrieved from GenBank and also form a distinct clade in the mtDNA SSU phylogenetic trees. Based on morphological and molecular data, we conclude that the studied taxa are conspecific. Among the names assigned to this species, G. fornicatum given to Asian collections has nomenclatural priority over the others. However, the type of G. fornicatum from Brazil is probably lost and a modern description based on the type lacks. The identification of the Asian collections to G. fornicatum therefore cannot be confirmed. To the best of our knowledge, G. orbiforme is the earliest valid name for use.

Isolation of the MAT1-1 mating type idiomorph and evidence for selfing in the Chinese medicinal fungus Ophiocordyceps sinensis.

Ophiocordyceps sinensis is one of the most valued medicinal fungi in China. Research on the mating system and sexual development is vitally important to this endangered species. Previous efforts devoted to investigate the mating type (MAT) locus of O. sinensis, however, resulted in an incomplete understanding. In this study, the MAT1-1 locus of O. sinensis was investigated. The conserved α-box and HMG-box regions of the MAT1-1-1 and MAT1-1-3 genes, respectively, and a conserved region of the DNA lyase gene were successfully amplified using degenerate PCR. A combination of TAIL-PCR and long-range PCR were used to connect these genes and obtain the sequence of the MAT1-1 locus. Screening of 22 single spore isolates by PCR demonstrated that both the MAT1-1-1 and MAT1-2-1 genes cooccurred within the same isolate. Additionally, both MAT1-1-1 and MAT1-2-1 are expressed in vegetative mycelia, providing evidence that O. sinensis is likely capable of selfing. DAPI (4,6-diamidino-2-phenylindole) staining of ascospores and hyphae showed that a majority of hyphal compartments are binucleate, suggesting that O. sinensis may be pseudohomothallic. Analyses of sequence diversity showed lower levels of genetic diversity in MAT1-1-1 compared to MAT1-2-1, indicating the possibility that different selective pressures act on the two MAT idiomorphs. The MAT1-1-1 sequences of O. sinensis and Tolypocladium inflatum cluster as a monophyletic group consistent with phylogenetic classification of Ophiocordycipitaceae. Comparison of the structure of the MAT1-1 locus across hypocrealean taxa showed that O. sinensis contains all three mating type genes (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and supported previous observations that of the four families in Hypocreales, MAT1-1-3 has undergone a lineage specific loss only in some members of the Cordycipitaceae.