Multiple reports have detailed the simultaneous detection of ribosomal DNA (rDNA) of Paecilomyces hepiali (Ph), Hirsutella sinensis and two genotypes of Ophiocordyceps sinensis (Os) from Cordyceps sinensis (Cs), and the altered phenotypes and chemical components of Cs during Cs maturation. In this study, we observed an increase in the biomass of Ph and two moieties of Os rDNA during Cs maturation. The GCand AT-biased genotypes of transition mutations in the Cs stroma were confirmed using MassARRAY SNP genotyping. In premature Cs, the AT-biased genotypes were not expressed in the caterpillar, but highly predominated in the stroma. The GC bias was expressed in an opposite manner in the premature Cs compartments. The differential expression of GC-and AT-biased mutants was altered during Cs maturation. The increased biomass of the inverse-oriented Os rDNA of the GC-genotype was associated with a maturational increase in stroma height. We also report evidence of transversion mutations within Cs genes. The dynamic alterations of Ph and Os mutant gene expressions along with Cs maturation may play an essential role in Cs germination and maturation, the key elements of Cs life cycle, and result in the varied therapeutic potency of Cs.

Objective To examine the differential occurrence of Ophiocordyceps sinensis genotypes in the stroma, stromal fertile portion (SFP) densely covered with numerous ascocarps, and ascospores of natural Cordyceps sinensis. Methods Immature and mature C. sinensis specimens were harvested. Mature C. sinensis specimens were continuously cultivated in our laboratory (altitude 2,200 m). The SFPs (with ascocarps) and ascospores of C. sinensis were collected for microscopic and molecular analyses using species-/genotype-specific primers. Sequences of mutant genotypes of O. sinensis were aligned with that of Genotype #1 Hirsutella sinensis and compared phylogenetically using a Bayesian majority-rule method. Results Fully and semiejected ascospores were collected from the same specimens. The semiejected ascospores tightly adhered to the surface of the asci as observed by the naked eye and under optical and confocal microscopies. The multicellular heterokaryotic ascospores showed uneven staining of nuclei. The immature and mature stromata, SFPs (with ascocarps) and ascospores were found to differentially contain several GC- and AT-biased genotypes of O. sinensis, Samsoniella hepiali, and an AB067719-type fungus. The genotypes within AT-biased Cluster-A in the Bayesian tree occurred in all compartments of C. sinensis, but those within AT-biased Cluster-B were present in immature and mature stromata and SPFs but absent in the ascospores. Genotype #13 of O. sinensis was present in semi-ejected ascospores and Genotype #14 in fully ejected ascospores. GC-biased Genotypes #13–14 featured large DNA segment substitutions and genetic material recombination between the genomes of the parental fungi (H. sinensis and the AB067719-type fungus). These ascosporic offspring genotypes combined with varying abundances of S. hepiali in the 2 types of ascospores participated in the control of the development, maturation and ejection of the ascospores. Conclusion Multiple genotypes of O. sinensis coexist differentially in the stromata, SFPs and 2 types of C. sinensis ascospores, along with S. hepiali and the AB067719-type fungus. The fungal components in different combinations and their dynamic alterations in the compartments of C. sinensis during maturation play symbiotic roles in the lifecycle of natural C. sinensis.

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Natural Cordyceps sinensis is a traditional Chinese medicine with a long history of use as a folk medicine in China. However, whether Ophiocordyceps sinensis is a single fungus or a collective name for multiple fungi remains controversial, as does the anamorph-teleomorph connection of O. sinensis. Although Hirsutella sinensis has been widely considered the sole anamorph of O. sinensis, direct evidence supporting this hypothesis is lacking. This paper reviews the molecular heterogeneity findings of C. sinensis studies and the issues with the hypotheses of the anamorph-teleomorph connection of O. sinensis. Molecular analyses have revealed the coexistence of multiple fungi and multiple genotypes of O. sinensis in natural C. sinensis. Mutant sequences from at least 11 O. sinensis genotypes are registered in GenBank and represent individual fungi distinct from the genome sequence of H. sinensis, which is inconsistent with the "sole anamorph" and "ITS pseudogenes" hypotheses. Together, the multicellular C. sinensis ascospores with mono-/bi-/trinucleate structures in each ascospore and the detection of at least 2 O. sinensis genotypes in the culture of heterokaryotic single-ascospore isolates challenge the study conclusions based on microcycle conidiation of C. sinensis ascospores. During C. sinensis maturation, the fungi that grow differentially in the caterpillar body and stroma of C. sinensis undergo asynchronous, dynamic alterations, supporting the integrated microecosystem hypothesis for natural C. sinensis proposed by Prof. Zongqi Liang.

Objective: To examine the differential occurrence of Ophiocordyceps sinensis genotypes in the stroma, stromal fertile portion (SFP), and ascospores of natural Cordyceps sinensis. Methods: Immature and mature C. sinensis were harvested. Mature C. sinensis were continuously cultivated in our laboratory (altitude 2,200 m). The SFPs (with ascocarps) and ascospores of C. sinensis were collected for microscopic and molecular analyses using species-/genotype-specific primers. Results: Fully and semi-ejected ascospores were collected from the same specimens. The semi-ejected ascospores tightly adhered to the surface of the asci. The multicellular heterokaryotic ascospores showed uneven staining of nuclei. The ascospores were found to contain several genotypes of O. sinensis, Samsoniella hepiali, and an AB067719-type fungus. The genotypes within AT-biased Cluster-A occurred in all compartments of C. sinensis, but those within AT-biased Cluster-B were absent in the ascospores. Genotypes #13?14 of O. sinensis occurred differentially in fully and semi-ejected ascospores, which featured DNA segment substitutions and genetic material recombination between the genomes of the parental fungi, Hirsutella sinensis and the AB067719-type fungus. These offspring genotypes combined with varying abundances of S. hepiali in the 2 types of ascospores participated in the control of the development, maturation and ejection of the ascospores. Conclusion: Multiple genotypes of O. sinensis coexist differentially in the SFP and 2 types of C. sinensis ascospores, along with S. hepiali and the AB067719-type fungus. The fungal components in different combinations and their dynamic alterations in the compartments of C. sinensis during maturation play symbiotic roles in the lifecycle of natural C. sinensis .