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.
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 .
Objective: To examine multiple genotypes of Ophiocordyceps sinensis in the stromal fertile portion (SFP) densely covered with numerous ascocarps and ascospores of natural Cordyceps sinensis. Methods: Mature C. sinensis specimens were harvested and continuously cultivated in our laboratory (altitude 2,200 m). The SFPs (with ascocarps) and fully and semi-ejected ascospores were collected for histological and molecular examinations. Biochip-based single nucleotide polymorphism (SNP) MALDI-TOF mass spectrometry (MS) was used to genotype multiple O. sinensis mutants in the SFPs and ascospores. Results: Microscopic analysis demonstrated distinct morphologies of the SFPs (with ascocarps) before and after ascospore ejection and the SFP of developmental failure, which, along with the fully and semi-ejected ascospores, were subjected to SNP MS genotyping analysis. Mass spectra showed the coexistence of GC- and AT-biased genotypes of O. sinensis that were genetically and phylogenetically distinct in the SFPs before and after ejection and of developmental failure and in fully and semi-ejected ascospores. The intensity ratios of MS peaks were dynamically altered in the SFPs and the fully and semi-ejected ascospores. Mass spectra also showed transversion mutation alleles of unknown sequences with altered intensities in the SFPs and ascospores. Genotype #5 of AT-biased Cluster-A maintained a high intensity in all SFPs and ascospores. An MS peak with a high intensity containing AT-biased Genotypes #6 and #15 in pre-ejection SFPs was significantly attenuated after ascospore ejection. The abundance of Genotypes #5‒6 and #16 of AT-biased Cluster-A was differentially altered in the fully and semi-ejected ascospores that were collected from the same C. sinensis specimens. Conclusion: Multiple O. sinensis genotypes coexisted in different combinations with altered abundances in the SFPs prior to and after ejection, the SFP of developmental failure and in the two types of ascospores of C. sinensis, demonstrating their genomic independence. Fungal metagenomic members present in different combinations and with dynamic alterations play symbiotic roles in different compartments of natural C. sinensis.
Objective To examine multiple genotypes of Ophiocordyceps sinensis in a semi-quantitative manner in the stromal fertile portion (SFP) densely covered with numerous ascocarps and ascospores of natural Cordyceps sinensis and to outline the dynamic alterations of the coexisting O. sinensis genotypes in different developmental phases. Methods Mature Cordyceps sinensis specimens were harvested and continuously cultivated in our laboratory (altitude 2,254 m). The SFPs (with ascocarps) and fully and semi-ejected ascospores were collected for histological and molecular examinations. Biochip-based single nucleotide polymorphism (SNP) MALDI-TOF mass spectrometry (MS) was used to genotype multiple O. sinensis mutants in the SFPs and ascospores Results Microscopic analysis revealed distinct morphologies of the SFPs (with ascocarps) before and after ascospore ejection and SFP of developmental failure, which, along with the fully and semi-ejected ascospores, were subjected to SNP MS genotyping analysis. Mass spectra showed the coexistence of GC- and AT-biased genotypes of O. sinensis that were genetically and phylogenetically distinct in the SFPs before and after ejection and of developmental failure and in fully and semi-ejected ascospores. The intensity ratios of MS peaks were dynamically altered in the SFPs and the fully and semi-ejected ascospores. Mass spectra also showed transversion mutation alleles of unknown upstream and downstream sequences with altered intensities in the SFPs and ascospores. Genotype #5 of AT-biased Cluster-A maintained a high intensity in all SFPs and ascospores. An MS peak with a high intensity containing AT-biased Genotypes #6 and #15 in pre-ejection SFPs was significantly attenuated after ascospore ejection. The abundance of Genotypes #5‒6 and #16 of AT-biased Cluster-A was differentially altered in the fully and semi-ejected ascospores that were collected from the same Cordyceps sinensis specimens. Conclusion Multiple O. sinensis genotypes coexisted in different combinations with altered abundances in the SFPs prior to and after ejection, the SFP of developmental failure, and the two types of ascospores of Cordyceps sinensis, demonstrating their genomic independence. Metagenomic fungal members present in different combinations and with dynamic alterations play symbiotic roles in different compartments of natural Cordyceps sinensis.
Objective: The goal of this study was to develop and validate density-weighted arithmetic methods for the analysis of the dynamic maturational changes in random amplified polymorphic DNA (RAPD) polymorphisms in Cordyceps sinensis containing multiple fungi. Methods: Ten random primers were used for PCR amplification to monitor changes in the RAPD molecular marker polymorphisms in caterpillar body, stroma and ascocarp portion samples of C. sinensis collected at different stages of maturation. We compared (1) the density-unweighted Nei-Li similarity equation and new similarity equations considering the densities of DNA bands and (2) the density-unweighted and weighted arithmetic methods for cluster analysis. Results: The algorithm using the Nei-Li similarity equation did not account for the differences in the density of the matched randomly amplified amplicons, whereas the new similarity equations were capable of integrating different amplicon densities into the similarity computation. With improvements in the similarity computation and cluster construction, the new methods revealed dynamic maturational changes in the RAPD polymorphisms of molecular markers in C. sinensis caterpillar bodies and stromata. The polymorphism analysis revealed similarities of 74%-88% between the RAPD polymorphisms of the ascocarp portion versus premature and mature C. sinensis stromata, but low similarities of<70% versus the caterpillar bodies and maturing stromata. Apparent dissimilarities (similarity <66%) were also found between Hirsutella sinensis and C. sinensis samples. Conclusions: These new algorithmic methods represent an advance in similarity computations when comparing polymorphisms of RAPD molecular markers in C. sinensis and provide accurate analytic tools to capture the molecular information related to the dynamic Am. J. Biomed. Sci. 2014, 6(2), 82-104; doi: 10.5099/aj140200082 © 2014 by NWPII. All rights reserved 83 changes in differential expression of C. sinensis intrinsic fungi during maturation. The apparent dissimilarity in RAPD polymorphisms between H. sinensis and C. sinensis suggests differences of mycological background and challenges the hypothesis of the anamorph-teleomorph connection between H. sinensis and Ophiocordyceps sinensis.
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