Altered GC- and AT-biased genotypes of Ophiocordyceps sinensis in the stromal fertile portions and ascospores of natural Cordyceps sinensis

Abstract

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.

Genetic and phylogenetic features of the repetitive ITS copies in theHirsutella sinensisgenome and the genomically independent AT-biasedOphiocordyceps sinensisgenotypes

“…Genotypes #4 and #15 of the AT-biased Cluster B of O. sinensis ( cf . Figure 2) were reported to exist in the stroma and stromal fertile portion (densely covered with numerous ascocarps) of natural C. sinensis but not in the ascospores (Li et al . 2023b, 2023c), consistent with the failure to identify Genotype #4 of AT-biased Cluster B in mono-ascospores as reported by Li et al .…”

“…, Natural C. sinensis ) collected from an alpine meadow of Nyingchi, Tibet Autonomous Region, China, but not the 3 pure GC-biased Genotype #1 H. sinensis strains reported by Wei et al . (2016), Li et al . (2023b, 2023c) reported that the fully ejected ascospores of natural C. sinensis contained GC-biased Genotypes #1 and #14, AT-biased O. sinensis Genotypes #5–6 and #16 within AT-biased Cluster A ( cf .…”

“…Li et al . (2023b, 2023c) used several pairs of genotype-specific primers, touch-down PCR, cloning-based PCR amplicon sequencing and MassARRAY SNP mass spectrometry genotyping techniques and identified Genotypes #5-6 and #16 of AT-biased Cluster A ( cf .…”

“…Li et al . (2023b) used the MassARRAY SNP mass spectrometry genotyping technique and found dynamic alterations of GC-biased Genotype #1 and several AT-biased genotypes of O. sinensis in the stromal fertile portion (densely covered with numerous ascocarps) before and after the ejection of C. sinensis ascospores and in fully and semiejected ascospores collected from the same C. sinensis specimens.…”

“…2013; Li et al . 2020b, 2022c, 2023b, 2023c), i.e. , the natural C. sinensis insect-fungal complex ≠ the entomopathogenic O. sinensis fungus.…”