Differential coexistence of multiple genotypes of Ophiocordyceps sinensis in the stromata, ascocarps and ascospores of natural Cordyceps sinensis

Abstract

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.

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

“…As shown in Tables 1‒2 and Figure 1, the sequences of GC-biased Genotypes #2–3 and #7–14 and of AT-biased Genotypes #4‒6 and #15‒17 of O. sinensis are absent in the genome assemblies ANOV00000000, JAAVMX000000000, LKHE00000000, LWBQ00000000, and NGJJ00000000 of H. sinensis Strains Co18, IOZ07, 1229, ZJB12195, and CC1406-203, respectively, and instead belong to the genomes of independent O. sinensis fungi (Stensrud et al 2007; Xiao et al 2009; Hu et al 2013; Zhu & Wu 2015; Li et al 2016a, 2016b, 2020a, 2022a, 2022c, 2023c; Zhu & Li 2017; Zhu et al 2018, 2019; Jin et al 2020; Liu et al 2020; Shu et al 2020). The genomic repetitive ITS copies were phylogenetically clustered into the GC-biased clades in blue alongside the Bayesian majority rule consensus tree in Figure 2 and were phylogenetically distant from the AT-biased clades containing Genotypes #4‒6 and #15‒17 of O. sinensis shown in red alongside in the tree, similar to what has previously been demonstrated in phylogenetic trees (Kinjo & Zang 2001; Stensrud et al 2007; Zhang et al 2013a; Zhu & Wu 2015; Li et al 2016b, 2020a, 2022a, 2022c, 2023c; Wei et al 2016; Zhu & Li 2017).…”

“…A Basic Local Alignment Search Tool (BLAST) search of the GenBank database revealed 668 O. sinensis ITS sequences under GenBank taxid:72228, 440 of which belong to GC-biased Genotypes #1‒3 and #7‒14 and 228 belong to AT-biased Genotypes #4‒6 and #15‒17 (Stensrud et al 2005,2007; Zhang et al 2013a; Li et al 2016b, 2022c, 2023b, 2023c).…”

“…Freshly collected C. sinensis specimens were washed thoroughly in running water with gentle brushing and surface sterilized in 0.1% mercuric chloride for 10 min, followed by washing 3 times with sterile water (Zhu et al 2010; Li et al 2023c). The thoroughly cleaned and sterilized C. sinensis specimens were immediately frozen in liquid nitrogen and kept frozen during transportation and storage prior to further processing.…”

“…The Chinese Pharmacopeia defines natural C. sinensis as an insect-fungal complex, which includes the remains of Hepialidae moth larva and >90 fungal species from >37 genera, with differential co-occurrence of some of 17 genotypes of Ophiocordyceps sinensis , i.e. , natural C. sinensis insect-fungal complex ≠ the entomopathogenic O. sinensis fungus (Engh 1999; Kinjo and Zang 2001; Chen et al 2004, 2011; Stensrud et al 2005, 2007; Wei et al 2006, 2016; Xiao et al 2009; Zhang et al 2009, 2010; Zhu et al 2010, 2012, 2018, 2019; Gao et al 2011, 2012; Yao et al 2011; Mao et al 2013; Xia et al 2015; Zhu and Wu 2015; Li et al 2016b, 2020a, 2022a, 2022c, 2023b, 2023c; Zhu and Li 2017). However, the Latin name C. sinensis has been indiscriminately used for both the wild insect-fungal complex and the teleomorph/holomorph of the C. sinensis fungus since the 1840s (Yao & Zhu 2016; Li et al 2022a).…”

“…Wei et al (2006) hypothesized that GC-biased Genotype #1 H. sinensis is the sole anamorph of O. sinensis . However, numerous studies have identified 17 mutant genotypes of O. sinensis in natural C. sinensis , which are indiscriminately referred to by the same Latin name (Zhu et al 2010, 2018, 2019; Yao & Zhu 2016; Li et al 2020a, 2022a, 2022c, 2023b, 2023c). Stensrud et al (2007) and Zhang et al (2013a) analyzed 71 and 397 ITS sequences, respectively, that were annotated as C. sinensis or O. sinensis in GenBank under taxid:72228 and confirmed the existence of 3 phylogenetic clades of mutant O. sinensis sequences: Group A (GC-biased Genotype #1) and Groups B‒C (AT-biased Genotypes #4‒5).…”