Multiple GC-biased repetitive ITS copies in the Hirsutella sinensis genome are not generated by RIP mutagenesis involving transition point mutations

It has been hypothesized that AT-biased genotypes of Ophiocordyceps sinensis are generated through repeat-induced point mutation (RIP) and coexist as permanently nonfunctional internal transcribed spacer (ITS) pseudogenes in the genome of Hirsutella sinensis (GC-biased Genotype #1 of O. sinensis). This study examined the H. sinensis genome, which contains multiple repetitive ITS copies (GC content: 64.7{+/-}0.33%) with multiple insertion/deletion and transversion alleles, which were not generated through RIP mutagenesis that theoretically causes cytosine-to-thymine (C-to-T) and guanine-to-adenine (G-to-A) transitions. The repetitive ITS copies in the H. sinensis genome were found to be genetically and phylogenetically distinct from the AT-biased O. sinensis genotypes (GC content: 51.1{+/-}1.69%), which possess multiple transition alleles. The sequences of Genotypes #2-17, both GC- and AT-biased, are absent from the H. sinensis genome; these genotypes belong to interindividual O. sinensis fungi and differentially occur in different compartments of natural Cordyceps sinensis, with dynamic alterations in abundance occurring in an asynchronous, disproportional manner during C. sinensis maturation. Metatranscriptomic analyses of natural C. sinensis revealed the transcriptional silencing of 5.8S genes in all C. sinensis- colonizing fungi, including H. sinensis. The transcription assay reported by Li et al. [1] provided unsound, controversial evidence indicating that the 5.8S genes of AT-biased genotypes are nonfunctional pseudogenes. In addition to the single ITS locus analysis, repetitive genomic copies were also examined at multiple loci in the H. sinensis genome, and approximately 8.2% of the authentic genes had repetitive copies, including various transitions, transversions, and insertions/deletions. The transcripts for the repetitive copies, regardless of the decreases, increases, or bidirectional changes in the AT content, were identified in the H. sinensis transcriptome. These results are inconsistent with those of RIP mutagenesis, which generates pseudogenic, nonfunctional, repetitive copies. In conclusion, AT-biased genotypes of O. sinensis might have evolved through evolutionary mechanisms from a common ancestor over the long course of evolution, in parallel with GC-biased Genotype #1 H. sinensis.