Non-concerted ITS evolution in fungi, as revealed from the important medicinal fungus Ophiocordyceps sinensis

• Non-concerted ITS evolution was reported in O. sinensis.
• AT-rich divergent ITS paralogs of O. sinensis were identified as pseudogenes.
• ITS pseudogenes were amplified from both single spore and tissue isolates.
• Hypothesis of cryptic species of O. sinensis and some other hypotheses were rejected.

The internal transcribed spacer (ITS) of nuclear ribosomal DNA (nrDNA) has been widely used as a molecular marker in phylogenetic studies and has been selected as a DNA barcode for fungi. It is generally believed that nrDNA conforms to concerted evolution in most eukaryotes; however, intraindividual–intraspecific polymorphisms of this region were reported in various organisms, suggesting a non-concerted evolutionary process. In Ophiocordyceps sinensis, one of the most valuable medicinal fungi, a remarkable variation of the ITS region has been revealed. Some highly divergent sequences were thought to represent cryptic species, different species or genotypes in previous studies. To clarify the unusual ITS polymorphisms observed in O. sinensis, specific primers were designed to amplify ITS paralogs from pure cultures of both single-ascospore and tissue isolates in this study. All of the available ITS sequences, including those generated by this group and those in GenBank, were analyzed. Several AT-biased ITS paralogs were classified as pseudogenes based on their nucleotide compositions, secondary structures and minimum free energies of their 5.8S rRNAs, substitution rates, phylogenetic positions and gene expression analyses. Furthermore, ITS pseudogenes were amplified with specific primers from 10 of the 28 strains tested, including eight single-ascospore and two tissue isolates. Divergent ITS paralogs were proved to coexist in individual genomes, suggesting a non-concerted mechanism of evolution in the ITS region of O. sinensis. The hypotheses that divergent ITS paralogs represent cryptic or other species or different genotypes were thus rejected.

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