Genomic analyses reveal evolutionary and geologic context for the plateau fungus Ophiocordyceps sinensis

Abstract

Background Ophiocordyceps sinensis, which is only naturally found in the high-elevation extreme environment of the Tibetan Plateau, has been used in traditional Chinese medicine. Information concerning the evolutionary and geologic context of O. sinensis remains limited, however. Methods We constructed the high-quality genome of O. sinensis and provided insight into the evolution and ecology of O. sinensis using comparative genomics. Results We mapped the whole genome of the anamorph/asexual form Hirsutella of O. sinensis using Illumina and PacBio sequencing technologies and obtained a well assembled genome of 119.2 Mbp size. Long-read Single Molecule Real Time (SMRT) sequencing technology generated an assembly with more accurate representation of repeat sequence abundances and placement. Evolutionary analyses indicated that O. sinensis diverged from other fungi 65.9 Mya in the Upper Cretaceous, during the uplift of the Tibetan Plateau. Gene family expansions and contractions in addition to genome inflation via long terminal repeat (LTR) retrotransposon insertions were implicated as an important driver of O. sinensis divergence. The insertion rate of LTR sequences into the O. sinensis genome peaked ~ 30–40 Mya, when the Tibetan Plateau rose rapidly. Gene Ontology (GO) enrichment analysis suggested that O. sinensis contained more genes related to ice binding compared to other closely related fungi, which may aid in their adaptability to the cold Tibetan Plateau. Further, heavy metal resistance genes were in low abundance in the O. sinensis genome, which may help to explain previous observations that O. sinensis tissues contain high levels of heavy metals. Conclusions Our results reveal the evolutionary, geological, and ecological context for the evolution of the O. sinensis genome and the factors that have contributed to the environmental adaptability of this valuable fungus. These findings suggest that genome inflation via LTR retrotransposon insertions in O. sinensis coincided with the uplift of the Tibetan Plateau. LTRs and the specific genetic mechanisms of O. sinensis contributed to its adaptation to the environment on the plateau.

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

“…Based on insufficient and contradictory evidence, these researchers overinterpreted that all ATbiased genotypes were "ITS pseudogene" components of the genome of Genotype #1 H. sinensis, in agreement with the sole anamorph hypothesis for H. sinensis [44]. However, AT-biased genotype sequences are not present in the 5 whole-genome assemblies (ANOV0000000, JAAVMX0000000000, LKHE00000000, LWBQ00000000 and NGJJ00000000) of Genotype #1 H. sinensis strains Co18, IOZ07, 1229, ZJB12195 and CC1406-203 [7][8][9][10]31,[51][52][53][54][55]. The genomic independence evidence disproved the "ITS pseudogene" hypothesis for the AT-biased genotypes of O. sinensis proposed by Li et al [61].…”

“…The sequences of Genotypes #2-17 of O. sinensis, however, were not present in the genome assemblies (ANOV00000000, JAAVMX000000000, LKHE00000000, LWBQ00000000 and NGJJ00000000) of 5 Genotype #1 H. sinensis strains, Co18, IOZ07, 1229, ZJB12195 and CC1406-203, and instead belonged to the genomes of independent O. sinensis fungi [7][8][9][10][11][12][27][28][30][31][51][52][53][54][55][56]69].…”

“…Wei et al [50] reported an industrial artificial cultivation project and demonstrated a mismatch between the inoculants of 3 GC-biased Genotype #1 H. sinensis strains and a sole teleomorphic fungus (AT-biased Genotype #4 of O. sinensis) in the fruiting body of cultivated C. sinensis. The sequences of the AT-biased genotypes of O. sinensis are absent in the genome of GC-biased Genotype #1 H. sinensis and belong to independent O. sinensis fungi [7][8][9][10][11][12]24,[30][31]43,[51][52][53][54][55][56]. Thus, according to the fourth criterion of Koch's postulates, the species contradiction between the anamorphic inoculants and the teleomorph in cultivated products disproves the sole anamorph hypothesis for H. sinensis that was proposed by Wei et al [44] of the same research group 10 years ago.…”

“…In contrast to the maturation pattern of Genotype #1, the abundance of Genotype #2 remained at a low level in the maturing stroma and increased markedly in the mature stroma to levels that greatly exceeded the abundance of Genotype #1. The cooccurrence of Genotypes #1 and #2 in the stroma with distinct maturation patterns indicates the genomic independence of the 2 GC-biased genotypes, which is proven by the absence of Genotype #2 sequences in the whole-genome sequences of 5 Genotype #1 H. sinensis strains[7][8][9][10][11][12][30][31][51][52][53][54][55][56].In this study, we did not detect GC-biased Genotypes #3 and #7-12 of O. sinensis in the compartments of natural C. sinensis specimens collected from the Hualong and Yushu areas of Qinghai Province.Genotype #3 was detected in natural C. sinensis specimens collected from Nyingchi in Tibet, where it was hypothesized to be the center of origin for Genotype #1 H. sinensis under the geographic hypothesis of the genetic evolution of H. sinensis[57]. The GC-biased Genotype #7 sequence AJ488254 of O. sinensis was detected in the stroma of natural C. sinensis specimen #H1023 collected from Qinghai Province of China, without the coexistence of Genotype #1 H. sinensis.…”

“…However, AT-biased Cluster-B genotypes were absent in ascospores but present in the SFP (with ascocarps). Pseudogymnoascus roseus, Geomyces pannorum, Tolypocladium sinensis, and Penicillium chrysogenum were not detected in the SFP (with ascocarps) and ascospores of C. sinensis, despite their differential predominance in the stroma and caterpillar body of natural C. sinensis found in 2 previous mycobiota studies[40][41].The sequences of Genotypes #2-17 of O. sinensis, however, were not present in the genome assemblies (ANOV00000000, JAAVMX000000000, LKHE00000000, LWBQ00000000 and NGJJ00000000) of 5 Genotype #1 H. sinensis strains, Co18, IOZ07, 1229, ZJB12195 and CC1406-203, and instead belonged to the genomes of independent O. sinensis fungi[7][8][9][10][11][12][27][28][30][31][51][52][53][54][55][56]69].…”