| Ophiocordyceps sinensis is a valuable Traditional Chinese Medicinal fungus. It parasitizes insects within the family Hepialidae to form a Traditional Chinese Medicine that is called "Dong Chong Xia Cao" in Chinese. Because of the unique medicinal effect, limited natural resources, rising prices, the fungus has been the center of recent researches. However, the artificial cultivation of the fungus is still far from being successful. A close parasitic relationship was formed between the fungus and its host insects, but due to the lack of the systematic study on their evolutionary relationship, people did not pay much attention to geographic origins of the fungal and insect materials used in cultivation practices, which might be one of the major obstacles for the large-scale artificial cultivation of the fungus on insect larvae.In this study, natural O. sinensis specimens was broadly collected from its production areas on the Tibetan Plateau. Novel molecular markers of O. sinensis were determined by constructing a shotgun genomic library, and markers of host insects were selected from gene fragments commonly used in insect molecular systematics. Phylogeneticc and genetic structures of both O. sinensis and host insects were analyzed systematically, and factors driving the observed genetic structures were investigated. Based on results of multigene sequence analyses, the coevolutionary relationship between O. sinensis and host insects was elucidated. Results of this study help to enrich contents of classification and evolution of both O. sinensis and its host insects, to instruct the reasonable resource protection of naturally-occurring O. sinensis, and to facilitate the fulfillment of the large-scale artificial cultivation of O. sinensis on ghost moth larvae.The main results are as follows:1) Over900O. sinensis specimens were collected from Tibet, Qinghai, Sichuan, Yunnan, Gansu, etc., and59isolates were obtained. These isolates were confirmed to be Hirsutella sinensis, the anamorph of O. sinensis, by both morphological examination and nrDNA ITS sequence analyses. 2) A shotgun genomic library was constructed using the O. sinensis isolate YN07-8. About260clones were randomly selected and sequenced. PCR primers for33different fragments (designated as OSRC) were designed and used to amplify from four distantly separated O. sinensis isolates. We tested the sequence variation of these fragments among these isolates and found several novel molecular marker genes, such as OSRC14and OSRC32, that have the potential of being used as markers for population genetic studies of O. sinensis.3) Population genetics of O. sinensis was investigated using121individuals and seven different marker genes, that is nrDNA ITS, MAT1-2-1, cspl, OSRC14, OSRC17, OSRC27, and OSRC32. We found that there was great genetic differentiation not only within populations but between populations. South Tibet might be the center of origin for O. sinensis because of the high genetic diversity there. There is a positive correlation between genetic distance and latitudinal distances, but not between genetic distance and longitudinal or altitudinal distances. Molecular dating and biogeographical inference found that O. sinensis might have originated22.59Mya at South Tibet and then transmitted to other regions. Multiple evolutionary events accompanied this process.4) Using identical materials in population genetic studies of O. sinensis, we extracted genomic DNA from the sclerotial section of natural O. sinensis. The DNA thus obtained was used to amplify genes of host insects. Three marker genes (COI, cytb, and wingless) were employed to investigate the genetic structures of host insects. We found that there is great genetic differentiation not only within populations but between populations. There is a positive correlation between genetic distance and horizontal distances but not with altitudinal distances. We found that host insects originated about6Mya from South Tibet and then dispersed to other regions.5) Significant correlation was found between genetic distances of O. sinensis and genetic distances of host insects. Some switching events were found besides cospeciation events. One fungal MLH can associate with multiple host insect MLHs, and one host insect MLH can associate with multiple fungal MLHs. |
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