The ascomycete Ophiocordyceps sinensis (Berk.) Sung, Sung, Hywel-Jones and Spatafora (syn. Cordyceps sinensis) , commonly known as the Chinese caterpillar fungus, has been widely used in traditional Chinese medicine for the treatment of asthma, bronchial, lung inflammation, and other diseases . The medicinal activity is from the complex of the moth caterpillar parasitized by the fungus and the fungal stroma biomass. The caterpillars, which belong to the family Hepialidae, live underground in soil burrows in the Tibetan plateau and feed on plant roots. When an infection is established, the fungus grows within body cavity of the insect larvae and eventually kills and mummifies them in the underground burrows. Fungal fruiting body emerges from the front end of the caterpillar and pokes out of the ground in spring and early summer .
The natural distribution of this fungus, however, is limited to alpine regions on the Tibetan Plateau and huge commercial demand has led to excessive harvest and a dramatic decline in its numbers . As a result, the price for natural O. sinensis has increased rapidly and it cost as much as $32,000 (USD) per kg for top quality in late 2006 . Although several fermented mycelial products from the anamorph (Hirsutella sinensis) of the fungus have been commercialized in China , the teleomorph, which is the component for traditional Chinese medicine, has not yet been commercially cultivated. The natural population of this fungus needs to be preserved for the fragile Tibetan Plateau ecosystem and for sustainable supply of this natural resource.
Ophiocordyceps sinensis occupies a diverse habitat on the Tibetan Plateau, which is the largest and highest plateau on Earth, covering more than 2.5 million km2 at an average elevation of over 4.5 km . It is surrounded by and interspersed with towering mountain ranges. Its complex geomorphology and climate generate substantial interregional variation which results in remarkable faunal and floral diversity and high levels of endemism [8–11]. O. sinensis is endemic to the Tibetan Plateau and occurs at elevations ranging from 3000 m up to the snow line . This distribution pattern suggests that the evolution of O. sinensis was significantly influenced by the tectonic movements during the strong uplift of the Tibetan Plateau. The spread of O. sinensis, depending on the shooting of ascospores, is limited to defined areas in a mountain and is unlikely through discontinuous mountains. Therefore, gene flow between different populations on discontinuous mountains is expected to be low, and divergence may have occurred .
Intraspecific genetic diversity of O. sinensis may also be generated by its wide host range. O. sinensis infects soil-borne larvae of more than 50 species of ghost moths, mostly in the genus Hepialus and to a lesser extent in the genera Hepialiscus, Forkalus, and Bipectilus [13, 14]. Coevolution between the fungus and its host insects may have originated a long time ago . Although the host insects in general have a limited distribution in the Tibetan Plateau and their distribution varies among different mountain ranges and even from different sides and habitats of the same mountain , the greater mobility of moths may also drive the genetic diversity of the fungus.
To date, genetic diversity of O. sinensis has only been studied at the local level of limited geographic areas. Three populations found in the north (Menyuan, Maqu, and Luqu), the middle (Yushu and Chengduo), and the south (Baima Snow Mountain, Renzhi Snow Mountain, Dacaodi, and Chongcaoxiawa) of the Tibetan Plateau, have been recognized from 29 samples based on RAPD analyses . Inter-simple sequence repeat (ISSR) analyses of O. sinensis from 11 counties in Qinghai Province also resulted in three groups (around the Qinghai Lake, central eastern Qinghai, and southern Qinghai) . Based on ITS sequences of samples from 11 localities in southwestern China, O. sinensis was divided into two populations (one from southwestern Sichuan to the northern slopes of the Himalayan Mountains, the other from northern Sichuan to Qinghai and Gansu) . However, ITS sequence analyses in another study did not indicate any subgroup for 17 O. sinensis samples from different geographical regions . Overall, the number of O. sinensis samples used in earlier studies was limited, and more samples from Tibet, a major production area, should be surveyed. In addition, other molecular markers, especially protein-encoding genes, should be employed to clarify the genetic diversity and to refine the phylogenetic origin of this fungus.
Mating-type genes play an important role in the evolution of fungal species. Sexual development is controlled by a single mating-type locus (MAT) in the fungi of Ascomycota. The term "idiomorph" instead of "allele" is usually used to describe the two alternate forms (MAT1-1 and MAT1-2) at the mating-type locus . Mating-type genes evolve at a faster rate than other sequences, such as ITS and glyceraldehyde 3-phosphate dehydrogenase . Although MAT1-2-1 sequences have been used to investigate the phylogenetic relationships among closely related species and usually provide high resolution , other studies found that the variability of MAT1-2-1 within species is low . A putative mating-type gene (MAT1-2-1) of O. sinensis was cloned in our laboratory. Similar to MAT1-2-1 orthologs of other ascomycetes, the O. sinensis MAT1-2-1 gene contained the conserved HMG motif. The sequence homologies to Cordyceps militaris and Cordyceps takaomontana were ~50% between cDNA sequences and ~40% between amino acid sequences (Zhang et al., unpublished data). During our preliminary experiment, several distinctive base changes within the MAT1-2-1 sequences were found among some O. sinensis isolates. Therefore, this gene was selected to investigate whether it can provide reasonable information on the genetic diversity of O. sinensis.
The objective of this study was to evaluate the population distribution of O. sinensis from geographically diverse regions of the Tibetan Plateau based on nrDNA ITS and MAT1-2-1 gene sequences. Understanding of the genetic diversity and genesis of O. sinensis should provide valuable information for the protection and sustainable utilization of this natural resource.