蟲草屬真菌為一群以昆蟲及蜘蛛為主要宿主的寄生真菌，文獻指出此屬真菌約有四百種且廣泛分佈于地球上各區域。在此屬真菌中有12種被用來作為中藥材，且在中國歷史上已有千年的歷史。因為藥用的價值，我們由九個菌種中心收集了約八十種蟲草屬及其相關真菌，建立完整的菌種保存條件及核糖體基因DNA序列資料庫。我們的蟲草屬真菌菌株資料庫為世界上比較完整的。研究顯示，其培養最適溫度介於18到24 ℃，且溫度超過37 ℃就會造成菌種的死亡。此屬真菌最適生長之酸鹼度在pH值為6左右，但是，經培養條件的測試在pH 3至13皆不會造成菌株的死亡。另外，在核糖體基因資料庫建立方面，研究發現此屬真菌其核糖體基因序列約8 Kb。我們利用收集來自六個菌種中心的二十九株蟲草屬真菌，其中包括二十三株Cordyceps屬、二株Beauveria屬、一株Paecilomyces屬、一株Phytocordyceps屬、一株Podostroma 屬與一株Tolypocladium屬的真菌。完成了二十九株蟲草屬真菌的核糖體基因，其中包括18 S、5.8 S與28 S前半部的選殖以及定序工作。在我們的研究中顯示，二十九株真菌其18 S核糖體基因相似度高達96%以上，這樣的結果可以得知，我們所收集的菌株其應為擁有相同祖先的一群菌株。另外，我們利用ITS1，5.8 S核糖體與ITS2區域來鑑別所收集的菌株其種與亞種之間的地位及相關性。發現，雖然所收集的菌株其18 S核糖體基因非常相似但仍然可以分類方法將其歸類為幾個主要的群。由不同地理位置所採集到的蟲草屬菌株，其中包括，加拿大、中國、法國、丹麥、比利時、瓜雅那、印度、日本、韓國、荷蘭、英國、美國及台灣皆與其基因的相似度相關。由分類分析得知，冬蟲夏草屬真菌在遷移過程中經歷多次的演化，這樣的演化最終受到最後棲息地的環境所影響，造成來自於相同起源的蟲草真菌各自獨立演化並出現基因上畸異。我們的研究結果也符合這樣的假說。

Ascomycetous genus Cordyceps widespread distributes on earth and they are endoparasitic fungi of insects and other arthropods. There are 12 species of genus Cordyceps fungi being used as herbal medicine for thousands of years in China. Because of the value of this fungi in medicial treatment, we collected about 80 isolates of this fungi from nine culture collection centers. We set up the culturing condition and sequenced the ribosoml DNA of Cordyceps spp. as database. In our study, we found the optimum culturing temperature of Cordyceps fungi is around 18 to 24 ℃. When the culturing temperature is over 37 ℃, the fungi could not survival. The optimum culturing pH value of Cordyceps fungi is around 6, but the fungi can keep alive when pH values changed from 3 to 13. In another hand, in our study on seting up the ribosomal DNA database, we found the ribosomal DNA of Cordyceps fungi is about 8 Kb. After cloned and sequenced about 50 isolates based on 18 S, 5.8 S and the first half of 28 S ribosomal DNA. We can study the phylogenetic relationship of Cordyceps fungi based on 18 S rDNA and ITS1, 5.8 S rDNA and ITS2 region. In order to understand the connection between evolution and geographical distribution of this fungi, we investigated the phylogenetic relationships of 29 isolates, 23 belongs to genus Cordyceps, 2 from genus Beauveria, one from genus Paecilomyces, one from genus Phytocordyceps, one from genus Podostroma and one from genus Tolypocladium, collected from six culture collection centers around the world, based on their 18 S rRNA gene and the ITS1, 5.8 S rRNA gene and ITS2 region. In our study, 18 S rRNA gene of all 29 isolates are very similar, up to 96 %, shows that these fungi collected form different locations on earth may be from the common ancestor. On the other hand, even their 18 S rRNA genes are similar, they can still be divided into several groups based on phylogenetic analysis of ITS1, 5.8 S rRNA gene and ITS2 region. Cordyceps isolated from different geographical positions including Canada, China, Guyana, India, Japan, Korea, Netherlands, Taiwan, U.K. and U.S. could be correlated to their genetic diversity. Cordyceps isolated from closed geographical positions have simulated features in DNA sequences. Phylogenetic analysis strongly suggests that fungi might evolute during traveling and affected by the ecology of the last defined. In the genus Cordyceps, many agencies and factors may affect it’s distribution, they have been isolated in different locations on earth and gave them different names, but they are the same species of fungi. In the fungal evolution, it departed from the origin circumstances by any possible way and exhibited the new environment and independently evoluted individual. Our study revealed the genus Cordyceps widespread distribution on earth should compliance with this hypothesis.

曹暉、劉玉萍。DNA分子標記技術：一種新的中藥分析方法。藥物分析雜誌， 19（5）：355－360。1999。

曾聰徹、陳瑞青。真菌學之最近發展。國科會生物科學中心專刊，12：207。1985。

蔡竹固。植物病原簡介 (二) 真菌。嘉義農專農藝學報， 21：19-40。1989。

劉祖惠。中草藥之技術發展現況與產業化策略。科技發展政策報導， SR9007:501-511。2001。

Alexopoulos, C. J. et. al. Introductory Mycology. John Wiley & Sons, Inc., New York, U. S. p.868, 1996.

Jensen, A. B., Gargas, A., Eilenberg, J., and Rosendahl, S. Relationships of the insect-pathogenic order entomophthorales (Zygomycota, Fungi) based on phylogenetic analysis of nuclear small subunit ribosomal DNA sequences (SSUrDNA). Fung. Genet. Biol. 24:325-334, 1998.

Arenal, F., Platas, G., Martin, J., Salazar, O., Pelaez, F. Evaluation of different PCR-based DNA fingerprinting techniques for assessing the genetic variability of isolate the fungus Epicoccum nigrum. J. Appl. Microbiol. 87:898-906, 1999.

Bowman, B. H., Taylor, J. W., Brownlee, A. G., Lee, J., Lu, S. D., and White, T. J. Molecular Evolution of the Fungi: Relationship of the Basidiomycetes, Ascomycetes, and Chytridiomycetes. Mol. Biol. Evol. 9:285-296, 1992.

Barnett, H. L., and Hunter, B. B. Illustrated genera of imperfect fungi 4th ed. Macmillan Pub. Co., New York, p218, 1987.

Berbee, M.L. and Taylor, J.W. Dating the evolutionary radiations of the true fungi. Can. J. Bot. 71:1114-1127, 1993.

Winnepenninckx, B., Steiner, G., Backeljau, T., and De Wachter, R. Details of gastropod phylogeny inferred from 18S rRNA sequences. Mol. Phylo. Evol. 9:55-63, 1998.

Blears, M. J., De Grandis, S. A., Lee, H., and Trevors, J. T. Amplified fragment length polymorphism (AFLP): a review of the procedure and its application. J. Ind. Microbiol. Biotech. 21:99-114, 1998.

Bridge, P. D., and Arora, D. K.Interpretation of PCR methods for species definition. In Applications of PCR in Mycology.Edited by Bridge, P. D., Arora, D. K., Reddy, C. A., and Elander, R. P. CAB International. pp.63-84, 1998.

Burnett, J. H.Fundamentals of Mycology, 2nd ed. Edward Arnold, London.pp.99-114, 1976.

Blker, M. Ustilago maydis – a valuable model system for the study of fungal dimorphism and virulence. Microbiology 147:1395-1401, 2001.

Borges-Walmsley, M. I. and Walmsley, A. R.cAMP signalling in pathogenic fungi: control of dimorphic switching and pathogenicity. Trends in Microbiology 8:133-141, 2000.

Buller, A. H. R. Researches on Fungi. Vol. 1. Longmans, Green and Co., London, p.274, 1909.

Buller, A. H. R. Researches on Fungi. Vol. 2. Longmans, Green and Co., London, p.492, 1922.

De Barros Lopes, M., Rainieri, S., Henschke, P.A., and Langridge, P. AFLP fingerprinting for analysis of yeast genetic variation. Int. J. Syst. Bacteriol. 49:915-924, 1999.

Chen, Y., Wang, W., Yang, Y., Su, B., Zhang, Y., Xiong, L., He, Z., Shu, C., and Yang, D. Genetic divergence of Cordyceps sinesis as estimated by random amplified polymorphic DNA analysis. I Chuan Hsueh Pao. 24:410-416, 1997.

Chen, Y., Zhang, Y.P., Yang, Y., and Yang, D. Genetic diversity and taxonomic implication of Cordyceps sinesis as revealed by RAPD markers. Biochemical Genetics 37:201-213, 1999.

Cheng, K. T., Su, C. H., Chang, H. C., and Huang, J. Y. Differentiation of genuines and counterfeits of Cordyceps species using random amplified ploymorphic DNA. Planta. Medica. 64:451-453, 1998.

Collins, T. J., Moerschbacher, B. M., and Read, N. D. Synergistic induction of wheat stem rust appressoria by chemical and topographical signals. Physiological and Molecular Plant Pathology 58:259-266, 2001.

Cuningham, K. G., Manson, W., Spring, E. S., and Hutchison, S. A. Cordycepin, a metabolic product isolated from cultures of Cordyceps militaris (Linn.). Nature 166:9-14, 1950.

Deitch, A., and Sawick, S. G. Effects of cordycepin on microtubules of cultured mammalian cells. Exp. Cell. Res. 118:1-13, 1979.

De Jong, E., Field, J. A., Spinnler, H. E., Wijnberg, J. B. P. A. and de Bont, J. A. M. Significant biogenesis of chlorinated aromatics by fungi in natural environments. Appl. Envir. Microbiology 60:264-270, 1994.

Felsenstein, J. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17:386-376, 1981.

Felsenstein, J. Distance methods for inferring phylogeneies: A justification. Evolution 38:16-24, 1984.

Glazer, R. I., and Kuo, J. F. Inhibition of effects of cordycepin on cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinases. Biochem. Pharmacol. 26:1287-1290, 1977.

Guzman, G. Inventorying the fungi of Mexico. Biodiv. Conserv. 7:369-384, 1998.

Guy R. B. Geographical Distribution of Fungi. The Botanical Review, The New York Botanical Garden. 9:466-482, 1943.

Higgins, D. G., Bleasby, A. J., and Fuchs, R. CLUSTAL V: improved software for multiple sequence alignment. CABIOS 8:189-191, 1992.

Hseu, R. S., Wang, H. H., Wang, H. F. and Moncalvo, J. M. Application of PCR-amplified DNA to differentiate the Ganoderma isolates. J. Chinese Agric. Chem. Soc. 34:129-143, 1996.

Ito, Y., and Hirano, T. The detremination of partial 18S ribosomal DNA sequence of Cordyceps species. Lett. Appl. Microbiol. 25:239-242, 1997.

Lee, J. S., Ko, K. S., and Jung, H. S. Phylogenetic analysis of Xylaria based on nuclear ribosomal ITS1-5.8S-ITS2 sequences. FEMS Microbiol. Lett. 187:89-93, 2000.

Kasahara, A. Influence of orography on the atmospheric general circulation. In “Orographic effects in planetary flows”, World Meteorological Organiztion. pp. 4-49, GARP Publications series, 1980.

Kimura, M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120, 1980.

Strimmer, K., and von Haeseler, A. Quartet Puzzling: A Quartet Maximum-Likelihood Method for Reconstructing Tree Topologies. Mol. Biol. Evol. 13:964-969, 1996..

Kredich, N. M., and Guarino, A. J. Homocitrullyami-noadenosine, a nucleoside isolated frorr Cordyceps militaris. J. Biol. Chem. 236:3300-3302, 1961.

Kuo, Y. C., Lin, C. Y., Tsai, W. J., Wu, C. L., Chen, C. F., and Shiao, M. S. The growth inhibitors against tumor cells in Cordyceps sinensis other than cordycepin and polysaccharides. Cancer Invest. 12:611-615, 1994.

Lin, C. Y., Ku, F. M., Kuo, Y. C., Chen, C. F., Chen, W. P., Chen, A., and Shiao, M. S. Inhibition of activated human mesangial cell proliferation by the natural product of Cordyceps sinesis (H1-A): an implication for treatment of IgA mesangial nephropathy. J. Lab. Clin. Med. 133:55-63, 1999.

Ling, L., Li, J., and Cang.. Y. PCR-Based Sensitive Detection of Medicinal Fungi Hericium Species fromRibosomal Internal Transcribed Spacer (ITS) Sequences. Biol. Pharm. Bull. 25:975-980, 2000.

Mari, L. S., Katherine, F. L., and Scott, O. R. Comparison of Ribosomal DNA ITS regions among geographic isolates of Cenococcum geophilum Curr. Genet. 35:527-535, 1999.

Mikosch, T. S. P., Sonnenberg, A. S. M., and Van Griensven, L. J. L. D. Isolation, characterization and expression patterns of a DMC1 homolog from the basidiomycete Pleurotus ostreatus. Fung. Genet. Biol. 33:59-66, 2001.

Montefiori, D. C., Sobol, R. W. Jr., Li, S. W., Reichen Bach, N. L., Sunhodolnik, R. J., Charubala, R., Pfleriderder, W., Modliszwski, A., Robinson, W. E. Jr., and Mitchill, W. M. Phosphorothioate and cordycepin analogues of 2'5'-oligoadenylate: inhibition of human immunodeficiency type I reverse transcriptase and infection in vitro. PNAS 86:7191-7194, 1998.

Moore, D. Fungal Morphogenesis. Cambridge University Press. 1998.

Mueller, U. G., Lipari, S. E., and Milgroom, M. G. Amplified fragment length polymorphism (AFLP) fingerprinting of symbiotic fungi cultured by the fungus-growing ant Cyphomyrmex minutus. Mol. Ecol. 5:119-122., 1996.

Muller, W. E. G., Weller, B. E., Charubala, R., Plferiderer, W., Lserman, L.,Sobol, R. W., Suhadolnik, R. J., and Schroder, H. C. Cordycepin analogues of 2'5'-oligoadenylate inhibit human immunodeficiency virus infection via inhibition of reverse transcriptase. Biochem. 30:2027-2033, 1990.

Mueller, U. G., Wolfenbarger, L. L. AFLP genotyping and fingerprinting. Tree. 14:389-394, 1999.

Muraguchi, H. and Kamada, T. A mutation in the eln2 gene encoding a cytochrome P450 of Coprinus cinereus affects mushroom Morphogenesis. Fung. Genet. Biol. 29:49-59, 2000.

Nikoh, N. and Fukatsu, T. Interkindom host jumoing underground: phylogenetic analysis of entomoparasitic fungi of the genus Cordyceps. Mol. Biol. Evol. 17: 629-638, 2000.

Noval-Fernandez, A., and Leroy, F. Inhibition of nucleic acid methylation by cordycepin. J. Biol. Chem. 255:7380-7385, 1980.

Ospina-Giraldo, M. D., Collopy, P. D., Romaine, C. P. and Royse, D. J. Classification of sequences expressed during the primordial and basidiome stages of the cultivated mushroom Agaricus bisporus. Fung. Genet. Biol. 29:81-94, 2000.

Pelczar, M. J., Chan, E. C. S., and Krieng, N. R. Microbiology 4th ed. McGraw-hill Book Co., New York, p.918, 1986.

Saitou, N., and M. Nei. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425, 1987.

Sawado, T. and Sakaguchi, K. A DNA polymerase alpha catalytic subunit is purified independently for the tissues at meiotic prometaphase I of a basidiomycete, Coprinus cinereus. Biochem. Biophy. Res. Commun. 232:454-460, 1997.

Shimizu, D. Color Iconography of Vegetable Wwasps and Plant Worns. Seibundo Shinkosha, Tokyo, 1994.

Stephen B. G., Larry D. D., and Victoria L. Z Phylogenetic Analysis of Cercospora and Mycosphaerella Based on the internal Spacer Region of Ribosomal DNA. Phytopathology 91:649-658, 2001.

Suh, S. O., Noda, H., and Blackwell, M. Insect Symbiosis: Derivation of Yeast-like Endosymbionts Within an Entomopathogenic Filamentous Lineage. Mol. Biol. Evol. 18: 995-1000, 2001.

Thompson, J. D., Higgins, D. G., and Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680, 1994.

T. O. Powers, Todd, T. C., Burnell, A. M.l, Murray, P. C. B., Fleming, C. C., Szalanski, A. L., Adams, B. A. and Harris, T. S. The rDNA Internal Transcribed Spacer Region as a Taxonomic Marker for Nematodes. J. Nematology 29:441-450, 1997.

Ukai, S., Kiho, T., Hara, C., Morita, M., Goto, A., Imaizumi, M., and Hasegawa, Y. Polysaccharides in fungi. XIII. Antitumor activity of various polysaccharides isolated from Dictyophora indusiata , Ganoderma japonicum, Cordyceps cicadae, Auricularia auriculajaudae and Auricilaria species. Chem. Pharm. Bull. 31:741-744, 1983.

Versalovic, J., de Bruijn, F. J., and Lupski, J. R. Repetitive sequence-based PCR (rep-PCR) DNA fingerprinting of bacterial genome. In Bacterial Genomes: Physical Structure and Analysis, Edited by de Bruijn, F. J., Lupski, J. R., Weinstock, G. M. New York: Chapman & Hall. pp.437-454, 1998.

Versalovic, J., Koeuth, T., and Lupski, J. R. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids. Res. 19: 6823-6831, 1991.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., and Zabeau, M. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23:4407-4414, 1995.

Williams, J. G. K., KubelikA. R., Livak, K. J., Rafalski, J. A., and Scott, V. J. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531-6535, 1990.

White, T. J., Bruns, T., Lee, S., and Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications, Edited by Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J. San Diego: Academic Press. pp.315-322, 1990.

Yanada, H. Structure and antitumor activity of alkali-soluble polysaccharides from Cordyceps ophioglossiodes. Carbonhydr. Res. 125:107-115, 1984.

Yang, L.Y., Chen, A., Kuo, Y.C., and Lin, C.Y. Efficacy of a pure compound H1-A extracted from Cordyceps sinesis on autoimmune disease of MRL lpr/lpr mice. J. Lab. Clin. Med. 134:492-500, 1999.

Yhompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. The Clustal-windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24: 4876-4882, 1997.

Zieve, G. W., and Roemer, E. J. Cordycepin rapidly collapse the intermediate filament networks into juxtanuclear caps in fibroblast and epidermal cells. Exp. Cell. Res. 177:19-26, 1988.