用线粒体基因序列探讨西部高原几种蝮蛇和云南半叶趾虎的分类学问题
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摘要
本研究基于线粒体基因序列探讨了西部高原几种蝮蛇的分类地位以及云南半叶趾虎(Hemiphyllodactylus yunnanensis)的种下分类，并根据12S rRNA基因序列对宜兴工商执法部门送检的5件野生动物样品进行了鉴定。
1 西部高原地区蝮蛇的分类
测定了高原蝮(Gloydius strauchii)、六盘山蝮(G. liupanensis)、秦岭蝮(G. qinlingensis)等9号标本的Cyt b基因319 bp片段的序列，对这4种蝮蛇的系统发生关系进行研究，获得的结果不支持G. monticola为Glovdius strauchii的亚种，相反支持前者为一个独立的物种；G. liupanensis为G. strauchii的同物异名，G. qinlingensis(?)是1个独立的物种。
2 云南半叶趾虎的种下分类
测定了云南半叶趾虎(Hemiphyllodactylus yunnanensis)4个亚种共20号标本的Cyt b 385 bp片段的序列，及ND2-CO Ⅰ基因485 bp片段的序列。根据Cyt b基因片段的序列定义了15个单元型。其中LLC1为龙陵和保山2个地方种群共享，未发现亚种间的共享单元型。根据ND2-CO Ⅰ基因片段的序列定义了12个单元型。其中KMN1为昆明和石林2个地方种群共享，也未发现亚种间的共享单元型。分子变异分析(AMOVA)和系统发生分析表明云南半叶趾虎的核苷酸多样性和单元型多样性都相当高。所有的分子系统树都强力支持云南的半叶趾虎与贵州的半叶趾虎组成姐妹群(自引导值至少95)。云南的半叶趾虎包括指名亚种和龙陵亚种2支。金平亚种绿春地方种群2个个体分别嵌入指名亚种和和龙陵亚种。不论单元型的分析还是系统发生关系的分析都支持龙陵亚种、独山亚种和指名亚种的分类地位。单元型分析也支持金平亚种是有效的，但系统发生分析研究未能解决其分类地位。
3 几种野生动物样品的DNA分子鉴定
用12S rDNA通用引物L1091和H1478扩增了宜兴工商执法部门送检的5件野生动物样品。测定其序列后，与GenBank的同源序列比对，并计算测定得到的序列与相关物
用线粒体基因序列探讨西部高原几种蝮蛇和么南半叶趾虎的分类学问题
种序列间的P值，作为种类鉴定的依据。分子鉴定的结果:2件样品为孟加拉巨晰(Varanus
bengalensis)、l件为黑熊(乙乃份us rh艺betanus)、l件为中国穿山甲(材。nis尸entadae如la)、
1件为穿山甲属未定种(人勿瓜ssP.)。
The present study discussed the classification of some pit-vipers {Gloydius) from the western plateau and intraspecific classification of Hemiphyllodactylus yunnanensis based on sequences of fragments of Cyt b and ND2-CO I genes, and identified five animal samples by means of molecular identification techniques using 12S rDNA sequences.
1. Classification of pit-vipers from the western plateau of China
The sequences of a 319 bp fragment of Cyt b gene were determined for 9 samples of Gloydius strauchii, G liupanensis and G. qinlingensis and the phylogenetic relationships among the 4 pit-vipers were studied. The results obtained do not support the opinion to refer G monticola as a subspecies of G. strauchii, and on the contrary support it to be a separate species; indicate G liupanensis would be a junior synonym of G strauchii, and G qinlingensis is a separate species.
2. Intraspecific classification of Hemiphyllodactylus yunnanensis
The sequences of a 385 bp fragment of Cyt b gene and 485 bp fragment of ND2-CO I gene for 20 samples belonging to 4 subspecies of Hemiphyllodactylus yunnanensis were determined. Fifteen haplotypes were defined according to the Cyt b sequences, among which Longling and Baoshan local populations share LLC1, no shared haplotypes is found between local populations. Twelve haplotypes were defined according to the ND2-CO I sequences, among which Kunming and Shilin local populations share KMN1, no shared haplotypes is found between local populations also. The results of AMOVA analysis and phylogenetic analyses indicate both high nucleotide diversity and haplotype diversity for Hemiphyllodactylus yunnanensis. All trees from sequence data derived from molecular phylogenetic analyses strongly support the sister relationship between the Yunnan population and Guizhou population of Hemiphyllodactylus yunnanensis (BCL values at least 95). The Yunnan population contains two clades: Hemiphyllodactylus yunnanensis yunnanensi
s and
Hemiphyllodactylus yunnanensis longlingensis. The two individuals of Lvchun local population of Hemiphyllodactylus yunnanensis jinpingensis nested into the nominal subspecies H. y. yunnanensis and H. y. longlingensis respectively. The validity of H. y. yunnanensis, H. y. longlingensis and H. y. dushanensis is supported by both haplotype analysis and phylogenetic analysis. The subspecific status of H. y. jinpingensis is also suppoted by haplotype analysis, but it has not been resolved by phylogenetic analyses.
3. DNA molecular identification of 5 animal samples
The Industry and Commerce Bureau of Yixing City sent us five animal samples for identification. Fragment of 12S rDNA was amplified from the five samples using the primer pair L1091 and H1478. Sequences obtained in the present study, and homologous sequences retrieved from GenBank were aligned by Clustal software. P-distance was assessed by MEGA software version 2.1., and was used as a criterion for identification. The results of the molecular identification indicate: there are two samples of Varanus bengalensis, one sample of Ursus thibetanus, one sample of Manis pentadactyla and one sample of unidentified species of pangolin {Manis sp.).
1 西部高原地区蝮蛇的分类
测定了高原蝮(Gloydius strauchii)、六盘山蝮(G. liupanensis)、秦岭蝮(G. qinlingensis)等9号标本的Cyt b基因319 bp片段的序列，对这4种蝮蛇的系统发生关系进行研究，获得的结果不支持G. monticola为Glovdius strauchii的亚种，相反支持前者为一个独立的物种；G. liupanensis为G. strauchii的同物异名，G. qinlingensis(?)是1个独立的物种。
2 云南半叶趾虎的种下分类
测定了云南半叶趾虎(Hemiphyllodactylus yunnanensis)4个亚种共20号标本的Cyt b 385 bp片段的序列，及ND2-CO Ⅰ基因485 bp片段的序列。根据Cyt b基因片段的序列定义了15个单元型。其中LLC1为龙陵和保山2个地方种群共享，未发现亚种间的共享单元型。根据ND2-CO Ⅰ基因片段的序列定义了12个单元型。其中KMN1为昆明和石林2个地方种群共享，也未发现亚种间的共享单元型。分子变异分析(AMOVA)和系统发生分析表明云南半叶趾虎的核苷酸多样性和单元型多样性都相当高。所有的分子系统树都强力支持云南的半叶趾虎与贵州的半叶趾虎组成姐妹群(自引导值至少95)。云南的半叶趾虎包括指名亚种和龙陵亚种2支。金平亚种绿春地方种群2个个体分别嵌入指名亚种和和龙陵亚种。不论单元型的分析还是系统发生关系的分析都支持龙陵亚种、独山亚种和指名亚种的分类地位。单元型分析也支持金平亚种是有效的，但系统发生分析研究未能解决其分类地位。
3 几种野生动物样品的DNA分子鉴定
用12S rDNA通用引物L1091和H1478扩增了宜兴工商执法部门送检的5件野生动物样品。测定其序列后，与GenBank的同源序列比对，并计算测定得到的序列与相关物
用线粒体基因序列探讨西部高原几种蝮蛇和么南半叶趾虎的分类学问题
种序列间的P值，作为种类鉴定的依据。分子鉴定的结果:2件样品为孟加拉巨晰(Varanus
bengalensis)、l件为黑熊(乙乃份us rh艺betanus)、l件为中国穿山甲(材。nis尸entadae如la)、
1件为穿山甲属未定种(人勿瓜ssP.)。
The present study discussed the classification of some pit-vipers {Gloydius) from the western plateau and intraspecific classification of Hemiphyllodactylus yunnanensis based on sequences of fragments of Cyt b and ND2-CO I genes, and identified five animal samples by means of molecular identification techniques using 12S rDNA sequences.
1. Classification of pit-vipers from the western plateau of China
The sequences of a 319 bp fragment of Cyt b gene were determined for 9 samples of Gloydius strauchii, G liupanensis and G. qinlingensis and the phylogenetic relationships among the 4 pit-vipers were studied. The results obtained do not support the opinion to refer G monticola as a subspecies of G. strauchii, and on the contrary support it to be a separate species; indicate G liupanensis would be a junior synonym of G strauchii, and G qinlingensis is a separate species.
2. Intraspecific classification of Hemiphyllodactylus yunnanensis
The sequences of a 385 bp fragment of Cyt b gene and 485 bp fragment of ND2-CO I gene for 20 samples belonging to 4 subspecies of Hemiphyllodactylus yunnanensis were determined. Fifteen haplotypes were defined according to the Cyt b sequences, among which Longling and Baoshan local populations share LLC1, no shared haplotypes is found between local populations. Twelve haplotypes were defined according to the ND2-CO I sequences, among which Kunming and Shilin local populations share KMN1, no shared haplotypes is found between local populations also. The results of AMOVA analysis and phylogenetic analyses indicate both high nucleotide diversity and haplotype diversity for Hemiphyllodactylus yunnanensis. All trees from sequence data derived from molecular phylogenetic analyses strongly support the sister relationship between the Yunnan population and Guizhou population of Hemiphyllodactylus yunnanensis (BCL values at least 95). The Yunnan population contains two clades: Hemiphyllodactylus yunnanensis yunnanensi
s and
Hemiphyllodactylus yunnanensis longlingensis. The two individuals of Lvchun local population of Hemiphyllodactylus yunnanensis jinpingensis nested into the nominal subspecies H. y. yunnanensis and H. y. longlingensis respectively. The validity of H. y. yunnanensis, H. y. longlingensis and H. y. dushanensis is supported by both haplotype analysis and phylogenetic analysis. The subspecific status of H. y. jinpingensis is also suppoted by haplotype analysis, but it has not been resolved by phylogenetic analyses.
3. DNA molecular identification of 5 animal samples
The Industry and Commerce Bureau of Yixing City sent us five animal samples for identification. Fragment of 12S rDNA was amplified from the five samples using the primer pair L1091 and H1478. Sequences obtained in the present study, and homologous sequences retrieved from GenBank were aligned by Clustal software. P-distance was assessed by MEGA software version 2.1., and was used as a criterion for identification. The results of the molecular identification indicate: there are two samples of Varanus bengalensis, one sample of Ursus thibetanus, one sample of Manis pentadactyla and one sample of unidentified species of pangolin {Manis sp.).
引文
[1] 郑乐怡．1987．动物分类原理与方法．北京：高等教育出版社．
[2] Cope, E. D., 1864. On the Character of the higher Groups of Reptilia Squamata-and especially of the Diploflossa. Proc. Acad. Naat. Sci. Phila. [ⅩⅥ]: 224～231.
[3] 陈俊才，彭先步，余多慰．1986．壁虎属三种壁虎的核型研究．两栖爬行动物学报．5(1)：24～29．
[4] 郭超文，李洁莉．1995．三种壁虎染色体核仁组织者的银染色观察与无蹼壁虎的核型．遗传．17(1)：16～18．
[5] Yann S. G, B. Heulin, C. P. Guillaume et al. 2001. Intraspecific Phylogeography of Lacrerta vivipara and the Evolution of Viviparity. Mol. phyl. Evol. 18(3): 449～459.
[6] 谭竹钧，韩雅莉．1996．正蚯蚓科(Lumbricidae)三属七种动物同工酶研究．内蒙古大学学报(自然科学版)．27(1)：111～116．
[7] Ruedi, M., C. Courvoisier, P. Vogel and F. M. Cateflis. 1996. Genetic differentiation and zoogeography of Asian Suncus murinus (Mammalia: Soricidae). Biological Journal of the Linnean Society. 57:307～316.
[8] 万发春，王慧，吴乃克．1999．血液中DNA提取方法的比较研究．山东畜牧兽医．(1)：6～7．
[9] 钟华，赖旭龙，魏荣平，刘中来．一种从大熊猫粪便中提取DNA的改进方法．动物学报．49(5)：670～674．
[10] Hss, M., M. Kohn and S. Pbo. 1992. Excrement analysis by PCR. Nature 359: 199.
[11] 李明，魏辅文，饶刚，方盛国，冯祚建．2001．非损伤性取样法在保护遗传学研究中的应用．动物学报．47(3)：338～342．
[12] 饶刚，李明，牛屹东，王静，魏辅文，方盛国．2001．陈旧皮张中DNA提取的新方法．动物学杂志．36(4)：53～57．
[13] 赵春江，李宁．2003．一种从毛发中提取DNA的简易方法．遗传．25(1)：69～70．
[14] 栗磊，王慧．1999．从动物毛发中提取DNA进行RAPD扩增的研究．山东畜牧兽医．(2)：1～2．
[15] Carranza, S., E. N. Arold, J. A. Mateo, and P. Geniez. 2002. Relationships and evolution of the North African geckos, Geckonia and Tarentola(Reptilia: Gekkonidae), based on mitochondrial and nuclear DNA sequences. Mol. phyl. Evol. 23:244～256.
[16] Levi, HW, von Eickstedt VDR. 1989. The Nephilinae spides of the neotropics. Mems Inst Butantan. 51: 43～56.
[17] Pan, H. C, K. Zhou, D. Song and Y. Qiu. 2004. Phylogenetic Placement of the Spider Genus Nephila(Araneae: Araneoidea) Inferred from rRNA and MaSp Ⅰ Gene Sequences. Zoological Science. 21: 343～351.
[18] Tang, B., K. Zhou, D. Song, G. Yang and A. Dai. 2003. Molecular systematics of the Asian mitten crabs genus Eriochier (Crustacea: Brachyura). Mol. phyl. Evol. 29: 309～316.
[19] Parkinson, C. L., K. R. Zamudio and H. W. Greene. 2000. Phylogeography of the pitviper clade Agkinstrodon: historical ecology, species status, and conservation of cantils. Molecular Ecology. 9:411～420.
[20] Harris, D. J., V. Batista, P. lymberakis, and M. A. Carretero. 2004. Complex estimates of evolutionary relationships in Tarentola mauritanica (Reptilia: Gekkonidae) derived from mitochondrial DNA sequences. Mol. phyl. Evol. 30: 855～859.
[21] Fitzsimmons, N. N., J. C. Buchan, P. V. Lam, G. Polet, T. T. Hung, N. Q. Thang and J. Gratten. 2002. Identification of Purebred Crocodylus siamensis for Reintroduction in Vietnam. J. of Experimental Zoology (Mol Dev Evol). 294: 373～381.
[22] Dalebout, M. L, A. V. Helden, K. van Waerebeek, C. S. Baker. 1998. Molecular genetic identification of southern hemisphere beaked whales (Cetacea:
Ziphiidae). Mol Ecol. 7(6): 687-94.
[23] Dalebout, M. L., J. G. Mead, C. S. Baker, A. N. Baker and A. L. V. Helden. 2002. A new species of beaked whale Mesoplodon perrni sp. n. (Cetacea: Ziphiidae) discovered through phylogenetic analysis of mitochondrial DNA sequence. Marine Mammal Science 18(3): 577-608.
[24] 吴平，周开亚，徐珞珊，滕健昌．1998．中药材龟甲的分子鉴定研究．药学学报．33(3)：304～309．
[25] Wang Y Q, Zhou K Y. 2002. Authentication of animal medicinal materials by DNA molecular markers. In: Shaw P C, Wang J, But PP-H eds. Authentication of Chinese Medicinal Materials by DNA Technology. World Scientific, New Jersey, London, Singapore, Hong Kong. 125～153.
[26] 孙红英，周开亚，陆健健，杨光．2002．中国大陆绒螯蟹线粒体16S rDNA序列变异与分子鉴定标记．自然科学进展．12(5)：485～490．
[27] 杨光，刘海，周开亚，季国庆．2002．用mtDNA序列鉴定一头小布氏鲸标本．动物学杂志．37(4)：35～38．
[28] 杨小军，周开亚．2004．几种野生动物样品的DNA分子鉴定．动物学杂志．39(1)：89～92．
[29] 周开亚．2001．两栖爬行动物的分子系统发生．动物学研究．22(5)：397～405．
[30] 周开亚，刘月珍，李德俊．1982．壁虎属三新种兼记铅山壁虎(蜥蜴目)[J]．动物分类学报．7(4)：438～444．
[31] Zhang, Yun-wu, Oliver A. Ryder, Zhang, Ya-ping. 2001. Genetic divergence of orangutan subspecies(Pocgo pygmaeus). J Mol Evol. 52:516～526.
[2] Cope, E. D., 1864. On the Character of the higher Groups of Reptilia Squamata-and especially of the Diploflossa. Proc. Acad. Naat. Sci. Phila. [ⅩⅥ]: 224～231.
[3] 陈俊才，彭先步，余多慰．1986．壁虎属三种壁虎的核型研究．两栖爬行动物学报．5(1)：24～29．
[4] 郭超文，李洁莉．1995．三种壁虎染色体核仁组织者的银染色观察与无蹼壁虎的核型．遗传．17(1)：16～18．
[5] Yann S. G, B. Heulin, C. P. Guillaume et al. 2001. Intraspecific Phylogeography of Lacrerta vivipara and the Evolution of Viviparity. Mol. phyl. Evol. 18(3): 449～459.
[6] 谭竹钧，韩雅莉．1996．正蚯蚓科(Lumbricidae)三属七种动物同工酶研究．内蒙古大学学报(自然科学版)．27(1)：111～116．
[7] Ruedi, M., C. Courvoisier, P. Vogel and F. M. Cateflis. 1996. Genetic differentiation and zoogeography of Asian Suncus murinus (Mammalia: Soricidae). Biological Journal of the Linnean Society. 57:307～316.
[8] 万发春，王慧，吴乃克．1999．血液中DNA提取方法的比较研究．山东畜牧兽医．(1)：6～7．
[9] 钟华，赖旭龙，魏荣平，刘中来．一种从大熊猫粪便中提取DNA的改进方法．动物学报．49(5)：670～674．
[10] Hss, M., M. Kohn and S. Pbo. 1992. Excrement analysis by PCR. Nature 359: 199.
[11] 李明，魏辅文，饶刚，方盛国，冯祚建．2001．非损伤性取样法在保护遗传学研究中的应用．动物学报．47(3)：338～342．
[12] 饶刚，李明，牛屹东，王静，魏辅文，方盛国．2001．陈旧皮张中DNA提取的新方法．动物学杂志．36(4)：53～57．
[13] 赵春江，李宁．2003．一种从毛发中提取DNA的简易方法．遗传．25(1)：69～70．
[14] 栗磊，王慧．1999．从动物毛发中提取DNA进行RAPD扩增的研究．山东畜牧兽医．(2)：1～2．
[15] Carranza, S., E. N. Arold, J. A. Mateo, and P. Geniez. 2002. Relationships and evolution of the North African geckos, Geckonia and Tarentola(Reptilia: Gekkonidae), based on mitochondrial and nuclear DNA sequences. Mol. phyl. Evol. 23:244～256.
[16] Levi, HW, von Eickstedt VDR. 1989. The Nephilinae spides of the neotropics. Mems Inst Butantan. 51: 43～56.
[17] Pan, H. C, K. Zhou, D. Song and Y. Qiu. 2004. Phylogenetic Placement of the Spider Genus Nephila(Araneae: Araneoidea) Inferred from rRNA and MaSp Ⅰ Gene Sequences. Zoological Science. 21: 343～351.
[18] Tang, B., K. Zhou, D. Song, G. Yang and A. Dai. 2003. Molecular systematics of the Asian mitten crabs genus Eriochier (Crustacea: Brachyura). Mol. phyl. Evol. 29: 309～316.
[19] Parkinson, C. L., K. R. Zamudio and H. W. Greene. 2000. Phylogeography of the pitviper clade Agkinstrodon: historical ecology, species status, and conservation of cantils. Molecular Ecology. 9:411～420.
[20] Harris, D. J., V. Batista, P. lymberakis, and M. A. Carretero. 2004. Complex estimates of evolutionary relationships in Tarentola mauritanica (Reptilia: Gekkonidae) derived from mitochondrial DNA sequences. Mol. phyl. Evol. 30: 855～859.
[21] Fitzsimmons, N. N., J. C. Buchan, P. V. Lam, G. Polet, T. T. Hung, N. Q. Thang and J. Gratten. 2002. Identification of Purebred Crocodylus siamensis for Reintroduction in Vietnam. J. of Experimental Zoology (Mol Dev Evol). 294: 373～381.
[22] Dalebout, M. L, A. V. Helden, K. van Waerebeek, C. S. Baker. 1998. Molecular genetic identification of southern hemisphere beaked whales (Cetacea:
Ziphiidae). Mol Ecol. 7(6): 687-94.
[23] Dalebout, M. L., J. G. Mead, C. S. Baker, A. N. Baker and A. L. V. Helden. 2002. A new species of beaked whale Mesoplodon perrni sp. n. (Cetacea: Ziphiidae) discovered through phylogenetic analysis of mitochondrial DNA sequence. Marine Mammal Science 18(3): 577-608.
[24] 吴平，周开亚，徐珞珊，滕健昌．1998．中药材龟甲的分子鉴定研究．药学学报．33(3)：304～309．
[25] Wang Y Q, Zhou K Y. 2002. Authentication of animal medicinal materials by DNA molecular markers. In: Shaw P C, Wang J, But PP-H eds. Authentication of Chinese Medicinal Materials by DNA Technology. World Scientific, New Jersey, London, Singapore, Hong Kong. 125～153.
[26] 孙红英，周开亚，陆健健，杨光．2002．中国大陆绒螯蟹线粒体16S rDNA序列变异与分子鉴定标记．自然科学进展．12(5)：485～490．
[27] 杨光，刘海，周开亚，季国庆．2002．用mtDNA序列鉴定一头小布氏鲸标本．动物学杂志．37(4)：35～38．
[28] 杨小军，周开亚．2004．几种野生动物样品的DNA分子鉴定．动物学杂志．39(1)：89～92．
[29] 周开亚．2001．两栖爬行动物的分子系统发生．动物学研究．22(5)：397～405．
[30] 周开亚，刘月珍，李德俊．1982．壁虎属三新种兼记铅山壁虎(蜥蜴目)[J]．动物分类学报．7(4)：438～444．
[31] Zhang, Yun-wu, Oliver A. Ryder, Zhang, Ya-ping. 2001. Genetic divergence of orangutan subspecies(Pocgo pygmaeus). J Mol Evol. 52:516～526.