白臀叶猴属（Pygathrix）走私样本的鉴定
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摘要
本研究的实验材料是被中国海关查获由越南走私入境,不知道确切来源地的28个白臀叶猴属(Pygathrix)干制样本。研究目的是为了确定该样本的来源地、种类和样本的性别,从而调查出野生动物非法贸易地。研究采用PCR方法扩增样本线粒体控制区高变区Ⅰ基因,利用测定序列与白臀叶猴的国外数据库进行比对鉴定样本来源地和种类。对测定序列进行分析,用MEGA4.0软件,以白头叶猴为外群构建系统树。通过性别鉴定的SRY和AMEL特异引物进行PCR扩增,鉴定个体的性别。同时利用微卫星标记的分析方法,对28个白臀叶猴样本进行亲缘关系的鉴定。
结果显示,白臀叶猴线粒体控制区的高变区Ⅰ序列均长401bp。在全部401个线粒体控制区位点中,其中有40个简约信息位点,和5个单态位点。共有374个不变位点,49个可变位点。平均碱基对存在4个A-G转换, 6个T-C转换,颠换较少,表现出转换偏倚,没有缺失和插入。通过与国外数据库比对(Roos,灵长类基因数据库),确定这28个样本均为黑腿白臀叶猴(Pygathrix nigripes)。20个走私样本都来自于越南庆和省(Khanh Hoa);3个走私样本来自越南平福省(Binh Phuoc)和柬埔寨的蒙多基里省(Mondulkiri);18号来自越南多乐省(Dak Lak)和林同省(Lam Dong);还有4个样本的确切来源地不清楚。性别鉴定结果表明,28个样本中,有17个雌性个体,11个雄性个体。其中来自于越南庆和省的20个样本中有12个雌性个体,7个雄性个体;来自于越南平福省和柬埔寨的蒙多基里省的3个样本中有1个雌性个体,2个雄性个体;来自越南多乐省和林同省的18号样为雄性;不知道确切来源地的4个样本全为雌性。利用8对白臀叶猴的微卫星引物,我们进行亲缘关系分析,发现来自庆和省的20个样本可能存在几个群,而来自平福省的样本很可能都是来自于同一个群。这说明来自庆和省的那些个体中有被偷猎者整群猎杀的,而来自其他地点的个体是被随机猎杀的。
本研究利用测定样本线粒体序列的分子生物学方法,成功鉴定了走私样本属于黑腿白臀叶猴。从而为海关鉴定走私白臀叶猴样本提供了一种灵敏而可靠的方法。样本来源地的确定,也为黑腿白臀叶猴的保护提供有力的支持。从越南地图可以看出庆和省临中国南海,水上交通发达;而平福省临近柬埔寨,是进出柬埔寨的陆地交通要道之一。这些有利地形为不法分子的走私活动提供了便利。确定走私白臀叶猴样本的来源地,为进一步调查出野生动物非法贸易地,准确打击濒危野生动物的走私活动,提供了可靠的依据。这对于白臀叶猴种群的国际保护很有参考价值,同时也为科学制定其保护策略有着重要的意义。
All materials which smuggled from Vietnam were captured by China customs, we must find out exact origins and species of these samples. The 28 dried samples belonged to genus Pygathrix, including hair, bones and muscle. This study based on mitochondrial control region HVⅠgene sequences, used sequence alignment with foreign gene bank of douc langur to identify the species and origins. Phylogenetic tree reconstructed by using neighbor-joining, minimum evolution, and maximum parsimony algorithm implemented in the computer software MEGA (molecular evolutionary genetic analysis) version 4.0. We used special primers did sex-typing. In order to study parentage of these 28 samples, we also used microsatellite DNA markers.
In the 401bp sequences of control region, a total of 49 variable sites, 374 constant sites, 5 singleton sites, and 40 parsimony informative sites had been found. Among the sequences, there were 4 A-G and 6 T-C transitions sites on average, and more transitions than transversions. It bias against transitions, and has no gaps and insertions. The information of phylogenetic tree revealed the topology of phylogenetic tree constructed by the Neighbor-Joining method was accordant with that of the Maximum Parsimony tree and Minimum Evolution tree. NJ tree, ME tree and MP tree all primarily suggested the twenty-nine samples can divide into three clusters. According to the genetic pairwise distances results and compared with foreign gene bank information, we concluded 20 samples most likely originated from Khanh Hoa province in Vietnam. Another three came from Binh Phuoc province in Vietnam or Cambodian Mondulkiri province, whereas one sample came from Dak Lak or Lam Dong provinces, Vietnam. The origin of four further samples was not clearly traceable. Based on sex-typing PCRs, 17 samples are from females and 11 from males. This information indicated the 28 individuals maybe have some nature groups. We used 8 polymorphic microsatellite DNA primers to do paternity testing. The result showed there were several natural groups in Khanh Hoa samples. We concluded some samples from Khanh Hoa were poached by whole group; but other samples from different places were poached randomly.
The smuggled samples were successfully identified by mtDNA sequences. This study provided a novel approach for the identification of douc langur samples in customs. Confirmed origins of smuggled samples were a strong support to protect the black-shanked Douc langur. Looking the map of Vietnam, we could easily find Khanh Hoa province facing to the South Sea of China, public transport was very convenience on the sea. Binh Phuoc province near by Cambodia closely, was one of the important main paths to Cambodia on the land. These advantageous geographic positions provided good conditions for the smuggler. After confirmed the origins, we could further observe illegal wildlife trade places and routes, provide trustworthy evidences to accurately beat smuggling activities of endanger Primates. This successful identification was due to international cooperation. The result of identification was important reference to international protection of douc langur. Furthermore, it was also significant for scientifically planning protection strategy of endanger Primates.
结果显示,白臀叶猴线粒体控制区的高变区Ⅰ序列均长401bp。在全部401个线粒体控制区位点中,其中有40个简约信息位点,和5个单态位点。共有374个不变位点,49个可变位点。平均碱基对存在4个A-G转换, 6个T-C转换,颠换较少,表现出转换偏倚,没有缺失和插入。通过与国外数据库比对(Roos,灵长类基因数据库),确定这28个样本均为黑腿白臀叶猴(Pygathrix nigripes)。20个走私样本都来自于越南庆和省(Khanh Hoa);3个走私样本来自越南平福省(Binh Phuoc)和柬埔寨的蒙多基里省(Mondulkiri);18号来自越南多乐省(Dak Lak)和林同省(Lam Dong);还有4个样本的确切来源地不清楚。性别鉴定结果表明,28个样本中,有17个雌性个体,11个雄性个体。其中来自于越南庆和省的20个样本中有12个雌性个体,7个雄性个体;来自于越南平福省和柬埔寨的蒙多基里省的3个样本中有1个雌性个体,2个雄性个体;来自越南多乐省和林同省的18号样为雄性;不知道确切来源地的4个样本全为雌性。利用8对白臀叶猴的微卫星引物,我们进行亲缘关系分析,发现来自庆和省的20个样本可能存在几个群,而来自平福省的样本很可能都是来自于同一个群。这说明来自庆和省的那些个体中有被偷猎者整群猎杀的,而来自其他地点的个体是被随机猎杀的。
本研究利用测定样本线粒体序列的分子生物学方法,成功鉴定了走私样本属于黑腿白臀叶猴。从而为海关鉴定走私白臀叶猴样本提供了一种灵敏而可靠的方法。样本来源地的确定,也为黑腿白臀叶猴的保护提供有力的支持。从越南地图可以看出庆和省临中国南海,水上交通发达;而平福省临近柬埔寨,是进出柬埔寨的陆地交通要道之一。这些有利地形为不法分子的走私活动提供了便利。确定走私白臀叶猴样本的来源地,为进一步调查出野生动物非法贸易地,准确打击濒危野生动物的走私活动,提供了可靠的依据。这对于白臀叶猴种群的国际保护很有参考价值,同时也为科学制定其保护策略有着重要的意义。
All materials which smuggled from Vietnam were captured by China customs, we must find out exact origins and species of these samples. The 28 dried samples belonged to genus Pygathrix, including hair, bones and muscle. This study based on mitochondrial control region HVⅠgene sequences, used sequence alignment with foreign gene bank of douc langur to identify the species and origins. Phylogenetic tree reconstructed by using neighbor-joining, minimum evolution, and maximum parsimony algorithm implemented in the computer software MEGA (molecular evolutionary genetic analysis) version 4.0. We used special primers did sex-typing. In order to study parentage of these 28 samples, we also used microsatellite DNA markers.
In the 401bp sequences of control region, a total of 49 variable sites, 374 constant sites, 5 singleton sites, and 40 parsimony informative sites had been found. Among the sequences, there were 4 A-G and 6 T-C transitions sites on average, and more transitions than transversions. It bias against transitions, and has no gaps and insertions. The information of phylogenetic tree revealed the topology of phylogenetic tree constructed by the Neighbor-Joining method was accordant with that of the Maximum Parsimony tree and Minimum Evolution tree. NJ tree, ME tree and MP tree all primarily suggested the twenty-nine samples can divide into three clusters. According to the genetic pairwise distances results and compared with foreign gene bank information, we concluded 20 samples most likely originated from Khanh Hoa province in Vietnam. Another three came from Binh Phuoc province in Vietnam or Cambodian Mondulkiri province, whereas one sample came from Dak Lak or Lam Dong provinces, Vietnam. The origin of four further samples was not clearly traceable. Based on sex-typing PCRs, 17 samples are from females and 11 from males. This information indicated the 28 individuals maybe have some nature groups. We used 8 polymorphic microsatellite DNA primers to do paternity testing. The result showed there were several natural groups in Khanh Hoa samples. We concluded some samples from Khanh Hoa were poached by whole group; but other samples from different places were poached randomly.
The smuggled samples were successfully identified by mtDNA sequences. This study provided a novel approach for the identification of douc langur samples in customs. Confirmed origins of smuggled samples were a strong support to protect the black-shanked Douc langur. Looking the map of Vietnam, we could easily find Khanh Hoa province facing to the South Sea of China, public transport was very convenience on the sea. Binh Phuoc province near by Cambodia closely, was one of the important main paths to Cambodia on the land. These advantageous geographic positions provided good conditions for the smuggler. After confirmed the origins, we could further observe illegal wildlife trade places and routes, provide trustworthy evidences to accurately beat smuggling activities of endanger Primates. This successful identification was due to international cooperation. The result of identification was important reference to international protection of douc langur. Furthermore, it was also significant for scientifically planning protection strategy of endanger Primates.
引文
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