日本伏翼（Pipistrellus abramus）微卫星位点的筛选及其种群遗传结构特征
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摘要
相对于欧洲和南美洲,第四纪冰川的气候变化对亚洲生物群落的分布和遗传结构形成的影响的研究报导至今还很少。在冰川后期的种群扩张期间,地理屏障对某个物种的扩散能力的促进或限制的作用同样对其种群遗传结构有重要的影响。日本伏翼(翼手目)在中国的很多地理区域包括海南岛和舟山群岛在内都有分布。近些年来,由于生态环境遭到严重破坏以及城乡建设的快速发展,对于主要栖息于居民老房里的翼手目动物类群,如日本伏翼,在某些地区其种群数量急剧下降。遗传多样性是种群长期生存繁衍和抵御疾病等外界胁迫的基础,种群数量的演变历史对种群的保护有很重要的指导作用。为全面了解日本伏翼的生存现状及其种群历史演替情况,并制定正确的、合理的动物保护措施,我们非常有必要对日本伏翼的遗传多样性以及种群间的基因流动等方面进行研究。
本文运用线粒体DNA和微卫星DNA两种不同分子遗传标记对日本伏翼的种群遗传结构和种群历史情况进行研究。其结果如下：
1、利用富集法对日本伏翼(Pipistrellus abramus)进行微卫星DNA文库第一次构建,共筛选到微卫星引物位点10对。在筛选到的这10对微卫星引物位点中,分别对来自广西桂林的37个日本伏翼个体种群进行扩增。每对引物扩增后的分析结果都呈现出多态性,所有微卫星位点的平均等位基因数目为10个,范围在7-13个不等。杂合度观测值和杂合度期望值范围分别为从0.486到0.971和.752到0.876。经过分析,三个微卫星位点(WW6、PA133和1-20)偏离了哈迪-温伯格平衡,但没有发现任何一个位点发生连锁不平衡。这些微卫星位点将对日本伏翼及本属的其他物种的种群遗传结构的研究具有非常重要的作用。
2、线粒体DNA数据和微卫星DNA(8对位点引物)数据都表明了日本伏翼在各种不同的地理种群均具有较高的遗传多样性。线粒体DNA单倍型种群多样性(h)从0.286到0.9,核苷酸多样性(∏)从0.00025到0.01878；微卫星DNA的种群杂合度观测值(HO)从0.58到0.89,杂合度期望值(HE)从0.67到0.82,种群的平均等位基因丰富度从4.13到10.36。
3、两种分子遗传标记的研究结果都说明了自然地理结构对日本伏翼种群结构的连续性产生明显的影响。特别地,两种分子标记的结果都说明了琼州海峡(把海南岛和中国大陆隔开)对日本伏翼的基因流具有很大的阻隔作用。同时,在两种遗传标记的结果中,所有种群的遗传距离与地理距离均呈显著相关(线粒体DNA,R2=0.27,P<0.001；微卫星DNA,R2=0.31,P<0.001)。
4、线粒体DNA序列单倍型的歧点分布分析(Mismatch distribution)说明了自从第四纪冰川以来大陆/舟山群岛蝙蝠种群和海南岛蝙蝠蝙蝠种群都经历了种群扩张事件,且前者呈现星状拓扑结构。线粒体DNA的数据分析结果揭示了虽然相互隔离了7000-9000年,但大陆/舟山群岛蝙蝠种群仍具有它们的共同祖先。虽然琼州海峡把海南岛与大陆分离至少有10000年的时间,但海南岛蝙蝠种群内没有出现共享的单倍型,说明没有发现共同的祖先或通过近期种群扩张而来。
5、微卫星DNA数据分析结果并没有完全支持线粒体DNA数据的分析结果,反之,发现在大陆/舟山群岛蝙蝠种群和海南岛蝙蝠蝙蝠种群之间存在一些差异,进一步说明了只有结合运用不同分子标记对种群遗传空间尺度的分析,才能得出更为正确而有效地评价物种种群遗传结构及其种群历史演化情况。
综合以上的研究结果得知,本文运用了线粒体DNA和微卫星DNA两种不同的分子标记比较分析了日本伏翼不同种群的遗传特征与地理分布的关系,为进一步研究该物种的谱系地理学特征奠定了基础。
Compared to Europe and North America, the influence of climatic oscillations during the Pleistocene in shaping the distribution and genetic structure of biota in Asia is not well known. The influence of geographical barriers in promoting or restricting the dispersal ability of a species during post-glacial expansions will also have a large impact on its population genetic structure. The Japanese pipistrelle bat, Pipistrellus abramus, is found over a wide range in China, including Hainan Island and the Zhoushan Archipelago. Recently, in some areas, the number individuals of some bat species have dramatically been in decline due to seriously destructive ecological environment and fast development of towns. Genetic diversity is the basis of population long-term survival and resisting the diseases. Demographic history analysis is vital to population protection. In order to comprehensively understand survival status and demographic history and establish accurate protection steps for Pipistrellus abramus, it plays an important role in estimation of genetic diversity and gene flow between populations.
We applied both mitochondrial (cytochrome b) and microsatellite markers to examine population genetic structure and demographic history in this species. The main results are as follows:
1. We describe the first set of ten microsatellite markers isolated in Pipistrellus abramus. The number of alleles per locus ranged from 7 to 13. The observed and expected heterozygosities values ranged from 0.486 to 0.971 and from 0.752 to 0.876, respectively. Three loci (WW6、PA133 and 1-20) revealed significant departure from Hardy-Weinberg equilibrium and no linkage disequilibrium was found between loci pairs. These infonnative microsatellite markers will be a powerful molecular tool for studying the population genetic structure of P. abramus, as well as other species of this genus.
2. Both results of mtDNA and microsatellite DNA (8 loci) revealed Pipistrellus abramus contain relatively high genetic diversities among different geographic populations. For the mitochondrial DNA (mtDNA) data, the haplotype diversity ranged from 0.286 to 0.9, and nucleotide diversity ranged from 0.00025 to 0.01878. For the microsatellite data, the observed heterozygosity (Ho) and expected (HE) heterozygosity ranged from 0.58 to 0.89, and from 0.67 to 0.82, respectively. The allelic richness (Rs) ranged from 4.13 to 10.36 among sampled populations.
3. Our results suggest that geographical features have had a clear impact on population connectivity in P. abramus. Specifically, both markers indicate that the Qiongzhou Strait, which separates Hainan Island from the continent, acts as a significant barrier to gene flow. Likewise, both genetic distance of mtDNA and microsatellite DNA were significant with natural geography distance (mtDNA, R2=0.27, P＜0.001; microsatellite DNA,R2=0.31, P＜0.001).
4. Mismatch distributions show that populations from continental China/Zhoushan Archipelago and Hainan Island appear to have undergone expansions since the Pleistocene, with evidence of a star-like topology in the former. MtDNA reveals evidence of common ancestry between the colonies sampled on the Zhoushan Archipelago and those on the continent, which are thought to have become isolated approximately 7000-9000 years ago. Although the Qiongzhou trait seperates the Hainan Island and mainland for over 10000 years, no haplotype was found to share among individuals sampled from the island, and it revealed that the bats sampled from the Hainan Island may colony into the island vita recent population expansion since the Pleistocene.
5. Microsatellite data did not fully support the mitochondrial data and instead detected some differentiation between the two groups, highlighting the importance of using different markers at different spatial scales.
In summary, we were explicit that the thesis data were combined with mitochondrial (cytochrome b) and microsatellite molecular markers for comparison of different geographical populations and geography distribution. Farther more, the results were the basis study of phylogeography of the Japanese pipistrelle bat, Pipistrellus abramus.
本文运用线粒体DNA和微卫星DNA两种不同分子遗传标记对日本伏翼的种群遗传结构和种群历史情况进行研究。其结果如下：
1、利用富集法对日本伏翼(Pipistrellus abramus)进行微卫星DNA文库第一次构建,共筛选到微卫星引物位点10对。在筛选到的这10对微卫星引物位点中,分别对来自广西桂林的37个日本伏翼个体种群进行扩增。每对引物扩增后的分析结果都呈现出多态性,所有微卫星位点的平均等位基因数目为10个,范围在7-13个不等。杂合度观测值和杂合度期望值范围分别为从0.486到0.971和.752到0.876。经过分析,三个微卫星位点(WW6、PA133和1-20)偏离了哈迪-温伯格平衡,但没有发现任何一个位点发生连锁不平衡。这些微卫星位点将对日本伏翼及本属的其他物种的种群遗传结构的研究具有非常重要的作用。
2、线粒体DNA数据和微卫星DNA(8对位点引物)数据都表明了日本伏翼在各种不同的地理种群均具有较高的遗传多样性。线粒体DNA单倍型种群多样性(h)从0.286到0.9,核苷酸多样性(∏)从0.00025到0.01878；微卫星DNA的种群杂合度观测值(HO)从0.58到0.89,杂合度期望值(HE)从0.67到0.82,种群的平均等位基因丰富度从4.13到10.36。
3、两种分子遗传标记的研究结果都说明了自然地理结构对日本伏翼种群结构的连续性产生明显的影响。特别地,两种分子标记的结果都说明了琼州海峡(把海南岛和中国大陆隔开)对日本伏翼的基因流具有很大的阻隔作用。同时,在两种遗传标记的结果中,所有种群的遗传距离与地理距离均呈显著相关(线粒体DNA,R2=0.27,P<0.001；微卫星DNA,R2=0.31,P<0.001)。
4、线粒体DNA序列单倍型的歧点分布分析(Mismatch distribution)说明了自从第四纪冰川以来大陆/舟山群岛蝙蝠种群和海南岛蝙蝠蝙蝠种群都经历了种群扩张事件,且前者呈现星状拓扑结构。线粒体DNA的数据分析结果揭示了虽然相互隔离了7000-9000年,但大陆/舟山群岛蝙蝠种群仍具有它们的共同祖先。虽然琼州海峡把海南岛与大陆分离至少有10000年的时间,但海南岛蝙蝠种群内没有出现共享的单倍型,说明没有发现共同的祖先或通过近期种群扩张而来。
5、微卫星DNA数据分析结果并没有完全支持线粒体DNA数据的分析结果,反之,发现在大陆/舟山群岛蝙蝠种群和海南岛蝙蝠蝙蝠种群之间存在一些差异,进一步说明了只有结合运用不同分子标记对种群遗传空间尺度的分析,才能得出更为正确而有效地评价物种种群遗传结构及其种群历史演化情况。
综合以上的研究结果得知,本文运用了线粒体DNA和微卫星DNA两种不同的分子标记比较分析了日本伏翼不同种群的遗传特征与地理分布的关系,为进一步研究该物种的谱系地理学特征奠定了基础。
Compared to Europe and North America, the influence of climatic oscillations during the Pleistocene in shaping the distribution and genetic structure of biota in Asia is not well known. The influence of geographical barriers in promoting or restricting the dispersal ability of a species during post-glacial expansions will also have a large impact on its population genetic structure. The Japanese pipistrelle bat, Pipistrellus abramus, is found over a wide range in China, including Hainan Island and the Zhoushan Archipelago. Recently, in some areas, the number individuals of some bat species have dramatically been in decline due to seriously destructive ecological environment and fast development of towns. Genetic diversity is the basis of population long-term survival and resisting the diseases. Demographic history analysis is vital to population protection. In order to comprehensively understand survival status and demographic history and establish accurate protection steps for Pipistrellus abramus, it plays an important role in estimation of genetic diversity and gene flow between populations.
We applied both mitochondrial (cytochrome b) and microsatellite markers to examine population genetic structure and demographic history in this species. The main results are as follows:
1. We describe the first set of ten microsatellite markers isolated in Pipistrellus abramus. The number of alleles per locus ranged from 7 to 13. The observed and expected heterozygosities values ranged from 0.486 to 0.971 and from 0.752 to 0.876, respectively. Three loci (WW6、PA133 and 1-20) revealed significant departure from Hardy-Weinberg equilibrium and no linkage disequilibrium was found between loci pairs. These infonnative microsatellite markers will be a powerful molecular tool for studying the population genetic structure of P. abramus, as well as other species of this genus.
2. Both results of mtDNA and microsatellite DNA (8 loci) revealed Pipistrellus abramus contain relatively high genetic diversities among different geographic populations. For the mitochondrial DNA (mtDNA) data, the haplotype diversity ranged from 0.286 to 0.9, and nucleotide diversity ranged from 0.00025 to 0.01878. For the microsatellite data, the observed heterozygosity (Ho) and expected (HE) heterozygosity ranged from 0.58 to 0.89, and from 0.67 to 0.82, respectively. The allelic richness (Rs) ranged from 4.13 to 10.36 among sampled populations.
3. Our results suggest that geographical features have had a clear impact on population connectivity in P. abramus. Specifically, both markers indicate that the Qiongzhou Strait, which separates Hainan Island from the continent, acts as a significant barrier to gene flow. Likewise, both genetic distance of mtDNA and microsatellite DNA were significant with natural geography distance (mtDNA, R2=0.27, P＜0.001; microsatellite DNA,R2=0.31, P＜0.001).
4. Mismatch distributions show that populations from continental China/Zhoushan Archipelago and Hainan Island appear to have undergone expansions since the Pleistocene, with evidence of a star-like topology in the former. MtDNA reveals evidence of common ancestry between the colonies sampled on the Zhoushan Archipelago and those on the continent, which are thought to have become isolated approximately 7000-9000 years ago. Although the Qiongzhou trait seperates the Hainan Island and mainland for over 10000 years, no haplotype was found to share among individuals sampled from the island, and it revealed that the bats sampled from the Hainan Island may colony into the island vita recent population expansion since the Pleistocene.
5. Microsatellite data did not fully support the mitochondrial data and instead detected some differentiation between the two groups, highlighting the importance of using different markers at different spatial scales.
In summary, we were explicit that the thesis data were combined with mitochondrial (cytochrome b) and microsatellite molecular markers for comparison of different geographical populations and geography distribution. Farther more, the results were the basis study of phylogeography of the Japanese pipistrelle bat, Pipistrellus abramus.
引文
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