【作者】 贝永建；

【导师】 李进华；

【作者基本信息】 安徽大学 ， 生态学， 2014， 博士

【摘要】 黑颈长尾雉(Syrmaticus humiae)属于世界珍稀濒危雉类,地理分布狭窄,仅分布在中国西南部、泰国北部、缅甸西北部和印度东北部。由于栖息地破碎化和人为捕猎,黑颈长尾雉种群分布范围和种群数量不断锐减,已被列为国家Ⅰ级保护动物,国际濒危物种贸易公约(CITES)附录Ⅰ,世界保护联盟(IUCN)将其定为近危种(Near Threatened)。本文以分布在广西-贵州两省的南盘江和红水河流域的6县(隆林县、西林县、田林县、乐业县、天峨县和罗甸县)的4个黑颈长尾雉地理种群为研究对象,综合运用线粒体DNA和核DNA微卫星为分子标记,检测黑颈长尾雉的种群遗传多样性和种群遗传结构,以期为针对该珍稀濒危物种保护策略的制定提供科学依据。主要研究结果如下：1、通过磁珠富集法构建了黑颈长尾雉微卫星文库,共筛选出12对具有多态性的微卫星引物。利用这12对微卫星引物对来自天峨县的16个黑颈长尾雉个体样品DNA进行PCR扩增并测序和分型。结果表明,12对引物扩增条带均呈现多态性,平均等位基因(Na)数目为9个(4-13),平均观察杂合度(Ho)和期望杂合度(He)分别为0.70(0.2～0.938)和0.81(0.303～0.925)。经进一步分析发现,有4个微卫星位点(ShuI-35, ShuI-62, ShuI-51和ShuI-15)偏离哈迪-温伯格平衡(P<0.05),但是没有发现位点间的连锁不平衡。这12个具有多态性微卫星位点将对黑颈长尾雉的保护遗传学或者长尾雉属的其它物种种群遗传学研究提供了很好的分析标记工具。2、线粒体DNA数据和微卫星DNA(12对微卫星引物扩增)数据分析表明,黑颈长尾雉的田林-隆林-西林种群(TLX)、乐业-下老种群(LX)、罗甸种群(LD)和坡结种群(PJ)均具有较高的遗传多样性。4个地理种群73个个体全部扩增成功,获得73条黑颈长尾雉mtDNA D-loop基因全序列(1143bp),检测到58个变异位点,并定义了39个单倍型。各地理种群的单倍型多样性(h)分别为0.812、0.926、0.722和0.905；核苷酸多样性(π)分别为0.00516、0.0075、0.00463和0.01；微卫星DNA的种群杂合度期望值(HE)分别为0.75、0.77、0.79和0.81,观察值杂合度(HO)分别为0.73、0.77、0.83和0.72。3、利用黑颈长尾雉线粒体DNA控制区序列以邻接法(NJ)构建的系统发生树和单倍型网络关系图显示黑颈长尾雉4个种群的单倍型并没有按照独自的地理种群聚集在一起,而是互相混合分散的分布在系统进化树上,没有表现出明显的系统地理格局。微卫星数据多元协调的主成分分析(principal coordinate analysis, PCA)也显示相同的结果,4个地理种群的73个体相互混合聚在一起,并形成出现独立进化的地理种群单元。通过用STRUCTURE软件对种群遗传结构分析发现,种群间不存在聚类关系,种群之间没有出现明显的遗传分化,说明了种群间个体交流频繁。3、对黑颈长尾雉种群的选择中性检测(neutrality test)进行分析,结果表明,各个种群的Tajima’s D、Fu and Li’s D、Fu and Li’s F、Fu’s Fs statistic等参数均不显著,线粒体DNA中性检测结果为Fu’s Fs=-0.971(p>0.05)和拟合优度检测结果SSD=0.78094表明黑颈长尾雉种群历史上并没有发生过显著的种群扩张。基于微卫星数据,采用杂合子检验法(Wilcoxon’s Test)及TPM突变模型检测种群近期动态及遗传瓶颈效应,结果表明种群未经历过近期的瓶颈效应,整个黑颈长尾雉种群处于一个稳定状态。4、在种群遗传分化检测中,线粒体DNA序列数据分析(AMOVA)结果表明,遗传分化主要来自于种群内部个体之间,达到了98.03%,而种群间的差异仅为1.97%,而微卫星数据分析(AMOVA)结果与线粒体DNA序列分析结果相似,种群内部个体之间的差异达到97%之多,而种群间的差异仅为3%,其结果进一步说明了黑颈长尾雉的种群间基因交流非常频繁(表3-10和表3-17)。另外,线粒体DNA和微卫星DNA遗传标记共同分析结果表明,323国道对TLX和LX两个种群之间并没有产生阻隔效应,南盘江、和红水河在LX和LD、LX和PJ、TLX和LD种群之间均没有形成天然的地理阻隔,南盘江和红水河两岸的种群基因交流频繁。此外,本文所研究的黑颈长尾雉4个地理种群之间没有遗传分化,因此,建议把这4个地理种群当做一个管理单元进行保护管理。5、问卷调查结果说明,民众野生动物保护意识薄弱以及大量的人为猎捕是导致黑颈长尾雉种群数量急剧减少的主要因素。为了有效遏制黑颈长尾雉种群数量不断减少的趋势,急需对黑颈长尾雉进行相应保护,主要措施为：(1)重视和加强偏远山区民众的野生动、植物保护科普和法律宣传教育,提高广大公众的保护意识和法制观念；(2)加强野生动物管理的监督和执法力度,对非法偷猎野生动物的不法分子给予坚决的查处,通过法律手段来震慑各种偷猎野生动物的不法分子；(3)加快偏远山区的经济建设和发展,尽早的让当地老百姓脱贫致富,改变他们的生活习惯和耕种模式,尽可能的减少对野生动物栖息地的破坏。更多还原

【Abstract】 The Hume’s pheasant (Syrmaticus humiae) is a rare and endangered species of pheasants in the world that used to be widely distributed in the mountain areas of north-eastern India, north and west Myanmar, south-west China and north Thailand. Due to human disturbance, poaching and habitat loss, the wild population size of this species has been decreasing dramatically in rencent years. Therefore, it was listed as a globally near-threatened species by IUCN and ranked as Grade I National Protected Wildlife in China and was also listed in Appendix I of the Convention on International Trade in Endangered Specicies(CITES).In this study,73Hume’s pheasant individual samples grouped into four geographical populations were collected from Longlin, Xilin, Leye, Tiane and Luodian counties crossed the Nanpanjiang river and red river valleys. Both mitochondrial control region (mtDNA D-loop) and microsatellite DNA markers were used to examine genetic diversity and genetic structure of the four geographical populations of S. humiae. These results will be contributed to provide important informations for reasonsable conservation and management strategies to devise for this bird species. The main results in this study are as follows:Firstly, we described the novel set of twelve polymorphic microsatellite loci isolated from the Hume’s pheasant genomic DNA using an enrichment protocol which is able to enrich AC and AG motifs. All the loci were screened from a group of16individuals sampled from Tiane County in Guangxi province. The results indicated that each locus showed a relatively high polymorphism (ranged from4to13alleles). The observed (Ho) and expected (He) heterozygosity values were ranged from0.2to0.938and from0.303to0.925, respectively. However, four loci (ShuI-35, ShuI-62, ShuI-51and ShuI-15) showed deviation from Hardy-Weinberg equilibrium (HWE) and no linkage disequilibrium was found between pairs of loci. These polymorphic microsatellite loci will be useful tools in studies of the conservation genetics of S. humiae and other species of this genus.Secondly, we investigated the population genetic structure of S. humiae using1143bp of mitochondrial DNA control region amplfied from73individuals sampled from TLX, LX, LD and PJ populations.39haplotypes were defined based on58 variable sites. The haplotype diversity (h) of the four populations was0.812、0.926、0.722and0.905, and the nucleotied diversity (ο) was0.00516、0.0075、0.00463and0.01, respectively. For the microsatellite data, the expected heterozygosity (He) and observed heterozygosity (Ho) ranged from0.75to0.79and from0.72to0.83, respectively. Both results of mt DNA control region and microsatellite DNA indicated that S. humiae expressed relatively high genetic diversities in each geographic population.Thirdly, the results of phylogenetic analyses showed that the haplotypes derived from the four putative populations did not cluster into separate geographic branches. A PCA plot of nuclear genetic variation also showed that the dividuals sampled from the four putative populations did not cluster into separate geographic branches. Moreover, the result based on the STRUCTURE also showed the four putative populations did not cluster into separate geographic branches too.Fourthly, results of the mismatch distribution and neutrality test (Fu’s Fs=-0.971, P>0.05) and goodness of fit test (SSD=0.78094, P<0.01) analyses indicated the four populations did not experience a range expansion over the course of their histories. The results of population bottleneck effect test indicated that all populations did not experience recent bottleneck effect under two-phase mutational (TPM) models.Further, using hierarchical analysis of molecular variance (AMOVA) based on mitochondrial DNA data. The results indicated that most molecular variances were originated from different individuals within populations (98.03%), whereas variances among populations (1.97%) were small. These results further indicated that gene flow (Nm) was frequently taken place among populations.The hierarchical analysis of molecular variance (AMOVA) based on microsatellite DNA data showed that variances among populations were only reached at1.97%. The gene flow between TLX and LX,between TLX and LD, between TLX and PJ, between Lx and LD, between Lx and PJ are4.11,4.61,8.35,11.36,9.72, respecively. The mating behaviour between populations took place at randomLy.Moreover, results of both mtDNA data and microsatellite DNA data sampled from the Hume’s pheasan indicated that no evidence was found that the national highway No.323, Nanpangjiang river and Red river were as to be geographical barriers to gene flow. The results further demonstrated that there are high levels of gene flow between the four populations. Therefore, we suggested that the four populations of Hume’s pheasan should be taken as one management unit (MU).Fifthly, through the questionnaire, the survey found that the people’s consciousness of wildlife protection is weak. Hunting are the main factors caused the Hume’s pheasant population decrease. In order to better protect the Hume’s pheasant, we suggest the following:(1) It is very important to promote wildlife conservation and legal sence through the education of public science popularization.(2) Our government shoud enhance a directive calling for increased protection of wild tigers through habitat management, public education and stronger law enforcement action.(3) Speed up the development and construction the economy in the remote mountainous areas, change their habits and mode of cultivation to reduce the destruction of wildlife habitat.更多还原