更新世-全新世大熊猫种群演化历史的古基因组研究

The giant panda (Ailuropoda melanoleuca) is known as the iconic species of world wildlife conservation. As the first species from which a de-novo genome was assembled by Chinese scientists, it is also a symbol for successful science in China. Fossil findings indicate that the ancestors of this species probably originated in the late Miocene in southwestern China, expanded their range during the early Pleistocene, and began to contract during the late Pleistocene. Nowadays, the giant panda is restricted to only six fragmented mountain ranges on the eastern edge of the Tibetan plateau. The dramatic population collapse of this endangered species has attracted both concerns and interests as evolutionary biology and population genetics...Palaeontological and biological studies were interpreted the giant panda an evolutionary dead end species. However, more recent morphological and biological characters suggest that it has several evolutionary advantages compared to other large mammals that went extinct during the late Pleistocene. Molecular studies on giant panda’s population history have resulted in controversial results with regard to its genetic diversity. By sequencing whole genomes of living giant panda populations, Zhao et al. reconstructed the panda’s demographic history that showed population peaks and bottlenecks. However, Zhao et al. were failed to reconstruct the more recently demographic history less than 20,000 years ago due to limitation of the approach used. ..However, none of the studies to date has yet reported an ancient genome of the giant panda, which is essential for investigating how much the genetic diversity has been changed over time. As the carrier of genetic information, ancient DNA can provide molecular evidence for tracing the evolutionary processes of species that have been influenced by climate change and human impacts. Therefore, we recently tried to amplify mitochondrial DNA segments from giant panda fossils and evaluated the relationship between its contracting population size and the genetic diversity. We found that there is a temporal lag between the reduction in population size and its genetic impacts. Bayesian skyline plot (BSP) analyses indicate that there was a decline of effective population size around 4.0 ka BP, which might be the result of a significant cold event occurred 4.2 ka BP in China. However, due to the short sequence data set, the decrease of population size is not statistically significant. Considering that paleogenetic studies based on partial mitochondrial sequences were either not capable of solving the phylogenetic controversial, or even guiding scientists towards incorrect conclusions，large DNA sequences data sets, such as complete paleogenome sequences are significantly important in addressing the evolutionary history of the giant panda...Therefore, this proposal aims at investigating the genetic structure and population history of the giant panda during the Pleistocene by sequencing the complete genome from fossil findings in China using state-of-the-art DNA-hybridization-capture and Next Generation Sequencing approaches that have revolutionized the fields of ancient DNA and evolutionary genetics. By bioinformatics analyses of the ancient genome sequences of giant panda specimens collected from various location and different stratigraphic distribution, we can explore the population genetic diversity dynamics during its evolution, which will result in a more comprehensive picture than one based on data from extant populations alone. Moreover, by analyzing the genome sequences in combination with the calibration points derived from the securely dated fossil specimens, we will reconstruct the recent evolutionary and demographic history of giant panda. This will provide us with a better understanding of both the evolutionary history and the causes of the species’ endangerment and thus allow us strengthening the management strategies for its long-term conservation.

大熊猫是我国特有的第四纪孑遗物种，自晚更新世开始的大熊猫栖居地退缩、种群衰退现象及原因是演化生物学关注热点之一。现生大熊猫的遗传多样性水平尚存争议，根据现生种群的遗传结构研究其演化历史存在局限。古DNA能为绝灭或死亡生物提供实时遗传分子信息。对大熊猫亚化石开展了古DNA预研究，初步结果显示全新世以来大熊猫种群遗传多样性较稳定，有效种群大小稍有缩减，且缩减与同期出现的降温事件有关。受样品年代、序列长度等因素限制，预研究工作无法系统重建更新世以来的大熊猫种群演化历史。本项目拟借助分子生物学方法，构建更新世中、晚期及全新世大熊猫古DNA测序文库，用新一代测序技术测定短片段DNA，组装得到其线粒体及核基因组，分析大熊猫种群遗传多样性的动态变化；结合样品年代信息，系统重建更新世以来在气候变化、人类活动影响下的大熊猫种群演化历史，为合理评估该濒危物种的种群现状并制定科学保护策略提供理论依据。

本项目按照项目计划及进度安排，四年内顺利完成了预期研究内容，实现了预期研究目标。针对大熊猫自晚更新世以来栖居地退缩过程中种群遗传结构尚不明确等演化生物学问题，本项目从我国多个化石地点采集了第四纪晚期大熊猫化石或亚化石样品，运用古DNA提取及二代测序技术，先后在国内和国际古DNA实验室提取并构建大熊猫二代测序文库，通过对测序数据的生物信息学分析，从采自云南腾冲的2个全新世大熊猫个体中得到其线粒体部分基因，并得到全世界首例古代（约5000年前）大熊猫全基因组。结合现生大熊猫基因组信息，对古代及现代大熊猫进行遗传多样性比对、系统发育分析及种群历史重建，发现云南腾冲的全新世大熊猫个体代表一个不同于现生大熊猫、现已绝灭的大熊猫遗传谱系；该谱系与现生大熊猫共同祖先种群的分化，早于现生大熊猫三个不同地理种群（即：秦岭种群、岷山种群、邛崃-大相岭-小相岭-凉山种群）的形成。对全新世大熊猫与现生大熊猫种群是否存在遗传交流进行D-统计分析，结果表明云南腾冲的大熊猫代表的古代种群在现生大熊猫的各祖先种群中留下了多少不一的基因成分。由此说明，在大熊猫物种演化过程中，伴随着其栖息地的退缩，其遗传多样性也有所丧失；通过与现生大熊猫祖先种群的基因交流，云南腾冲江东山的古代大熊猫绝灭谱系，部分基因得以在现生大熊猫基因库中幸存。本研究提供的古代大熊猫实时基因组信息，可为合理制定大熊猫保护方案提供科学依据和借鉴。此外，作为项目的延伸内容，本项目在执行过程中还对采自我国东北的最后斑鬣狗化石、鹿科动物化石及采自北京周口店的豪猪化石进行了古DNA研究，现已从多个斑鬣狗及鹿科动物化石样品中得到其较完整的线粒体基因组数据，验证了研究的可行性及分析方法的科学性。若能继续深入进行上述样品的核基因组研究，将有望从多物种角度探究第四纪晚期我国大型哺乳动物的种群迁移演化历史等具体科学问题，探究其分子地理谱系及其对环境气候变化的分子响应。