| The origin and evolution of life in the natural sciences has been received great concern. Paleontologists, evolutionary biologists normally use the animal and plant fossils or sub fossils as well as relics to explore the causes of their evolution and extinction. However, the traditional fossil-analyzing method which based on anatomy and morphology can not totally solve the problems about biological evolution due to the limitation of the fossil records. By modern molecular biology techniques to study ancient DNA in paleontological remains or relics, it can reveal a strong molecular evidence for the evolutionary paleontology, and broaden the field of the traditional paleontology research in object and method.The giant panda, Ailuropoda melanoleuca known as the "living fossil", is one of the famous endangered species. Fossil data indicates that this species originated in late Miocene, probably expanded during the middle Pleistocene, and reached its peak in the late Pleistocene.Once a widely distributed species to Yangtze River, Pearl River basin and north of China, giant panda distributed to the most northern boundary was40°N (first point of Zhoukoudian fossil sites), towards the south, it once extended to Vietnam, Laos, Myanmar, and arrived in the southeast coastal areas. It begun to shrink the distribution in the late Quaternary glacial. Due to the effects of climate change and human activities, the Giant panda is living in small and isolated populations with highly fragmented distributions at present, while the living giant pandas are mainly restricted in Sichuan, Shaanxi, and Gansu provinces.The problem of the giant Panda classification has always been a hot spot of debate. Threre were three viewpoints on its classfication in1980ago, it was considered that giant panda belongs to the bear family, the raccoon family and the giant panda family (Independent). Mivart studied the skull, limbs, teeth, foot type, kidney, hair texture, offal and some fossils of the giant panda, he also carefully studied the fourth upper promolars which it is important for the evolution, with giant pandas and raccoons animals in the skull structure, teeth, internal organs and other aspects of similarity,especially crack teeth (P4) crown-shaped of tooth crown. He concluded that the giant panda should belong to the raccoon family. Davis published the monograph "Study on morphology and evolution mechanism of the giant panda" based on the comparative results of50giant panda organ systems, he asserted that each morphological characteristics of the giant panda showed they were merely a highly specialized Bear, so it can be put in the bear family, or a separate family. Afterward,,the taxonomic status of giant panda was gradually formed two factions:the bear family or the giant panda family. Lin Feng et al analysised a common113kb RAPD fragment of the giant panda,the lesser panda,the sun bear and raccoon using PCR and Southern hybridization methods,the result showed that sun bears has not corresponding hybridization band, which implied that such a genetic relationship to the giant panda and the sun bear should more closer than the lesser panda and raccoon, he advocated that the giant panda should be designated to bear family. To construct the phylogenetic tree using Cyt b,12s rRNA gene sequences and genome of mitochondrial,we can find the giant panda and bear family on the same branch. Zhang Yaping et al thought that the DNA sequence similarity to giant panda and bears can not be a evidence as the giant panda belong to bear family, and if the partial cyt b sequences were translated into amino acid sequences, the results displayed that the giant panda is closer to the lesser panda, so as the similarity of chimpanzee and human DNA sequence is98%, but it can not be classified as human subjects, the giant panda should became a separate family.In recent years, with the discovery of more giant panda ancestors’ fossils, scholars thought that the giant panda and bear ancestors had already appeared a parallel development in the Late Miocene. The giant panda and bear ancestors have the relationship to each other, but the giant panda morphology has more difference with the species of bear family, so scholars advocated the giant panda should be classified into a separate family Ailuropodinae.Previous studies suggested that genetic diversity of the giant panda has been declined obviously. Pan analysised genetic pressure of the giant panda population in Qinling, and drawed the conclusion that the population will suffer inbreeding loss of genetic diversity with the rate of0.5456%in each generation. Li et al., Su Bing et al. studied the blood isozymes and plasma protein. They drew the conclusion that the species diversity of the giant panda population is poor. Zhang et al. explored the panda mitochondrial tRNA genes and D-loop region sequences. They found9haplotypes within21experimental samples and derived that the giant panda has a low level of genetic differentiation. Fang et al. used DNA fingerprinting technique to analyze the genetic diversity of the giant panda population and thought that the genetic diversity was seriously poor in five mountains areas, while there was a significant genetic differentiation among the mountains. However, recent studies have shown that the genetic diversity of extant giant panda remains moderate or a higher level. Lu et al. studied the mitochondrial DNA control region sequences of the giant panda in Minshan, Qionglai and Qinling and detected17individual haplotypes from36giant pandas. These haplotypes have no obvious correlation to the geographical distribution of the giant panda. They analyzed genetic diversity of giant pandas by restriction endonuclease and microsatellite sequences probes that and believed it is on the middle level. By comparing with other bears, Zhang et al. believed that the giant panda was not reach the end of evolution. They analyzed the genetic diversity of the giant panda populations in five mountains and found that the giant panda population has high level of diversity in terms of the control region and microsatellite loci.. Li et al. sequenced the whole genome of the giant panda and found that the giant panda populations still have a high genetic diversity. Hu et al. studied the mitochondrial control region sequences of the giant panda to explore the genetic diversity of of the giant panda population in Liangshan region. The result showed that the genetic diversity of the giant panda at the middle level in this region. In this study, ancient DNA were extracted from five fossil specimens using the silicon-GuSCN method. These specimens were collected in Tengchong and Zhenxiong, Yunnan Province,Tiandong,Guangxi Province,Sangzhi,Hunan Province. The numbers of these sample are05001,97001,14162, GXPB,HNSZ, respectively. By the molecular method such as multiplex PCR, electrophoresis, purification, cloning and sequencing, Samples GXPB, HNSZ, and14162have not gotten reliable ancient DNA sequences, but we got the ancient DNA sequences from samples97001and05001. We reconstructed phylogenetic trees of giant panda and bear family species based on the mitochondrial Cyt b,12s rRNA,16s rRNA, ND1and D-Loop genes from two samples (97001and05001) together with homologous sequences in GenBank. We explored the genetic diversity of giant panda based on the D-loop sequence dataset. The progresses made in this thesis are as follows:(1) We obtained ancient DNA sequences from two out of five fossil samples of giant panda. These two samples has been dated at8740±45a B.P.(97001) and5025±35a B.P.(05001), respectively. Total length of5025bp mitochondrial DNA sequences obtained from sample97,001, and5142bp sequences obtained from sample05001.The results proved that it is feasible using the silicon-GuSCN-proteinase K method and multiplex PCR method to study ancient fossil samples in southern China, and also illustrated that it can get ancient DNA from the fossils about ten thousand years old from southern China where is hot and humid.(2) Using14pairs of primers, we obtained the Cyt b gene sequence (1140bp) from two giant panda samples.,The average content of A,T,G,C is29.3%,31.3%,14.2%,25.2%, respectively in two sequences, which content of A+T(60.7%) is higher than G+C(39.3%). When we aligned sequences deriving from GenBank on extant giant panda, brown bear, polar bear, sun bear, sloth bear, asiatic black bear, American black bear, spectacled bear, lesser panda and raccoon, the result showed that the ratio of transition and transversion is4.2. We also calculated the genetic distance between the nucleotide sequences. The giant panda has the least genetic distance with the bear family while it has larger distance with lesser panda and raccoon. Using15pairs of primers we amplified the complete sequence of12s rRNA gene(966bp).The average content of A,T,G,C is35.4%,24.6%,18.4%,21.6%, respectively in two sequences(05001and97001). The content of A+T(60.0%) is higher than C+G(40%). We analyzed the nucleotide sequence differences with extant giant panda, seven species of the bear family, lesser pandas, and raccoon. The ratio of transition and transversion is3.6. Saturation analysis conducted that these sequence did not reach saturation. To calculate the genetic distances between sequences, the giant panda and the bear family have the smallest discrepancy of sequences and genetic distances. To obtain the D-loop gene sequences(932bp), we used13pairs of primers and got a continuous fragment of683bp. The average content of A, T, G, C is28.8%,30.2%,15.7%,25.3%. The content of A+T(59.1%) is significantly higher than G+C content(40.9%). Sequence analysis showed the ratio of transition and transversion is0.9. Saturation analysis indicated that the sequence will reach saturation. We calculated the genetic distance between the two sequences. The results showed that the average genetic distance between giant panda and bear family was less than the lesser panda. Using15pairs of primers we got the16s rRNA gene partial sequences of97001(1064bp) and05001(1181bp). The sequence differences and genetic distance demonstrated that the ratio of transition and transversion is3.0, the genetic distance is closer between giant panda and lesser panda. We obtained923bp sequences by10primer pairs and830bp is continuous fragment. The average content of A+T(61.5%) is higher than G+C(38.5%). We compared the newly obtainded sequences and its homologous sequences. The result displayed that the ratio of transition transversion is4.4. Saturation analysis conducted the sequence did not reach saturation.To calculate the genetic distances between sequences, the result derived the giant panda and the spectacle bear have the least genetic distance. It has been different to the discrepancy of sequences and genetic distance by5genes aligning with homologous sequences, which may be related with the degree of gene conservation.(3) Ancient DNA sequences obtained in this study are reliable. Ancient DNA extraction, PCR mixture preparation and cloning were carried out in an independent laboratory. We strictly operated accordance with the requirements of ancient DNA experiment, from the extraction, PCR amplification and molecular cloning have all set the appropriate control experiments to monitor contamination. For each of the primer pairs, we have performed substantially more than two dependent amplification, cloning and sequencing to determine the nagetive sequence. The obtained sequence were used to blast in GenBank and the results showed that the sequences has the highest similarity with extant giant pandas. In this study, the DNA sequences were confirmed by at least twice repeatability experiments in the laboratory operated by different researchers. Moreover, the repeated experiments were carried out at the Australian Center for Ancient DNA, University of Adelaide in Australia. Identical results were obtained with China University of Geosciences (Wuhan). In summary, the obtained ancient DNA sequences are true and reliable in this study.(4) Based on the complete Cyt b gene and12s rRNA gene, partial16s rRNA gene sequences, partial ND1and D-Loop sequences, we aligned the datasets using MEGA software.We performed different datasets of Cyt b+12s rRNA, Cyt b+D-loop, Cyt b+12s rRNA+D-Loop, Cyt b+12s rRNA+16s rRNA+NDl and Cyt b+12s rRNA+16s rRNA+ND1+D-loop, respectively. We used NJ, MP, and ME method to reconstruct the phylogenetic trees. Trees had the same topology with the datasets of Cyt b,16s rRNA, ND1and Cyt b+12s rRNA+16s rRNA+NDl, while the bootstrap values are different in the main branches. It is not exactly the same topology on the phylogenetic tree by other single and combined sequences, but the positions of brown bear, polar bears, giant panda, lesser panda and raccoon are same on phylogenetic tree, only slightly different to the bootstrap values. The results showed that the D-loop sequences involved in phylogenetic trees have obvious difference with the other sequences constructed phylogenetic tree on the topology. Therefore, we suggests that D-loop sequences are not suitable for phylogenetic investigate in the phylogenetic relationship of giant panda and the bear family. Double-exploited species homologous sequences of individual phylogenetic tree, the results showed that a single individual species used to build phylogenetic trees is possible to exclude the effection of phylogenetic tree species in a single individual. Adding of the ancient DNA sequences in phylogenetic analysis has improved the accuracy of the phylogenetic tree that includes the bear family, giant panda, lesser panda,and raccoon. It has been further clarified that raccoons first separated, followed by the lesser panda, and then the giant panda. The bear family and giant panda have a closer relationship.(5) We calculated the divergence time between species by the mcmctree program of the PAML software based on combined data of Cyt b+12s rRNA+16s rRNA+ND1sequences. The divergence time was17.12Ma between the lesser Panda and the giant panda It was12.68Ma between the giant panda and species of Ursidae which was similar to the divergence time based on the fossil record (12Ma). To analyze the phylogenetic trees and species divergence time, the results suggested that the giant panda has diverged very early with bear family, and carried out an independent evolution and phylogeny, so we suggested that the giant panda should be classified as an independent family Ailuropodinae.(6) We aligned the655bp mitochondrial D-loop sequences of the two ancient panda samples together with40homologous sequences of extant panda which were obtained from GenBank by the MEGA. We then imported the alignment into DnaSP5.0software to analyze the haplotype. The result showed that the42sequences are belonging to42haplotypes. According to the analysis of base change for655bp homologous sequences and haplotype distribution in different regions, we constructed the simple network diagram by Network4610and the phylogenetic tree of haplotypes by DNASTAR.The results showed that the giant panda haplotype diversity is very rich, and there is a variety of shared haplotypes in different regions, indicating they had extensive gene flow between different regions in the distribution of giant pandas, so the giant panda could always maintaining genetic diversity. The haplotype of ancient giant panda at5000years ago in Yunnan had been spread to the mountain of Qinling, Qionglai, Liangshan and minshan through the gene flow.Using the Bayesian skyline plot (BSP) of BEAST1.7.4program assessmented the giant panda effective populations changes in the past8700years, the results showed that the giant panda population declined began at5000years ago, and now their population is keeping a stable level. At this stage we should build a "green corridor" among the giant panda habitats and thusto increase mutual communication improve population heterozygosity rate, and enrich their genetic diversity. |
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