【作者】 杨志松；

【导师】 刘迺发；

【作者基本信息】 兰州大学 ， 动物学， 2007， 博士

【摘要】 大石鸡(Alectoris magna)是我国特有种，分布区狭窄，分化为两个亚种，即指名亚种(A. m. magna)和兰州亚种(A. m. lanzhouensis)。石鸡(Alectoris chukar)是广布种，亚种分化众多。两种石鸡属鸟类均是我国北方干早、半干旱荒漠环境的指示鸟类。青藏高原的隆升是造成我国北方干早环境的重要因素。伴随着更新世冰期的作用，可能对生活于干旱环境的石鸡属两种鸟类的进化历史和系统地理结构有重要影响。目前线粒体DNA分子标记普遍用于分析物种系统进化和遗传结构。本文结合线粒体DNA细胞色素b基因(Cytb)和线粒体DNA控制区基因(CR)作为分子标记，通过PCR直接测序方法研究大石鸡和石鸡的遗传和系统地理结构，以期达到如下目的：(1)说明西宁盆地的大石鸡的亚种归属问题；(2)比较大石鸡和石鸡Cytb基因的遗传差异；(3)阐明大石鸡整个分布区内的系统地理结构，说明采样范围内石鸡的系统地理结构；(4)推演它们的历史起源进化进程；(5)推测杂交发生的时间，进一步确定杂交范围；(6)比较两种分子标记在同一物种的差别。本文共采集了大石鸡106个样本，石鸡48个样本用于细胞色素b序列测定分析；采集了大石鸡133个样本用于CR基因序列分析。Cytb基因序列碱基含量和CR基因差异显著，后者的A+G含量明显高于前者；后者的序列变异率是前者的9倍。大石鸡和石鸡的Cytb基因密码子偏倚指数CBI表明这两种石鸡的Cytb基因密码子存在偏倚，偏倚程度相近。两者总的碱基含量差异显著，密码子的三个位点中，最保守的密码子第二位点的碱基含量在种间没有显著差异。大石鸡13个地理种群间的碱基含量差异显著，三个种群组间的碱基含量差异也显著。石鸡种群中，单因素方差分析仅有密码子第三位点中的C、A、G含量在8个地理种群间差异显著，3个亚种之间的碱基含量差异不显著。大石鸡Cytb基因序列828bp有8个多态位点，序列变异率为0.97％；8个替代中有6个是同义替代；T—C转换为A—G转换的1.5倍。石鸡Cytb基因序列有14个多态位点，序列变异率为1.69％：15个替代中有11个是同义替代；T—C转换(8次)略低于A—G转换，这和石鸡CR基因序列的嘧啶变异更高的结果不一致(黄族豪等，2004)。大石鸡和石鸡Cytb基因突变的方向主要是G向A、T向C突变。从遗传距离和系统树看，西宁种群组的大石鸡应该属于兰州亚种，两者之间有一定的遗传分化。根据Cytb基因序列分析碱基含量、遗传多样性，所采样区域内石鸡种群遗传分化比大石鸡更不明显，各种群没有形成明显的地理分化，它们之间较大石鸡有更大的基因流。由于早更新世冰期和柴达木盆地的进一步内陷的作用，石鸡和大石鸡大约在190万年前发生分歧。大石鸡柴达木种群组和西宁种群组、兰州种群组之间分歧时间为27万年，西宁种群组和兰州种群组之间为19万年。推测大石鸡的扩散路线为从柴达木盆地向东扩散至兰州盆地，最后形成了以兰州盆地为中心向四周辐射逐步扩散的模式。西宁种群组的大石鸡是由兰州种群组返回扩散形成的。大石鸡所有种群都经历过种群扩张。兰州种群组扩张所经历的时间为10.7万年，西宁种群组扩张的时间为11.7万年。石鸡肃北、祁丰和贺兰山种群可能经历过瓶颈效应，而后经历过种群爆发过程。大石鸡种群分布属于“系统发生不连续，具有空间隔离”的地理格局。西宁种群组和兰州种群组属于“系统发生连续，具有部分空间隔离”的地理格局。从Cytb基因分化看，石鸡各地理种群间属于“系统发生连续，没有空间隔离”的地理格局。大石鸡和石鸡种群间属于“系统发生不连续，具有空间隔离”的地理格局。8个大石鸡样本的Cytb基因呈石鸡的基因模式，但是在石鸡中没有发现大石鸡的基因模式。石鸡的基因向大石鸡单向渗透，为种间杂交的渐渗杂交类型。推算发生在六盘山周缘到礼县的杂交带长度大约为400km。杂交带宽度约为130km。发生杂交的时间(9万年)正是大石鸡兰州种群组扩散的时间(10.7万年)之后。从Cytb基因序列表明，渐渗杂交的发生对大石鸡遗传多样性没有显著影响。杂种低的遗传多样性和单一的单倍型可能表明石鸡雌性个体和大石鸡雄性个体间的交配选择不是随机的，可能具有某种特定基因型的石鸡雌性才会选择同大石鸡雄性进行交配，从而造成杂种后代单一的基因型。不过这一推测尚需进一步研究。更多还原

【Abstract】 Przevalski partridge （Alectoris magna） is endemic in China with limited range, and was identified as two subspecies, a nominate subspecies A. m. magna residing in the Chaidamu Basin and A. m. lanzhouensis residing around the Lanzhou Basin. Chukar partridge （Alectoris chukar）, however has numerous subspecies with broad distribution. Both of them are indicative birds of arid and semiarid environments in northern China. The uplift of Tibetan Plateau is the important factor influencing climate severity and desert evolution in northwestern China, and it maybe affect historic evolution and phylogeographic structure of the two partridges severely accompanying with Pleistocene glaciation. At present, mtDNA marker was used for analyzing phylogeny and genetic structure of species. We used polymerase chain reaction （PCR） and direct sequencing methods to infer their genetic and phylogeographic structure based on mitochondrial cytochrome b （Cytb） and mtDNA control region （CR） data. The aims are: （1） to analyze Przevalski partridge in the Xining should belong to which subspecies; （2） to compare genetic variation within and among local populations between Przevalski partridge and Chukar Partridge; （3） to illuminate their phylogeographic relationship; （4） to infer their historic demography; （5） to speculate the time of hybridization happening, and confirm the geographic range of hybridization; （6） to compare the differences between two markers.We sampled 106 Przevalski partridges and 48 Chukar partridges for Cytb analyzing, also sampled 133 Przevalski partridges for CR analyzing.There were significant differences in nucleotide compositions between Cytb and CR gene, and the number of G + C was larger in the latter. The frequency of sequences variation of the latter was 9 times higher than the former.The Codon Bias Index （CBI） showed that Cytb gene had compositional bias within two partridges, and they had similar CBI. There were significant differences in total nucleotide compositions and no significant differences in 2^nd positions of codons between two partridges. The nucleotide compositions differed significantly among 13 local populations of Przevalski partridge, but no significant differences among 8 populations and subspecies of Chukar partridge (there were only significant differences in Cytosine, Adenine and Guanine of the 3^rd positions of condons among 8 populations).8 variable sites defined 9 haplotypes of Cytb gene in 98 Przevalski partridges. There was 0.97% sequences variation; 6 synonymous substitutions in 8 substitutions; the number of transitions between T and C was 1.5 times higher than that between A and G. 14 variable sites defined 12 haplotypes of Cytb gene in 133 Chukar partridges. There was 1.69% sequences variation; 11 synonymous substitutions in 15 substitutions; the number of transitions between T and C was less than that between A and G slightly, which was different from the nucleotide variation of CR gene in Chukar partridge in which there was higher pyridine variation （Huang et al., 2004）. The mutation direction of Cytb gene in two partridges was mainly from G to A and from T to C.As viewed from genetic distances and phylogenetic tree, we could speculate that the Przevalski partridges in Xining pop-group should belong to the Lanzhou subspecies （A. magna lanzhouensis）. There was partly genetic divergence between the Xining pop-group and the Lanzhou pop-group.According to nucleotide compositions and genetic diversity of Cytb sequences, Chukar partridges had less genetic divergence and more frequently gene flow than Przevalski partridge. There was no distinctly genetic divergence among local Chukar partridge populations.Calibrated rates of molecular evolution suggested that Przevalski partridge and Chukar partridge could have speciated about 1.90 million years ago, affected by the Pleistocene second frigid and the form of Chaidamu Basin. The divergence time was about 0.27 Myr between the Chaidamu pop-group and the other two pop-groups （xining pop-group and Lanzhou pop-group）, and about 0.19 Myr between Xining pop-group and Lanzhou pop-group, we speculated that the dispersal route was that Alectoris magna dispersed from Chaidamu Basin east to Lanzhou Basin, at last formed a dispersal star-like mode surrounding Lanzhou Basin step by step, and the Xining pop-group was formed by the Lanzhou pop-group dispersing back to west.All of the Przevalski partridge populations accepted sudden expansion model. the Lanzhou pop-group expansion began at 0.107Myr, and the Xining pop-group expansion began at 0.117. among Chukar partridge populations, the SB, QF and HLS populations were possible to suffer bottleneck effect, and sudden population expansion later.Lack of shared haplotypes of CR suggested that the phylogeographic structure of Przevalski partridge belonged to "Phylogenetic distinuity, spatial separation" pattern. There was "Phylogenetic continuity, partial spatial separation" geographic pattern between Lanzhou pop-group and Xining pop-group. To deduce from divergence of Cytb gene, the phylogeographic structure of eight Chukar partridge populations belonged to "Phylogenetic distinuity, lack of spatial separation". There were both lack of shared hayplotyes of CR and Cytb between Przevalski partridge and Chukar partridges, which had a "Phylogenetic distinuity, spatial separation" geographic pattern.There were 8 Przevalski partridges from five populations having Chukar partridge Cytb genotype. However, there were no Chukar partridge with Przevalski partridge’s genotype. Thus, we could make sure that the hybridization between two partridges was introgression hybridization, maybe as result of sexual selection. The introgression hybridization was about 130km wide and 400km long from the surrounding of the Liupan Mountains to Lixian County. The time of hybridization happening was about 90 thousand years, which showed that the hybridization happened after the Lanzhou Przevalski partridge pop-group expanded to encounter with Chukar partridge population （the expansion time of Przevalski partridge was 0.107Myr ago）.Analysis of Cytb gene indicated that introgression hybridization had no significant influence on genetic diversity of Przevalski partridge population. The low genetic diversity of hybrids and single haplotypes might indicated that mating selection between Chukar partridge female and Przevalski partridge male was not stochastic. It is possible that those Chukar partridges with specific genotype could mate with przevalski partridges, as a result, the hybrid offspring had single genotype. However, it just is a speculation, looking forward to advanced studies.更多还原