【作者】 周蓉；

【导师】 刘迺发；

【作者基本信息】 兰州大学 ， 生物地理学， 2012， 博士

【摘要】 大石鸡(Alectoris magna)隶属于鸟纲(Aves)、鸡形目(Galliforms)、雉科(Phasianidae)、石鸡属(Alectoris),是我国的特有种,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)。本文以线粒体DNA (mtDNA)的控制区(CR)和细胞色素b (Cytb)以及微卫星(SSR)为分子标记,研究了我国12个地理种群的大石鸡分子生态学以及大石鸡与石鸡之间的渐渗杂交。本文的主要目的是：(1)采用不同分子标记检测大石鸡不同地理种群的遗传多样性,揭示不同的环境因子对大石鸡遗传多样性的影响；(2)通过分子手段构建大石鸡整个分布区内的系统地理结构,检验其是否存在明显的系统地理分化；(3)分析比较不同分子标记构建的系统地理结构之间是否存在差异；(4)分析种群的遗传分化和种群历史动态,阐述更新世冰川作用和气候变化对大石鸡系统发生的影响；(5)通过分析杂交种群的杂交率和杂交种群的遗传多样性,检测渐渗杂交对大石鸡种群遗传多样性的影响；(6)进一步确定大石鸡与石鸡渐渗杂交的范围,推测杂交时间；(7)从遗传学角度探讨大石鸡的保护问题。本研究中,分子生态学的部分共对大石鸡12个地理种群的258个样本进行了分析,其中包括173个样本的mtDNA CR部分序列(1127bp)、230个样本的mtDNA Cytb部分序列(807bp)以及257个样本的8个多态性微卫星位点。渐渗杂交的部分采用了大石鸡分布区内15个地理种群的287个具大石鸡形态的样本的CR部分序列(473bp)和267个具大石鸡形态的样本的Cytb部分序列(807bp),以及宋森毕业论文中25个地理种群的285个具石鸡形态的样本的CR部分序列以及杨志松毕业论文中8个的地理种群的48个具石鸡形态的样本的Cytb部分序列。获得的主要研究结果如下：1.大石鸡mtDNA CR和mtDNA Cytb的碱基含量都显示A+T的含量超过了一半,符合1ntDNA控制区是“A和T丰富区”。173个样本的mtDNA CR序列中共发现33个变异位点,定义了44种单倍型,核苷酸多样性和单倍型多样性分别为0.00449和0.916。230个样本的mtDNA Cytb序列中共发现13个变异位点,定义了14种单倍型,核苷酸多样性和单倍型多样性分别为0.00216和0.738。8对微卫星位点平均等位基因数为18.875,平均期望杂合度和观察杂合度分别为0.90和0.88,总体遗传多样性低于其它分布于青藏高原和黄土高原的鸟类。2.MtDNA CR的核苷酸多样性与降水量显著负相关,说明气候因子中降水量是影响大石鸡遗传多样性的一个非常重要的因素,降水量越小,大石鸡遗传多样性越高。微卫星的观测杂合度与相对湿度变异系数呈显著正相关,说明气候越不稳定,大石鸡的遗传多样性越高。3.mtDNA CR、Cytb和拼接序列的贝叶斯树和网络图显示大石鸡12个地理种群的样本可以分为三个平行的进化分支,这三个分支没有显示出明显的地理分化,微卫星分析也获得了相同的结果,表明大石鸡并没有明显的系统地理结构。4.不同分子标记的AMOVA的结果显示,虽然大多数的遗传变异存在于种群内,但是地理群组内种群间以及地理群组间也存在显著的遗传,只是分化程度比较低,Fst值也显示大多种群间的遗传分化都显著。1mtDNA控制区的单倍型组成表明柴达木盆地与其它群组之间只有一个共享单倍型,说明柴达木盆地的种群与其它两个地理群组的种群之间具有一定的遗传分化,造成遗传分化的主要原因可能是更新世冰期的冰川作用,但由于冰期持续时间较短,进入间冰期之后,冰川消融,产生隔离作用的屏障消失,柴达木盆地的种群与其它种群之间又会发生广泛的基因交流,减弱了因冰期隔离所造成的遗传分化,使得地理群组之间的遗传分化水平低,而且缺乏明显的系统地理结构。5.柴达木盆地的种群分布于网络图的两端,位于网络图右端的这一部分柴达木盆地的个体在CR和Cytb的网络图中都与其它群组的样本没有共享单倍型,推测这一部分个体在物种形成之初就已经存在于柴达木盆地,经历了中更新世以来的多次冰川的隔离作用,尤其是规模最大的倒数第三次冰期,被隔离的时间比较早,它们和其它群组的个体之间的遗传分化比较明显。位于控制区和拼接序列网络图左端的这一部分柴达木盆地的个体,在CR的网络图中与其它群组的个体之间有一个共享单倍型,但在Cytb网络图中,这一部分个体全部与其它群组的个体共享一个单倍型,推测这一部分样本是从陇中黄土高原扩散过去的种群所演化而来的,被隔离的时间比较晚,因此与其他群组的遗传分化不明显。6.错配分布和中性检验的结果支持大石鸡兰州亚种经历过种群扩张,扩张的时间为0.23-0.18Mya,这段时期在青藏高原冰期划分上属于倒数第二次冰期(0.33Mya-0.13Mya),而在第四纪气候划分上属于第四寒冷期(0.24-0.15Mya),气候特征为干冷多风,植被类型属于荒漠型植被,草原的发育和森林的退缩是造成大石鸡种群扩张的主要原因。7.大石鸡和石鸡之间的杂交是石鸡基因向大石鸡的单向渗透,属于渐渗杂交。仅在六盘山地区的大石鸡种群中发现了杂种,基本都位于六盘山山脉周缘到礼县的位置的长度大约为400km,宽度约为130km的杂交带中。杂种具有石鸡的线粒体基因型和大石鸡的核基因型。渐渗杂交对大多数大石鸡种群的遗传多样性并没有产生明显的影响,但靠近六盘山的种群杂交率很高,甚至达到了100%,基因的不对称渗透可能导致这些地区的大石鸡种群发生遗传灭绝。根据mtDNA CR部分序列计算的杂交时间为0.049-0.062Myr,与宋森计算的大多数石鸡种群的扩张时间(0.027-0.060Mya)相对应。8.线粒体DNA控制区和细胞色素b的贝叶斯分析均显示大石鸡在系统发生树上没有互为单系发生的种群存在,因此应该将大石鸡作为一个独立的进化显著单元(ESU)来对待。AMOVA结果显示大部分遗传变异分布于种群内,但种群间和群组间也存在显著的遗传变异,除了民和、靖远、会宁、礼县和海原种群外,其它种群间的Fst均显著,所以应该把民和、靖远、会宁、礼县和海原种群作为一个管理单元,其它种群分别作为独立的管理单元(MU)来实现短期内的管理和保护。更多还原

【Abstract】 The rusty-necklaced partridge (Alectoris magna) is an endemic species of birds in China belonging to Aves, Galliformes, Phasianidae, Alectoris with two described subspecies A.m.magna and A.m.lanzhouensis. In this study, the molecular ecology of A.magna was studied from12populations across the entire distributional ranges based on mitochondrial DNA (CR+Cytb) and microsatellite markers. Introgression between A.magna and Alectoris chukar was also studied using mtDNA (CR+Cytb) and microsatellite markers. The aims of this study are to:(1) examine the effects of environmental factors on population genetic diversity of the12A.magna populations;(2) reconstruct the phylogeographic relationship of A.magan and detect whether there exists obvious phylogeographic differentiation in this species;(3) compare the phylogeographic structures based on different molecular markers;(4) demonstrate the roles of Pleistocene glaciation and climate oscillation played in the population structure, based on the analyses of genetic differentiation and population demography;(5) detect the effects of hybridization on the genetic diversity of A.magna;(6)confirm the geographic ranges of hybridization and speculate the time when the hybridization happened;(7)discuss the genetic conservation of A.magna.In this study, partial mtDNA CR sequences (1127bp) of173specimens, partial mtDNA Cytb sequences(807bp) of230specimens, and eight microsatellite loci for257specimens from12populations were used to analyze the molecular ecology of A.magna. Partial mtDNA CR sequences(473bp)of287specimens and Partial mtDNA Cytb sequences(807bp)of267specimens from15populations of A.magna, as well as partial sequences (473bp) of285specimens from25populations of Alectoris chukar and partial sequences (807bp) of48specimens from eight populations of A. chukar, were used to analyze the hybridization. The main results include:1. There were significant differences in base composition among12A.magna populations. The value of A+T preponderated over a half of the total base content, which was consistent with mtDNA control—region including abundance A and T. Forty-four haplotypes were defined by34variable sites with the mean haplotype diversity of0.916and the nucleotide diversity of0.00449based on mtDNA CR. Fourteen haplotypes were defined by14variable sites with the mean haplotype diversity of0.738and the nucleotide diversity of0.00216based on mtDNA Cytb. The average expected and observed heterozygosity was0.90and0.88, respectively. The genetic diversity of A.magna was lower than other birds distributed on Tibetan Plateau and Loess Plateau.2. A significantly negtive correlation was observed between nucleotide diversity of mtDNA CR and rainfall, indicating the more abundant the precipitation, the lower the genetic diversity. Precipitation played an important role in genetic diversity of A.magna. A significantly positive correlation was observed between expeted heterozygosity (HE) and variation coefficients of annual mean relative humidity (VCH), indicating the more unstable the climate, the higher the genetic diversity.3. For mtDNA CR, Cytb and combined sequences, all of the haplotypes formed three paraphyletic groups without evident spatial clustering. The similar results was also observed in the analysis based on microsatellites markers, indicating the lack of phylogeographic structure in A.magna.4. AMOVA analyses suggested that the variation among individuals within populations (ΦST) was maximized and the variation among regional groups(ΦCT) was minimized. However, ΦST,ΦCT and the variation among populations (ΦSC) were all statistically significant. Most of populations pairwise Fst-value was also statistically significant. Only one haplotype was shared by the specimens from Qaidam Basin and the other regional groups, indicating the low genetic differentiation between the populations from Qaidam Basin and the other regional groups. The populations in Qaidam Basin might be isolated from the other populations by the glaciation occurred in Pleistocene glacial period. However, the glacial ablation in the following interglacial period resulted in the disappearance of barriers. Extensive gene flow occurred between all populations of A.magna, decreasing the genetic differentiation among populations.5. For the CR and combined data set, the specimens from Qaidam Basin were distributed in both ends of the networks. The Qaidam specimens distributed in the right end have no shared haplotype with specimens from the other regional groups in all of the networks based on CR、 Cytb and combined sequences. They might be native to Qaidam Basin. However, the Qaidam specimens distributed in the left ends have shared haplotypes with specimens from the other regional groups in all of the three haplotype network. They might be derived from the specimens from the other regioanl groups and be isolated more recently than the specimens native to Qaidam Basin. Thus, they showed lower genetic differentiation with specimens from other regional groups. 6. The analysis of mismatch distribution and test of selective neutrality indicated that subspecies A.m.lanzhouensis had experienced population expansion at0.23-0.18Mya, co-occurring with the Penultimate Glaciation(0.33-0.13Mya) and the fifth cold period (0.24-0.15Mya), when the climate was dry and cold and the desert-steppe extended.7. The A.chukar’s mitochondrial genotype could be found in A.magna, but A.magna’s mitochondrial genotype could not be found in A.chukar. Thus, we can confirm that there existed introgression hybridization between A.magna and A.chukar. The introgression hybridization was only found in the surronding of the Liupan Mountains to Lixian County. Only A.chukar’s mitochondrial genotype and A.magna’s nuclear genotype could be observed in the hybrids. High level of introgression was observed in populations near to Liupan Mountains. The hybrid rate have reached100%in Longde. The asymmetrical introgression may result in the extinction of the population of A.magna distrubuted near to Liupan Mountains. The time when hybridization happened was about0.049-0.062Myr, which is consistent with the population expansion time of A.chukar (0.027-0.060Mya).8. Bayes analysis based on CR and Cytb revealed limited phylogenetic distinction between geographically distant populations of the rusty-necklaced partridge, and tentative support for a single evolutionarily significant unit (ESU) range. AMOVA revealed that the most genetic variation was distributed within populations. However, the genetic variations among groups and poulations were also statistically significant. The population pairwise Fst-value was not statistically significant among Minghe(MH), Jingyuan(JY), Huining(HN), Li country(LX) and Haiyuan(HY), but statistically significant among the other populations. According to the criteria of Mus, MH, JY, HN, LX and HY should be treated as a MU and the other populations should be treated as independent MUs.更多还原