Knowledge Resource Center for Ecological Environment in Arid Area
| 虫纹麻蜥线粒体基因组特征、种群遗传分化及群体动态研究 | |
| 其他题名 | Characterization of the Complete Mitochondrial Genome of Eremias vermiculata and Its Population Genetic Divergence and Historical Population Dynamics |
| 周天和 | |
| 出版年 | 2016 |
| 学位类型 | 硕士 |
| 导师 | 朱震达 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 摘 要线粒体基因常作为进化分析如多样性评估、系统发育和谱系地理检验的首选分子标记。对线粒体基因及其基因组的研究也一直是生命科学领域的热点之一，有重要的理论和实践意义。蜥蜴，作为地球上广泛分布的爬行动物之一，由于相对有限的扩散能力和特殊的栖息地要求，通常具有较强的种群遗传结构，常被作为验证生物地理假设和探讨自然种群的演化过程的理想类群。本文我们将以西北干旱荒漠区广泛分布的虫纹麻蜥 (Eremias vermiculata)为对象，对其天山南北的代表个体线粒体基因组进行比较分析；以此筛选分子标记，进而利用谱系地理学(Phylogeography)和生态位模型（Ecological niche modeling）的理论和方法研究其种群分化和谱系地理格局，重建该种的演化历史，阐明其演化模式，深入了解天山的构造隆升、西北地区的荒漠化、更新世冰期-间冰期循环以及环境气候变化对该地区物种分布格局和历史动态的影响机制。此外，筛选部分核基因并进行初步的谱系地理学分析，期望能够对后续研究提供借鉴。主要研究结果如下：1.虫纹麻蜥线粒体基因组对比分析对天山以北的1号样品（吉木乃县，准噶尔盆地，标本号GUO3027）及天山以南的1号样品（若羌县，塔克拉玛干沙漠，标本号WGXG08403）的线粒体基因组进行测定分析，其GenBank 登录号分别为KP981389、KP981388。发现这两线粒体基因组中的基因数量、排列顺序与典型脊椎动物的线粒体基因组一致。除控制区外，序列中有553个位点发生了变异，变异比例为3.59%。编码蛋白基因的起始密码子中除了ND3基因外（GUO3027为ATG，WGXG08403为GTG），在其余基因中南北之间没有差异，对应基因的终止密码子均相同。与大多数脊椎动物相同，虫纹麻蜥的D-loop (控制区)位于tRNA- Phe与tRNA- Pro之间。从D-loop长度上对比，天上以北为4097 bp，天山以南样品为4409 bp。结构上对比，D-loop内发生了长片段的插入缺失现象，有两类重复单元： (Type I长为55 bp：5’-TTT TGT CCA CTT CTC ACC CTA TTT TCT CTA CTT CAC ACG CCG CTT CTA GCG GCT T-3’)； Type II长为56 bp: 5’-TCT AGA ACA CAA ACG AAT TAA CAA AGA ACA ATT CGT TTG TAA TTT ACC TAA GTT TC-3’。值得注意的是，在重复序列中所含的以上重复单元在个体内和个体序列间都存在少量变异。两条序列相比较，在第一部分重复区中GUO3027比WGXG08403多一个重复单元；在第二个重复区中GUO3027比WGXG08403少3个重复单元。重复单元内序列的多态性与重复单元数量的变化都可能具有潜在的分子标记价值。本研究为其他物种的线粒体基因分析，特别是对长D-loop区的分析提供参考。同时，对编码蛋白基因序列分别进行对比，选择变异度适中，且易于扩增的Cyt b基因和COI基因片段进行后续谱系地理研究。2.基于线粒休DNA的虫纹麻蜥种群分化与群体动态研究通过PCR扩增和测定了69个样点、共456个个体的Cyt b基因序列 (1143 bp)和COI片段（660 bp）。将两个基因进行合并分析（共1803 bp），共鉴定识别200个单倍型。基于线粒体基因单倍型构建的BI, ML和MP树拓扑结构一致，得到四个有显著地理结构的支系，其中来自准噶尔盆地和阿拉善高原等地的样本（Phylogroup A）与来自吐鲁番-哈密盆地的样本（Phylogroup B）形成姐妹支系，来自敦煌盆地的样本(Phylogroup C)与来自塔里木盆地和焉耆盆地的（Phylogroup D）形成姐妹支系。这种谱系地理格局也得到了分子变异分析（AMOVA）和单倍型网络图分析(NETWORK)的支持。有趣的是，发现准噶尔盆地和阿拉善高原等地的样本间有共享单倍型，且共享的单倍型多为“较古老”（或祖先）类型，提示由于分化时间太短，它们之间可能有谱系不完全分选 (incomplete lineage sorting)。这些结果支持了天山、北山对其基因流的阻隔效应。虫纹麻蜥各个支系的分化时间估算结果表明：四个支系(A, B, C, D)的共祖时间为1.13 Ma (95% CI, 0.536-2.08)。准噶尔盆地-阿拉善高原与吐-哈盆地种群（A- B）的分化时间约为0.35 Ma (95% CI, 0.126-0.67 Ma)。敦煌盆地与塔里木盆地支系(C- D)分化时间约为0.31 Ma (95% CI, 0.117-0.603 Ma)。Phylogroup A共祖时间约为0.0916 Ma (95% CI, 0.03-0.18 Ma)。 Phlogroup B共祖时间约为0.18 Ma（95% CI, 0.06-0.345 Ma）。Phylogroup C共祖时间约为0.16 Ma（95% CI, 0.05-0.309 Ma）。Phylogroup D共祖时间约为0.12 Ma（95% CI, 0.04-0.23 Ma）。这些时间节点与更新世天山、北山的隆升时间相符。准噶尔盆地-阿拉善高原支系(Phylogroup A)、塔里木盆地支系(Phylogroup D)和敦煌盆地支系(Phylogroup C)的Tajima's D与Fu's Fs均为极显著负值，说明不能拒绝该地区的虫纹麻蜥在历史上经历过扩张。失配分布分析(mismatch distributions)也支持准噶尔盆地-阿拉善高原支系虫纹麻蜥经历过扩张。贝叶斯轮廓图分析(Bayesian Skyline Plot)也显示出塔里木盆地支系约在0.04 Ma至0.05 Ma期间种群出现了快速的扩增，而吐-哈盆地虫纹麻蜥从大约0.18 Ma开始有效种群大小一直处于恒定状态，但在大约0.01 Ma时出现了快速的种群收缩。准噶尔盆地-阿拉善高原支系也在0.04至0.05 Ma期间处于扩增状态。对虫纹麻蜥不同气候历史时期的分布情况进行生态位模型分析，结果显示：在末次盛冰期（LGM）时期，虫纹麻蜥的分布区最广，包括了阿拉善高原绝大部分地区、河西走廊西北部分、整个库姆塔格沙漠、吐-哈盆地绝大部分、焉耆盆地绝大部分、准噶尔盆地西南部地区、巴里坤和伊吾北部地区、塔里木盆地东部中部及西部边缘部分、蒙古南部与中国接壤部分地区，东南方向上适生分布区到达宁夏西北少部分地区。而且，上述地区的大部分均具有较高适生性指数；在末次间冰期（LIG）时期，与LGM时期相比适合虫纹麻蜥分布的区域都较小。其中在塔里木盆地中部和西部地区、塔-哈盆地、焉耆盆地和阿拉善高原东北部地区适生区域在三个时期中范围最小。在巴里坤和伊吾北部地区和准格尔盆地西南部地区适生区也较小。然而，该时期分布区向东南方向延伸，到达了如今的陕西省；现今虫纹麻蜥模拟分布区与LGM时期的相比，在塔里盆地中部西部地区和阿拉善高原东北部地区有较小的收缩。在塔里木盆地东部地区有较小扩张。在蒙古南部地区有小幅扩张。而在，罗布泊地区、焉耆盆地和吐-哈盆地适合分布区明显减小。由此可见，生态位模型分析支持了GM扩张模型对耐干旱的虫纹麻蜥的适用性。3.基于核基因的虫纹麻蜥谱系地理结构的初步分析对虫纹麻蜥结蛋白样蛋白（Cingulin like 1，CGNL1）、微管相关蛋白1A（Microtubule-associated protein 1A， MAP1A）基因部片段及纤维蛋白原β内含子7（β-fibrinogen intron 7）进行了扩增与分析。共筛选扩增了181个个体的CGNL1基因片段（812 bp），139个个体的MAP1A基因片段（1695 bp），55个个体的β-fibint 7 片段（608 bp）。利用核基因分别构建单倍型网络图，结果并没有显示出明显谱系群（Phylogroup）结构。运用单倍型序列构建系统发育树也未能得到稳健的结果。这可能是由于相比线粒体基因，核基于更易出现不完全谱系分选现象，即各个支系间分化时间短的情况下，与线粒体基因相比，核基因替代速率较小，而有效种群更大，从而引起谱系不完全分选。 |
| 英文摘要 | AbstractOver the last three decades, mitochondrial DNA (mtDNA) has been the most popular marker for evolutionary analysis such as assessing molecular diversity, testing phylogenetic and phylogeographic hypotheses. Research on mtDNA and the mitochondrial genome has also been one of the hotspots in life science, with important significance in theory and practice. Lizards, as one kind of widely distributed reptiles, tend to be highly structured genetically because of their relatively low dispersal, strict habitat specificity and physiological requirements. They have been model organisms for testing biogeographic hypotheses and exploring insights into what is happening in natural populations. In this study, we will investigate the characteristics of mitohondrial genome and evolutionary history of the Variegated Racerunner (Eremia vermiculata ) by using mtDNA and three nuclear DNA (nuDNA) loci, which is widespread in the arid desert regions in Northwest China. Samples are collected across the entire region of this lizard. We will combine the phylogenetic-based phylogeographic analyses and coalescent-based population genetic models as well as GIS-based ecological niche modeling technique to infer the phylogeographic pattern and historical demography. The objectives are (i) to determine and compare the mitogenomes of two individuals from northern and southern Tianshan Mountains; (ii) to reveal the phylogeographic structure of E. vermiculata; (iii) to explore the relative contribution of geographic barriers (e.g., Tianshan Mountains, Beishan Mountains) versus geographic distance in the observed population divergence. (iv) to test GM expansion model by unraveling the historical population dynamics of this lizard; (v) to evaluate the utility of three nuclear markers for phylogeographic analysis of this lizard. In addition, we will discuss how climatic oscillations in the Quaternary and the aridification of Arid Central Asia affected shifts in this species’ distributions. The main results are as follows.1.Comparison of the mitochondrial genomes of two individuals Two mitochondrial genomes have been determined: one sample from northern Tianshan Mountains (Jeminay County, Junggar Basin, voucher No., GUO3027), and the other from southern Tianshan Mountains (Ruoqiang County, Taklimakan Desert, voucher No., WGXG08403). They are deposited in the GenBank with accession numbers KP981389 and KP981388, respectively. The number and orgnization of the genes were in accordance with typical mitochondrial genes of vertebrates. Compared with each other, it was observed that there were 553 variable sites, with 3.59% of variable percentage in the noncontrol-region. Except for ND3 gene (ATG for GUO3027, GTG for WGXG08403), the initiation codons of each protein coding genes were the same from the north and south of Tianshan Mountains. All the termination codons of each protein coding gene of the two genomes were the same. In line with most vertebrates, D-loop (Control Region ) of Eremias vermiculata was between tRNA- Phe and tRNA- Pro. The D-loops of GUO3027 and WGXG08403 were 4097 bp and 4409 bp. There were several inserted and deleted fragments in D-loops. Two kinds of repeating regions were observed, with type I of 55 bp in lengh (5’-TTTTGTCCACTTCTCACCCTATTTTCTCTACTTCACACGCCGCTTCTAGCGGCTT-3’), type II of 56 bp in length (5’-TCTAGAACACAAACGAATTAACAAA GAACAATTCGTTTGTAATTTACCTAAGTTTC-3’). It should be noted that all the repeating units were not completely identical, with some variable sites in some units. In type I repeating region, one more repeating unit was observed in GUO3027 than WGXG08403. In type II repeating region, there were three more repeating units in WGXG08403 than GUO3027. With rich repeating units and highly variable sites, the control region showed its potential utility for population geneitc analyais. In addition, Cyt b gene and COI gene, easily amplified and sequenced, were chosen to study the range-wide phylogeographic analysis of Eremias vermicualta. 2.Population genetic divergence and historical population dynamics inferred from mtDNA A total of 456 specimens from 69 locations were sampled from throughout the species’ distribution. Two mitochondrial DNA genes (Cyt b, 1143 bp; COI, 660 bp) were amplified and sequenced. The two fragments were concatenated into one data set, and 200 haplotypes were defined. The topologies from BI, ML and MP analyses are identical with one antoher, with four allopatric phylogroups: ((A, B), (C, D)). Phylogroup A consisted of haplotypes from Junggar Basin, Alxa Plateau, and adjacent Kazakhstan and Mogolia; Phylogroup B consisted of Turpan-Hami Basin. Phylogroup C comprised haplotypes from Dunhuang Basin; and Phylogroup D comprised haplotypes from Tarim Basin and Yanqi Basin. The four allopatric phylogroups were recovered by the analyses of molecular variance (AMOVA) and network analysis. Interestingly, there were shared ancestral polymorphisms between the samples from Alxa Plateau and Junggar Basin. This observation provides evidence for incomplete lineage sorting (deep coalescence) between them, due to more recent divergence time. Taken together, the barrier effects of Tianshan Mountains and Beishan Mountains to gene flow are supported. The mean time of most recent common ancestor (TMRCA) of all haplotypes was estimated at about 1.13 Ma (95% CI, 0.536, 2.08). Divergence time of Turpan-Hami Basin clade and Junggar basin+Alxa Plateau clade (A-B) was estimated at about 0.35 Ma (95% CI, 0.126-0.67 Ma); Divergence time of Dunhuang basin clade and Tarim Basin+Yanqi Basin clade (C-D) was estimated at about 0.31 Ma (95% CI, 0.117-0.603 Ma). TMRCA of haplotypes from Junggar Basin+Alxa Plateau clade (Phylogroup A) was estimated at about 0.0916 Ma (95% CI, 0.03-0.18 Ma); from Turpan-Hami Basin clade (Phylogroup B) was estimated at about 0.18 Ma (95% CI, 0.06-0.345 Ma); from Dunhuang basin clade (Phylogroup C) was estimated at about 0.16 Ma (95% CI, 0.05-0.309 Ma); from Tarim Basin+Yanqi basin clade (Phylogroup D) was estimated at about 0.12 Ma (95% CI, 0.04-0.23 Ma). These time scales coincide with the uplift of Tianshan Mountains and Beihan Mountains in Pleistocene.Tajima’s D and Fu's Fs tests implied that Phylogroup A, Phylogroup C and Phylogroup D had undergone expansions in recent history. Analyses of mismatch distributions also supported Phylogroup A had been expansions. Analyses of data using Bayesian skyline plots suggested that: Phylogroup D had undergone rapid expansion during 0.04 Ma to 0.05 Ma; Phylogroup A had been in expansion during 0.04 Ma to 0.05 Ma as well. The effective population size in Phylogroup B has been stable from 0.18 Ma to about 0.01 Ma, and then populations went through an extreme contraction. Suitability regions of ecological niche modeling in different climate histories showed that suitability regions of Eremias vermiculata were the biggest in the LGM. Compared with suitability regions in the LIG, most of suitability regions had taken expansions in the LGM, except for southeast of the regions which contracted at that time. Compared with suitability regions in the LGM, the regions reduced in present. The results of ecological niche modeling and estimates of past population sizes support a model that the GM expansion model is suitable for arid-adapted Varigated Racerunner Eremias vermiculata. These data reject the universal use of a GM contraction model for arid biota.3.A preliminary phylogeographic analysis of three nuclear lociWe also investigated three nuclear-gene fragments: CGNL1 (Cingulin like 1), MAP1A (Microtubule-associated protein 1A) and FIB-7 (β-fibrinogen intron 7) to infer this lizard phlogeographic pattern. The CGNL1 gene fragments (812 bp) of 181 samples, MAP1A gene fragments (1695 bp) of 139 samples and FIB-7 fragments (608 bp) of 55 samples were sequenced. For the genealogical trees inferred from the three nuclear loci, no signifciantly geographic structure was recovered. The haplotypes networks do not show clear phylogeographic structure either. This is not surprising given the slower divergence rates of the nuDNA markers and usually higher effective population size, which could result in the prevalence of incomplete lineage sorting. |
| 中文关键词 | 虫纹麻蜥 ; 线粒体基因 ; 系统发育 ; 种群分化 ; 种群历史动态 ; 核基因 ; 天山 ; 北山 ; 沙漠 |
| 英文关键词 | Eremias vermiculata mitochondrial DNA Phylogeny Population divergence demographical history Tianshan Mountains Beishan Mountains Desert |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 动物学 |
| 来源机构 | 中国科学院成都生物研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287636 |
| 推荐引用方式 GB/T 7714 | 周天和. 虫纹麻蜥线粒体基因组特征、种群遗传分化及群体动态研究[D]. 中国科学院大学,2016. |
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