【作者】 李郊；

【导师】 雷光春；

【作者基本信息】 北京林业大学 ， 自然保护区学， 2014， 博士

【摘要】 生物多样性分布格局及其成因是生物地理学研究的核心内容之一,对于理解生物多样性的演化规律、物种形成的生态学过程与进化路径、制定物种保护与管理对策等具有重要意义。两栖动物是全球生物多样性受到最严重威胁的物种类群,濒危物种比例居各种生物类群之首。同时,两栖类物种的低迁移能力、生境敏感性以及高家域忠实度,是研究谱系生物地理学与生物多样性演化规律的理想物种。我国东北、华北地区共记载了蛙科(Ranidae)林蛙属(Rana)6个物种,分别是黑龙江林蛙(Rana amurensis Booulenger,1886)、昆嵛林蛙(Rana kunyuensis Lu et Li,2002).东北林蛙(Rana dybowskii Gunther,1876)、中国林蛙隐存种(研究中称为“中国林蛙近似种”,Rana cf. chensinensis)、桓仁林蛙(Rana huanrenensis Liu, Zhang et Liu,1993)、徂徕林蛙(Rana culaiensis Li, Lu and Li,2008),其中,黑龙江林蛙(Rana amurensis Booulenger,1886)、东北林蛙(Rana dybowskii Gunther,1876)、中国林蛙隐存种(研究中称为“中国林蛙近似种”,Rana cf.chensinensis)、为广泛分布种,昆嵛林蛙(Rana kunyuensis Lu et Li,2002)为山东昆嵛山特有种,其它两种特别稀有,本次野外调查没有采集到任何标本,因此本研究以这4个物种为研究对象,聚焦以下几个关键科学问题：(1)我国东北、华北地区四种林蛙与已知蛙科物种的线粒体基因组全序列结构是否一致?有何重要特征?(2)三种广泛分布林蛙(黑龙江林蛙、东北林蛙、中国林蛙近似种)的种群空间遗传结构及其种群的历史动态是否呈现相同趋势?(3)导致三种林蛙种群遗传多样性空间结构及种群动态变化的因素有哪些?是否符合气候隔离假说、河流障碍假说、山脊障碍假说?(4)如何根据林蛙的种群的历史变化规律制定物种保护对策?为此,本研究通过在东北、华北地区广泛调查采样,通过分子生物学方法获得序列信息,利用线粒体基因和核基因为分子标记,重建系统发育关系及分子钟估算,结合古冰期气候信息和地质事件证据开展研究,得到如下主要研究结果：(1)四个林蛙属物种线粒体基因组全序列特征通过黑龙江林蛙(Rana amurensis)、昆嵛林蛙(R. kunyuensis)、东北林蛙(R.dybowskii)、中国林蛙近似种(R. cf. chensinensis)四种林蛙标本线粒体基因组全序列的测试,发现林蛙属中存在着新型全序列结构。黑龙江林蛙(R. amurensis)和昆嵛林蛙(R. kunyuensis)的全长分别为20,564bp和22,255bp,均包含13个蛋白质编码基因、22个转运RNA、2个核糖体RNA基因、2段控制区,与己知蛙科物种的线粒体全序列存在差异,体现在2段控制区以及tRNALeu(CUN)和ND5的易位。东北林蛙(R. dybowskii)和中国林蛙近似种(R. cf. chensinensis)的全长分别是18,864bp和18,808bp,均包含13个蛋白质编码基因、22个转运RNA、2个核糖体RNA基因、1段控制区,与己知蛙科物种的线粒体基因组全序列结构一致。根据四个物种序列长度较长的8个蛋白质编码基因以及D-loop基因的分析,ND1/ND2/ND4、ND5、cytb较COⅠ、COⅡ、COⅢ变异程度高,可以提供了更多了系统发育信息。(2)三种广泛分布林蛙的种群空间遗传结构及其种群的历史演变规律黑龙江林蛙、东北林蛙、中国林蛙近似种等三种林蛙的遗传多样性空间分布格局清晰,即遗传谱系分化明显,地理分布区域相互隔离。黑龙江林蛙(Rana amurensis)的2个谱系分别分布在大兴安岭的高纬度高海拔区域,以及嫩江主干及以东的低海拔区域。东北林蛙(R. dybowskii)2个谱系分别分布在小兴安岭南麓,以及长白山、大兴安岭、小兴安岭。中国林蛙近似种(R. cf. chensinensis)共划分为4个谱系,谱系A分布于黄河中游(由北向南段)右岸、毛乌素沙地以南至渭河区域,谱系B分布于毛乌素沙地区域,谱系C分布于黄河中游(由北向南段)左岸,及海河流域,谱系D分布于燕山以北的东北区域。不同物种的种群历史动态稍有不同：黑龙江林蛙(R. amurensis)2个地理组在晚更新世至今存在种群扩张势；东北林蛙(R. dybowskii)3个地理组在历史上种群稳定,中更新时末期至今呈现种群扩张趋势；中国林蛙近似种(R. cf. chensinensis)4个谱系中,谱系A、B、D在历史上种群平稳,晚更新世至今存在扩张趋势,谱系C在历史上种群平稳,近期存在多次扩张过程。(3)导致三种林蛙种群空间结构变化的主要原因影响我国东北、华北地区林蛙遗传多样性空间的成因包括三种,分别是气候变化、河流和山脊。黑龙江林蛙的谱系地理结构受到希夏邦马冰期气候振荡以及大兴安岭山脊障碍的影响；东北林蛙的谱系地理结构受到古乡冰期气候振荡,以及大小兴安岭、长白山三座山脊的障碍作用。中国林蛙近似种的谱系地理结构受到更新世冰期气候变化的影响,同时黄河和燕山也分别起到了河流障碍和山脊障碍作用。(4)物种保护对策本研究的三种广布种均存在生物冰期避难所,建议建立自然保护区或者保护地。黑龙江林蛙2个谱系内部分别存在一个生物冰期避难所,分别位于海拉尔盆地和松花江中游；根据生物避难所及特有单倍型物种分布区域的重要程度,东北林蛙栖息地的重要性依次为长白山中部、小兴安岭南麓、小兴安岭北麓、大兴安岭东麓；中国林蛙近似种4个谱系内部各存在一个生物避难所,分别位于渭河流域、毛乌素沙地、太行山区域以及东北的低纬度区域,建议作为重点保护区域加以管理。综上所述,本研究探讨了四种林蛙的线粒体基因组全序列信息及特征,并且基于线粒体基因和核基因的分子证据,通过东北、华北地区三种林蛙的系统发育关系重建及分子钟估算,明确了三种林蛙的谱系地理格局,并结合地质学证据和古气候信息,揭示形成上述格局的成因包括历史气候振荡、河流障碍以及山脊障碍,为我国两栖动物的保护提供了科学依据。在本研究的基础上,后续研究可在如下几个方面开展：中国林蛙及隐存种物种确定及蛙科系统发育研究、利用生态学方法解析生物多样性空间格局、特有种的形成机理研究、我国北方地区比较生物地理学研究、未来不同环境变化情景下的物种分布格局模拟等。更多还原

【Abstract】 Biodiversity pattern and its formation mechanism is one of the key subjects of the biogeographic studies. It is the basis to understand the evolution of biodiversity, reveal the ecological and evolutionary process of speciation, and thus is critical important for setting biodiversity conservation and management strategies. Amphibians have been identified as the most threatened vertebrate group due to the highest percentage of the threated species among all species groups. Meanwhile, amphibian is the ideal species for phylogeographical and evolution of biodiveristy studies due to its low individual mobility, sensitive to habitat quality and its high loyalty to home range. Six Rana species are recorded in Northeast and North China, e.g., Rana amurensis Booulenger,1886, Rana kunyuensis Lu et Li,2002, Rana dybowskii Gunther,1876, a cryptic species of R. chensinensis(henceforth referred to as R. cf. chensinensis), Rana huanrenensis Liu, Zhang et Liu,1993, Rana culaiensis Li, Lu and Li,2008. Among the six known species to the region,Rana amurensis Booulenger,1886, Rana dybowskii Gunther,1876, a cryptic species of R. chensinensis(henceforth referred to as R. cf. chensinensis) are the widely distributed species, whereas Rana kunyuensis Lu et Li,2002is endemic to KunyuMountains in Shandong province. The other two species are extremely rare, and they were not found in the field survey, and thus, the study focus on the four Rana species (Ranidae) to address the following questions:(i) Whether the mtDNA mitochondrial genome structure and novel feature of the four Rana species is different from the known Rana species?(ii) What are the genetic structure and its demography of three widely distributed Rana species;(iii) What are the causes of phylogeographical patterns of three Rana species,? and whether the Climate barrier hypothesis, Riverrine barrier hypothesis and Ridge barrier hypothesis can explain such pattern?(iv) How to develop conservation strategies using the findings of this study?In order to address these questions, this study surveyed northest and northen China for the collection of experimental samples; applied mitochondrial genes and nuclear genes mark to obtain full gene sequences of the four species; reconstructed the phylogenetic relationship and population history using molecular clock, as well as the geological evidence, and obtained the following results:(i) Feature of complete mitochondrial genome structure of four Rana speciesThe mitogenome length of R. amurensis and R. kunyuensis were20,564bp and22,255bp, respectively, including13protein-coding genes,22transfer RNA genes,2ribosomal RNA genes, and2control region (D-loop).We first found a novel gene order arrangement with the translocation of tRNALeu(CUN) and ND5, and duplicated D-loop genes from this two species. The length of R. dybowskiiand R. cf. chensinensiswere18,864bp and18,808bp,respectively, including13protein-coding genes,22transfer RNA genes,2ribosomal RNA genes, and1control region (D-loop). The genes structure of above two species displayed a similar pattern to that of known Rana species. According to analysis of the8protein-coding genes and D-loop gene, we found that ND1, ND2, ND4, ND5and cytb contain more phylogenetic information than COI, COⅡ and COⅢ.(ii) The genetic structure and population demography of three common Rana speciesThe distribution pattern of genetic diversity showed phylogenetic discontinuities and spatial separation amongRana amurensis, R. dybowskii, and R. cf. chensinensis,. For R. amurensis, Clade A was distributed in the high-altitude and high-latitude of GreatHinganMountain, Clade B was distributed in NenjiangRiver and Songnen Plain. For R. dybowskii, Clade A was restricted to south slope of LessHinganMountain, Clade B was distributed in east slope of Great Hingan Mountain, north and south of Less Hingan Mountain, and Changbai Mountain. Four Clades were identified in R. cf. chensinensis. Clade A was distributed in Weihe River, Clade B was distributed in Mu us derset, Clade C was distributed in the left bank of Yellow River and Hai River Basin, and Clade D was distributed to north of Yan Mountain.There are some difference in the population demography among three Rana species. Population expansion of R. amurensis, R. dybowskii and clade A, B and D of R. cf. chensinensis were occurred since late-Pleistocene, mid-Pleistocene and late-Pleistocene, respectively. Clade C of R. cf. chensinensis have been indicated relative stable population demography.(iii) Factors that contributed to the phylogeographical patterns of three Rana species Climatic events, large river and mountain ridge are the major factors that influence the phylogeographic parttern of the three Rana species. The major divergence of Rana amurensis was trigged by climate oscillation of Xixiabangma Glaciation and the isolation of Great Hingan Mountains. The phylogeographical pattern of R. dybowskii was influenced by climate oscillation of Guxiang Glaciation and the isolation of Great Hingan Mountains, Less Hingan Mountains and Changbai Mountain. The phylogeographical pattern of R.cf.chensinensis indicated that the divergence time of clades were consistent withclimate oscillation of Xixiabangma Glaciation and the penultimate glaciation. Meanwhile, both Yellow River and Yan Mountain played an important role in shaping the phylogeograpohical pattern of R. cf.chensinensis.(iv) Conservation strategies for the three Rana speciesThe glacial refugia should be protected as wildlife habitat areas. The refugia of Rana amurensis are located in Hailar Basin and middle reach of Songhua River. The refugia and regions of unique haplotype for R. dybowskii are situated in middle Changbai Mountain, southern slopes of Less Hingan Mountains, northern slopes of Less Hingan Mountains, and eastern slopes of Great Hingan Mountains. Four refugia of R. cf. chensinensis are as follows: Weihe river, Mu us desert, Taihang Mountain and low-latitude area of Northeast China.In summary, this study determined the mitogenome of four Rana species, described phylogeographical pattern for the three widely distributed species, and analyzed causes for the spatial genetic distribution of the three studies species based on Mitochondrial DNA genes and nuclear genes. Clear clades were identified in three species distribution range, and climate oscillation, river and mountain ridge were found important in the phylogeographical pattern formulation. However, due to limited time and resources, a number of key questions have not yet addressed in this study, which include diversification history of R. chensinensis and R. cf. chensinensis, the phylogenetic study of Rana species, the spatial patterns of genetic diversity through ecological method, the speciation mechanism of endemic species in China, comparative biogeographical study in North region scale, and modeling of the species distribution patterns in the future environmental scenarios.更多还原