【作者】 龚杰；

【导师】 方盛国； 万秋红；

【作者基本信息】 浙江大学 ， 动物学， 2012， 博士

【摘要】 广域分布的两栖类无尾目动物,一直被视为分子支系地理学研究的模式物种。近年来,两栖类全球性的衰退问题已引起广泛关注。作为我国分布最广、数目最多的两栖类动物——黑斑侧褶蛙(Pelophylax nigromaculatus,简称黑斑侧褶蛙),也如同其他许多两栖类动物一样,正处于严重的衰退中。为此,本文在筛选SSR以建立检测标记,并配合使用线粒体控制区标记,对黑斑侧褶蛙的群体检测,分析种群的遗传结构特征,以期为相关领域开展黑斑侧褶蛙及其近缘类群衰退问题的研究,提供有用的信息。本研究主要结果如下：(1)以磁珠富集法筛选了13个多态的微卫星座位(Rnh1～Rnh13),自浙江金华的35个黑斑侧褶蛙个体检测到的等位基因值为3-14,观察杂合度和期望杂合度分别为0.171-0.941和0.422-0.912。各位点多态信息含量为0.341-0.891,位点累计的个体识别率达到0.9999。(2)以古北区区域内23个种群的321个个体的样品为材料,通过线粒体D-loop区约630bp的序列分析,定义了114个单倍型,并基于邻接法(neighbor joining, NJ)将这些单倍型分为两个明显的支系,依据其分布的地域命名为：中原支系和东北支系。除长白山脉一带的黑斑侧褶蛙种群属于东北支系外,其余中国境内的古北区黑斑侧褶蛙皆属于中原支系。其中,中原支系的在Bayes树、NeighborNet网络、Median-joining网络中都大致可以分为两支。这两支的分布基本是重叠的,这可能暗示着中原支系在扩散初期就存在着多态的祖先单倍型。(3)按照无尾目物种线粒体控制区的普遍进化速率(3.5% per million years),可推算出中原支系群体和东北支系群体分别在约0.070MYA和0.025MYA经历了种群扩张事件。这两个时间都处于第四纪的更新世。这暗示,黑斑侧褶蛙的群体扩张事件,可能与第四纪晚期的气候变化存在联系。但由于缺乏近缘物种的化石校正点,因此在具体的时间上,可能存在一些出入。(4)与线粒体的分析结果一样,基于微卫星座位数据的STRUCTURE分析将群体聚为中原支系和东北支系两支。另外,发现还一个位点仅在中原支系中扩增,这再次暗示了两个支系之间遗传分化。但由于缺乏其他分子标记的证据(特别是适应性的分子标记),两个支系是否能构成显著进化单元,仍有待于进一步的讨论。其中,东北支系的相关种群基于微卫星STRUCTURE分析又被明确地分为两组,这可能由于长白山脉的地理阻隔有关。但在线粒体方面,这两组群体并无明确分化,这可能是由于微卫星具备更高的分辨率,又或者可能与偏性扩散有关。(5)用mantel test检测遗传分化和地理距离之间的关系发现：如果把两个支系视为一个整体,则遗传分化与地理距离之间无明显的相关性(P值分别为0.462和0.280)。这可能是主要是由于两个支系缺乏基因交流造成的。而如果将两个支系分开考虑：中原支系无论在微卫星标记和线粒体标记上,都表现出了遗传分化与地理距离的显著正相关性(相关系数分别0.561和0.265,P值分别为0.001和0.015)。这可能是因为中原支系在古北区内主要分布于华北平原和东北平原,并不存在阻隔基因流的特殊因素,所以地理距离成了种群之间最主要的隔离因素。所以,邻近的种群之间可能依然存在一定的基因交流,这点在STRUCTURE分析的结果中也得到了验证。而东北支系则在两套标记上都表现为相关性并不显著(P值分别为0.552和0.160)。这说明地理因素并非东北支系各种群之间产生遗传隔离的主要原因。从STRUCTURE分析的结果中可知,东北支系的种群分为两支,这两支之间有长白山脉阻隔。综上可知,至少对于黑斑侧褶蛙而言,地形因素是影响遗传格局的重要因素(6)对从微卫星标记和线粒体标记中的所得的数据进行遗传多态性分析。发现无论是微卫星标记,还是线粒体标记,中原支系群体都比东北支系群体表现出了更高的遗传多态性。在线粒体标记方面,中原支系群体的单倍型多态性和核苷酸多态性分别为0.958和0.01268,而东北支系群体分别对应为0.859和0.00492。在微卫星标记方面,中原支系群体的有效等位基因数目、等位基因丰富度、观察杂合度分别为5.451、9.29、0.676、0.734,而东北支系群体对应为1.883、4.45、0.342、0.367。就中原支系群体而言,古北区的种群在微卫星标记和线粒体标记方面,都表现出了“南高北低”格局的遗传多样性。而在东北支系群体中,毗邻于朝鲜半岛的种群具有更高的遗传多样性。这种遗传格局可能和冰期后的种群扩散有关。(7)通过对各地种群进行近期瓶颈效应的检测,发现存在多个种群经历了显著的的近期瓶颈效应。这些近期经历了瓶颈效应的种群,并不连成区域,邻近的种群常表现不同情况。而且近期经历了瓶颈效应的种群,从南到北都有零散分布。这暗示,黑斑侧褶蛙种群衰退的主因可能并非是流行性疾病或全球气候变化,而是人为捕杀或生境破坏。因为由疾病或气候变化引起来的两栖类种群衰退,通常表现为区域性种群全面衰退。而这个结果也与宏观调查的结论相符合。更多还原

【Abstract】 Widespread anuran amphibians are regarded as good models for phylogeographic research. And global population decline of amphibians has attracted the attention of conservation biologist all over the world. As the most widespread and common amphibian, black-spotted frog (Pelophylax nigromaculatus) is also suffering from serious population decline. The work on phylogeography and conservation genetics was not only important for basic research, but also provided essential information for conservation strategies of amphibians.We developed a set of microsatellite loci for black-spotted frog which were immediately applied to the research of phylogeography and conservation genetics of black-spotted frog with a fragment of D-loop. The major results were as follow:(1) We isolated 13 polymorphic microsatellite loci according manetic beads enrichment protocol. Analysis of 35 muscle samples collected from Jinhua in Zhejiang showed the number of alleles from 3-14, observed heterozygosity and expected heterozygosity were 0.171-0.941 and 0.422-0.912, respectively. The polymorphic information content of these loci was 0.341-0.891, and combined identity exclusion probability reached 0.9999.(2) We collected 607 samples from 23 populations in Palearctic Realm. There were 321 individuals detected by the primers which could amplify a 630 bp fragment of D-loop. We identified 114 haplotypes which were clustered 2 clades based neighbor-joining method. The two lineages were named Central Plain Lineage and Northeast Lineage according distribution. The Central Plain Lineage was split into two clades based Bayes tree, NeighborNet network and Median-joining network. The two clades were approximate sympatry. It suggested the Central Plain Lineage was polymorphic before dispersal.(3) According to the universal evolutionary rate of anuran D-loop (3.5% per million years), we estimated the occurrence of population expansion began approximately 0.070MYA for Northeast Lineage and 0.025MYA for Central Plain Lineage. But due to lack of fossil calibration, there might be some error in temporal estimation.(4) As high degree of population genetic structure, only 7 microsatellite loci could be amplified among all population. Rnh5 could be amplified in the population belong to Central Plain Lineage. It suggested that high nuclear gene differentiation of two lineages, and it was verified by STRUCTURE analysis. But it was unknown that whether the lineages were two Evolutionarily Significant Units due to lack of evidence from other molecular markers, especially adaptive molecular markers. The population of Northeast Lineage could be divided into two groups, by STRUCTURE analysis. But it could not be verified by mtDNA. Maybe it explained by sex-biased dispersal or higher resolution of microsatellites.(5) There was a significant correlation between geogeraphic isolation and genetic differentiation in Central Plain Lineage (P-values were 0.001 and 0.015, respectively) reflected by both mtDNA and microsatellites. Because the major Northe lineage distributed in flatland of north China, and there was nothing separating populations of different regions. But there was no correlation between geogeraphic isolation and genetic differentiation in Northeast Lineage (P-values were 0.552 and 0.160, respectively). Because the major Northeast Lineage distributed in Changbai mountain range, and elevation could hinder gene flow of different populations. It suggested that the genetic distribution of black-spotted frog were impacted by landscape.(6) There was more genetic diversity in the populations of Central Plain Lineage according both nuclear genes and mtDNA markers. The haplotype diversity and nucleotide diversity of mtDNA were 0.958 and 0.01268 in populations of Central Plain Lineage, while 0.859 and 0.00492 in the populations of Northeast Lineage, respectively. The number of effective alleles, allelic richness, observed heterozygosity and expected heterozygosity of microsatellites in the populations of Central Plain Lineage were 5.451,9.29,0.676,0.734, while 1.883,4.45,0.342, 0.367 in the populations of Northeast Lineage. In the populations of Central Plain Lineage in Palearctic Realm, the genetic diversity of south populations was higher than north populations. In the populations of Northeast Lineage, the populations adjacent to korean peninsula had higher genetic diversity. The pattern of black-spotted frog might be related to population dispersal after glacial period.(7) Many black-spotted frog populations had experienced bottleneck in recent years. These populations were distributed widely, and the adjacent populations did not have unanimous demographic history in recent years. It suggested that the major causes of black-spotted frog might be habitat destruction or/and over exploitation, rather than epidemic or climate change.更多还原