【作者】 张明；

【导师】 马建章；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2010， 博士

【摘要】 东北林蛙(Rana dybowskii)是主要分布于我国东北小兴安岭和长白山地区的重要经济物种,雌性林蛙的输卵管(林蛙油)是传统的名贵中药材,已有几千年的利用历史,目前仍被广泛使用。为了保护野生种群,人们开始在它的自然分布区内进行人工野外包山封沟养殖林蛙。作为解决种源问题的方法,常常在不同地区之间相互引种,这就给本地种群遗传结构的改变埋下了隐患。本文利用14个微卫星位点检测东北林蛙的遗传多样性,以及人为干扰下10个种群遗传结构的变异水平。主要分析种群间的等位基因频率、特有等位基因(PA)、杂合度(H)、多态信息含量(PIC)、基因分化系数(FST)、基因流(NM),根据Nei’sDA遗传距离构建NJ系统树,并通过分子方差分析(AMOVA)、主成份分析(PCA)和贝叶斯聚类分析(Bayesian Analysis)方法用STRUCTURE软件来推导种群的遗传结构。得出以下研究结果和结论：1.本文从同为Rana属的物种已发表的50个微卫星位点中,筛选了14个能稳定扩增我国东北林蛙300个样本的多态位点,核心序列均由3个或4个碱基组成,成功率仅为28%。共检测到87个等位基因,各位点等位基因数最低为2个,最高为12个,平均为6.2个；平均多态信息含量(PIC)的值为0.57。所有位点的观测杂合度(Ho)介于0.156-0.603之间,平均值为0.319；期望杂合度(HE)为0.315到0.856,平均值为0.596。10个种群的遗传多样性较为丰富,期望杂合度(HE)介于0.568-0.621之间,其中,佳木斯种群最高,蛟河种群最低。另外,在通化和铁力种群中各检测到一个特有等位基因。2.14个多态位点的Hardy-Weinberg平衡检验结果显示：位点Rpi107、RsyC52、RsAAT23、RP23和Rpi100在10个种群的检测结果完全或基本符合Hardy-Weinberge平衡(P>0.05)；另外5个位点分别在3-8个种群中处于不平衡状态(P<0.01)；其余4个位点(RsyC41、RsyD25、RsyD40和RsyD88)在所有种群中都显著不平衡(P<0.01)。由于所研究的部分种群存在异地引种现象,不平衡可能与人工选择有关,也可能存在无效等位基因。3.通过基因分化系数(FST)的结果,可以看出除已知的异地引种种群外,小兴安岭和长白山地区的东北林蛙种群分化显著。较低的基因流(NM=3.17-8.28)、相对较远的遗传距离(DA=0.087-0.175),在NJ系统树上聚为两个完全不同的分支,遗传结构存在明显的差异等都证明这两大分布区之间产生了明显的遗传分化。基于保护遗传学的原理,确定为两个单独的管理单元来保护和管理东北林蛙。4.研究结果还显示人为异地引种已经改变了佳木斯和本溪本地种群的遗传结构。另外,鞍山历史上并没有东北林蛙分布,现在的种群完全是人为异地引种杂交后,在二三十年的养殖过程中逐步建立起来的人工种群。可见,异地引种已经对东北林蛙种群的遗传结构产生了严重的影响,而这种影响是不可逆的,很容易导致种群退化,甚至会威胁物种的生存和繁衍。5.虽然这些种群遗传结构的改变原因并没有完全得到证实,但依据上述遗传数据的分析结果,本文提出如下保护建议：(1)依据保护遗传学原理,将小兴安岭和长白山两大东北林蛙的分布区确立为两个单独的管理单元,严格控制和监管在不同管理单元间相互引种的行为；(2)从保护物种基因多态性,特别是保护优质基因库的角度,建议将通化、鹤北和铁力种群作为优先保护种群；(3)对于遗传结构已经发生改变的种群,应限制种蛙对外销售,防止对周边种群造成更大的影响；(4)对于鞍山种群,应将其与相邻的本地种群完全隔离,减少对本地种群基因污染的潜在可能。更多还原

【Abstract】 The Dybowski’s frog (Rana dybowskii) is an importnat economic species mainly distributing in Lesser Khingan Mountains and Changbai Mountains in northeast China, the oviducts of female Dybowski’s frog has been used as traditional Chinese medicine for thousands of years and now still being heavily harvested. In order to protect the wild populations, local people cultivate this species in closing hillsides field in their natural habitats. As a measure, frogs are frequently translocated amongst allopatric populations. This potentially alters the genetic structure of local populations.Fourteen microsatellite loci were used to investigate genetic diversity, genetic structure and variation level of 10 populations under human disturbance. Allele frequency, private allele, heterozygosity (H), polymorphic information content (PIC), genetic differentiation coefficient (FST) and gene flow (NM) were calculated, NJ phylogenetic trees was constructed based on Nei’s DA genetic distance. The genetic structure was further estimated using analysis of molecular variance (AMOVA), principal components analysis (PCA) and Bayesian analysis by STRUCTURE program. Some major results and conclusions were as follows:1. We selected 14 polymorphism microsatellite loci with three or four nucleotide repeat, which could steadily amplified 300 samples of Dybowski’s frog from 50 loci published in Rana, the proportion was 28%. A total of 87 alleles were observed. The number of allele per locus ranged from 2 to 12 with an average of 6.2, and mean PIC was 0.57. The Ho of these loci ranged from 0.156 to 0.603 (average=0.319) and HE ranged from 0.315 to 0.856 (average=0.596). Genetic diversity was high in 10 populations with estimation of mean expected heterozygosity was from 0.568 to 0.621, which of JMS population was highest, while the lowest was JH population. In addition, one private allele in TH and TL populations respectively were found.2. Results of Hardy-Weinberg equilibrium tests showed that loci Rpi107, RsyC52, RsAAT23, RP23 and Rpi100 were in Hardy-Weinberg equilibrium (P>0.05) in entirely or most populations; another five loci in 3 to 8 populations showed deviation from significant Hardy-Weinberg equilibrium (P<0.01); besides, the other four loci RsyC41, RsyD25, RsyD40 and RsyD88 presented signifieant deviation from Hardy-Weinberg equilibrium in all populations (P<0.01). In our study, some frogs were translocated in different populations, the deviations from Hardy-Weinberg equilibrium of these loci were probably selected by mankind and null alleles maybe exist.3. According to pair-wise FST, we could see that population differentiation was significant between Lesser Khingan Mountains and Changbai Mountains, except the known allopatry introduced populations. Low gene flow was between 3.17-8.28, DA genetic distance varied from 0.087 to 0.175, gathered into two different clades, and genetic structure of them was obviously differed from each other, all of these demonstrated that genetic differentiation were highly significant between populations in two distribution areas. Based on the principles of conservation geneties, we identified two distinctive management units (MUs) to protect and manage Dybowski’s frog.4. The results suggested that two local populations (JMS and BX) were dramatically introgressed due to introduction from allopatric populations. Meanwhile, a hybrid breeding population AS was established outside the natural range of this species, and was consisted of genetic resources from allopatric populations in resently two or three decades. Introduction from allopatry had threatened the genetic structure of Dybowski’s frog, because of, and the effect was irreversible, that can easily induce population degradation, even affact the existence and proliferation of species.5. Although the consequence of such genetic alterations was not definitely confirmed, we proposed conservation recommendations according to the results above those dentifying two distinctive management units (MUs), namely Lesser Khingan Mountains and Changbai Mountains, to avoid frogs translocated between them. From viewpoint of gene polymorphism conservation, especiaily conservation of high quality gene pools that TH, HB and TL populations should be preserved prior to the others. Introduction from allopatry should not be performed unless these populations were proved genetically homologous, and the extent of introduction should be restricted to a level not impacting the genetic structure. To reduce the potential impacts on local gene pool, the AS population outside natural range should be strictly isolated from natural populations.更多还原