【作者】 李辛雷；

【导师】 孙振元；

【作者基本信息】 中国林业科学研究院 ， 园林植物与观赏园艺， 2012， 博士

【摘要】 杜鹃红山茶（Camellia azalea Wei）为山茶科特有珍稀濒危植物，仅在我国广东省阳春市鹅凰嶂自然保护区内分布。在杜鹃红山茶被发现利用后，原有资源遭到了严重的人为破坏，目前其分布范围不断缩小，种群数量逐渐减少，已处于极度濒危状态。本研究进行了广泛的资源调查，通过形态特征、DNA水平的AFLP与SSR分析，研究了杜鹃红山茶种群的遗传多样性水平与遗传结构状况。从形态解剖学、种群生态学、繁殖生物学方面揭示了杜鹃红山茶濒危机制。结合核基因组DNA水平AFLP分析和叶绿体DNA片段序列分析提出了杜鹃红山茶系统分类地位。主要结果如下：1、杜鹃红山茶种群数量少，人为干扰破坏严重；种群沿河流呈狭窄的带状分布，生境片段化；分布地属热带北缘雨林气候，生长土壤缺少磷、钾元素，养分含量较低。2、杜鹃红山茶种群结构呈纺锤型，幼苗严重不足，种群存活曲线属于Deevey Ⅰ型，有衰退的趋势；不同生境的种群因所处微环境各异，结构上表现出一定差异。空间分布格局主要表现为集群分布，生长过程中从聚集型向随机型转变。3、杂交指数（OCI）、花粉胚珠比（P/O）和授粉试验等表明，杜鹃红山茶繁育系统为异交型，部分自交亲和，需要传粉者。风媒传粉测定及访问昆虫的观察表明，杜鹃红山茶为虫媒花，兼有少量风媒传粉，有效传粉昆虫为蜂类和蝶类。4、杜鹃红山茶土层内种子及幼苗较少，自然更新能力差。果实2～3个月成熟，自然开裂，种子靠重力散落。杜鹃红山茶饱满种子占总数43.29%，其中有活力种子占35.00%，质量较差；种子萌发率2.05%，后期死亡率59.26%。5、杜鹃红山茶始花期和盛花期为挥发性成分释放的主要时期；花瓣和雄蕊为挥发性成分释放的主要部位；挥发性成分释放的日变化与传粉昆虫活动规律相符。挥发性成分能够吸引昆虫，对杜鹃红山茶传粉具有重要作用。6、杜鹃红山茶花芽分化能持续不断进行，分化期约30d，花芽分化期间能观察到处于不同分化阶段的花芽；花芽分化过程分为生理分化期、花原基分化期、萼片原基分化期、花瓣原基分化期、雄蕊原基和雌蕊原基分化期。7、杜鹃红山茶种群15个表型性状中，9个具差异显著性，总变异系数25.137%；表型性状的变异幅度在不同亚居群间具较大差异。杜鹃红山茶遗传多样性的AFLP分析表明，多态位点百分率56.760％，期望杂合度0.129，Shannon信息指数0.203；总遗传变异中有24.687%发生在亚居群间，75.313%的变异发生在亚居群内；不同年龄级间遗传多样性无明显差异，1.468%的遗传变异存在于龄级间。杜鹃红山茶种群遗传多样性的SSR分析表明，观测杂合度为0.303，期望杂合度为0.465，Shannon信息指数为0.882；亚居群间的遗传分化系数为0.099，基因流为2.264；不同繁殖群体及龄级间遗传多样性无明显差异，不同龄级间的遗传分化系数为0.058，基因流为4.034。综合AFLP、SSR分析结果，杜鹃红山茶种群遗传多样性较低，不同繁殖群体及龄级间遗传多样性差异不大；遗传分化低，遗传变异主要存在于亚居群内；杜鹃红山茶SSR分析遗传多样性高于AFLP。8、杜鹃红山茶在核基因组水平上的AFLP分析及叶绿体psbA-trnH序列分析结合形态特征研究表明，杜鹃红山茶属于山茶属中独立一支，且与红山茶组相似系数最高、亲缘关系最近。研究结果表明，杜鹃红山茶种群分布范围不断缩小，种群数量持续下降，种群遗传多样性低，遗传分化小，对环境的适应性差。杜鹃红山茶濒危原因主要有：第一、种群分布范围狭窄、生境片段化及严重的人为干扰破坏，导致杜鹃红山茶资源受到破坏后，短时间内难以恢复。第二、杜鹃红山茶沿河流呈带状分布，果实成熟后种子靠重力散布，雨季时多被流水冲走，旱季时种子由于环境的恶劣及缺少适宜湿度，难以萌发。第三、杜鹃红山茶果实成熟期短，种子质量较差，萌发率极低，且幼苗后期会大量死亡，从而导致种群幼苗严重缺乏，更新困难。更多还原

【Abstract】 Camellia azalea Wei, an endemic species, is distributed only in the E’huangzhangprovincial nature reserve of Guangdong which located in Yangchun city, Guangdong province.The resource of C. azalea was severely destroyed after it was found and used. Today, it was anextremely endangered species with the gradual reduction of amount and distribution area. Thepopulation was widely surveyed, and its genetic diversity and genetic structure were studied bymorphological and AFLP and SSR analysis. The population ecology, reproductive biology andplant anatomy were investigated. The phylogenetic relationship of C. azalea was reconstructedby AFLP and cpDNA analysis. Based on these results, The endangered mechanism wererevealed. The results were summarized as follows.1. C. azalea, few individuals in population, was severely destroyed by human. C. azalea isdistributed as narrow bangs along a stream, and its habitat is fragmental. The distributionregion belongs to rainforest climate of tropical northern fringe. The nutrient content of soil islow, lacking P, K element.2. The population structure of C. azalea was spindle types in the natural distributionregion, and the survival curves was Deevey Ⅰtype. The population was declining due to lackof seedlings. The population structure difference among the different habitats was definitelydue to the different micro environments. The spatial distribution pattern of C. azaleapopulation was clump distribution, and changed from clump to random with the developmentof population.3. The results of outcrossing index(OCI), pollen-ovule ration(P/O) and pollinationtreatments showed the breeding system of C.azalea is outcrossing type, partly self-compatible,requiring pollinators. The results of determination of wind pollination and observation ofpollination insects showed C. azalea were insects pollination and weak wind pollination.Effective pollinators were bees and butterflies. 4. Natural regeneration of C. azalea was poor with the lack of seeds and seedling in thesoil. With the maturation and natural crack of fruit after2to3months, seeds of C. azaleascattered with gravity. The seed quality of C. azalea was poor, with43.29%full seeds, and thepercentage of full seeds with vitality was35%. Germination rate of seeds was2.05%, and deathrate of seedlings was59.26%in the late.5. The main volatile releasing period of flower of C. azalea were first flowering and fullflowering stage. The main flower parts of volatile releasing were petals and stamens. Diurnalvariation of the main volatile releasing was consistent with the activity patterns of pollinationinsects. The volatile attracts insects and plays an important role in the pollination.6. The flower bud differentiation of C. azalea progressed continually, and the flower budsin different differentiation phases could be observed in the period. The process of flower buddifferentiation was about thirty days, and was divided into six phases: physiologicaldifferentiation, flower primordium differentiation, sepal primordium differentiation, petalprimordium differentiation, stamen and pistil primordium differentiation phases.7.15phenotypic traits of C. azalea population were investigated. The result showedaverage coefficient of variation was25.137%, and9phenotypic traits were significantlydifferent. Variation range of phenotypic traits had large differences among subpopulation.According to AFLP data, expected heterozygosity（He）, Shannon’s diversity index（I）and thepercentage of polymorphic loci(P) were0.129,0.203and56.760％, respectively. Geneticvariation among and within subpopulation were24.687%and75.313%, respectively. Geneticdiversity of different age class was not significantly different, and genetic variation among theage class only was1.468%. According to SSR data, expected heterozygosity, Shannon’sdiversity index and observed heterozygosity(Ho) were0.465,0.882and0.303, respectively.Coefficient of genetic differentiation among subpopulation were0.099, and gene flow was2.264. Genetic diversity of different age class had not significant difference, and the same wasdifferent reproductive population. Coefficient of genetic differentiation among the age classwere0.058, and gene flow was4.034. The result of AFLP and SSR analysis showed that thegenetic diversity of C. azalea was low, and that had not significantly different among age class or reproductive population. Genetic differentiation was low, and main genetic variation waswithin subpopulation.Genetic diversity of SSR analysis was higher than that of AFLP.8. The result of morphological, AFLP and cpDNA analysis showed that C. azaleabelonged to an independent branch of the Camellia genus, and the phylogenetic relationshipwas near to Section Camellia with high similarity coefficient.The results showed that the distribution range of C. azalea population is continuouslyreducing, and population is decreasing. Genetic diversity and genetic differentiation of C.azalea population was low, that led to poor adaptability to environment. On the basis of aboveresults, it is suggested that there are three aspects for the endangered mechanisms of C. azalea.(1) Narrow distribution range, habitat fragmentation and hunman disturbance have led to theextinction of a great number of populations, and it is difficult to restore population in shorttime.(2) C. azalea distributes as narrow bangs along a stream. Seeds of C. azalea scatteredwith gravity after fruit maturation and natural crack. Most seeds scattering along the streamwere carried away by the water in the rainy season. With the bad environments and the lack ofsuitable humidity, the seeds were difficult to germinate in the dry season.(3)The seed quality ofC. azalea was poor with shorter fruit ripening, that resulted in poor germination rate of seedsand mass mortality of seedling in the late. For these reasons, C. azalea population severelylacks of seedling, and it is difficult to regenerate the population.更多还原