横断山区大型地衣特有种及其内生真菌遗传多样性研究

With the dramatic differences in topography, climate and vegetation, the Hengduan Mountains region has evolved a cluster of distinctive mini-hotspots of biodiversity, each with its own unique flora and fauna. This region is the most botanically rich temperate region in the world with the highest percent endemism, even though its species richness is not fully documented. This region is the center of origin for some plant species and the center of differentiation for many of the world larger genera, it provides a refugium for several ancient plant species found nowhere else in the world. The Hengduan Mountains region has also many lichens endemic to China, however, little is known on the distribution, genetic diversity and endolichenic fungi of them at present. Lichens are the mutual symbiotic organisms of special fungi with green algae and/or cyanobacteria. Endolichenic fungi live in close association with lichenized mycobiont and photobiont(s) inside intact lichen thalli and play very important roles on the host lichens. We will collect about 1500 samples (individuals or pure mats) for about ten endemic lichens in Lecanorales from 12 zones in 4 different mountains (Mts. Gaoligongshan, Laojunshan, Yulongshan in Yunnan, and Mt. Gonggashan in Sichuan) from small- to middle-scale areas (50-400 km). The techniques and protocols of DNA extraction, PCR amplification, and DNA sequencing will be used in our project. The sequence data of SSU of the nuclear ribosome DNA, the protein genes RPB1 and RPB2 for ten endemic lichen species will be obtained to determine their position in the Fungal Tree of Life and to estimate their divergent times. Meanwhile, the sequence data of ITS, LSU D1D2 regions of the nuclear ribosome DNA for about 500 samples of endemic lichens and more samples of endolichenic fungi will be obtained. The important intraspecific haplotypes of ITS and LSU D1D2 regions for the endemic lichens and associated endolichenic fungi including both cultivable and uncultivable will be detected. Basing on the data, we will document the genetic diversity of the endemic lichenized and endolichenic fungi obtained by reconstructing the haplotype networks，and determine the predominate haplotypes. Combining the special secondary metabolic chemotypes of the lichens examined, we will explore the relationship between the endemic lichens and the endolichenic fungi. Furthermore, we will explore potential ecological and chemical roles of endolichenic fungi based on a systematic sampling of endolichenic fungi from the endemic macrolichens occurring in Hengduan Mountains region and phylogenetic analyses. We hope to provide the phylogenetic evidence of Hengduan Mountains' endemism in lichenized fungi and provide support for the hypothesis that Hengduan Mountains region may serve as one of the centers for the colonization of lichen speciation.

横断山区因存在地貌、气候及植被方面的巨大差异，成为许多世界性植物大属的分布分化中心和一些类群的起源中心，具有温带地区最丰富的植物区系及最高比例的特有种类。该地区的地衣特有现象十分突出，但对其地衣特有种类的分布、遗传多样性及内生真菌知之甚少。地衣是与藻类或蓝细菌互惠共生的特殊真菌，在完整的地衣体中，除构成其主体的地衣型真菌及光合生物体外，还存在多种对其特有性、多样性和化学型产生重要影响的内生真菌。本研究拟查清横断山区大型地衣特有种的状况，以核糖体SSU及RPB2、RPB1基因序列确定其在真菌生命树中的位置及分化时间；以核糖体ITS及LSUD1D2区序列为分子标记，发现重要特有种内的基因单体型与化学型的关系；以分离培养和序列测定相结合研究其内生真菌，揭示大型地衣特有种的内生真菌遗传多样性，发现不同种类地衣的专一性内生真菌，探讨内生真菌对宿主的化学及生态作用，为横断山区地衣物种分化中心提供证据。

横断山地区具有温带地区最丰富的植物区系及最高比例的特有种类，形成了一系列拥有独特动植物区系的微型生物多样性热点区域；是许多世界性植物大属的分布分化中心和一些类群的起源中心。研究显示，该地区的地衣特有现象也十分突出，但对地衣特有种类的分布、遗传多样性和内生真菌知之甚少。本项目执行期间，采集和收集了该地区茶渍目的石蕊科、梅衣科、树花科，地卷目的地卷科，石耳目的石耳科等5科6属的12种特有地衣的1600份样品。对这些样品进行较为详细的形态学和化学研究，结合DNA提取、PCR扩增、DNA测序等分子生物学技术和方法，获得特定位点的序列数据。从300多份样品的近10000个组织块中分离获得约50种地衣内生真菌的1120个菌株；从56份样品中通过以nrDNA ITS2为分子标记的高通量测序获得近200个地衣内生真菌操作分类单元(OTUs)；对地衣样品及分离获得的地衣内生真菌菌株的完整nrDNA ITS, 部分 LSU, 部分 SSU, 和部分MitSSU基因位点进行测序，共获得2000多条序列。使用多种方法对这些数据进行系统进化分析，推测特有种类的分化时间及确定地衣内生真菌的系统位置。拟将桔色金丝亚属提升为属Chlorea，为横断山区的特有属，其分布可向北延伸至秦岭。基于核糖体小亚基序列及新的分析方法，估计茶渍亚纲各目的分化时间为155~278 百万年前，建议在茶渍亚纲中建立2个新目Ramalinales 和Scoliciosporales。 根据已获得的斑叶衣属种类的ITS及LSU序列的分析结果，确定ITS序列界定该属物种的适合标记。并以该属为例，检验了我们提出的核苷酸球形替代模型（Sphere Substitution Model, SSM）。依据SSM模型，设计位点比对计分集(0, 0.5, 1, 2, 3) ，计分值分别对应未变、延伸缺失、起始缺失、转换及颠换状态。提出用于进化标记的DNA分子有序的随机变化假说，认为DNA复制遵循随机的有序，而具有进化标记特征的DNA的变异遵循有序的随机。一些地衣内生真菌具有抗植物病原真菌活性，有成为新型生防菌剂的潜力。对5名硕士研究生和2名博士研究生进行了培训；5人次参加国内学术交流并做报告；发表研究论文9篇，申请发明专利1件；发表了4个新种，2个中国新纪录，5个新种待发表。项目收集的地衣样品及分离的地衣内生真菌菌株用于后续研究。