植物与传粉者的互作往往受到是否存在植物近缘种的影响，近缘种对传粉者资源的竞争会导致花部性状替代现象的发生，即同域居群种间的性状差异大于异域居群种间的差异。性状替代是生物多样性形成和维持的重要机制，普遍在生物多样性富集地区的辐射进化类群中发生，然而植物的案例却较少，并且缺乏与生殖隔离研究的有效整合。项目选取兰科玉凤花属夜间蛾类传粉（但花粉块在昆虫体表的携带位置有分化）的两个近缘种宽药隔玉凤花和长距玉凤花为研究对象，开展同域和异域多居群的性状替代的研究。1）通过花部性状和表型选择差异的比较分析，构建不同的零模型，确证是否有性状替代发生；2）通过传粉昆虫观察、花粉干扰实验和性状操控实验，验证性状替代是否是竞争传粉昆虫所导致，是否与携粉位置的竞争相关；3）通过生殖隔离的定量研究，解析性状替代是否加强生殖隔离。研究将阐明直接作用（植物与传粉者）和间接作用（竞争）对花部性状的塑造和对物种共存的影响。

Plant-pollinator interaction is always variable among habitats at different geographic scales. Co-evolution of plant-pollinator is also influenced by co-existing of related species in the same genus，because related species which more likely sharing similar ecological niche compete for limited pollinator resources. To stably coexist in sympatry, closely related species may evolve divergent characters driven by in situ competition, a process termed as character displacement. Character displacement usually leads to greater inter-specific trait divergence in sympatry than inter-specific trait divergence in allopatry. Therefore, character displacement as one of important mechanism to promote species diversification and maintenance is intensely studied in animal taxa in these biodiversity hotspots, however, there are limited evidences in plants possibly due to the intensely studies on reproductive isolation in plant systems, but both research frames (character displacement and reproductive isolation) are rarely combined. In this project, we propose to study character displacement among two closely related orchid species Habenaria limprichtii and H. davidii. Both species are co-occurred and co-blooming in several places in Yunnan province. Previous pollination study suggested that H. limprichtii with a distinctive floral column with the great distance between the two pollinium sacs pollinated by hawkmoth carrying the pollinaria on their eyes. The long rostellum arms and two, widely spaced viscidia, may be co-adaptive paralleling the distance between those eyes of settling moths and hawkmoths. Our primary study on H. davidii with a much narrow spaced viscidia suggest it should be pollinated by hawkmoth targeting the bases of proboscides as the sites of pollinaria attachment. Furthermore, our multiple population survey on both species suggested that the inter-specific difference of spur length and the distance between viscidia is enlarged in the sympatry compared with their allopatry populations suggesting a pattern of character displacement. It is possibly driven by competition for pollinaria attachment sites for avoiding inter-specific pollinaria transfer. Therefore, in this project, we aimed to test this hypothesis by addressing the following questions. 1) Is there floral character displacement among these two closely related species? If it is, does avoiding inter-specific pollinaria transfer or hybridization drive the evolution of character displacement? 2) What is the relationship between reproductive isolation and character divergence? Does character displacement reinforce the isolating barriers and promote the co-existence of sympatry populations. To answer these questions, we propose a multi-population and multi- multidisciplinary study with floral trait measurement/comparison, pollination observation, trait manipulation, hand intra- and inter-specific pollination experiment and statistic analysis with null model. Research subdivided into three overlapping programs: 1) Measurement, analysis floral and plant traits among species and among sympatry and allopatry populations to test floral character displacement; 2) Pollinator observation, morphology comparison between flowers with pollinator, the sites of pollinaria attachment are characterized; Trait selection are tested by male (pollinaria removal) and female (pollinaria deposition) reproductive success in the sympatric populations; Trait manipulation experiments including shorten spur and pollinaria staining technique are applied to test the competition of attachment sites for same or different pollinator species. 3) Quantification of six reproductive isolating barriers among two species to test the relationship between reproductive isolation and character displacement. The results from this project will provide evidences for understanding the floral trait evolution and the co-existence mechanisms of closely related species.