The evolution of apomixis in autopolyploid alpine plants

自然种群中无融合生殖的起源仍不是很清楚。无融合生殖通常与在较冷的气候中发生有关。在这里，我们想要检验的假设是，低温通过触发二倍体中未减少的卵细胞形成，直接影响无融合生殖的起源。未减少的雌配子形成可通过雌三倍体桥导致多倍化，而与孤雌生殖的耦合将导致完全功能的无融合生殖种子形成。另一方面，多倍体可能与适应较冷气候的特征有关，如高山矮生。我们认为，DNA甲基化对同一基因繁殖模式的自发转变和对环境条件的快速适应都起着重要作用。在前期项目中，我们研究了高山二倍体-同源四倍体系统库普菲猫(Ranunculus Kuepferi)在阿尔卑斯天然种群中的繁殖方式和生态位分化。我们观察到兼性四倍体种群的生态位显著向低温转移，二倍体中无融合生殖的频率很低，但在四倍体中无融合生殖占主导地位，以及繁殖方式的温度依赖性。不同细胞型之间的遗传差异很小，表明参与了表观遗传控制机制。在本项目中，我们想要在气候生长箱中实验检验以下假设：1)冷诱导二倍体和同源四倍体植物在逆温处理下无融合生殖种子形成的稳定性(通过流式细胞仪种子筛选进行定量测量)，2)二倍体中的无融合生殖作为通过雌性三倍体桥梁多倍化的第一步，而雄性未减少配子形成不那么重要(流式细胞仪种子筛选和花粉倍性)；4)四倍体在低温处理下通过减少细胞数量比二倍体更能减少它们的生长，并且更耐寒；5)DNA甲基化是依赖温度的变化，并且在倍性水平、无融合生殖/植物和有性征之间存在差异；在这项任务中，我们将比较试验性相互处理中甲基化敏感的AFLP模式与野生模式，并与繁殖/倍性/生长模式相关。该项目将有助于将繁殖方式和多倍体对无融合生殖进化的影响联系在一起。无融合生殖的出现被认为是冷处理在二倍体水平上发生的结果，而多倍化将在较冷的气候条件下建立各自的种群。对无融合生殖种子形成的内源选择和对冷适应多倍体基因型的外源选择相结合，导致了无融合生殖多倍体在阿尔卑斯山较高海拔地区的优势。

The origin of apomixis in natural populations is still not well understood. Apomixis is often associated to to occurrence in colder climates. Here we want to test hypotheses that low temperature has a direct effect on origin of apomixis by triggering unreduced egg cell formation in diploids. Unreduced female gamete formation can lead to polyploidization via a female triploid bridge, and coupling to parthenogenesis will result in fully functional apomictic seed formation. Polyploidy, on the other hand, is probably associated to adaptive features to colder climates like alpine dwarf growth. We suppose that DNA methylation profiles play a major role for both spontaneous shifts in mode of reproduction of the same genotype, and for rapid adaptations to environmental conditions. In pre-projects, we studied the mode of reproduction and ecological niche differentiation of the alpine diploid-autotetraploid system Ranunculus kuepferi in natural populations of the Alps. We observed a significant niche shift towards low temperatures in facultative tetraploid populations, low frequencies of apomixis in diploids but predominance of apomixis in tetraploids, and a temperature dependence of mode of reproduction. Low genetic divergence between cytotypes suggested involvement of epigenetic control mechanisms. In the present project we want to test experimentally in climate growth chambers the following hypotheses: 1) stability of cold-induced apomictic seed formation in diploid and autotetraploid plants in reciprocal temperature treatments (measured quantitatively via flow cytometric seed screening), 2) apomixis in diploids as the first step towards polyploidization via a female triploid bridge, while male unreduced gamete formation is less important (flow cytometric seed screening and pollen ploidy); 4) tetraploids can reduce their growth under cold treatments better than diploids by reducing cell numbers, and are more frost-tolerant; 5) DNA methylation variation is temperature-dependent and differs between ploidy levels, apomictic/sexual plants and growth syndromes; for this task we will compare patterns of methylation-sensitive AFLPs in experimental reciprocal treatments to the patterns in the wild, and correlate to mode of reproduction / ploidy / growth. The project will help to entangle effects of mode of reproduction and of polyploidy for the evolution of apomicts. The emergence of apomixis is thought to happen on diploid level as a consequence of cold treatments, while polyploidization would have the effect of establishing the respective populations in colder climates. A combination of endogenous selection for apomictic seed formation and exogenous selection for cold-adapted polyploid genotypes resulted in the predominance of apomictic polyploids in higher altitudes of the Alps.