Data from: Why do mixotrophic plants stay green? A comparison between green and achlorophyllous orchid individuals in situ

Some forest plants adapt to shade by mixotrophy, i.e., they obtain carbon both from photosynthesis and from their root mycorrhizal fungi. Fully achlorophyllous species using exclusively fungal carbon (the so-called mycoheterotrophic plants) have repeatedly evolved from such mixotrophic ancestors. However, adaptations for this evolutionary transition, and the reasons why it has happened a limited number of times, remain unknown. We investigated this using achlorophyllous variants (i.e., albinos) spontaneously occurring in Cephalanthera damasonium, a mixotrophic orchid. In two populations, we compared albinos with co-occurring green individuals in situ. We investigated vegetative traits, namely, shoot phenology, dormancy, CO2 and H2O leaf exchange, mycorrhizal colonization, degree of mycoheterotrophy (using 13C abundance as a proxy), and susceptibility to pathogens and herbivores. We monitored seed production (in natural or experimental crosses) and seed germination. Albinos displayed (1) more frequent shoot drying at fruiting, possibly due to stomatal dysfunctions, (2) lower basal metabolism, (3) increased sensitivity to pathogens and herbivores, (4) higher dormancy and maladapted sprouting, and, probably due to the previous differences, (5) fewer seeds, with lower germination capacity. Over the growing season, green shoots shifted from using fungal carbon to an increasingly efficient photosynthesis at time of fruiting, when fungal colonization reached its minimum. Conversely, the lack of photosynthesis in fruiting albinos may contribute to carbon limitation, and to the above-mentioned trends. With a 103× fitness reduction, albinos failed a successful transition to mycoheterotrophy because some traits inherited from their green ancestors are maladaptive. Conversely, mycoheterotrophy requires at least degeneration of leaves and stomata, optimization of the temporal pattern of fungal colonization and shoot sprouting, and new defenses against pathogens and herbivores. Transition to mycoheterotrophy likely requires progressive, joint evolution of these traits, while a sudden loss of photosynthesis leads to unfit plants. We provide explanations for the evolutionary stability of mixotrophic nutrition and for the rarity of emergence of carbon sinks in mycorrhizal networks. More broadly, this may explain what prevents the emergence of fully heterotrophic taxa in the numerous other mixotrophic plant or algal lineages recently described.部分森林植物通过混合营养（mixotrophy）适应荫蔽环境，即同时通过光合作用与根部菌根真菌获取碳源。完全无叶绿素、仅专一利用真菌碳源的物种（即所谓菌异养植物（mycoheterotrophic plants））已多次从这类混合营养祖先类群中演化而来。然而，这一演化过渡所需的适应性特征，以及该过渡仅少量发生的原因，仍未明确。我们以混合营养型兰科植物长叶头蕊兰（Cephalanthera damasonium）中自然产生的无叶绿素变异株（即白化株）为研究材料，对该演化过渡展开探究。我们在两个种群中，对原位生长的白化株与伴生的绿色个体开展了对照研究：测定了包括枝条物候、休眠状态、叶片CO₂与H₂O气体交换、菌根定殖程度、以¹³C丰度作为替代指标的菌异养水平，以及对病原菌和植食者的易感性在内的多项营养体性状；同时监测了自然授粉或人工杂交后的种子产量与种子萌发率。研究结果显示，白化株呈现出如下特征：(1) 结果期枝条干枯现象更为频发，这可能由气孔功能异常所致；(2) 基础代谢水平更低；(3) 对病原菌与植食者的敏感性升高；(4) 休眠程度更高且萌芽适应性不良；而上述差异可能进一步导致(5) 种子数量更少、萌发能力更弱。在整个生长季中，绿色枝条的碳源获取模式从依赖真菌碳源逐渐转向高效光合作用，至结果期时菌根定殖程度达到最低。与之相反，结果期的白化株因缺乏光合作用，可能出现碳限制，进而引发上述一系列性状差异。白化株的适合度较绿色个体下降了10³倍，未能成功完成向菌异养营养方式的过渡，这是因为其继承自绿色祖先的部分性状并不适配新的营养模式。反之，演化至完全菌异养营养方式，至少需要实现叶片与气孔的退化、菌根定殖与枝条萌芽的时间模式优化，以及针对病原菌和植食者的新型防御机制。向菌异养营养的过渡可能需要这些性状的渐进式协同演化，而光合作用的突然丧失只会产生适应性不良的个体。本研究为混合营养方式的进化稳定性，以及菌根网络中碳汇（carbon sinks）出现频次稀少的原因提供了解释。从更广泛的视角来看，这也可以阐明为何近期被报道的众多其他混合营养型植物或藻类类群中，并未出现完全异养类群（heterotrophic taxa）。