Functional and evolutionary significance of symbiotic fungal associations in lower land plants

450多万年前光合作用的陆地生物的起源和殖民是地球历史上影响最深远的篇章之一，并在高[二氧化碳]大气中上演。Pirozynski&Malloch(1975)在30多年前提出共生丛枝菌根真菌(AM)在植物入侵土地中起关键作用的假说。这一想法后来在古植物学和分子研究的支持下日益确立。然而，至关重要的是，尽管在细胞水平上对植物-真菌界面有了极其重要的新见解，但尚未对‘低等’陆地植物中植物-真菌相互作用的功能性质进行调查；其假定状态是通过与高等植物的类比得出的。然而，最近的关键证据证实，许多低等植物具有AM真菌联系，包括与非绿叶配子体的物种被认为依赖于它们的真菌伙伴来获得C。此外，AM真菌是依赖于自养植物的碳的专性共生体。因此，我们重塑了Pirozynski&Malloch(1975)的论点，提出了一个新的假设，即“较低”植物对陆地的成功入侵，以及它们此后在陆地栖息地的持续存在，需要AM类型的共生来提供双重好处。首先，通过改善光合作用优势世代的矿质营养。第二，通过共用菌丝网络(CMN)连接世代(亲本培养)或种间(表生寄生)，通过AM真菌提供的C对配子体世代进行培养。我们选择了四种“较低”的陆地植物，它们具有良好的AM真菌组合，代表了植物进化树中的关键节点(地钱、丛生苔藓、“较低”和“较高”的蕨类)。这些系统提供了一系列强大的模型系统，可以对它们整个生命周期中的C交换和营养关系进行实验和定量的功能分析，并包括从配子体到孢子体优势的转换，这是古生代植物进化的一个主要轴线。我们的主要实验研究计划旨在严格评估我们扩展的AM真菌在植物“绿化土地”中的作用的研究假说。我们将量化当代(约400ppm)和古生代(1500ppm)CMN[CO2]在供应养分和C方面的作用，以促进配子体和孢子体世代的萌发和建立。实验将基于包含天然植物群落、真菌群落和土壤群落的整个草坪，通过干预操作世代之间和物种之间的CMN。这些菌丝网络的功能将使用复杂的稳定(15N)和放射性同位素(14C，33P)示踪方法进行量化，这些示踪方法与插入草皮中的土壤填充的网状核心相结合，将在其中生长配子体萌发。通过允许一些核心由周围草坪上的菌丝体定植，同时定期旋转其他核心以切断生长中的菌丝体，可以控制草坪物种和实验配子体之间CMN的形成。这使得通过跨世代连接的CMN(亲本培养)或物种之间(表生寄生)的C和营养通量得以量化。将通过对关键真菌配对的序列分析进行基于DNA的分子鉴定，以确定世代之间和物种之间的真菌保真度。我们的建议是一个令人振奋的发展，在早期分析了‘较低’植物中AM真菌协会的结构，并通过共同的菌丝体网络在亲本培养和外植性的背景下重新构建了辩论。它将有助于对地球上最古老的共生生物之一的共同进化的基本知识和理解，这一主题与NERC的地球系统科学战略密切相关。

The origin and colonization of the land by photosynthetic terrestrial organisms over 450Myr ago was one of the most far-reaching chapters in Earth history, and played out in a high [CO2] atmosphere. Pirozynski & Malloch's (1975) hypothesized over thirty years ago that symbiotic arbuscular mycorrhizal (AM) fungi played a pivotal role in plants' 'invasion of the land'. This idea has subsequently become increasingly established with support from both palaeobotanical and molecular investigations. Critically, however, in spite of extremely important new insights into the plant-fungal interfaces at the cellular scale, the functional nature of plant-fungus interactions in 'lower' land plants has not yet been investigated; its presumed status is drawn by analogy with higher plants. However, recent critical evidence confirms that many lower plants have AM fungal associations, including species with achlorophyllous gametophytes presumed to depend on their fungal partners for C. Furthermore, AM fungi are obligate symbionts dependant upon autotrophic plants for carbon. We have therefore recast Pirozynski & Malloch's (1975) argument by proposing the novel hypothesis that the successful invasion of the land by 'lower' plants, and their persistence in terrestrial habitats ever since, required an AM-type symbiosis to provide a double benefit. First, through improved mineral nutrition of the dominant photosynthetic generation. Second, through nurture of the gametophyte generation by C supplied through AM fungi via a common mycelial network (CMN) linking across generations (parental nurture) or between species (epiparasitism). We have selected four 'lower' land plant species with well-documented AM fungal associations that represent key nodes across the plant evolutionary tree (liverworts, clubmosses, 'lower' and 'higher' ferns). These provide a powerful spectrum of model systems amenable to experimentation and quantitative functional analyses of C exchange and nutrient relationships across their entire life-cycles, and encompass the switch from gametophyte to sporophyte dominance, a major plant evolutionary axis during the Palaeozoic. Our major experimental research programme is designed to rigorously evaluate our extended research hypothesis for the role of AM fungi in allowing plants to 'green the land'. We will quantify the effects of CMNs in contemporary (ca. 400 ppm) and Palaeozoic (1500 ppm) [CO2] in supplying nutrients and C to enable germination and establishment of gametophyte and sporophyte generations. Experiments will be based on whole turfs containing natural plant, fungal and soil communities with the CMNs linking between generations and between species being manipulated by intervention. The functioning of these mycelial networks will be quantified using sophisticated stable (15N) and radioisotope (14C, 33P) tracer methods coupled to soil-filled mesh-cores inserted into the turfs and in which gametophyte gerations will be grown. By allowing some cores to be colonised by mycelia from the surrounding turf, while regularly rotating others to sever in-growing mycelia, the formation of CMNs between turf-species and experimental gametophytes can be controlled. This allows C and nutrient fluxes through CMNs linking across generations (parental nurture) or between species (epiparasitism) to be quantified. DNA-based molecular identification by sequence analysis of the critical fungal partners will be undertaken to determine fungal fidelity between generations and between species. Our proposal is an exciting development following earlier analyses of the structure of AM fungal associations in 'lower' plants, and reframes the debate in the context of parental nurture and epiparisitism through common mycelial networks. It will contribute fundamental knowledge and understanding on the co-evolution of one of the most ancient symbioses on Earth, a topic closely aligned with NERC's Earth system science strategy.