Unveiling an asymmetric plant–fungal symbiosis: morphological, cytogenetic, and molecular characterization of a haploid <em>Epichloë festucae</em> strain associated with three polyploid cytotypes of the Iberian endemic grass <em>Festuca rothmaleri</em>

The ecological and evolutionary outcomes of plant–fungal interactions are strongly influenced by genome size and ploidy, yet the ploidy level of both partners is rarely assessed simultaneously. Epichloë symbioses with Pooideae grasses are established model systems for exploring these dynamics, but associations between polyploid hosts and haploid endophytes remain poorly documented. In this study, the association of the Iberian endemic Festuca rothmaleri—which includes tetraploid, hexaploid, and octoploid cytotypes—with Epichloë fungal endophytes is documented for the first time. An integrative, method-rich framework combining cytogenetics, morphometrics, and multilocus phylogenetics revealed a strikingly asymmetric interaction, with all cytotypes harboring a single haploid strain of Epichloë festucae. Two methodological innovations were developed: (i) an image-based tool for automated measurement of asexual structures, including the novel metric “conidial area,” and (ii) a flow cytometr..., Sampling and characterization of host plants Sampling and taxonomic identification. Individuals of Festuca rothmaleri representing three putative ploidy levels (tetraploid, hexaploid, and octoploid) were collected from three mountainous sites in northwestern Spain, Montemayor del Río (Mon), Candelario (Can), and El Cabaco (Cab) (Salamanca province), located 20–50 km apart and separated by mountain ranges (Fig. S1). Between 20 and 35 individuals were sampled per site during two consecutive flowering seasons (June 2022 and June 2023). To reduce the likelihood of clonal sampling due to the rhizomatous growth habit of the host species, individuals were collected at a minimum distance of 3 meters from one another. Plants were transplanted into pots with universal substrate (Blumenerde, Gramoflor) and maintained in a greenhouse under relatively constant temperature conditions (22–27 °C) and watering regimes (adjusted according to seasonal needs, on average three times per week) throughout the..., # Unveiling an asymmetric plant–fungal symbiosis: morphological, cytogenetic, and molecular characterization of a haploid Epichloë festucae strain associated with three polyploid cytotypes of the Iberian endemic grass Festuca rothmaleri ### FrothmaleriEpichloe.zip The project is organized under the main directory FrothEpi/, structured into two major modules: Plant_host/ and Fungal_endophyte/. Each module contains data, scripts, and outputs corresponding to specific stages of the analysis. Folder system as follows: ``` FrothEpi/ │ ├── Plant_host/ │ └── data/ │ ├── chromcount_host.xlsx # Chromosome count data for the three cytotypes of Festuca rothmaleri. │ │ Variables: EndophyteID (sample identification code); │ │ IndshortID (short name for each isolate); │ │ repID (replicate code within each isolate); │ │ ...,植物-真菌互作的生态与进化结果强烈受基因组大小与倍性水平影响，但目前极少有研究同时评估宿主与真菌双方的倍性状态。香柱菌属（Epichloë）与早熟禾亚科（Pooideae）草本的共生体系，是探索此类互作动态的经典模型系统，但多倍体宿主与单倍体内生真菌之间的共生关联仍鲜有文献记载。本研究首次对伊比利亚特有种罗特马利羊茅（Festuca rothmaleri）——该物种包含四倍体、六倍体及八倍体细胞型——与香柱菌属真菌内生菌的共生关系进行了系统记录。本研究采用整合式多方法分析框架，结合细胞遗传学、形态测量学与多位点系统发育学手段，揭示了一类显著不对称的互作模式：所有宿主细胞型均仅携带单一单倍体的羊茅草香柱菌（Epichloë festucae）菌株。本研究开发了两项方法学创新：其一，搭建了基于图像的无性结构自动化测量工具，并提出全新形态指标“分生孢子面积（conidial area）”；其二，开发了流式细胞术（flow cytometry）相关技术（原文此处存在截断）。 ### 宿主植物的采样与表征 #### 采样与分类学鉴定 本研究从西班牙西北部萨拉曼卡省的三处山地样地——蒙特马约尔德尔里奥（Montemayor del Río，简称Mon）、坎德拉里奥（Candelario，简称Can）与埃尔巴卡科（El Cabaco，简称Cab）——采集了罗特马利羊茅的三个推定倍性水平（四倍体、六倍体、八倍体）的个体，三地间距20至50千米，且被山地分隔（附图S1）。在两个连续的开花季（2022年6月与2023年6月），每个样地采集20至35株个体。为避免因宿主物种的根状茎生长习性导致克隆采样，个体间的最小采集间距设置为3米。采集的植株被移栽至装有通用基质（Blumenerde、Gramoflor品牌）的花盆中，并在温室中维持相对恒定的温度条件（22至27摄氏度）与浇水周期（根据季节需求调整，平均每周三次）直至[原文此处截断]。 # 揭示不对称植物-真菌共生：关联伊比利亚特有草本罗特马利羊茅三个多倍体细胞型的单倍体羊茅草香柱菌菌株的形态、细胞遗传学与分子表征 ### FrothmaleriEpichloe.zip 本项目以主目录`FrothEpi/`进行组织，分为两大核心模块：`Plant_host/`（宿主植物模块）与`Fungal_endophyte/`（真菌内生菌模块）。每个模块均包含对应分析阶段的数据、脚本与输出结果。文件夹结构如下： FrothEpi/ ├─ Plant_host/ │ └─ data/ │ ├─ chromcount_host.xlsx # 罗特马利羊茅三个细胞型的染色体计数数据 │ │ 变量说明：EndophyteID（样本识别码）； │ │ IndshortID（单分离株的短名称）； │ │ repID（单分离株内的重复样本码）； │ │ ...,