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); │ │ ...,