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

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. <i>Epichloë</i> symbioses with <i>Pooideae</i> 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 <i>Festuca rothmaleri</i>-which includes tetraploid, hexaploid, and octoploid cytotypes-with <i>Epichloë</i> 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 <i>Epichloë festucae</i>. 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 cytometry protocol for estimating fungal genome size. Despite morphological variability, all fungal isolates shared similar genome sizes and formed a well-supported monophyletic lineage in a coalescent species tree based on nuclear loci sequences (<i>actG</i>, <i>CalM</i>, ITS, <i>tefA</i>, <i>tubB</i>). This work provides the first comprehensive characterization of a haploid <i>Epichloë</i> endophyte spanning multiple naturally distributed host ploidy levels and highlights a rare but promising system for future evolutionary, physiological, and ecological studies of plant-fungal interactions.