DOI: 10.5061/dryad.hx3ffbgsb
title: 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>

description: 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
 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 (actG, CalM, ITS, tefA, tubB). This
 work provides the first comprehensive characterization of a haploid
 Epichloë endophyte spanning multiple naturally distributed host polyploid
 levels and highlights a rare but promising system for future evolutionary,
 physiological, and ecological studies of plant–fungal interactions.
description: <strong>Sampling and characterization of host
 plants</strong> <strong>Sampling and
 taxonomic identification</strong>. Individuals of <em>Festuca
 rothmaleri</em> 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 study.
 Taxonomic identification of the host plants was performed based
 on morphoanatomical examination and measurements of vegetative and
 reproductive structures, following the diagnostic criteria and
 identification keys provided by De la Fuente and Sánchez (1987),
 Al-Bermani et al. (1992), De la Fuente et al. (2001), Loureiro et al.
 (2007), and Devesa et al. (2020).
 <strong>Cytogenetic characterization.</strong> The
 host plant genome size was determined by flow cytometry (Ploidy Analyzer,
 Sysmex) in fresh mature leaf tissue, following the protocol and reagents
 (Otto I and Otto II) described by Doležel et al. (2007). A preliminary
 analysis confirmed the existence of three genome size categories
 corresponding to different ploidy levels. Based on these results, up to 15
 individuals were selected per population of origin and ploidy level
 (Mon4x, Can4x, Cab6x, and Can8x), analyzing two technical replicates in
 each case. Measurements were made on more than 5,000 nuclei with a
 coefficient of variation (CV) &lt; 3%. Primary standards included
 <em>Solanum lycopersicum</em> ‘Stupické polní rané’ (1.96
 pg/2C) for tetraploids, <em>Pisum sativum</em> ‘Ctirad’ (9.09
 pg/2C) for octoploids, and <em>Secale cereale</em>
 ‘Daňkovské’ (16.19 pg/2C) for hexaploids according to Doležel et al.
 (2007). Chromosome number was determined from
 meristematic cells of the root tip using the protocol of Jenkins and
 Hasterok (2007). For each ploidy level, five independent counts were
 performed for each of three randomly selected individuals from the
 genome-size dataset, resulting in a total of 15 counts per detected ploidy
 level. Images were obtained at 400× magnification using a Zeiss Axio
 Lab.A1 phase-contrast microscope equipped with a Canon EOS 2000D digital
 camera. <strong>Comprehensive multi-method
 characterization of <em>Epichloë</em>
 endophytes</strong> <strong>Detection,
 isolation and incidence.</strong> The presence of the endophyte in
 the host's aerial tissues was initially detected by aniline blue
 staining (Florea et al. 2015). In parallel, fragments of floral stems or
 the bases of vegetative tillers from all collected specimens, with the
 surface disinfected, were cultured on potato dextrose agar (PDA, Potato
 Dextrose Agar EP/USP/BAM, Condalab) plates containing chloramphenicol
 (Chloramphenicol BioChemica, PanReac AmpliChem; 25 µg/ml) to inhibit
 bacterial growth. When an <em>Epichloë</em>-like endophyte
 emerged from the host tissue, it was isolated on PDA plates and cultured
 at room temperature (22–25°C) in the dark. The incidence of *Epichloë
 *endophytes in <em>Festuca</em> hosts varies by holobiont and
 locality (Zabalgogeazcoa et al. 1999; Saikkonen et al. 2000; Clement et
 al. 2001; Bazely et al. 2007; Gundel et al. 2014). The proportion of
 infected individuals was estimated by sampling site (Mon, Can, Cab), host
 ploidy level (4x, 6x, 8x), and their combination (Mon4x, Can4x, Cab6x,
 Can8x), considering both detection methodologies.
 <strong>Exploratory mating-type composition
 screening.</strong> During field collections, no stromata were
 observed in <em>F. rothmaleri</em> individuals. Therefore, the
 sexual reproduction potential of the endophyte was evaluated through
 exploratory PCR screening for the MAT1-1 (785 bp) and MAT1-2 (215 bp)
 idiomorphs (Florea et al. 2015) in 4–6 <em>F. rothmaleri</em>
 individuals per sampling site (Mon, Cab, Can). Sample sizes were adjusted
 to ensure detection of both idiomorphs at each location, using total DNA
 extracted from leaf tissue. Primer sequences and PCR conditions are
 provided in Table S2. <strong>Morphological
 analyses and growth rate.</strong> Asexual reproductive structures
 (conidia and conidiophores) have proven to be a valuable taxonomical trait
 for the identification of <em>Epichloë</em> endophytes
 (Gentile et al. 2005; Leuchtmann and Schardl 2005; McCargo et al. 2014;
 Campbell et al. 2017; Thünen et al. 2022). The dimensions of these
 structures have also been correlated with the putative ploidy level of the
 endophytes (Kuldau et al. 1999). To assess the natural variability in
 these traits and their potential link to ploidy, four
 <em>Epichloë</em> isolates from hosts with differing source
 populations and ploidy levels (i.e., Mon4x, Can4x, Cab6x, Can8x) were
 analyzed, with three biological replicates per isolate. Microscopic
 preparations consisted of 1 mm² sections of mycelium, grown for three
 weeks on water agar (WA; European Bacteriological Agar, Condalab), which
 were carefully melted and sealed under a coverslip. The length and width
 of 10 conidia, along with the total length and width at the base of 10
 conidiophores, were recorded for each replicate, yielding a total of 30
 entries per isolate. To morphometrically characterize these endophytes, a
 novel software –Epichloë conidia– was developed using Matlab version 9.3
 (R2017b; see Appendix S1) to automatically detect and estimate conidial
 area. Only spores photographed in sagittal plane were analyzed to ensure
 comparability, using both ImageJ v154g (Schneider et al. 2012) and custom
 software, with a fixed scale of 15.8097 pixels/μm. Morphometric
 differences among groups were assessed using PERMANOVA based on Euclidean
 distances with 10,000 permutations (R package ‘vegan’ v2.6.10; Oksanen et
 al. 2025), which does not assume multivariate normality and is suitable
 for nested designs. Pairwise permutation tests (R package ‘coin’ v1.4.3,
 Hothorn et al. 2008) were also performed with 10,000 resamples,
 considering the clustering structure of the study: sampling sites (n = 3),
 host ploidy levels (n = 3), individuals per source population and host
 ploidy level (n = 4), and replicates within individuals (n = 10). P-values
 were adjusted using the Holm-Bonferroni method, with significance set at
 p* &lt; 0.05. Additionally, exploratory
 multivariate analyses, including Principal Component Analysis (PCA, R
 package ‘FactoMineR’ v2.11, Lê et al. 2008) and Linear Discriminant
 Analysis (LDA, R package ‘MASS’ v7.3.60.2), were carried out to identify
 potential patterns of endophyte diversification associated with population
 origin, host ploidy level, or their interaction. These analyses were
 performed under three data clustering schemes: (i) individual-level
 mean-pooled measurements (n = 16); (ii) replicate-level mean-pooled
 measurements (n = 48); and (iii) resampling-level individual measurements
 (n = 469). This hierarchical structuring of sample sizes was designed to
 help identify the main sources of variability within the system. To
 statistically evaluate the morphological variation revealed by principal
 component analysis (PCA), linear models (LM) and analyses of variance
 (ANOVA) were developed on the scores of the first two principal components
 (PC1 and PC2), including host ploidy level, population of origin, and
 their interaction as explanatory variables, using the R package “stats”
 v4.4.1. The performance of the LDA models was assessed using
 cross-validation procedures implemented with the R package “caret” v6.0.94
 (Kuhn 2008). Since the growth rate of fungal cultures
 on PDA medium has been used as a trait to characterize
 <em>Epichloë</em> species, 1.5 mm<sup>2</sup>
 sections of four two-week-old isolates per population were simultaneously
 grown on PDA plates at room temperature in the dark for 24 days to
 estimate endophyte growth rate. Three technical replicates per isolate
 were analyzed. Culture growth was monitored every eighth day by taking
 pictures and measuring the diameter of the culture. Growth front was
 considered the external limit for this measure and a digital vernier
 caliper was used to record culture diameter. Growth rate (GR; mm/day) was
 calculated for each individual sample following the equation GR =
 (Diameter<sub>t´</sub> - Diameter<sub>t</sub>) /
 Δ<sub>t</sub>, where Diameter<sub>t</sub> is the
 initial diameter of the culture, Diameter<sub>t’</sub> is the
 final diameter and Δ~t ~is the total number of days. Mixed-effect models
 were fitted due to the dependency between the measurements registered
 (i.e., diameter measured through time) and the hierarchical structure of
 the experimental design using the R package ‘lme4’ v1.1.35.5 (Bates et al.
 2015). The effect of the source population, host ploidy level, and their
 interaction (fixed effects) on fungal growth rate (mm/day) was analyzed,
 including individuals or replicates as random effects to account for
 intra-individual variability and make more precise estimates without
 inflating standard errors. ANOVAs and subsequent pos-hoc pairwise tests
 assessing differences by marginal means (EMMs) applying Tukey’s HSD
 correction were carried out to test the significance of these models and
 detect potential sources of variability using the R package ‘emmeans’
 v1.10.5 (Lenth 2024). All these analyses were performed in R v4.4.1 (R
 Core Team 2024) using a custom script (script1.Rmd) created with R
 Markdown (rmarkdown’ v2.29; Allaire et al. 2024).
 <strong>Genome size estimation.</strong> DAPI-stained
 slides were prepared to confirm that the endophyte conidia were
 uninucleate before performing genome size estimations. Two-week-old 1 mm2
 slices of mycelium were grown on a nutrient-deficient medium, consisting
 of water agar, for 15 days at room temperature and in the dark to
 facilitate sporulation. Photographs were taken with a fluorescence
 microscope (Miotic BA410) equipped with the Miotic MoticamPro 285D
 camera. Existing protocols for other fungal species
 were adapted to the endophytes under study by flow cytometry (Ploidy
 Analyzer, Sysmex) to determine their genome sizes and infer the ploidy
 levels of the endophytes isolated from the different <em>F.
 rothmaleri</em> cytotypes. <em>Colletotrichum
 acutatum</em> strain PT812 (68 Mb or ~ 0.069 pg/1C) was included as
 primary standard following Talhinhas et al. (2017). Each
 <em>Epichloë</em> isolate and the standard were ground with a
 sterile mortar and pestle in 700 µl of ddH<sub>2</sub>O and
 grown for 7–10 days in PDA plates with a sterile cellophane disk to
 increase the availability of active young mycelia. A 0.5
 cm<sup>2</sup> portion was extracted and grounded in a glass
 petri dish with 1 ml of LB01 buffer (Doležel et al. 2007) using a sharp
 blade. The nuclear suspension was filtered through a 20 μm nylon mesh
 filter before being stained with 50 μl of propidium iodide. After a brief
 incubation on ice (3–5 minutes), samples were analyzed using the Ploidy
 Analyzer flow cytometer (Sysmex). Sample processing was carried out on ice
 (~ 4ºC) to ensure nuclear stability. Three isolates were considered to
 establish the genome sizes of endophytes from each source population and
 the ploidy level of the host (Mon4x, Can4x, Cab6x, Can8x), measuring 4
 technical replicates in each case (n=12). Here, a minimum threshold of
 5000 nuclei was set for each measurement, and coefficients of variation
 (CV) were considered acceptable when they were below 10%, following
 previous flow cytometry analysis standards in other fungi (Bourne et al.
 2014). <strong>Molecular and phylogenetic
 analyses.</strong> <em>Epichloë</em> isolates were grown
 for 7–10 days on PDA plates covered with sterile cellophane disks to
 facilitate mycelial recovery. DNA was extracted using a modified version
 of the CTAB-based DNA extraction protocol (Doyle and Doyle 1987) (see
 Appendix S2). Quality and concentration were assessed using a Biodrop
 spectrophotometer (µLite) and a Qubit 3.0 fluorometer. PCR amplified
 products from four nuclear loci [γ-actin (<em>actG</em>),
 calmodulin (<em>CalM</em>), translation elongation factor 1-α
 (<em>tefA</em>), and β-tubulin (<em>tubB</em>)],
 as well as the ITS region of rDNA, were subjected to bidirectional Sanger
 sequencing (Moon et al. 2004; McCargo et al. 2014; Chen et al. 2019;
 Thünen et al. 2022; Wang et al. 2022), using optimized primers and cycling
 programs detailed in Table S2. Two isolates per combination of source
 population and host ploidy level (Mon4x, Can4x, Cab6x, Can8x) were
 selected for molecular characterization. Sequences were
 trimmed and aligned in Geneious Prime version 2024.0.5 (Biomatters Ltd,
 New Zealand) using MAFFT algorithm v7.490 (Katoh and Standley 2013) and
 subsequently processed manually. Single nucleotide polymorphisms (SNPs)
 calling was performed with a threshold value of a minimum variant
 frequency of 25% due to the small sample size of our dataset. Reference
 sequences for the five studied loci were obtained from published
 <em>Epichloë</em> genomes and two <em>Claviceps
 purpurea</em> strains that were used as outgroups (Table S3). Only
 haploid genomes (i.e., single-copy loci) were included, verified using a
 custom bash script (script2.sh). Single-gene maximum likelihood
 phylogenetic trees were obtained using IQ-TREE 2 (Minh et al. 2020) with
 1000 UltraFast Bootstrap (BS) replicates (Hoang et al. 2017). For
 visualization, the lengths of all branches were transformed using
 x<sup>¼</sup>. Subsequently, both the concatenated maximum
 likelihood tree and a coalescence-based species tree were obtained using
 IQ-TREE 2 and ASTRAL-III (Zhang et al. 2018), respectively. The
 likelihood-based site concordance factor (sCFL) was calculated using 1000
 replicates for the concatenated ML tree (Mo et al. 2023), and quartet
 scores for the species tree and its two alternative topologies (Sayyari
 and Mirarab 2016) were inferred using ASTRAL-III. All trees were
 generated, edited, and formatted with two custom scripts created with bash
 and R Markdown (script3.sh, script4.Rmd).
description: # 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); │ │ Plo_pop (interaction between the population
 of origin and ploidy level); │ │ pictureID (picture identification code).
 │ │ │ └── flow_cytometry_host.xlsx # Genome size estimations via flow
 cytometry for F. rothmaleri cytotypes. │ Variables: Sample_ID (sample
 identification code); │ Plo_pop (interaction between the population of
 origin and ploidy level); │ measurement_ID (measurement identification
 code); │ nucleids (number of particles counted in the sample); │
 nucleids_STD (number of particles counted in the standard); │ mean
 (arithmetic mean of the fluorescence intensities of all particles included
 in the sample); │ mean_STD (arithmetic mean of the fluorescence
 intensities of all particles included in the standard); │ CV (coefficient
 of Variation of the sample); │ CV_STD (coefficient of Variation of the
 standard); │ pg/2C (estimated genome size of the sample); │ pg/2C_STD
 (known genome size of the standard); │ meanGS_Sample (mean genome size per
 isolate); │ SD_Sample (Standard deviation per isolate); │ meanGS_Plopop
 (genome size value per population of origin and host ploidy level); │
 SD_Plopop (standard deviation value per population of origin and host
 ploidy level). │ │ │ │ ├── Fungal_endophyte/ │ ├── data/ │ │ ├──
 outputs_script1/ # Output files of the morphological analyses (from
 script1.Rmd) │ │ ├── outputs_script3/ # Single-gene, species tree and
 concatenated ML files (from script3.sh) │ │ ├── outputs_script4/ #
 Formatted phylogenetic trees (from script4.Rmd) │ │ ├──
 Phylogenetic_analysis/ │ │ │ │ │ │ │ ├── MSA/ # Cleaned multiple sequence
 alignments and originals (FASTA format) │ │ │ │ │ │ │ ├──
 PP_values_ASTRAL_SPTREE.xlsx # Posterior probability values for each node
 of the ASTRAL phylogenetic tree. Input script4.Rmd. │ │ │ ├──
 Q_values_ASTRAL_SPTREE.xlsx # Quartet values for each node of the ASTRAL
 phylogenetic tree. Input script4.Rmd. │ │ │ ├── UFBoot_values_actG.xlsx #
 UltraFast bootstrap values for each node in the actG phylogenetic tree.
 Input script4.Rmd. │ │ │ ├── UFBoot_values_CalM.xlsx # UltraFast bootstrap
 values for each node in the CalM phylogenetic tree. Input script4.Rmd. │ │
 │ ├── UFBoot_values_ITS.xlsx # UltraFast bootstrap values for each node in
 the ITS phylogenetic tree. Input script4.Rmd. │ │ │ ├──
 UFBoot_values_tefA.xlsx # UltraFast bootstrap values for each node in the
 tefA phylogenetic tree. Input script4.Rmd. │ │ │ ├──
 UFBoot_values_tubB.xlsx # UltraFast bootstrap values for each node in the
 tubB phylogenetic tree. Input script4.Rmd. │ │ │ └──
 UFBoot_and_scfl_values_MLtree # UltraFast bootstrap and SCFL values for
 each node in the ML phylogeentic tree. Input script4.Rmd. │ │ │ │ │ ├──
 culture_growth_dataset.csv # Dataset for culture growth rate analysis
 (used in script1.Rmd). │ │ │ This file is semicolon-delimited and uses a
 comma as the decimal separator. │ │ │ Variables: IndID (measurement
 identification code); │ │ │ Ind (short name for each isolate); │ │ │
 Ploidy (host ploidy level); │ │ │ Pop (Population of origin); │ │ │ Rep
 (replicate code within each isolate); │ │ │ Day (Day the measurement was
 made); │ │ │ Diameter (diameter of the culture in mm) │ │ │ │ │ ├──
 spores_dataset.csv # Morphometric data of asexual reproductive structures
 (used in script1.Rmd). │ │ │ This file is semicolon-delimited and uses a
 comma as the decimal separator. │ │ │ Variables: SampleID (measurement
 identification code); │ │ │ Ind (short name for each isolate); │ │ │
 conidW (conidial width in μm); │ │ │ conidL (conidial length in μm); │ │ │
 conidiophL (conidiophore length in μm); │ │ │ conidiophW (conidiophore
 width in μm); │ │ │ conidA (conidial area in μm²); │ │ │ Pop (Population
 of origin); │ │ │ Ploidy (host ploidy level). │ │ │ │ │ └──
 flow_cytometry_endophyte.xlsx # Genome size estimations of Epichloë
 festucae via flow cytometry. │ │ Variables: Plo_pop (interaction between
 the population of origin and ploidy level); │ │ Sample_ID (sample
 identification code); │ │ Measurement_ID (measurement identification
 code); │ │ nucleids (number of particles counted in the sample); │ │
 nucleids_STD (number of particles counted in the standard); │ │ mean
 (arithmetic mean of the fluorescence intensities of all particles included
 in the sample); │ │ mean_STD (arithmetic mean of the fluorescence
 intensities of all particles included in the standard); │ │ CV
 (coefficient of Variation of the sample); │ │ CV_STD (coefficient of
 Variation of the standard); │ │ pg/1C (estimated genome size of the
 sample); │ │ pg/1C_STD (known genome size of the standard); │ │
 meanGS_Sample (mean genome size per isolate); │ │ SD_Sample (Standard
 deviation per isolate); │ │ global_GS (global genome size value); │ │
 global_SD (global standard deviation). │ │ │ └── scripts/ │ ├──
 script1.Rmd # RMarkdown: Morphological analysis of fungal endophyte. │ ├──
 script2.sh # Shell script: Detection of multiple gene copies in reference
 genomes. │ ├── script3.sh # Shell script: Phylogenetic inference using
 IQ-TREE2 and ASTRAL. │ └── script4.Rmd # RMarkdown: Visualization and
 editing of phylogenetic trees. └ ````