Pilar Catalán

Understanding how fungal symbionts diversify across host lineages requires the characterization of associations involving phylogenetically informative hosts. Festuca triflora is an early-diverging broad-leaved Loliinae grass, yet its associated Epichloë endophytes have remained unexplored. Here, we characterize the F. triflora–Epichloë symbiosis using an integrative approach combining endophyte isolation, multilocus phylogenetics, genome size estimation, morphological characterization, growth analyses, and PCR screening of alkaloid biosynthetic genes.Epichloë endophytes were detected in 58.3% of the sampled plants. All isolates were consistently assigned to a cohesive lineage within E. festucae across all analyses performed, indicating that they represent a cohesive, non-hybrid haploid. In contrast, they exhibited a distinctive cultural phenotype with compact colonies and markedly reduced growth rates relative to other E. festucae lineages under comparable conditions.These findings reveal a previously undocumented E. festucae lineage associated with F. triflora, broadening the current knowledge of Festuca–Epichloë diversity. Given the phylogenetically informative position of its host, this newly described symbiosis provides a promising framework for investigating host specificity, lineage diversification, and the evolutionary processes shaping grass–endophyte associations.

SUMMARY: Natural allopolyploids with multiple origins are powerful systems for analyzing genome evolution; however, population-level whole-genome studies of wild-type recurrent polyploids remain scarce. We investigated the origins and evolutionary dynamics of the allotetraploid grass Brachypodium hybridum and its diploid progenitors ( B. distachyon , B. stacei ) by combining whole-genome sequencing of 307 accessions from across the circum-Mediterranean region with phylogenomics, population genomics, and comparative subgenomic analyses. Nuclear and plastome phylogenies reveal three independent allopolyploidization events: an ancient Iberian origin (∼1.78 Ma) and two more recent origins in the western (∼0.56 Ma) and eastern (∼0.24 Ma) Mediterranean. Each subgenome (D and S) evolved independently with minimal recombination. All B. hybridum lineages carry higher deleterious loads than their diploid progenitors, and the Ancient lineage carries a disproportionately heavy burden, particularly in the S subgenome. Population structure identifies three genetic groups; while the Ancient lineage remained isolated, recent western and eastern lineages exchanged migrants in the eastern Mediterranean contact zone. Brachypodium hybridum exemplifies how recurrent allopolyploidization, minimal subgenomic recombination, and environmental filtering generate and maintain genetic diversity, establishing it as a model for polyploid evolution and ecological adaptation in grasses worldwide.

SUMMARY: Epichloë fungal endophytes form widespread symbioses with temperate grasses, yet the extent to which diversity within endophyte species is shaped by host association remains poorly understood. Here, we characterized naturally occurring Festuca–Epichloë symbioses across diverse Iberian ecosystems using an integrative framework combining ecological, cytogenetic, phenotypic, molecular and chemical analyses. Novel associations of Epichloë festucae with Festuca trichophylla , F. lambinonii and F. yvesii were documented, together with substantial variation in infection incidence and mating-type composition among host-associated populations. Morphological traits, vegetative growth and alkaloid profiles differentiated strains according to host identity. Furthermore, multilocus phylogenetic analyses assigned all fine-leaved Festuca host isolates to Epichloë festucae , but identified a recurrent host-associated genetic structure, along with a deeper evolutionary signal, that largely corresponds to the host phylogeny. By contrast, genome size estimates varied little among Epichloë festucae strains, with all isolates exhibiting haploid genomes. Alkaloid content across the four major classes of Epichloë compounds (pyrrolopyrazines, 1-aminopyrrolizidines, ergot alkaloids and indole-diterpenes) showed only partial concordance with the presence of biosynthetic genes, indicating that functional outcomes are influenced by regulatory and environmental factors beyond biosynthetic gene presence. Chemotypic profiles clearly differentiated Epichloë festucae from E. coenophiala while demonstrating considerable functional diversity among E. festucae strains. Collectively, these complementary datasets reveal two interconnected signatures of diversification: pervasive host-associated differentiation across multiple biological dimensions and a deeper historical signal retained in phylogenetic relationships. These findings provide a foundation for future genomic, evolutionary and systematic studies to determine whether these lineages represent ongoing fungal divergence and speciation. Significance statement: Understanding how diversity is generated and maintained within plant-associated symbionts remains a fundamental challenge in biology. By integrating ecological, phylogenetic, phenotypic and chemical evidence across natural Festuca–Epichloë symbioses, including newly documented host associations, we reveal two complementary signatures of host-associated diversification within a widespread fungal endophyte. Beyond expanding the known diversity of these symbioses, our findings highlight natural Festuca – Epichloë associations as a valuable framework for exploring diversification in plant–microbe interactions.

Brachypodium is a powerful model system for investigating grass genome evolution, yet genomic resources remain concentrated in the three annual species, whereas perennial species are less sampled. Here, we present chromosome-level assemblies for two perennial species, Brachypodium mexicanum and B. arbuscula , which represent the earliest-diverging lineages of the genus and the earliest-diverging lineage of the core perennial clade, respectively. Synteny-based phylogenomics indicate that B. mexicanum is a meso-allotetraploid composed of two closely related but temporally distinct x=10 subgenomes, here designed as P and U, each carrying subgenome-specific chromosome rearrangements. We further show that the unusually large B. mexicanum genome, in contrast to the reduced genomes of most other Brachypodium species, is primarily due to transposable elements distributed across all chromosomal regions. By contrast, the diploid genome of the earliest-diverging core perennial, B. arbuscula , contains few transposable elements, whereas the most recent diverged diploid perennial B. sylvaticum shows evidence of a secondary TEs proliferation. Comparisons of lineage-specific and functionally enriched orthogroups among B. mexicanum , core perennial species and annual species suggest that ancestral hybridization between annual and perennial lineages may have contributed to the origin of allotetraploid B. mexicanum . These assemblies provide a framework for testing how polyploidy, descending dysploidy, transposable-element turnover, and life-history evolution jointly shaped genome architecture in Brachypodium .

Brachypodium sylvaticum is a perennial woodland grass selected as a model species for perenniality, which is widely distributed across the Palearctic. This plant forms a symbiosis with the endophytic fungus Epichloë sylvatica. Despite its widespread distribution and ecological importance, the evolutionary history of the B. sylvaticum complex and the role of its fungal symbiont remain poorly understood, and no integrative phylogeographic study of the grass-endophyte holobionts has been conducted to date. We hypothesize that niche dynamics and host-fungal interactions shaped the diversification and current distribution of the complex. We integrate whole-genome phylogenomics, plastome analysis, environmental niche modeling (ENM), and coevolutionary analyses to investigate the diversification of B. sylvaticum and its fungal symbiont. Using 94 representative individuals spanning Eurasia and North Africa, we recovered two deeply divergent sister lineages (Eastern and Western Palearctic), with cytonuclear discordances suggesting historical plastid capture events in the western group. Admixture analysis revealed four genetic clusters, including signatures of secondary contact and hybridization in the Western lineage. Filtered ITS sequences of E. sylvatica recovered from holobiont genome skimming reads enabled phylogenetic reconstruction, revealing two fungal clades that broadly mirror their host's evolutionary history in the West. Parafit and Procrustes Application to Cophylogenetic (PACO) analyses supported partial co-divergence between hosts and endophytes. ENM projections identified climatically stable glacial refugia for both B. sylvaticum main lineages during the Last Glacial Maximum and asymmetric postglacial expansion, with moderate niche shifts in the West and stronger turnover in the East. Evidence of niche overlap and similarity indicated niche conservatism among clades, suggesting that geographic isolation, rather than adaptive divergence, was the primary driver of lineage splitting. IBD and IBE patterns significantly influenced divergences in the Western, but not the Eastern, group, highlighting contrasting demographic and ecological dynamics. Our results provide the first evidence of coevolutionary and ecological structuring in B. sylvaticum–E. sylvatica holobionts across their Western native range, highlighting how this ubiquitous host–endophyte association may have contributed to the ecological success, persistence, and expansion of the complex under Quaternary climatic fluctuations.

The grass subtribe Loliinae has great ecological and economic importance, as it includes community-dominant species of mountain grasslands and the most extensively cultivated pasture, fodder, and turf grasses (fescues, ryegrasses). Resolving the phylogeny of recently evolved Loliinae lineages has proven challenging due to frequent introgressions and polyploidizations that occurred throughout their history. Here, we present the first target capture phylogeny of Loliinae using 270 orthologous single-copy nuclear coding loci for a large sample of 132 representative taxa, covering all its 29 evolutionary lineages. Additionally, we assembled plastome sequences to complement the inferred hybrid speciation history of the Loliinae. Concatenated maximum likelihood and multispecies coalescent trees of ortho-homeolog single-copy genes showed well-supported relationships for major lineages, which were generally consistent across analyses and genomes, and with previous taxonomic and phylogenetic findings. However, they also revealed high levels of both nuclear and cytonuclear discordances estimated to be caused by hybridization and incomplete lineage sorting. We complemented this with a phylogenetic network analysis with representative samples from the main clades to infer reticulation events in the evolution of these grasses. Furthermore, we performed gene tree–species tree reconciliation methods using gene duplication and loss models and multi-labeled trees for polyploidy analysis to estimate the proportion of duplicated genes and the nature of polyploidy of the major Loliinae lineages. These analyses agreed that the fine-leaved (FL) Loliinae clade could have evolved from hybridization between more ancestral broad-leaved (BL) Loliinae lineages and that both groups underwent ancestral and recent hybridizations. The time-calibrated phylogeny of for the main Loliinae clades supports an early Miocene origin for Loliinae and Mid-Late Miocene splits for its main BL and FL lineages, whereas current species-rich groups radiated in the Late Miocene. Hybridization tests of nuclear data and topological incongruence assays between the nuclear single-copy genes and plastome-based trees using various approaches and different sampling subsets confirmed the rampant hybridization experienced by Loliinae at deep and shallow nodes. However, hybridization rates differed from lineage to lineage within the major clades and were not correlated with time or ploidy level, but rather depended on their different propensities to hybridize with species within and/or outside their own clade. Our analyses detected high hybridization rates in four BL (Subulatae-Hawaiian, Tropical-South African, Mexico-Central-South American [MCSA I–II], and Leucopoa p.p.) and five FL Loliinae lineages (American II, Aulaxyper, Afroalpine, American-Neozeylandic, and Australia-Tasmania) containing rogue species that probably originated from trans-clade crosses and are more likely to hybridize greatly. In contrast, they recovered low hybridization rates in four BL (Schedonorus-Lolium, Subbulbosae, Drymanthele-Pseudoscariosae-Lojaconoa, and Leucopoa p.p.) and six fine-leaved lineages (Festuca, Psilurus-Vulpia, American I, Exaratae-Loretia, American-Vulpia-Pampas, and Eskia), with species derived from single ancestors that hybridize only with close congeners. Inferences from gene duplications and allopolyploidizations, along with inheritance probabilities from the phylogenetic network, point to the BL Loliinae lineages as ancient hybrids or paleo-allopolyploids, whereas the FL lineages, especially those of the core FL clade, correspond to more recent meso- or neo-allopolyploids.

Comparative genomics and plastome phylogenomics have advanced significantly in recent years, highlighting the diversity, possible admixture, and non-neutra

Polyploidization plays a fundamental role in plant evolution and crop domestication. However, due to the high similarity of genomic sequences between some homologous or homeologous chromosomes, the assembly of some polyploid genomes is extremely difficult, frequently resulting in erroneous assemblies, such as sequence chimeras and sequence collapse. The genus Brachypodium is an important model system for the study of polyploidy in grasses. However, high-quality reference genomes are still lacking for its complex polyploid perennial species. In this study, we developed a bioinformatic pipeline for the accurate assembly of high-quality reference genomes at the chromosomal level for two representative perennial Brachypodium species with conflicting collapsed segments, the allotetraploid B. phoenicoides (2n = 4x = 28) and the autohexaploid B. boissieri (2n = 6x = 48). We developed an innovative methodology (CollapsedChrom) that uses depth-of-read profiling and relies on prior karyotypic information to systematically detect and rescue collapsed regions. This depth-sensitive curation strategy successfully recovered 328.9 Mb and 195.8 Mb of previously collapsed sequences in the genomes of B. phoenicoides and B. boissieri, respectively. Comprehensive quality assessments demonstrated the high quality of our final assemblies. Our chromosomal-level assemblies fully capture the genomic architectures of these species. These robust genomic resources overcome long-standing challenges in polyploid assembly and provide an essential foundation for future research on the evolutionary dynamics, subgenomic interactions, and functional biology of complex polyploid plant genomes.

Pooideae represents a diverse subfamily of grasses with over 4000 species classified into 12 tribes and 216 genera. Over 67 million years of evolution shaped their dynamic genomes, resulting in their enormous phenotypic and ecological diversity. During this time, many members of the Pooideae have further co-evolved with fungal endophytes of Epichloë (family Clavicipitaceae). Epichloë display long-term associations with their temperate grass hosts and are naturally restricted within the Pooideae to a host genus or closely related grass genera within a tribe. Epichloë grow systemically within the above ground organs of their grass hosts and form a continuum of interactions from vertically transmitted mutualists (asexual species) to horizontally transmitted antagonists (sexual species). While sexual Epichloë species are haploid, most asexual species are alloploids, interspecific hybrids having ancestries involving two or more distinct Epichloë lineages. Comparative genomic and phylogenomic analyses of Pooideae and Epichloë species make these symbioses an excellent model for tracing coevolution through diversification within a hologenomic framework. We hypothesized that the predominant vertical transmission of these endophytes would favour a Pooideae-Epichloë coevolutionary scenario, although horizontal transmission, both sexual and asexual, of endophytes can alter this. We reconstructed the phylogeny of Pooideae host species using single-copy nuclear genes and that of Epichloë species using nuclear barcoding markers and available sequenced genomes. We also filtered and assembled Epichloë genes from host genome skimming data. Phylogenetic reconstructions were performed using maximum likelihood and coalescence-based approaches. Potential coevolutionary trends of Pooideae grasses and their Epichloë fungal endophytes were analysed statistically, based on host-parasite associations, and through concordance scores of host-endophyte topological conflicts. We performed coevolutionary analyses for the main tribal and subtribal representatives of Pooideae and described Epichloë species, and then focused on the more recently evolved Loliinae grasses (e.g., Festuca and Lolium) and their Epichloë symbionts. We found diffuse and lineage-specific coevolutionary trends for some groups of Pooideae and their endophytes, also explored through co-genomic approaches. The more detailed analysis of ~200 Loliinae species confirmed that at least 15% of them associate with Epichloë endophytes. Furthermore, we detected 20 lineage-specific relationships for temperate and tropical mountain fescues and their endophytes. Our analyses revealed significant coevolution for Loliinae and their symbionts at individual scale for two lineages, the fine-leaved F. sect. ovina clade (sheep fescues) with E. festucae strains, and the broad-leaved Schedonorus-Lolium clade (meadow and tall fescues, ryegrasses) with E. typhina, E. hybrida, E. occultans, E. siegelii, and E. uncinata strains. Interestingly, these two groups of Loliinae showed high intraclade hybridization rates, but low interclade rates. Our results highlight the impact of the narrow vs. broad hybridogenous nature of the host plant in the strong vs. weak lineage-specific coevolutionary trends with their respective Epichloë endophytes.