Inside daisies' evolutionary tangle: new insights into the hybridogenic genus Anacyclus (Asteraceae, Anthemideae).

BACKGROUND: The genus Anacyclus, distributed across the western Mediterranean Basin, has gained attention as a model system for studying reproductive biology and evolutionary genomics. The genus displays a remarkable diversity in capitulum morphologies, ranging from rayed to rayless forms, while also exhibiting substantial variation in genome size within a context of extensive homoploid hybridisation. However, previous studies are based on fragmented sampling, with the northern Iberian Peninsula underrepresented, and often rely on single individuals per species for genome size estimates. METHODS: To generate a more comprehensive understanding of evolutionary patterns in Anacyclus, nuclear ITS and plastid rpL32-trnL and ycf1-rps15 sequences were analysed from 80 individuals representing 47 populations across the distribution range of the genus. Phylogenetic relationships were also reconstructed using complete plastomes and 35S rDNA sequences. Nuclear DNA content was estimated by flow cytometry in 258 individuals from 45 populations. Capitulum morphology was characterised in 75 individuals of A. clavatus, A. valentinus, and mixed populations. Sampling encompassed all eight recognised species, with particular emphasis on populations from northern Spain. RESULTS: Re-examination of Anacyclus specimens previously identified as A. homogamos clarified the debated presence of this species in Catalonia (northeastern Spain), showing that these individuals belong to A. valentinus. Nuclear genetic clusters largely correspond to species or species' groups, although they fail to resolve intraspecific structure and allow only limited detection of hybrids. Genome size analyses confirmed substantial variation of 2C-values across the genus and identified two groups within A. clavatus that parallel genetic groups previously identified based on microsatellite data, although a direct correspondence cannot yet be confirmed. Phylogenetic reconstructions based on plastome and nuclear data were strongly discordant, likely reflecting extensive interspecific hybridisation. CONCLUSIONS: Our findings underscore the complexity of Anacyclus evolution and emphasise the importance of integrated, population-level studies that combine genomic and phenotypic approaches to better elucidate evolutionary processes in hybridogenic plant lineages.