Hybridization and cryptic speciation in the Iberian endemic plant genus Phalacrocarpum (Asteraceae-Anthemideae)

Understanding the role and impact of reticulation in phylogenetic inquiry has improved with extended use of high throughput sequencing data. Yet, due to the dynamism of genomes over evolutionary time, disentangling old hybridization events remains a serious challenge. Phalacrocarpum (DC.) Willk. is one of the 27 Iberian endemic plant genera, currently considered monotypic but including three subspecies. Its uncertain phylogenetic relationships within tribe Anthemideae (Asteraceae) point to an Early Miocene divergence from its sister group, and its persistent taxonomic instability has been proposed to be due to hybridization. We aim at understanding the evolutionary history of this genus using SNPs called from a genotyping-by-sequencing (GBS) analysis, Sanger sequences-from three plastid DNA regions (psbJ-petA, petB-petD, trnH-psbA) and the nuclear ribosomal ITS regions (cloned)-as well as leaf morphometric multivariate analysis. SNP data and Sanger sequences strongly support the unforeseen existence of a cryptic species in the eastern populations of P. oppositifolium subsp. anomalum. Broad molecular and morphometric patterns of variation found in conflictive populations from the Sanabria Valley region convincingly identify a recent previously undocumented hybrid zone. By contrast, evidence is less conclusive on relationships between subspecies hoffmannseggii, oppositifolium and a second conflictive group distributed along the Galician-Portuguese border (Orense massifs). Although genetic clustering analysis of SNP data suggests that the former subspecies was the maternal progenitor in hybridization events that gave rise to the other two groups, we found considerable uniqueness of ITS ribotypes and plastid haplotypes in them. This result, in the context of Pleistocene climatically-driven range shifts in NW Iberian Peninsula, can be due to periods of isolation, genetic bottlenecks and drift superimposed on old hybridization events. Our study confirms the idea that unravelling old hybridization events may be compromised by the suite of evolutionary processes accumulated subsequently, particularly in areas with a history of climatic instability.