Our research investigates the overall diversity of Pulmonaria hirta complex in the Italian peninsula, by applying traditional and geometric morphometrics analyses of basal leaf and flower features, as well as karyological analysis, AFLP and phylogenetic analysis.. Different chromosome numbers, but slight morphological differentiation and genetic admixture among populations of Pulmonaria hirta complex (Boraginaceae)

Hybridization and introgression have had a significant impact on the taxonomically controversial genus Pulmonaria. For example, the P. hirta complex shows puzzling systematic relationships among P. hirta s.str. (2n = [22, 26]28), P. apennina (2n = 22[26, 28]), and P. vallarsae (2n = 22), with range overlaps and mixed phenotypes in Southern Europe. We conducted morphometric analyses of basal leaves and flower features along with AFLP characterization of 236 plants belonging to 11 populations within the complex and 1 population of P. officinalis. We also implemented an already available phylogeny with sequences from our target species, and characterized the karyotype of each population. For all the populations within the complex, we found molecular evidence of a hybrid origin involving species belonging to different clades (angustifolia clade and officinalis clade). However, there is a certain morphological differentiation between some populations on one hand (“hirtoid” morph) and some populations (“vallarsoid” morph) on the other, but with individuals or entire populations having intermediate features. According to our results, hybridization and/or backcrossing/introgression have occurred, and gene flow is currently taking place among these "taxa". Following the hybridization event(s), we can elaborate three possible scenarios: 1) one hybrid species, "vallarsoid" (2n = 22), spread across Italian peninsula and originated the "hirtoid" morph (2n = 28) through dysploidy; 2) two geographically distinct hybrid events produced both "vallarsoid" and "hirtoid" morphs; 3) one "hirtoid" alloploid hybrid species originated and backcrossed with P. officinalis generating "vallarsoid" plants. Under scenarios 1 and 2, the different morphs met again in C Italy, with massive current gene flow. Under scenario 3, “vallarsoid” plants spread across Italian peninsula, but further backcrossed with "hirtoid" plants in C Italy, leaving pure lineages of "vallarsoid" plants only in the extreme north and south of their range. This scenario is supported by populations with 2n = 22, 26 having asymmetry indices intermediate between those of P. officinalis and those of the populations with 2n = 28. Irrespective of the evolutionary dynamics, today a single lineage showing three cytotypes occurs throughout the Italian peninsula, supporting the circumscription of a single polymorphic species, namely P. hirta.