• cpDNA
• AFLP
• Pulmonaria
• dysploidy
• hybridization
• introgression
• ITS
• morphometry

Pulmonaria is a taxonomically controversal genus, in which hybridization and introgression are common and played a significant role in the evolution and diversification of species. For example, the P. hirta complex shows puzzling systematic relationships among P. hirta s.str., P. apennina, and P. vallarsae, with overlaps in distribution and mixed phenotypes in Southern Europe. Pulmonaria vallarsae is endemic to northeast Italy, while P. apennina extends along the whole Ap- ennine, and has been also treated as a subspecies of the former. To the north of its range, P. apennina overlaps with P. hirta, which is mostly distributed on the north-western side of the penin- sula, from Liguria to the upper Lazio. The highly morphological convergence and the intermediate chromosome numbers (2n = 24, 25, 26) have been observed between P. hirta and P. apennina in C Italy. The present thesis investigates the overall diversity of P. hirta complex in the Italian peninsu- la, by applying traditional and geometric morphometrics analyses of basal leaf and flower features, as well as karyological analysis, AFLP and phylogenetic analysis.
236 plants belonging to 3 populations of P. hirta, 2 populations of P. apennina, 2 populations of P. vallarsae, 4 from areas where taxonomically critical populations occured and 1 population of P. officinalis were sampled and cultivated in the Botanical Garden of Pisa. Chromosome number and karyotype were analysed for each population. Phylogenetic reconstruction of the genus Pulmonaria was performed based on both cpDNA (trnh-psbA, rpl16, and rps16) and nrDNA (ITS) markers, with sequences of 60 individuals obtained from our target species. AFLP analysis was conducted on all cultivated individuals and successfully performed on 225 individuals.
Pulmonaria vallarsae and P. apennina has shown 2n = 22 chromosomes, while chromosome num- bers 2n = 22, 26, and 28 have been found in the other populations of P. hirta s.l. However, a certain morphological differentiation could be found between some populations of P. hirta s.str. (“hirtoid” morph) on one hand, and some others of P. apennina/P. vallarsae (“vallarsoid” morph) on the other hand, but with single individuals or entire populations displaying intermediate features. Generally P. apennina/P. vallarsae (“vallarsoid” morph) showed more rounded and slightly or no spotted leaves with base and petiole being more distinct than in P. hirta s.str. (“hirtoid” morph), which had instead a more spotted, lanceolate leaf shape with a winged base running into the petiole. According to our
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results, hybridization and/or backcrossing/introgression have occurred and gene flow is currently taking place among these "taxa". For all the populations within the complex, I found molecular evi- dence of a hybrid origin involving species belonging to different clades (angustifolia clade and officinalis clade). Since then, I can elaborate three possible scenarios: 1) a unique resulting hybrid species, "vallarsoid" with 2n = 22 chromosomes, spread across Italian peninsula and gave rise to the "hirtoid" morph (2n = 28) through dysploidy; 2) two geographically distinct hybrid events produced both "vallarsoid" (2n = 22) and "hirtoid" (2n = 28) morphs; 3) a unique "hirtoid" (2n = 28) alloploid hybrid species originated, which then produced the "vallarsoid" (2n = 22) plants through backcross- ing with P. officinalis. In scenarios 1 and 2, the different morphs met again in C Italy, with massive current gene flow. In 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 the karyotype asymmetry of populations with 2n = 22, 26 chromosomes, which showed values intermediate between those of P. officinalis and those of the populations with 2n = 28 chromosomes. Irrespective of the evolutionary dynamics, today a unique lineage showing three cytotypes (2n = 22, 26, 28) is widely distributed throughout the Italian peninsula. On taxonomic grounds, I propose to consider these plants as be- longing to a single polymorphic species, namely P. hirta, so that P. apennina and P. vallarsae should be treated as synonyms of P. hirta.