The phylogenetic relationships and phylogeography of two relatively rare Macaronesian Lactuca species, Lactuca watsoniana (Azores) and L. palmensis (Canary Islands), were, until this date, unclear. Karyological information of the Azorean species was also unknown. For this study, a chromosome count was performed and L. watsoniana showed 2n = 34. A phylogenetic approach was used to clarify the relationships of the Azorean endemic L. watsoniana and the La Palma endemic L. palmensis within the subtribe Lactucinae. Maximum parsimony, Maximum likelihood and Bayesian analysis of a combined molecular dataset (ITS and four chloroplast DNA regions) and molecular clock analyses were performed with the Macaronesian Lactuca species, as well as a TCS haplotype network. The analyses revealed that L. watsoniana and L. palmensis belong to different subclades of the Lactuca clade. Lactuca watsoniana showed a strongly supported phylogenetic relationship with North American species, while L. palmensis was closely related to L. tenerrima and L. inermis, from Europe and Africa. Lactuca watsoniana showed four single-island haplotypes. A divergence time estimation of the Macaronesian lineages was used to examine island colonization pathways. Results obtained with BEAST suggest a divergence of L. palmensis and L. watsoniana clades c. 11 million years ago, L. watsoniana diverged from its North American sister species c. 3.8 million years ago and L. palmensis diverged from its sister L. tenerrima, c. 1.3 million years ago, probably originating from an African ancestral lineage which colonized the Canary Islands. Divergence analyses with *BEAST indicate a more recent divergence of the L. watsoniana crown, c. 0.9 million years ago. In the Azores colonization, in a stepping stone, east-to-west dispersal pattern, associated with geological events might explain the current distribution range of L. watsoniana.
Keywords: Azores; Canary Islands; Divergence time; Karyology; Lactuca; Macaronesia; Molecular clock; Phylogeny; Phylogeography.