Study of the evolutionary relationships among Limonium species (Plumbaginaceae) using nuclear and cytoplasmic molecular markers.

The genus Limonium, due to the patchiness of the natural habitats of its species as well as the high frequency of hybridization and polyploidy and the possibility of reproduction by apomixis, provides an example of all the principal mechanisms of rapid speciation of plants. As an initial study of evolution in this genus, we have analyzed intra- and interspecific variability in 17 species from section Limonium, the largest in the genus, based on RFLPs of cpDNA and nuclear rDNA ITS sequences. In the cpDNA analysis, 21 restriction enzymes were used, resulting in 779 fragments, 490 of which were variable and 339 parsimony informative. L. furfuraceum exhibited two relatively divergent cpDNA haplotypes. The relationships found among the species based on cpDNA restriction fragments were coincident using different methods of phylogenetic analysis. Due to the presumed reticulate evolution in the genus Limonium, the comparison of these results with data from the nuclear DNA was necessary; ITS sequences were analyzed. The final alignment contained 488 characters, of which 198 were variable and 156 parsimony informative. Two relatively divergent ITS types were present at the intraindividual level in L. delicatulum, a triploid species. Each type was related to ITS from different groups of diploid Limonium species, one with a base haploid chromosome number n = 8 (represented by L. cossonianum) and the other with n = 9 (represented by L. minutum). The different phylogenetic inference methods used for the analysis of ITS sequences rendered very similar topologies. In general, the relationships among the species studied were coincident with those obtained with the chloroplast genome. Both nuclear and cytoplasmic markers support the polyphyly of section Limonium, with at least two species, L. narbonense and L. vulgare, clearly divergent from the rest. Moreover, the remaining subsections into which section Limonium is currently divided seem to be artificial.