Multiple hybridization origin of Ranunculus cantoniensis (4x): evidence from trnL-F and ITS sequences and fluorescent in situ hybridization (FISH)

ITS sequences of Ranunculus cantoniensis apparently an allotetraploid were polymorphic at ten nucleotide sites. ITS-based phylogeny of the complex and its allied species showed that ITS clones of the tetraploid were clustered with R. silerifolius var. silerifolius, R. chinensis, R. silerifolius var. dolicathus and R. trigonus. Chloroplast trnL-F phylogeny showed that the complex is a natural group, in which the tetraploid shared the same clade with R. silerifolius var. dolicathus and R. silerifolius var. silerifolius, whose genetic distances were zero. rDNA FISH showed that the longest rDNA-chromosome of the tetraploid was similar to that of R. silerifolius var. dolicathus exclusively. Combining trnL-F, ITS and FISH data, it is suggested that the most probable parents of the tetraploid were R. silerifolius var. silerifolius, R. chinensis and R. silerifolius var. dolicathus, among them R. silerifolius var. silerifolius donated most. Evidences from DNA sequences and chromosome FISH indicated that the tetraploid was most probably a homoploid hybrid. Thus, a scenario of the tetraploid formation is proposed: the tetraploid was synthesized by two rounds of hybridization. The first round was between two pairs of diploids, forming two tetraploids. The second round was between the two primary tetraploids, producing the allotetraploid, R. cantoniensis, eventually.