Cytotype variation, cryptic diversity and hybridization in Ranunculus sect. Batrachium revealed by flow cytometry and chromosome numbers

Water-crowfoots (Ranunculus sect. Batrachium) are one of the taxonomically most intricate groups of aquatic plants. Investigation of its species is complicated by morphological reduction and phenotypic plasticity, as well as by the frequent occurrence of polyploidy and hybridization, giving rise to numerous intergrading or morphologically undetectable taxa. We used, for the first time in this group, flow cytometry to gain an insight into evolutionary processes and genome size differentiation in Ranunculus sect. Batrachium. Flow cytometry complemented by confirmation of chromosome counts was applied to an extensive dataset of 3354 individuals from 612 localities in 13 mainly central-European countries, of which the genome size was estimated for 1032 individuals. In total, 34 Batrachium cytotypes of five ploidy levels ranging from diploids to hexaploids were detected. The results indicate that the genome size is a reliable marker for distinguishing most of the traditionally recognized species, including those with identical chromosome numbers. Although variation in chromosome numbers is reported for six of eight central-European species, we detected only two taxa variable at the ploidy level (R. fluitans 2x + 3x, R. penicillatus 4x + 6x). However, the situation is much more challenging due to the presence of numerous hybrids and cryptic lineages. Cryptic variation was revealed within R. trichophyllus, in which three distinct tetraploid cytotypes were detected, which differ in their ecology and distribution. The allopolyploid complex of running-water taxa referred to as R. penicillatus is even more complex, including six different cytotypes. We also revealed 16 cytotypes of hybrid origin, which accounted for ca 15% of the individuals studied. Seven of the hybrid cytotypes were identified as F1 hybrids and the rest are of unknown or uncertain origin. In the R. penicillatus group, the occurrence of a large hybrid swarm was documented. Hybrids with variable genome sizes frequently arise also from hybridization of the tetraploids R. peltatus and R. trichophyllus in which the gene flow tends to be largely unidirectional towards R. peltatus.