Intricate evolutionary history of Callitriche (Plantaginaceae) taxa elucidated by a combination of DNA sequencing and genome size

The widespread aquatic plant genus Callitriche is taxonomically very challenging, but noteworthy in many evolutionary aspects including a high overall diversity, extensive phenotypic plasticity, remarkable reproductive systems and a large variation in ploidy levels and chromosome numbers. We conducted a multi-level systematic study on 346 individuals of 25 taxa from 21 mostly European countries. Flow cytometric estimation of genome size, chromosome counting and direct sequencing of ITS and trnT-trnL DNA markers combined with RFLPs of the ITS region were applied in order to unravel the phylogenetic relationships among Callitriche taxa and to clarify the origin of polyploid species and hybrids. Additionally, ITS sequences from a recent worldwide phylogenetic study of the genus were included for comparison. We demonstrate that most of the traditionally recognized European Callitriche taxa are well defined by a combination of genome size and molecular markers. Several species showed remarkable intraspecific genetic variation; previously unknown cryptic taxa were revealed within C. stagnalis, C. truncata and North American C. heterophylla. The origin of selected polyploid taxa was investigated in detail. Diploid C. cophocarpa was confirmed to be the parental species of tetraploid C. platycarpa, but we did not find direct evidence for the putative allopolyploid origin of this species. The complex of C. brutia included three taxa; of these, C. hamulata is probably an allooctoploid derivative of C. brutia var. brutia and C. cophocarpa/C. platycarpa. The third member, C. brutia var. naftolskyi, was newly reclassified at the subspecies level; for the first time, chromosome numbers are provided for this poorly known taxon. For a single triploid sample, our results suggested an autopolyploid origin from C. stagnalis. Four Callitriche hybrids were revealed, two ofwhich are newly described and validated here as C. ×nyrensis and C. brutia nothosubsp. neglecta. A tentative intrageneric concept of two sections (Callitriche, Pseudocallitriche) is adopted, with the need for a more detailed evaluation in the future.