Flow cytometric histogram showing simultaneous analysis of 6 <i>Callitriche</i> taxa: <i>C. cophocarpa</i> (2n = 10), <i>C. palustris</i> (2n = 20), hybrid <i>C. cophocarpa × C. platycarpa</i> (<i>C. ×vigens</i>, 2n = 15), <i>C. platycarpa</i> (2n = 20), hybrid from the Tichá Orlice River (2n = 29) and <i>C. hamulata</i> (2n = 38).

<p>Nuclei of all samples were isolated, stained with propidium iodide and analysed simultaneously.</p

Flow cytometric analysis of <i>Callitriche lenisulca</i> with <i>Bellis perennis</i> as an internal standard, using propidium iodide staining.

<p>Flow cytometric analysis of <i>Callitriche lenisulca</i> with <i>Bellis perennis</i> as an internal standard, using propidium iodide staining.</p

Ecological preferences of 594 <i>Callitriche</i> samples of 9 species and 2 hybrids and co-ocurrence of particular taxa in mixed populations.

<p>* For details, see Materials and Methods.</p><p>Ecological preferences of 594 <i>Callitriche</i> samples of 9 species and 2 hybrids and co-ocurrence of particular taxa in mixed populations.</p

Map of sampled localities of diploid <i>Callitriche cophocarpa</i>, tetraploid <i>C. platycarpa</i> and their triploid hybrid <i>C.</i> ×<i>vigens</i> in north-western Czech Republic.

<p>Two populations with co-occurrence of two taxa are shown as two-colour bisected symbols.</p

Box-and-whisker plots showing the monoploid genome sizes (1Cx-values) for 9 <i>Callitriche</i> taxa.

<p>The species <i>C. hamulata</i> (2n = 38) and the hybrid from the Tichá Orlice River (2n = 29) were not included due to aneuploid chromosome counts.</p

Box-and-whisker plots showing the holoploid genome sizes (2C-values) for 9 <i>Callitriche</i> species and two hybrids: <i>C. cophocarpa</i> × <i>C. platycarpa</i> (<i>C.</i> ×<i>vigens</i>) and a hybrid (probably <i>C. hamulata</i> × unreduced gamete of <i>C. cophocarpa</i>) from the Tichá Orlice River, Czech Republic (Orlice).

<p>Taxa with different chromosome numbers are separated by vertical lines. For <i>C. obtusangula</i>, values for samples from Italian (IT) and north-western Europe were plotted separately, due to significantly different genome sizes.</p

Genome Size as a Key to Evolutionary Complex Aquatic Plants: Polyploidy and Hybridization in <i>Callitriche</i> (Plantaginaceae)

<div><p>Despite their complex evolutionary histories, aquatic plants are highly underrepresented in contemporary biosystematic studies. Of them, the genus <i>Callitriche</i> is particularly interesting because of such evolutionary features as wide variation in chromosome numbers and pollination systems. However, taxonomic difficulties have prevented broader investigation of this genus. In this study we applied flow cytometry to <i>Callitriche</i> for the first time in order to gain an insight into evolutionary processes and genome size differentiation in the genus. Flow cytometry complemented by confirmation of chromosome counts was applied to an extensive dataset of 1077 <i>Callitriche</i> individuals from 495 localities in 11 European countries and the USA. Genome size was determined for 12 taxa. The results suggest that many important processes have interacted in the evolution of the genus, including polyploidization and hybridization. Incongruence between genome size and ploidy level, intraspecific variation in genome size, formation of autotriploid and hybridization between species with different pollination systems were also detected. Hybridization takes place particularly in the diploid – tetraploid complex <i>C. cophocarpa</i> – <i>C. platycarpa</i>, for which the triploid hybrids were frequently recorded in the area of co-occurrence of its parents. A hitherto unknown hybrid (probably <i>C. hamulata</i> × <i>C. cophocarpa</i>) with a unique chromosome number was discovered in the Czech Republic. However, hybridization occurs very rarely among most of the studied species. The main ecological preferences were also compared among the taxa collected. Although <i>Callitriche</i> taxa often grow in mixed populations, the ecological preferences of individual species are distinctly different in some cases. Anyway, flow cytometry is a very efficient method for taxonomic delimitation, determination and investigation of <i>Callitriche</i> species, and is even able to distinguish homoploid taxa and identify introduced species.</p></div

Chromosome numbers of 8 <i>Callitriche</i> species counted in this study.

<p>*For samples details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105997#pone.0105997.s002" target="_blank">Table S2</a>.</p><p>Chromosome numbers of 8 <i>Callitriche</i> species counted in this study.</p