Karyological evolution in Cistaceae, with emphasis on Cistus and Helianthemum

Abstract

Karyological changes, both in number and structure, are defined as the onset of the speciation processes and may promote the evolutionary dynamics of ribosomal RNA gene families, 5S and 45S rDNAs. Numerous 45S rDNA repeats are tandemly arranged in one or more loci in the genome called nucleolar-organizer regions (NORs). Knowledge about the number of rDNA loci, together with their chromosomal distribution and structure, provides clues about organismal and molecular evolution at various phylogenetic levels. To elucidate the evolutionary dynamics of rDNA loci number and position in diploid plant lineages, we have established the 45S rDNA loci for 30 taxa of Cistus, 28 taxa of Helianthemum and 9 species of the closely related genera Fumana (8) and Tuberaria (1) using the Fluorescence In Situ Hybridisation (FISH) method. Moreover, we have assessed their nucleolar activity through the AgNOR staining. Lastly, we have reconstructed the evolutionary history of 45S rDNA loci number based on maximum likelihood and maximum parsimony analysis using available phylogenies as framework. In Cistus, the little variation found in number and position of 45S rDNA sites agree with the high karyotypic uniformity observed. The nucleolar activity of NOR loci doesn’t change significantly. The reconstruction of the ancestral rDNA state shows congruence between the loci number and the tree topology with well-supported clades, indicating different rates of changes in the most recent lineages. We suggest that the organismal evolutionary history has affected the patterns of the 45S rDNA loci distribution. As for Helianthemum, we found that the few changes in the 45S rDNA loci number and position observed are not associated to the chromosome base number variability (four cytotypes recognized) but are more likely related to the differentiation of taxa. Indeed, the ancestral state reconstruction highlights that the number of 45S rDNA loci changes in correspondence of clades which converge with the sectional taxonomic division. We also find few differences in number and size of nucleoli. The results prove that species diversification of Helianthemum has been driven, at least partly, by chromosomal restructuring. At a smaller scale, such as a single-species level, we have used the same cytogenetic approach to asses the stability of the two nuclear rDNA families (5S and 45S rDNAs) of 118 accessions including wild and in vitro micropropagated individuals and bi-directional artificial crosses between wild and in vitro-generated plants of the endangered species Cistus heterophyllus. Plants regenerated by micropropagation show a lower size of the FISH signals in a 45S rDNA site, but this heterozygous condition is not observed in the wild accessions. Three new cytogenetic variants have been found in 36% of the experimental progeny, involving the amplification of one additional 45S rDNA site and the presence of heteromorphic nucleoli, proving a rDNA-based genomic instability present after sexual reproduction between wild and in vitro-generated plants.