Genome size variation and species relationships in Hieracium sub-genus Pilosella (Asteraceae) as inferred by flow cytometry

Abstract

Background and aims: Hieracium sub-genus Pilosella (hawkweeds) is a taxonomically complicated group of vascular plants, the structure of which is substantially influenced by frequent interspecific hybridization and polyploidization. Two kinds of species, 'basic' and 'intermediate' (i.e. hybridogenous), are usually recognized. In this study, genome size variation was investigated in a representative set of Central European hawkweeds in order to assess the value of such a data set for species delineation and inference of evolutionary relationships.

Methods: Holoploid and monoploid genome sizes (C- and Cx-values) were determined using propidium iodide flow cytometry for 376 homogeneously cultivated individuals of Hieracium sub-genus Pilosella, including 24 species (271 individuals), five recent natural hybrids (seven individuals) and experimental F(1) hybrids from four parental combinations (98 individuals). Chromosome counts were available for more than half of the plant accessions. Base composition (proportion of AT/GC bases) was cytometrically estimated in 73 individuals.

Key results: Seven different ploidy levels (2x-8x) were detected, with intraspecific ploidy polymorphism (up to four different cytotypes) occurring in 11 wild species. Mean 2C-values varied approx. 4.3-fold from 3.53 pg in diploid H. hoppeanum to 15.30 pg in octoploid H. brachiatum. 1Cx-values ranged from 1.72 pg in H. pilosella to 2.16 pg in H. echioides (1.26-fold). The DNA content of (high) polyploids was usually proportional to the DNA values of their diploid/low polyploid counterparts, indicating lack of processes altering genome size (i.e. genome down-sizing). Most species showed constant nuclear DNA amounts, exceptions being three hybridogenous taxa, in which introgressive hybridization was suggested as a presumable trigger for genome size variation. Monoploid genome sizes of hybridogenous species were always between the corresponding values of their putative parents. In addition, there was a good congruency between actual DNA estimates and theoretical values inferred from putative parental combinations and between DNA values of experimental F(1) hybrids and corresponding established hybridogenous taxa.

Conclusions: Significant differences in genome size between hawkweed species from hybridogenous lineages involving the small-genome H. pilosella document the usefulness of nuclear DNA content as a supportive marker for reliable delineation of several of the most problematic taxa in Hieracium sub-genus Pilosella (including classification of borderline morphotypes). In addition, genome size data were shown to have a good predictive value for inferring evolutionary relationships and genome constitution (i.e. putative parental combinations) in hybridogenous species.

Fig. 1.

Genome size determination in Hieracium sub-genus Pilosella species with the smallest and the largest C-values. (A) Diploid H. hoppeanum (accession 781 HO) + Zea mays as an internal reference standard (mean sample 1C-value = 1·77 pg; peak ratio, 1·529; CVs, 2·08 and 2·01 %). (B) Octoploid H. brachiatum (accession 1025 BRA) + Pisum sativum as an internal reference standard (mean sample 1C-value = 7·53 pg; peak ratio, 1·698; CVs, 2·28 and 2·09 %). Nuclei of the analysed species and internal standard were isolated, stained with propidium iodide and measured simultaneously.

Fig. 2.

2C-value variation in 46 cytotypes belonging to 24 species of Hieracium sub-genus Pilosella. Arrows indicate basic (non-hybridogenous) species.

Fig. 3.

Evolutionary relationships among the studied Hieracium sub-genus Pilosella species inferred from their phenotypes (according to Schuhwerk and Fischer, 2003; corrected) together with corresponding Cx-values (in DNA picograms). The number of plants analysed for each ploidy level is given in parentheses. Basic species are displayed in solid boxes, intermediate species on interconnecting lines; dashed box denotes uncertain species classification. Exclamation marks indicate intraspecific variation in genome size (>3·5 %). The central taxon, H. pilosella, possesses the smallest genome.

Fig. 4.

Flow cytometric histogram documenting intraspecific variation in genome size between two tetraploid accessions of H. brachiatum (BRA 642 and BRA 495). Peak ratio 1 : 1·040; CV 1·25 and 1·02 %, respectively. Nuclei of both accessions were isolated, stained with DAPI and analysed simultaneously.