Intraspecific DNA content variability in Festuca pallens on different geographical scales and ploidy levels

Abstract

Background and aims: Intraspecific genome size variability of Festuca pallens occurring on relict rocky steppes in Central Europe was studied on two ploidy levels and three geographical scales: (1) local scale of 24 populations, (2) landscape scale of three transects in river canyons or hill systems, and (3) global scale of 160 samples covering the whole distribution area.

Methods: DAPI flow cytometry of homogeneously cultivated samples (>or=1 year), measured randomly with two internal standards, Lycopersicon esculentum and Pisum sativum. Differences in DNA content were confirmed (1) by the double peaks of simultaneously measured samples, (2) based on measurements carried out in different seasons, and (3) by additional measurements with propidium iodide.

Key results: On a global scale, the relative DNA content ranged between 1.170-fold in diploids and 1.164-fold in tetraploids. A maximum difference of 1.088-fold between the mean relative DNA content of nearby populations was found. In 16 of 24 populations significant variability was shown (P<0.001, 1.121-fold as maximum). For both ploidy levels, the relative genome size had the same range and geographical pattern, correlated with geographical coordinates (P<0.01). Diploids with larger genomes occur on relict habitats (P<0.01), and in areas of periglacial steppes (20,000 years ago; P<0.02). In tetraploids, the relative DNA content differs among the three previously recognized geographical types (Alpine, Pannonian and Scabrifolia, P<0.001). Tetraploids have a relative DNA content smaller than twice that of the diploids (P<0.001). An influence of microhabitat on DNA content variation was not confirmed.

Conclusions: Genome size variability occurs over all spatial scales: intrapopulation, landscape and global. Correlation between geographical coordinates and palaeovegetation type, concomitant with diploids and tetraploids, and no influence of microhabitat were found. Genome size decreases in tetraploids. Lower CVs, and thus higher accuracy, resolution and reproducibility, favour DAPI measurements for the study of intraspecific genome size variability.

Fig. 1.

Comparison of the relative DNA content estimated with the AT-selective DAPI dye and intercalary PI dye in diploid (2x) and tetraploid (4x) Festuca pallens (Lycopersicon esculentum as the standard). Samples with known chromosome number (Šmarda and Kočí, 2003) are marked with closed symbols, those with unknown chromosome number with open ones. In ploidy levels together (A) and also in both ploidy levels separately (B, C), strong linear correlations appear between both dyes (full lines). The slope of the regression lines is around 1·58 in all three diagrams, if the five diploid samples with the highest relative DNA content are excluded (A, C - full lines, B - dashed line). These five samples appear to have a slightly higher AT content and decrease the slope of regression line of all diploids (B, full line). ^aIntercept non-significant (P > 0·05) and set to be zero.

Fig. 2.

Reproducibility of measurements: comparison of DAPI measurements of diploid and tetraploid Festuca pallens carried out in autumn 2004 and spring 2006. Strong linear correlation of both indicates high reproducibility. No seasonal changes were detected (paired t-test, P > 0·05).

Fig. 3.

Observed intrapopulation and landscape pattern in relative DNA content on three investigated transects: circles represent diploids, squares tetraploids. Dark segment of symbols shows relative DNA content, and symbol size indicates position of the sample within the total observed range for the respective ploidy level (open: lower limit, closed: upper limit).

Fig. 4.

Difference in relative DNA content of two simultaneously measured diploid Festuca pallens samples: (A) maximal difference of diploid samples of two localities separated by 2 km from a transect along the Rokytná river; (B) maximal difference of tetraploid samples from the same population from Mikulov (Pálava hills transect); 10 000 cells were counted.

Fig. 5.

Relative genome size of separate ploidy levels of Festuca pallens. Relative genome size of higher polyploids (4x and 5x) is significantly smaller (t-test, P < 0·001). The numbers below the box plots give the total range of variation within the respective ploidy level. Relative genome size within all samples of all ploidy levels varied 1·201-fold. *Value calculated together with additional data from uncultivated population samples.

Fig. 6.

Observed difference in relative DNA content in the whole distribution area of Festuca pallens. Open symbols represent diploids, closed symbols tetraploids. The size of symbols reflects the relative DNA content divided into the five categories in a particular ploidy level with the 6th given to the extremely large diploid from Vadu Crişului. The natural range is marked by a dashed line. The generally higher DNA amounts in the south and east part of the natural range and in areas of steppes 20 000 years ago are of particular note.

Fig. 7.

Differences in relative DNA content of diploid plants from the different phytogeographical regions. Phytogeographical regions indicated with the same letter above the box-plots do not differ significantly (Bonferoni post-hoc comparison, P = 0·05).

Fig. 8.

Relative DNA content of separate tetraploid types. One-way ANOVA revealed significant differences among these types (P < 0·001). The relative DNA content of the Scabrifolia type is significantly smaller than in the two others (t-test, P < 0·001).