Chromosome counts and mode of reproduction
Chromosome numbers for plants of 43 populations analysed in the present paper belonging to 28 species were published elsewhere (Chrtek et al., 2007
), and counts for the remaining 46 populations are presented here (Table ). A new ploidy is reported for H. gymnocephalum
= 18). Other counts confirmed previously published chromosome numbers. All diploids studied were found to be sexual and allogamous, and all polyploids (3x
) were agamospermous (data not provided).
Flow cytometric analyses yielded high-resolution histograms with CVs of G0/G1 peaks for Hieracium samples ranging from 0·83 to 5·76% (mean 2·28%), the values for internal reference standards were 0·97 to 5·0% (mean 2·19%). Generally, CVs of Pisum sativum were lower than those of Zea mays.
Nuclear DNA content: within-species variation
Intraspecific variation was assessed in 40 of 42 species sensu Zahn. Variation within accessions (populations) was generally low (Table ). Mean values with standard errors (ranges for 2C and means for 1Cx genome sizes) for each population and mean values with standard errors and ranges for 1Cx for each species are summarized in Table . Variation in 1Cx values between conspecific accessions (in both homo- and multiploid species) ranged from 0·24% in H. gymnocephalum and H. heterogynum to 7·2% in H. pannosum. Variation exceeding the approximate measurement inaccuracy threshold of 3·5% was detected in seven species, namely H. amplexicaule, H. bupleuroides, H. laevigatum, H. pannosum, H. pictum, H. piliferum and H. prenanthoides.
However, variation in the more naturally delimited (without heterogeneity in ETS sequences and inter-population genome size; see Materials and methods) H. bupleuroides was below the threshold of 3·5% variation. Variation within the separately treated populations of morphologically heterogeneous H. pannosum was also below 3·5% (Table ). In further paragraphs, these ‘narrower’ taxa (a total of 46 taxa) are used.
C-values in the total set of ‘basic’ species
The mean 2C values differed up to 2·37-fold among different species (from 7·03 pg in diploid H. stelligerum to 16·67 in a tetraploid accession of H. pannosum). The 1Cx values varied 1·22-fold between 3·51 pg in H. stelligerum and 4·34 pg in H. virosum (mean 1Cx value of 3·87, s.d. 0·27; Fig. ). The 1Cx values of diploids (including diploid accessions of multiploid species, means for species/cytodemes) varied 1·22-fold between 3·51 pg in H. stelligerum and 4·29 pg in H. plumulosum (mean 1Cx value 3·92 pg, s.d. 0·30), in triploids [including triploid accessions of multiploid species (means for species/cytodemes)] 1·23-fold between 3·53 pg in H. bifidum and 4·35 pg in H. virosum (mean 1Cx value of 3·81 pg, s.d. 0·25), and in tetraploids 1·17-fold between 3·56 pg in H. humile and 4·17 pg in H. pannosum II (mean 1Cx value of 3·79, s.d. 0·19).
1Cx-value variation in 46 taxa of Hieracium subgenus Hieracium.
Correlation between genome size, ploidy and breeding system
Diploids differed significantly in their 1Cx values from both triploids (t = 2·71, d.f. = 196, P = 0·007) and tetraploids (t = 2·01, d.f. = 109, P = 0·047), but triploids did not differ significantly from tetraploids (t = 0·72, d.f. = 119, P = 0·476) (Fig. ). The value of the Spearman non-parametric rank order correlation coefficient was r = –0·179, P = 0·009. The mean 1Cx value was 3·93 pg in diploids, 3·82 pg in triploids and 3·78 pg in tetraploids, suggesting a trend towards smaller genome size with increasing ploidy.
Fig. 2. Variation of genome size among diploids, triploids and tetraploids (all samples). 1Cx values of all accessions are shown. Differences between diploids and triploids, and between diploids and tetraploids are significant. The box indicates the interquartile (more ...)
In multiploid species, there was no general trend to either genome downsizing or upsizing. In H. prenanthoides 2x/3x there was 2·47% upsizing, in H. villosum 3x/4x 0·06% downsizing, in H. tomentosum 2x/3x 0·36% upsizing, in H. humile 3x/4x 0·36% upsizing and in H. alpinum 2x/3x 0·17% upsizing.
Comparison between 1Cx values of sexually reproducing plants (i.e. all diploids; polyploids were exclusively apomictic) and apomicts (triploids and tetraploids) revealed significant differences at α = 0·01 (t-test, t = 3·04, d.f. = 213, P = 0·003); the mean 1Cx value in sexuals was 3·93 pg, whereas in apomicts it was 3·82 pg, corresponding to the value for triploids due to the low number of tetraploid accessions.
Molecular phylogenetics of Hieracium
Analysis of ETS data including all sequenced accessions resulted in the same tree with both methods. It indicates monophyly of Hieracium
, but species relationships remained completely unresolved as reflected by a large polytomy with only two small subclusters that received low support (Fig. ). However, as two major species groups could be identified by visual inspection of the alignment and many sequences showed additive patterns indicative of hybridization involving both groups, these accessions were deleted from subsequent analysis, because reticulation is known to collapse branches (Feliner et al., 2001
; Soltis et al., 2008
). With the reduced data set, a clear separation into two major clades with strong statistical support was found with both methods (Fig. ). These lineages were designated ‘eastern’ and ‘western’ clade because they contained species of predominantly eastern or western European origin. A large number of accessions (18) showed ETS variants of both clades in either equal proportion or with the ‘eastern’ or ‘western’ sequence type dominating as indicated in Fig. . Details of these analyses will be given in a parallel paper (J. Fehrer et al., unpubl. res.
Fig. 3. Phylogenetic analysis of ETS sequences based on all accessions. A Bayesian consensus tree of 3002 saved trees is shown with posterior probabilities above branches. The maximum likelihood tree has the same topology; bootstrap values are indicated below (more ...)
Fig. 4. Phylogenetic analysis of ETS sequences excluding interclade hybrid accessions. A Bayesian consensus tree of 1502 saved trees is shown with posterior probabilities above branches. The maximum likelihood tree has the same topology; bootstrap values are (more ...)
Correlation of genome size with phylogenetic signal
The ‘western’ clade included 15 accessions: 2C values ranged from 7·03 pg in diploid H. stelligerum to 14·25 pg in a tetraploid accession of H. humile; 1Cx values ranged from 3·51 pg in H. stelligerum to 4·28 pg in H. transylvanicum (mean ± s.d.: 3·61 ± 0·19 pg; with H. transylvanicum excluded: up to 3·74 pg in H. tomentosum, 3·57 ± 0·06 pg). The ‘eastern’ clade also comprised 15 accessions: 2C values ranged from 7·78 pg in diploid H. porrifolium to 15·71 pg in a tetraploid accession of H. villosum; 1Cx values ranged from 3·63 pg in H. naegelianum to 4·35 pg in H. virosum (4·02 ± 0·20 pg). Significant differences in 1Cx values were found between the clades at α = 0·001 (Student's t-test), with (t = –5·71, d.f. = 28, P < 0·001) and without (t = –8·23, d.f. = 27, P < 0·001) H. transylvanicum.
Differences in 1Cx values between accessions of the ‘western’ (W) and ‘eastern’ (E) clades and of interclade hybrid accessions (X) are significant, independent of the inclusion of H. transylvanicum (F = 13·79, d.f. = 45, P < 0·001 with H. transylvanicum, F = 20·87, d.f. = 44, P < 0·001 without H. transylvanicum; Fig. A). However, post hoc comparison (Scheffé test) revealed only two groups at α = 0·05, the first comprising all ‘western’ accessions, and the second embracing ‘eastern’ and ‘hybrid’ accessions. Thus, ‘eastern’ and ‘hybrid’ accessions do not differ significantly from each other. Significant differences were also found between five groups, i.e. after splitting the bulk of hybrids into three groups, namely hybrids with intermediate position (X) and hybrids with strongly dominating ‘western’ [X(W)] or ‘eastern’ [X(E)] ETS sequences (F = 17·07, d.f. = 43, P < 0·001 with H. transylvanicum, F = 28·86, d.f. = 42, P < 0·001 without H. transylvanicum). The Scheffé test revealed only two groups at α = 0·05, the first including W and X(W) accessions, the second X, X(E) and E accessions (Fig. B). A significant correlation (Spearman rank coefficient r = 0·705, P < 0·001) between phylogenetic signal and hybrid origin [all five groups – W, E, X, X(W) and X(E)] and the pattern of genome size variation was found. Hieracium piliferum (1Cx = 3·9) occupies an isolated position, and it was identified as a hybrid between an ‘eastern’ clade taxon and ‘Hieracium’ intybaceum (1Cx = 3·76).
Fig. 5. Correlation of 1Cx values with phylogeny. Only accessions for which sequence data were available are included. (A) W1, ‘western’ clade accessions without H. transylvanicum; W2, ‘western’ clade accessions including H. transylvanicum (more ...)
Evolution of genome size
Maximum likelihood method
For the complete data set (all species), a directional model of evolution (model B) did not result in significantly higher likelihood scores than the drift model of evolution (model A; 74·856 vs. 75·746), indicating that there is no general trend to either genome size increase or decrease. Scaling parameters leading to the highest likelihood for 1Cx values were λ = 0·908, δ = 0·819 and κ = 1·035 in model A and λ = 0·701, δ = 0·637 and κ = 1·201 in model B, respectively. For both models, the values of scaling parameters did not differ significantly from 1 (the null expectation, LR test), indicating that the phylogenetic tree correctly predicts the pattern of covariance among species on the trait (1Cx) and that there is no evidence of accelerated evolution.
For the western clade, likelihood scores of models A and B did not differ significantly (44·061 vs. 44·790). The maximum likelihood values for λ (<1; 0·550 in model A, 0·523 in model B) show a role of adaptive response to some external factors. Values of δ and κ are >1 in both models (not shown) indicating that longer paths contribute more to 1Cx evolution (accelerated evolution as time progresses) and that longer branches contribute more to the trait evolution. Likelihoods of the null model (with scaling paramaters set to 1·0) are significantly lower in both models, indicating that scaling parameters improve the fit of the data to the models.
For the eastern clade, likelihood scores of models A and B also did not differ significantly (40·915 vs. 42·141). Scaling parameters are not significantly different from 1 (the null expectation, Brownian motion, data not shown) indicating that the phylogenetic tree correctly predicts the pattern of covariance among species and that there is no evidence of accelerated evolution.
For the complete data set, comparison of harmonic means of log maximum likelihoods of models A and B showed a somewhat higher value in the latter (82·544 vs. 84·670). The model with estimated scaling parameters is a better fit than the null model (with scaling parameters set to 1) for both models A and B, showing that the scaling parameters improve the fit of the data to the model. The values of λ did not differ significantly from 1, and relatively high values of κ (3·379 and 3·606, respectively) indicate that longer branches contribute more to genome size evolution.
For the western clade only, the harmonic mean of model B is also higher than that of model A (49·888 vs. 45·494) and the harmonic mean of the null model is significantly lower than that for the model with estimated scaling parameters. Scaling parameters are similar to those found with the maximum likelihood method, i.e. λ and δ < 1, κ > 1 (for interpretation see above).
For the eastern clade, harmonic means of models A and B do not differ significantly. Values of λ and δ do not differ from 1, higher values of κ (1·988 in model A and 2·212 in model B) again indicate accelerated rates of evolution within long branches.
Correlation between genome size and ecogeographic features
The genome size of particular accessions was significantly correlated with their geographic position (longitude) in a west–east direction, both in the complete set of accessions (Spearman rank coefficient r = 0·562, P < 0·001; Fig. A) and after exclusion of widely distributed species (r = 0·617, P < 0·001; i.e. without H. bifidum, H. lachenalii, H. laevigatum, H. murorum, H. sabaudum and H. umbellatum; Fig. B). The correlation was stronger in the second case due to the strong dependence on the part of the geographic area from which the target plants of widespread species were sampled.
Fig. 6. Distribution of 1Cx values versus longitudinal position of collection sites: (A) based on the complete set of accessions/populations (Spearman rank coefficient r = 0·562, P < 0·001); (B) based on a subset after excluding accessions (more ...)
No correlation between genome size and latitude (r = 0·049, P = 0·646) or genome size and altitude (r = –0·224, P = 0·034) was found (complete set of accessions, results not shown). Also, no significant correlation was found between genome size and selected ecological parameters (Ellenberg's indicator values), namely temperature (r = 0·194, P = 0·427) and light (r = –0·236, P = 0·331) in a subset of species occurring in central Europe (results not shown).
Distinction between longitudinal and phylogenetic correlation
In order to determine whether the increase in genome size towards the east/‘eastern’ clade is based on geographic distribution or on species relationships, the longitudinal correlation was re-analysed for those accessions for which molecular data were available. Even stronger correlation was found between longitude and genome size in a set of accessions with known ETS sequences independent of including (r = 0·656, P < 0·001) or excluding (r = 0·688, P < 0·001) accessions of widely distributed species (Fig. A, B). In contrast, no significant correlation was found either among species of the ‘western’ (r = 0·161, P = 0·567) or among species of the ‘eastern’ (r = 0·394, P = 0·245) clade when tested separately (Fig. C and D). These results reveal that the evolutionary history due to eastern or western origin of the species is the dominant parameter affecting genome size in Hieracium rather than longitude.
Fig. 7. Longitudinal component of genome size variation for accessions of known phylogenetic origin: (A) based on a complete set of ‘western’ and ‘eastern’ accessions/populations analysed by molecular methods (excluding interclade (more ...)