3.2 Genetic variation in S-locus genes and their paralogs
Our results showed that, as expected due to S-locus hemizygosity,
πS was lower in S-locus genes of thrums (0.0012 ± 0.0006
[mean ± SE]) than in their paralogs in pins and thrums (0.0034 ±
0.0008; Table 3A). Moreover, πS in homostyles was zero
for both S-locus genes and their paralogs, except forGLOT and CYP734A51 , where
πS was extremely low (0.0012 and 0.0008, respectively;
Table 3B), thus supporting the prediction that the shift to predominant
selfing should be associated with lower πS in homostyles
than in heterostyles.
Furthermore, our results indicated that, on average,
πN/πS values were higher for S-locus
genes of thrums than for their paralogs in pins and thrums (1.01 ± 0.37
vs. 0.53 ± 0.29, respectively; Table 3A), implying lower purifying
selection in S-locus genes. Additionally,
πN/πS was lower in KFB1 than inKFBT (πN/πS =
0.23 and 1.83, respectively), but higher in CCM1 than inCCMT (πN/πS =
1.5 and 0.91, respectively; Table 3A), implying stronger and weaker
purifying selection on the two paralogs than on their respective S-locus
genes, respectively. Finally, we found that, within the S-locus,
πN/πS was higher forCCMT , KFBT , andPUMT (πN/πS =
0.91, 1.83, and 10.36, respectively; Table 3A) than forCYPT (πN/πS =
0.28), whereas πN/πS inGLOT was not calculated due to the lack of
variation at synonymous sites in this gene. In homostyles,
πN/πS was zero for most S-locus genes
and their paralogs, except for CYP734A51 , due to the lack
variation at synonymous and non-synonymous sites (Table 3B).
3.3 S- locus genotypes in natural populations of P.
vulgaris
Relative S-locus sequencing
depth (RelS-locus depth ) allowed us to determine
S-locus ploidy in the analyzed thrums and homostyles. Of the 37 thrums
collected from six dimorphic and two trimorphic populations across our
sampling range, 34 had a haploid S-locus (i.e., S/0) and three had a
diploid S-locus (S/S; Figure 4). Of these three thrums, two were
homozygous for the functional copy of CYPT(CYPT-1/CYPT-1 ; i.e., S/S)
and belonged to one trimorphic and one dimorphic population each,
respectively (Table 1), while one thrum was heterozygous and carried one
functional and one disrupted copy of CYPT(CYPT-1/CYPT-2 ; i.e., S/S*).
Of the 31 homostyles collected from two trimorphic (EN4-T and EN5-T) and
one monomorphic population (EN6-M) in England, 10 (32%) had a haploid
S-locus (S*/0) and 21 (68%) had a diploid S-locus (S*/S*) (Figure 4).
Specifically, S*/0- homostyles represented 40% and 60% of
homostyles in the trimorphic populations EN4-T and EN5-T, respectively,
while all tested homostyles of the monomorphic population EN6-M had the
S*/S*- genotype (Table 1; purple triangles in Figure 4).
We also calculated expected frequencies and observed frequencies of 0/0,
S/0-, S*/0-, and S*/S*-genotypes under different assumptions for
viability of S*/S*-genotypes compared to the other three genotypes and
after different numbers of generations following the origin of
homostyles (Table 4). The results of chi-squared tests showed that
non-significant differences were found only in seven cases, of which
four occurred in the monomorphic, homostylous population EN6-M and three
in the two trimorphic populations EN5-T and EN4-T. Specifically, in
EN6-M, observed frequencies matched expected frequencies at generations
30 and 40 under the assumption of equal and slightly lower viability (v
= 1 and 0.9, respectively) for the S*/S*-homostyles. In EN5-T, observed
frequencies matched expected frequencies at 20 generations under lower
viability (v = 0.8) of S*/S*-homostyles. Finally, in EN4-T, observed
frequencies matched expected genotypic frequencies at generations 20 and
30 under levels of viability for S*/S*-homostyles close or equal to
those of the model in Figure 1C (i.e., v = 0.70 and 0.65, respectively).