Molecular response to reciprocal hybridizations of <i>C</i>. <i>rubella</i> × <i>C</i>. <i>grandiflora</i> is similar to interploidy hybridizations in Arabidopsis.

<p>(A) Genes up- and downregulated in <i>C</i>. <i>rubella</i> × <i>C</i>. <i>grandiflora</i> reciprocal hybrid seeds compared to both parents overlap with genes deregulated in Arabidopsis interploidy seeds. The Arabidopsis <i>osd1</i> mutant produces unreduced gametes, mimicking an interploidy hybridization when crossed with wild-type (WT). P values reflecting significance of overlap were calculated using a hypergeometric test. (B) Heatmap of expression log2 fold changes of selected <i>AGL</i> genes between samples. Capsella <i>AGLs</i> with several homologs in Arabidopsis are marked by small letters.</p

Cross direction-dependent incompatibility affects development of <i>Capsella rubella</i> and <i>C</i>. <i>grandiflora</i> hybrid seeds.

<p>Percentage (A) and phenotypes (B) of aborted and non-aborted seeds of <i>C</i>. <i>rubella</i>, <i>C</i>. <i>grandiflora</i> and reciprocal hybrids of both species. Scale bars reflect 1 mm. (C) Percentage of germinated seeds of indicated crosses. (D) Seedlings of indicated crosses 10 days after germination. Seed area (E) and seed weight (F) of indicated crosses. Error bars show standard deviation. Significance was determined by t test analysis. * P < 0.05, ** P < 0.01 ns, not significant. In all graphs numbers on top of the bars correspond to number of analyzed seeds.</p

Non-reciprocal Interspecies Hybridization Barriers in the Capsella Genus Are Established in the Endosperm

<div><p>The transition to selfing in <i>Capsella rubella</i> accompanies its recent divergence from the ancestral outcrossing <i>C</i>. <i>grandiflora</i> species about 100,000 years ago. Whether the change in mating system was accompanied by the evolution of additional reproductive barriers that enforced species divergence remained unknown. Here, we show that <i>C</i>. <i>rubella</i> and <i>C</i>. <i>grandiflora</i> are reproductively separated by an endosperm-based, non-reciprocal postzygotic hybridization barrier. While hybridizations of <i>C</i>. <i>rubella</i> maternal plants with <i>C</i>. <i>grandiflora</i> pollen donors resulted in complete seed abortion caused by endosperm cellularization failure, the reciprocal hybridization resulted in the formation of small seeds with precociously cellularized endosperm. Strikingly, the transcriptomic response of both hybridizations mimicked respectively the response of paternal and maternal excess hybridizations in <i>Arabidopsis thaliana</i>, suggesting unbalanced genome strength causes hybridization failure in both species. These results provide strong support for the theory that crosses between plants of different mating systems will be unbalanced, with the outcrosser behaving like a plant of increased ploidy, evoking a response that resembles an interploidy-type seed failure. Seed incompatilibity of <i>C</i>. <i>rubella</i> pollinated by <i>C</i>. <i>grandiflora</i> followed the Bateson-Dobzhansky-Muller model, involving negative genetic interaction of multiple paternal <i>C</i>. <i>grandiflora</i> loci with at least one maternal <i>C</i>. <i>rubella</i> locus. Given that both species only recently diverged, our data suggest that a fast evolving mechanism underlies the post-zygotic hybridization barrier(s) separating both species.</p></div

<i>C</i>. <i>rubella</i> × <i>C</i>. <i>grandiflora</i> hybrid seed incompatibility involves multiple genetic loci.

<p>(A) Frequency of F2 plants derived from crosses of <i>C</i>. <i>grandiflora</i> × <i>C</i>. <i>rubella</i> producing defined rates of seed abortion when backcrossed to <i>C</i>. <i>rubella</i> maternal plants. (B) Theoretical distribution of F2 plants producing defined rates of seed abortion when assuming that one maternal locus from <i>C</i>. <i>rubella</i> negatively interacts with two or three paternal loci from <i>C</i>. <i>grandiflora</i>. Experimental data correspond to data shown in (A). Significance of the observed distributions with the predictions of the models has been tested by Chi-square and P>0.05 is marked by an asterisk. Ns, non significant. (C) LOD scores for paternal <i>C</i>. <i>grandiflora</i> QTLs affecting abortion of <i>C</i>. <i>rubella</i> × <i>C</i>. <i>grandiflora</i> hybrid seeds. The purple line (top) represents the LOD score of each marker and the purple line (bottom) represents the effects. The red line represents the significance threshold as estimated by 1,000 permutations.</p

Hybrid seed incompatibility between <i>C</i>. <i>rubella</i> and <i>C</i>. <i>grandiflora</i> correlates with endosperm cellularization defects.

<p>Sections of <i>C</i>. <i>rubella</i>, <i>C</i>. <i>grandiflora</i> and reciprocal hybrid seeds at 4–7 days after pollination (DAP). Scale bar 100μm.</p