Based on the biological species concept, two species are considered distinct if reproductive barriers prevent gene flow between them. In Central Europe, the diploid species Arabidopsis lyrata and Arabidopsis arenosa are genetically isolated, thus fitting this concept as "good species." Nonetheless, interspecific gene flow involving their tetraploid forms has been described. The reasons for this ploidy-dependent reproductive isolation remain unknown. Here, we show that hybridization between diploid A. lyrata and A. arenosa causes mainly inviable seed formation, revealing a strong postzygotic reproductive barrier separating these two species. Although viability of hybrid seeds was impaired in both directions of hybridization, the cause for seed arrest differed. Hybridization of A. lyrata seed parents with A. arenosa pollen donors resulted in failure of endosperm cellularization, whereas the endosperm of reciprocal hybrids cellularized precociously. Endosperm cellularization failure in both hybridization directions is likely causal for the embryo arrest. Importantly, natural tetraploid A. lyrata was able to form viable hybrid seeds with diploid and tetraploid A. arenosa, associated with the reestablishment of normal endosperm cellularization. Conversely, the defects of hybrid seeds between tetraploid A. arenosa and diploid A. lyrata were aggravated. According to these results, we hypothesize that a tetraploidization event in A. lyrata allowed the production of viable hybrid seeds with A. arenosa, enabling gene flow between the two species.interspecies hybrid seed failure | Arabidopsis | reproductive isolation | endosperm cellularization | EBN

After meiosis, an unequal cell division generates the male gamete lineage in flowering plants. The generative cell will undergo a second division, giving rise to the two gametes, i.e. the sperm cells. The other cell will develop into the vegetative cell that plays a crucial role in pollen tube formation and sperm delivery. Recently, the vegetative cell has been suggested to be important for programming of the chromatin state in sperm cells and/or the resulting fertilization products. Blocking the initial unequal division genetically, we first highlight that the default differentiation state after male meiosis is a vegetative fate, which is consistent with earlier work. We find that uni-nucleated mutant microspores differentiated as wild-type vegetative cells, including chromatin remodeling and the transcriptional activation of transposable elements. Moreover, live-cell imaging revealed that this vegetative cell is sufficient for the correct guidance of the pollen tube to the female gametes. Hence, we conclude that vegetative cell differentiation and function does not depend on the formation or presence of the actual gametes but rather on external signals or a cell-autonomous pace keeper.