Flow cytometric seed screen data are consistent with models of chromosome inheritance in asymmetrically compensating allopolyploids.

Angiosperms have evolved a mechanism of double fertilization, which results in the production of a separate embryo (new individual) and endosperm (nutritive tissue). The flow cytometric seed screen (FCSS) was developed to infer plant reproduction modes based on endosperm-to-embryo DNA content ratio (Pind ). A ratio of 1.5 indicates sexual reproduction, whereas higher values of ≥2.0 are consistent with apomixis. Although FCSS has been successfully applied to the study of sexual and asexual plants, the limits of FCSS and particularly its potential for determination of reproduction modes in hemisexual plants have not been explored. Here, we evaluated the application of FCSS to the study of reproduction modes in two asymmetrically compensating allopolyploids (ACAs), Onosma arenaria and Rosa canina. These two species are characterized by the presence of asexually inherited univalent-forming and sexually inherited bivalent-forming chromosome sets. They both use asymmetric meiosis, which eliminates univalent-forming chromosome sets from the male gamete and retains them in the female gamete. Different chromosomal behavior in male and female meiosis in these plants is reflected in different theoretically derived Pind values, which deviate from a sexual 1.5 value. Here, we determined Pind FCSS-based values in seeds of ACAs, and compared the results to sexual species. As expected, we determined that the mean Pind is 1.51, 1.52, and 1.52 in the sexual plants, that is, Capsella bursa-pastoris, Crataegus monogyna, and O. pseudoarenaria, respectively. In the ACAs, different mean Pind values were determined for O. arenaria (1.61) and R. canina (1.82). These values are consistent with the theoretical Pind values determined based on models of chromosome inheritance. This study highlights the precision of flow cytometry in determining DNA content and it's utility in screening reproduction modes. Additionally, it advocates for more in-depth investigations into rapid screening of accessions where the Pind ratio has deviated from the 1.5 value typical of sexual species, which may indicate meiotic irregularities.

Pollen precedence in sexual Potentilla puberula and its role as a protective reproductive barrier against apomictic cytotypes.

Cross-pollination is a major factor determining the demographic dynamics of mixed-ploidy populations. Typically, rare cytotypes are suppressed due to reduced female fertility by losing gametes in heteroploid crosses (i.e., through minority cytotype exclusion). In species with reproductive differentiation into sexual and apomictic cytotypes, sexuals might be reproductively suppressed by apomicts (or transformed due to introgression of apomixis genes). Pollen precedence potentially acts as a post-pollination pre-fertilization barrier protecting sexuals against their apomictic counterparts. We estimated the role of pollen precedence as a barrier against cross-fertilization of tetraploid sexuals by penta- and heptaploid gametophytic apomicts in Potentilla puberula (Rosaceae) by means of controlled crosses, and inference of the paternity through DNA ploidy estimation of embryos. Individuals from five regions spanning an elevational and biogeographic gradient were used to account for the variation in the relative frequencies of reproductive modes across the study area. We tested (1) whether the application of heteroploid pollen (sexual × apomictic) causes a reduction of seed yield compared to homoploid crosses (sexual × sexual), and (2) if so, whether pollen precedence recovers the seed yield in simultaneous applications of pollen from sexuals and apomicts (mixed-ploidy). Seed yield was significantly lower in hetero- than in homoploid crosses. We found clear evidence for precedence of homoploid pollen, despite a 13% to 15% of embryos experienced a change in ploidy due to heteroploid fertilizations. Thus, our study indicates that pollen precedence operates as a barrier against intercytotype fertilization in P. puberula, promoting the integrity of the sexual cytotype and their co-existence with apomictic individuals.

Pathways to polyploidy: indications of a female triploid bridge in the alpine species Ranunculus kuepferi (Ranunculaceae).

Polyploidy is one of the most important evolutionary processes in plants. In natural populations, polyploids usually emerge from unreduced gametes which either fuse with reduced ones, resulting in triploid offspring (triploid bridge), or with other unreduced gametes, resulting in tetraploid embryos. The frequencies of these two pathways, and male versus female gamete contributions, however, are largely unexplored. Ranunculus kuepferi occurs with diploid, triploid and autotetraploid cytotypes in the Alps, whereby diploids are mostly sexual, while tetraploids are facultative apomicts. To test for the occurrence of polyploidization events by triploid bridge, we investigated 551 plants of natural populations via flow cytometric seed screening. We assessed ploidy shifts in the embryo to reconstruct female versus male gamete contributions to polyploid embryo and/or endosperm formation. Seed formation via unreduced egg cells (BIII hybrids) occurred in all three cytotypes, while only in one case both gametes were unreduced. Polyploids further formed seeds with reduced, unfertilized egg cells (polyhaploids and aneuploids). Pollen was highly variable in diameter, but only pollen >27 µm was viable, whereby diploids produced higher proportions of well-developed pollen. Pollen size was not informative for the formation of unreduced pollen. These results suggest that a female triploid bridge via unreduced egg cells is the major pathway toward polyploidization in R. kuepferi, maybe as a consequence of constraints of endosperm development. Triploids resulting from unreduced male gametes were not observed, which explains the lack of obligate sexual tetraploid individuals and populations. Unreduced egg cell formation in diploids represents the first step toward apomixis.