Stage-Dependent Persistence of Nucleated Endosperm Cells in Seeds of Limonium Sexual and Apomictic Species with Autonomous Endosperm Formation.

Limonium Mill. species present a polymorphic sexual system associated with flower polymorphisms like ancillary pollen and stigma, with sexual and/or apomictic reproduction. The aim of this study was to investigate the reproductive traits, test for autonomous apomixis, and assess seed formation in triploid Limonium algarvense and Limonium daveaui. Pollen-stigma combinations were determined and the number of flowers and seeds counted. Single-seed flow cytometry was performed using seeds in three phenological stages: immature (stage I), early maturing (stage II) and mature seeds (stage III). The findings revealed that all triploid plants were self-sterile and produced seeds in the absence of pollination. Despite L. daveaui having a higher number of flowers than L. algarvense, a significantly higher ratio of seeds/flowers was observed in the latter species. Stage-dependent endosperm developmental patterns were observed, with nucleated cells present in stage II seeds with a light brown or pinkish coat, and an embryo peak and an endosperm peak with the double ploidy level. Stage III seeds, with a dark brown coat, presented only an embryo peak. Additionally, a single hexaploid endosperm peak was detected in stage I seeds, revealing early initiation of the endosperm with nucleated cells prior to embryo development. The single 6C endosperm peak was always associated with shrunken and wrinkled or underdeveloped stage I seeds but was never detected in stage II seeds. Overall, our results support reproduction via asexually formed seeds with pollen-independent endosperm formation and allow the identification of phenological development stages and seed coat morphological markers associated with single-seed flow cytometric screening patterns in apomictic species.