Apomixis is a form of asexual reproduction that consists in cloning through seeds. In Limonium (Plumbaginaceae) species present a pollen-stigma dimorphism linked to a sporophytic self-incompatibility system associated with sexual and/ or apomictic reproductive modes. Previous work in other genera suggests that the emergence of apomixis is associated with hybridization and/or polyploidy. In this study, our goal was to test the ability of diploid and tetraploid species to hybridize and to evaluate the variate outcomes from these crosses. To achieve this, sexual diploid (L. nydeggeri, L. ovalifolium) and facultative apomict tetraploid (L. binervosum, L. dodartii) plants from cultivated material, previously cytogenetically and reproductively characterized, were used for experimental intra-and interspecific crosses. Genome sizes, ploidy levels and morphology were examined in the resulting progenies. Results showed a high production of viable seeds in particular in plants from tetraploid × diploid (heteroploid) crosses. In these crosses, some seedlings exhibited pleiocotyly (tricotyl, tetracotyl), while others showed polyembryony. In both homoploid (diploid × diploid) and heteroploid (tetraploid × diploid) crosses, most of the offspring plants were morphologically and in their ploidy similar to the female receiver, although some morphological abnormalities were found. Molecular progeny tests using the nrDNA ITS1-ITS2 sequence demonstrated an astounding range of diploid offspring plants originated from diploid × diploid crosses that were either genetically similar or distinct from parental plants. Although in intraspecific crosses most of the resulting progeny was diploid, one triploid plant was formed. Moreover, in homoploid interspecific crosses, neopolyploids (two tetraploid plants) were produced. Progeny plants from heteroploid crosses always showed nrDNA ITS1-ITS2 sequences identical to the parental plant used as female receiver. In conclusion, diploid homoploid crosses presented genetically diverse offspring arising from sexual reproduction. By contrast, heteroploid crosses generated clonal, maternal (apomictic) offspring. (Tables S1-S3, Fig. S1) and DNA sequence alignments (used to compare parental and progeny plants of nine homoploid and/or interploid crosses) are available from https://doi. Supplementary Material The Electronic Supplement

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The genus Limonium (sea lavenders) is characterized by various taxonomically challenging complexes with great karyological diversity, and chromosomes ranging in size from diminutive to large. In this biosystematic study, our goal was to investigate cytogenetic features of twelve sexual and asexual taxa in a particular part of their distribution range. Genome size, chromosome number variation, and other cytological features were investigated in a representative set of sea lavenders with distinct leaf venation, present in the coastal areas of the Iberian Peninsula, Morocco, France, and the Channel Islands. Flow cytometric genome size determinations were made in plants from natural populations. Cell and nuclear areas of epidermal cells, as well as, chromosome numbers and length were measured in plants from ex situ collections. Our findings revealed positive and significant correlations between holoploid genome size and cell size, nuclei area and chromosome total length. Tetraploid taxa with pinnately veined leaves had significant lower holoploid and monoploid genome sizes along with lower cell and nuclei areas and chromosome sizes than tetraploid taxa with parallelveined leaves. The results obtained are discussed in the context of breeding relationships known or suspected among these taxa. In conclusion, our results provide a basis for future evolutionary studies, and support that pollenstigma dimorphisms and genome size allied with chromosome counts might be important taxonomic traits in Limonium in apomictic/sexual groups.

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Ex situ plant collections established from seeds of natural populations are key tools for understanding mating systems of intricate taxonomic complexes, as in the Limonium Mill. genus (sea lavenders, Plumbaginaceae). Plants show a polymorphic sexual system associated to flower polymorphisms such as ancillary pollen and stigma and/or heterostyly that prevents self and intramorph mating. The main objectives of this study were to investigate the significance of pollen-stigma dimorphisms and the role of flower visitors in the reproductive output of hybrids arising from sexual diploids of Limonium ovalifolium complex and apomicts tetraploids of L. binervosum complex in an open cross-pollination experiment. Results showed that, similarly to parental plants, hybrids present inflorescence types, self-incompatible flowers, and produced regular pollen grains with the typical exine patterns, with medium to high viability. By contrast, apomicts show floral polymorphisms, inflorescences, and pollen grains of maternal phenotype but with low stainability. Several insects’ species visited the inflorescences of parental plants and both hybrids and apomicts and some of these insects carried A and/or B pollen grains on their bodies, especially Clepsis coriacana (Rebel) and Tapinoma sp. Insects’ floral visits to hybrids and apomicts seem to be independent of pollen fertility and plants’ reproductive modes. Both hybrids and apomicts were able to produce fertile seeds, although the latter showed more seedlings with developmental anomalies than the first plants. The findings demonstrate that there is a weak reproductive barrier between the diploid species of L. ovalifolium complex as they can hybridize and produce fertile hybrids, provided there is pollen transport by pollinator insects. This study supports that apomixis is a strong reproductive barrier between both L. ovalifolium and L. binervosum complexes but did not allow us to exclude reproductive interferences of apomict pollen into sexuals.