Ovarian self-incompatibility, including pre-and post-zygotic reactions, is a complex mechanism for which we still lack many details relating to its function and significance. The joint presence of ovarian self-incompatibility with style polymorphism is a rare combination that is found in the genus Narcissus. Usually, style polymorphic species have heteromorphic (diallelic and linked to style length locus) incompatibility, which prevents fertilization between individuals of the same morph, thereby helping to maintain equal proportions of floral morphs in populations. However, when present, self-incompatibility in Narcissus is not linked to style polymorphism and cross-fertilization within each morph is possible. Hence, self-incompatibility in Narcissus is of particular interest when attempting to unravel the nature of the rejection reaction and aiming to assess possible cryptic differences in the fertilization process in intra-and inter-morph crosses, which might ultimately explain the wide variation of morph-ratio in the field. We examined the breeding system of Narcissus papyraceus, a style-dimorphic species that has biased morph ratios in most of its populations. We studied pollen-tube growth in the pistil and ovule fate after experimentally controlled hand pollinations. The growth of pollen tubes in self-and intra-and inter-morph crosses was similar up to the point of micropyle penetration in both morphs but, subsequently, a pre-zygotic failure appeared to affect male and female gametophytes in selfed pistils. A high proportion of ovules from self-pollinated flowers showed signs of collapse and self-pollen tubes were blocked or behaved abnormally before entering the embryo sac. Selfincompatibility was stronger in the long-styled morph than in the short-styled morph. We did not find any conclusive sign of differential functioning between intra-and inter-morph cross-pollinations in any morph. These results enable us to rule out the possible effects of pollen-pistil interactions in N. papyraceus as a cause of morph-ratio biases and confirm the exceptional nature of the self-incompatibility mechanism in this polymorphic species.

Heterostyly and related polymorphisms (e.g. stigma-height dimorphism) have been used as model systems for studying the origin and maintenance of plant population variability. Stigma-height dimorphism frequently occurs in Narcissus and is associated with a particular flower shape. In the present study, we describe a new, peculiar case of stigma-height dimorphism in Narcissus broussonetii, a species on the margin of the geographical distribution of the genus. We determined the stylar condition of N. broussonetii and its variation across populations, analyzed perianth morphology and its relationship with stylar variation, and compared this species with other stylar dimorphic species of the genus. We also studied the incompatibility system and pollination ecology of the species. Narcissus broussonetii is a style-dimorphic species, as suggested in early studies that were subsequently neglected, and displays unusual flower morphology, with a long floral tube and a virtual absence of a corona. The species shows a late-(ovarian) acting incompatibility system and crosses within and between morphs are fertile. We observed short-tongued diurnal and long-tongued nocturnal pollinators. Our findings confirm that the presence of the observed dimorphism across populations is most probably the result of the joint action of a nonheteromorphic incompatibility system, extremely long and narrow floral tubes, and a combined role of short-and long-tongued pollinators.

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In flowering plants, the shift from outcrossing to selfing is associated with a set of correlated changes in morphological and reproductive features known as the ‘selfing syndrome’. Species of the sub-endemic Mediterranean genus Centaurium Hill (Gentianaceae) exhibit a wide array of flower traits related to pollination biology and different ploidy levels. We explored if the evolutionary transitions of seven flower traits and life cycle, typically associated with the selfing syndrome, are related to polyploidy, diversification patterns, divergence times and the geological and climatic history of the Mediterranean Basin. Using 26 species of Centaurium we reconstructed a phylogenetic tree, inferred the ancestral states of the selected traits, estimated their phylogenetic signal and tested the correlative evolution among them. We found a significant increase in diversification rates during the Quaternary. Anther length, flower size, herkogamy and polyploidy undergo rapid state transitions without phylogenetic signal that could be the result of adaptation for selfing. Changes in character states do not show evidence of correlative evolution among them, as would be predicted during the evolution of selfing syndrome. The evolution of reproductive traits in Centaurium has probably relied on a more diverse array of drivers than just reproductive assurance or polyploidy events.

The floral phenotype plays a main role in the attraction and fit of pollinators. Both perianth traits and the positioning of sex organs can be subjected to natural selection and determine nonrandom mating patterns in populations. In stylar-polymorphic species, the Darwinian hypothesis predicts increased mating success between individuals with sex organs at equivalent heights (i.e. with higher reciprocity).We used paternity analyses in experimental populations of a stylar-dimorphic species. By comparing the observed mating patterns with those expected under random mating, we tested the effects of sex organ reciprocity and perianth traits on mating success. We also analysed phenotypic selection on perianth traits through female and male functions.The (dis)similarity of parental perianth traits had no direct effects on the mating patterns. Sex organ reciprocity had a positive effect on mating success. Narrow floral tubes increased this effect in upper sex organs. Perianth traits showed little signs of phenotypic selection. Female and absolute fitness measures resulted in different patterns of phenotypic selection.We provide precise empirical evidence of the Darwinian hypothesis about the functioning of stylar polymorphisms, demonstrating that mating patterns are determined by sex organ reciprocity and only those perianth traits which are critical to pollinator fit.