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Non-random mating in plant populations can be influenced by numerous reproductive and demographic factors, including floral morphology and inter-plant distance. Here, we investigate patterns of outcrossed mating through male function in Narcissus triandrus, a tristylous, bee-pollinated wild daffodil from the Iberian Peninsula, to test pollen transfer models which predict that floral morphology promotes asymmetrical mating and biased morph ratios. Unlike other tristylous species, N. triandrus has an incompatibility system that permits intra-morph mating and long-level rather than mid-level stamens in the L-morph. Incomplete sex-organ reciprocity should result in significant intra-morph mating in the L-morph. We measured mating patterns in two L-biased populations--dimorphic (two style morphs) and trimorphic (three style morphs)--using multilocus genotyping and maximum-likelihood-based paternity analysis. We also examined the spatial distribution of style morphs and neutral markers to investigate the potential consequence of spatially restricted mating on morph ratios. As predicted, we detected significant amounts of intra-morph mating in the L-morph in both populations. Pollen transfer coefficients generally supported predictions based on the Darwinian hypothesis that anthers and stigmas of equivalent level promote pollinator-mediated cross-pollination in heterostylous populations. There was evidence of significant spatial aggregation of both style morphs and neutral markers in populations of N. triandrus, probably as a result of restricted pollen and seed dispersal. Our results provide empirical support for theoretical models of pollen transfer, which indicate that the commonly observed L-biased morph ratios in Narcissus species result from significant intra-morph mating in the L-morph because of its atypical floral morphology.

We investigated cpDNA sequence and nuclear microsatellite variation among populations of the wild daffodil Narcissus triandrus to examine the role of historical vs. contemporary forces in shaping population structure, morphological differentiation and sexual-system evolution. This wide-ranging heterostylous species of the Iberian Peninsula is largely composed of two allopatric varieties (vars. cernuus and triandrus), and populations with either stylar trimorphism or dimorphism. Dimorphic populations only occur in var. triandrus, are mainly restricted to the northwestern portion of the species range, and uniformly lack the mid-styled morph (M-morph). Chloroplast DNA (cpDNA) sequence variation revealed strong geographical structuring and evidence for a fragmentation event associated with differentiation of the two varieties. In var. triandrus, population fragmentation, restricted gene flow and isolation-by-distance were also inferred. Significant differences in genetic diversity and population structure between the two varieties likely reflect historical and contemporary differences in demography and gene flow among populations. Discordance between cpDNA markers and both microsatellite and morphological variation indicate that hybridization has occurred between the two varieties at contact zones. There were no differences in genetic diversity or population structure between dimorphic and trimorphic populations, and chloroplast haplotypes were not associated with either sexual system, indicating transitions in morph structure within each maternal lineage. M-morph frequencies were positively correlated with differentiation at microsatellite loci, indicating that the evolutionary processes influencing these neutral markers also influence alleles controlling the style morphs.

Mating patterns in plant populations are influenced by interactions between reproductive traits and ecological conditions, both factors that are likely to vary geographically. Narcissus triandrus, a wide-ranging heterostylous herb, exhibits populations with either two (dimorphic) or three (trimorphic) style morphs and displays substantial geographical variation in demographic attributes and floral morphology. Here, we investigate this variation to determine if demography, morphology, and mating system differ between the two sexual systems. Our surveys in Portugal and NW Spain indicated that dimorphic populations were less dense, of smaller size, and had larger plants and flowers compared to trimorphic populations. Outcrossing rates estimated using allozyme markers revealed similar outcrossing rates in dimorphic and trimorphic populations (t(m) dimorphic=0.759; t(m) trimorphic=0.710). All populations experienced significant inbreeding in progeny (mean F=0.143). In contrast, parental estimates of inbreeding were not significantly different from zero (mean F=0.062), implying that few inbred offspring survive to reproductive maturity due to inbreeding depression. Although the majority of inbreeding results from selfing, significant levels of biparental inbreeding were also detected in eight of the nine populations (mean s(s)-s(m)=0.081). Density was negatively associated with levels of selfing but positively associated with biparental inbreeding. Population size was positively associated with outcrossing but not biparental inbreeding. There were no consistent differences among the style morphs in outcrossing or biparental inbreeding indicating that the maintenance of trimorphism vs dimorphism is unlikely to be associated with inbreeding of maternal parents.

The spatial context of reproduction is of crucial importance to plants because of their sessile habit. Since pollen and seed dispersal is often restricted, mating success is likely to depend on the quantity and quality of mates in local neighbourhoods. Here we use neighbourhood models to investigate the spatial ecology of pollination and mating in Narcissus assoanus, a sexually polymorphic plant with two mating morphs that differ in style length. By mapping individuals in eight populations from southwestern France, we investigated the influence of the density and morph identity of plants at different spatial scales on variation in female fertility. By using inferences on the expected patterns of pollen transfer based on floral morphology, we were able to predict the quantitative relations between local morph ratios and variation in fertility. Our analyses revealed differences in the spatial clustering of morphs and in their response to plant density and morph identity within local neighbourhoods. Mating success in N. assoanus was characterized by both density- and frequency-dependent processes, a condition that may be a general feature of the spatial ecology of plant mating.

In tristylous populations, mating-type frequencies are governed by negative frequency-dependent selection typically resulting in equal morph ratios at equilibrium. However, Narcissus triandrus generally exhibits long-styled (L)-biased populations with a deficiency of the mid-styled (M)-morph. Here we used a pollen-transfer model and measurements of female fertility in natural populations to investigate whether these uneven morph ratios were associated with variation in female reproductive success. Our theoretical analysis demonstrated that morph ratio bias can result from maternal fitness differences among the morphs, and that these effects were magnified by asymmetrical mating. In nine out of 15 populations of N. triandrus, seed set differed significantly among the morphs, but pollen limitation occurred in only two of 11 populations investigated. Average seed set of the M-morph was positively associated with its frequency in populations. Flower size was negatively correlated with the seed set of the M-morph. Our results suggest that interactions between mating patterns and female fertility are responsible for variation in morph frequencies and loss of the M-morph from tristylous populations of N. triandrus.

Background and Aims: Floral design in self-compatible plants can influence mating patterns. This study investigated Narcissus longispathus, a self-compatible bee-pollinated species with wide variation in anther-stigma separation (herkogamy), to determine the relationship between variation in this floral trait and the relative amounts of cross- and self-fertilization., Methods: Anther-stigma separation was measured in the field in six populations of N. longispathus from south-eastern Spain. Variation in herkogamy during the life of individual flowers was also quantified. Multilocus outcrossing rates were estimated from plants differing in herkogamy using allozyme markers., Key Results: Anther-stigma separation varied considerably among flowers within the six populations studied (range = 1-10 mm). This variation was nearly one order of magnitude larger than the slight, statistically non-significant developmental variation during the lifespan of individual flowers. Estimates of multilocus outcrossing rate for different herkogamy classes (t(m) range = 0.49-0.76) failed to reveal a monotonic increase with increasing herkogamy., Conclusions: It is suggested that the lack of a positive relationship between herkogamy and outcrossing rate, a result that has not been previously documented for other species, could be mostly related to details of the foraging behaviour of pollinators.

Narcissus, the daffodil genus, exhibits an unusual diversity of sexual systems, with populations that are monomorphic, dimorphic or trimorphic for style length. Associated with this variation are striking differences among species in floral morphology and pollination biology. This diversity provides an opportunity to investigate the evolution of mating polymorphisms, and to determine how floral morphology promotes transitions among sexual systems. Because of the absence of heteromorphic incompatibility in Narcissus, floral morphology plays a key role in governing patterns of outcrossed mating. Phylogenetic evidence indicates that stylar monomorphism is ancestral in the genus, with multiple origins of stylar polymorphism, including independent origins of stigma-height dimorphism, distyly and tristyly. Sexual polymorphisms have evolved only in lineages with narrow floral tubes that are pollinated by Lepidoptera and/or long-tongued bees. Populations of polymorphic Narcissus species are typically dominated by the long-styled morph and display imperfect reciprocity in the positions of sexual organs. These features are consequences of the unusual association between stylar polymorphism and a self-incompatibility system that permits intramorph mating.

Floral traits that reduce self-pollination in hermaphroditic plants have usually been interpreted as mechanisms that limit the genetic consequences of self-fertilization. However, the avoidance of sexual conflict between female and male function (self-interference) may also represent an important selection pressure for the evolution of floral traits, particularly in self-incompatible species. Here, we use experimental manipulations to investigate self-interference in Narcissus assoanus, a self-incompatible species with a stigma-height dimorphism in which the degree of spatial separation between sex organs (herkogamy) differs strikingly between the long- and short-styled morphs (hereafter L- and S-morphs). We predicted that weak herkogamy in the L-morph would cause greater self-pollination and hence self-interference. Experimental self-pollination reduced seed set when it occurred prior to, or simultaneously with, cross-pollination in the L-morph, but only if it occurred prior to cross-pollination in the S-morph. In the field, autonomous self-pollination was greater in the L-morph than the S-morph, but we found no evidence that self-interference reduced maternal or paternal fitness in either morph. One-day-old flowers of the L-morph have reduced stigma receptivity and hence exhibit protandry, whereas stigma receptivity and anther dehiscence are concurrent in the S-morph. This suggests that the two style morphs have alternative strategies for reducing self-interference: dichogamy in the L-morph and herkogamy in the S-morph. These results provide insight into the mechanisms that reduce sexual conflict in hermaphrodite plants and are of significance for understanding the evolution and maintenance of sexual polymorphisms.

In sexually polymorphic species, reproductive morphology governs mating patterns and the character of negative frequency-dependent selection. If local environmental conditions cause sexual morphs to differ between populations, then frequency-dependent selection should create corresponding geographic variation in morph frequencies. We investigate this relation with a model of morph-ratio evolution and analysis of geographic variation in the heterostylous plant Narcissus triandrus. Unlike other tristylous species, N. triandrus possesses both imperfect reciprocity among morphs in sex-organ position and a self-incompatibility system that permits outcrossing within and between morphs. We sampled 137 populations throughout the Iberian Peninsula for floral-morph ratios, and measured floral morphology in 31 populations. Morph ratios exhibited three atypical features: (1) predominance of the long-styled (L) morph; (2) absence of the mid-styled (M) morph from 17.5% of populations; and (3) a negative relation between the frequencies of the L and M morphs among populations. Morph ratios varied geographically, with decreasing frequency of the M morph from the southeast to the northwest of the species' range. Much of this variation accompanied allometric change in the positions of sex organs, especially the mid-level organs, with the M morph declining in frequency and ultimately being lost in large-flowered populations. Using multivariate multiple regression, we demonstrate that variation in floral morphology among populations predicts this geographic variation in morph frequencies. Our theoretical analysis illustrates that patterns of pollen transfer governed by imperfect sex-organ reciprocity can select for unequal equilibrium morph ratios like those observed for N. triandrus. We interpret the L-biased morph ratios and the unusual morphology of N. triandrus as a consequence of its atypical intramorph compatibility system.

Despite the importance of Narcissus to ornamental horticulture, there have been no population genetic studies of wild species, many of which have narrow distributions. Here, we measure selfing rates and levels of genetic diversity at allozyme loci in six populations of Narcissus longispathus, a self-compatible daffodil endemic to a few mountain ranges in southeastern Spain. The populations were distributed among four distinct river valleys encompassing two main watersheds in the Sierra de Cazorla mountains. Selfing rates averaged 0.37 (range 0.23-0.46), resulting in significant inbreeding coefficients for the progeny (f = 0.324). In contrast, estimates of inbreeding in parental genotypes were not significantly different from zero (f = 0.001), indicating that few selfed offspring survive to maturity because of inbreeding depression. Species-wide estimates of genetic diversity for the six populations were P(s) = 0.38, H(es) = 0.119 and A(s) = 1.27 with significant genetic differentiation among populations theta = 0.15. The observed patterns of genetic differentiation among populations are likely influenced by the mating system, and a combination of local topography, watershed affinities and gene flow., (March 2004)

Negative frequency-dependent selection is a major selective force maintaining sexual polymorphisms. However, empirical demonstrations of frequency-dependent reproductive success are rare, particularly in plants. We investigate this problem by manipulating the frequencies of style morphs in a natural population of Narcissus assoanus, a self-incompatible herb with style-length dimorphism and intra-morph compatibility. We predicted that the reproductive success of morphs would vary negatively with their frequency because of the effects of morph-specific differences in sex-organ position on patterns of pollen transfer. This prediction was generally supported. The fruit and seed set of the two morphs did not differ significantly in plots with 1 : 1 morph ratios. However, short-styled plants produced significantly fewer seeds than long-styled plants in monomorphic plots, and significantly more seeds than long-styled plants in plots with 'long-biased' morph ratios. These patterns indicate that in the absence of physiological barriers to intra-morph mating, negative frequency-dependent selection contributes to the maintenance of stylar polymorphism through inter-morph pollen transfer. Our experimental results also provide insights into the mechanisms governing the biased style-morph ratios in populations of Narcissus species.