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Plant populations invading new environments might compromise their fitness contribution to the next generation, because of the lack of native specialist pollinators and/or potential mates. Thus, changes in plant mating system and traits linked to it are expected in populations colonising new environments where selection would favour selfing and floral traits that maximise reproductive output. To test this, we studied native (Mexico) and non-native (Spain) populations of the obligate sexual reproducing annual weed Datura stramonium. Flower size, herkogamy, total number of seeds per plant, number of visits by and type of pollinators, and inbreeding depression were assessed in native and non-native populations. Finally, we measured phenotypic selection on corolla size and herkogamy in each population. Flower size and herkogamy showed wide and similar variation in both ranges. However, the largest average flower size was found in one non-native population whereas the highest average positive herkogamy was detected in one native population. On average, flowers in the native range received more visits by pollinators. Hawkmoths were the main visitors in the native populations while only bees were observed visiting flowers in Spain's populations. Only in the native range was inbreeding depression detected. Selection to reduce herkogamy was found only in one native population. Absence of both inbreeding depression and selection on floral traits suggest a change in mating system of D. stramonium in a new range where generalist pollinators may be promoting high reproductive success. Selection against deleterious alleles might explain the reduction of inbreeding depression, promoting the evolution of selfing.

Spatial (herkogamy) or temporal (dichogamy) separation of sex organs are mechanisms considered to restrict self-pollination and promote outcrossing. Additionally, avoidance of self-interference is proposed to be the driving force for the evolution of these mechanisms, particularly in self-incompatible species. However, species with anthers and stigmas at different levels may increase the rate of imprecise pollen transfer, resulting in pollen discounting. Non-reciprocal stylar dimorphism has been considered a transitional, unstable stage towards the evolution of reciprocal style dimorphism (distyly), to simultaneously avoid interference and lack of precision. In this study we investigate the spatial and temporal separation of sex organs in a population of the style dimorphic and self-incompatible Narcissus broussonetii and their consequences in the reciprocity between the sex organs of morphs and their fecundity. First, we evaluated the relative growth of sex organs after anthesis. Then, we studied the stigma receptivity along the flower lifespan including its effect on seed production in both morphs. Finally, given the weak reciprocity between the sex organs of morphs of this species, we estimated population genetic diversity parameters in Long- and Short-styled plants to explore differences between them as a result of rates of inbreeding due to different mating strategies. We observed that Long-styled plants and Short-styled plants present different strategies to avoid sexual interference and both of them had negative consequences in the reciprocity between the sex organs of morphs. Long-styled plants exhibited a delay in stigma receptivity and a higher growth rate of the style after anthesis, while Short-styled plants presented higher herkogamy and no delay in stigma receptivity. These findings suggest that the avoidance of self-interference, in stylar dimorphic Narcissus species, seems to be more critical than improving of reciprocity between the sex organs of morphs. This might explain why reciprocal herkogamy (distyly) is rare in the genus., In this study we investigated the spatial and temporal separation of sex organs in a population of the style dimorphic and self-incompatible Narcissus broussonetii and their consequences in the reciprocity between the sex organs of morphs and their fecundity. We observed that Long-styled plants and Short-styled plants present different strategies to avoid sexual interference and both of them had negative consequences in the reciprocity between the sex organs of morphs. Long-styled plants exhibited a delay in stigma receptivity and a higher growth rate of the style after anthesis, while Short-styled plants presented higher herkogamy and no delay in stigma receptivity. These findings suggest that the avoidance of self-interference in stylar dimorphic Narcissus species seems to be more critical than improving reciprocity between the sex organs of morphs.

Studies of fruit or seed heteromorphism (the presence of two or more fruit or seed morphs within an individual) provide valuable information about the influence of environmental variation on the evolution of reproductive strategies. Such heteromorphisms are more likely to be favoured by selection in annual species, because short-lived plants are in a disadvantaged position to gather information about environmental conditions their offspring are likely to face when they germinate, compared with perennial counterparts. Here, we describe fruit heteromorphism of Tithonia diversifolia, a perennial shrub found in habitats with contrasting environmental conditions. We tested whether fruit morphs differ in weight and germination success, and whether populations differed in fruit morph ratio. To do this, we first determined fruit morph ratio of three populations from contrasting ecosystems (evergreen high forest, evergreen medium forest and oak-pine forest), and compared germination success in a common-garden setting. In all populations, two fruit morphs were consistently distinguishable within the same capitulum. Although fruit morph ratios differed significantly across populations. Furthermore, fruit morphs differed in weight and germination success, but the direction of the difference was dependent on the population. These results suggest that fruit heteromorphism in the perennial T. diversifolia might represent distinct reproductive strategies allowing it to adapt to uncertain environmental conditions.

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.

•Heterostyly is a sex polymorphism that has challenged evolutionary biologists eversince Darwin. One of the lineages where heterostyly and related stylar conditionsappear more frequently isLinum(Linaceae). This group is particularly suitable fortesting competing hypotheses about ancestral and transitional stages on the evolution-ary building up of heterostyly.•We generated a phylogeny ofLinumbased on extensive sampling and plastid andnuclear DNA sequences, and used it to trace the evolution of character states of stylepolymorphism. We also revised available data on pollination, breeding systems, andpolyploidy to analyse their associations.•Our results supported former phylogenetic hypotheses: the paraphyly ofLinumandthe non-monophyly of current taxonomic sections. Heterostyly was common in thegenus, but appeared concentrated in the Mediterranean Basin and the South AfricanCape. Ancestral character state reconstruction failed to determine a unique state as themost probable condition for style polymorphism in the genus. In contrast, approachherkogamy was resolved as ancestral state in some clades, giving support to recenthypotheses. Some traits putatively related to heterostyly, such as life history and poly-ploidy, did show marginal or non-significant phylogenetic correlation, respectively.Although pollinator data are limited, we suggest that beeflies are associated with speci-fic cases of heterostyly.•The consistent association between style polymorphism and heteromorphic incompat-ibility points to ecological factors as drivers of the multiple evolution of style polymor-phism inLinum. Albeit based on limited evidence, we hypothesised that specialisedpollinators and lack of mating opportunities drive evolution of style polymorphismand loss of the polymorphism, respectively. Ministerio de Economía, Industria y Competitividad de España (MINECO)-CGL2013-45037-P, CGL2010-11379-E, CGL2009-12565 y CGL2006-13847-CO2-01

Narcissus papyraceus is a style dimorphic species with two floral forms, with anthers at similar height and stigmas above (long-styled L) and below (short-styled S) the anther level. The species is self-incompatible, but intra- and inter-morph compatible. Populations are either dimorphic (including both morphs) in the region of the Strait of Gibraltar, or L-monomorphic (with only L plants) in the inland of the Iberian Peninsula. This variation correlates with the most common floral visitors, being primarily long-tongued and short-tongued pollinators, respectively, a rare condition in Mediterranean plants. The maintenance of S-flowers relies on long-tongued insects, as only those deliver pollen to short-styled stigmas. Narcissus flowers present a long and narrow tube, at the bottom of which nectar accumulates, and a floral corona, which has been proposed as an important trait for the attraction of pollinators. Here we tested the importance of the corona on pollination of L and S flowers. We described UV reflectance patterns of the corona and tepals, and characterised VOCs in intact flowers and flowers with trimmed coronas. We also conducted a field experiment in the dimorphic and monomorphic region to estimate the importance of corona removal on seed production in stands with solitary plants and in groups to control for compatible pollen limitation. Reflectance was higher in the tepals than the corona, although both traits presented a reflectance peak around 450 nm wavelength. L- and S-flowers produced similar volatiles, regardless of the manipulation of the corona. Across dimorphic and monomorphic regions, S-flowers with the corona removed suffered a reduction in seed production of ca. 50%, while seed production remained similar in L flowers both with the corona intact and removed. Plants in solitary stands suffered a strong reduction in seed production, which was more pronounced in the monomorphic region. Our results suggest that the corona in Narcissus is more important for the pollination of S-flowers, which generally have lower seed production compared to L-flowers. Taken together, these results suggest that the floral corona indirectly plays an important role for maintenance of the polymorphism. Junta de Andalucía P09-RNM-5280, PAIDI, RNM210 Dirección General de Investigación Científica y Técnica PB98-1144, REN2001-4738-E, BOS 2003-07924-CO2-01, CGL2004-22246- E, CGL2006-13847-CO2, CGL2009-12565, CGL2013-45037-P

Summary In hermaphroditic flowers, reciprocal herkogamy, for example heterostyly, enhances pollen transfer between floral morphs (disassortative pollination) while avoiding self-interference between sexual organs. By contrast, disassortative pollination might be compromised in style-dimorphic flowers, which lack perfect reciprocity between the two floral morphs. This suboptimal functioning has been considered to explain why stylar dimorphism is rare in nature. Some style-polymorphic species receive a wide array of floral visitors, including long-tongued insects that feed on nectar and short-tongued insects that feed on pollen. Differences in the morphology and behaviour of these two insect types could be manifested as different pollination patterns in each floral morph. By observing individual pollinators and pollen deposition and delivery in small field-based experimental arrays with emasculated and intact flowers, we studied pollination patterns mediated by different insect types (long- and short-tongued) in the two floral morphs (long- and short-styled) of the style-dimorphic Narcissus papyraceus. We investigated the patterns of pollen transfer between and within style morphs in cross-pollinations, as well as self-pollination and pollen removal rates, for each floral morph mediated by each insect type. Long-tongued insects were efficient pollinators of both floral morphs as they removed little pollen from the anthers but deposited comparatively large amounts on the stigmas. Although disassortative pollen transfer and assortative pollen transfer were equally high to the long-styled morph, the former prevailed in the short-styled morph. Short-tongued insects removed large amounts of pollen from the anthers but deposited only a few pollen grains on the long-styled stigmas and a negligible number of grains on short-styled stigmas regardless of the morph of the donor. Synthesis. In this study, we provide empirical support for the hypothesis that, under the action of long-tongued pollinators, pollen transfer patterns in the stylar-dimorphic Narcissus papyraceus resemble in part those of heterostylous species. In addition, we found that short-tongued insects act mostly as pollen thieves, thereby limiting the male fitness of both style morphs, besides depleting the female fitness of S-morph plants. In view of these results, we propose that the differing pollination efficiencies of floral visitors, in addition to their frequency, are key in determining the morph ratio of populations in this Narcissus.

Darwin's early work on heterostyly and related style polymorphisms (the presence of two or three style morphs within a population) generated much interest to understand how precise interactions between ecological and genetic mechanisms influence the evolution of floral diversity. Here we tested three key hypotheses proposed to explain the evolution of heterostyly: (i) the presence of self-incompatibility; (ii) the role of pollinators in promoting dissasortative mating; and (iii) floral architecture, which restricts pollinators' movements and ensures more exact pollen deposition on their bodies. We combined data from experiments, field observations, and published studies to test whether evolution of style polymorphism in Narcissus is driven by the incompatibility system, pollinator guilds, or floral architecture, within a phylogenetic framework. Neither differences in pollinator environment nor the presence of genetic self-incompatibility were correlated with presence of style polymorphism. However, our results indicate that the evolution of style polymorphism was driven by the presence of a narrow and long floral tube.

Aim Our aims were: (1) to reconstruct the phylogenetic relationships of daffodils (Narcissus), focusing on the lowland subgenus Hermione and the mountain section Apodanthi; (2) to estimate the temporal setting of diversification; (3) to reconstruct the migration patterns of the lineages; and (4) to examine the microevolutionary differentiation of the wide-ranging Narcissus tazetta group across the Mediterranean. Location The Mediterranean Basin. Methods Plastid (trnT–L, trnL–F and ndhF) sequences were obtained from 63 populations representing 23 species of Narcissus and combined with published data from 16 species. Phylogenetic relationships and dating were inferred by Bayesian analysis based on geological events and divergence estimates of closely related taxa. A dispersal–extinction–cladogenesis analysis was performed using maximum likelihood methods to infer ancestral geographical distributions, and phylogeographical reconstruction was performed using coalescence analysis. Results Subgenus Hermione is not recognized as a monophyletic group because two of the nine species were found to have a close relationship with the subgenus Narcissus. The results on section Apodanthi confirmed previous findings of its monophyly and phylogenetic relationships within this mountain group. Molecular dating and ancestral range reconstructions suggest that the ancestor of Narcissus originated in the Iberian Peninsula during the Late Oligocene–Early Miocene. Eastward expansion of the lineage range proceeded from the western Mediterranean and involved colonization of mountain ranges in northern Africa. The phylogeography of the N. tazetta group revealed a widespread distribution of certain haplotypes, suggesting wide dispersal and a high level of colonization in the Mediterranean Basin. Main conclusions Our study points to the role of three key historical events in Narcissus diversification: tectonic shifts of the Alboran domain in the western Mediterranean, the Messinian salinity crisis, and the onset of the Mediterranean climate followed by periods of repeated glaciation. Diversification of section Apodanthi probably resulted from allopatric speciation, while subgenus Hermione may have shown more sympatric speciation and high dispersal, despite the lack of apparent adaptations to long-distance dispersal. This is best exemplified by the presence of both ancestral and recent haplotypes of N. tazetta across the Mediterranean.

Different pollinators can exert different selective pressures on floral traits, depending on how they fit with flowers, which should be reflected in the patterns of variation and covariation of traits. Surprisingly, empirical evidence in support of this view is scarce. Here, we have studied whether the variation observed in floral phenotypic integration and covariation of traits in Narcissus species is associated with different groups of pollinators. Phenotypic integration was studied in two style dimorphic species, both with dimorphic populations mostly visited by long-tongued pollinators (close fit with flowers), and monomorphic populations visited by short-tongued insects (loose fit). For N. papyraceus , the patterns of variation and correlation among traits involved in different functions (attraction and fit with pollinators, transfer of pollen) were compared within and between population types. The genetic diversity of populations was also studied to control for possible effects on phenotypic variation. In both species, populations with long-tongued pollinators displayed greater phenotypic integration than those with short-tongued pollinators. Also, the correlations among traits involved in the same function were stronger than across functions. Furthermore, traits involved in the transfer of pollen were consistently more correlated and less variable than traits involved in the attraction of insects, and these differences were larger in dimorphic than monomorphic populations. In addition, population genetic parameters did not correlate with phenotypic integration or variation. Altogether, our results support current views of the role of pollinators in the evolution of floral integration.

†Background and Aims Heterostyly and related style polymorphisms are suitable model systems to evaluate the importance of functional pollinators in the maintenance of population variability. In Narcissus papyraceus different functional pollinators, incompatibility system and flower morphology have been proposed to influence the maintenance of polymorphism through their effect on disassortative mating. Here a test is done to find out if the visitation rate of long- versus short-tongued pollinators correlates with the morph ratio and if the latter is related to other flower traits of the species across its main geographic range. †Methods Floral traits from 34 populations in the south-west of the Iberian Peninsula and in north-west Africa were measured, perianth variation was described and a comparison was made of allometric relationships between sex organs and floral tube. Correlations between pollinator guilds, stigma ‐anther separation of reciprocal morphs (our proxy for disassortative mating) and morph-ratio variation were analysed. Finally, the incompatibility system of the species in the northern and southern borders of its distribution are described. †Key Results Flowers from southern populations were significantly larger than flowers from centre and northern populations. The abundance of short-styled plants decreased gradually with increasing distance from the core region (the Strait of Gibraltar), with these disappearing only in the northern range. Although there was a significant difference in stigma‐anther separation among populations, morph ratio was not associated with reciprocity or floral tube length. Long-style morph frequency increased with short-tongued pollinator visitation rate. Populations from both edges of the distribution range were self-incompatible and within- and between-morph compatible. †Conclusions The style morph ratio changed gradually, whereas perianth trait variation showed abrupt changes with two morphotypes across the range. The positive relationship between the visitation rate of short-tongued pollinators and the decrease of the short-style morph supports our initial hypothesis. The results highlight the importance of different pollinators in determining the presence of style polymorphism.