Pollinator shifts and the loss of style polymorphism in Narcissus papyraceus (Amaryllidaceae)

Darwin proposed that the driving force for the evolution of style polymorphisms is the promotion of cross‐pollination between style morphs, through accurate placement of pollen on the pollinator’s body. This hypothesis has received much attention, but the effect of different pollinators in the fitness of morphs remains poorly understood. Narcissus papyraceus is a style dimorphic species (long ‐L‐ and short ‐S‐ styled) with isoplethic (1 : 1) and L‐monomorphic populations, mainly visited by long‐tongued (LT) nocturnal and short‐tongued (ST) diurnal pollinators, respectively. We studied natural female fertility of morphs, and assessed the role of diurnal and nocturnal pollinators. We also quantified female fertility of the morphs in experimental populations with different morph ratio, exposed to predominately long‐ or short‐tongued pollinators. We found that with LT pollinators, both morphs were successfully pollinated in all morph ratio conditions, suggesting that these insects could be involved in maintenance of the polymorphism, although other factors may also play a role. However, with ST pollinators, S‐plants displayed less fertility than L‐plants, and mating among L‐plants was favoured, implying that the polymorphism is lost. These results underscore the role of pollinators on variations in style polymorphism.     

Geographic variation of flower traits in Narcissus papyraceus (Amaryllidaceae): do pollinators matter?

Aim The geographic clinal variation of traits in organisms can indicate the possible causes of phenotypic evolution. We studied the correlates of flower trait variation in populations of a style‐dimorphic plant, Narcissus papyraceus Ker‐Gawl., within a region of high biogeographical significance, the Strait of Gibraltar. This species shows a geographic gradient in the style‐morph ratio, suggested to be driven by pollinator shifts. We tested whether parallel geographic variation of perianth traits also exists, concomitant with vegetative trait variation or genetic similarity of plant populations. Location The Strait of Gibraltar region (SG hereafter, including both south‐western Iberian Peninsula and north‐western Morocco). Methods We used univariate and multivariate analyses of flower and vegetative traits in 23 populations. We applied Mantel tests and partial Mantel correlations on vegetative and flower traits and geographic locations of populations to test for spatial effects. We used Moran’s autocorrelation analyses to explore the spatial structure within the range, and performed the analyses with and without the Moroccan samples to test for the effects of the SG on spatial patterns. Amplified fragment length polymorphism data were used to estimate the genetic distance between populations and to ascertain its relationship with morphometric distance. Results There was high variation between and within populations in both flower and vegetative traits. Mantel correlations between geographic and morphometric distances were not significant, but the exclusion of Moroccan populations revealed some distance effect. Partial Mantel correlation did not detect a significant correlation between flower and vegetative morphometric distances after controlling for geographic distance. There were opposite trends in spatial autocorrelograms of flower and vegetative traits. The genetic distance between pairs of populations was directly correlated with geographic distance; however, flower morphometric and genetic distances were not significantly correlated. Main conclusions The SG had some influence on phenotypes, although the causes remain to be determined. The opposite trend of variation in flower and vegetative traits, and the lack of correlation between genetic distance and dissimilarity of flower phenotypes favour the hypothesis of pollinator‐mediated selection on flower morphology, although this may affect only particular traits and populations rather than overall phenotypes. Although stochastic population processes may have a small effect, other factors may account for the high flower variation within and between populations.     

Disassortative mating prevails in style‐dimorphic Narcissus papyraceus despite low reciprocity and compatibility of morphs

Evolution to reduce inbreeding can favor disassortative (intermorph) over assortative (intramorph) mating in hermaphroditic sexually polymorphic plant species. Heterostyly enhances disassortative pollination through reciprocal placement of stigmas and anthers of morphs and appropriate pollinators. Stylar dimorphism in which there is not reciprocal anther placement may compromise disassortative mating, particularly when there is not intramorph incompatibility. Variable rates of disassortative mating along with differential female fecundity or siring success among floral morphs could lead to variation in morph ratio. We investigated mating patterns, female fecundity, and siring success of style‐length morphs in Narcissus papyraceus, a self‐incompatible but morph‐compatible species with dimorphic (long‐ and short‐styled) and monomorphic (long‐styled) populations in central and north regions of its range, respectively. We established experimental populations in both regions and exposed them to ambient pollinators. Using paternity analysis, we found similar siring success of morphs and high disassortative mating in most populations. Female fecundity of morphs was similar in all populations. Although these results could not completely explain the loss of dimorphism in the species’ northern range, they provided evidence for the evolutionary stability of stylar dimorphism in N. papyraceus in at least some populations. Our findings support the hypothesis that prevailing intermorph mating is key for the maintenance of stylar dimorphism.     

Breeding systems in Cyrtanthus (Amaryllidaceae): variation in self‐sterility and potential for ovule discounting

• Breeding systems of plants determine their reliance on pollinators and ability to produce seeds following self‐pollination. Self‐sterility, where ovules that are penetrated by self‐pollen tubes that do not develop into seeds, is usually considered to represent either a system of late‐acting self‐incompatibility or strong early inbreeding depression. Importantly, it can lead to impaired female function through ovule or seed discounting when stigmas receive mixtures of self and cross pollen, unless cross pollen is able to reach the ovary ahead of self pollen (‘prepotency’). Self‐sterility associated with ovule penetration by self‐pollen tubes appears to be widespread among the Amaryllidaceae.
• We tested for self‐sterility in three Cyrtanthus species – C. contractus, C. ventricosus and C. mackenii – by means of controlled hand‐pollination experiments. To determine the growth rates and frequency of ovule penetration by self‐ versus cross‐pollen tubes, we used fluorescence microscopy to examine flowers of C. contractus harvested 24, 48 and 72 h after pollination, in conjunction with a novel method of processing these images digitally. To test the potential for ovule discounting (loss of cross‐fertilisation opportunities when ovules are disabled by self‐pollination), we pollinated flowers of C. contractus and C. mackenii with mixtures of self‐ and cross pollen.
• We recorded full self‐sterility for C. contractus and C. ventricosus, and partial self‐sterility for C. mackenii. In C. contractus, we found no differences in the growth rates of self‐ and cross‐pollen tubes, nor in the proportions of ovules penetrated by self‐ and cross‐pollen tubes. In this species, seed set was depressed (relative to cross‐pollinated controls) when flowers received a mixture of self and cross pollen, but this was not the case for C. mackenii.
• These results reveal variation in breeding systems among Cyrtanthus species and highlight the potential for gender conflict in self‐sterile species in which ovules are penetrated and disabled by pollen tubes from self pollen.

     

Breakdown of distyly and pin‐monomorphism in Psychotria carthagenensis Jacq. (Rubiaceae)

Rubiaceae contains the largest number of distylous species in the Angiosperms, but the evolution and control of distyly is poorly understood in this group. Atypical distylous populations and species have been described in this family and associated with disturbance, pollination failure and impaired reproductive success. Some of these atypical taxa belong to Psychotria, the largest genus in the family. In the present study, we investigate the pollination and breeding biology of Psychotria carthagenensis Jacq., a widely distributed species that has been described as typically distylous. Fieldwork was carried out mainly at the Panga Ecological Station, in Uberlândia, MG, Brazil. We carried out phenological observations, floral biology studies and hand pollination experiments within the Panga Ecological Station population. We complemented these data with herbarium and field surveys from elsewhere in Brazil. The studied population bloomed at the beginning of the rains (September–December). The main pollinators were Bombus and small Halictidae bees. The Panga Ecological Station population presented pin‐monomorphism with only two thrum individuals in more than 100 flowering individuals surveyed. Hand pollinations at the Panga Ecological Station showed self‐compatibility and intramorph compatibility. Typical distyly and isoplethy were found in other areas and both morphs appeared in herbarium material. However, pin‐monomorphism was also observed elsewhere in Brazil. Typical distyly, pin‐monomorphism and homostyly observed concurrently in Brazilian populations of P. carthagenensis indicate that alternative reproductive strategies, probably favoring selfing, appear to have developed independently in this species.     

Unravelling the stylar polymorphism in Melochia (Malvaceae): reciprocity and ancillary characters

Heterostyly is a genetic polymorphism in which plant populations possess two (distyly) or three (tristyly) morphs with flowers differing reciprocally in stigma and anther height. Sex organ deployment has been described as being highly variable among and within species of several distylous taxa belonging to different taxonomic groups. However, the number of studies considering within‐species disparities is still limited. For a better overview of the existing amount of variation that can occur within and between heterostylous species, we sampled 46 populations of six Melochia spp., a style‐polymorphic genus in Cuba. We characterized the floral morphology in all populations and described a set of ancillary characters per species. All of these Melochia spp. are distylous, except for the monomorphic M. nodiflora. The S‐morph produces fewer, larger pollen than L‐morphs, and has verrucose ornamentation. The L‐morph produces reticulate pollen and has larger stigmatic papillae than the S‐morph. The monomorphic M. nodiflora shows ancillary characteristics that are similar to the L‐morph individuals in the related species. As expected, there are differences in ancillary characters among species and also dissimilarities in reciprocity among and within species of Melochia. Our results highlight the importance of considering intraspecific variation in the morphometric characterization of heterostylous taxa. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 147–158.     

An experimental evaluation of self-interference in Narcissus assoanus: functional and evolutionary implications

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.     

Experimental insights on the function of ancillary pollen and stigma polymorphisms in plants with heteromorphic incompatibility

Most heterostylous plants possess a reciprocal arrangement of stigmas and anthers (reciprocal herkogamy), heteromorphic self‐incompatibility, and ancillary polymorphisms of pollen and stigmas. The topographical complementarity hypothesis proposes that ancillary polymorphisms function in the rejection of incompatible pollen thus promoting disassortative pollination. Here, we test this hypothesis by investigating patterns of pollen transfer and capture in populations of dimorphic Armeria maritima and A. pubigera and distylous Limonium vulgare (Plumbaginaceae), and by studying pollen adherence and germination patterns in A. maritima following controlled hand‐pollinations. Armeria lacks reciprocal herkogamy allowing the evaluation of the extent to which ancillary polymorphisms affect the composition of pollen loads. We compared the amounts of compatible and incompatible pollen on stigmas in natural populations and calculated the proficiencies of pollen transfer for each mating type. We detected disassortative pollination in each species, and mating types did not differ in compatible pollen capture, although cob stigmas captured more incompatible pollen. Controlled hand‐pollinations revealed the failure of incompatible pollen to adhere and germinate on stigmas. Our results provided evidence that, while structural in nature, pollen‐stigma dimorphisms are tightly associated with heteromorphic incompatibility and likely function to promote disassortative pollination, especially in the absence of reciprocal herkogamy.     

Ovarian self‐incompatibility in Narcissus papyraceus (Amaryllidaceae) is the result of a pre‐zygotic response

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. Self‐incompatibility 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. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 629–643.     

Effect of plant traits and population structure on the female reproductive success of the endemic Primula elatior subsp. bergidensis (Primulaceae)

This study explores the possible causes of variation in female reproductive success of the subspecific taxon Primula elatior subsp. bergidensis, a distylic endemic to the north-western Iberian Peninsula, by analysing both vegetative and reproductive traits. In three populations, we marked vegetative and reproductive individuals either by mapping the spatial position of every individual (in one population), or by establishing permanent quadrats (in the remainder two populations). We recorded floral morph (pin or thrum), width and length of the largest leaf, scape length, and number of flowers produced; all individuals were monitored to estimate the number of fruits and seeds produced. The results show that the morph ratio did not differ significantly from 1:1 in any of the populations. The number of flowers per plant varied between populations, and longer scape length was associated with higher fruit set in all populations. Plant size, scape length, and population spatial structure all had major effects on reproductive success, but the strength—and in some cases the direction—of the effects varied among populations.     

Unsuccessful introduced biocontrol agents can act as pollinators of invasive weeds: Bitou Bush (Chrysanthemoides monilifera ssp. rotundata) as an example

The extent of self‐compatibility and reliance on pollinators for seed set are critical determinants of reproductive success in invasive plant species. Seed herbivores are commonly used as biocontrol agents but may also act as flower visitors, potentially resulting in pollination. However, such contrasting or potentially counterproductive interaction effects are rarely considered or evaluated for biological control programs. We investigated the breeding system and pollinators of Bitou Bush (Chrysanthemoides monilifera ssp. rotundata), an invasive species in Australia that has been the subject of biocontrol programs since 1987. We found the species to be obligate outcrossing in all six populations tested. From 150 video hours, we found 21 species of potential pollinators, including Mesoclanis polana, the Bitou Seedfly, native to South Africa and released in Australia as a biocontrol agent in 1996. Mesoclanis polana transferred pollen to stigmas and was the most common pollinator (52% of pollinator visits), followed by the syrphid fly Simosyrphus grandicornis (9%) and introduced honeybee, Apis mellifera (6.5%). Fruit‐to‐flower ratios ranged from 0.12 to 0.45 and were highest in the population with the greatest proportion of Mesoclanis polana visits. In an experimental trial, outside the naturalized range, the native bee Homalictus sphecodoides and the native syrphid Melangyna viridiceps were the primary pollinators, and fruit‐to‐flower ratios were 0.35, indicating that Bitou Bush would have ready pollinators if its range expanded inland. Synthesis. Invasive Bitou Bush requires pollinators, and this is effected by a range of generalist pollinators in eastern Australia including the Bitou Seedfly, introduced as a biocontrol agent, and the major pollinator detected in this study. Fruit‐to‐flower ratios were highest when the Bitou Seedfly was in high abundance. This study underscores the importance of evaluating the pollination biology of invasive species in their native ranges and prior to the introduction of biocontrol agents.     

Loss of floral polymorphism in heterostylous Luculia pinceana (Rubiaceae): a molecular phylogeographic perspective

Both deterministic and stochastic forces determine the representation and frequency of floral morphs in heterostylous plant populations. Phylogeographic analysis of molecular variation can provide information on the role of historical factors, including founder events, in affecting population morph structure. Here, we investigate geographical patterns of floral morph variation in a distylous shrub Luculia pinceana (Rubiaceae) by examining the relations between floral polymorphism and molecular (cpDNA and microsatellite) variation in 25 populations sampled throughout the distribution of the species in southwest China and adjacent countries. In 19 of the 25 populations, the frequency of floral morphs was not significantly different from the expected 1:1 ratio. The remaining populations were either L‐morph biased (2) or monomorphic (4) for this form and were morphologically differentiated from the remaining populations in several floral traits, that is, corolla tube length, sex organ position and stigma‐anther separation. Phylogeographic analysis supports the hypothesis that L. pinceana was initially split into west‐central and eastern lineages in the Early Pleistocene (～1.982 Mya). A centrally located lineage composed of morph‐biased and monomorphic populations appears to have been subsequently derived from the west‐central lineage, perhaps by a founder event after the last glacial maximum. Hypotheses to explain why these populations have not returned to equilibrium morph frequencies are considered.     

Self‐ and intra‐morph incompatibility and selection analysis of an inconspicuous distylous herb growing on the Tibetan plateau (Primula tibetica)

There is discussion over whether pollen limitation exerts selection on floral traits to increase floral display or selects for traits that promote autonomous self‐fertilization. Some studies have indicated that pollen limitation does not mediate selection on traits associated with either pollinator attraction or self‐fertilization. Primula tibetica is an inconspicuous cross‐fertilized plant that may suffer from pollen limitation. We conducted a selection analysis on P. tibetica to investigate whether pollen limitation results in selection for an increased floral display in case the evolution of autonomous self‐fertilization has been difficult for this plant. The self‐ and intra‐morph incompatibility features, the capacity for autonomous self‐fertilization, and the magnitude of pollen limitation were examined through hand‐pollination experiments. In 2016, we applied selection analysis on the flowering time, corolla width, stalk height, flower tube length, and flower number in P. tibetica by tagging 76 open‐pollinated plants and 37 hand‐pollinated plants in the field. Our results demonstrated that P. tibetica was strictly self‐ and intra‐morph incompatible. Moreover, the study population underwent severe pollen limitation during the 2016 flowering season. The selection gradients were found to be significantly positive for flowering time, flower number, and corolla width, and marginally significant for the stalk height. Pollinator‐mediated selection was found to be significant on the flower number and corolla width, and marginally significant on stalk height. Our results indicate that the increased floral display may be a vital strategy for small distylous species that have faced difficulty in evolving autonomous self‐fertilization.     

Sex dimorphism and evidence of sexually selected traits: A case study on the killifish Aphaniops stoliczkanus (Day, 1872)

Sex dimorphism is found in many organisms and is an important source of intraspecific variation. Among freshwater fishes, all members of the Aphaniidae (killifishes, Cyprinodontiformes) are known for their pronounced sex dimorphism, and it has been proposed that sexual selection has played a role in their diversification. However, few studies have assessed their sex dimorphism in detail. Here, we use Aphaniops stoliczkanus (Day, 1872) to analyse morphological variability within and between sexes and within the total sample using statistical analyses of body morphometry, meristic traits and otolith data. We found that sex dimorphism (i) is significant in many body morphometric variables, but (almost) absent in meristic traits and otolith morphometry, (ii) is most marked in anal- and dorsal-fin lengths (larger in males), and in anal- and dorsal-fin positions (more anteriorly positioned in males) and (iii) does not alter the overall variance of the population unless the character concerned is highly divergent. Our data also suggest that anal- and dorsal-fin lengths in males of A. stoliczkanus are sexually selected traits. Together with literature data, this reinforces the idea that sexual selection plays a role in the diversification of aphaniid species. Our results are also relevant to work on fossils, in which morphological variability is often difficult to interpret.     

Differential ovule development following self- and cross-pollination: the basis of self-sterility in Narcissus triandrus (Amaryllidaceae)

Self-pollination results in significantly lower seed set than cross-pollination in tristylous Narcissus triandrus. We investigated structural and functional aspects of pollen–pistil interactions and ovule–seed development following cross- and self-pollination to assess the timing and mechanism of self-sterility. Ovule development within an ovary was asynchronous at anthesis. There were no significant differences in pollen tube behavior following cross- vs. self-pollination during the first 6 d of growth, regardless of style morph type. Double fertilization was significantly higher following cross- vs. self-pollination. Aborted embryo development was not detected following either pollination type up to seed maturity. Prior to pollen tube entry, a significantly greater number of ovules ceased to develop following self- vs. cross-pollination. These results indicate that self-sterility in N. triandrus operates prezygotically but does not involve differential pollen tube growth typical of many self-incompatibility (SI) systems. Instead, low seed set following self-pollination is caused by a reduction in ovule availability resulting from embryo sac degeneration. We hypothesize that this is due to the absence of a required stimulus for normal ovule development. If this is correct, current concepts of SI may need to be broadened to include a wider range of pollen–pistil interactions.     

Migration rates, frequency-dependent selection and the self-incompatibility locus in Leavenworthia (brassicaceae)

Loci subject to negative frequency-dependent selection are expected to exhibit higher effective migration rates compared to reference loci. Although the number of gene copies transferred between populations by migration is the same for all genes, those subject to negative frequency-dependent selection are more likely to be retained in the immigrant population because rare alleles are selectively favored. So far, evidence for this prediction has been indirect, based on summary statistics rather than on migration rate estimates. Here, we introduce an approximate Bayesian procedure to jointly estimate migration rates at two predefined sets of loci between two populations. We applied the procedure to compare migration rate estimates at the self-incompatibility locus (S-locus) with that at 10 reference loci in two plant species, Leavenworthia alabamica and L. crassa (Brassicaceae). The maximum likelihood estimate for the proportion of migrants (m) was four times higher at the S-locus than at reference loci, but the difference was not statistically significant. Lack of significance might be due to insufficient data, but perhaps also to the recent divergence of the two species (311 ka), because we also show using simulations that the effective migration rate at the S-locus is expected to increase with increasing divergence time. These findings aid in understanding the evolutionary dynamics of negative frequency-dependent selection and they suggest that divergence time should be accounted for when employing migration rates to help detect negative frequency-dependent selection.