Selection on floral design in Polemonium brandegeei (Polemoniaceae): female and male fitness under hawkmoth pollination

Plant-pollinator interactions promote the evolution of floral traits that attract pollinators and facilitate efficient pollen transfer. The spatial separation of sex organs, herkogamy, is believed to limit sexual interference in hermaphrodite flowers. Reverse herkogamy (stigma recessed below anthers) and long, narrow corolla tubes are expected to promote efficiency in male function under hawkmoth pollination. We tested this prediction by measuring selection in six experimental arrays of Polemonium brandegeei, a species that displays continuous variation in herkogamy, resulting in a range of recessed to exserted stigmas. Under glasshouse conditions, we measured pollen removal and deposition, and estimated selection gradients (β) through female fitness (seeds set) and male fitness (siring success based on six polymorphic microsatellite loci). Siring success was higher in plants with more nectar sugar and narrow corolla tubes. However, selection through female function for reverse herkogamy was considerably stronger than was selection through male function. Hawkmoths were initially attracted to larger flowers, but overall preferred plants with reverse herkogamy. Greater pollen deposition and seed set also occurred in reverse herkogamous plants. Thus, reverse herkogamy may be maintained by hawkmoths through female rather than male function. Further, our results suggest that pollinator attraction may play a considerable role in enhancing female function.     

Enantiostyly in Chamaecrista ramosa (Fabaceae‐Caesalpinioideae): floral morphology,pollen transfer dynamics and breeding system

Enantiostyly is a form of reciprocal herkogamy, in which floral morphs present reciprocal differences in the position of sexual elements, and occurs in monomorphic and dimorphic forms. This polymorphism maximises cross‐pollination and reduces self‐pollination, being very common within the subtribe Cassiinae (Fabaceae). Nevertheless, few studies have investigated the functionality of enantiostyly, particularly in this plant group. The present study aimed to investigate enantiostyly and its functionality in Chamaecrista ramosa, a monomorphic enantiostylous shrub, in an area of coastal vegetation in northeast Brazil. Pollen deposition and capture on the body of floral visitors, the relationship of these data with floral biology and breeding system, and morph ratio were evaluated. Pollen deposition and capture occurred in specific sites of the floral visitor body, showing the functionality of enantiostyly. The floral architecture, associated with the floral visitor behaviour, resulted in indirect pollen deposition on the floral visitor body. This occurred through a loop made by the pollen upon the inner petal surface, similar that generally reported for other Cassiinae. Chamaecrista ramosa is self‐compatible, although no fruit set was observed through spontaneous self‐pollination. The occurrence and number of floral morphs was similar within clumps. Enantiostyly seems to be advantageous for this species, as it results in efficient pollen capture and deposition, reduces the chances of autogamy and maximises intermorph pollen flow.     

Patterns of pollen removal and deposition in tristylous Pontederia cordata L. (Pontederiaceae)

In tristylous Pontederia cordata (Pontederiaceae), conspicuous differences in the size of pollen grains and discrete variation in the length of reproductive organs provide a suitable experimental system for the study of fine-scale pollination events. At a population of P. cordata at Pothole Lake, Ontario, the majority of flowers are visited by bumble bees which remove on average 45% of the pollen during single visits to previously unvisited flowers. The amount and proportion of pollen removed are significantly different among floral morphs and stamen levels. Deposition of the three pollen types on the bodies of Bombus spp., Apis mellifera and Melissodes apicata is non-random: large- and medium-size pollen tends to remain in greatest concentrations where it is initially deposited, whereas small-size pollen is displaced from the proboscis to more posterior body parts, probably as a result of grooming activities. Stigmatic pollen loads of individual flowers following single bumble bee visits indicate that the mid-styled morph captures the largest total pollen load, and the short-styled morph the smallest. The largest proportion of compatible pollen grains is deposited on stigmas of the long-styled morph. Pollen load data from "single visit" flowers is in general agreement with previously published population surveys involving multiply-visited flowers.     

Feeding friend and foe: ample pollen mitigates the effects of pollen theft for a gynodioecious plant,Polemonium foliosissimum (Polemoniaceae)

Background and AimsMost angiosperms rely on pollinators to transport pollen and effect fertilization. While some floral visitors are effective pollinators, others act as thieves, consuming pollen but effecting little pollination in return. The importance of pollen theft in male and female reproductive success has received little attention. Here, we examined if pollen consumption by flies altered pollen receipt and exacerbated pollen limitation for a bumblebee-pollinated plant, Polemonium foliosissimum (Polemoniaceae).MethodsTo examine the effect of pollen-thieving flies, we took a three-pronged approach. First, we used single-visit observations to quantify pollen removal and pollen deposition by flies and bumblebees. Second, we manipulated pollen in the neighbourhood around focal plants in two years to test whether pollen reduction reduced pollen receipt. Third, we combined pollen reduction with hand-pollination to test whether pollen thieving exacerbated pollen limitation. Polemonium foliosissimum is gynodioecious in most populations in the Elk Mountains of central Colorado, USA. Thus, we also tested whether pollen theft affected hermaphrodites and females differently.ResultsFlies removed significantly more pollen and deposited less pollen per visit than did bumblebees. Reduction of pollen in the neighbourhood around focal plants reduced pollen receipt in both years but only nearly significantly so in 2015. In 2016, plants were significantly pollen-limited; hand-pollination significantly increased seeds per fruit for both hermaphrodites and females. However, the reduction of pollen around focal plants did not exacerbate pollen limitation for either hermaphrodites or females.ConclusionsOur results suggest that plants tolerate significant consumption of pollen by thieves and pollinators by producing ample pollen to feed both and fertilize available ovules. Our results demonstrate that pollen limitation in P. foliosissimum is driven by lack of effective pollinators rather than lack of pollen. Teasing out these effects elucidates the relative importance of drivers of reproductive success and thus the expected response to selection by different floral visitors.     

Patterns and consequences of self-pollen deposition on stigmas in heterostylous Persicaria japonica (Polygonaceae)

In order to test the "anti-interference" hypothesis for adaptive significance of reciprocal herkogamy, patterns of illegitimate pollination and effects of self-pollen on the legitimate pollination and fertility were investigated in a naturally pollinated experimental population of distylous Persicaria japonica. Pollen deposition was compared among the emasculation treatments, i.e., emasculation of a single flower from individual inflorescences, emasculation of all the flowers of individual inflorescences, and no emasculation control. In both morphs, considerable illegitimate pollination was found to occur in all the treatments, and there was no significant difference in incompatible pollen load among the treatments. Therefore, it is suggested that herkogamy of P. japonica can sufficiently reduce both intraflower and intra-inflorescence self-pollinations, but not interinflorescence geitonogamous and-or interclonal illegitimate pollination. Measurements of pollen load after the repetitive pollen addition showed that space on the stigma surface may not limit the legitimate pollination under natural pollination conditions. Seed sets after legitimate pollination following prior self-pollination did not differ from the controls without self-pollination. Therefore, there is little possibility that incompatible pollen load interferes with either pollination or fertilization by compatible pollen, suggesting that "anti-interference" is unimportant for adaptive significance of reciprocal herkogamy at least in P. japonica.     

Influence of pollen limitation and inbreeding depression in the maintenance of incomplete dichogamy in Salvia elegans

The widespread presence of incomplete dichogamy (i.e., partial separation in time between male and female phases) in flowering plants is a long‐standing question in floral evolution. In this study, we proposed four scenarios in which depending on the particular combination of pollen limitation and inbreeding depression, the presence of complete dichogamy, incomplete dichogamy, or adichogamy may be favored. Moreover, we evaluated the role of pollen limitation and inbreeding depression in a natural population of Salvia elegans to test the validity of our predicted scenarios. Our results indicate that S. elegans is partially protandrous as pollen viability and stigma receptivity overlap in the last days of life of the flower. Furthermore, through pollination treatments, we found no evidence of pollen limitation or inbreeding depression in any of the evaluated fitness components. As expected by one of the proposed scenarios, incomplete dichogamy seems to be favored in plants with absence of inbreeding depression and pollen limitation as a way to diminish interference between male and female functions.     

The role of pollinator shifts in the floral diversification of Iochroma (Solanaceae)

Differences in floral traits among plant species have often been attributed to adaptation to pollinators. We explored the importance of pollinator shifts in explaining floral divergence among 15 species of Iochroma. We examined four continuously varying floral traits: corolla length, nectar reward, display size, and flower color. Pollinator associations were characterized with a continuously varying measure of pollinator importance (the product of visitation and pollen deposition) for four groups of pollinators: hummingbirds, Hymenoptera, Lepidoptera, and Diptera. A phylogenetic generalized least squares approach was used to estimate correlations between pollinator groups and floral traits across a sample of Bayesian trees using different models of trait evolution. Multivariate analyses were also employed to identify suites of traits associated with each pollinator group. We found that nonphylogenetic models typically fit the data better than phylogenetic models (Brownian motion, Ornstein-Uhlenbeck), and thus results varied little across trees. Our results indicated that species with high nectar reward and large displays are significantly more likely to be pollinated by hummingbirds and less likely to be pollinated by all groups of insects. Corolla length and flower color did not show any consistently significant associations with pollinator groups. For these two traits, we discuss alternative evolutionary forces, including phylogenetic inertia and community-level factors.     

Heterostylous morph differences in pollen transfer and deposition patterns in Primula sieboldii on a visitation by a queen bumblebee, measured with a semi-natural experimental system

Abstract Under a semi-natural setting the between-morph pollen exchange patterns were studied in distylous Primula sieboldii flowers by measuring pollen removal from the anthers on a single visit by a Bombus diversus tersatus queen, and stigmatic pollen deposition along the sequence of the visitation of the opposite-morph flowers by the bee. Despite the twofold larger number of pollen grains produced in a single flower of the long-styled morph compared to that of the short-styled morph, no significant difference in pollen removal from a flower was found between the morphs. The stigmas of the long-styled morph received significantly more opposite-morph pollen grains than those of the short-styled morph on a single visit by the bee. Sufficient legitimate pollen grains, surpassing the ovule number, were loaded on the stigmas of 27% and 17% of visited flowers of the long- and short-styled morphs, respectively. The short-styled morph could more efficiently donate pollen to the opposite morph stigmas than the long-styled morph.     

Pollen limitation,fruit abortion,and autonomous selfing in three populations of the perennial herb Ruellia nudiflora

Multiple factors determine plant reproductive success and their influence may vary spatially. This study addresses several factors influencing female reproductive success in three populations of Ruellia nudiflora, specifically we: (i) determine if fruit set is pollen‐limited and if pollinator visitation rates are related to this condition; (ii) estimate fruit set via autonomous self‐pollination (AS) and relate it to the magnitude of herkogamy; and (iii) evaluate if fruit abortion is a post‐pollination mechanism that determines the magnitude of pollen limitation. At each site we marked 35 plants, grouped as: unmanipulated control (C) plants subjected to open pollination, plants manually cross‐pollinated (MP), and plants excluded from pollinators and only able to self‐pollinate autonomously (AS). Fruit set was greater for MP relative to C plants providing evidence for pollen limitation, while a tendency was observed for lower fruit abortion of MP relative to C plants suggesting that fruit set is influenced not only by pollen delivery per se, but also by subsequent abortion. In addition, although pollinator visits varied significantly among populations, the magnitude of pollen limitation did not, suggesting that pollinator activity was not relevant in determining pollen limitation. Finally, fruit set tended to decrease with the degree of herkogamy for AS plants, but this result was inconclusive. These findings have contributed to identify which factors influence reproductive success in populations of R. nudiflora, with potentially relevant implications for population genetic structure and mating system evolution of this species.     

Patterns and sources of variation in pollen deposition and pollen tube formation in flowers of the endemic monoecious shrub Cnidoscolus souzae (Euphorbiaceae)

Pollen deposition and pollen tube formation are key components of angiosperm reproduction but intraspecific variation in these has rarely been quantified. Documenting and partitioning (populations, plants and flowers) natural variation in these two aspects of plant reproduction can help uncover spatial mosaics of reproductive success and underlying causes. In this study, we assess variation in pollen deposition and pollen tube formation for the endemic monoecious shrub Cnidoscolus souzae throughout its distribution range in Mexico, and determine how this variation is structured among populations, plants and flowers. We also infer the relative importance of pollen quantity and quality in determining pollination success in this species. While we found no evidence suggesting that pollen receipt limits C. souzae reproduction across 19 populations, we did find extensive variation in pollen load size and pollen tube number per flower. Total variation in pollen receipt and pollen tube number was mostly explained by intra‐individual and among‐population variance. Furthermore, pollen load size had a stronger effect on the number of pollen tubes at the base of the style than pollen germination rate, suggesting that pollen quantity may be more important than quality for pollen tube success in C. souzae. Our results suggest that both small within‐plant flower differences and broad‐scale differences in community attributes can play an important role in determining pollination success. We emphasise the need to evaluate patterns and sources of variation in pollen deposition and pollen tube formation as a first step in understanding the causes of variation in pollination success over broad spatial scales.     

Wind‐mediated horseweed (Conyza canadensis) gene flow: pollen emission,dispersion, and deposition

Horseweed (Conyza canadensis) is a problem weed in crop production because of its evolved resistance to glyphosate and other herbicides. Although horseweed is mainly self-pollinating, glyphosate-resistant (GR) horseweed can pollinate glyphosate-susceptible (GS) horseweed. To the best of our knowledge, however, there are no available data on horseweed pollen production, dispersion, and deposition relative to gene flow and the evolution of resistance. To help fill this knowledge gap, a 43-day field study was performed in Champaign, Illinois, USA in 2013 to characterize horseweed atmospheric pollen emission, dispersion, and deposition. Pollen concentration and deposition, coupled with atmospheric data, were measured in a source field (180 m by 46 m) and its surrounding areas up to 1 km downwind horizontally and up to 100 m vertically. The source strength (emission rate) ranged from 0 to 140 pollen grains per plant per second (1170 to 2.1×10⁶ per plant per day). For the life of the study, the estimated number of pollen grains generated from this source field was 10.5×10¹⁰ (2.3×10⁶ per plant). The release of horseweed pollen was not strongly correlated to meteorological data and may be mainly determined by horseweed physiology. Horseweed pollen reached heights of 80 to100 m, making long-distance transport possible. Normalized (by source data) pollen deposition with distance followed a negative-power exponential curve. Normalized pollen deposition was 2.5% even at 480 m downwind from the source edge. Correlation analysis showed that close to or inside the source field at lower heights (≤3 m) vertical transport was related to vertical wind speed, while horizontal pollen transport was related to horizontal wind speed. High relative humidity prevented pollen transport at greater heights (3–100 m) and longer distances (0–1000 m) from the source. This study can contribute to the understanding of how herbicide-resistance weeds or invasive plants affect ecology through wind-mediated pollination and invasion.     

Absence of conspecific pollen advantage in the dynamics of an Ipomopsis (Polemoniaceae) hybrid zone

The frequency of hybrid formation in angiosperms depends on how often heterospecific pollen is transferred to the stigma and on the success of that heterospecific pollen at fertilizing ovules. Even if heterospecific pollen is capable of effecting fertilization it may perform poorly when conspecific pollen is also available on the stigma. We applied pollen mixtures to stigmas to determine how pollen interactions affect siring success and the frequency of hybrid formation between two species of Ipomopsis (Polemoniaceae) in Colorado. Plants of both parental species and natural hybrids were pollinated with I. aggregata and I. tenuituba pollen in ratios of 100:0, 80:20, 50:50, 20:80, and 0:100 by mass. Plants were homozygous for different alleles at an isozyme marker, allowing us to distinguish the type of pollen parent for 2166 viable seeds from 273 fruits. In contrast to studies of many other hybridizing taxa, there was no evidence of an advantage to conspecific pollen, nor did composition of the stigmatic pollen load affect seed set. Instead, the frequency of seeds sired by a given species was proportional to its representation in the pollen load. In this hybrid zone, both the frequency of first-generation hybrid formation and the relative male fitness of the two parental species should be predictable from the rates of pollen transfer to stigmas.     

The conserved nature and taxonomic utility of pollen morphology in Cantua (Polemoniaceae)

Pollen exine morphology of nine of the ten species of Cantua (Polemoniaceae) is examined using light and scanning electron microscopy. A total of 28 specimens of C. bicolor, C. buxifolia, C. candelilla, C. cuzcoensis, C. flexuosa, C. pyrifolia, C. quercifolia, C. volcanica, and an as yet undescribed species (Cantua sp. nov.) are examined using either fresh or herbarium material. Pollen grains are found to be spheroidal, pantoporate, and quite large; mean diameter varies from 62 to 87?μm. Mean number of pores varies from 4.5 to 21.2 and mean pore size varies from 4.86 to 12.40?μm. Pollen grains of all species have insulate semitectate sexines. This feature distinguishes the pollen of Cantua species from the remainder of the Polemoniaceae. Insulae are evenly distributed over the surface of the pollen grain, with the exception of C. flexuosa and occasionally C. buxifolia, where insulae are more sparsely and haphazardly distributed. The majority of the species examined have irregularly shaped tectal insulae, with the exception of the large rounded insulae in C. quercifolia and the elongated narrow insulae in C. volcanica. Cantua quercifolia and C. volcanica have supratectal verrucae, a possible synapomorphy. In comparison to close relatives, the pollen grains of Cantua are evolutionarily conserved, and show little variation among species.     

Pollination in small islands by occasional visitors: the case of <Emphasis Type="Italic">Daucus carota</Emphasis> subsp. <Emphasis Type="Italic">commutatus</Emphasis> (Apiaceae) in the Columbretes archipelago,Spain

This study investigates the pollination ecology and related floral traits of the species Daucus carota subsp. commutatus in the isolated archipelago of Columbretes, E. Spain, where bees are absent. Two populations were studied: a small population found on a relatively large island (Grossa) inhabited nowadays by three people; and a larger population on a smaller uninhabited island (Foradada). The plant, found also in other rocky coastal enclaves of the west Mediterranean, is totally self-compatible, andromonoecious, exhibiting strong protandrous dichogamy and sequential flowering of umbel orders, with limited probability of self pollination at the plant level. Pollination on both study islands is carried out solely by flies, mainly by Eristalis tenax (Syrphidae), a long distance visitor and efficient pollinator, but irregular migrant to the archipelago; and Lucilia sericata (Calliphoridae), an abundant flower visitor but less efficient pollinator dependent on human and avian wastes for its larval growth. The small population on Grossa appears not to suffer pollen limitation. Seed set is reduced, however, in the large population on Foradada, which we attribute to the low pollinator capital of this island, which is too small to attract and sustain migrant hoverflies and where no human and avian wastes are available to sustain blowfly growth. We conclude that in isolated environments with reduced or unpredictable pollinator capital, long distance migrant pollinators, such as E. tenax, play an invaluable conservation and perhaps evolutionary role for the native plants. Because these islands have been always practically uninhabited we argue that the absence of adequate local pollinator capital is perhaps compensated by the selection of particular floral traits, which differentiate the study subspecies from other conspecifics. Such traits are: the exposure of nectar during male versus female stage to enhance quick pollen flow; the extended stigma receptivity period (up to 10 days) resulting in a total flower life span of up to 22 days, enabling an adequate pollen deposition on stigmas even under low pollen transfer rates. In this respect, the low flower sex ratio (hermaphrodite: staminate) compared to other conspecifics studied elsewhere may be also relevant.     

The differential contributions of herkogamy and dichogamy as mechanisms of avoiding self‐interference in four self‐incompatible Epimedium species

Self‐interference is one of the most important selective forces in shaping floral evolution. Herkogamy and dichogamy both can achieve reductions in the extent of self‐interference, but they may have different roles in minimizing self‐interference in a single species. We used four self‐incompatible Epimedium species to explore the roles of herkogamy and dichogamy in avoiding self‐interference and to test the hypothesis that herkogamy and dichogamy may be separated and become selected preferentially in the taxa. Two species (E. franchetii and E. mikinorii) expressed strong herkogamy and weak protogyny (adichogamy), whereas another two species (E. sutchuenense and E. leptorrhizum) expressed slight herkogamy and partial protandry. Field investigations indicated that there was no physical self‐interference between male function and female function regarding pollen removal and pollen deposition in all species. Self‐pollination (autonomous or facilitated) was greater in species with slight herkogamy than in those with strong herkogamy. Artificial pollination treatments revealed that self‐pollination could reduce outcrossed female fertility in all species, and we found evidence that self‐interference reduced seed set in E. sutchuenense and E. leptorrhizum in the field, but not in E. franchetii and E. mikinorii. These results indicate that well‐developed herkogamy is more effective compared with dichogamy in avoiding self‐interference in the four species. In genus Epimedium, herkogamy instead of dichogamy should be selected preferentially and evolved as an effective mechanism for avoiding self‐interference and might not need to evolve linked with dichogamy.     

Evaluation of pollinator effectiveness based on pollen deposition and seed production in a gynodieocious alpine plant,Cyananthus delavayi

Examining variations in pollinator effectiveness can enhance our understanding of how pollinators and plants interact. Pollen deposition and seed production after a single visit by a pollinator are often used to estimate pollinator effectiveness. However, seed production is not always directly related to pollen deposition because not all pollen grains that are deposited on a stigma are compatible or conspecific. In the field, we tested pollinator effectiveness based on pollen deposition and the resulting seed production after single visits by different pollinator groups in a gynodieocious alpine plant Cyananthus delavayi (Campanulaceae). Our results showed that mean pollen deposition was generally inconsistent with mean seed production when comparisons were performed among different pollinator groups and sexes. In general, the correlations were not significant between pollen deposition and seed production in both perfect and female flowers after single visits by halictid bees, bumble bees, and hoverflies. We suggest seed set of virgin flowers after single visits is a more reliable indicator of pollinator effectiveness than pollen deposition and would be a better indicator of pollinator effectiveness for future studies.     

Secondary pollen presentation and the temporal dynamics of stylar hair retraction and style elongation in Campanula trachelium (Campanulaceae)

To increase the accuracy of pollen capture and transfer by pollinators some plant species have developed secondary pollen presentation structures. Because the presence of secondary pollen presentation structures at the pistil may reduce the spatial separation between the sexual functions and increase the risk of self‐interference and selfing, temporal segregation of the sexual organs, triggered by visiting insects, can be expected to occur. We investigated secondary pollen presentation and the temporal dynamics of the sexual phases in combination with the physiological self‐incompatibility system in Campanula trachelium, a protandrous insect‐pollinated herb. Stylar hair retraction (male function) and curling of the stigmatic lobes (female function) were modelled using Gompertz growth functions. Finally, we performed pollination experiments in the lab and field to assess seed set and pollen limitation under natural conditions. About 68% of the total pollen load was captured by stylar hairs. Manual manipulation of the stylar hairs, mimicking pollinator visitation, significantly shortened the male phase and accelerated the female phase, resulting in a significant decline in temporal overlap between the two sexual functions. Conversely, when pollinators and/or manual manipulations were lacking, the male phase was substantially prolonged and sexual overlap was maximal. This suggests that spreading of the sexual phases and thus the risk of sexual interference are largely determined by the interaction between stylar hairs and visiting pollinators. Natural seed set was high and not pollinator limited. Overall, these results indicate that secondary pollen presentation and partial protandry resulted in efficient pollen capture, transfer and deposition.