Pollen Capture Research Articles

Diversity in bread and durum wheat stigma morphology and linkage of increased stigma length to dwarfing gene Rht14

Key messageThe dwarfing allele Rht14 of durum wheat associates with greater stigma length, an important trait for hybrid breeding, whilst major dwarfing alleles Rht-B1b and Rht-D1b showed little to no effect.Although much understudied in wheat, the stigma is a crucial component for attaining grain set, the fundamental basis for yield, particularly in hybrid production systems where successful grain set relies on wind-driven pollen dispersal by the male parent and effective pollen capture by the female parent. Females with long stigma that exsert early are thought to be advantageous. Using glasshouse-grown lines, we examined variation in Total Stigma Length (TSL) across diverse panels comprising 27 durum and 116 bread wheat genotypes. Contrasting genotypes were selected for population development and genetic analysis. Quantitative trait loci (QTL) analysis was performed on a durum F2 population and a bread wheat recombinant inbred line (RIL) population. Contrasting with studies of anther length, we found no large effect on TSL of the GA-insensitive semi-dwarfing genes Rht-B1 and Rht-D1 in either durum or bread wheat. However, in durum cultivar Italo, we identified a region on chromosome 6A which is robustly associated with larger TSL and contains the Rht14 allele for reduced plant height, a trait that is favourable for female line development in hybrid systems. This dual effect locus explained 25.2 and 19.2% of TSL phenotypic variation in experiments across two growing seasons, with preliminary results suggesting this locus may increase TSL when transferred to bread wheat. In a bread wheat, RIL population minor QTL on 1A and 2A was indicated, but the strongest association was with Ppd-B1. Methods developed here, and the identification of a TSL-enhancing locus provides advances and further opportunities in the study of wheat stigma.

Investigating sexual and asexual modes of reproduction in Palmer Amaranth (Amaranthus palmeri)

Abstract Palmer amaranth (Amaranthus palmeri S. Watson), a dioecious wind-pollinated plant, is one of the most troublesome crop weeds in the United States and is spreading northwards. The prodigious production of seed contributes to establishment of populations and spread across the landscape. Sexual reproduction via outcrossing is likely the primary mode of seed production for this dioecious plant. However, A. palmeri may also be capable of autonomous asexual seed production (apomixis), which could be beneficial during colonization. We conducted two studies of female isolation from pollen to investigate the propensity for autonomous seed production in 19 populations across eastern North America. In the first we observed low frequency seed production on many isolated females. Using flow cytometry of seed samples (FCSS) we primarily found patterns of ploidy consistent with sexual reproduction; no significant differences in ploidy between seeds produced on isolated females (putative apomicts) and non-isolated females (putatively sexual) were detected. We also investigated patterns of DNA content and found no evidence for polyploidy in 153 samples, which is often observed in apomictic species. The second female isolation trial utilized sex-specific molecular markers to identify and remove males prior to flowering, and we observed zero seed production. Overall, we did not detect evidence in support of apomixis in these populations of A. palmeri, suggesting that apomixis is unlikely to have played a role in the northward advance of this species in eastern North America. We also investigated whether there is variation between females and males in size and secondary reproductive traits. We found evidence for sexual dimorphism in three of six traits investigated: females are taller at senescence, produce longer secondary branches, and more axillary flowers than males. Differences in cost of reproduction and strategies for pollen release vs. pollen capture are likely factors shaping the evolution of sexual dimorphism in this wind-pollinated dioecious plant.

Extra-stigmatic pollen germination and pistil elongation: a novel strategy towards reproductive assurance in Dodonaea viscosa subsp. angustifolia

Context Reproductive success in dioecious plant species may be limited by severe pollen limitation owing to their separate sexes and pollination barriers. Aims Dodonaea viscosa subsp. angustifolia (Jacq.) (Sapindaceae) is a dioecious and wind-pollinated species that has a long flowering period. This study sought to determine the relationship between its reproductive behaviour and pollen availability during different flowering phases. Methods Extra-stigmatic pollen germination and reproductive performance during different phases of flowering were investigated and correlated with pistil elongation under natural conditions. Results The species offers whole stigmatic and stylar surfaces for pollen to land and germinate under natural conditions. During pollen-limiting conditions, the length of the pistil increases significantly to enhance pollen capture. Depending on where on the pistil pollen lands, the timing of pollen tube arrival at the ovary varies. Conclusions Dodonaea viscosa subsp. angustifolia is the first reported wild species in the Sapindaceae where pistil elongation is regulated by pollination conditions and extra-stigmatic pollen germination ensures reproduction during phases of differing pollen availability. Our results indicated that the flexibility of female function and reproductive behaviour in Dodonaea viscosa subsp. angustifolia adds to the evolutionary possibilities to overcome pollination constraints. Implications This unique strategy for increasing female fitness through pollen presentation in D. viscosa subsp. angustifolia could be explored in other subspecies of D. viscosa on the Australian subcontinent.

The fertilization process in Lithocarpus dealbatus (Fagaceae) and its implication on the sexual reproduction evolution of Fagales.

The pistillate flowers of Lithocarpus dealbatus show two pollen tube (PT) arresting sites (the style-joining and micropyle) within the pistil during the postpollination-prezygotic stage. The PT, arrested at the pre-ovule stage, enhanced PT competition allowing the most compatible PTs to enter the ovary to ensure the highest fertilization success. During the shift from animal pollination to wind pollination, plants require a series of changes in reproductive traits. The mode of pollination is striking labile in Fagaceae. Lithocarpus is insect pollinated and is closely related to wind-pollinated Quercus. Little is known about the sexual reproduction of Lithocarpus. This study aimed to reveal the sexual reproduction of Lithocarpus dealbatus and to explore the evolutionary pattern of the key sexual reproduction traits to better understand their possible role in labile pollination. We found that after pollination, L. dealbatus PTs grew slowly in the style reaching style-joining in mid-January of the second year; then PT growth was arrested at style-joining for four months. Only two to three PTs resumed growth in mid-May to reach the micropyle, where PT growth ceased for one month before one PT resumed growth and passed through the micropyle to the embryo sac. Fagaceae showed a generalized mating system. Vast pollen production, small-sized pollen grains, long stigmatic receptive time, and reduced perianth were compatible with beetle pollination syndrome, representing the plesiomorphic status in Fagaceae. A large stigmatic surface and dry pollen grains linked to wind pollination might be independently derived several times in fagaceous lineages. Beetle pollination syndrome can cope with the uncertainty of pollinators to ensure conspecific pollen capture, which represents pre-adaptation status and has a selective advantage when conditions change, favouring wind pollination. The arrest of the PT at style-joining is a unique mechanism in later derived fagaceous lineages to enhance PT competition and promote outcrossing.

Cytoskeleton Remodeling in Arabidopsis Stigmatic Cells Following Pollination.

In plants, the first interaction that occurs between the male gametophytes (pollen grains) and the stigmatic epidermis of the female organ is crucial for successful reproduction. The stigma consists of a dome of flask-shaped cells specialized in pollen capture. In these stigmatic cells, the cytoskeleton network (cortical microtubules and actin microfilaments) actively responds to pollen contact and undergoes dynamic remodeling required for successful pollen acceptance to occur. Here, we have designed several microscopy mountings to monitor stigmatic cytoskeleton dynamics. These designs are based on the constraints linked to the tightly regulated pollen-stigma interaction and depend upon the experimental goal, either a static view or live-cell imaging.

Vibrational response of the male ‘peter’ pistachio branch organs to facilitate artificial pollination

Artificial pollination may be a promising practice to both improve pistachio fruit production by mitigating the losses caused by phenological gaps or by adverse meteorological events and establish new production areas. Artificial pollination requires large quantities of pollen and manual collection is not economically viable. Mechanical capture of pollen, for subsequent storage and application, could achieved in two ways: by releasing mature pollen from the inflorescences and capturing it in the air, or detaching mature male inflorescences for taking pollen in laboratory. In this work, the dynamic behaviour of branches of the male ‘Peter’ variety was analysed under controlled laboratory conditions. A wide range of vibration frequencies was used for branch excitation, and the responses of the stem and its inflorescences were analysed. The stem and inflorescences had clearly differentiated dynamic responses according to the vibration frequency. The stem attained maximum acceleration transmissibility values at specified frequency values (8, 13.5, 20.5–22 Hz). At these values, the vibration applied amplified the stem movement and could clearly contribute to pollen release to the atmosphere. At the highest frequency values (45 Hz), the stem damped the vibration, but the inflorescences amplified the response. In the 54–59 Hz range, the inflorescences amplified the acceleration received by an average 5-fold. In this range, detachment of the inflorescences is facilitated without exciting the stems. Thus, current commercial trunk shakers must be adapted if they are used to collect pollen from pistachio trees owing to the higher transmission values of trees without leaves. • Stem and male inflorescences of the Peter variety have a different dynamic response. • Precise vibration frequency applied to branch enables a selective organ excitation. • Trunk shakers can be adapted for mass pollen detachment. • Absence of leaves during the pollination period may increase damage to shaken trees .

Patterns of pollen dispersal and pollen capture in the hybridizing cattails,Typha latifoliaandTypha angustifolia

Pollen dispersal regulates the formation of the invasive, wind-pollinated hybrid cattail Typha × glauca Godron, the F1offspring of the broadleaf ( Typha latifolia L.) and narrowleaf ( Typha angustifolia L.) cattail. An earlier study suggested that pollen dispersal by T. latifolia might be spatially restricted, with most dispersal occurring over distances less than 2 m. Restricted pollen dispersal would imply that hybrid formation primarily occurs within mixed stands of cattails. Hybrid formation might also be affected by preferential receipt of conspecific pollen, but this has not been investigated for cattails. We compared patterns of pollen dispersal for T. latifolia and T. angustifolia using a wind tunnel. We then tested whether patterns of pollen receipt were biased toward the capture of conspecific versus heterospecific pollen using monospecific cattail stands with a single local pollen source. Results from the wind tunnel partially supported the previous finding of spatially restricted pollen dispersal for T. latifolia, the paternal parent of F1hybrids. Pollen receipt by T. angustifolia was biased toward the capture of conspecific pollen. Localized pollen dispersal by T. latifolia and preferential conspecific pollen capture by T. angustifolia should reduce rates of hybrid formation below that expected under random mating.

An integrated system for automated measurement of airborne pollen based on electrostatic enrichment and image analysis with machine vision

Here we describe an automated and compact pollen detection system that integrates enrichment, in-situ detection and self-cleaning modules. The system can achieve continuous capture and enrichment of pollen grains in air samples by electrostatic adsorption. The captured pollen grains are imaged with a digital camera, and an automated image analysis based on machine vision is performed, which enables a quantification of the number of pollen particles as well as a preliminary classification into two types of pollen grains. In order to optimize and evaluate the system performance, we developed a testing approach that utilizes an airflow containing a precisely metered amount of pollen particles surrounded by a sheath flow to achieve the generation and lossless transmission of standard gas samples. We studied various factors affecting the pollen capture efficiency, including the applied voltage, air flow rate and humidity. Under optimized conditions, the system was successfully used in the measurement of airborne pollen particles within a wide range of concentrations, spanning 3 orders of magnitude.

The neglected other half - role of the pistil in plant heat stress responses.

Heat stress coinciding with reproductive stage leads to a significant loss in reproductive organs viability, resulting in lower seed-set and crop productivity. Successful fertilization and seed formation are determined by the viability of male and female reproductive organs. The impact of heat stress on the male reproductive organ (pollen) is studied more often compared to the female reproductive organ (pistil). This is attributed to easier accessibility of the pollen coupled with the notion that the pistil's role in fertilization and seed-set under heat stress is negligible. However, depending on species and developmental stages, recent studies reveal varying degrees of sensitivity of the pistil to heat stress. Remarkably, in some cases, the vulnerability of the pistil is even greater than the pollen. This article summarizes the current knowledge of the impact of heat stress on three critical stages of pistil for successful seed-set, that is, female reproductive organ development (gametogenesis), pollen-pistil interactions including pollen capture on stigma and pollen tube growth in style, as well as fertilization and early embryogenesis. Further, future research directions are suggested to unravel molecular basis of heat stress tolerance in pistil, which is critical for sustaining crop yields under predicted warming scenarios.

Efficiency of Herkogamy in Narcissus bulbocodium (Amaryllidaceae).

Within the theoretical framework of the correlation pleiades, floral phenotypic integration has been proposed as a consequence of selection mediated by pollinators acting on floral characters. Here, we analyzed that assumption by studying the floral biology and pollination of the late-winter species Narcissus bulbocodium L. We found that the flowers of N. bulbocodium are pollinator-dependent (mainly on Bombus terrestris) in terms of achieving optimal levels of seed production (xenogamy mean seed-to-ovule ratio 64%). Flowers are phenotypically integrated, and only the inclusion of the stigma within the corona seems to have a positive and significant influence on the deposition of the pollen. It has been hypothesized that by including the stigma within the corolla, the flower has some control over the contact between stigma and pollinators that could lead to an “ordered herkogamy” as a way to promote outcross and avoid self-interference. Therefore, herkogamy was also studied, and while most previous studies have assessed the evolutionary significance of herkogamy by considering its relationship with outcrossing rates, we approach this phenomenon from a novel direction assessing the relationship between a proxy for herkogamy and the precision of the pollination process. Our results seem to support the existence of an optimal herkogamy distance that could maintain maximum levels of both pollen export and (cross) pollen capture. On the basis of the broad variability of herkogamy that we have found in N. bulbocodium and other data in the literature, we discuss the universality of the adaptive origin of herkogamy.

Functional Analysis of the Rice Type-B Response Regulator RR22.

The phytohormone cytokinin plays a critical role in regulating growth and development throughout the life cycle of the plant. The primary transcriptional response to cytokinin is mediated by the action of the type-B response regulators (RRs), with much of our understanding for their functional roles being derived from studies in the dicot Arabidopsis. To examine the roles played by type-B RRs in a monocot, we employed gain-of-function and loss-of-function mutations to characterize RR22 function in rice. Ectopic overexpression of RR22 in rice results in an enhanced cytokinin response based on molecular and physiological assays. Phenotypes associated with enhanced activity of RR22 include effects on leaf and root growth, inflorescence architecture, and trichome formation. Analysis of four Tos17 insertion alleles of RR22 revealed effects on inflorescence architecture, trichomes, and development of the stigma brush involved in pollen capture. Both loss‐ and gain-of-function RR22 alleles affected the number of leaf silica-cell files, which provide mechanical stability and improve resistance to pathogens. Taken together, these results indicate that a delicate balance of cytokinin transcriptional activity is necessary for optimal growth and development in rice.

Comparing methods for controlled capture and quantification of pollen in Cannabis sativa.

PremisePrecise pollen collection methods are necessary for crop breeding, but anemophilous pollen is notoriously difficult to capture and control. Here we compared a variety of methods for the controlled capture of cannabis pollen, intended to ease the process of cross‐fertilization for breeding this wind‐pollinated plant, and measured the utility of light spectroscopy for quantifying relative pollen yield.Methods and ResultsIn two independent trials, we compared a control method of pollen collection (hand collection) to either vacuum‐, water‐, or bag‐collection methods. We used visible light spectroscopy to quantify relative pollen yield, and validated this approach using microscopic pollen counts. We determined that pollen yield was highest when using hand collection or vacuum collection, but efficiency did not differ significantly among methods.ConclusionsTo maximize yield, pollen should be collected by hand or vacuum, but all collection methods were equally efficient in a relative sense because yield increased with collection time. We also found that light spectroscopy is an accurate and rapid method of quantifying pollen abundance (R 2 = 0.86) in a liquid suspension.

Bumblebee hairs as electric and air motion sensors: theoretical analysis of an isolated hair.

Foraging bumblebees are electrically charged. Charge accumulation has been proposed to enable their ability to detect and react to electrical cues. One mechanism suggested for bumblebee electro-sensing is the interaction between external electric fields and electric charges accumulating on fine hairs on the cuticular body. Such hairs exhibit several functional adaptations, for example, thermal insulation, pollen capture and notably, the sensing of air motion such as flow currents or low frequency sound particle velocity. Both air motion and electric fields are ubiquitous in the sensory ecology of terrestrial arthropods, raising the question as to whether cuticular hairs respond to both stimuli. Here, a model-theoretical approach is taken to investigate the capacity of bumblebee filiform hairs as electric sensors and compare it to their response to air motion. We find that oscillating air motion and electric fields generate different mechanical responses, depending on stimulus frequency and body geometry. Further, hair morphology can enhance one sensing mode over the other; specifically, higher surface area favours electric sensitivity. Assuming a maximum stable charge on the hair that is limited only by electric breakdown of air, it is expected that an applied oscillating electric field strength of approximately 300 V m−1 produces comparable mechanical response on the hair as a 35 mm s−1 air flow oscillating at 130 Hz—an air disturbance signal similar to that produced by wingbeats of insects within a few bodylengths of the bumblebee. This analysis reveals that bumblebee filiform hairs can operate as bi-modal sensors, responding to both oscillating electric and air motion stimuli in the context of ecologically relevant scenarios.

Canopy-Mediated Hydrodynamics Contributes to Greater Allelic Richness in Seeds Produced Higher in Meadows of the Coastal Eelgrass Zostera marina

Seagrass meadows, which mediate ocean acidity and turbidity, sequester carbon, and increase biodiversity by providing shelter for larvae and small fish, are among the fastest disappearing ecosystems worldwide. Seagrasses are ecosystem engineers, creating distinct regions of enhanced and diminished flow and turbulent mixing, dependent upon canopy physical parameters, such as canopy density and blade morphology, which in turn impact the transport of pollen, sediment, and nutrients. The health and resilience of seagrass meadows increase with intraspecies genetic diversity, which depends on successful sexual reproduction and the transport of pollen particles between reproductive shoots, which in turn depends on the hydrodynamic conditions created by the meadow. This paper explored the transport of pollen grains in seagrass meadows using a random walk model. The model was parameterized with profiles of mean velocity and eddy diffusivity derived as functions of shoot density, canopy height, canopy shear velocity, canopy drag coefficient, and blade width, and validated with experimental measurements of a tracer plume evolving in a submerged model canopy. Model results showed that release at the top of the canopy led to significantly greater dispersal than release within the canopy, which was consistent with observed patterns of genetic diversity in Zostera marina seeds collected from coastal Massachusetts meadows. Specifically, seeds produced from upper inflorescences had greater allelic richness than seeds from lower inflorescences on the same reproductive shoot, and were the product of a greater number of fathers, reflecting the greater in-canopy pollen movement farther from the bed. Pollen grains modeled with a realistic elongated shape experienced significantly higher rates of capture by the canopy relative to spherical grains of the same volume. The effect of submergence depth (the ratio of water depth to canopy height) on pollen dispersal depended on the nature of the surface boundary: when pollen reflected off the water surface, the mean travel distance before pollen capture decreased with decreasing submergence depth. In contrast, when pollen accumulated at the water surface, surface transport increased pollen dispersal distances, especially at low submergence depths.

Type-B response regulators of rice play key roles in growth, development and cytokinin signaling.

Cytokinins are plant hormones with crucial roles in growth and development. Although cytokinin signaling is well characterized in the model dicot Arabidopsis, we are only beginning to understand its role in monocots, such as rice (Oryza sativa) and other cereals of agronomic importance. Here, we used primarily a CRISPR/Cas9 gene-editing approach to characterize the roles of a key family of transcription factors, the type-B response regulators (RRs), in cytokinin signaling in rice. Results from the analysis of single rr mutants as well as higher-order rr21/22/23 mutant lines revealed functional overlap as well as subfunctionalization within members of the gene family. Mutant phenotypes associated with decreased activity of rice type-B RRs included effects on leaf and root growth, inflorescence architecture, flower development and fertilization, trichome formation and cytokinin sensitivity. Development of the stigma brush involved in pollen capture was compromised in the rr21/22/23 mutant, whereas anther development was compromised in the rr24 mutant. Novel as well as conserved roles for type-B RRs in the growth and development of a monocot compared with dicots were identified.

The Evolution of Sexual Fluids in Gymnosperms From Pollination Drops to Nectar.

A current synthesis of data from modern and fossil plants paints a new picture of sexual fluids, including nectar, as a foundational component of gymnosperm reproductive evolution. We review the morpho-anatomical adaptations, their accompanying secretions, and the functional compounds involved. We discuss two types of secretions: (1) those involved in fertilization fluids produced by gametophytes and archegonia of zooidogamous gymnosperms, i.e., Ginkgo and cycads, and (2) those involved in pollen capture mechanisms (PCMs), i.e., pollination drops. Fertilization fluids provide both liquid in which sperm swim, as well as chemotactic signals that direct sperm to the egg. Such fertilization fluids were probably found among many extinct plants such as ancient cycads and others with swimming sperm, but were subsequently lost upon the evolution of siphonogamy (direct delivery of sperm to the egg by pollen tubes), as found in modern gnetophytes, conifers, and Pinaceae. Pollination drops are discussed in terms of three major types of PCMs and the unique combinations of morphological and biochemical adaptations that define each. These include their amino acids, sugars, calcium, phosphate and proteins. The evolution of PCMs is also discussed with reference to fossil taxa. The plesiomorphic state of extant gymnosperms is a sugar-containing pollination drop functioning as a pollen capture surface, and an in ovulo pollen germination medium. Additionally, these drops are involved in ovule defense, and provide nectar for pollinators. Pollination drops in anemophilous groups have low sugar concentrations that are too low to provide insects with a reward. Instead, they appear to be optimized for defense and microgametophyte development. In insect-pollinated modern Gnetales a variety of tissues produce sexual fluids that bear the biochemical signature of nectar. Complete absence of fluid secretions is restricted to a few, poorly studied modern conifers, and is presumably derived. Aspects of pollination drop dynamics, e.g., regulation of secretion and retraction, are reviewed. Lastly, we discuss pollination drops’ control of pollen germination. Large gaps in our current knowledge include the composition of fertilization fluids, the pollination drops of Podocarpaceae, and the overall hydrodynamics of sexual fluids in general.

Phylogenetic and functional signals in gymnosperm ovular secretions.

Gymnosperms are either wind-pollinated (anemophilous) or both wind- and insect-pollinated (ambophilous). Regardless of pollination mode, ovular secretions play a key role in pollen capture, germination and growth; they are likely also involved in pollinator reward. Little is known about the broad-scale diversity of ovular secretions across gymnosperms, and how these may relate to various reproductive functions. This study analyses the sugar and amino acid profiles of ovular secretions across a range of ambophilous (cycads and Gnetales) and anemophilous gymnosperms (conifers) to place them in an evolutionary context of their possible functions during reproduction. Ovular secretions from 13 species representing all five main lineages of extant gymnosperms were sampled. High-performance liquid chromatography techniques were used to measure sugar and amino acid content. Multivariate statistics were applied to assess whether there are significant differences in the chemical profiles of anemophilous and ambophilous species. Data were compared with published chemical profiles of angiosperm nectar. Chemical profiles were placed in the context of phylogenetic relationships. Total sugar concentrations were significantly higher in ovular secretions of ambophilous species than wind-pollinated taxa such as Pinaceae and Cupressophyta. Ambophilous species had lower amounts of total amino acids, and a higher proportion of non-protein amino acids compared with anemophilous lineages, and were also comparable to angiosperm nectar. Results suggest that early gymnosperms likely had ovular secretion profiles that were a mosaic of those associated with modern anemophilous and ambophilous species. Ginkgo, thought to be anemophilous, had a profile typical of ambophilous taxa, suggesting that insect pollination either exists in Gingko, but is undocumented, or that its ancestral populations were insect-pollinated. Chemical profiles of ovular secretions of ambophilous gymnosperms show a clear signal of pollinator-driven selection, including higher levels of carbohydrates than anemophilous taxa, lower levels of amino acids, and the presence of specific amino acids, such as β-alanine, that are known to influence insect feeding behaviour and physiology.

Honey bee hairs and pollenkitt are essential for pollen capture and removal

While insect grooming has been observed and documented for over one hundred years, we present the first quantitative analysis of this highly dynamic process. Pollinating insects, like honey bees, purposely cover themselves with millions of pollen particles that, if left ungroomed, would make sensing and controlled flight difficult. How do they get clean? We show that the hairs on insect eyes are tuned to the pollen they collect; namely, the hairs are spaced so that they suspend pollen above the body for easy removal by the forelegs. In turn, hair spacing on the foreleg dictates the leg’s ability to store the pollen removed during each swipe. In tests with wax-covered honey bees, we show that hairy forelegs are necessary for pollen removal. Moreover, the viscous fluid found on the surface of pollen grains, or pollenkitt, greatly enhances adhesion. We find that bees accumulate twice as much pollen if pollenkitt is present. This study may help further understand pollination, as well as inform designs for mechanically-sensitive functional surfaces with micro- and nano-structures that are easier to keep clean.

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.

The role of pollination drops in animal pollination in the Mediterranean gymnosperm Ephedra fragilis (Gnetales)

The pollination drop (PD) is a sweet secretion produced by female cones of many gymnosperms whose main function is associated with the capture and transport of pollen to the nucellar surface of the ovule. Due to its high sugar content, PDs appear to be highly suitable for the attraction of animals that might act as potential pollinators for gymnosperms. In the present study, we evaluated the attraction of animals to female and male PDs (sugar-rich rewards) in plants of the Mediterranean shrub Ephedra fragilis in addition to their contribution to plant fitness. Animal exclusion experiments were conducted in Sa Dragonera Islet Natural Park (Western Mediterranean, Spain). In addition, a link between PD investment (PD concentration) and plant fitness was evaluated on plants of E. fragilis. Lizards and insects were the principal visitors to PDs offered by E. fragilis plants. Animals effectively contributed to plant fitness. Nevertheless, the fitness contribution was not as elevated as the contribution from wind pollination (~30 vs. 70 %, respectively). Increased plant investment on PDs significantly elevated fitness in plants of E. fragilis. The present study provides evidence that E. fragilis relies primarily on wind pollination, with pollination and fitness being supplemented by animal visitation. Additionally, besides its main role as pollen capture and germination, PDs act as a pollinator reward that benefits plant reproduction.