Heteranthery as a solution to the demand for pollen as food and for pollination – Legitimate flower visitors reject flowers without feeding anthers

• Heteranthery, the presence of feeding and pollinating anthers in the same flower, seems to mediate the evolutionary dilemma for plants to protect their gametes and yet provide food for pollinators. This study aims to elucidate the role of heteranthery in the buzz‐pollinated Senna reniformis.
• The fecundity of pollen from long‐, medium‐ and short‐sized anthers was determined by hand cross‐pollination experiments, and the quantity, size, ornamentation and viability of pollen of different anthers were compared. Rates of flower rejection by bees were measured in anther removal experiments to assess the preferences of flower visitors for feeding or pollinating anthers.
• Large bees, which were the effective pollinators of self‐incompatible S. reniformis, avoided flowers without short feeding anthers, but not those without medium or long anthers. Illegitimate small and medium‐sized bees were unresponsive to anther exclusion experiments. Long anthers deposited pollen on the back and short anthers on the venter of large bees. Pollen from long anthers had higher in vitro viability and higher fruit and seed set after cross‐pollination than pollen from other sized anthers.
• Short anthers produce feeding pollen to effective pollinators and long anthers are related to pollination of S. reniformis. Bee behaviour and size was found to directly influence the role of anthers in the ‘division of labour’. Only large bee pollinators that carry the pollinating pollen from long anthers in ‘safe sites’ associated short anthers with the presence of food. In the absence of these larger bee pollinators, the role of heteranthery in S. reniformis would be strongly compromised and its function would be lost.

     

Morphological specialization of heterantherous Rhynchanthera grandiflora (Melastomataceae) accommodates pollinator diversity

• The tropical Melastomataceae are characterized by poricidal anthers which constitute a floral filter selecting for buzz‐pollinating bees. Stamens are often dimorphic, sometimes with discernible feeding and pollinating functions. Rhynchanthera grandiflora produces nectarless flowers with four short stamens and one long stamen; all anthers feature a narrow elongation with an upwards facing pore.
• We tested pollen transfer by diverse foraging bees and viability of pollen from both stamen types. The impact of anther morphology on pollen release direction and scattering angle was studied to determine the plant's reproductive strategy.
• Medium‐sized to large bees sonicated flowers in a specific position, and the probability of pollen transfer correlated with bee size even among these legitimate visitors. Small bees acted as pollen thieves or robbers. Anther rostrum and pore morphology serve to direct and focus the pollen jet released by floral sonication towards the pollinator's body. Resulting from the ventral and dorsal positioning of the short and long stamens, respectively, the pollinator's body was widely covered with pollen. This improves the plant's chances of outcrossing, irrespective of which bee body part contacts the stigma. Consequently, R. grandiflora is also able to employ bee species of various sizes as pollen vectors.
• The strategy of spreading pollen all over the pollinator's body is rather cost‐intensive but counterbalanced by ensuring that most of the released pollen is in fact transferred to the bee. Thus, flowers of R. grandiflora illustrate how specialized morphology may serve to improve pollination by a functional group of pollinators.

     

Bumble bee-initiated vibration release mechanism of rhododendron pollen

Although Rhododendron spp. anthers have apical pores and should be expected to be buzz pollinated, bees do not normally sonicate them to release pollen. Stamens were examined to determine their pollen release mechanism; the filaments were shown to have natural vibration modes that cause pollen to be forcibly ejected by vibration, providing the pollen is mature. The first natural vibration mode of the stamens was found to generate centrifugal force sufficient to throw the pollen toward the apical pore and out of the anther, so that it adheres to the anther tip and hangs in the bee's path to the nectaries. These vibrations may be triggered by bees brushing past the long stamens when foraging for nectar, so that the flowers retain their pollen until insect vectors begin foraging on them. The low frequency vibration modes of the viscin threads, which hold the pollen together, may contribute towards the pollen ejection by low frequency stamen vibration. Vibration transmission of dehiscent anthers containing pollen was good from 50 Hz to 850 Hz, suggesting that sonication should be an efficient method of pollen collection, although this rarely occurs. Vibratory ejection occurs at a constant energy level, rather than the constant force levels found in previous research on Actinidia.     

Division of labour within flowers: heteranthery,a floral strategy to reconcile contrasting pollen fates

In many nectarless flowering plants, pollen serves as both the carrier of male gametes and as food for pollinators. This can generate an evolutionary conflict if the use of pollen as food by pollinators reduces the number of gametes available for cross‐fertilization. Heteranthery, the production of two or more stamen types by individual flowers reduces this conflict by allowing different stamens to specialize in ‘pollinating’ and ‘feeding’ functions. We used experimental studies of Solanum rostratum (Solanaceae) and theoretical models to investigate this ‘division of labour’ hypothesis. Flight cage experiments with pollinating bumble bees (Bombus impatiens) demonstrated that although feeding anthers are preferentially manipulated by bees, pollinating anthers export more pollen to other flowers. Evolutionary stability analysis of a model of pollination by pollen consumers indicated that heteranthery evolves when bees consume more pollen than should optimally be exchanged for visitation services, particularly when pollinators adjust their visitation according to the amount of pollen collected.     

Ricochet pollination in Senna (Fabaceae) – petals deflect pollen jets and promote division of labour among flower structures

• Naturalists Fritz and Hermann Müller hypothesised that heteranthery often leads to a division of labour into ‘feeding’ and ‘pollinating’ stamens; the latter often being as long as the pistil so as to promote successful pollination on the bees’ back. In many buzz‐pollinated species of Senna, however, the so‐called pollinating stamens are short and not level with the stigma, raising the question of how pollen is shed on the bees’ back. Here we explore a mechanism called ‘ricochet pollination’. We test whether division of labour is achieved through the interaction between short lower stamens and strongly concave ‘deflector petals’.
• We studied the arrangement and morphology of the floral organs involved in the ricochet pollination, functioning of the flowers through artificial sonication and observed the interactions between bees and flowers in the field.
• The middle stamens are adapted to eject pollen downwards, which can be readily collected on the bee mid legs. Most of the pollen is ejected towards the deflector petal(s). Pollen from this set of stamens is more likely to contribute to pollination. The pollen grains seem to ricochet multiple times against the deflector petals to eventually reach the bee's back.
• The pollen ricochet mechanism promotes a division of labour by involving additional floral organs, such as petals, reinforcing the Müllers’ division‐of‐labour hypothesis. However, alternative, non‐multiexclusive hypotheses could be explored in genus Senna and other angiosperm species.

     

A biophysical model for buzz pollination in angiosperms

The stamens of most of the world's flowering plants are longitudinally dehiscent, releasing their pollen passively, whereupon floral visitors may collect it. In nearly 400 genera in 65 plant families, the anthers dehisce by means of short apical slits or true pores. In these forms, the small light pollen can only be efficiently released by native bees capable of vibrating these stamens. This intrafloral behavior propels pollen out of the pores striking the bees on their venters. It is then collected for use in larval cell provisions. Aspects of the historical development of this novel pollination syndrome, known as “buzz” or vibratile (equals vibrational) pollination, are presented including a discussion and figures of a poricidal anther, a buzzing bee and the model system.A biophysical model for the pollen/locule wall interactions resulting in pollen expulsion upon bee or artificial vibration is developed. The model was created with the morphology of anthers of Solanum (Solanaceae) in mind, but the results obtained are generally applicable to any apically dehiscent flower which is vibrated by bees to release pollen.The anthers were modeled as a tall rectangular box with an apical pore and containing numerous small particles. As the box vibrates, particles striking the walls rebound elastically. If a pollen grain strikes a receding wall, it loses energy. If a grain strikes an advancing wall, it gains energy in the collision. In each oscillation, there is a net gain in the energy of the particles. As the anther (box) is shaken, vibrational energy is transmitted from the pterothorax of the bees to the flower, the pollen grains gaining significant energy. As the energy increases and the particles begin to move about more and more vigorously, they will begin to escape through the hole in the box (or stamina] pore). The rate at which particles leave the box and time required to empty the box are calculated as functions of the geometry of the model system and the frequency of vibration.In order to test the influence of air currents, Bernolli effects and viscous drag, the flowers were mecahnically vibrated in vacuum. The pollen cloud thus produced was virtually unchanged ans so it seems unlikely that air plays any significant role in the phenomenon of vibrational pollen release.Finally, variables such as: inelastic interactions, electrostatic forces, slightly sticky pollen due to presence of “pollenkitt”, duration and types of bee buzzes are discussed in relation to the mathematical model presented.     

Shifts from specialised to generalised pollination systems in Miconieae (Melastomataceae) and their relation with anther morphology and seed number

Most species in Melastomataceae have poricidal anthers related to specialised bee buzz‐pollination, while some have anthers with large openings associated to non‐bee pollination systems. We tracked the evolution of anther morphology and seed number on the Miconieae phylogenetic tree to understand the evolutionary shifts in such pollination systems. Anther morphometric data and seed number were recorded for 54 taxa. Pollinators (bees, flies, wasps) were recorded for 20 available species. Ancestral state reconstruction was made using Maximum Likelihood from nrITS sequences. We used phylogenetic eigenvector regressions to estimate phylogenetic signal and the adaptive component for these traits. Species pollinated by bees or bees and wasps tend to have smaller pores and fruits with more seeds. Species pollinated by flies or flies and bees and/or wasps tend to have larger pores and fruits with less seeds. Independent evolution occurred three times for anthers with large pores and twice for fruits with few seeds. We detected a phylogenetic signal in both traits, and negative correlated evolution between them. In actinomorphic small‐flowered Miconieae, changes in anther morphology can be related to generalisation in the pollination system incorporating flies and wasps as pollinators and lessening the importance of buzzing bees in such process. Differences in pollen removal and deposition may explain differences in anther morphology and seed number in Miconieae.     

Darwin's vexing contrivance: a new hypothesis for why some flowers have two kinds of anther

Heteranthery, the presence of two or more anther types in the same flower, is taxonomically widespread among bee-pollinated angiosperms, yet has puzzled botanists since Darwin. We test two competing hypotheses for its evolution: the long-standing ‘division of labour'' hypothesis, which posits that some anthers are specialized as food rewards for bees whereas others are specialized for surreptitious pollination, and our new hypothesis that heteranthery is a way to gradually release pollen that maximizes pollen delivery. We examine the evolution of heteranthery and associated traits across the genus Clarkia (Onagraceae) and study plant–pollinator interactions in two heterantherous Clarkia species. Across species, heteranthery is associated with bee pollination, delayed dehiscence and colour crypsis of one anther whorl, and movement of that anther whorl upon dehiscence. Our mechanistic studies in heterantherous species show that bees notice, forage on and export pollen from each anther whorl when it is dehiscing, and that heteranthery promotes pollen export. We find no support for division of labour, but multifarious evidence that heteranthery is a mechanism for gradual pollen presentation that probably evolved through indirect male–male competition for siring success.     

Shakers and head bangers: differences in sonication behavior between Australian <Emphasis Type="Italic">Amegilla murrayensis</Emphasis> (blue-banded bees) and North American <Emphasis Type="Italic">Bombus impatiens</Emphasis> (bumblebees)

Many bees collect pollen by grasping the anthers of a flower and vibrating their flight muscles at high frequencies—a behavior termed sonication, or buzz-pollination. Here we compare buzz-pollination on Solanum lycopersicum (cherry tomatoes) by two bees that fill similar niches on different continents—in Australia, Amegilla murrayensis (blue-banded bee), and in North America, Bombus impatiens (bumblebee). We collected audio recordings of buzz-pollination and quantified the frequency and length of buzzes, as well as the total time spent per flower. We found that A. murrayensis buzzes at significantly higher frequencies (~350 Hz) than B. impatiens (~240 Hz) and flaps its wings at higher frequencies during flight. There was no difference in the length of a single buzz, but A. murrayensis spent less time on each flower, as B. impatiens buzzed the flower several times before departing, whereas A. murrayensis typically buzzed the flower only once. High-speed videos of A. murrayensis during buzz-pollination revealed that its physical interaction with the flower differs markedly from the mechanism described for Bombus and other bees previously examined. Rather than grasping the anther cone with its mandibles and shaking, A. murrayensis taps the anther cone with its head at the high buzzing frequencies generated by its flight muscles. This unique behavior, combined with its higher buzzing frequency and reduced flower visit duration, suggests that A. murrayensis may be able to extract pollen more quickly than B. impatiens, and points to the need for further studies directly comparing the pollination effectiveness of these species.     

Could learning of pollen odours by honey bees (Apis mellifera) play a role in their foraging behaviour?

Abstract. The role of pollen odour cues in the foraging behaviour of honey bees (Apis mellifera L.) is poorly understood. Using classical conditioning of the proboscis extension response, in which bees learn to associate an odour with a sucrose reward, the present study tests whether odours of bee-collected pollen from the hive environment or odours of fresh pollen on the anthers of flowers could be used in pollen foraging. Honey bees efficiently learn odours from field-bean (Vicia faba) bee-collected pollen and oilseed-rape (Brassica napus) bee-collected pollen, hand-collected pollen, anthers and whole flowers, demonstrating that honey bees can learn pollen odours associatively in biologically realistic concentrations. Honey bees learn pollen odours of oilseed rape better than field bean and, although they generalize these two odours, they easily distinguish between them in discrimination tests, suggesting that pollen odours may be used in species recognition/discrimination. There is little evidence that honey bees can recognize whole flowers based on previous experience of bee-collected pollen odour. However, they generalize the odours of oilseed-rape anthers and whole flowers, suggesting that anther pollen in situ may play a more prominent role than bee-collected pollen in foraging behaviour.     

Assessment of pollen reward and pollen availability in Solanum stramoniifolium and Solanum paniculatum for buzz‐pollinating carpenter bees

The two widespread tropical Solanum species S. paniculatum and S. stramoniifolium are highly dependent on the visits of large bees that pollinate the flowers while buzzing them. Both Solanum species do not offer nectar reward; the rewarding of bees is thus solely dependent on the availability of pollen. Flower visitors are unable to visually assess the amount of pollen, because the pollen is hidden in poricidal anthers. In this study we ask whether and how the amount of pollen determines the attractiveness of flowers for bees. The number of pollen grains in anthers of S. stramoniifolium was seven times higher than in S. paniculatum. By contrast, the handling time per five flowers for carpenter bees visiting S. paniculatum was 3.5 times shorter than of those visiting S. stramoniifolium. As a result foraging carpenter bees collected a similar number of pollen grains per unit time on flowers of both species. Experimental manipulation of pollen availability by gluing the anther pores showed that the carpenter bees were unable to detect the availability of pollen by means of chemical cues before landing and without buzzing. Our study shows that the efficiency of pollen collecting on S. paniculatum is based on large inflorescences with short between‐flower search times and short handling time of individual flowers, whereas that of S. stramoniifolium relies on a large amount of pollen per flower. Interestingly, large carpenter bees are able to adjust their foraging behaviour to drastically different strategies of pollen reward in otherwise very similar plant species.     

The effect of wild radish floral morphology on pollination efficiency by four taxa of pollinators

The effects of floral morphology on rates of pollen removal and deposition by different pollinators in generalist plant species are not well known. We studied pollination dynamics in wild radish, Raphanus raphanistrum, a plant visited by four groups of pollinators: honey bees, small native bees, butterflies, and syrphyd flies. The effects of anther position and other factors on pollen removal during single visits by all four pollinator taxa were measured. Flowers with high anther exsertion (i.e., anthers placed higher above the opening of the corolla tube) tended to have the highest numbers of pollen grains removed, but this effect was strongest for honey bees and butterflies. For all pollinator taxa, pollen removal increased with the number of pollen grains available on a flower and whowed a positive, decelerating relationship with the duration of the visit. The effects of stigma position and other factors on pollen deposition during single visits by honey bees and butterflies were also studied. The nectar-feeding butterflies had a higher pollination efficiency (percentage of pollen grains removed from anthers that were subsequently deposited on a stigma) than the nectar- and pollen-feeding honey bees. Flowers with intermediate stigma exsertion had the highest numbers of pollen grains deposited on their stigmas by butterflies, but stigma exsertion had no effect on deposition by honey bees. For both butterflies and honey bees, pollen deposition on the recipient flower increased with the amount of pollen removed from the donor flower, and there was a positive, decelerating relationship between deposition and time spent at the flower; these results are analogous to those for pollen removal. The effects of anther and stigma exsertion on pollen removal and denosition did not fit predictions based on patterns of floral correlations, but results for morphology, pollen availability, time spent per visit, and pollinator efficiency are in broad agreement with previous studies, suggesting the possible emergence of some general rules of pollen transfer.     

POLLINATION AND THE FUNCTION OF FLORAL PARTS IN CHAMAECRISTA FASCICULATA (FABACEAE)

Chamaecrista fasciculata has a rigid upper petal that curves over nine of the ten anthers. The single anther is deflexed, as is the style. An experimental pollination study was designed to test the hypotheses that: 1) the rigid petal acts as flight guide to ensure pollination; and 2) the nine grouped anthers serve as fodder anthers, whereas the single deflexed anther functions in pollination. The rigid petal was removed from 97 flowers. Only 5% of the manipulated flowers set fruit in comparison to a fruit set of 47% for the control group. The results of the study support the flight guide hypothesis. Pollen from both sets of anthers is viable and germinates on receptive stigmas. A pollen flow experiment using powdered metals, backscatter scanning electron microscopy, and X-ray microanalysis revealed that there is no difference in the frequency of pollen distribution from either set of anthers. Therefore, this study refutes the fodder/pollinating anther hypothesis.     

Floral Sonication is an Innate Behaviour in Bumblebees that can be Fine-Tuned with Experience in Manipulating Flowers

Bumblebees demonstrate an extensive capacity for learning complex motor skills to maximise exploitation of floral rewards. This ability is well studied in nectar collection but its role in pollen foraging is less well understood. Floral sonication is used by bees to extract pollen from some plant species with anthers which must be vibrated (buzzed) to release pollen. Pollen removal is determined by sonication characteristics including frequency and amplitude, and thus the ability to optimise sonication should allow bees to maximise the pollen collection. We investigated the ability of the buff-tailed bumblebee (Bombus terrestris) to modify the frequency and amplitude of their buzzes with increasing experience manipulating flowers of the buzz-pollinated plant Solanum rostratum. We analysed flight and feeding vibrations generated by naïve workers across feeding bouts. Feeding buzzes were of a higher frequency and a lower amplitude than flight buzzes. Both flight and feeding buzzes had reduced amplitudes with increasing number of foraging trips. However, the frequency of their feeding buzzes was reduced significantly more than their flight buzzes as bumblebee workers gained experience manipulating flowers. These results suggest that bumblebees are able to modify the characteristics of their buzzes with experience manipulating buzz-pollinated flowers. We discuss our findings in the context of bumblebee learning, and the current understanding of the optimal sonication characteristics for releasing pollen in buzz-pollinated species. Our results present a tantalising insight into the potential role of learning in floral sonication, paving the way for future research in this area.     

POLLINATION ECOLOGY OF VACCINIUM STAMINEUM (ERICACEAE: VACCINIOIDEAE)

Successful fruit set by deerberry, Vaccinium stamineum (Ericaceae: Vaccinioideae) requires floral visitation by insects, and in particular by bees that collect its pollen by sonication of anthers (“buzz-pollination”). A diversity of bees visit deerberry flowers, but most species are either 1) infrequent, 2) polylectic (broad host range for pollen), or 3) principally nectar-seeking, and so are unimportant as pollen vectors. Both sexes of the solitary, oligolectic bee Melitta americana⁵ (Melittidae) are abundant at flowering deerberry bushes in central New York State. Female M. americana sonicate the deerberry anthers while imbibing nectar, and carry virtually pure scopal loads of deerberry pollen, unlike the other common visitor, Xylocopa virginica (Anthophoridae). Comparative analyses of bee 1) abundances, 2) buzz-pollination behaviors, 3) pollen fidelities, and 4) pollination efficiencies, all indicate that Melitta americana is the primary pollinator of Vaccinium stamineum.     

Floral reward presentation favored the expression of male function in the pollen‐only flower Melastoma malabathricum

Abstract Animal‐pollinated plant species modulate the presentation of pollinator rewards to maximize reproductive success. In plants providing pollen as the only reward for pollinators, it is usually difficult to unravel the dual roles of reward presentation and the realization of male and female functions (pollen removal and deposition). Exploiting the two types of anther in the androecia of Melastoma malabathricum L., we examined whether the removal of pollen for reward is regulated primarily to favor male function or female function. Pollen removal by carpenter bees from the feeding and pollination anthers, as well as pollen deposition on the stigmas, were quantified during anthesis of M. malabathricum. There was no significant difference in pollen removal rates from the feeding and pollination anthers of M. malabathricum between the onset of anthesis and flower wilting. The stigmatic pollen loads exceeded the ovule number after three sonication bouts, and female function was satisfied earlier than male function. The results support the hypothesis that the presentation of pollination reward in this species is regulated primarily to favor the expression of male function, rather than female function, in agreement with the pollen‐donation hypothesis. A cooperative relationship between the feeding and pollination anthers was demonstrated in heterantherous flowers, which optimizes the balance in investments between pollinator rewards and “functional pollen” for gene transfer.     

Environmental control and evidence for predetermination of pollen embryogenesis inNicotiana tabacum pollen

Summary The effect of daylenght and temperature for the donor plants (Nicotiana tabacum var. Badischer Burley) on the formation of pollen competent for embryogenesis (P-pollen) by the three possible routes (during normal flower developmentin situ (pollen dimorphism), during cold-treatment of excised flower buds, in cultured anthers) was studied. In all three routes, P-pollen frequency (premitotic pollen, before 1. sporophytic division, PPF) was affected in essentially the same way. At 24 °C and long days, PPF was low and short days had only a slightly increasing effect. At 18 °C and long days, PPF was higher and short days further increased it. Correlated with PPF under the different growth regimes was the percentage of units with more than one vegetative-type nucleus (normal embryos + abortive embryos + multinucleate pollen) in 3 weeks old anther cultures. Under greenhouse conditions, PPF was generally higher than at 24° in growth rooms and showed a maximum in the winter months. Plant age did not affect PPF. These results give further evidence that pollen embryogenesis is predetermined before excision and culture of the pollen or anthers.     

Pollen production and release in <Emphasis Type="Italic">Mesua ferrea</Emphasis> Linn. (Guttiferae): a spatio-temporal pattern

Based on a five-year study of pollen production and release in two different natural populations of Mesua ferrea from Indo-Burma region of Northeast India, we determined that pollen output follows a spatio-temporal pattern. Pollen productivity determinations considered various sources of variability, including the number of flowers per branch, flowers per tree, anthers per tree and pollen grains per tree. Each of these parameters revealed significant year-to-year and population effects. Anthesis follows a forenoon pattern, whereas anther dehiscence pursues the diurnal pattern. The former was significantly correlated with the timing of floral visitation and pollen deposition on stigmas. The latter, however, had significant relationship with the deposition of pollen grains on microscopic slides. The Apis and Xylocopa bees are the efficient pollinators to achieve the reproductive success in M. ferrea. Annual production of pollen per tree varied from averages of 1.07 ± 0.10 × 10¹⁰ and 3.24 ± 0.16 × 10¹⁰ in years of low production, with alternate high years, producing 3.85 ± 0.34 × 10¹⁰ and 8.22 ± 0.76 × 10¹⁰ pollen grains per tree.     

Dynamic changes in insoluble polysaccharides and neutral lipids in the developing anthers of an endangered plant species, <Emphasis Type="Italic">Pancratium maritimum</Emphasis>

Dynamic changes in the distribution of lipid and insoluble polysaccharide reserves of Pancratium maritimum L. (Amaryllidaceae) anthers were investigated throughout the successive stages of pollen development, using cytochemical methods, to determine whether the synthesis, transformation, and mobilization of reserve materials in developing anthers follow the regular pathway in angiosperms and support the physiological activities in developing pollen. Polysaccharides and lipid reserves exhibited a variable pattern of distribution from the sporogenous cell stage to the anthesis. Starch granules and lipid bodies were scarce in the cytoplasm of sporogenous cells, but their number increased significantly at the premeiotic stage. Conversely, starch and lipid reserves of meiocytes reduced at the early prophase of the first meiotic division, and then their amount showed fluctuations during the microsporogenesis. The cytoplasm of free and vacuolated microspores was poor regarding the polysaccharide and lipid reserves. However, at the late vacuolated microspore stage, small insoluble polysaccharides began to appear in the microspore cytoplasm, and their number increased remarkably in the cytoplasm of the bicellular pollen grain. During the maturation of pollen grains, polysaccharide reserves were replaced with lipids. The starch and lipid reserves of the staminal envelope also showed variations at different stages of the anther development. The dynamic changes in the polysaccharide and lipid reserves of P. maritimum anthers were consistent with the physiological activities such as differentiation, cell division and material synthesis that occur in the anther tissue at different stages of the male gametophyte development, and supported the normal pollen development.