Does the morphological fit between flowers and pollinators affect pollen deposition? An experimental test in a buzz‐pollinated species with anther dimorphism

Some pollination systems, such as buzz‐pollination, are associated with floral morphologies that require a close physical interaction between floral sexual organs and insect visitors. In these systems, a pollinator's size relative to the flower may be an important feature determining whether the visitor touches both male and female sexual organs and thus transfers pollen between plants efficiently. To date, few studies have addressed whether in fact the “fit” between flower and pollinator influences pollen transfer, particularly among buzz‐pollinated species. Here we use Solanum rostratum, a buzz‐pollinated plant with dimorphic anthers and mirror‐image flowers, to investigate whether the morphological fit between the pollinator's body and floral morphology influences pollen deposition. We hypothesized that when the size of the pollinator matches the separation between the sexual organs in a flower, more pollen should be transferred to the stigma than when the visitor is either too small or too big relative to the flower. To test this hypothesis, we exposed flowers of S. rostratum with varying levels of separation between sexual organs, to bumblebees (Bombus terrestris) of different sizes. We recorded the number of visits received, pollen deposition, and fruit and seed production. We found higher pollen deposition when bees were the same size or bigger than the separation between anther and stigma within a flower. We found a similar, but not statistically significant pattern for fruit set. In contrast, seed set was more likely to occur when the size of the flower exceeded the size of the bee, suggesting that other postpollination processes may be important in translating pollen receipt to seed set. Our results suggest that the fit between flower and pollinator significantly influences pollen deposition in this buzz‐pollinated species. We speculate that in buzz‐pollinated species where floral morphology and pollinators interact closely, variation in the visitor's size may determine whether it acts mainly as a pollinator or as a pollen thief (i.e., removing pollen rewards but contributing little to pollen deposition and fertilization).     

Buzz-pollination in Neotropical bees:genus-dependent frequencies and lack of optimal frequency for pollen release

Over 50 genera of bees release pollen from flower anthers using thoracic vibrations,a phenomenon known as buzz-pollination.The efficiency of this process is directly affected by the mechanical properties of the buzzes,namely the duration,amplitude,and frequency.Nonetheless,although the effects of the former two properties are well described,the role of buzz frequency on pollen release remains unclear.Furthermore,nearly all of the existing studies describing vibrational properties of natural buzz-pollination are limited to bumblebees(Bombus)and carpenter bees(Xvlocopa)constraining our current understanding of this behavior and its evolution.Therefore,we attempted to minimize this shortcoming by testing whether flower anthers exhibit optimal frequency for pollen release and whether bees tune their buzzes to match these(optimal)frequencies.If true,certain frequencies will trigger more pollen release and lighter bees will reach buzz frequencies closer to this optimum to compensate their smaller buzz amplitudes.Two strategies were used to test these hypotheses:(i)the use of(artificial)vibrational playbacks in a broad range of buzz frequencies and amplitudes to assess pollen release by tomato plants(Solarium Ivcopersicum L.)and(ii)the recording of natural buzzes of Neotropical bees visiting tomato plants during pollination.The playback experiment indicates that although buzz frequency does affect pollen release,no optimal frequency exists for that.In addition,the recorded results of natural buzz-pollination reveal that buzz frequencies vary with bee genera and are not correlated with body size.Therefore,neither bees nor plants are tuned to optimal pollen release frequencies.Bee frequency of buzz-pollination is a likely consequence of the insect flight machinery adapted to reach higher accelerations,while flower plant response to buzz-pollination is the likely result of its pollen granular properties.     

One for all and all for one: retention of colour‐unchanged old flowers increases pollinator attraction in a hermaphroditic plant

• Long‐lived flowers increase pollen transfer rates, but these entail high water and carbon maintenance costs. The retention of pollinated and reward‐free old flowers enhances pollinator visitation to young receptive flowers by increasing floral display size. This mechanism is associated with acropetal inflorescences or changes in flower colour and openness, but the retention of unchanging solitary flowers remains overlooked.
• We examined pollination‐dependent variation in floral longevity and determined stigmatic receptivity, pollen viability and pollen removal rates among flower ages in Kielmeyera regalis, a Neotropical savanna shrub. We also evaluated the effects of floral display size on pollinator visitation rates. Lastly, we determined whether old flowers are unvisited and exclusively increase pollinator attraction to young flowers through flower removal experiments.
• Regardless of pollination treatment, flowers lasted fully open with no detectable physical changes for 3 days. Over time, stigmas remained receptive but >95% of pollen was removed. Pollinator visitation significantly increased with floral display size and intermediate percentages (15–30%) of newly opened flowers. Accordingly, the retention of reward‐free and unvisited old flowers increased young flower–pollinator interaction.
• Our results reveal the importance of a prolonged floral longevity in increasing pollinator attraction toward newly opened receptive flowers without changes in flower colour and form. We conclude that the retention of pollinated, reward‐free and unvisited colour‐unchanged old flowers in K. regalis is a strategy that counteracts the water use costs associated with the maintenance of large flowers with increased mate opportunities in a pollen‐limited scenario.

     

Does body size predict the buzz‐pollination frequencies used by bees?

Body size is an important trait linking pollinators and plants. Morphological matching between pollinators and plants is thought to reinforce pollinator fidelity, as the correct fit ensures that both parties benefit from the interaction. We investigated the influence of body size in a specialized pollination system (buzz‐pollination) where bees vibrate flowers to release pollen concealed within poricidal stamens. Specifically, we explored how body size influences the frequency of buzz‐pollination vibrations. Body size is expected to affect frequency as a result of the physical constraints it places on the indirect flight muscles that control the production of floral vibrations. Larger insects beat their wings less rapidly than smaller‐bodied insects when flying, but whether similar scaling relationships exist with floral vibrations has not been widely explored. This is important because the amount of pollen ejected is determined by the frequency of the vibration and the displacement of a bee's thorax. We conducted a field study in three ecogeographic regions (alpine, desert, grassland) and recorded flight and floral vibrations from freely foraging bees from 27 species across four families. We found that floral vibration frequencies were significantly higher than flight frequencies, but never exceeded 400 Hz. Also, only flight frequencies were negatively correlated with body size. As a bee's size increased, its buzz ratio (floral frequency/flight frequency) increased such that only the largest bees were capable of generating floral vibration frequencies that exceeded double that of their flight vibrations. These results indicate size affects the capacity of bees to raise floral vibration frequencies substantially above flight frequencies. This may put smaller bees at a competitive disadvantage because even at the maximum floral vibration frequency of 400 Hz, their inability to achieve comparable thoracic displacements as larger bees would result in generating vibrations with lower amplitudes, and thus less total pollen ejected for the same foraging effort.     

Using geometric morphometrics to determine the “fittest” floral shape: A case study in large-flowered,buzz-pollinated Meriania hernandoi

Premise

Floral shape (relative arrangement and position of floral organs) is critical in mediating fit with pollinators and maximizing conspecific pollen transfer particularly in functionally specialized systems. To date, however, few studies have attempted to quantify flowers as the inherently three-dimensional (3D) structures they are and determine the effect of intraspecific shape variation on pollen transfer. We here addressed this research gap using a functionally specialized system, buzz pollination, in which bees extract pollen through vibrations, as a model. Our study species, Meriania hernandoi (Melastomataceae), undergoes a floral shape change from pseudocampanulate corollas with more actinomorphically arranged stamens (first day) to open corollas with a more zygomorphic androecium (second day) over anthesis, providing a natural experiment to test how variation in floral shape affects pollination performance.

Methods

In one population of M. hernandoi, we bagged 51 pre-anthetic flowers and exposed half of them to bee pollinators when they were in either stage of their shape transition. We then collected flowers, obtained 3D flower models through x-ray computed tomography for 3D geometric morphometric analyses, and counted the pollen grains remaining per stamen (male pollination performance) and stigmatic pollen loads (female pollination performance).

Results

Male pollination performance was significantly higher in open flowers with zygomorphic androecia than in pseudo-campanulate flowers. Female pollination performance did not differ among floral shapes.

Conclusions

These results suggest that there is an “optimal” shape for male pollination performance, while the movement of bees around the flower when buzzing the spread-out stamens results in sufficient pollen deposition regardless of floral shape.

     

Anther and stigma morphology in mirror-image flowers of Chamaecrista chamaecristoides (Fabaceae): implications for buzz pollination

Enantiostyly (mirror-image flowers) is usually associated with buzz pollination. In buzz-pollinated flowers, pollen is released through terminal pores after bees vibrate the stamens. Several studies have evaluated the function of 'buzzing' in pollen release, but less attention has been paid to the effect of buzzing on pollen capture and deposition on stigmas. Evaluating the mechanism of pollen dispersion in buzz-pollinated flowers is important because it may affect mating patterns and reproductive success. In this study, we analysed the morphology of sexual organs (anther and stigma) using electron microscopy, and determined the relationship between sexual organ structure and pollen capture function through experimental manipulations of buzz-pollinated flowers of Chamaecrista chamaecristoides, as well as vibration frequencies on floral visitors. Pollen release occurs through two terminal pores at the tip of the stamens. However, unlike most angiosperms that have their stigmatic surface exposed, C.?chamaecristoides presents a stigmatic surface inside a cavity covered by trichomes. Experimental manipulations showed that effective fertilisation is only achieved when the style is vibrated, suggesting that buzzing is not only important for pollen release but also for pollen capture and deposition on the stigma. This result, in addition to vibration frequency analysis, suggests that although all floral visitors buzz flowers only those that buzz at higher frequencies achieve effective fertilisation. The anatomical features of sexual organs in flowers of C.?chamaecristoides demonstrate that this species possesses a highly specialised, elaborate morphology, with both genders selected for traits that promote buzz pollination.     

The bees don't know and the flowers don't care: the effect of heterospecific pollen on reproduction in co‐occurring Thysanotus species (Asparagaceae) with similar flowers

Species integrity relies on the maintenance of reproductive isolation, particularly between closely related species. Further, it has been hypothesized that the presence of heterospecific pollen on flower stigmas adversely affects plant reproduction with increasing effect in closely related species. Using two pairs of co‐occurring buzz‐pollinated Thysanotus spp. in the Mediterranean climate region of Perth, Western Australia, we quantified the effect of heterospecific pollen on fruit and seed set. We first determined the mating systems of the two focal species using self‐ and outcross pollen, followed by separate treatments with heterospecific pollen within each species pair. Of the two species receiving pollen, Thysanotus triandrus had a mixed mating system, but with significantly lower fruit and seed set from self‐pollen relative to outcross pollen. Thysanotus tenellus was autogamous with no difference in fruit or seed set between autogamous, self‐ or outcross pollinations. Heterospecific pollen had no effect on fruit or seed set of either focal species. These outcomes point to post‐pollination reproductive isolation, consistent with a floral morphology that causes low specificity of pollen placement and thus a poor capacity for pre‐pollination discrimination. Negative effects of heterospecific pollen, therefore, do not appear to play a role in the reproduction in this group of buzz‐pollinated flowers.     

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.

     

Pollination and breeding system of the enigmatic South African parasitic plant Mystropetalon thomii (Mystropetalaceae): rodents welcome,but not needed

• Unrelated plants adapted to particular pollinator types tend to exhibit convergent evolution in floral traits. However, inferences about likely pollinators from ‘pollination syndromes’ can be problematic due to trait overlap among some syndromes and unusual floral architecture in some lineages. An example is the rare South African parasitic plant Mystropetalon thomii (Mystropetalaceae), which has highly unusual brush‐like inflorescences that exhibit features of both bird and rodent pollination syndromes.
• We used camera traps to record flower visitors, quantified floral spectral reflectance and nectar and scent production, experimentally determined self‐compatibility and breeding system, and studied pollen dispersal using fluorescent dyes.
• The dark‐red inflorescences are usually monoecious, with female flowers maturing before male flowers, but some inflorescences are purely female (gynoecious). Inflorescences were visited intensively by several rodent species that carried large pollen loads, while visits by birds were extremely rare. Rodents prefer male‐ over female‐phase inflorescences, likely because of the male flowers’ higher nectar and scent production. The floral scent contains several compounds known to attract rodents. Despite the obvious pollen transfer by rodents, we found that flowers on both monoecious and gynoecious inflorescences readily set seed in the absence of rodents and even when all flower visitors are excluded.
• Our findings suggest that seed production occurs at least partially through apomixis and that M. thomii is not ecologically dependent on its rodent pollinators. Our study adds another species and family to the growing list of rodent‐pollinated plants, thus contributing to our understanding of the floral traits associated with pollination by non‐flying mammals.

     

Horizontal orientation of zygomorphic flowers: significance for rain protection and pollen transfer

• Floral traits are recognized to have evolved under selection for abiotic and biotic factors. Complex zygomorphic flowers usually face horizontally. It has been proved that a horizontal orientation facilitates pollinator recognition and pollination efficiency, but its significance in adaptation to abiotic factors remains unknown.
• The floral orientation of Abelia × grandiflora naturally varies around horizontal (with an angle of ?30 to +33° between the floral main axis and the horizontal). We examined whether three different floral orientations affected flower thermal conditions, response to rain and pollination.
• Results showed that floral orientation had no effect on diurnal variations in flower temperature. The anthers of all three flower orientations were wetted by rainfall, but the inclined upward‐facing flowers contained significantly more rainwater. The horizontal flowers received significantly higher visitation by hawkmoths and had a higher stigmatic pollen load. In contrast, the upward flower orientation reduced pollination precision, while downward‐facing flowers had decreased pollinator attraction.
• This study indicates that horizontal flowers may have evolved as a trade‐off between rain protection and pollination. Zygomorphic flowers that deviate from a horizontal orientation may have lower fitness because of flower flooding by rainwater and decreased pollen transfer.

     

High specialisation in the pollination system of Mandevilla tenuifolia (J.C. Mikan) Woodson (Apocynaceae) drives the effectiveness of butterflies as pollinators

Butterfly pollination in the tropics is considered somewhat effective or solely effective in a few plant species. In the present study, we tested the hypothesis that Mandevilla tenuifolia (Apocynaceae), which has floral attributes associated with psychophily, has strategies adapted to pollination by butterflies, restricting other floral visitors and making these insects act as efficient pollinators. We analysed the floral and reproductive biology of M. tenuifolia, as well as the frequency and efficiency of its flower visitors. M. tenuifolia is an herb whose flowers have strong herkogamy and secondary pollen presentation on the style head, which corresponds to 60.4% of pollen on the anthers. Flower longevity and the long period of receptivity of the stigmatic region associated with the large amount of pollen removed in the first visits suggest that flowers remain functionally female during part of anthesis. Butterflies, mainly of the families Nymphalidae and Pieridae, are the only pollinators of M. tenuifolia. Despite being self‐compatible, M. tenuifolia depends on biotic vectors for fruit production. A non‐significant difference in fruit set between controlled treatments and natural conditions suggests that the pollinators are efficient. The inclination resulting from the landing of butterflies on flowers, together with flower morphology, guiding the insect proboscis inside the floral tube, as well as the frequency and efficiency of butterfly visits, are evidence of the close relationship between butterflies and M. tenuifolia, and also of the efficiency of these insects as pollinators.     

Beyond the pollination syndrome: nectar ecology and the role of diurnal and nocturnal pollinators in the reproductive success of Inga sessilis (Fabaceae)

Inga species present brush‐type flower morphology allowing them to be visited by distinct groups of pollinators. Nectar features in relation to the main pollinators have seldom been studied in this genus. To test the hypothesis of floral adaptation to both diurnal and nocturnal pollinators, we studied the pollination ecology of Inga sessilis, with emphasis on the nectar secretion patterns, effects of sequential removals on nectar production, sugar composition and the role of diurnal and nocturnal pollinators in its reproductive success. Inga sessilis is self‐incompatible and pollinated by hummingbirds, hawkmoths and bats. Fruit set under natural conditions is very low despite the fact that most stigmas receive polyads with sufficient pollen to fertilise all ovules in a flower. Nectar secretion starts in the bud stage and flowers continually secreting nectar for a period of 8 h. Flowers actively reabsorbed the nectar a few hours before senescence. Sugar production increased after nectar removal, especially when flowers were drained during the night. Nectar sugar composition changed over flower life span, from sucrose‐dominant (just after flower opening, when hummingbirds were the main visitors) to hexose‐rich (throughout the night, when bats and hawkmoths were the main visitors). Diurnal pollinators contributed less than nocturnal ones to fruit production, but the former were more constant and reliable visitors through time. Our results indicate I. sessilis has floral adaptations, beyond the morphology, that encompass both diurnal and nocturnal pollinator requirements, suggesting a complementary and mixed pollination system.     

Effectiveness of buzz pollination in Pedicularis chamissonis: significance of multiple visits by bumblebees

Pollination success of plants is highly susceptible to the frequency of visits and foraging behavior of pollinators. Pollination of the nectarless flowers of Pedicularis species depends on bumblebee workers collecting pollen by vibrating the anthers (buzz pollination). However, little is known about the efficiency of the pollination system. Foraging behavior, pollen removal from anthers and pollen deposition on stigmas of P. chamissonis were studied to assess the effectiveness of buzz pollination in an alpine snowbed population of northern Japan. Although bumblebees tended to visit most of the flowers open at a given time within inflorescences during a single visit, pollen removal rate at the first visit was about 20%, and buzzing period decreased with increasing number of previous visits, resulting in a decreasing proportion of pollen removed per visit as the number of visits increased. These trends enable plants to provide pollen for more pollinators. The number of pollen grains deposited on stigmas was not saturated during the first visit and increased with additional visits. Irrespective of weak self-compatibility, evidence of interference between self and outcross pollen was lacking for seed production. Therefore, buzz pollination in P. chamissonis acts as a mechanism that improves the chance of cross-pollination upon multiple visits if pollinator visitation is frequent.     

Cryptic self‐incompatibility and distyly in Hedyotis acutangula Champ. (Rubiaceae)

Distyly, floral polymorphism frequently associated with reciprocal herkogamy, self‐ and intramorph incompatibility and secondary dimorphism, constitutes an important sexual system in the Rubiaceae. Here we report an unusual kind of distyly associated with self‐ and/or intramorph compatibility in a perennial herb, Hedyotis acutangula. Floral morphology, ancillary dimorphisms and compatibility of the two morphs were studied. H. acutangula did not exhibit precise reciprocal herkogamy, but this did not affect the equality of floral morphs in the population, as usually found in distylous plants. Both pin and thrum pollen retained relatively high viability for 8 h. The pollen to ovule ratio was 72.5 in pin flowers and 54.4 in thrum flowers. Pistils of pin flowers remained receptive for longer than those of thrum flowers. No apparent difference in the germination rate of pin and thrum pollen grains was observed when cultured in vitro, although growth of thrum pollen tubes was much faster than that of pin pollen tubes. Artificial pollination revealed that pollen tube growth in legitimate intermorph crosses was faster than in either intramorph crosses or self‐pollination, suggesting the occurrence of cryptic self‐incompatibility in this species. Cryptic self‐incompatibility functioned differently in the two morphs, with pollen tube growth rates after legitimate and illegitimate pollination much more highly differentiated in pin flowers than in thrum flowers. No fruit was produced in emasculated netted flowers, suggesting the absence of apomixis. Our results indicate that H. acutangula is distylous, with a cryptic self‐incompatibility breeding system.     

Geographic variation of reproductive traits and competition for pollinators in a bird‐pollinated plant

Geographic variation in the reproductive traits of animal‐pollinated plants can be shaped by spatially variable selection imposed by differences in the local pollination environment. We investigated this process in Babiana ringens (Iridaceae), an enigmatic species from the Western Cape region of South Africa. B. ringens has evolved a specialized perch facilitating cross‐pollination by sunbirds and displays striking geographic variation in perch size and floral traits. Here, we investigate whether this variation can be explained by geographic differences in the pollinator communities. We measured floral and inflorescence traits, and abiotic variables (N, P, C, and rainfall) and made observations of sunbirds in populations spanning the range of B. ringens. In each population, we recorded sunbird species identity and measured visitation rates, interfloral pollen transfer, and whether the seed set of flowers was pollen limited. To evaluate whether competition from co‐occurring sunbird‐pollinated species might reduce visitation, we quantified nectar rewards in B. ringens and of other co‐flowering bird‐pollinated species in local communities in which populations occurred. Variation in abiotic variables was not associated with geographical variation of traits in B. ringens. Malachite sunbirds were the dominant visitor (97% of visits) and populations with larger‐sized traits exhibited higher visitation rates, more between‐flower pollen transfer and set more seed. No sunbirds were observed in four populations, all with smaller‐sized traits. Sunbird visitation to B. ringens was not associated with local sunbird activity in communities, but sunbird visitation was negatively associated with the amount of B. ringens sugar relative to the availability of alternative nectar sources. Our study provides evidence that B. ringens populations with larger floral traits are visited more frequently by sunbirds, and we propose that visitation rates to B. ringens may be influenced, in part, by competition with other sunbird‐pollinated species.     

Impact of pre‐ and post‐pollination barriers on pollen transfer and reproductive isolation among three sympatric Pedicularis (Orobanchaceae) species

• Pedicularis is the largest genus in the Orobanchaceae (>300) with many species co‐occurring and co‐blooming in subalpine to alpine meadows in the Himalayas. Although it is well known that different Pedicularis species place pollen on different parts of the same bumblebee's body, thus reducing interspecific pollen transfer, it is not known whether post‐pollination components also contribute to reproductive isolation (RI).
• In this study, we quantified the individual strengths and absolute contributions of six pre‐ and post‐pollination components of RI between three sympatric species in two pairs; Pedicularis gruina × Pedicularis tenuisecta (gru × ten) and Pedicularis comptoniifolia × Pedicularis tenuisecta (com × ten).
• All three Pedicularis species shared the same Bombus species. Individual foragers showed a high, but incomplete, floral constancy for each species. Therefore, pre‐pollination barriers were potentially ‘leaky’ as Bombus species showed a low but consistent frequency of interspecific visitation. The RI strength of pre‐pollination was lower in com × ten than in gru × ten. In contrast, post‐pollination barriers completely blocked gene flow between both sets of species pairs. Two post‐pollination recognition sites were identified. Late acting rejection of interspecific pollen tube growth occurred in com♀ × ten♂, while seeds produced in bi‐directional crosses of gru × ten failed to germinate.
• We propose that, although floral isolation based on pollen placement on pollinators in the genus Pedicularis is crucial to avoid interspecific pollen transfer, the importance of this mode of interspecific isolation may be exaggerated. Post‐pollination barriers may play even larger roles for currently established populations of co‐blooming and sympatric species in this huge genus in the Himalayas.

     

Floral specialization for different pollinators and divergent use of the same pollinator among co‐occurring Impatiens species (Balsaminaceae) from Southeast Asia

Floral variation among closely related species is thought to often reflect differences in pollination systems. Flowers of the large genus Impatiens are characterized by extensive variation in colour, shape and size and in anther and stigma positioning, but studies of their pollination ecology are scarce and most lack a comparative context. Consequently, the function of floral diversity in Impatiens remains enigmatic. This study documents floral variation and pollination of seven co‐occurring Impatiens spp. in the Southeast Asian diversity hotspot. To assess whether floral trait variation reflects specialization for different pollination systems, we tested whether species depend on pollinators for reproduction, identified animals that visit flowers, determined whether these visitors play a role in pollination and quantified and compared key floral traits, including floral dimensions and nectar characteristics. Experimental exclusion of insects decreased fruit and seed set significantly for all species except I. muscicola, which also received almost no visits from animals. Most species received visits from several animals, including bees, birds, butterflies and hawkmoths, only a subset of which were effective pollinators. Impatiens psittacina, I. kerriae, I. racemosa and I. daraneenae were pollinated by bees, primarily Bombus haemorrhoidalis. Impatiens chiangdaoensis and I. santisukii had bimodal pollination systems which combined bee and lepidopteran pollination. Floral traits differed significantly among species with different pollination systems. Autogamous flowers were small and spurless, and did not produce nectar; bee‐pollinated flowers had short spurs and large floral chambers with a wide entrance; and bimodally bee‐ and lepidopteran‐pollinated species had long spurs and a small floral chamber with a narrow entrance. Nectar‐producing species with different pollination systems did not differ in nectar volume and sugar concentration. Despite the high frequency of bee pollination in co‐occurring species, individuals with a morphology suggestive of hybrid origin were rare. Variation in floral architecture, including various forms of corolla asymmetry, facilitates distinct, species‐specific pollen‐placement on visiting bees. Our results show that floral morphological diversity among Impatiens spp. is associated with both differences in functional pollinator groups and divergent use of the same pollinator. Non‐homologous mechanisms of floral asymmetry are consistent with repeated independent evolution, suggesting that competitive interactions among species with the same pollination system have been an important driver of floral variation among Impatiens spp.     

Temperature and water stress affect plant–pollinator interactions in Borago officinalis (Boraginaceae)

Climate change alters the abiotic constraints faced by plants, including increasing temperature and water stress. These changes may affect flower development and production of flower rewards, thus altering plant–pollinator interactions. Here, we investigated the consequences of increased temperature and water stress on plant growth, floral biology, flower‐reward production, and insect visitation of a widespread bee‐visited species, Borago officinalis. Plants were grown for 5 weeks under three temperature regimes (21, 24, and 27°C) and two watering regimes (well‐watered and water‐stressed). Plant growth was more affected by temperature rise than water stress, and the reproductive growth was affected by both stresses. Vegetative traits were stimulated at 24°C, but impaired at 27°C. Flower development was mainly affected by water stress, which decreased flower number (15 ± 2 flowers/plant in well‐watered plants vs. 8 ± 1 flowers/plant under water stress). Flowers had a reduced corolla surface under temperature rise and water stress (3.8 ± 0.5 cm² in well‐watered plants at 21°C vs. 2.2 ± 0.1 cm² in water‐stressed plants at 27°C). Both constraints reduced flower‐reward production. Nectar sugar content decreased from 3.9 ± 0.3 mg/flower in the well‐watered plants at 21°C to 1.3 ± 0.4 mg/flower in the water‐stressed plants at 27°C. Total pollen quantity was not affected, but pollen viability decreased from 79 ± 4% in the well‐watered plants at 21°C to 25 ± 9% in the water‐stressed plants at 27°C. Flowers in the well‐watered plants at 21°C received at least twice as many bumblebee visits compared with the other treatments. In conclusion, floral modifications induced by abiotic stresses related to climate change affect insect behavior and alter plant–pollinator interactions.     

Intensified wind pollination mediated by pollen dimorphism after range expansion in an ambophilous biennial Aconitum gymnandrum

Pollination systems and associated floral traits generally differ between core and marginal populations of a species. However, such differences are rarely examined in plants with a mixed wind‐ and bumblebee‐pollination system, and the role of wind pollination during range expansion in ambophilous plants remains unclear. We compared floral traits and the contributions of bumblebee and wind pollination in refugium and marginal populations of the ambophilous plant Aconitum gymnandrum. We found that most floral traits differed between the two populations, and those traits associated with the shift to wind pollination were pronounced in the marginal population. Bumblebee visitation rates varied significantly, but were generally low in the marginal population. Wind pollination occurred in both populations, and the efficiency was lower than that of bumblebee pollination. Two types of pollen grains, namely round and fusiform pollen, were transported to a stigma by bumblebees and wind, but fusiform pollen contributed to wind pollination to a larger degree, especially in the marginal population. Our results suggest that wind pollination was enhanced by pollen dimorphism in the marginal population of A. gymnandrum, and wind pollination may provide reproductive assurance when bumblebee activity is unpredictable during range expansion, indicating that ambophily is stable in this species and shift in pollination system could be common when plants colonize new habitats.     

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.