Relationship of visual and olfactory signal parameters in a food-deceptive flower mimicry system

Pollinators such as bees are attracted to flowers by their visualdisplay and their scent. Although most flowers reinforce visitsby providing pollen and/or nectar, there are species—notablyfrom the orchid family—that do not but do resemble rewardingspecies. These mimicry relationships provide ideal opportunitiesfor investigating the evolution of floral signals and theirimpact on pollinator behavior. Here, we have reanalyzed a caseof specialized food mimicry between the orchid Orchis israeliticaand its model, the lily Bellevalia flexuosa. Based on currentknowledge of insect sensory physiology, we were able to characterizeboth the visual and olfactory signals of model and mimic, aswell as of two phylogenetically related orchids. By using acolor vision model, we mapped each species' visual signals tothe perceptual space of honeybees and found an apparent shiftof the mimic's visual signals towards the model. We confirmthat visual mimicry is present. We analyzed the flower odorsby using gas chromatography/mass spectroscopy. We related thesesignals to the perceptual space of the pollinators by testingthe scent extracts physiologically, using in vivo brain imaging.We found no evidence of olfactory mimicry. The results indicatethat evolutionary pressure acts on the visual, but not olfactory,traits of O. israelitica toward a higher similarity to its model.Apparently, odor mismatch does not prevent a bee from landingon a flower that has the expected visual display. The resultstherefore argue for the dominance of visual stimuli in short-distanceflower choice. The orchid may still depend on long-distanceolfactory attraction originating from neighboring model plants.     

Relative role of flower color and scent on pollinator attraction: experimental tests using F1 and F2 hybrids of daylily and nightlily

The daylily (Hemerocallis fulva) and nightlily (H. citrina) are typical examples of a butterfly-pollination system and a hawkmoth-pollination system, respectively. H. fulva has diurnal, reddish or orange-colored flowers and is mainly pollinated by diurnal swallowtail butterflies. H. citrina has nocturnal, yellowish flowers with a sweet fragrance and is pollinated by nocturnal hawkmoths. We evaluated the relative roles of flower color and scent on the evolutionary shift from a diurnally flowering ancestor to H. citrina. We conducted a series of experiments that mimic situations in which mutants differing in either flower color, floral scent or both appeared in a diurnally flowering population. An experimental array of 6 × 6 potted plants, mixed with 24 plants of H. fulva and 12 plants of either F1 or F2 hybrids, were placed in the field, and visitations of swallowtail butterflies and nocturnal hawkmoths were recorded with camcorders. Swallowtail butterflies preferentially visited reddish or orange-colored flowers and hawkmoths preferentially visited yellowish flowers. Neither swallowtail butterflies nor nocturnal hawkmoths showed significant preferences for overall scent emission. Our results suggest that mutations in flower color would be more relevant to the adaptive shift from a diurnally flowering ancestor to H. citrina than that in floral scent.     

Priority of color over scent during flower visitation by adult <Emphasis Type="Italic">Vanessa indica</Emphasis> butterflies

Most flower visitors innately prefer a particular color and scent, and use them as cues for flower recognition and selection. However, in most cases, since color and scent serve as a combined signal, not only does the preference for an individual cue, but also the preference hierarchy among different cues, influence their flower visitation. In the present study, we attempted to reveal (1) the chromatic and (2) the olfactory cues that stimulate flower visiting, and (3) the preference hierarchy between these cues, using the naïve adult butterfly Vanessa indica. When we offered 12 different-colored (six chromatic and six achromatic) paper flower models, V. indica showed a color preference for yellow and blue. When we examined the proboscis extension reflex (PER) of V. indica towards 16 individual compounds identified in the floral scents from two nectar plants belonging to the family Compositae, Taraxacum officinale and Cirsium japonicum, six compounds were found to have relatively high PER-eliciting activities, including benzaldehyde, acetophenone, and (E+Z)-nerolidol. When we combined color and scent cues in two-choice bioassays, where butterflies were offered flower models that were purple (a relatively unattractive color), the models scented with these active compounds were significantly more attractive than the odorless controls. In addition, synthetic blends mimicking the floral scents of T. officinale and C. japonicum (at doses equivalent to that of ten flowers) enhanced the number of visits to the scented models. However, the effect of odorizing was not conspicuous in parallel bioassays when yellow flower models were used, and the butterflies also significantly preferred odorless yellow models to scented purple models. These results demonstrate that V. indica depends primarily on color and secondarily on scent during flower visitation.     

Colour vision in diurnal and nocturnal hawkmoths

Diurnal and nocturnal hawkmoths (Sphingidae, Lepidoptera) havethree spectral types of receptor sensitive to ultraviolet, blueand green light. As avid flower visitors and pollinators, theyuse olfactory and visual cues to find and recognise flowers.Moths of the diurnal species Macroglossum stellatarum and thenocturnal species Deilephila elpenor, Hyles lineata and Hylesgallii use and learn the colour of flowers. Nocturnal speciescan discriminate flowers at starlight intensities when humansand honeybees are colour-blind. M. stellatarum can use achromatic,intensity-related cues if colour cues are absent, and this isprobably also true for D. elpenor. Both species can recognisecolours even under a changed illumination colour.     

Variation in floral characters,particularly floral scent,in sapromyophilous Stemona species

Flowers or inflorescences often deploy various signals, including visual, olfactory, and gustatory cues, that can be detected by their pollinators. In many plants, these cues and their functions are poorly understood. Deciphering the interactions between floral cues and pollinators is crucial for analyzing the reproductive success of flowering plants. In this study, we examined the composition of the fetid floral scents produced by several Stemona species, including nine S. tuberosa populations from across China, using dynamic headspace adsorption, gas chromatography, and mass spectrometry techniques. We compared variations in floral phenotype, including floral longevity, nectar rewards, pollinator behavior, and flower length and color among the Stemona species. Of the 54 scent compounds identified, the major compounds include fetid dimethyl disulfide, dimethyl trisulfide, 1‐pyrroline, butyric acid, p‐cresol, isoamyl alcohol, and indole. We detected striking differentiation in floral scent at both the species and population level, and even within a population of plants with different colored flowers. Floral characteristics related to sapromyophily and deceptive pollination, including flower color mimicking livor mortis and a lack of nectar, were found in five Stemona species, indicating that Stemona is a typical sapromyophilous taxon. Species of this monocot genus might employ evolutionary tactics to exploit saprophilous flies for pollination.     

The Complexity of Background Clutter Affects Nectar Bat Use of Flower Odor and Shape Cues

Given their small size and high metabolism, nectar bats need to be able to quickly locate flowers during foraging bouts. Chiropterophilous plants depend on these bats for their reproduction, thus they also benefit if their flowers can be easily located, and we would expect that floral traits such as odor and shape have evolved to maximize detection by bats. However, relatively little is known about the importance of different floral cues during foraging bouts. In the present study, we undertook a set of flight cage experiments with two species of nectar bats (Anoura caudifer and A. geoffroyi) and artificial flowers to compare the importance of shape and scent cues in locating flowers. In a training phase, a bat was presented an artificial flower with a given shape and scent, whose position was constantly shifted to prevent reliance on spatial memory. In the experimental phase, two flowers were presented, one with the training-flower scent and one with the training-flower shape. For each experimental repetition, we recorded which flower was located first, and then shifted flower positions. Additionally, experiments were repeated in a simple environment, without background clutter, or a complex environment, with a background of leaves and branches. Results demonstrate that bats visit either flower indiscriminately with simple backgrounds, with no significant difference in terms of whether they visit the training-flower odor or training-flower shape first. However, in a complex background olfaction was the most important cue; scented flowers were consistently located first. This suggests that for well-exposed flowers, without obstruction from clutter, vision and/or echolocation are sufficient in locating them. In more complex backgrounds, nectar bats depend more heavily on olfaction during foraging bouts.     

Pollination of the “carrion flowers” of an African stapeliad (<Emphasis Type="Italic">Ceropegia mixta</Emphasis>: Apocynaceae): the importance of visual and scent traits for the attraction of flies

Flowers that mimic carrion or faeces exhibit unusual traits, the evolution and functional significance of which remain poorly understood. Odour is an important pollinator attractant, but visual traits and interactions between visual and scent traits have seldom been considered. We studied pollination of the “carrion flowers” of Ceropegia mixta [= Orbea variegata], analysed floral traits and used manipulative experiments to explore the contributions of visual and scent traits to pollinator attraction. Flowers were pollinated primarily by Musca domestica (Muscidae), with lesser contributions by Calliphoridae and Sarcophagidae flies. The floral odour (analysed using gas chromatography–mass spectrometry) was dominated by oligosulphides and phenol. Comparison of floral and abiotic background colours (analysed using reflectance spectrometry) using a fly colour vision model suggested that flowers would be chromatically indistinguishable from the background. Comparison of fly arrival rates at concealed (but still scented) versus exposed flowers showed that flies can locate flowers without visual cues, but visitation was higher when the flowers were visible. Experiments using model flowers with odour supplied by real flowers (to explore the significance of dark flowers and dark spots on a pale background, which both occur frequently in flowers that mimic carrion or faeces) showed that scented black flowers attracted significantly more flies than similarly scented human-yellow flowers, while the presence or size of black spots on the corolla had no effect on the attraction of flies. Our results suggest that there is a visual component to fly attraction, but some traits, such as the mottled patterning, may not have evolved to enhance pollinator attraction.     

Specialized pollination in the African milkweed Xysmalobium orbiculare: a key role for floral scent in the attraction of spider-hunting wasps

Specialized pollination by prey-hunting wasps is poorly documented in rewarding plants. Furthermore, the mechanisms of achieving specialization are not clear since flowers typically produce exposed nectar and have no morphological adaptations (such as long spurs) to exclude non-pollinating visitors. We investigated the pollination of Xysmalobium orbiculare and explored the functional roles of floral scent and nectar in attracting pollinators and deterring nectar robbers. Floral visitor observations showed that this milkweed is visited almost exclusively by pompilid wasps in the genus Hemipepsis. These wasps were the only insects to carry pollinia, and a cage experiment confirmed their effectiveness in removing and inserting pollinia on flowers. Hand-pollinations showed that plants are genetically self-incompatible and thus reliant on pollinators for seed set. Palatability experiments with honeybees showed that nectar is distasteful to non-pollinating insects and is therefore likely to play a functional role in deterring nectar thieves. Choice experiments in the field showed that the wasp pollinators are attracted primarily by floral scent rather than visual cues. Analysis of spectral reflectance of flowers revealed that flowers are dull colored and are unlikely to stand out from the background vegetation. We conclude that X. orbiculare is specialized for pollination by spider-hunting wasps in the genus Hemipepsis and utilizes floral scent to selectively attract its pollinators and unpalatable nectar to deter non-pollinating visitors.     

Evidence of red sensitive photoreceptors in Pygopleurus israelitus (Glaphyridae: Coleoptera) and its implications for beetle pollination in the southeast Mediterranean

A very well-documented case of flower-beetle interaction is the association in the Mediterranean region between red bowl-shaped flowers and beetles of the family Glaphyridae. The present study examines the visual mechanisms by which Pygopleurus israelitus (Glaphyridae: Scarabaeoidea: Coleoptera) would perceive the colors of flowers they visit by characterizing the spectral sensitivity of its photoreceptors. Our measurements revealed the presence of three types of photoreceptors, maximally sensitive in the UV, green and red areas of the spectrum. Using color vision space diagrams, we calculated the distribution of beetle-visited flower colors in the glaphyrid and honeybee color space and evaluated whether chromatic discrimination differs between the two types of pollinators. Respective color loci in the beetle color space are located on one side of the locus for green foliage background, whereas in the honeybee the flower color loci surround the locus occupied by green foliage. Our results represent the first evidence of a red sensitive photoreceptor in a flower-visiting coleopteran species, highlighting Glaphyridae as an interesting model group to study the role of pollinators in flower color evolution.     

Discrimination of Unrewarding Flowers by Bees; Direct Detection of Rewards and Use of Repellent Scent Marks

Bumblebees and honeybees deposit short-lived scent marks on flowers that they visit when foraging. Conspecifics use these marks to distinguish those flowers that have recently been emptied and, so, avoid them. The aim of this study was to assess how widespread this behavior is. Evidence for direct detection of reward levels was found in two bee species: Agapostemon nasutus was able to detect directly pollen availability in flowers with exposed anthers, while Apis mellifera appeared to be able to detect nectar levels of tubular flowers. A third species, Trigona fulviventris, avoided flowers that had recently been visited by conspecifies, regardless of reward levels, probably by using scent marks. Three further bee/flower systems were examined in which there was no detectable discrimination among flowers. We argue that bees probably rely on direct detection of rewards where this is allowed by the structure of the flower and on scent marks when feeding on flowers where the rewards are hidden. However, discrimination does not always occur. We suggest that discrimination may not always make economic sense; when visiting flowers with a low handling time, or flowers that are scarce, it may be more efficient to visit every flower that is encountered.     

Location and Color Learning in Bumblebees in a Two-Phase Conditioning Experiment

Bees use spatial and visual cues that characterize flowers to make dietary choices. If two such cues always appear together nonambiguously, they provide identical information. In such cases, do bees base dietary choices on one cue and ignore the other, or do they consider both cues? We allowed bumblebees to forage on two patches of artificial flowers that differed in location, color, and reward presence in a two-phase experiment. We switched either the display color, the location, or both the color and the location associated with the rewarding patch between phases. We tested for the effects of the switch on the bees' choices. Immediately following a switch in the location or both the location and the color of the rewarding patch, the bees' performance decreased, as they continued to visit the patch that was previously rewarding. This decrease did not occur when only the color of the rewarding patch was changed or in no-change controls. We suggest that the bees' foraging choices were guided mostly by a location cue when both the location and the color conveyed the same information.     

Visual and Olfactory Floral Cues of Campanula (Campanulaceae) and Their Significance for Host Recognition by an Oligolectic Bee Pollinator

Oligolectic bees collect pollen from a few plants within a genus or family to rear their offspring, and are known to rely on visual and olfactory floral cues to recognize host plants. However, studies investigating whether oligolectic bees recognize distinct host plants by using shared floral cues are scarce. In the present study, we investigated in a comparative approach the visual and olfactory floral cues of six Campanula species, of which only Campanula lactiflora has never been reported as a pollen source of the oligolectic bee Ch. rapunculi. We hypothesized that the flowers of Campanula species visited by Ch. rapunculi share visual (i.e. color) and/or olfactory cues (scents) that give them a host-specific signature. To test this hypothesis, floral color and scent were studied by spectrophotometric and chemical analyses, respectively. Additionally, we performed bioassays within a flight cage to test the innate color preference of Ch. rapunculi. Our results show that Campanula flowers reflect the light predominantly in the UV-blue/blue bee-color space and that Ch. rapunculi displays a strong innate preference for these two colors. Furthermore, we recorded spiroacetals in the floral scent of all Campanula species, but Ca. lactiflora. Spiroacetals, rarely found as floral scent constituents but quite common among Campanula species, were recently shown to play a key function for host-flower recognition by Ch. rapunculi. We conclude that Campanula species share some visual and olfactory floral cues, and that neurological adaptations (i.e. vision and olfaction) of Ch. rapunculi innately drive their foraging flights toward host flowers. The significance of our findings for the evolution of pollen diet breadth in bees is discussed.     

Odour and colour information in the foraging choice behaviour of the honeybee

1. Honeybees Apis mellifera ligustica were trained to work on a patch with artificial rewarding and non-rewarding flowers, coupled to an air extractor. The perceptual colour distance between the rewarding and the non-rewarding flowers was varied and the flower choice and the repellent scent-marking behaviour of the bees were recorded. 2. The discrimination between rewarding and non-rewarding flowers depended on their colour distance, improving with a greater colour difference. This task was guided thus visually and was not affected by activating the air extractor. 3. The scent-marking activity was only observable when the colour information of both groups of flowers was the same or very similar. This thus represents the first reported case of a modulation of an olfactory activity through the visual input provided by colour distances. When the air extractor was activated, rejections associated with the scent-marking behaviour disappeared, thus confirming the olfactory nature of this behaviour. 4. Honeybees are thus capable of using one or more sensory cues to enhance their foraging efficiency, according to the environmental situation. This great plasticity allows them to attain an enhanced efficiency while foraging. 5. We successfully applied the model of colour choice behaviour of the honeybee. Since the original theory was developed for Apis mellifera carnica, this work also constitutes the first attempt to describe the behaviour of the honeybee race, Apis mellifera ligustica, using the postulated model, and reaffirms thus its generality.     

Bumblebees require visual pollen stimuli to initiate and multimodal stimuli to complete a full behavioral sequence in close‐range flower orientation

Flower visits are complex encounters, in which animals are attracted by floral signals, guided toward the site of the first physical contact with a flower, land, and finally take up floral rewards. At close range, signals of stamens and pollen play an important role to facilitate flower handling in bees, yet the pollen stimuli eliciting behavioral responses are poorly known. In this study, we test the response of flower‐naive bumblebees (Bombus terrestris) toward single and multimodal pollen stimuli as compared to natural dandelion pollen. As artificial pollen stimuli, we used the yellow flavonoid pigment quercetin, the scent compound eugenol, the amino acid proline, the monosaccharide glucose, and the texture of pollen‐grain‐sized glass pellets as a tactile stimulus. Three test stimuli, dandelion pollen, one out of various uni‐ and multimodal stimulus combinations, and a solvent control were presented simultaneously to individual bumblebees, whose response was recorded. The results indicate that bumblebees respond in an irreversible sequence of behavioral reactions. Bumblebees approached the visual stimulus quercetin as often as natural dandelion pollen. An additional olfactory stimulus resulted in slightly more frequent landings. The multimodal stimulus combinations including visual, olfactory, gustatory, and tactile stimuli elicited approaches, antennal contacts, and landings as often as natural pollen. Subsequent reactions like proboscis extension, mandible biting, and buzzing were more often but not regularly observed at dandelion pollen. Our study shows that visual signals of pollen are sufficient to trigger initial responses of bumblebees, whereas multimodal pollen stimuli elicit full behavioral response as compared to natural pollen. Our results suggest a major role of pollen cues for the attraction of bees toward flowers and also explain, why many floral guides mimic the visual signals of pollen and anthers, that is, the yellow and UV‐absorbing color, to direct bumblebees toward the site where they access the floral rewards.     

Context-dependent reproductive isolation mediated by floral scent and color

Reproductive isolation due to pollinator behavior is considered a key mode of speciation in flowering plants. Although floral scent is thought to mediate pollinator behavior, little is known about its effects on pollinator attraction and floral visitation in the wild. We used field experiments with wild hawkmoths and laboratory experiments with naïve hawkmoths to investigate attraction to and probing of flowers in response to indole, a volatile emitted by Ipomopsis tenuituba but not its close relative I. aggregata, both alone and in combination with floral color differences. We demonstrated that indole attracts wild hawkmoths to flowers, but has little effect on the rate at which those attracted moths probe flowers. In contrast, white flower color did not influence hawkmoth attraction in the field, but caused more attracted moths to probe flowers. Thus, the moths require both scent and high visual contrast, in that order, to feed at flowers at dusk. Their preference for indole-scented flowers is innate, but species-specific preference is mitigated by previous experience and plant spatial patterning. This context-dependent behavior helps explain why these Ipomopsis species show geographical variation in the extent of hybridization and may potentially explain formation of hybrid bridges in other systems of hawkmoth-pollinated plants.     

Recognition of scent marks in solitary bees to avoid previously visited flowers

Knowing how floral visitors forage efficiently among flowers is important to understanding plant-pollinator interactions. When bees search for rewarding flowers, they use several visual cues to detect the available floral resources. In addition to these cues, bees can recognize scent marks, which are olfactory cues left on flowers foraged by previous visitors. This behavior is well known in social bees, such as honeybees and bumblebees. Although solitary bees do not need to give information about which flowers were foraged to conspecifics, several pieces of evidence have indicated the use of scent marks. However, it is unknown whether the behavior is widely used in many different bee species. We investigated whether four different solitary bees, Colletes patellatus (Colletidae), Andrena prostomias (Andrenidae), Osmia orientalis (Megachilidae), and Tetralonia mitsukurii (Apidae), can recognize flowers that have been foraged previously by visitors within 3 min. All four bees showed rejection responses to flowers foraged by conspecifics. However, our results showed that responses to foraged flowers varied among bee species. The tendency of A. prostomias and T. mitsukurii to reject the foraged flowers was pronounced, while in C. patellatus and O. orientalis it was weak. In both A. prostomias and T. mitsukurii, the rejection rate of flowers foraged by conspecifics decreased as the time lag after the last visit increased. Both bees visited the flowers from which pollen or nectar had been artificially removed. We suggest that A. prostomias and T. mitsukurii would recognize scent marks left by previous visitors, while the other two bees would not recognize them so strongly. It is likely that the decision to use scent marks is dependent either on the richness of resources or on the complexity of floral structure.     

Genetic and linkage analysis of purple-blue flower in soybean

Flower color of soybean is primarily controlled by genes W1, W3, W4, Wm, and Wp. In addition, the soybean gene symbol W2, w2 produces purple-blue flower in combination with W1. This study was conducted to determine the genetic control of purple-blue flower of cultivar (cv). Nezumisaya. F(1) plants derived from a cross between Nezumisaya and purple flower cv. Harosoy had purple flowers. Segregation of the F(2) plants fitted a ratio of 3 purple:1 purple-blue. F(3) lines derived from F(2) plants with purple-blue flowers were fixed for purple-blue flowers, whereas those from F(2) plants with purple flowers fitted a ratio of 1 fixed for purple flower:2 segregating for flower color. These results indicated that the flower color of Nezumisaya is controlled by a single gene whose recessive allele is responsible for purple-blue flower. Complementation analysis revealed that flower color of Nezumisaya is controlled by W2. Linkage mapping revealed that W2 is located in molecular linkage group B2. Sap obtained from banner petals of cvs. with purple flower had a pH value of 5.73-5.77, whereas that of cvs. with purple-blue flower had a value of 6.07-6.10. Our results suggested that W2 is responsible for vacuolar acidification of flower petals.     

Qualitative and quantitative analyses of flower scent in Silene latifolia

The quantitative and qualitative variability in floral scent of 98 specimens of the dioecious species Silene latifolia belonging to 15 European and 19 North American populations was determined. Floral scent was collected from single flowers using dynamic headspace methods, and analysed by Micro-SPE and GC-MS methods. The flowers showed a nocturnal rhythm, and scent was emitted only at night. The amount of emitted volatiles varied greatly during the season, from 400 ng/flower/2 min in June to 50 ng/flower/2 min in August and September. The qualitative variability in the floral scent was high and different chemotypes, characterised by specific scent compounds, were found. Female and male flowers emitted the same type and amount of volatiles. The differences in floral scent composition between European and North American populations were small. Typical compounds were isoprenoids like lilac aldehyde isomers, or trans-beta-ocimene, and benzenoids like benzaldehyde, phenyl acetaldehyde, or veratrole. Some of these compounds are known to attract nocturnal Lepidoptera species. The high qualitative variability is discussed in relation to the pollination biology of S. latifolia, and the results are compared with other studies investigating intraspecific variability of flower scent.     

Flower Choice and Learning in Foraging Bumblebees: Effects of Variation in Nectar Volume and Concentration

Bees collect food from flowers that differ in morphology, color, and scent. Nectar‐seeking foragers can rapidly associate a flower's cues with its profitability, measured as caloric value or ‘net energy gain,’ and generally develop preferences for more profitable species. If two flower types are equally easy to discover and feed from, differences in profitability will arise from differences in the volume or the sugar concentration of their nectar crops. Although there has been much study of how bees respond to one or the other of these two kinds of nectar variation, few studies have considered both at once. We presented free‐foraging bumblebees with two different types of equally rewarding artificial flowers. After a period of familiarization, we made one type more rewarding than the other by increasing its nectar concentration, volume, or both. Bees responded more rapidly to a change in the reward's sugar concentration than to a change in its volume, even if the profitability differences were approximately equal. Sucrose concentration differences (40% vs. 13%) caused bees to virtually abandon the more dilute flower type, whether both types offered the same volume (2 μl) or the less concentrated reward offered higher volume (7 μl vs. 0.85 μl). When the two types of flower differed only in nectar volume (7 μl vs. 0.85 μl), the less rewarding type continued to receive 22% of the visits. We propose three different hypotheses to explain the stronger response of the bees to changes in sugar concentration: (i) their response threshold to sucrose concentration might change; (ii) less time is needed to assess the concentration of a reward than its volume; and (iii) a smaller sample size may be needed for reliable estimation of profitability when flowers differ in concentration.     

Pollinator-Mediated Selection on Flower Color,Flower Scent and Flower Morphology of Hemerocallis: Evidence from Genotyping Individual Pollen Grains On the Stigma

To trace the fate of individual pollen grains through pollination processes, we determined genotypes of single pollen grains deposited on Hemerocallis stigmas in an experimental mixed-species array. Hemerocallis fulva, pollinated by butterflies, has diurnal, reddish and unscented flowers, and H. citrina, pollinated by hawkmoths, has nocturnal, yellowish and sweet scent flowers. We observed pollinator visits to an experimental array of 24 H. fulva and 12 F2 hybrids between the two species (H. fulva and H. citrina) and collected stigmas after every trip bout of swallowtail butterflies or hawkmoths. We then measured selection by swallowtail butterflies or hawkmoths through male and female components of pollination success as determined by single pollen genotyping. As expected, swallowtail butterflies imposed selection on reddish color and weak scent: the number of outcross pollen grains acquired is a quadratic function of flower color with the maximum at reddish color, and the combined pollination success was maximal at weak scent (almost unrecognizable for human). This explains why H. fulva, with reddish flowers and no recognizable scent, is mainly pollinated by swallowtail butterflies. However, we found no evidence of hawkmoths-mediated selection on flower color or scent. Our findings do not support a hypothesis that yellow flower color and strong scent intensity, the distinctive floral characteristics of H. citrina, having evolved in adaptations to hawkmoths. We suggest that the key trait that triggers the evolution of nocturnal flowers is flowering time rather than flower color and scent.