Hawkmoth Pollination Research Articles

Phenotypic selection mosaic for flower length influenced by geographically varying hawkmoth pollinator proboscis length and abiotic environment.

Biotic and abiotic context may affect the intensity of interspecific interactions and subsequently drive locally particular phenotypic selection patterns on interacting traits. We evaluated the geographical variation of matching traits of the brush-type flowers of Caesalpinia gilliesii and of the proboscis length of its guild of hawkmoth pollinators, as well as their relationship with environmental variables. We assessed the geographical variation of interacting traits (style and filament vs mean proboscis length of the guild of hawkmoths) across seven populations and estimated phenotypic selection on the plant side. Interacting traits showed similar relationships with environmental variables. Phenotypic selection on the plant side was influenced by proboscis length and by environmental conditions. Mean proboscis length of the guild was shorter than previously recorded for the same study area, thus probably shifting the selective optima of flower length. We observed two presumptive coevolutionary cold spots where one-sided negative directional selection is acting on style length. The lack of selection on the pollinator side should be further confirmed. We provided joint evidence, mostly lacking, about the geographical variation of selective pressures on the plant side associated with both proboscis length and abiotic conditions. We suggest that recent environmental change may be shifting floral length optima.

Hawkmoth Pollination Facilitates Long-distance Pollen Dispersal and Reduces Isolation Across a Gradient of Land-use Change

Land-use change is among the top drivers of global biodiversity loss, which impacts the arrangement and distribution of suitable habitat for species. Population-level effects include increased isolation, decreased population size, and changes to mutualistic and antagonistic interactions. However, the extent to which species are impacted is determined by life history characteristics including dispersal. In plants, mating dynamics can be changed in ways that can negatively impact population persistence if dispersal of pollen and/or seed is disrupted. Long-distance dispersal has the potential to buffer species from the negative impacts of land-use change. Biotic vectors of long-distance dispersal have been less frequently studied, though specific taxa are known to travel great distances. Here, we describe population genetic diversity and structure in a sphingophilous species that is experiencing habitat fragmentation through land-use change, Oenothera harringtonii W. L. Wagner, Stockh. & W. M. Klein (Onagraceae). We use 12 nuclear and four plastid microsatellite markers and show that pollen dispersal by hawkmoths drives high gene flow and low population differentiation despite a range-wide gradient of land-use change and habitat fragmentation. By separating the contributions of pollen and seed dispersal to gene flow, we show that most of the genetic parameters are driven by hawkmoth-facilitated long-distance pollen dispersal, but populations with small, effective population sizes experience higher levels of relatedness and inbreeding. We discuss considerations for conservation efforts for this and other species that are pollinated by long-distance dispersers.

Pollination of the long-spurred African terrestrial orchid Bonatea steudneri by long-tongued hawkmoths, notably Xanthopan morganii

Charles Darwin correctly predicted the existence of an extraordinarily long-tongued hawkmoth in Madagascar, based on the length of the floral spur of an Angraecum orchid. This hawkmoth, Xanthopan morganii, is also the longest-tongued hawkmoth on the African mainland, but its ecological role as a pollinator has been virtually unknown outside of Madagascar. We investigated whether X. morganii pollinates Bonatea steudneri, an orchid with floral spurs that vary from 10 to 21 cm in length among populations. At a forest locality in central Kenya, we observed X. morganii individuals with tongues ca. 16 cm in length pollinating B. steudneri plants with spurs ca. 15 cm in length. At other localities in Kenya, we observed visits by the hawkmoths Agrius convolvuli (tongue c. 12 cm) and Coelonia fulvinotata (tongue ca. 10 cm). Pollinaria of B. steudneri are attached to the eyes of hawkmoth visitors. Flowers of B. steudneri show several adaptations for hawkmoth pollination, including anthesis at dusk and nocturnal emission of scent dominated by the volatile compound linalool. This study highlights the potential for predicting certain highly specialized interactions among species by matching of their respective morphological dimensions.

Plant-pollinator interactions in East Asia: a review

Pollination studies in East Asia have been developing rapidly in recent decades. East Asia may provide important information on many aspects of plant-pollinator interactions because of the rich fauna and flora and highly heterogeneous environments that occur there. In this review, plant-pollinator interactions in East Asia were summarized. Bumblebees are important pollinators of many plant species in East Asia, as well as in Europe and North America. Native honeybees may also have important roles in pollination in East Asia. Bird pollination and hawkmoth pollination may be less common in East Asia than in North America. Geographic variation in pollination interactions is expected because several types of pollinators are rare or absent in some habitats or geographic regions. For example, specialized nectar-feeding vertebrates like sunbirds and pteropodid bats are absent from most of East Asia except for some areas in its southern part. Opportunistic nectar-feeding vertebrates may have important roles in pollination where specialized nectar-feeding vertebrates are absent. Human impacts on plant pollinator interactions are understudied in this region. However, climate change, habitat degradation, and invasive species may have negative impacts on plant-pollinator interactions and thus plant reproductive success there. The information available on the plant-pollinator interactions in East Asia is still limited because many plant and pollinator taxa and many types of habitats are understudied.

Floral adaptations of two lilies: implications for the evolution and pollination ecology of huge trumpet-shaped flowers.

Evolutionary transitions among floral morphologies, many of which provide evidence for adaptation to novel pollinators, are common. Some trumpet-shaped flowers are among the largest flowers in angiosperms, occurring in different lineages. Our goal was to investigate the role of pollinators in the evolution of these flowers using Lilium. We investigated floral traits and pollinators of L. primulinum var. ochraceum and L. brownii var. viridulum and reviewed reports of visitors to huge trumpet-shaped flowers. Using a published phylogeny of Lilium, we reconstructed ancestral floral morphological states in Lilium to elucidate the origins of trumpet-shaped lilies. Both lilies are largely self-incompatible and show floral syndromes indicative of hawkmoth pollination. The short trumpet-shaped lily can be pollinated by short-tongued (<40 mm) but not long-tongued hawkmoths (>65 mm), while the huge trumpet-shaped lily can be pollinated by both. A literature review including 22 species of trumpet-shaped flowers suggests that their pollinator guilds commonly include both short- and long-tongued moths. A phylogenetic reconstruction indicates that trumpet-shaped lilies possibly have multiple origins from tepal-reflexed ancestors, at least six of which have evolved huge flowers (>50 mm). Adaptation to short-tongued hawkmoths may have initiated the evolution of trumpet-shaped lilies. Huge trumpet-shaped lilies may have evolved as a response to selection by long-tongued hawkmoths, without excluding the short-tongued ones. This evolutionary pathway leads to a functionally more generalized pollination system instead of an increasingly specialized one and is not necessarily associated with pollinator shifts.

Morphospace exploration reveals divergent fitness optima between plants and pollinators.

The obligate mutualism and exquisite specificity of many plant-pollinator interactions lead to the expectation that flower phenotypes (e.g., corolla tube length) and corresponding pollinator traits (e.g., hawkmoth proboscis length) are congruent as a result of coevolution by natural selection. However, the effect of variation in flower morphology on the fitness of plants and their pollinators has not been quantified systematically. In this study, we employed the theoretical morphospace paradigm using a combination of 3D printing, electronic sensing, and machine vision technologies to determine the influence of two flower morphological features (corolla curvature and nectary diameter) on the fitness of both parties: the artificial flower and its hawkmoth pollinator. Contrary to the expectation that the same flower morphology maximizes the fitness of both plant and pollinator, we found that the two parties have divergent optima for corolla curvature, with non-overlapping fitness peaks in flower morphospace. The divergent fitness optima between plants and pollinators could lead to evolutionary diversification in both groups.

Flower movement balances pollinator needs and pollen protection.

Flower signaling and orientation are key characteristics that determine a flower's pollinator guild. However, many flowers actively move during their daily cycle, changing both their detectability and accessibility to pollinators. The flowers of the wild tobacco Nicotiana attenuata orientate their corolla upward at sunset and downward after sunrise. Here, we investigated the effect of different flower orientations on a major pollinator of N. attenuata, the hawkmoth Manduca sexta. We found that although flower orientation influenced the flight altitude of the moth in respect to the flower, it did not alter the moth's final flower choice. These behavioral observations were consistent with the finding that orientation did not systematically change the spatial distribution of floral volatiles, which are major attractants for the moths. Moreover, hawkmoths invested the same amount of time into probing flowers at different orientations, even though they were only able to feed and gather pollen from horizontally and upward‐oriented flowers, but not from downward‐facing flowers. The orientation of the flower was hence crucial for a successful interaction between N. attenuata and its hawkmoth pollinator. Additionally, we also investigated potential adverse effects of exposing flowers at different orientations to natural daylight levels, finding that anther temperature of upward‐oriented flowers was more than 7°C higher than for downward‐oriented flowers. This increase in temperature likely caused the significantly reduced germination success that was observed for pollen grains from upward‐oriented flowers in comparison to those of downward and horizontally oriented flowers. These results highlight the importance of flower reorientation to balance pollen protection and a successful interaction of the plant with its insect pollinators by maintaining the association between flower volatiles and flower accessibility to the pollinator.

Experimental sympatry reveals geographic variation in floral isolation by hawkmoths.

Under a widely accepted model of pollinator-driven speciation, geographic variation in pollinator assemblage drives floral divergence and automatically causes reproductive isolation. Yet it is unclear whether divergent floral adaptation initially confers strong reproductive isolation, or whether that comes at later stages of speciation and requires other forms of reproductive isolation. This study uses a pair of recently diverged, interfertile and parapatric species in the genus Clarkia to determine whether adaptation to hawkmoths, a novel pollinator functional group, would automatically confer floral isolation upon sympatric contact. First, genetically based differences in floral traits between C. breweri and C. concinna that would be maintained upon migration are quantified. Then scenarios of experimental sympatry are constructed in which arrays of flowers are exposed to the novel pollinator, the hawkmoth Hyles lineata, and pollinator preference and heterospecific pollen transfer are assessed. Source populations from across the ranges of each species are used to understand how geographic variation in floral traits within species may affect floral isolation. Although H. lineata has never been observed visiting C. concinna in the wild, it regularly moves between species in experimental floral arrays. Hawkmoth preference and heterospecific pollen transfer vary both among moths and among geographic source locations of C. concinna. The strength of floral isolation in this system is related to variation in flower size, especially hypanthium tube width, and nectar reward among C. concinna forms. Although C. breweri has adopted a novel hawkmoth pollination system, both ethological and mechanical floral isolation by hawkmoths are incomplete and vary according to the specific phenotype of the C. concinna source population. The results suggest that strong floral isolation is not automatically conferred by a pollinator shift and may require additional evolution of deterrent floral traits and habitat isolation that reduces the immediate spatial co-occurrence of young species.

Pollination in Lilium sargentiae (Liliaceae) and the first confirmation of long‐tongued hawkmoths as a pollinator niche in Asia

AbstractThe geographical distribution of pollinators is an important factor determining the biogeographical pattern of floral evolution and diversification. In Africa, a guild of plant species has converged in a floral syndrome for pollination by long‐tongued hawkmoths (predominantly Agrius convolvuli). It is hypothesized that such floral convergence could track the geographical distribution of long‐tongued hawkmoths, so it may not be confined to Africa. We investigated the pollination biology of Lilium sargentiae E. H. Wilson, which is endemic to China and exhibits traits suggestive of long‐tongued hawkmoth pollination. Lilium sargentiae was visited by A. convolvuli as well as small beetles (Nitidulidae) but pollination was mostly or totally effected by the moth. It was consistent with other hawkmoth‐pollinated plants in terms of floral tube length, nectar traits, tepal reflectance, and scent composition. We present the first experimental evidence for the hypothesis proposed above and for hawkmoth pollination in the widespread and ornamentally and economically important genus Lilium L. in a natural habitat. Our findings imply that long‐tongued hawkmoths (especially the extremely widespread A. convolvuli) as a distinctive pollinator niche may have underlain the evolution of long‐tubed (&gt;8 cm) flowers across different continents.

Floral Trait Variations Among Wild Tobacco Populations Influence the Foraging Behavior of Hawkmoth Pollinators

Most pollinators visit flowers in the search of nectar rewards. However, as the floral nectar can often not be directly detected by pollinators, many flower visitors use secondary metabolites such as odor- or taste-proxies to anticipate nectar quantity and quality. Plants might exploit these sensory inferences of the pollinator to increase their pollination rates without increasing their caloric investment into their floral rewards. Here we investigated the effects of natural variation in certain primary and secondary floral metabolites in three populations of the wild tobacco, Nicotiana attenuata, on the pollination behavior of the hawkmoth Manduca sexta. Although offering the same caloric value per flower, the plants of these populations differ in the compositions and concentrations of sugars within the nectar. Moreover, the flowers of these plants emitted highly contrasting levels of attractive floral volatiles (benzyl acetone), but did not differ in the amounts of defensive nectar metabolites (nicotine). In wind tunnel assays with M. sexta moths, plants from those populations that released the largest amount of benzyl acetone as well as those that had a higher ratio of nectar sucrose were more frequently visited and re-visited by the hawkmoth. High emissions of benzyl acetone additionally correlated with a higher time investment of the moths into individual flowers on each visit, leading to the largest foraging success of the moths on those flowers that were most strongly scented. We propose that it is the variation of flower metabolites and their detection by the pollinator rather than the actual caloric value of the nectar, which determines pollinator visitations to a certain flower population. Hence, plants could potentially create a specialist pollinator community by altering their floral signals, either by producing volatiles that pollinators prefer or by providing nectar sugars that pollinators are most sensitive to, while at the same time keeping the caloric value of their nectar rewards at a constant level. These findings highlight the importance of variations among floral signals and their detection by specific pollinators for plant diversification as well as for the evolution of specific plant-pollinator interactions.

Do honeybees act as pollen thieves or pollinators of &lt;i&gt;Datura wrightii&lt;/i&gt;?

Datura wrightii (Solanaceae), a common shrub in the southwestern United States, bears massive, white, night-blooming flowers that attract and reward hawkmoth pollinators. However, Apis mellifera (honeybee) foragers are often observed on its flowers, especially at dusk and dawn hours. Their foraging activities are focused on the anthers, suggesting they could be pollen thieves. We used a series of observations and manipulative experiments to determine if honeybees are detrimental or beneficial to D. wrightii. We found that honeybees were the most frequent visitors to D. wrightii flowers at both dusk and dawn, and that they removed and carried large amounts of D. wrightii pollen. Flowers were capable of being pollinated at dusk and dawn and a single visit by a honeybee was sufficient to pollinate the flowers and produce fruit. There was no evidence that restricting visitation to diurnal hours yielded fruit set that was different from when we restricted visitation (likely by hawkmoths) to evening hours. These results suggest that honeybees are capable of effective pollination of D. wrightii. Although honeybees might interfere with pollen transmission mediated by their highly specialized hawkmoth pollinators, they may also increase plant fitness by pollinating D. wrightii when hawkmoths are not present.

Genetic Mapping Reveals an Anthocyanin Biosynthesis Pathway Gene Potentially Influencing Evolutionary Divergence between Two Subspecies of Scarlet Gilia (Ipomopsis aggregata).

Immense floral trait variation has likely arisen as an adaptation to attract pollinators. Different pollinator syndromes-suites of floral traits that attract specific pollinator functional groups-are repeatedly observed across closely related taxa or divergent populations. The observation of these trait syndromes suggests that pollinators use floral cues to signal the underlying nectar reward, and that complex trait combinations may persist and evolve through genetic correlations. Here, we explore pollinator preferences and the genetic architecture of floral divergence using an extensive genetic mapping study in the hybrid zone of two Ipomopsis aggregata subspecies that exhibit a hummingbird and a hawkmoth pollinator syndrome. We found that natural selection acts on several floral traits, and that hummingbirds and hawkmoths exhibited flower color preferences as predicted by their respective pollinator syndromes. Our quantitative trait loci (QTL) analyses revealed 46 loci affecting floral features, many of which colocalize across the genome. Two of these QTL have large effects explaining >15% of the phenotypic variance. The strongest QTL was associated with flower color and localized to a SNP in the anthocyanin biosynthesis pathway gene, dihydroflavonol-4-reductase (DFR). Further analysis revealed strong associations between DFR SNP variants, gene expression, and flower color across populations from the hybrid zone. Hence, DFR may be a target of pollinator-mediated selection in the hybrid zone of these two subspecies. Together, our findings suggest that hummingbirds and hawkmoths exhibit contrasting flower color preferences, which may drive the divergence of several floral traits through correlated trait evolution.

Multiple factors contribute to reproductive isolation between two co-existing Habenaria species (Orchidaceae).

Reproductive isolation is a key feature that forms barriers to gene flow between distinct plants. In orchids, prezygotic reproductive isolation has been considered to be strong, because their associations with highly specific pollinators. In this study, the reproductive ecology and reproductive isolation of two sympatric Habenaria species, H. davidii and H. fordii, was investigated by floral phenology and morphology, hand-pollination experiments and visitor observation in southwest China. The two species were dependent on insects for pollination and completely self-compatible. A number of factors have been identified to limit gene flow between the two species and achieved full reproductive isolation. Ecogeographic isolation was a weak barrier. H. fordii and H. davidii had completely overlapped flowering periods, and floral morphology plays an important role in floral isolation. The two species shared the same hawkmoth pollinator, Cechenena lineosa, but the pollinaria of the two orchids were attached on different body parts of pollinators. Prezygotic isolation was not complete, but the interspecific pollination treatments of each species resulted in no seed sets, indicating that unlike many other orchid species, in which the postzygotic reproductive isolation is very weak or complete absence, the post-zygotic isolation strongly acted in the stage of seed production between two species. The results illustrate the reproductive isolation between two species involves multiple plant life-history stages and a variety of reproductive barriers can contribute to overall isolation.

Genetic and environmental integration of the hawkmoth pollination syndrome in Ruellia humilis (Acanthaceae).

The serial homology of floral structures has made it difficult to assess the relative contributions of selection and constraint to floral integration. The interpretation of floral integration may also be clouded by the tacit, but largely untested, assumption that genetic and environmental perturbations affect trait correlations in similar ways. In this study, estimates of both the genetic and environmental correlations between components of the hawkmoth pollination syndrome are presented for chasmogamous flowers of Ruellia humilis , including two levels of control for serial homology. A greenhouse population for quantitative genetic analysis was generated by a partial diallel cross between field-collected plants. An average of 634 chasmogamous flowers were measured for each of eight floral traits that contribute to the hawkmoth syndrome. Genetic correlations (across parents) and environmental correlations (across replicate flowers) were estimated by restricted maximum likelihood. Stigma height, anther height and floral tube length were very tightly integrated in their responses to both genetic and environmental perturbations. The inclusion of floral disc width as a control for serial homology suggests this integration is an adaptive response to correlational selection imposed by pollinators. In contrast, integration of non-homologous traits was low. Furthermore, when comparisons between the dimensions of serially homologous structures were excluded, the genetic and environmental correlation matrices showed little congruence. The results suggest that hawkmoths have imposed strong correlational selection on floral traits involved in the deposition and removal of pollen, and that this is a consequence of stabilizing selection on the relative positions of stigmas and anthers in the face of substantial flower size variation. Low integration of other floral traits, and conflicting patterns of genetic and environmental correlations among these traits, suggest weak or no correlational selection within the range of variability expressed within a population.

Stefan Vogel's analysis of floral syndromes in the South African flora: An appraisal based on 60 years of pollination studies

The concept of floral syndromes has been controversial since it was first proposed by Delpino in the nineteenth century. Some have derided the concept for being typological and inconsistent with the generalized nature of many pollination systems. However, others have lauded it for providing a testable framework for the study of convergent evolution in plants showing specialization to particular functional groups of pollinators. During the 1950s, Stefan Vogel assigned floral syndromes to 810 plant species in South Africa. Now, 60 years later, data on the pollination systems of 272 of these species allow us to test whether the floral syndromes Vogel assigned to them are consistent with their actual pollinators. The overwhelming majority (99.6%) of species in Vogel’s sample that have been investigated in the field appear to be ecologically specialized for pollination by a particular functional pollinator group. We found that Vogel’s syndrome classification accurately predicted pollinators for almost 82% of species. The highest accuracy (93%) was for the prediction of bird pollinators from the floral syndrome of “ornithophily”, while the lowest accuracy (68%) was for the prediction of hawkmoth pollination from the floral syndrome of “sphingophily”. The degree of accuracy of pollinator predictions based on floral syndromes did not vary significantly among plant families, despite differences in floral construction and levels of ecological specialization among these lineages. This study provides support for the concept of floral pollination syndromes by demonstrating the existence of strong associations between floral phenotypes and pollinators in a region of high biodiversity.

Gain and Loss of Floral Scent Production through Changes in Structural Genes during Pollinator-Mediated Speciation

The interactions of plants with their pollinators are thought to be a driving force in the evolution of angiosperms. Adaptation to a new pollinator involves coordinated changes in multiple floral traits controlled by multiple genes. Surprisingly, such complex genetic shifts have happened numerous times during evolution. Here we report on the genetic basis of the changes in one such trait, floral scent emission, in the genus Petunia (Solanaceae). The increase in the quantity and complexity of the volatiles during the shift from bee to hawkmoth pollination was due to de novo expression of the genesencoding benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) and benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase (BPBT) together with moderately increased transcript levels for most enzymes of the phenylpropanoid/benzenoid pathway. Loss of cinnamate-CoA ligase (CNL) function as well as a reduction in the expression of the MYB transcription factor ODO1 explain the lossof scent during the transition from moth to hummingbird pollination. The CNL gene in the hummingbird-adapted species is inactive due to a stop codon, but also appears to have undergone further degradation over time. Therefore, we propose that loss of scent happened relatively early inthe transition toward hummingbird pollination, and probably preceded the loss of UV-absorbing flavonols. The discovery that CNL is also involved in the loss of scent during the transition from outcrossing to selfing in Capsella (Brassicaceae) (see the accompanying paper) raises interesting questions about the possible causes of deep evolutionary conservation of the targets of evolutionary change.

The long and the short of it: a global analysis of hawkmoth pollination niches and interaction networks.

1. Proboscis length has been proposed as a key dimension of plant pollination niches, but this niche space has not previously been explored at regional and global scales for any pollination system. Hawkmoths are ideal organisms for exploring pollinator niches as they are important pollinators in most of the biodiverse regions of the earth and vary greatly in proboscis length, with some species having the longest proboscides of all insects. 2. Using datasets for nine biogeographical regions spanning the Old and New World, we ask whether it is possible to identify distinct hawkmoth pollination niches based on the frequency distribution of proboscis length, and whether these niches are reflected in the depths of flowers that are pollinated by hawkmoths. We also investigate the levels of specialization in hawkmoth pollination systems at the regional and community level using data from interaction network studies. 3. We found that most regional hawkmoth assemblages have bimodal or multimodal distributions of proboscis length, and that these are matched by similar distributions of floral tube lengths. Hawkmoths, particularly those with longer proboscides, are polyphagous and at the network level show foraging specialization equivalent to or less than that of bees and hummingbirds. In the case of plants, shorter-tubed flowers are usually visited by numerous hawkmoth species, while those that are longer-tubed tend to exclude shorter-proboscid hawkmoths and thus become ecologically specialized on longer-proboscid hawkmoth species. Longer-tubed flowers tend to have greater nectar rewards and this promotes short-term constancy by long-proboscid hawkmoths. 4. Our results show that pollinator proboscis length is a key niche axis for plants and can account for patterns of evolution in functional traits such as floral tube length and nectar volume. We also highlight a paradoxical trend for nectar resource niche breadth to increase according to proboscis length of pollinators, while pollinator niche breadth decreases according to the tube length of flowers.

Hawkmoths evaluate scenting flowers with the tip of their proboscis.

Pollination by insects is essential to many ecosystems. Previously, we have shown that floral scent is important to mediate pollen transfer between plants (Kessler et al., 2015). Yet, the mechanisms by which pollinators evaluate volatiles of single flowers remained unclear. Here, Nicotiana attenuata plants, in which floral volatiles have been genetically silenced and its hawkmoth pollinator, Manduca sexta, were used in semi-natural tent and wind-tunnel assays to explore the function of floral scent. We found that floral scent functions to increase the fitness of individual flowers not only by increasing detectability but also by enhancing the pollinator's foraging efforts. Combining proboscis choice tests with neurophysiological, anatomical and molecular analyses we show that this effect is governed by newly discovered olfactory neurons on the tip of the moth's proboscis. With the tip of their tongue, pollinators assess the advertisement of individual flowers, an ability essential for maintaining this important ecosystem service.

Reproductive isolation between Zaluzianskya species: the influence of volatiles and flower orientation on hawkmoth foraging choices.

Floral trait differences between related species may play a key role in reproductive isolation imposed by pollinators. Volatile emissions can influence pollinator choice, but how they act in combination with traits such as flower orientation is rarely studied. We compared flower-opening patterns, morphology, colour, orientation and volatile emissions for two closely related species of Zaluzianskya and their natural hybrids. Hawkmoth pollinators were tested for preference between flowers of the two species, and between flowers with manipulations of volatiles or orientation. Flowers of Z.natalensis and Z.microsiphon open at night and day, respectively, but they overlap during early evening, when hawkmoths showed a strong preference for Z.natalensis. The species have similar flower size and colour, but Z.natalensis emits more floral volatiles in the evening and presents flowers vertically face-up as opposed to horizontally in Z.microsiphon, whereas natural hybrids are intermediate. Adding methyl benzoate and linalool to flowers of Z.microsiphon did not increase hawkmoth attraction, but re-orientation of flowers to face vertically increased attraction when scent cues were present, whereas re-orientation of Z.natalensis flowers to face horizontally decreased attraction. This study highlights the importance of flower orientation in imposing reproductive isolation.

Scent emission profiles from Darwin’s orchid – Angraecum sesquipedale: Investigation of the aldoxime metabolism using clustering analysis

The display of scent is crucial for plants in attracting pollinating insects to flowers and ensuring successful pollination and reproduction. The large number of aldoxime volatile species present in the scent of the Madagascan orchid Angraecum sesquipedale has been suggested to play a primary role in attracting the sphingid moth Xanthopan morgani praedicta. By solid phase micro-extraction (SPME) coupled with gas chromatography–mass spectrometry (GC–MS), we monitored the scent release from different flowers of a single orchid, day and night throughout the entire flowering period. In separate experiments, the diurnal release was monitored in 3h intervals and the tissue specific release from the different floral parts was tracked. Numerous novel compounds related to the aldoxime metabolism not previously detected in A. sesquipedale were identified and positioned into a proposed pathway for aldoxime metabolism. From the results, we hypothesize that (E/Z)-phenylacetaldoxime and its derivatives could be important attractants for the pollinating moth X. morgani praedicta. By applying an untargeted Partitioning Around Medoids (PAM) cluster analysis to the metabolite profiles in the scent, the proposed pathways for the formation of aldoximes were substantiated. With this study, we demonstrate the powerful utility of a bioinformatics tool to aid in the elucidation of the routes of formation for volatiles and provide a benchmark and guidelines for future detailed observations of hawkmoth pollination of Angraecum species, and in particular A. sesquipedale, in the wild.