Flower orientation in Gloriosa superba (Colchicaceae) promotes cross-pollination via butterfly wings.

BACKGROUND AND AIMS: Complex modifications of angiosperm flowers often function for precise pollen placement on pollinators and to promote cross-pollination. We explore the functional significance of the unusually elaborate morphology of Gloriosa superba flowers, which are divided into one hermaphrodite meranthium and five male meranthia (functional pollination units of a single flower). METHODS: We used controlled pollination experiments, floral measurements, pollen load analyses and visitor observations in four populations of G. superba in South Africa to determine the breeding system, mechanism of pollination and role of flower in the promotion of cross-pollination. KEY RESULTS: We established that G. superba is self-compatible, but reliant on pollinators for seed production. Butterflies, in particular the pierid Eronia cleodora, were the primary pollinators (>90 % of visitors). Butterflies brush against the anthers and stigma during nectar feeding and pollen is carried on their ventral wing surfaces. Butterfly scales were positively correlated with the number of pollen grains on stigmas. We demonstrate that the styles were orientated towards clearings in the vegetation and we confirm that the highest proportion of initial visits was to hermaphrodite meranthia pointing towards clearings. CONCLUSIONS: The flower morphology of G. superba results in effective pollen transfer on the wings of butterfly visitors. The style-bearing hermaphrodite meranthium of the flowers orientates towards open spaces in the vegetation, thus increasing the probability that butterflies land first on the hermaphrodite meranthium. This novel aspect of flower orientation is interpreted as a mechanism that promotes cross-pollination.

A pollinator shift explains floral divergence in an orchid species complex in South Africa.

BACKGROUND AND AIMS: Floral diversification driven by shifts between pollinators has been one of the key explanations for the radiation of angiosperms. According to the Grant-Stebbins model of pollinator-driven speciation, these shifts result in morphologically distinct 'ecotypes' which may eventually become recognizable as species. The current circumscription of the food-deceptive southern African orchid Eulophia parviflora encompasses a highly variable monophyletic species complex. In this study, two forms were identified within this complex that differ in distribution, floral morphology, scent chemistry and phenology, and a test was made of whether these differences represent adaptations for different pollinators. METHODS AND RESULTS: Multivariate analysis of floral and vegetative traits revealed that there are at least two discrete morphological forms in the species complex. Field observations revealed that each form is pollinated by a different insect species, and thus represent distinct ecotypes. The early-flowering coastal form which has long spurs and floral scent dominated by sesquiterpene compounds is pollinated exclusively by the long-tongued bee Amegilla fallax (Apidae, Anthophorinae), while the late-flowering inland form with short spurs and floral scent dominated by benzenoid compounds is pollinated exclusively by the beetle Cyrtothyrea marginalis (Cetoniinae; Scarabaeidae). Choice experiments in a Y-maze olfactometer showed that beetles are preferentially attracted to the scent of the short-spurred form. A spur-shortening experiment showed that long spurs are required for effective pollination of the bee-pollinated form. Although it was initially thought likely that divergence occurred across a geographical pollinator gradient, plants of the long-spurred form were effectively pollinated when transplanted to an inland locality outside the natural coastal range of this form. Thus, the underlying geographical basis for the evolution of ecotypes in the E. parviflora complex remains uncertain, although early flowering in the long-spurred form to exploit the emergence of naïve bees may restrict this form to coastal areas where there is no frost that would damage flower buds. Later flowering of the short-spurred form coincides closely with the emergence of the pollinating beetles following winter frosts. CONCLUSIONS: This study identifies a shift between bee and beetle pollination as the main driver of floral divergence in an orchid species complex. Floral scent and spur length appear to be key traits in mediating this evolutionary transition.

Structural anther mimics improve reproductive success through dishonest signaling that enhances both attraction and the morphological fit of pollinators with flowers.

Numerous studies have identified traits associated with anther mimicry; however, the processes underlying floral deception remain poorly documented for these structures. We studied the importance of pollinator attraction and mechanical fit of anther mimics in Tritonia laxifolia (Iridaceae) and their relative contributions to reproductive success. To determine anther mimics role in attraction, we offered bees' binary choices to flowers painted with UV-absorbent and UV-reflecting paints. We also conducted preference experiments between flowers with excised anther mimics and unmanipulated controls, from which mechanical fit was assessed by allowing single visits. Anther mimics' effects on female reproductive success were determined using similar treatments, but on rooted plants. Bees preferred UV-absorbent over UV-reflecting anther mimics. Bees did not discriminate between flowers with and without three-dimensional anther mimics. Single visits resulted in more pollen deposition on unmanipulated controls over flowers with their anther mimics excised, which was directly linked to pollen-collecting behavior. Controls with unmanipulated anther mimics had higher seed set than those with their anther mimics excised. This study provides insights into pollinator-mediated selection on deceptive floral signals and shows that three-dimensional anther mimics increases reproductive success through both attraction and pollen-collecting behaviors.

Changing How Biologists View Flowers-Color as a Perception Not a Trait.

Studying flower color evolution can be challenging as it may require several different areas of expertise, ranging from botany and ecology through to understanding color sensing of insects and thus how they perceive flower signals. Whilst studies often view plant-pollinator interactions from the plant's perspective, there is growing evidence from psychophysics studies that pollinators have their own complex decision making processes depending on their perception of color, viewing conditions and individual experience. Mimicry of rewarding flowers by orchids is a fascinating system for studying the pollinator decision making process, as rewarding model flowering plants and mimics can be clearly characterized. Here, we focus on a system where the rewardless orchid Eulophia zeyheriana mimics the floral color of Wahlenbergia cuspidata (Campanulaceae) to attract its pollinator species, a halictid bee. Using recently developed psychophysics principles, we explore whether the color perception of an insect observer encountering variable model and mimic flower color signals can help explain why species with non-rewarding flowers can exist in nature. Our approach involves the use of color discrimination functions rather than relying on discrimination thresholds, and the use of statistical distributions to model intraspecific color variations. Results show that whilst an experienced insect observer can frequently make accurate discriminations between mimic and rewarding flowers, intraspecific signal variability leads to overlap in the perceived color, which will frequently confuse an inexperienced pollinator. This new perspective provides an improved way to incorporate pollinator decision making into the complex field of plant-pollinator interactions.

Reproductive biology of Acrolophia cochlearis (Orchidaceae): estimating rates of cross-pollination in epidendroid orchids.

BACKGROUND AND AIMS: Pollen fates strongly influence mating success in plants but are difficult to quantify. By promoting foraging constancy in pollinators, floral rewards such as nectar may enhance the overall efficiency of pollen transfer. However, this can also lead to high levels of geitonogamy. Pollen fates were studied in Acrolophia cochlearis, a member of a terrestrial epidendroid orchid genus that includes both rewarding and deceptive species. METHODS: Pollinator observations were conducted. Pollen transfer efficiency (PTE), the proportion of removed pollinia deposited on stigmas, was measured in a large population at regular intervals throughout the 5-month flowering season. The level of cross-pollination in two populations was estimated from the percentage of seeds with embryos in naturally pollinated fruits. KEY RESULTS: Acrolophia cochlearis (and a congener A. micrantha) produce minute but concentrated nectar rewards. Observations showed that A. cochlearis is pollinated exclusively by a solitary bee species, Colletes claripes. Although both sexes visited flowers, only males carried pollinaria. Overall levels of pollination and PTE of the rewarding A. cochlearis were much higher than in a deceptive congener, A. capensis. Seeds resulting from self-fertilization had a significantly lower probability of containing viable embryos than did those from cross-fertilization. This dichotomy in fruit quality was used to estimate that cross-pollination occurred in approx. 66 % of A. cochlearis flowers in a large dense population and approx. 10 % in a small sparse population. Traits of A. cochlearis that limit geitonogamy include pollinarium reconfiguration that exceeds the visit time of pollinators and rapid flower senescence following visitation. CONCLUSIONS: Presence of a nectar reward in Acrolophia cochlearis results in high levels of PTE. It is estimated that approx. 33-90 % of fruits in natural populations arise from self-pollination in this species.

A global test of the pollination syndrome hypothesis.

BACKGROUND AND AIMS: 'Pollination syndromes' are suites of phenotypic traits hypothesized to reflect convergent adaptations of flowers for pollination by specific types of animals. They were first developed in the 1870s and honed during the mid 20th Century. In spite of this long history and their central role in organizing research on plant-pollinator interactions, the pollination syndromes have rarely been subjected to test. The syndromes were tested here by asking whether they successfully capture patterns of covariance of floral traits and predict the most common pollinators of flowers. METHODS: Flowers in six communities from three continents were scored for expression of floral traits used in published descriptions of the pollination syndromes, and simultaneously the pollinators of as many species as possible were characterized. KEY RESULTS: Ordination of flowers in a multivariate 'phenotype space' defined by the syndromes showed that almost no plant species fall within the discrete syndrome clusters. Furthermore, in approximately two-thirds of plant species, the most common pollinator could not be successfully predicted by assuming that each plant species belongs to the syndrome closest to it in phenotype space. CONCLUSIONS: The pollination syndrome hypothesis as usually articulated does not successfully describe the diversity of floral phenotypes or predict the pollinators of most plant species. Caution is suggested when using pollination syndromes for organizing floral diversity, or for inferring agents of floral adaptation. A fresh look at how traits of flowers and pollinators relate to visitation and pollen transfer is recommended, in order to determine whether axes can be identified that describe floral functional diversity more successfully than the traditional syndromes.

Mimics and magnets: the importance of color and ecological facilitation in floral deception.

Plants that lack floral rewards can attract pollinators if they share attractive floral signals with rewarding plants. These deceptive plants should benefit from flowering in close proximity to such rewarding plants, because pollinators are locally conditioned on floral signals of the rewarding plants (mimic effect) and because pollinators are more abundant close to rewarding plants (magnet effect). We tested these ideas using the non-rewarding South African plant Eulophia zeyheriana (Orchidaceae) as a study system. Field observations revealed that E. zeyheriana is pollinated solely by solitary bees belonging to a single species of Lipotriches (Halictidae) that appears to be closely associated with the flowers of Wahlenbergia cuspidata (Campanulaceae), a rewarding plant with which the orchid is often sympatric. The pale blue color of the flowers of E. zeyheriana differs strongly from flowers of its congeners, but is very similar to that of flowers of W. cuspidata. Analysis of spectral reflectance patterns using a bee vision model showed that bees are unlikely to be able to distinguish the two species in terms of flower color. A UV-absorbing sunscreen was applied to the flowers of the orchid in order to alter their color, and this resulted in a significant decline in pollinator visits, thus indicating the importance of flower color for attraction of Lipotriches bees. Pollination success in the orchid was strongly affected by proximity to patches of W. cuspidata. This was evident from one of two surveys of natural populations of the orchid, as well as experiments in which we translocated inflorescences of the orchid either into patches of W. cuspidata or 40 m outside such patches. Flower color and location of E. zeyheriana plants relative to rewarding magnet patches are therefore key components of the exploitation by this orchid of the relationship between W. cuspidata and Lipotriches bee pollinators.

The effects of nectar addition on pollen removal and geitonogamy in the non-rewarding orchid Anacamptis morio.

It has been suggested that the absence of floral rewards in many orchid species causes pollinators to probe fewer flowers on a plant, and thus reduces geitonogamy, i.e. self-pollination between flowers, which may result in inbreeding depression and reduced pollen export. We examined the effects of nectar addition on pollinator visitation and pollen transfer by tracking the fate of colour-labelled pollen in Anacamptis morio, a non-rewarding orchid species pollinated primarily by queen bumble-bees. Addition of nectar to spurs of A. morio significantly increased the number of flowers probed by bumble-bees, the time spent on an inflorescence, pollinarium removal and the proportion of removed pollen involved in self-pollination through geitonogamy, but did not affect pollen carryover (the fraction of a pollinarium carried over from one flower to the next). Only visits that exceeded 18 s resulted in geitonogamy, as this is the time taken for removed pollinaria to bend into a position to strike the stigma. A mutation for nectar production in A. morio would result in an initial 3.8-fold increase in pollinarium removal per visit, but also increase geitonogamous self-pollination from less than 10% of pollen depositions to ca. 40%. Greater efficiency of pollen export will favour deceptive plants when pollinators are relatively common and most pollinaria are removed from flowers or when inbreeding depression is severe. These findings provide empirical support both for Darwin's contention that pollinarium bending is an anti-selfing mechanism in orchids and for the idea that floral deception serves to maximize the efficiency of pollen export.

A temporal dimension to the influence of pollen rewards on bee behaviour and fecundity in Aloe tenuior.

The net effect of pollen production on fecundity in plants can range from negative--when self-pollen interferes with fecundity due to incompatibility mechanisms, to positive--when pollen availability is associated with increased pollinator visitation and fecundity due to its utilization as a reward. We investigated the responses of bees to pollen and nectar rewards, and the effects of these rewards on pollen deposition and fecundity in the hermaphroditic succulent shrub Aloe tenuior. Self-pollinated plants failed to set fruit, but their ovules were regularly penetrated by self-pollen tubes, which uniformly failed to develop into seeds as expected from ovarian self-incompatibility (or strong early inbreeding depression). Bees consistently foraged for pollen during the morning and early afternoon, but switched to nectar in the late afternoon. As a consequence of this differential foraging, we were able to test the relative contribution to fecundity of pollen- versus nectar-collecting flower visitors. We exposed emasculated and intact flowers in either the morning or late afternoon to foraging bees and showed that emasculation reduced pollen deposition by insects in the morning, but had little effect in the afternoon. Despite the potential for self-pollination to result in ovule discounting due to late-acting self-sterility, fecundity was severely reduced in artificially emasculated plants. Although there were temporal fluctuations in reward preference, most bee visits were for pollen rewards. Therefore the benefit of providing pollen that is accessible to bee foragers outweighs any potential costs to fitness in terms of gender interference in this species.

The invasive 'mothcatcher' (Araujia sericifera Brot.; Asclepiadoideae) co-opts native honeybees as its primary pollinator in South Africa.

BACKGROUND AND AIMS: Successful invasive plants such as Araujia sericifera usually either are capable of automatic self-pollination or maintain pollinator services by having generalized pollination systems to make use of local pollinators in the invaded range. Alternatively, plants must co-opt new pollinators with similar morphology to native pollinators or reproduce asexually. We aimed to document the pollination biology of A. sericifera in South Africa. Given the success of this species as an invader, we predicted that sexual reproduction occurs either through self-pollination or because A. sericifera has successfully co-opted native insect pollinators. METHODOLOGY: We examined the pollination biology of the South American A. sericifera in South Africa. We documented the effective pollinators including a comparison of the efficacy of nocturnal versus diurnal pollinators as well as the breeding system and long-term natural levels of the pollination success of this species. PRINCIPAL RESULTS: We found that native honeybees (Apis mellifera) were the main pollinators of A. sericifera in South Africa. Visiting moths are unimportant pollinators despite being attracted by the pale colour and nocturnal scent of the flowers. Plants from the Grahamstown population were incapable of autonomous self-pollination but pollinator-mediated self-pollination does occur. However, the highest fruit initiation resulted from out-crossed pollination treatments. The high pollen transfer efficiency of this species was comparable to other hymenopteran-pollinated exotic and native milkweeds, suggesting that A. sericifera maintains pollinator services at levels experienced by indigenous asclepiad species. CONCLUSIONS: Araujia sericifera reproduces successfully in South Africa due to a combined ability of this species to attract and exploit native honeybees as its pollinators and of individual plants to set fruit from pollinator-mediated self-pollination.

Pollinators, "mustard oil" volatiles, and fruit production in flowers of the dioecious tree Drypetes natalensis (Putranjivaceae).

The Putranjivaceae is an enigmatic family, notable for being the only lineage outside the Capparales to possess the glucosinolate biochemical pathway, which forms the basis of an induced chemical defense system against herbivores (the "mustard oil bomb"). We investigated the pollination biology and floral scent chemistry of Drypetes natalensis (Putranjivaceae), a dioecious subcanopy tree with flowers borne on the stem (cauliflory). Flowering male trees were more abundant than female ones and produced about 10-fold more flowers. Flowers of both sexes produce copious amounts of nectar on disc-like nectaries accessible to short-tongued insects. The main flower visitors observed were cetoniid beetles, bees, and vespid wasps. Pollen load analysis indicated that these insects exhibit a high degree of fidelity to D. natalensis flowers. Insects effectively transfer pollen from male to female plants resulting in about 31% of female flowers developing fruits with viable seeds. Cetoniid beetles showed significant orientation toward the scent of D. natalensis flowers in a Y-maze olfactometer. The scents of male and female flowers are similar in chemical composition and dominated by fatty acid derivatives and isothiocyanates from the glucosinolate pathway. The apparent constitutive emission of isothiocyanates raises interesting new questions about their functional role in flowers.

Anther cap retention prevents self-pollination by elaterid beetles in the South African orchid Eulophia foliosa.

BACKGROUND AND AIMS: Pollination by insects that spend long periods visiting many flowers on a plant may impose a higher risk of facilitated self-pollination. Orchids and asclepiads are particularly at risk as their pollen is packaged as pollinia and so can be deposited on self-stigmas en masse. Many orchids and asclepiads have adaptations to limit self-deposition of pollinia, including gradual reconfiguration of pollinaria following removal. Here an unusual mechanism--anther cap retention--that appears to prevent self-pollination in the South African orchid Eulophia foliosa is examined. METHODS: Visits to inflorescences in the field were observed and pollinators collected. Visitation rates to transplanted inflorescences were compared between a site where putative pollinators were abundant and a site where they were rare. Anther cap retention times were determined for removed pollinaria and atmospheric vapour pressure deficit was recorded concurrently. Anther cap anatomy was examined using light microscopy. KEY RESULTS: Eulophia foliosa is pollinated almost exclusively by Cardiophorus obliquemaculatus (Elateridae) beetles, which remain on the deceptive inflorescences for on average 301 s (n = 18). The anther cap that covers the pollinarium is retained for an average of 512 s (n = 24) after pollinarium removal by beetles. In all populations measured, anther cap dimensions are greater than those of the stigmatic cavity, thus precluding the deposition of self-pollinia until after the anther cap has dropped. An anatomical investigation of this mechanism suggests that differential water loss from regions of the anther cap results in opening of the anther cap flaps. This is supported by observations that as atmospheric vapour pressure deficits increased, the duration of anther cap retention was reduced. CONCLUSIONS: Flowers of E. foliosa are specialized for pollination by elaterid beetles. Retention of anther caps for a period exceeding average visit times by beetles to inflorescences appears to prevent facilitated self-pollination in E. foliosa effectively.

Doing the twist: A test of Darwin's cross-pollination hypothesis for pollinarium reconfiguration.

Mating success in plants depends largely on the efficiency of pollen dispersal. For hermaphrodite plants, self-pollination, either within or among flowers, can reduce mating opportunities because of pollen and ovule discounting and inbreeding depression. Self-pollination may be particularly detrimental in plants such as orchids and asclepiads that package each flower's pollen into one or more pollinia which, together with accessory structures, comprise a pollinarium. Darwin proposed that physical reconfiguration of pollinaria serves as a mechanism for reducing the likelihood of self-pollination. To be effective, the time taken for pollinarium reconfiguration would need to exceed that spent by a pollinator on a plant. We investigated pollinarium reconfiguration (including pollinarium bending, pollinium shrinking and anther cap retention) in 19 species and found a strong positive relationship between reconfiguration time and the duration of pollinator visits. Reconfiguration times were also consistently longer than pollinator visit times. These results provide strong support for Darwin's idea that this mechanism promotes cross-pollination.