Pollinator Shifts Research Articles

Nuclear phylogenomics of Eperua (Leguminosae) highlights the role of habitat and morphological lability in dispersal and diversification across Amazonia and in the Caatinga-Cerrado ecotone.

Eperua is a genus of Neotropical trees that forms a major component of tropical lowland forests in Amazonia, especially in the Guiana Shield and on white-sand forests. One species occurs in the Cerrado-Caatinga ecotone, and the genus also inhabits riverine and terra firme forests. Species in Eperua exhibit one of two drastically different floral architectures and inflorescence types, each associated with distinct pollinators. Prior phylogenetic studies of Eperua have revealed an unstructured topology concerning floral architectures and inflorescence types. In addition, no investigation has been conducted on how the evolution of these traits and habitat preferences influenced the dispersal and diversification of Eperua. Using target capture sequencing, we inferred the most comprehensive phylogeny for Eperua to date, sampling all 19 known species, five for the first time. We used coalescence, concatenation, and network methods to infer the Eperua phylogeny and investigate sources of incongruence impacting resolution and support. We reconstructed the biogeographic history and ancestral states for the flower architecture, inflorescence type, and habitat preference. Our phylogenomic analyses successfully resolved relationships within Eperua, attributing conflicts between the species tree and concatenated tree to gene tree discordance linked to reticulation events. Biogeographical analyses indicate that Eperua originated and initially diversified in the white-sand forests of the Guiana Shield. A subsequent adaptation to riverine and terra firme forests enabled Eperua to expand into new habitats and regions. Still, its historical preference for white-sand forests probably accounts for its absence in the southern and western parts of Amazonia. Ancestral geographic areas and corolla morphotype reconstructions suggest that speciation in Eperua has occurred in sympatry, likely driven by pollinator shifts mediated by drastic changes in floral architecture.

1116. Rhododendron boninense Nakai

SummaryRhododendron boninense Nakai, illustrated here, is a critically endangered endemic rhododendron from the Ogasawara Islands, whose population was once reduced to a single individual. Since the 1980s, Koishikawa Botanical Garden has made efforts to propagate and reintroduce this species to its original habitat, resulting in a current population of 60 mature plants and several natural seedlings. The pure white corolla, which lacks a nectar guide, is distinctive among related rhododendrons, suggesting that a pollinator shift occurred after colonisation of the Ogasawara Islands, where the ancestral pollinators are absent.

Pollen transfer efficiency in Erica depends on type of pollinator.

Pollen transfer efficiency (PTE; the proportion of pollen removed from flowers that reaches conspecific stigmas) is expected to vary with the type of pollinator and flower morphology, and to influence male siring success. Many species in the genus Erica are pollinated by bees (which consume pollen and should thus lower PTE) but during its radiation in the Cape, several independent shifts to both sunbird and long-proboscid fly (LP fly) pollinators, which do not consume pollen have taken place. Improvements in PTE could be one of the factors driving these pollinator shifts. PTE data for 15 Erica species (five for each of the three pollinator types) were collected and compared in relation to type of pollinator and anther exsertion. LP fly- and bird-pollinated species had higher PTE in comparison with bee-pollinated species. Species with inserted anthers had higher PTE than those with exserted anthers. This suggests that sunbirds and LP flies are more efficient pollinators than bees. Additionally, the study suggests that insertion of anthers within the corolla tube can reduce pollen losses.

The only purple-flower species in Actaea L. is pollinated by a hornet

Abstract Examining the pollination biology of plant species is not only crucial for enhancing our understanding of their reproductive biology, but also essential for elucidating their adaptation and evolutionary history. Here, we investigated the breeding system and pollination biology of two closely related species in Actaea. The flower of A. purpurea is unique in the genus with purple and chartaceous (paper-like) sepals, fewer stamens with yellow anthers and purple filaments. Through three seasons of field observation and exclusion experiments, we determined that A. purpurea was primarily pollinated by a hornet species, Vespa bicolor, which also served as the most efficient pollinator. In contrast, A. japonica was primarily pollinated by large flies. Actaea purpurea exhibited a significantly higher cumulative nectar volume than A. japonica, which could be a crucial factor in attracting V. bicolor. A control experiment further demonstrated that removing the nectar leaf (petal) significantly decreased the visiting frequency of V. bicolor. Breeding system studies revealed that both species were self-compatible, yet they primarily underwent outcrossing in natural habitats. Our study presents a compelling case of possible pollinator shift in A. purpurea accompanied by morphological divergence. A more in-depth investigation of this system would offer crucial insights into the extent to which pollinators are involved in the plant speciation process and whether they contribute to reproductive isolation between closely related species.

Recent evolutionary divergence in a plant ring-species is not accompanied by floral phenology or pollinator shifts

Background and aims – The study of factors and processes involved in evolutionary divergence can inform how biodiversity is generated and maintained. We evaluate shifts in phenology or in pollination systems as potential barriers to gene exchange and thus promoters of divergence at the population-species boundary in the plant ring-species Euphorbia tithymaloides in the Caribbean. Material and methods – Combining collections-based and field-based observations and measurements, we evaluate evidence supporting that shifts in tempo of reproductive activity (floral phenology) or pollinator guilds (using visitation as a proxy) could be acting as mechanisms promoting divergence in E. tithymaloides. We focus on the geographic region where evolutionary divergence in this species has been documented: Greater and Lesser Antilles. Phenology data were derived from herbaria and online databases, for a total of 376 records across the Greater and Lesser Antilles. We quantified and characterized reward (nectar n = 13 sites) and gathered visitation data using direct observation (n = 12 sites) for a total of over 133 hours of observation/site. Key results – The peak of floral activity of E. tithymaloides is in winter, when days are short (~late October–late May). Under natural conditions, plants in the Antilles produce up to 22.4 µL of nectar, with mean sugar concentrations of ~ 46.5 ºBrix that amount to up to 10.3 mg of total sugars, with no significant differences observed between plants of the Lesser and Greater Antilles. Hummingbirds are the main floral visitors of E. tithymaloides in both areas: Greater Antilles: 61%, Lesser Antilles: 85%, and network analyses support a floral visitor community turnover across islands/countries. Conclusion – Evolutionary divergence in Caribbean E. tithymaloides along the Greater and Lesser Antilles is not accompanied by shifts in floral phenology or pollinator systems. Other factors, like pollinator turnover or pollinator-plant trait matching, might be at play. We outline hypotheses to this effect.

Pollinator shift ensures reproductive success in a camouflaged alpine plant.

There are intrinsic conflicts between signalling to mutualists and concealing (camouflaging) from antagonists. Like animals, plants also use camouflage as a defence against herbivores. However, this can potentially reduce their attractiveness to pollinators. Using Fritillaria delavayi, an alpine camouflaged plant with inter-population floral colour divergence, we tested the influence of floral trait differences on reproduction. We conducted pollination experiments, measured floral morphological characteristics, estimated floral colours perceived by pollinators, analysed floral scent and investigated reproductive success in five populations. We found that the reproduction of F. delavayi depends on pollinators. Under natural conditions, a flower-camouflaged population had 100 % fruit set and similar seed set to three out of four yellow-flowered populations. Bumblebees are important pollinators in the visually conspicuous yellow-flowered populations, whereas flies are the only pollinator in the flower-camouflaged population, visiting flowers more frequently than bumblebees. The camouflaged flowers cannot be discriminated from the rock background as perceived by pollinators, but may be located by flies through olfactory cues. Collectively, our results demonstrate that the flower-camouflaged population has different reproductive traits from the visually conspicuous yellow-flowered populations. A pollinator shift from bumblebees to flies, combined with high visitation frequency, compensates for the attractiveness disadvantage in camouflaged plants.

Strong habitat and seasonal phenology effects on the evolution of self-compatibility, clonality and pollinator shifts in Lachenalia (Asparagaceae: Scilloideae).

Plants employ a diversity of reproductive safeguarding strategies to circumvent the challenge of pollen limitation. Focusing on southern African Lachenalia (Asparagaceae: Scilloideae), we test the hypothesis that the evolution of reproductive safeguarding traits (self-compatibility, autonomous selfing, bird pollination and clonal propagation) is favoured in species occupying conditions of low insect abundance imposed by critically infertile fynbos heathland vegetation and by flowering outside the austral spring insect abundance peak. We trace the evolution of these traits and selective regimes on a dated, multi-locus phylogeny of Lachenalia and assess their evolutionary associations using ordinary and phylogenetic regression. Ancestral state reconstructions identify an association with non-fynbos vegetation and spring flowering as ancestral in Lachenalia, the transition to fynbos vegetation and non-spring flowering taking place multiple times. They also show that self-compatibility, autofertility, bird pollination and production of multiple clonal offsets have evolved repeatedly. Regression models suggest that bird pollination and self-compatibility are selected for in fynbos and in non-spring flowering lineages, with autofertility being positively associated with non-spring flowering. These patterns support the interpretation of these traits as reproductive safeguarding adaptations under reduced insect pollinator abundance. We find no evidence to support the interpretation of clonal propagation as a reproductive safeguarding strategy.

High floral disparity without pollinator shifts in buzz-bee-pollinated Melastomataceae.

Shifts among functional pollinator groups are commonly regarded as sources of floral morphological diversity (disparity) through the formation of distinct pollination syndromes. While pollination syndromes may be used for predicting pollinators, their predictive accuracy remains debated, and they are rarely used to test whether floral disparity is indeed associated with pollinator shifts. We apply classification models trained and validated on 44 functional floral traits across 252 species with empirical pollinator observations and then use the validated models to predict pollinators for 159 species lacking observations. In addition, we employ multivariate statistics and phylogenetic comparative analyses to test whether pollinator shifts are the main source of floral disparity in Melastomataceae. We find strong support for four well-differentiated pollination syndromes ('buzz-bee', 'nectar-foraging vertebrate', 'food-body-foraging vertebrate', 'generalist'). While pollinator shifts add significantly to floral disparity, we find that the most species-rich 'buzz-bee' pollination syndrome is most disparate, indicating that high floral disparity may evolve without pollinator shifts. Also, relatively species-poor clades and geographic areas contributed substantially to total disparity. Finally, our results show that machine-learning approaches are a powerful tool for evaluating the predictive accuracy of the pollination syndrome concept as well as for predicting pollinators where observations are missing.

A shift between bee and wasp pollinators explains floral divergence in the Duvernoia clade of Justicia (Acanthaceae)

Abstract Floral divergence among sister taxa can arise from pollinator shifts and play a role in speciation. We investigate a potential case of pollinator-driven divergence in the Duvernoia clade of Justicia (Acanthaceae), which includes two species with markedly divergent floral morphology. Pollinator observations and pollen load analyses showed that Justicia aconitiflora is pollinated by vespid wasps, while its sister taxon Justicia adhatodoides is confirmed to be pollinated exclusively by large carpenter bees. Reciprocal nectar-feeding experiments show that wasps and bees are not deterred by the taste of nectar of non-preferred species. Floral morphology matches body and tongue morphology of these two insect groups, and this phenotypic matching mediates pollen transfer and physical access to nectar. Controlled pollination experiments showed that both species are self-compatible, yet dependent on pollinator visits for fruit-set. Historical reconstruction of pollination system evolution in Justicia indicates that the direction of the shift in the Duvernoia clade was from bee to wasp pollinators. This study provides a compelling example of a transition between bee and wasp pollination, and highlights the effects of floral architecture on specialization in plant pollination systems.

Floral colour variation across life history and geography in Mimulus ringens (Phrymaceae)

Abstract Floral and life history traits play important roles in plant speciation. The genus Mimulus is a model system for studying speciation. It includes examples of species in which floral colour facilitates isolation through pollinator shifts, as well as life history changes that result in temporal or ecogeographic isolation. We investigate the possibility that both floral colour and life history have shifted together in a recently described, genetically distinct group within the species Mimulus ringens. Using a large, range-wide citizen science dataset, we test for geographic trends in flower colour and flowering time. We combine this with greenhouse studies in populations of known life history to test for differences in flower colour with life history. We show that darker-flowered plants are more common at higher latitudes, that annual-like populations have darker flowers, and that flowering time varies with latitude only in the subset of populations that have lighter flowers. This suggests that annual-like populations (with the earlier flowering time typical of this life history) are restricted to the northern part of the species range and may have distinct trends in flowering date.

Hybrid Mimulus flowers attract a new pollinator.

Hybridization is common in flowering plants and is believed to be an important force driving adaptation and speciation. The flowers of hybrids often exhibit new trait combinations, which, theoretically, could attract new species of pollinators. In this study, we found that the hybrids between a hummingbird-pollinated species Mimulus cardinalis and a self-pollinated species Mimulus parishii attract bumblebees (Bombus impatiens), a pollinator not attracted to either of the progenitor species. This novel attraction is explained by new combinations of floral traits in hybrids, including, most importantly, petal color, in addition to nectar concentration and corolla size. To understand how petal color variation is perceived by bumblebees, we performed reflectance spectroscopy and multispectral imaging to model the flower appearance in bee vision. This analysis showed that color variation would impact the ease of detection. We also found that YUP, the genetic locus responsible for a large portion of floral color variation and previously shown to be important in bee interactions with other Mimulus species, also played an important role in this novel attraction. These results together suggest that the attraction of new pollinators to hybrid plants could be an underexplored avenue for pollinator shift and speciation.

A new orchid species expands Darwin’s predicted pollination guild in Madagascar

The world-renowned pollination system of the long-spurred orchid Angraecum sesquipedale Thouars and the long-tongued hawkmoth Xanthopan praedicta (Rothschild & Jordan, 1903), from Madagascar, is the best-known example of the predictive power of evolutionary ecology1,2, yet its actual degree of specialisation remains poorly described due to the incompleteness of the pollination record of X. praedicta. Here, we describe another species from Madagascar, an angraecoid orchid distantly related to the genus Angraecum Bory, that has evolved these extreme adaptations to a single pollinator after a pollinator shift. It bears the longest spur of any flowering plant, relative to flower diameter, reaching 33 cm. The discovery of a species with such an exceptionally long spur is a rare event, the most recent dating to 19653. This novelty is described here as Solenangis impraedicta (Figure 1A-F) and discussed in a phylogenetic framework. Its conservation status is assessed as Endangered.

Biogeography of orchids and their pollination syndromes in small Mediterranean islands

AbstractAimsDespite the research on orchid in insular conditions, few studies are focused on the spatial distribution of their reproductive syndromes across complex insular systems. By using island species–area relationships (ISAR), we explore orchid biogeography in the Central Western‐Mediterranean islands. In this study, we aim to investigate variation in ISARs using orchid pollination mechanisms as proxies to establish permanent populations explaining how the c and z parameters of ISARs vary among island types and pollination strategies and defining the most influential factors in shaping orchids' distribution.LocationMediterranean Basin.TaxonOrchidaceae.MethodsChecklist of native orchids was obtained for 112 islands of Central‐Western Mediterranean Basin. The Arrhenius power function () was used to fit ISARs for the total number of orchids as well as for functional groups defined by the pollination strategies, across different island types. We used GLM to investigate the relation between pollination syndromes with area and isolation as well as elevation, island origin, taxa richness of the source area and habitat diversity.ResultsWe found that ISARs differ between continental and volcanic islands depending on isolation. The z‐value was found to be higher for more specialized strategies while the c‐value increases from autogamy to allogamy, supporting the role of these two parameters in understanding distributional dynamics. Distance from the mainland is a negative predictor for all the strategies except when deception is decoupled; island area is a positive predictor only for allogamic, deceptive and food deceptive strategies, while habitat diversity is a positive predictor for allogamic, rewarding and deceptive strategies.Main ConclusionsPollination syndromes contribute in explaining the distribution of orchids in insular conditions. Furthermore, we identified differences in ISARs across pollination syndromes in which the intercept increases when the pollination shifts from a generalist to a more specialized one.

Local adaptation to hummingbirds and bees in Salvia stachydifolia: insights into pollinator shifts in a Southern Andean sage.

Differences among populations in pollinator assemblages can lead to local adaptation mosaics in which plants evolve different floral morphologies and attractive traits. Mountain habitats may promote local adaptation because of differences in environmental conditions with altitude, causing changes in pollinators, and because mountaintops can act as isolated habitats. We studied if the differences in floral shape, size and nectar traits in Salvia stachydifolia can be attributed to variations in the relative contribution of hummingbirds and insects. We studied eight populations of S. stachydifolia in natural and under common garden conditions, to assess whether population differences have a genetic component. We recorded pollinators, their behaviour and visitation rates, and characterized pollinator assemblages. In addition, we measured nectar volume and concentration, and collected flowers to describe floral shape and size variation using geometric morphometric methods. We then applied an unsupervised learning algorithm to identify ecotypes based on morphometric traits. Finally, we explored whether populations with different pollinator assemblages had different climatic and/or elevation preferences. We found that variation in the identity of the main pollinators was associated with differences among populations in all traits, as expected under a local adaptation scenario. These differences persisted in the common garden, suggesting that they were not due to phenotypic plasticity. We found S. stachydifolia populations were pollinated either by bees, by hummingbirds or had mixed pollination. We identified two ecotypes that correspond to the identity of the main pollinator guilds, irrespective of climate or altitude. Variation in S. stachydifolia floral traits did not follow any evident association with bioclimatic factors, suggesting that populations may have diverged as the product of historical isolation on mountaintops. We suggest that differences among populations point to incipient speciation and an ongoing pollinator shift.

Evolution of a non-flying mammal-dependent pollination system in Asian Mucuna (Fabaceae).

Pollinator shifts are often related to speciation in angiosperms, and the relationship between them has been discussed in several plant taxa. Although information on plants pollinated by non-flying mammals in Central and South America and Africa is limited, related research has not been conducted in Asia. Herein, I summarize the available knowledge of pollination in Asian Mucuna (Fabaceae), a genus mainly distributed in the tropics, and discuss the evolution of plants pollinated by non-flying mammals in Asia. Nineteen pollinator species have been recorded and pollination systems have been categorized into four types. An examination of the relationship between Mucuna species and their pollinators from the lineage perspective revealed that all species in M. subgenus Macrocarpa, which are distributed in Asia, are pollinated exclusively by non-flying mammals. Additionally, plants pollinated by non-flying mammals were found to have diverged from bat-pollinated and non-flying mammal-pollinated plants, while plants pollinated by non-flying mammals have evolved multiple times. This is a unique example of evolutionary transition. I hypothesize that the diversification of squirrel species in tropical Asia may have led to the speciation and diversification of Mucuna in Asia. Furthermore, the behavioral and ecological characteristics of bats and birds in Asia differ from the characteristics of those in other regions, implying that Asian Mucuna species do not rely on bat or bird pollinators. The adaptation of floral characteristics to pollinators is not well understood in Asia. Mammal-pollinated plants in Asia may have evolved differently from those in other regions and have unique pollination systems.

Insect pollination for most of angiosperm evolutionary history.

Most contemporary angiosperms (flowering plants) are insect pollinated, but pollination by wind, water or vertebrates occurs in many lineages. Though evidence suggests insect pollination may be ancestral in angiosperms, this is yet to be assessed across the full phylogeny. Here, we reconstruct the ancestral pollination mode of angiosperms and quantify the timing and environmental associations of pollination shifts. We use a robust, dated phylogeny and species-level sampling across all angiosperm families to model the evolution of pollination modes. Data on the pollination system or syndrome of 1160 species were collated from the primary literature. Angiosperms were ancestrally insect pollinated, and insects have pollinated angiosperms for c. 86% of angiosperm evolutionary history. Wind pollination evolved at least 42 times, with few reversals to animal pollination. Transitions between insect and vertebrate pollination were more frequent: vertebrate pollination evolved at least 39 times from an insect-pollinated ancestor with at least 26 reversals. The probability of wind pollination increases with habitat openness (measured by Leaf Area Index) and distance from the equator. Our reconstruction gives a clear overview of pollination macroevolution across angiosperms, highlighting the long history of interactions between insect pollinators and angiosperms still vital to biodiversity today.

Genetic architecture of a pollinator shift and its fate in secondary hybrid zones of two Petunia species

BackgroundTheory suggests that the genetic architecture of traits under divergent natural selection influences how easily reproductive barriers evolve and are maintained between species. Divergently selected traits with a simple genetic architecture (few loci with major phenotypic effects) should facilitate the establishment and maintenance of reproductive isolation between species that are still connected by some gene flow. While empirical support for this idea appears to be mixed, most studies test the influence of trait architectures on reproductive isolation only indirectly. Petunia plant species are, in part, reproductively isolated by their different pollinators. To investigate the genetic causes and consequences of this ecological isolation, we deciphered the genetic architecture of three floral pollination syndrome traits in naturally occurring hybrids between the widespread Petunia axillaris and the highly endemic and endangered P. exserta.ResultsUsing population genetics, Bayesian linear mixed modelling and genome-wide association studies, we found that the three pollination syndrome traits vary in genetic architecture. Few genome regions explain a majority of the variation in flavonol content (defining UV floral colour) and strongly predict the trait value in hybrids irrespective of interspecific admixture in the rest of their genomes. In contrast, variation in pistil exsertion and anthocyanin content (defining visible floral colour) is controlled by many genome-wide loci. Opposite to flavonol content, the genome-wide proportion of admixture between the two species predicts trait values in their hybrids. Finally, the genome regions strongly associated with the traits do not show extreme divergence between individuals representing the two species, suggesting that divergent selection on these genome regions is relatively weak within their contact zones.ConclusionsAmong the traits analysed, those with a more complex genetic architecture are best maintained in association with the species upon their secondary contact. We propose that this maintained genotype–phenotype association is a coincidental consequence of the complex genetic architectures of these traits: some of their many underlying small-effect loci are likely to be coincidentally linked with the actual barrier loci keeping these species partially isolated upon secondary contact. Hence, the genetic architecture of a trait seems to matter for the outcome of hybridization not only then when the trait itself is under selection.

Color strategies of camellias recruiting different pollinators

Most ornithophilous plants have red flowers; this has been associated with ‘the bee avoidance hypothesis’, in which ornithophilous flowers may bear colors that are less conspicuous to bees than melittophilous flowers. In the genus Camellia, C. rusticana and C. japonica bear red flowers and yet recruit different pollinators; the former is entomophilous, while the latter is ornithophilous. C. japonica is considered to have been speciated from a common ancestor later than C. rusticana, accompanying a pollinator shift from insects to birds. Nevertheless, factors explaining the pollinator difference in camellias remain rudimentary. In this study, the color traits of the two camellias were investigated, to determine their color strategy to allure different pollinators.The behavior of bees towards the two camellias was examined by a two-choice assay. Flower color characteristics of the two camellias were analyzed with diffuse reflectance and fluorescence spectra. Based on the visual sensory system of bees and birds, the achromatic contrast, chromatic contrast, intensity, and spectral purity of the two species were evaluated, testing the bee avoidance hypothesis. Furthermore, the compounds responsible for the fluorescence, likely serving as a visual attractant, were identified by NMR and MS.Bees visited C. rusticana flowers almost exclusively and C. japonica hardly at all. Reflectance spectral data showed that C. rusticana petals are more conspicuous to bees than birds due to a UV-reflection secondary peak; and that C. japonica petals exhibited crucially low chromatic contrast against a leaf background to bees, suggesting them to be almost indistinguishable. On the other hand, C. japonica flowers appeared conspicuous to birds. The anthers of C. rusticana exhibited blue fluorescence derived from two anthranilates, while those of C. japonica did not.The two camellias offer different color strategies to be conspicuous to their respective pollinators, and C. japonica seemed to have evolved to avoid bees. Alterations in these color traits may have played a role in pollinator shift.

Did early shifts to bird pollination impose constraints on Salvia flower evolution?

A recent article by Kriebel et al. (2020) examines the relationship between floral shape evolution and pollination shifts in Salvia, a plant genus almost worldwide distributed, but particularly diverse in the New World. Kriebel et al. (2020) argue that a major shift to bird pollination at the origin of the subg. Calosphace (~20 million years ago [Mya]) imposed a legacy of constraints, resulting in significant differences in flower morphology between New World and Old World Salvia. However, reanalyses of the data using hidden states to account for the heterogeneity in evolutionary rates do not support an early origin of bird pollination in this group. Instead, bird pollination may have appeared after the arrival of modern hummingbirds to North America (15.5-12 Mya), as in other North American plant clades. The use of more complex models of ancestral state reconstruction into comparative analyses provides a different perspective to explain morphological differences within Salvia. Our results indicate that bird pollination did not impose constraints on corolla shape evolution. Evolutionary constraints in anther connective and style shapes may have arisen at the origin of Calosphace but they were not associated with shifts to hummingbird pollination, being more likely the product of contingent evolution.

Single gene mutation in a plant MYB transcription factor causes a major shift in pollinator preference

Understanding the molecular basis of reproductive isolation and speciation is a key goal of evolutionary genetics. In the South American genus Petunia, the R2R3-MYB transcription factor MYB-FL regulates the biosynthesis of UV-absorbing flavonol pigments, a major determinant of pollinator preference. MYB-FL is highly expressed in the hawkmoth-pollinated P.axillaris, but independent losses of its activity in sister taxa P.secreta and P.exserta led to UV-reflective flowers and associated pollinator shifts in each lineage (bees and hummingbirds, respectively). We created a myb-fl CRISPR mutant in P.axillaris and studied the effect of this single gene on innate pollinator preference. The mutation strongly reduced the expression of the two key flavonol-related biosynthetic genes but only affected the expression of few other genes. The mutant flowers were UV reflective as expected but additionally contained low levels of visible anthocyanin pigments. Hawkmoths strongly preferred the wild-type P.axillaris over the myb-fl mutant, whereas both social and solitary bee preference depended on the level of visible color of the mutants. MYB-FL, with its specific expression pattern, small number of target genes, and key position at the nexus of flavonol and anthocyanin biosynthetic pathways, provides a striking example of evolution by single mutations of large phenotypic effect.